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Implications of Glycosylation in Alzheimer’s Disease

Implications of Glycosylation in Alzheimer’s Disease fnins-14-625348 January 2, 2021 Time: 16:0 # 1 REVIEW published: 13 January 2021 doi: 10.3389/fnins.2020.625348 Implications of Glycosylation in Alzheimer’s Disease Henriette Haukedal and Kristine K. Freude Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark Alzheimer’s disease (AD) is the most common cause of dementia, affecting millions of people worldwide, and no cure is currently available. The major pathological hallmarks of AD are considered to be amyloid beta plaques and neurofibrillary tangles, generated by respectively APP processing and Tau phosphorylation. Recent evidence imply that glycosylation of these proteins, and a number of other AD-related molecules is altered in AD, suggesting a potential implication of this process in disease pathology. In this review we summarize the understanding of glycans in AD pathogenesis, and discuss how glycobiology can contribute to early diagnosis and treatment of AD, serving as potential biomarkers and therapeutic targets. Furthermore, we look into the potential link between Edited by: the emerging topic neuroinflammation and glycosylation, combining two interesting, and David Blum, INSERM U1172 Centre de Recherche until recent years, understudied topics in the scope of AD. Lastly, we discuss how new Jean Pierre Aubert, France model platforms such as induced pluripotent stem cells can be exploited and contribute Reviewed by: to a better understanding of a rather unexplored area in AD. Tony Lefebvre, Lille University of Science Keywords: Alzheimer’s disease, glycans, neuroinflammation, AD biomarkers, AD therapeutics, iPSCs and Technology, France Maud Gratuze, Washington University in St. Louis, INTRODUCTION United States *Correspondence: Alzheimer’s Disease Kristine K. Freude Life expectancy has been steadily increasing over the past 200 years, first due to improvements kkf@sund.ku.dk within housing, sanitation and education, followed by the revolutionary development of vaccines Specialty section: and antibiotics. Although these improvements have significantly reduced the early/mid-life This article was submitted to mortality, together with an exponentially aging population we are facing new health challenges Neurodegeneration, (American Association for the Advancement of Science, 2008). More than 50 million people a section of the journal worldwide are affected by dementia, and the numbers are expected to reach 150 million in 2050, Frontiers in Neuroscience making it not only a global health concern, but also an economical burden. Approximately 70% Received: 02 November 2020 of all dementia cases are caused by Alzheimer’s disease (AD), making it the most prevalent of all Accepted: 17 December 2020 neurodegenerative disorders (Alzheimer’s Association, 2018). AD is characterized by an irreversible Published: 13 January 2021 loss of neurons, particularly in the cortex and hippocampus, causing a progressive decline in Citation: memory. There is currently no cure for AD, and the disease typically runs its course over 5–10 years Haukedal H and Freude KK (2021) after symptoms appear. However, it is believed that AD initiated in the brain decades before clinical Implications of Glycosylation in symptoms appear and a diagnosis can be made, making timely medical intervention substantially Alzheimer’s Disease. more challenging. Research within the area is therefore crucial to develop new early-on diagnostic Front. Neurosci. 14:625348. doi: 10.3389/fnins.2020.625348 tools and potential new therapeutic approaches. Frontiers in Neuroscience | www.frontiersin.org 1 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 2 Haukedal and Freude Implications of Glycosylation in AD Alzheimer’s disease can be categorized as familial (fAD) Thus, targeting these cells will have to be directed toward the or sporadic (sAD). sAD is by far the most common form detrimental effects, whilst maintaining the protective properties. of AD, accounting for approximately 95% of all cases, and Another issue, as previously mentioned, is that AD is often far is known to be a multifactorial disease where genetic risk progressed when diagnosed. Therefore, it is important to identify factors in combination with adverse environmental factors play new preclinical biomarkers for AD together with potential a role in disease development. Although age is recognized as new targets. Recent studies have suggested that glycosylation is the most common non-modifiable risk factor, genetic factors implicated in AD, and considering the fact that most known AD- such as the +4 allele of Apolipoprotein E (APOE4) are known related molecules are either modified with glycans, or play a role to increase the risk of developing sAD. More recent, genetic in glycan regulation, glycobiology represent an interesting new profiles related to the innate immune system have been shown insight into the understanding of AD, and a potential for new to increase the risk of sAD, emphasizing the potential role of therapeutic approaches. non-neuronal cells and neuroinflammation in neurodegenerative disorders (Efthymiou and Goate, 2017). fAD on the other GLYCOSYLATION hand, is caused by specific mutations in Presenilin 1 (PSEN1), Presenilin 2 (PSEN2) or Amyloid precursor protein (APP), Biological information is transcribed from DNA to RNA, and affecting APP processing, thus supporting the currently widely can be further translated into proteins. Following translation, accepted “Ab cascade hypothesis.” APP can be processed these proteins are often additionally modified. An important through the amyloidogenic or the non-amyloidogenic pathway, post-translational modification is the attachment of glycans to where the former leads to production of amyloid beta (Ab) such proteins, providing important roles in recognition, energy peptides (Figure 1). In AD there is an imbalance in terms metabolism, signaling and structure. Such modifications also of production and clearance of Ab, and APP processing is provide the possibility of functional diverse products from a shifted toward the amyloidogenic pathway. APP is initially limited number of genes. Glycobiology is the study of the cleaved by b-secretase, followed by g-secretase, causing excessive structure, biology, function and evolution of carbohydrates, production of Ab peptides. These peptides can aggregate into or glycans and their associated aglycone (proteins, lipids or insoluble Ab plaques in the brain (Hardy and Higgins, 1992). any other kind of molecule). All cells and a substantial Extracellular Ab plaques is the main characteristic of AD, number of molecules carry glycans, underlying their biological followed by the formation of intracellular neurofibrillary tangles impact. Glycosylation is known to be the most common post- (NFTs) caused by Tau hyperphosphorylation, and together these translational modification, and more than 50% of all proteins phenotypes are considered the neuropathological hallmarks of are glycosylated. Not surprisingly, it has therefore been suggested AD (Figure 2). to be implicated in a number of diseases. In this review Although the pathology described above is still considered we will give a brief introduction to glycosylation, but for representative of the main AD features, the precise underlying extended information we refer to textbooks such as Essentials of mechanisms have yet to be identified. Other hypotheses have Glycobiology (Varki et al., 2015). recently gained increasing interest. Those include organelle Carbohydrates can be classified as monosaccharides, pathology such as Golgi and endoplasmic reticulum (ER) oligosaccharides or polysaccharides. Monosaccharides are the alterations, as well as neuroinflammation and the role of non- simplest form of carbohydrates and can be linked together neuronal cells such as microglia and astrocytes in AD progression through glycosidic linkages to form the higher saccharide (Kinney et al., 2018). AD patients present with hyperactivation classes. Typically, oligosaccharides consist of less than 20 of the immune cells, most likely due to the inability to clear monosaccharides, while more complex structures are referred Ab plaques, causing a chronic activated state with release of to as polysaccharides. The term glycan refers to carbohydrate pro-inflammatory and neurotoxic factors, further promoting structures that are attached to a protein, lipid or other molecule, neurodegeneration (Figure 2). Several therapeutic approaches forming a glycoconjugate. The complexity of a glycan can be have been suggested in the search for an AD cure, targeting highly variable depending on how many different types of components in each of these hypotheses. However, most of monosaccharides it contains. Furthermore, glycans themselves these have failed in clinical trials, and treatment today is can be modified by phosphorylation, acylation, methylation highly focused on symptom management (Liu et al., 2019). or sulfation, ensuring great diversity in terms of glycan One of the challenges in terms of potential targets in AD function and mechanisms. treatment is that most molecules are not only implicated in AD but have important functions in other pathways, as Mechanisms and Major Types of seen with the approach of targeting the secretases involved Glycosylation in APP processing. While inhibition of these can reduce the amount of Ab peptides, it will also impair Notch signaling, Glycoconjugates are formed when sugar chains are added another g-secretase substrate, essential in proper neurogenesis to proteins, lipids or other molecules, and in mammals 17 (Wolfe, 2012). The same can be seen for neuroinflammation. monosaccharides are commonly found in such glycan structures. Microglia are crucial in maintaining a healthy homeostasis and Glycosylation can occur through various mechanisms, and defeating potential threats in the brain. On the other hand, includes addition of glycans to both proteins and lipids. There are chronic activation leads to pro-inflammatory and toxic effects. two major types of protein glycosylation; the addition of N -linked Frontiers in Neuroscience | www.frontiersin.org 2 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 3 Haukedal and Freude Implications of Glycosylation in AD FIGURE 1 | Amyloid precursor protein processing. APP can be processed either through the non-amyloidogenic (left) or the amyloidogenic pathway (right). In the non-amyloidogenic processing pathway, APP is initially cleaved by a-secretase generating sAPPa and C83. C83 is further processed by g-secretase producing AICD and the non-toxic p3 fragment. In contrary, APP is first processed by b-secretase in the amyloidogenic pathway, leading to formation of sAPPb and C99, where C99 is further cleaved by g-secretase generating AICD and Ab peptides. These peptides are prone to aggregate into the toxic Ab plaques, characteristic for AD. FIGURE 2 | Complex AD Pathology. AD is characterized by the formation of extracellular amyloid beta plaques and intracellular Tau tangles. Additionally, chronic activation of microglia due to an inability to clear such plaques through phagocytosis, leads to a state of neuroinflammation in these patients, further contributing to disease progression. These neuropathological hallmarks ultimately lead to neurodegeneration and atrophy of the brain. glycans and O-linked glycans (Figure 3). N -glycosylation takes N -glycosylation refers to addition of a N -acetylglucosamine place in the ER and Golgi apparatus. Complex O-glycans are also (GlcNAc) to the peptide backbone, via a b-1N linkage to the mainly built in the Golgi, whereas O-GlcNAcylation occurs in the nitrogen atom of an Asparagine (Asn). The initial process takes cytoplasm, nucleus and mitochondria. place in the ER, where the N -glycan precursor is synthesized Frontiers in Neuroscience | www.frontiersin.org 3 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 4 Haukedal and Freude Implications of Glycosylation in AD FIGURE 3 | Common Glycan Structures. N-glycans consist of the same core consisting of GlcNAc and mannose, and can further be trimmed and modified from this core structure, often with galactose, fucose or sialic acids. High mannose, complex, hybrid and bisecting glycans are common types of such modified N-glycans. O-glycans on the other hand have varying core structures, consisting of either GalNAc or GlcNAc. O-GalNAc are referred to as Mucin-type O-glycans, and are usually found on membrane-bound, extracellular and secreted proteins. O-GalNAcylation is a complex form of O-glycosylation, and four common core structures are shown. O-GlcNAc on the other hand, are usually found on intracellular proteins. from GlcNAc and Mannose. Mannose and glucose units (OGT) and O-GlcNAcase (OGA). O-GlcNAc are attached to are further added to form a glycan structure consisting of Thr and Ser residues, and no further sugars are added. This 14 sugars. The sugar chain is then transferred to a protein is a dynamic process where GlcNAc is rapidly added and containing the sequence Asn-X-Serine (Ser)/Threonine (Thr). removed from various protein substrates (Varki et al., 2015; Following, the added glycan is trimmed by mannosidases Reily et al., 2019). and glucosidases in the ER, and further modified by Increasing evidence indicate that both N -glycosylation and glycosyltransferases in the Golgi apparatus to ensure glycan O-glycosylation are implicated in AD, emphasized by the fact maturity (Reily et al., 2019). that the two hallmark proteins APP and Tau carry both potential When studying glycosylation, N -glycans, have the advantage N -glycosylation and O-glycosylation sites. that they are all comprised of a common core. This is however Regulation of Glycosylation not the case in terms of O-glycosylation, a highly diverse form of glycosylation. O-glycans are covalently linked to a Ser/Thr, and in The process of glycosylation is mainly regulated by humans these are commonly GlcNAc or N -acetylgalactosamine the key enzymes glucosidases and glycosyltransferases. (GalNAc). GalNAc-linked glycans are often referred to as mucin- Glucosidases function by hydrolyzing glycan linkages, whereas type O-glycans, and the glycosylation process takes place in glycosyltransferases synthesize the glycan chain. Together they the cis-, medial- and trans-Golgi compartments. In the case determine the structure of the glycan outcome. Mechanisms that of such complex O-glycosylation, trimming of existing sugar regulate the gene expression of these enzymes, or the availability chain does not take place, unlike the pre- and post-processing of specific substrates are therefore crucial in regulating the glycan steps seen in N -glycosylation. Modification of the O-glycan composition of a cell. chains instead occur by specific glycosyltransferases with addition Glycosyltransferases and glucosidases are regulated both at of galactose, GlcNAc, sialic acids or fucose. Such O-glycans a post-transcriptional and post-translational level, and multiple are often found on extracellular and secreted glycoproteins. mechanisms can contribute in altering the expression, activity In contrast, intracellular glycoproteins often contain GlcNAc- or structure of such enzymes. First of all, the formation linked glycans, where the biosynthesis usually takes place of glycans can be regulated by the transcription levels of in the cytoplasm, regulated by O-linked GlcNAc transferase glycosidases and glycosyltransferases. Furthermore, mechanisms Frontiers in Neuroscience | www.frontiersin.org 4 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 5 Haukedal and Freude Implications of Glycosylation in AD FIGURE 4 | Glycosylation regulation. The process of glycosylation can be regulated at several stages; the transcription level of glycosyltransferases and glucosidases, transport and synthesis of sugar donors to the ER and Golgi, trafficking and localization of glycosyltransferases and glucosidases and substrate competition of these enzymes, phosphorylation of glycosyltransferase and glucosidase and total glycan turnover. that control localization and trafficking of these enzymes is roles of these highly diverse macromolecules, amongst others an important regulatory factor, as it influences the access within the fields of cancer, infection and immunity, drug to potential substrates. Transport and synthesis of sugar and pharmaceuticals, fertilization and blood types. Glycans donors to both the Golgi and ER influence the availability are often present at the surface of cells, indicating a role in of substrates, contributing to control of glycan formation. signaling and recognition. However, alongside the elaborate Additionally, the activity of glycosylation enzymes can potentially glycan diversity, comes a great range of functions. The biological be modified through phosphorylation of the cytoplasmic tails role of these macromolecules can be broadly placed into four (Ohtsubo and Marth, 2006), and enzymes competing for the distinct categories. This includes structural and modulatory same substrate will overall have an effect on the glycome roles, extrinsic recognition, intrinsic recognition and host glycan of a given cell (Okada et al., 2015). Moreover, proteolysis mimicry (Table 1; Varki, 2017). that occur in the Golgi apparatus and total glycan turnover The modulatory and structural importance of glycans is through endocytosis at the cell surface can impact glycan underlined by the glycocalyx coating of eukaryotic cells and formation. Thus, multiple regulatory mechanisms are involved polysaccharide layer surrounding a number of prokaryotes, in modulation of glycan expression, contributing to the representing protective and stabilizing effects by providing a highly diverse pool of glycans and their function (Figure 4; physical barrier. Furthermore, proteoglycans can contribute Ohtsubo and Marth, 2006). to maintenance of tissue integrity and overall organization, and ensure correct folding of newly synthesized proteins. Function of Glycans Additionally, glycans modulate protein interactions, and in Although glycobiology has been a field of many mysteries, many cases ligand binding has been seen do be glycosylation- research the past decades have revealed important functional dependent. They can also function as a storage center for essential Frontiers in Neuroscience | www.frontiersin.org 5 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 6 Haukedal and Freude Implications of Glycosylation in AD TABLE 1 | Important glycan functions. the responsible converting enzyme GnT-III (Akasaka-Manya et al., 2010). Studies in neuroblastoma cells have shown an Glycan Function upregulation of GnT-III in response to Ab treatment, and reduced Structural and Intrinsic Extrinsic Host mimicry Ab load in GnT-III transfected cells, hypothesizing a protective Modulatory Recognition Recognition effect of N -glycans, and upregulation of GnT-III as an adaptive response in AD brains (Akasaka-Manya et al., 2010). On the Protection Intracellular signaling Immunity Adaption of glycan other hand, reports have shown that increased levels of bisecting surface N -glycans can promote AD pathogenesis by delaying BACE1 Stabilizer Intracellular protein Antigen uptake Invasive pathogens trafficking degradation. Loss of bisecting N -glycans have been shown to Tissue Integrity intracellular Protein Viral, Bacterial, reduce Ab generation and slow down AD progression in mice Folding Fungal Adhesins (Kizuka et al., 2015). Protein folding SAMPs/DAMPs PAMPs Protein sialylation has been seen to be altered in AD, Storage center Clearance receptors and a decrease in sialyltransferase (ST) has been identified in AD patients, observed in both serum (Maguire et al., 1994) and postmortem brain samples (Maguire and Breen, molecules, providing easy access and proper activity of such 1995). Sialylation is a terminal glycosylation process involving under specific conditions. attachment of sialic acids to glycans/glycoproteins, catalyzed Glycans can act as specific ligands for cell-cell recognition by ST. These sialic acids can play an important role in and cell-matrix interactions, also known as intrinsic recognition. several cellular function by acting as recognition molecules, and Intrinsic glycan functions can impact intracellular glycoprotein N -acetylneuraminic acid (NeuAc) is the most abundant type folding, trafficking and degradation, and play an important role of sialic acids in mammalian cells (Schedin-Weiss et al., 2014). in intracellular signaling. There is evidence that glycans can Furthermore, altered sialylation profiles have been revealed in act as self-associated molecular patterns (SAMPs) to maintain cerebrospinal fluid (CSF) of AD patients (Fodero et al., 2001), a non-activated state of immune cells, or as danger-associated increasing the evidence toward an implicated sialylation and molecular patterns (DAMPs), overall ensuring cell homeostasis. glycosylation process in AD pathogenesis. To elucidate how this In an extrinsic recognition matter, many glycoconjugates are implication contributes to the progression of AD, a number of acting as pathogen-associated molecular patterns (PAMPs) and studies have been conducted, looking into glycosylation of the facilitate antigen recognition and uptake, further emphasizing most common AD-related proteins, which will be described in the immune-related role of glycans. These functions have not the following sections. surprisingly led to host glycan mimicry, with pathogens adapting their glycan surfaces nearly identical to the host, thereby blocking APP recognition of underlying antigens, to increase their invasive Amyloid precursor protein is the precursor protein that when capacities, providing yet another functionality of glycans (Varki cleaved by b-secretase in the amyloidogenic pathway form et al., 2015; Varki, 2017). the toxic, aggregating Ab peptides seen in AD patients. Both The same glycans can also act differently within the same N -glycosylation and O-glycosylation sites have been identified organism, and a glycan that serves a specific function in one in this protein, and both N- and O-glycosylated APP has been tissue, can have a completely different role in another. Given the detected in the CSF of AD patients (Saito et al., 1993). APP highly diverse functional properties of glycans, it is not surprising has two potential sites for N -glycosylation, located at Asn467 that altered glycosylation can play an important role in health and and Asn496 (Påhlsson et al., 1992). APP N -glycans have been disease. Glycan dysregulation have been identified in a number of suggested to play a role in regulating the production of Ab, as neurodegenerative disorders, and in AD the glycosylation profile manipulation of such glycan structures have been observed to of key disease regulators, such as APP, Tau, beta-secretase 1 implicate APP transport and trafficking (Kizuka et al., 2017). (BACE1) and Nicastrin, have been shown to be altered. Inhibiting N -glycan formation or maturity has been shown to reduce the secretion of APP in various models (Tienari et al., 1996; McFarlane et al., 1999, 2000). Furthermore, sialylation of GLYCOSYLATION ALTERATIONS IN APP-linked N -glycans seems to affect APP processing, whereas enhanced sialylation leads to an increase in APP secretion ALZHEIMER’S DISEASE and Ab production (Nakagawa et al., 2006), together with an A lot of key proteins affected in AD are either glycosylated increase in the bisecting GlcNAc level. Such alterations have themselves or have an effect on the glycosylation of other been identified in relation to the familial AD causing mutations proteins (Figure 5 and Table 2), supporting the hypothesis of in the APP gene, such as the London and Swedish mutations, a possible link between glycans and AD. AD patients present known to cause increased Ab42/Ab40 ratio, highlighting the with an abnormal glycan profile, and several studies have shown potential link between glycosylation and AD pathogenesis altered glycosylation patterns in these patients. Increased levels (Schedin-Weiss et al., 2014). of bisecting GlcNAc, which is a special N -glycan structure highly A number of O-glycosylation sites, both for mucin-linked expressed in the brain, have been revealed in AD patients O-glycans and O-GlcNAc, have been identified in APP. (Wang et al., 2019), together with an increased expression of O-GalNAcylation at sites Thr291, Thr292 and Thr576 have been Frontiers in Neuroscience | www.frontiersin.org 6 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 7 Haukedal and Freude Implications of Glycosylation in AD FIGURE 5 | Glycosylation Sites of major AD molecules. Two N-glycosylation sites have been identified in APP, together with eight sites for O-glycosylation and sites for O-GlcNAcylation. Tau can be modified with O-GlcNAc at four potential sites, and only O-GlcNAcylation is observed in healthy brains. However, N-glycosylation of Tau have been observed in the case of AD, at three different sites. BACE1 only undergoes N-glycosylation, with four identified sites, whereas Nicastrin contains 16 potential N-glycan sites. No glycosylation sites have been observed in the presenilins (PSEN1/2), but studies suggest that these might play a regulatory role in glycosylation of other proteins, such as Nicastrin. The sporadic AD linked molecules APOE and TREM2 contains seven O-glycosylation sites and two N-glycosylation sites, respectively. TABLE 2 | Glycosylation in Alzheimer’s disease. of APP, potentially by increasing non-amyloidogenic alpha- secretase (a-secretase) processing, the level of soluble APPa Gene Glycosylation Type Comment (sAPPa) and thus reducing the secretion of Ab (Jacobsen APP N-glycosylation Altered N-glycosylation can reduce APP and Iverfeldt, 2011). This has been investigated in vitro O-glycosylation processing and secretion. O-glycosylation in HeLa cells transfected with the Swedish APP mutant. reduce Ab secretion Inhibition of O-GlcNAcase in these transfected cells leads BACE1 N-glycosylation Modifications with bisecting GlcNAc increase to increased levels of a-secretase products and decreased b- Ab production, and is increased in AD. secretase processing. Increased O-GlcNacylation of APP can Tau O-GlcNAcylation O-glycosylation potentially play a protective potentially affect the localization of the protein, promoting N-glycosylation role, decreased in AD patients. trafficking to the plasma membrane and decreasing endocytosis N-glycosylation of Tau is only observed in AD conditions, not in healthy controls. (Chun et al., 2015). Nicastrin N-glycosylation Immature and mature version, but effects of The link between O-GlcNAcylation and APP and Ab is N-glycosylation is poorly understood supported by in vivo studies in rats, showing that treatment PSEN1 None Potential regulatory role in glycosylation of with Ab25-35 reduce the level O-GlcNAcylation in these rats. other proteins These findings correlate with up-regulation of glycogen synthase APOE O-glycosylation Alterations could potentially be linked to kinase-3-beta (GSK3b) and increased Tau phosphorylation increased Ab42 level (Lozano et al., 2017). Studies in 5xFAD mice models further TREM2 N-glycosylation Altered N-glycosylation could affect ligand highlight the relationship between O-GlcNAcylation and Ab binding properties and contribute to AD pathogenesis load, showing that inhibiting O-GlcNAcase, and thus enhancing O-GlcNAcylation, reduced the Ab generation by lowering the activity of g-secretase (Kim et al., 2013). In transgenic TAPP mice expressing both mutant human Tau and APP, O-GlcNAcase observed in APP695 in Chinese hamster ovary cells (CHO) (Perdivara et al., 2009). Additionally, complex O-glycosylation inhibition has also been thoroughly investigated, pointing toward a correlation between increased O-GlcNAcylation and reduced sites in APP770 have been revealed at Ser597, Ser606, Ser662, Ser611, Ser680, Thr616, Thr635 and Thr635 in human CSF cognitive decline (Yuzwa et al., 2014). Although studies indicate (Halim et al., 2011). Furthermore, APP has been seen to undergo that there is a potential link between O-GlcNAcylation of APP and the Ab load in AD patients, it is important to bear in O-GlcNAcylation (Griffith et al., 1995), and it has been suggested that such simple O-glycosylation can implicate the processing mind that other AD-related molecules are O-glycosylated, and Frontiers in Neuroscience | www.frontiersin.org 7 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 8 Haukedal and Freude Implications of Glycosylation in AD could thus contribute to the effects seen by altered glycosylation to complex glycosylation, and a mature version carrying such (Schedin-Weiss et al., 2014). complex N -glycans. It has been hypothesized that the maturity level is dependent on presenilins (Yang et al., 2002), and in BACE1 PSEN1/2 knock-out cells the mature form of nicastrin cannot In the amyloidogenic pathway, APP is initially processed by be identified, potentially due to impaired intracellular trafficking. BACE1, producing soluble b-APP fragments (sAPPb) and C99. However, the maturity level does not seem to affect its function C99 is further cleaved by g-secretase to produce Ab peptides (Herreman et al., 2003). Specific glycan structures related to of various lengths, with Ab42 being the most toxic variant, nicastrin in neurons should be further elucidated to evaluate its prone to aggregate into the senile plaques’ characteristic for role in nicastrin function. an AD brain. BACE1 can be N -glycosylated at four potential sites, and modifications at these sites have been suggested Presenilin 1 to affect the activity of the enzyme. On the other hand, no As previously stated, PSEN1 and 2 is the catalytic subunit of g- O-glycosylation sites have been detected in BACE1. It has secretase. Although no glycosylation sites have been identified, been shown that the maturity and correct folding of BACE1 several studies have shown a regulatory role of presenilins is highly dependent on N -glycan modifications, and that the in terms of glycosylation of other proteins, such as nicastrin number of such N -glycans correlates directly with folding, discussed in the previous section. PSEN1 and 2 have also been secretion rates and activity of the enzyme (Vanoni et al., 2008). observed to affect the glycosylation and sialylation of the neural BACE1 has been reported to be highly modified with bisecting cell adhesion molecule (NCAM), essential for brain function GlcNAc, and as previously discussed, in vivo studies in mice (Farquhar et al., 2003), and have an impact on the receptor show that knock-out of the Mgat3-gene, encoding the GnT- Tyrosine-related kinase B, known to play a role in neural III enzyme, improve cognitive impairment and reduce Ab differentiation (Naruse et al., 1998). It has thus been suggested deposition (Kizuka et al., 2015). In vitro studies with Mgat- that the Presenilins can affect the glycosylation process in a 3 knock-out cells have revealed a shift in BACE1 localization number of proteins, either directly or by affecting the cellular toward late endosomes/lysosomes and thus leading to increased location of proteins (Schedin-Weiss et al., 2014). degradation. BACE1 localization in endosomal compartments is required for APP processing, and a shift toward lysosomal Tau localization is suggested to be the cause of the drastic Ab Together with Ab plaques, neurofibrillary tangles (NFTs) reduction observed in knock-out Mgat3 studies (Tan and Evin, are considered the neuropathological hallmark of AD. These 2012). As bisecting GlcNAc on BACE1 is upregulated in AD are found intracellularly and they are caused by abnormal patients, and could potentially also be linked to the oxidative hyperphosphorylation of the Microtubule-associated protein stress observed in AD (Kizuka et al., 2016), inhibiting the (MAP) Tau, which causes intraneuronal accumulation of paired GnT-III might be an interesting approach to reduce the Ab helical filaments (PHF) eventually forming the NFTs (Iqbal et al., load, indirectly targeting the BACE1 activity, yet circumventing 2005). Tau is a cytosolic protein, and has been seen to undergo the issues of adverse effects seen with BACE1 inhibitors both N -glycosylation and O-GlcNAcylation, which is interesting (Kizuka et al., 2017). given the fact that N -glycans are usually a modification seen BACE1 can also be linked to the altered protein sialylation with extracellular proteins, or at membrane-bound proteins seen in AD patients. One of the BACE1 substrates is known to extracellular domain. However, such N -glycosylation has been be b-galactoside a2,6-sialyltransferase-1 (ST6GaI1), and BACE1 identified in AD patients, but not in healthy control brains, processing of this protein is required to generate the soluble ST indicating an altered glycosylation process in such patients. form. BACE1 can thus affect sialylation of glycoproteins, and These N -glycans have been identified at three sites; Asn359- enhancement of these processes have been linked to increased Ile-Thr, Asn167-Ala-Thr and Asn410-Val-Ser (Sato et al., 2001; APP secretion and Ab production (Nakagawa et al., 2006). Liu et al., 2002; Losev et al., 2020), and have been proposed to affect the aggregation of Tau (Losev et al., 2019). Tau also g-Secretase (Nicastrin) undergoes O-GlcNAcylation, and four sites have been mapped to After APP is cleaved by b-secretase, Ab peptides are generated human Tau; Thr-123, Ser-208, Ser-400, Ser-409/Ser-412/Ser-413 through further processing of the C99 fragment via g-secretase. (Zhu et al., 2014). In contrast to N -glycosylation, the level of This cleaving enzyme consists of four subunits; nicastrin, PSEN1 O-GlcNAcylation has been observed to be decreased in AD and PSEN2, Presenilin enhancer 2 (Pen-2) and Anterior pharynx- brains compared to controls (Liu et al., 2004). It has thus defective 1. Amongst these subunits, nicastrin has been suggested been suggested that O-GlcNAcylation can play a protective to be involved in g-secretase substrate interactions (Bolduc et al., role against the pathological hyperphosphorylation seen in 2016), and it is the only subunit of g-secretase that is known AD. This is potentially due to the fact that O-GlcNAcylation to be N -glycosylated, containing as much as 16 potential sites. and phosphorylation of Tau are two competing processes. Nicastrin contributes to g-secretase activity by interacting with Abnormal Tau phosphorylation could be caused by decreased PSEN1 and PSEN2, the catalytic subunits of the enzyme, and O-GlcNAcylation, which again could be linked to the complex glycosylation of nicastrin has been seen to be dependent metabolic alterations/deficiencies observed in AD and other on these presenilins (Yu et al., 2000). Two forms of nicastrin have neurodegenerative disorders, underlying the potential role been identified, an immature form with N -glycans not subjected of glycosylation in disease progression (Liu et al., 2004; Frontiers in Neuroscience | www.frontiersin.org 8 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 9 Haukedal and Freude Implications of Glycosylation in AD Zhu et al., 2014). The link between glycosylation, metabolic in mode of action remains, the +4 genotype has shown disorders and AD is further highlighted in a study investigating to increase both the intraneuronal Ab accumulation and a mice model of Diabetes mellitus (DM). DM has been seen plaque deposition in postmortem AD brains (Yamazaki to increase the risk of cognitive dysfunction, and in these et al., 2019). Interestingly, APOE is as many other proteins mice impaired learning and memory is seen together with modified by glycosylation. O-glycosylation of APOE was obesity and hyperglycemia. These findings correlate with first identified at Thr194, and newly secreted APOE was decreased O-GlcNAcylation and increased Tau phosphorylation, found to be highly sialylated (Wernette-Hammond et al., whereas hypoglycemic therapy improved these phenotypes, 1989). More recently, a number of O-glycosylation sites causing increased levels of O-GlcNAc transferase (Huang R. have been identified, and the glycan profile has been seen et al., 2020). Transgenic mice models of AD show the same to differ in the lipid-binding domain of APOE in CSF and trend, with an upregulation of Tau phosphorylation and plasma, indicating tissue specific glycoforms. Sialylated reduced O-GlcNAcylation in the hippocampus, supporting glycans are more abundant on the lipid-binding domain of the findings of an imbalance between O-GlcNAcylation and CSF APOE, and could indicate that glycosylation plays a Tau phosphorylation (Gatta et al., 2016). This imbalance role in the flexibility of lipoprotein-binding (Flowers et al., could potentially also affect the localization of Tau proteins 2020). APOE is known to interact with Ab, and it has been (Lefebvre et al., 2003), and an overall affect on both function suggested that the sialic moiety of APOE affects this interaction, of Tau and formation of neurofibrillary tangles seen in AD thus being an important contributor in AD development patients (Shane Arnold et al., 1996). These findings highlight the (Sugano et al., 2008). Altered glycosylation profile of ApoE potential of O-GlcNAcase inhibitors in treatment of AD, with has been observed in a mouse model of Niemann-Pick the aim to prevent the pathological hyperphosphorylation of Type C (NPC), a cholesterol-storage disorder that causes Tau (Yuzwa et al., 2014). However, increased insight into the neurodegeneration, which shares some of the pathological specific mechanisms of O-GlcNAcylation of Tau is required to mechanisms seen in AD, including Ab deposition. In this elucidate if the hyperphosphorylation in AD is in fact a cause or study, they identified a potential link between changes in ApoE a consequence of decreased O-GlcNAcylation. glycosylation and increased level of the toxic Ab42 peptide The altered glycosylation of the main AD-related molecules (Chua et al., 2010). described above provides an evident implication of glycans in Although the precise mechanisms of how APOE glycosylation AD. Furthermore, organelle pathology such as ER stress and contributes to AD pathogenesis have yet to be elucidated, Golgi fragmentation have been observed in AD conditions, and there is clear indication that this process is implicated in could be a very interesting link to the glycan alterations seen the disease. Although the APOE +4 allele is known as the in AD. A plausible hypothesis could be that the excessive Ab strongest genetic risk factor for developing sAD, genome accumulation and Tau phosphorylation cause a fragmentation wide association studies (GWAS) have identified a number of the Golgi, by causing inactivation of major Golgi proteins of genes that confers an increased risk of sAD. These are and cytoskeleton disruption, respectively (Joshi et al., 2015). especially genes linked to the innate immune system, such Golgi fragmentation will most likely affect glycosylation, as as Triggering receptor expressed on myeloid cells-2 (TREM2), this is the location for a majority of these processes, and highlighting the role of microglia and neuroinflammation in AD could thus contribute to the glycan alterations seen in AD (Efthymiou and Goate, 2017). Altered glycosylation pattern of patients. Consequently, as the altered glycosylation can affect TREM2 has been identified in AD, and provides an interesting APP processing and Ab load, together with Tau phosphorylation, link between glycosylation and neuroinflammation. This link it is not unlikely that it becomes a vicious cycle, enhancing is further emphasized by the fact that glycosylation plays each of these phenotypes. Elucidating the mechanisms behind an important role in cell-to-cell, as well as cell-environment this pathway would therefore be an interesting approach in the interactions, which the immune system is highly dependent search for an AD cure. on. Neuroinflammation and glycosylation will therefore be In addition to the molecules described above, glycosylation elaborated in the following section. has been observed to affect APOE, identified as a risk factor for developing sAD. NEUROINFLAMMATION AND APOE and Sporadic AD GLYCOSYLATION ApoE is a cholesterol carrier important in transport of lipids and injury repair in the brain, and genetic variants Until recently, AD research has been focused on neural within the APOE gene have been identified as the strongest pathology. However, increasing evidence support an important genetic determinants of sAD risk (Liu et al., 2013). Three role of glial cells, such as microglia and astrocytes in disease polymorphic alleles have been identified, with the +4 allele pathogenesis. Neuroinflammation is a common characteristic being known to cause increased risk of developing AD. seen in AD brains, and is now considered a highly interesting On the other hand, the +2 allele has a protective role, topic within the field of neurodegenerative disorders (Fakhoury, whereas the most common +3 variant has a neutral effect 2017). Especially the role of microglia has become evident, and (Chartier-Harlin et al., 1994). Studies have shown a clear link the cells and genes related to the innate immune systems can be between APOE genotype and Ab, and although uncertainties affected by glycans. Frontiers in Neuroscience | www.frontiersin.org 9 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 10 Haukedal and Freude Implications of Glycosylation in AD explaining the late-onset in AD versus the early-onset seen in Microglia and Glycosylation Alterations NHD (Li and Zhang, 2018). Recently, studies have shown that Microglia are the resident macrophages of the brain, responsible TREM2 activity can be modulated by interactions with TMEM59. for the innate immune system in the central nervous system TMEM59 regulates complex glycosylation, secretion and cell (CNS). Although microglia only accounts for about 5% of the surface expression of APP, with overexpression causing defects in glial cell population in the cerebral cortex (Lawson et al., 1990), glycan maturation (Ullrich et al., 2010). Furthermore, TMEM59 they play a vital role in terms of brain homeostasis and protection could play an important role in immunity, as downregulation against potential threats (Mandrekar-Colucci and Landreth, has resulted in anti-inflammatory effects, potentially mediating 2012). In AD however, microglia have been observed to be microglia activity, functioning as a self-defense in microglia. associated with Ab plaques, presenting with a proinflammatory TREM2 have been observed to interact with TMEM59, and phenotype. As they are unable to clear such plaques through potentially mediating the degradation of the protein. TREM2 phagocytosis, microglia in the condition of AD enters a state deficiency cause impaired microglial survival and phagocytic of chronic activation. These microglia release proinflammatory activities in TREM2 knock-out mice, together with elevated levels factors such as IL-1b, IL-6 and TNFa, which can be detrimental to of TMEM59. Downregulation of TMEM59 in these knock-out surrounding neurons, further enhancing the neurodegenerative mice however, reversed such impairments, indicating a role of progression of the disease. Neuroinflammation can therefore elevated TMEM59 in microglial defects. The fact that TMEM59 be of large impact in AD pathogenesis (Mandrekar-Colucci have an effect on both glycosylation and neuroinflammation in and Landreth, 2012; Sarlus and Heneka, 2017). GWAS studies AD provides yet another potential connection between the two have revealed a number of genes related to the innate immune mechanisms (Liu et al., 2020). system as risk factors for developing AD, and lot of these genes are either highly or exclusively expressed by microglia, SIGLECs and Galectins indicating an important microglial role in AD (Verheijen Lectins are carbohydrate binding proteins that can interact and Sleegers, 2018). Among the identified risk factors are specifically with selected sugar structures, and which is a highly the genes TREM2 and CD33 (Karch et al., 2012; Guerreiro exploited feature used in glycoanalysis, as discussed in the et al., 2013). Altered glycosylation profile have been detected in following section (Van Damme, 2011). SIGLECs are lectins that TREM2 variants associated with sAD, providing a potential link play an important role in regulation of the immune response, between microglial neuroinflammation and glycosylation in AD and an important member of the SIGLEC family is CD33, also progression. Furthermore, sialylation, which is altered in AD, known as Siglec-3 (Crocker and Varki, 2001). Together with have been suggested to play an important role in microglia- the evidence of altered sialylation in AD, such as decreased ST mediated neuroinflammation. CD33 is a sialic acid-binding levels in these patients, desialylation of the microglial surface has receptor, within the sialic-acid-binding immunoglobulin-type been shown to be induced by activating stimuli such as LPS, Ab lectin (SIGLEC) family, known to bind sialylated glycans, and and Tau, which can in turn enhance the complement receptor 3 plays an important role in microglia activation (Estus et al., (CR3) mediated phagocytosis of neurons (Allendorf et al., 2020). 2019), highlighting the potential implication of glycosylation CD33 is expressed on microglia, and activation of this receptor and sialylation in AD and other neurodegenerative disorders. can inhibit the phagocytic activity of microglia. On the other Exploitation of these pathways could potentially serve as new hand, sialylation of neurons can also contribute in inhibiting target sites for therapeutic intervention (Puigdellívol et al., 2020). microglial phagocytosis of these neurons, protecting against neurodegeneration. When microglia are activated, such as in AD, TREM2 they release a sialidase activity that can act by desialylating both TREM2 is expressed by microglia, and plays a role in modulating neurons and microglia, promoting phagocytosis of neurons, and the inflammatory response and phagocytosis (Li and Zhang, thus neurodegeneration. Microglial release of galectin-3 (Gal- 2018). Loss of TREM2 has been shown to reduce phagocytosis 3), another lectin member, binding to galactose residues (Dong (Kawabori et al., 2015), whereas TREM2 overexpression has et al., 2018), further activates microglia by Toll-like receptor 4 resulted in increased phagocytic activity (Takahashi et al., 2005). (TLR4) and TREM2 binding, as well as binding to desialylated Genetic variations of TREM2 have been associated with increased neurons and promoting phagocytosis of such (Puigdellívol et al., risk of AD, and especially the identified missense mutation 2020). These finding, together with the GWAS identification R47H have been linked to increased AD risk. Interestingly, of CD33 as a risk factor for AD, underline the connection studies have shown that the glycosylation pattern in this rare between altered sialylation of glycans and neuroinflammation in disease-associated TREM2 variant differs from the one seen in AD pathogenesis, providing new potential targets in preventing wild-type TREM2, having increased terminal glycosylation with neuroinflammation and degeneration. complex oligosaccharides in the Golgi and decreased solubility, On an interesting note, inflammation in AD can be linked potentially affecting the function and ligand binding of the to the mitochondrial dysfunction observed in the disease, receptor, and in this way contribute to AD pathogenesis (Park by increasing the production of reactive oxygen species et al., 2017). Furthermore, the TREM2 variants Y38C and (ROS), and such oxidative stress has been implicated in T66M, linked with the early-onset disease Nasu-Hakola disease AD pathogenesis (Perry et al., 2002). Although glycosylation (NHD) display differences in the N -glycosylation profile, but of mitochondrial proteins is a rather unexplored field, a varies from the profile seen within the R47H variant, potentially number of such proteins have been suggested as targets for Frontiers in Neuroscience | www.frontiersin.org 10 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 11 Haukedal and Freude Implications of Glycosylation in AD glycan modification, and may affect mitochondrial dysfunction animal models. Given that these enzymes are the main regulators and the oxidative stress response. The connection between of glycans, targeted inhibitors of these could make an interesting mitochondrial dysfunction, glycosylation and inflammation has therapeutic approach to cope with the abnormal glycosylation for example been reviewed in relation to Parkinson’s disease (PD) observed in diseases (Rempel and Withers, 2008; Videira et al., (Videira and Castro-Caldas, 2018). Furthermore, alterations of 2018). Glycoanalysis is however more challenging in terms of the glycosylation pattern of mitochondrial proteins have been O-linked glycans, due to the lack of a common core structure, observed in the cerebral cortex of a rat sAD model, indicating and further optimization of tools for studying these is needed. an implication of such processes also in AD (Yu et al., 2017), a potentially important future research area. Detection of AD Biomarkers and Diagnostic Properties GLYCANS AS BIOMARKERS IN Although there are currently no validated glycan biomarkers for AD, several candidates are being investigated in ongoing ALZHEIMER’S DISEASE studies. These biomarkers can be studied in various ways and Treatment of AD is challenging, partially because the disease is can be broadly classified as biochemical (CSF-, blood sample), often far progressed when diagnosed. Pathologically, the course neuroanatomical (CT-, MRI scan), metabolic (PET, SPECT scan), of AD starts decades before the clinical onset, further limiting genetic (e.g., APP/PSEN/APOE profile) and neuropsychological the already restricted treatment options, emphasizing the need (e.g., Memory) (Wattamwar and Mathuranath, 2010). In terms for new biomarkers and diagnostic tools. Even though the field of glycan biomarkers, studies are usually performed in samples of biomarkers in AD has advanced over the past few years, most from postmortem brains, CSF or blood (Figure 5). Identifying studies have focused on markers for Ab and Tau pathology, these biomarkers in serum from patients would be a great together with neurodegeneration, inflammation and synaptic advantage in AD diagnostic, making the procedure as less deficiencies, as reviewed in Zetterberg and Bendlin (2020). Given invasive as possible. the observations of aberrant glycome profiles in AD, these could Several studies have investigated the glycan profile in CSF potentially be used as new early stage biomarkers of AD. In this from AD patients compared to healthy controls. One issue with section tools used to identify and study glycans will be discussed, this approach has been the fact that the CSF contains a rather together with potential glycan biomarkers that can be beneficial low concentration of proteins, and a large volume is often in AD diagnosis and treatment strategies. required. However, recent studies have shown the possibility of performing CSF glycoanalysis with a very small amount of Glycoanalysis fluid (Cho et al., 2019). Increased levels of fucosylated and There are several approaches to glycoanalysis (Figure 6), bisecting GlcNAc in AD CSF has been observed in several including investigation of intact glycoproteins and analysis of studies, which has also been confirmed in postmortem brains. glycan structures after cleavage from their respective protein. In Furthermore, the altered bisecting glycan profile correlates with terms of studying intact glycoproteins as well as the localization CSF levels of phosphorylated and total Tau (Schedin-Weiss et al., of such, lectins are commonly used (Zou et al., 2017). Lectins 2020). A study of the CSF N -glycome in both mild cognitive recognizes and bind specific glycans and can be used for impairment (MCI) and AD patients identifying 90 N -glycan purification by methods such as fluorescence microscopy or structures by mass spectrometry revealed both a significant enzyme-linked immunosorbent assay (ELISA) (Belický et al., increase in bisected N -glycans together with a decrease in overall 2016). Another highly exploited method for intact glycoprotein sialylation. Interestingly, the MCI patients that progressed to assessment is mass spectrometry (MS) (Domínguez-Vega et al., AD all showed such abnormalities in glycan profile, indicating 2018), whereas nuclear magnetic resonance spectroscopy (MRS) that glycan alterations might precede the clinical onset of AD, can be used if a large amount of purified glycans is obtained highlighting their potentials as biomarkers for early AD diagnosis (Zhang et al., 2016). (Palmigiano et al., 2016). Many glycome studies have focused on Removal of glycans, to investigate cleaved glycans, can N -glycans. However, the interplay between various glycosylation be performed either enzymatically or chemically, by peptide pathways have also been assessed both in brain tissue and serum. N -glycosidase F (PNGase F) and hydrazinolysis, respectively. A simultaneous analysis of N - and O-glycomes revealed global PNGase F cleaves N -linked glycans between the inner GlcNAc alterations of protein glycosylation in both MCI and AD patients. at the Asn residues, thus releasing the entire glycan structure. Furthermore, the altered glycosylation pattern appeared to be Furthermore, these released glycans can be analyzed using region-specific in the brain, with O-GlcNAcylation observed to methods such as liquid chromatography (LC), porous graphitic be decreased in the frontal lobe of AD brains, whereas it was carbon chromatography (PGC) or capillary electrophoresis. Prior increased in the hippocampus. Similar changes were observed to the LC analysis, glycans are usually fluorescently labeled. Often in the serum of AD patients, which presented a unique glycol- the advanced LC technique known as ultra-high performance LC fingerprint, that could be exploited to develop a new class (UPLC) is used to enhance the process (Regan et al., 2019). of biomarkers for AD diagnosis, that thus could be obtained To investigate the function of glycoproteins, inhibitors of by a simple blood sample. This profile appears to be specific the regulating enzymes glycosidases and glycosyltransferases are for AD, and differed from other neurodegenerative disorders commonly used, and this technique is often exploited in cell- and (Frenkel-Pinter et al., 2017). Furthermore, blood plasma from Frontiers in Neuroscience | www.frontiersin.org 11 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 12 Haukedal and Freude Implications of Glycosylation in AD FIGURE 6 | Glycoanalysis. Samples from postmortem brains, CSF and blood can all be obtained for glycoanalysis. Blood samples are especially of interest in terms of biomarker identification and AD diagnosis, due to the preferable less invasive procedure. Glycoproteins can be extracted from these samples, and either intact or released glycans can be investigated. Intact glycoproteins is commonly studied by lectin arrays and ELISA or fluorescence microscopy or by mass spectrometry (MS) or nuclear magnetic resonance spectrometry (MRS). To evaluate released glycoproteins, these can be either enzymatically or chemically cleaved, labeled and detected through liquid chromatography (LC), LC-MS, porous graphitic carbon chromatography (PGC) or ultra-high performance LC (UPLC). AD patients has shown alterations in glycosylation of specific such trials have decreased due to the constant failures, pointing immunoglobulins (IgG), providing increased evidence of a toward a shift in treatment strategy, and need for novel targets. correlation between neuroinflammation and glycosylation in AD Aberrant glycosylation can be identified in AD patients (Lundström et al., 2014). Decreased ST activity and altered before the clinical disease onset. Thereby, re-establishing the glycosylation of transferrin, an iron transport mediator, together glycosylation homeostasis could potentially be of interest in drug with clusterin (CLU), an important player in debris clearance development. As previously described, glycosylation is mainly and apoptosis, both being genetically linked to AD, have been regulated by the action of glycosidases and glycotransferases. observed in AD blood samples (Maguire et al., 1994; Van Modulating the function of these enzymes could thus be a Rensburg et al., 2004; Liang et al., 2015), making them potential potential therapeutic strategy (Wang et al., 2019). Furthermore, blood biomarker candidates. An overview of potential glycan the increase in level of bisecting GlcNAc and decreased biomarkers in AD is presented in Table 3. ST activity, together with the abnormal glycosylation linked to neuroinflammation presents potential new targets in AD treatment (Table 4). GLYCANS IN TREATMENT OF Novel Targets ALZHEIMER’S DISEASE Glycosyltransferase Inhibitors and Glycosidase Inhibitors Today, only symptomatic treatment options exist for AD, and five FDA-approved prescription drugs are currently The activity of glycosyltransferases and glycosidases could be available. This includes the cholinesterase inhibitors Aricept modulated directly or indirectly, and several methods can be (donepezil), Exelon (rivastigmine) and Razadyne (galantamine), used. One example is to inhibit the metabolism of glycan and the N -methyl D-aspartate (NMDA) antagonist Namenda precursors or the glycan transport in the Golgi and ER. Other (memantine). Additionally, in moderate to severe cases of types of inhibition include blocking the addition of N -glycans to AD, Namzaric, a combination of donepezil and memantine, is glycoproteins, for instance by using the inhibitor tunicamycin, commonly used. All of these can only manage symptoms of or to use glycosidase or mannosidase inhibitors that prevents AD for a certain period of time, but cannot reverse or stop the the formation of mature, complex glycoproteins. A suggested disease progression, highlighting the need of new drug targets glycosyltransferase target in AD treatment is the GnT-III enzyme, (Yiannopoulou and Papageorgiou, 2013). Several candidates responsible for the formation of bisecting glycans, observed to are being investigated in ongoing clinical trials, and a lot of be upregulated in AD patients. GnT-III have been shown to these are focusing on the amyloid-, tau- and neuroinflammatory reduce the Ab deposition in mice, by acting on BACE1, without hypotheses, as reviewed in Huang L. K. et al. (2020). Although compromising the activity of the enzyme. BACE1 has several many anti-amyloid trials are still being conducted, the number of substrates besides APP, important for neural functions, making Frontiers in Neuroscience | www.frontiersin.org 12 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 13 Haukedal and Freude Implications of Glycosylation in AD TABLE 3 | Potential biomarkers for AD diagnosis. of these mice, protecting against brain atrophy (Wang et al., 2020). Inhibitors of the glycosylation enzymes could in this way Biomarker Alteration in AD Detected in target the pathological effects of AD related molecules, while Bisecting GlcNAc Increased Postmortem brains, CSF maintaining the normal functions, thereby minimizing potential and Serum side effects. However, inhibition of OGA will most likely affect a Fucosylated GlcNAc Increased Postmortem brains, CSF great number of proteins, potentially causing chronic O-GlcNAc and Serum elevation. It would therefore be crucial to understand the exact ST activity Decreased Postmortem brains, CSF mechanisms of such inhibitors and the consequences of elevated and Serum glycan levels, before implementing those in human clinical trials Overall sialylation Decreased Postmortem brains, CSF (Wang et al., 2020). and Serum O-GlcNAcylation Region-specific Postmortem brains Sialylation Modulators: Siglecs and Galectins alteration As previously discussed, protein sialylation is implicated in AD, IgG Decreased Plasma complex and decreased levels of ST has been identified in the serum of AD glycosylation and patients. ST are a large group of enzymes that attach sialic acids to sialylation glycoproteins, whereas the removal of such residues is performed Transferrin Decreased CSF, serum by sialidases. High sialylation has been indicated to protect sialylation against neurodegeneration, and targeting the sialylation enzymes Clusterin Decreased Plasma could therefore be of interest in drug development, either by N-glycosylation increasing the sialylation process, or reducing desialylation. Microglia initiate sialidase activity when activated, that desialylate TABLE 4 | Potential therapeutic targets of glycosylation and drug both microglia and neurons causing increased phagocytosis candidates for AD. of surrounding neurons and in that way could contribute Drug Candidates Mode of Action to neurodegeneration. Targeting sialidases could therefore contribute to prevent the neuroinflammation observed in AD. GnT-III inhibitors Reduce the level of bisecting GlcNAc and Ab deposition Furthermore, lectins have been shown to be implicated in O-GlcNAcase inhibitors Reduce deglycosylation and phosphorylation of Tau AD. Gal-3 is released by activated microglia, and contribute to ST modulators Increase sialylation, potentially preventing phagocytosis of neurons, further promoting Ab aggregation. Gal- neuroinflammation 3 could thus be another potential therapeutic target. The sialic Sialidase modulators Reduce desialylation, potentially preventing acid binding microglial receptor CD33, that has been genetically neuroinflammation linked to late onset AD, is yet another promising drug candidate. Gal-3 modulators Reduce phagocytosis of neurons and Ab Single nucleotide polymorphisms in this gene have been shown to production increase the risk of AD. On the other hand, reduced expression of CD33 modulators Increase uptake and phagocytosis of Ab deposits the CD33 sialic acid-binding domain has been shown to confer GM1 Increase Ab clearance and improve memory protection against AD, introducing the potential beneficial use of CD33 inhibitors in AD treatment. Crystal structures of the direct inhibition of the enzyme challenging and presented with CD33 protein have recently been studied, providing structural a number of side effects and abnormalities in mouse models. insights into the sialic binding sites and ligands that can increase Knocking out the GnT-III gene in such models has however phagocytosis and Ab uptake, pointing out the possibility of not shown the same difficulties, and the mice remain healthy in therapeutic intervention at this binding site to promote Ab these studies, indicating that the pathological effects of BACE1 clearance (Miles et al., 2019). could be selectively regulated by GnT-III glycosylation. GnT-III Gangliosides inhibitors could thus potentially be safer drug targets for AD treatment, circumventing the potential side effects seen with In this review, the focus has been on glycosylation of proteins. BACE1 inhibition (Kizuka and Taniguchi, 2018). Inhibition of However, glycosylation of lipids is another common process. glycosidases on the other hand, have been proven beneficial in Gangliosides are a type of glycosphingolipids containing one or terms of Tau pathology. Inhibiting the O-GlcNAcase, as shown more sialic acids, and play an important role in development with the use of compound Thiamet-G (Yuzwa et al., 2008), and protection of the CNS. Ganglioside metabolism is reported has reduced Tau phosphorylation, potentially by reducing the to be associated with AD pathology, and changes in ganglioside deglycosylation of the protein. Tau is modified by O-GlcNAc, profile have been observed in AD patients. Especially the GM1 and increasing the glycosylation could potentially compete ganglioside have been Investigated, and show neuroprotective with and thus reduce the pathological hyperphosphorylation of roles, making it a potential therapeutic target in a number of Tau seen in AD (Bojarová and Køen, 2009). A recent study neurodegenerative disorders, and such beneficial effects have further highlights this potential, presenting a promising new and already been described from clinical trials in the case of both selective OGA inhibitor; MK-8719. In vivo studies in a mouse stroke and PD (Magistretti et al., 2019). In AD, GM1 is observed model of tauopathy has shown that this compound can increase to interact with Ab, and in AD mouse models increased GM1 the level of O-GlcNAc and reduce pathologic Tau in the brains showed reduced Ab accumulation and improved neuropathology Frontiers in Neuroscience | www.frontiersin.org 13 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 14 Haukedal and Freude Implications of Glycosylation in AD (Bernardo et al., 2009). Beneficial effects of GM1 administration have also been observed in an AD rat model, improving memory deficits and spatial learning, potentially by mediating oxidative stress and lipid peroxidation (Yang et al., 2013). The protective actions of GM1 has been suggested to be initiated by increasing autophagy and promoting Ab clearance by microglia (Yuyama et al., 2014; Dai et al., 2017). Further studies are however needed to confirm the GM1 mode of action in AD, establishing the therapeutic potential of the candidate in clinical use. Research within neurological disorders can be challenging, due to the lack of sufficient models. Postmortem brains can only give insight into late stages of the diseases, whereas animal models often fall short in mimicking the precise human pathologies. A number of drugs that have proved to be potent in animal models, have failed to show the same efficacy in human trials. This issue is for instance evident with the potential drug target CD33, where differences in human and mouse properties of this protein is apparent (Bhattacherjee et al., 2019). Induced pluripotent stem cells could therefore provide an advantage in both disease modeling and therapeutic testing for glycosylation related disorders. FIGURE 7 | Induced pluripotent stem cells disease modeling. Somatic cells can be obtained from a patient, reprogrammed into induced pluripotent stem cells (iPSC), and further differentiated into the cell type of interest, such as neurons in the case of AD. These cell models could then be used for INDUCED PLURIPOTENT STEM CELL identifying disease mechanisms, drug screening to develop new therapeutic MODELING AND GLYCOSYLATION candidates. The potential of reprogramming somatic cells into induced pluripotent stem cells (iPSC) revolutionized the scientific world, CONCLUDING REMARKS AND FUTURE when introduced by Takahashi and Yamanaka (2006), Takahashi et al. (2007), providing a new platform for disease modeling DIRECTIONS and personalized medicine, whilst circumventing the ethical issues of embryonic stem cells. Since then, iPSC technology Glycosylation affects both lipids and proteins, the latter accounts has been a widely exploited tool for modeling of a number of for the most common post-translational modification. Given diseases and genetic disorders (Figure 7). iPSC allows study the fact that more than 50% of all proteins are thought to of cellular mechanisms within the same genetic background be glycosylated, it is not surprising that this process could be as the patient themselves, and the differentiation potential implicated in a number of diseases. This is highlighted by the of such stem cells provides the possibility of investigating fact that major AD-related molecules such as APP, BACE1 and cell-type specific phenotypes, such as neurons and glial cells Tau are all modified by glycosylation and abnormalities of glycan in AD. The implication of patient genetics can further be pattern have been observed on several levels in AD patients. explored in these cell models using gene editing techniques Furthermore, modulating the key enzymes glycotransferases and such as CRISPR/Cas9, underlining the unique possibilities of glycosidases have been shown to affect the Ab load in AD, iPSC in disease modeling (Hsu et al., 2014). The features of the main pathological hallmark of the disease, indicating that iPSC are being exploited in modeling of both glycosylation targeting the glycan balance could be a beneficial approach disorders as well as neurodegenerative disorders (Berger et al., for AD intervention. In the scope of AD treatment, inhibitors 2016), and can be of great benefit to test the potential drug of the APP processing molecules BACE1 and g-secretase have candidates identified in other model systems, on a human been thoroughly investigated as drug candidates. However, level. Implications of glycosylation that for instance have been such inhibitors will not only affect the pathogenic actions of observed in transgenic mice with mutations in APP and these proteins, but will also implicate normal functions crucial PSEN1 can be further investigated using iPSCs either from for neural development. By targeting the glycan structures patients with the same mutations, or by introducing these attached to AD-related molecules, and not the protein itself, one mutations into healthy cells. This will be a great advantage could potentially inhibit the harmful effects whilst maintaining to potentially validate disease phenotypes and therapeutic the normal function of these proteins. With this approach targets in a human model, which is an important step before one could potentially minimize side effects seen in current translating it into clinical use. These cell models also provide clinical trials. a great test platform for potential drug candidates, which Furthermore, altered glycosylation and sialylation has been will evaluate the safety and potential side effects of drugs observed in relation to the recently identified genetic AD risk in a human matter. factors TREM2 and CD33. These are both linked to microglia Frontiers in Neuroscience | www.frontiersin.org 14 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 15 Haukedal and Freude Implications of Glycosylation in AD activity and neuroinflammation, presenting a possible link role of glycosylation in AD pathogenesis, glycans remain as between neuroinflammation and glycosylation in AD. promising new biomarkers for early diagnosis and drug targets Although studies have indicated that glycans could serve in AD treatment. as new and reliable biomarkers for AD, and could be great targets for medical intervention, further research is necessary to confirm these potentials. One challenge is the AUTHOR CONTRIBUTIONS fact that although modulators of the glycosylation process have been beneficial, precise mechanistic insights are still HH wrote the manuscript and prepared the figures. KF wrote lacking, and due to the fact that multiple proteins are being and edited the manuscript. Both authors approved the final modified in a similar matter, it is difficult to predict if the version. Both authors contributed to the article and approved the effects of treatments is caused by one or more proteins. submitted version. Studying the potential link between glycosylation, ER stress and Golgi fragmentation could be an interesting new aspect in future AD research, to potentially elucidate these mechanisms. FUNDING iPSC modeling provides a potential new platform for glycan research in neurodegenerative disorders, and could be a This work was supported by Innovation Fund Denmark great addition accompanying the animal and postmortem (BrainStem and NeuroStem), Alzheimer Foundation Denmark, studies. Whilst more knowledge is needed to confirm the and Novo Nordisk Foundation (GliAD – NNF1818OC0052369). Chua, C.-C., Lim, M.-L., and Wong, B.-S. (2010). 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Conflict of Interest: The authors declare that the research was conducted in the doi: 10.1074/jbc.M114.577213 absence of any commercial or financial relationships that could be construed as a Yuzwa, S. A., Macauley, M. S., Heinonen, J. E., Shan, X., Dennis, R. J., He, Y., potential conflict of interest. et al. (2008). A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat. Chem. Biol. 4, 483–490. doi: 10.1038/ Copyright © 2021 Haukedal and Freude. This is an open-access article distributed nchembio.96 under the terms of the Creative Commons Attribution License (CC BY). The use, Yuzwa, S. A., Shan, X., Jones, B. A., Zhao, G., Woodward, M. L., Li, X., et al. distribution or reproduction in other forums is permitted, provided the original (2014). Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive author(s) and the copyright owner(s) are credited and that the original publication decline and amyloid plaque formation in bigenic tau/APP mutant mice. Mol. in this journal is cited, in accordance with accepted academic practice. No use, Neurodegeneration 9:42. doi: 10.1186/1750-1326-9-42 distribution or reproduction is permitted which does not comply with these terms. Frontiers in Neuroscience | www.frontiersin.org 18 January 2021 | Volume 14 | Article 625348 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Neuroscience Pubmed Central

Implications of Glycosylation in Alzheimer’s Disease

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fnins-14-625348 January 2, 2021 Time: 16:0 # 1 REVIEW published: 13 January 2021 doi: 10.3389/fnins.2020.625348 Implications of Glycosylation in Alzheimer’s Disease Henriette Haukedal and Kristine K. Freude Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark Alzheimer’s disease (AD) is the most common cause of dementia, affecting millions of people worldwide, and no cure is currently available. The major pathological hallmarks of AD are considered to be amyloid beta plaques and neurofibrillary tangles, generated by respectively APP processing and Tau phosphorylation. Recent evidence imply that glycosylation of these proteins, and a number of other AD-related molecules is altered in AD, suggesting a potential implication of this process in disease pathology. In this review we summarize the understanding of glycans in AD pathogenesis, and discuss how glycobiology can contribute to early diagnosis and treatment of AD, serving as potential biomarkers and therapeutic targets. Furthermore, we look into the potential link between Edited by: the emerging topic neuroinflammation and glycosylation, combining two interesting, and David Blum, INSERM U1172 Centre de Recherche until recent years, understudied topics in the scope of AD. Lastly, we discuss how new Jean Pierre Aubert, France model platforms such as induced pluripotent stem cells can be exploited and contribute Reviewed by: to a better understanding of a rather unexplored area in AD. Tony Lefebvre, Lille University of Science Keywords: Alzheimer’s disease, glycans, neuroinflammation, AD biomarkers, AD therapeutics, iPSCs and Technology, France Maud Gratuze, Washington University in St. Louis, INTRODUCTION United States *Correspondence: Alzheimer’s Disease Kristine K. Freude Life expectancy has been steadily increasing over the past 200 years, first due to improvements kkf@sund.ku.dk within housing, sanitation and education, followed by the revolutionary development of vaccines Specialty section: and antibiotics. Although these improvements have significantly reduced the early/mid-life This article was submitted to mortality, together with an exponentially aging population we are facing new health challenges Neurodegeneration, (American Association for the Advancement of Science, 2008). More than 50 million people a section of the journal worldwide are affected by dementia, and the numbers are expected to reach 150 million in 2050, Frontiers in Neuroscience making it not only a global health concern, but also an economical burden. Approximately 70% Received: 02 November 2020 of all dementia cases are caused by Alzheimer’s disease (AD), making it the most prevalent of all Accepted: 17 December 2020 neurodegenerative disorders (Alzheimer’s Association, 2018). AD is characterized by an irreversible Published: 13 January 2021 loss of neurons, particularly in the cortex and hippocampus, causing a progressive decline in Citation: memory. There is currently no cure for AD, and the disease typically runs its course over 5–10 years Haukedal H and Freude KK (2021) after symptoms appear. However, it is believed that AD initiated in the brain decades before clinical Implications of Glycosylation in symptoms appear and a diagnosis can be made, making timely medical intervention substantially Alzheimer’s Disease. more challenging. Research within the area is therefore crucial to develop new early-on diagnostic Front. Neurosci. 14:625348. doi: 10.3389/fnins.2020.625348 tools and potential new therapeutic approaches. Frontiers in Neuroscience | www.frontiersin.org 1 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 2 Haukedal and Freude Implications of Glycosylation in AD Alzheimer’s disease can be categorized as familial (fAD) Thus, targeting these cells will have to be directed toward the or sporadic (sAD). sAD is by far the most common form detrimental effects, whilst maintaining the protective properties. of AD, accounting for approximately 95% of all cases, and Another issue, as previously mentioned, is that AD is often far is known to be a multifactorial disease where genetic risk progressed when diagnosed. Therefore, it is important to identify factors in combination with adverse environmental factors play new preclinical biomarkers for AD together with potential a role in disease development. Although age is recognized as new targets. Recent studies have suggested that glycosylation is the most common non-modifiable risk factor, genetic factors implicated in AD, and considering the fact that most known AD- such as the +4 allele of Apolipoprotein E (APOE4) are known related molecules are either modified with glycans, or play a role to increase the risk of developing sAD. More recent, genetic in glycan regulation, glycobiology represent an interesting new profiles related to the innate immune system have been shown insight into the understanding of AD, and a potential for new to increase the risk of sAD, emphasizing the potential role of therapeutic approaches. non-neuronal cells and neuroinflammation in neurodegenerative disorders (Efthymiou and Goate, 2017). fAD on the other GLYCOSYLATION hand, is caused by specific mutations in Presenilin 1 (PSEN1), Presenilin 2 (PSEN2) or Amyloid precursor protein (APP), Biological information is transcribed from DNA to RNA, and affecting APP processing, thus supporting the currently widely can be further translated into proteins. Following translation, accepted “Ab cascade hypothesis.” APP can be processed these proteins are often additionally modified. An important through the amyloidogenic or the non-amyloidogenic pathway, post-translational modification is the attachment of glycans to where the former leads to production of amyloid beta (Ab) such proteins, providing important roles in recognition, energy peptides (Figure 1). In AD there is an imbalance in terms metabolism, signaling and structure. Such modifications also of production and clearance of Ab, and APP processing is provide the possibility of functional diverse products from a shifted toward the amyloidogenic pathway. APP is initially limited number of genes. Glycobiology is the study of the cleaved by b-secretase, followed by g-secretase, causing excessive structure, biology, function and evolution of carbohydrates, production of Ab peptides. These peptides can aggregate into or glycans and their associated aglycone (proteins, lipids or insoluble Ab plaques in the brain (Hardy and Higgins, 1992). any other kind of molecule). All cells and a substantial Extracellular Ab plaques is the main characteristic of AD, number of molecules carry glycans, underlying their biological followed by the formation of intracellular neurofibrillary tangles impact. Glycosylation is known to be the most common post- (NFTs) caused by Tau hyperphosphorylation, and together these translational modification, and more than 50% of all proteins phenotypes are considered the neuropathological hallmarks of are glycosylated. Not surprisingly, it has therefore been suggested AD (Figure 2). to be implicated in a number of diseases. In this review Although the pathology described above is still considered we will give a brief introduction to glycosylation, but for representative of the main AD features, the precise underlying extended information we refer to textbooks such as Essentials of mechanisms have yet to be identified. Other hypotheses have Glycobiology (Varki et al., 2015). recently gained increasing interest. Those include organelle Carbohydrates can be classified as monosaccharides, pathology such as Golgi and endoplasmic reticulum (ER) oligosaccharides or polysaccharides. Monosaccharides are the alterations, as well as neuroinflammation and the role of non- simplest form of carbohydrates and can be linked together neuronal cells such as microglia and astrocytes in AD progression through glycosidic linkages to form the higher saccharide (Kinney et al., 2018). AD patients present with hyperactivation classes. Typically, oligosaccharides consist of less than 20 of the immune cells, most likely due to the inability to clear monosaccharides, while more complex structures are referred Ab plaques, causing a chronic activated state with release of to as polysaccharides. The term glycan refers to carbohydrate pro-inflammatory and neurotoxic factors, further promoting structures that are attached to a protein, lipid or other molecule, neurodegeneration (Figure 2). Several therapeutic approaches forming a glycoconjugate. The complexity of a glycan can be have been suggested in the search for an AD cure, targeting highly variable depending on how many different types of components in each of these hypotheses. However, most of monosaccharides it contains. Furthermore, glycans themselves these have failed in clinical trials, and treatment today is can be modified by phosphorylation, acylation, methylation highly focused on symptom management (Liu et al., 2019). or sulfation, ensuring great diversity in terms of glycan One of the challenges in terms of potential targets in AD function and mechanisms. treatment is that most molecules are not only implicated in AD but have important functions in other pathways, as Mechanisms and Major Types of seen with the approach of targeting the secretases involved Glycosylation in APP processing. While inhibition of these can reduce the amount of Ab peptides, it will also impair Notch signaling, Glycoconjugates are formed when sugar chains are added another g-secretase substrate, essential in proper neurogenesis to proteins, lipids or other molecules, and in mammals 17 (Wolfe, 2012). The same can be seen for neuroinflammation. monosaccharides are commonly found in such glycan structures. Microglia are crucial in maintaining a healthy homeostasis and Glycosylation can occur through various mechanisms, and defeating potential threats in the brain. On the other hand, includes addition of glycans to both proteins and lipids. There are chronic activation leads to pro-inflammatory and toxic effects. two major types of protein glycosylation; the addition of N -linked Frontiers in Neuroscience | www.frontiersin.org 2 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 3 Haukedal and Freude Implications of Glycosylation in AD FIGURE 1 | Amyloid precursor protein processing. APP can be processed either through the non-amyloidogenic (left) or the amyloidogenic pathway (right). In the non-amyloidogenic processing pathway, APP is initially cleaved by a-secretase generating sAPPa and C83. C83 is further processed by g-secretase producing AICD and the non-toxic p3 fragment. In contrary, APP is first processed by b-secretase in the amyloidogenic pathway, leading to formation of sAPPb and C99, where C99 is further cleaved by g-secretase generating AICD and Ab peptides. These peptides are prone to aggregate into the toxic Ab plaques, characteristic for AD. FIGURE 2 | Complex AD Pathology. AD is characterized by the formation of extracellular amyloid beta plaques and intracellular Tau tangles. Additionally, chronic activation of microglia due to an inability to clear such plaques through phagocytosis, leads to a state of neuroinflammation in these patients, further contributing to disease progression. These neuropathological hallmarks ultimately lead to neurodegeneration and atrophy of the brain. glycans and O-linked glycans (Figure 3). N -glycosylation takes N -glycosylation refers to addition of a N -acetylglucosamine place in the ER and Golgi apparatus. Complex O-glycans are also (GlcNAc) to the peptide backbone, via a b-1N linkage to the mainly built in the Golgi, whereas O-GlcNAcylation occurs in the nitrogen atom of an Asparagine (Asn). The initial process takes cytoplasm, nucleus and mitochondria. place in the ER, where the N -glycan precursor is synthesized Frontiers in Neuroscience | www.frontiersin.org 3 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 4 Haukedal and Freude Implications of Glycosylation in AD FIGURE 3 | Common Glycan Structures. N-glycans consist of the same core consisting of GlcNAc and mannose, and can further be trimmed and modified from this core structure, often with galactose, fucose or sialic acids. High mannose, complex, hybrid and bisecting glycans are common types of such modified N-glycans. O-glycans on the other hand have varying core structures, consisting of either GalNAc or GlcNAc. O-GalNAc are referred to as Mucin-type O-glycans, and are usually found on membrane-bound, extracellular and secreted proteins. O-GalNAcylation is a complex form of O-glycosylation, and four common core structures are shown. O-GlcNAc on the other hand, are usually found on intracellular proteins. from GlcNAc and Mannose. Mannose and glucose units (OGT) and O-GlcNAcase (OGA). O-GlcNAc are attached to are further added to form a glycan structure consisting of Thr and Ser residues, and no further sugars are added. This 14 sugars. The sugar chain is then transferred to a protein is a dynamic process where GlcNAc is rapidly added and containing the sequence Asn-X-Serine (Ser)/Threonine (Thr). removed from various protein substrates (Varki et al., 2015; Following, the added glycan is trimmed by mannosidases Reily et al., 2019). and glucosidases in the ER, and further modified by Increasing evidence indicate that both N -glycosylation and glycosyltransferases in the Golgi apparatus to ensure glycan O-glycosylation are implicated in AD, emphasized by the fact maturity (Reily et al., 2019). that the two hallmark proteins APP and Tau carry both potential When studying glycosylation, N -glycans, have the advantage N -glycosylation and O-glycosylation sites. that they are all comprised of a common core. This is however Regulation of Glycosylation not the case in terms of O-glycosylation, a highly diverse form of glycosylation. O-glycans are covalently linked to a Ser/Thr, and in The process of glycosylation is mainly regulated by humans these are commonly GlcNAc or N -acetylgalactosamine the key enzymes glucosidases and glycosyltransferases. (GalNAc). GalNAc-linked glycans are often referred to as mucin- Glucosidases function by hydrolyzing glycan linkages, whereas type O-glycans, and the glycosylation process takes place in glycosyltransferases synthesize the glycan chain. Together they the cis-, medial- and trans-Golgi compartments. In the case determine the structure of the glycan outcome. Mechanisms that of such complex O-glycosylation, trimming of existing sugar regulate the gene expression of these enzymes, or the availability chain does not take place, unlike the pre- and post-processing of specific substrates are therefore crucial in regulating the glycan steps seen in N -glycosylation. Modification of the O-glycan composition of a cell. chains instead occur by specific glycosyltransferases with addition Glycosyltransferases and glucosidases are regulated both at of galactose, GlcNAc, sialic acids or fucose. Such O-glycans a post-transcriptional and post-translational level, and multiple are often found on extracellular and secreted glycoproteins. mechanisms can contribute in altering the expression, activity In contrast, intracellular glycoproteins often contain GlcNAc- or structure of such enzymes. First of all, the formation linked glycans, where the biosynthesis usually takes place of glycans can be regulated by the transcription levels of in the cytoplasm, regulated by O-linked GlcNAc transferase glycosidases and glycosyltransferases. Furthermore, mechanisms Frontiers in Neuroscience | www.frontiersin.org 4 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 5 Haukedal and Freude Implications of Glycosylation in AD FIGURE 4 | Glycosylation regulation. The process of glycosylation can be regulated at several stages; the transcription level of glycosyltransferases and glucosidases, transport and synthesis of sugar donors to the ER and Golgi, trafficking and localization of glycosyltransferases and glucosidases and substrate competition of these enzymes, phosphorylation of glycosyltransferase and glucosidase and total glycan turnover. that control localization and trafficking of these enzymes is roles of these highly diverse macromolecules, amongst others an important regulatory factor, as it influences the access within the fields of cancer, infection and immunity, drug to potential substrates. Transport and synthesis of sugar and pharmaceuticals, fertilization and blood types. Glycans donors to both the Golgi and ER influence the availability are often present at the surface of cells, indicating a role in of substrates, contributing to control of glycan formation. signaling and recognition. However, alongside the elaborate Additionally, the activity of glycosylation enzymes can potentially glycan diversity, comes a great range of functions. The biological be modified through phosphorylation of the cytoplasmic tails role of these macromolecules can be broadly placed into four (Ohtsubo and Marth, 2006), and enzymes competing for the distinct categories. This includes structural and modulatory same substrate will overall have an effect on the glycome roles, extrinsic recognition, intrinsic recognition and host glycan of a given cell (Okada et al., 2015). Moreover, proteolysis mimicry (Table 1; Varki, 2017). that occur in the Golgi apparatus and total glycan turnover The modulatory and structural importance of glycans is through endocytosis at the cell surface can impact glycan underlined by the glycocalyx coating of eukaryotic cells and formation. Thus, multiple regulatory mechanisms are involved polysaccharide layer surrounding a number of prokaryotes, in modulation of glycan expression, contributing to the representing protective and stabilizing effects by providing a highly diverse pool of glycans and their function (Figure 4; physical barrier. Furthermore, proteoglycans can contribute Ohtsubo and Marth, 2006). to maintenance of tissue integrity and overall organization, and ensure correct folding of newly synthesized proteins. Function of Glycans Additionally, glycans modulate protein interactions, and in Although glycobiology has been a field of many mysteries, many cases ligand binding has been seen do be glycosylation- research the past decades have revealed important functional dependent. They can also function as a storage center for essential Frontiers in Neuroscience | www.frontiersin.org 5 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 6 Haukedal and Freude Implications of Glycosylation in AD TABLE 1 | Important glycan functions. the responsible converting enzyme GnT-III (Akasaka-Manya et al., 2010). Studies in neuroblastoma cells have shown an Glycan Function upregulation of GnT-III in response to Ab treatment, and reduced Structural and Intrinsic Extrinsic Host mimicry Ab load in GnT-III transfected cells, hypothesizing a protective Modulatory Recognition Recognition effect of N -glycans, and upregulation of GnT-III as an adaptive response in AD brains (Akasaka-Manya et al., 2010). On the Protection Intracellular signaling Immunity Adaption of glycan other hand, reports have shown that increased levels of bisecting surface N -glycans can promote AD pathogenesis by delaying BACE1 Stabilizer Intracellular protein Antigen uptake Invasive pathogens trafficking degradation. Loss of bisecting N -glycans have been shown to Tissue Integrity intracellular Protein Viral, Bacterial, reduce Ab generation and slow down AD progression in mice Folding Fungal Adhesins (Kizuka et al., 2015). Protein folding SAMPs/DAMPs PAMPs Protein sialylation has been seen to be altered in AD, Storage center Clearance receptors and a decrease in sialyltransferase (ST) has been identified in AD patients, observed in both serum (Maguire et al., 1994) and postmortem brain samples (Maguire and Breen, molecules, providing easy access and proper activity of such 1995). Sialylation is a terminal glycosylation process involving under specific conditions. attachment of sialic acids to glycans/glycoproteins, catalyzed Glycans can act as specific ligands for cell-cell recognition by ST. These sialic acids can play an important role in and cell-matrix interactions, also known as intrinsic recognition. several cellular function by acting as recognition molecules, and Intrinsic glycan functions can impact intracellular glycoprotein N -acetylneuraminic acid (NeuAc) is the most abundant type folding, trafficking and degradation, and play an important role of sialic acids in mammalian cells (Schedin-Weiss et al., 2014). in intracellular signaling. There is evidence that glycans can Furthermore, altered sialylation profiles have been revealed in act as self-associated molecular patterns (SAMPs) to maintain cerebrospinal fluid (CSF) of AD patients (Fodero et al., 2001), a non-activated state of immune cells, or as danger-associated increasing the evidence toward an implicated sialylation and molecular patterns (DAMPs), overall ensuring cell homeostasis. glycosylation process in AD pathogenesis. To elucidate how this In an extrinsic recognition matter, many glycoconjugates are implication contributes to the progression of AD, a number of acting as pathogen-associated molecular patterns (PAMPs) and studies have been conducted, looking into glycosylation of the facilitate antigen recognition and uptake, further emphasizing most common AD-related proteins, which will be described in the immune-related role of glycans. These functions have not the following sections. surprisingly led to host glycan mimicry, with pathogens adapting their glycan surfaces nearly identical to the host, thereby blocking APP recognition of underlying antigens, to increase their invasive Amyloid precursor protein is the precursor protein that when capacities, providing yet another functionality of glycans (Varki cleaved by b-secretase in the amyloidogenic pathway form et al., 2015; Varki, 2017). the toxic, aggregating Ab peptides seen in AD patients. Both The same glycans can also act differently within the same N -glycosylation and O-glycosylation sites have been identified organism, and a glycan that serves a specific function in one in this protein, and both N- and O-glycosylated APP has been tissue, can have a completely different role in another. Given the detected in the CSF of AD patients (Saito et al., 1993). APP highly diverse functional properties of glycans, it is not surprising has two potential sites for N -glycosylation, located at Asn467 that altered glycosylation can play an important role in health and and Asn496 (Påhlsson et al., 1992). APP N -glycans have been disease. Glycan dysregulation have been identified in a number of suggested to play a role in regulating the production of Ab, as neurodegenerative disorders, and in AD the glycosylation profile manipulation of such glycan structures have been observed to of key disease regulators, such as APP, Tau, beta-secretase 1 implicate APP transport and trafficking (Kizuka et al., 2017). (BACE1) and Nicastrin, have been shown to be altered. Inhibiting N -glycan formation or maturity has been shown to reduce the secretion of APP in various models (Tienari et al., 1996; McFarlane et al., 1999, 2000). Furthermore, sialylation of GLYCOSYLATION ALTERATIONS IN APP-linked N -glycans seems to affect APP processing, whereas enhanced sialylation leads to an increase in APP secretion ALZHEIMER’S DISEASE and Ab production (Nakagawa et al., 2006), together with an A lot of key proteins affected in AD are either glycosylated increase in the bisecting GlcNAc level. Such alterations have themselves or have an effect on the glycosylation of other been identified in relation to the familial AD causing mutations proteins (Figure 5 and Table 2), supporting the hypothesis of in the APP gene, such as the London and Swedish mutations, a possible link between glycans and AD. AD patients present known to cause increased Ab42/Ab40 ratio, highlighting the with an abnormal glycan profile, and several studies have shown potential link between glycosylation and AD pathogenesis altered glycosylation patterns in these patients. Increased levels (Schedin-Weiss et al., 2014). of bisecting GlcNAc, which is a special N -glycan structure highly A number of O-glycosylation sites, both for mucin-linked expressed in the brain, have been revealed in AD patients O-glycans and O-GlcNAc, have been identified in APP. (Wang et al., 2019), together with an increased expression of O-GalNAcylation at sites Thr291, Thr292 and Thr576 have been Frontiers in Neuroscience | www.frontiersin.org 6 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 7 Haukedal and Freude Implications of Glycosylation in AD FIGURE 5 | Glycosylation Sites of major AD molecules. Two N-glycosylation sites have been identified in APP, together with eight sites for O-glycosylation and sites for O-GlcNAcylation. Tau can be modified with O-GlcNAc at four potential sites, and only O-GlcNAcylation is observed in healthy brains. However, N-glycosylation of Tau have been observed in the case of AD, at three different sites. BACE1 only undergoes N-glycosylation, with four identified sites, whereas Nicastrin contains 16 potential N-glycan sites. No glycosylation sites have been observed in the presenilins (PSEN1/2), but studies suggest that these might play a regulatory role in glycosylation of other proteins, such as Nicastrin. The sporadic AD linked molecules APOE and TREM2 contains seven O-glycosylation sites and two N-glycosylation sites, respectively. TABLE 2 | Glycosylation in Alzheimer’s disease. of APP, potentially by increasing non-amyloidogenic alpha- secretase (a-secretase) processing, the level of soluble APPa Gene Glycosylation Type Comment (sAPPa) and thus reducing the secretion of Ab (Jacobsen APP N-glycosylation Altered N-glycosylation can reduce APP and Iverfeldt, 2011). This has been investigated in vitro O-glycosylation processing and secretion. O-glycosylation in HeLa cells transfected with the Swedish APP mutant. reduce Ab secretion Inhibition of O-GlcNAcase in these transfected cells leads BACE1 N-glycosylation Modifications with bisecting GlcNAc increase to increased levels of a-secretase products and decreased b- Ab production, and is increased in AD. secretase processing. Increased O-GlcNacylation of APP can Tau O-GlcNAcylation O-glycosylation potentially play a protective potentially affect the localization of the protein, promoting N-glycosylation role, decreased in AD patients. trafficking to the plasma membrane and decreasing endocytosis N-glycosylation of Tau is only observed in AD conditions, not in healthy controls. (Chun et al., 2015). Nicastrin N-glycosylation Immature and mature version, but effects of The link between O-GlcNAcylation and APP and Ab is N-glycosylation is poorly understood supported by in vivo studies in rats, showing that treatment PSEN1 None Potential regulatory role in glycosylation of with Ab25-35 reduce the level O-GlcNAcylation in these rats. other proteins These findings correlate with up-regulation of glycogen synthase APOE O-glycosylation Alterations could potentially be linked to kinase-3-beta (GSK3b) and increased Tau phosphorylation increased Ab42 level (Lozano et al., 2017). Studies in 5xFAD mice models further TREM2 N-glycosylation Altered N-glycosylation could affect ligand highlight the relationship between O-GlcNAcylation and Ab binding properties and contribute to AD pathogenesis load, showing that inhibiting O-GlcNAcase, and thus enhancing O-GlcNAcylation, reduced the Ab generation by lowering the activity of g-secretase (Kim et al., 2013). In transgenic TAPP mice expressing both mutant human Tau and APP, O-GlcNAcase observed in APP695 in Chinese hamster ovary cells (CHO) (Perdivara et al., 2009). Additionally, complex O-glycosylation inhibition has also been thoroughly investigated, pointing toward a correlation between increased O-GlcNAcylation and reduced sites in APP770 have been revealed at Ser597, Ser606, Ser662, Ser611, Ser680, Thr616, Thr635 and Thr635 in human CSF cognitive decline (Yuzwa et al., 2014). Although studies indicate (Halim et al., 2011). Furthermore, APP has been seen to undergo that there is a potential link between O-GlcNAcylation of APP and the Ab load in AD patients, it is important to bear in O-GlcNAcylation (Griffith et al., 1995), and it has been suggested that such simple O-glycosylation can implicate the processing mind that other AD-related molecules are O-glycosylated, and Frontiers in Neuroscience | www.frontiersin.org 7 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 8 Haukedal and Freude Implications of Glycosylation in AD could thus contribute to the effects seen by altered glycosylation to complex glycosylation, and a mature version carrying such (Schedin-Weiss et al., 2014). complex N -glycans. It has been hypothesized that the maturity level is dependent on presenilins (Yang et al., 2002), and in BACE1 PSEN1/2 knock-out cells the mature form of nicastrin cannot In the amyloidogenic pathway, APP is initially processed by be identified, potentially due to impaired intracellular trafficking. BACE1, producing soluble b-APP fragments (sAPPb) and C99. However, the maturity level does not seem to affect its function C99 is further cleaved by g-secretase to produce Ab peptides (Herreman et al., 2003). Specific glycan structures related to of various lengths, with Ab42 being the most toxic variant, nicastrin in neurons should be further elucidated to evaluate its prone to aggregate into the senile plaques’ characteristic for role in nicastrin function. an AD brain. BACE1 can be N -glycosylated at four potential sites, and modifications at these sites have been suggested Presenilin 1 to affect the activity of the enzyme. On the other hand, no As previously stated, PSEN1 and 2 is the catalytic subunit of g- O-glycosylation sites have been detected in BACE1. It has secretase. Although no glycosylation sites have been identified, been shown that the maturity and correct folding of BACE1 several studies have shown a regulatory role of presenilins is highly dependent on N -glycan modifications, and that the in terms of glycosylation of other proteins, such as nicastrin number of such N -glycans correlates directly with folding, discussed in the previous section. PSEN1 and 2 have also been secretion rates and activity of the enzyme (Vanoni et al., 2008). observed to affect the glycosylation and sialylation of the neural BACE1 has been reported to be highly modified with bisecting cell adhesion molecule (NCAM), essential for brain function GlcNAc, and as previously discussed, in vivo studies in mice (Farquhar et al., 2003), and have an impact on the receptor show that knock-out of the Mgat3-gene, encoding the GnT- Tyrosine-related kinase B, known to play a role in neural III enzyme, improve cognitive impairment and reduce Ab differentiation (Naruse et al., 1998). It has thus been suggested deposition (Kizuka et al., 2015). In vitro studies with Mgat- that the Presenilins can affect the glycosylation process in a 3 knock-out cells have revealed a shift in BACE1 localization number of proteins, either directly or by affecting the cellular toward late endosomes/lysosomes and thus leading to increased location of proteins (Schedin-Weiss et al., 2014). degradation. BACE1 localization in endosomal compartments is required for APP processing, and a shift toward lysosomal Tau localization is suggested to be the cause of the drastic Ab Together with Ab plaques, neurofibrillary tangles (NFTs) reduction observed in knock-out Mgat3 studies (Tan and Evin, are considered the neuropathological hallmark of AD. These 2012). As bisecting GlcNAc on BACE1 is upregulated in AD are found intracellularly and they are caused by abnormal patients, and could potentially also be linked to the oxidative hyperphosphorylation of the Microtubule-associated protein stress observed in AD (Kizuka et al., 2016), inhibiting the (MAP) Tau, which causes intraneuronal accumulation of paired GnT-III might be an interesting approach to reduce the Ab helical filaments (PHF) eventually forming the NFTs (Iqbal et al., load, indirectly targeting the BACE1 activity, yet circumventing 2005). Tau is a cytosolic protein, and has been seen to undergo the issues of adverse effects seen with BACE1 inhibitors both N -glycosylation and O-GlcNAcylation, which is interesting (Kizuka et al., 2017). given the fact that N -glycans are usually a modification seen BACE1 can also be linked to the altered protein sialylation with extracellular proteins, or at membrane-bound proteins seen in AD patients. One of the BACE1 substrates is known to extracellular domain. However, such N -glycosylation has been be b-galactoside a2,6-sialyltransferase-1 (ST6GaI1), and BACE1 identified in AD patients, but not in healthy control brains, processing of this protein is required to generate the soluble ST indicating an altered glycosylation process in such patients. form. BACE1 can thus affect sialylation of glycoproteins, and These N -glycans have been identified at three sites; Asn359- enhancement of these processes have been linked to increased Ile-Thr, Asn167-Ala-Thr and Asn410-Val-Ser (Sato et al., 2001; APP secretion and Ab production (Nakagawa et al., 2006). Liu et al., 2002; Losev et al., 2020), and have been proposed to affect the aggregation of Tau (Losev et al., 2019). Tau also g-Secretase (Nicastrin) undergoes O-GlcNAcylation, and four sites have been mapped to After APP is cleaved by b-secretase, Ab peptides are generated human Tau; Thr-123, Ser-208, Ser-400, Ser-409/Ser-412/Ser-413 through further processing of the C99 fragment via g-secretase. (Zhu et al., 2014). In contrast to N -glycosylation, the level of This cleaving enzyme consists of four subunits; nicastrin, PSEN1 O-GlcNAcylation has been observed to be decreased in AD and PSEN2, Presenilin enhancer 2 (Pen-2) and Anterior pharynx- brains compared to controls (Liu et al., 2004). It has thus defective 1. Amongst these subunits, nicastrin has been suggested been suggested that O-GlcNAcylation can play a protective to be involved in g-secretase substrate interactions (Bolduc et al., role against the pathological hyperphosphorylation seen in 2016), and it is the only subunit of g-secretase that is known AD. This is potentially due to the fact that O-GlcNAcylation to be N -glycosylated, containing as much as 16 potential sites. and phosphorylation of Tau are two competing processes. Nicastrin contributes to g-secretase activity by interacting with Abnormal Tau phosphorylation could be caused by decreased PSEN1 and PSEN2, the catalytic subunits of the enzyme, and O-GlcNAcylation, which again could be linked to the complex glycosylation of nicastrin has been seen to be dependent metabolic alterations/deficiencies observed in AD and other on these presenilins (Yu et al., 2000). Two forms of nicastrin have neurodegenerative disorders, underlying the potential role been identified, an immature form with N -glycans not subjected of glycosylation in disease progression (Liu et al., 2004; Frontiers in Neuroscience | www.frontiersin.org 8 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 9 Haukedal and Freude Implications of Glycosylation in AD Zhu et al., 2014). The link between glycosylation, metabolic in mode of action remains, the +4 genotype has shown disorders and AD is further highlighted in a study investigating to increase both the intraneuronal Ab accumulation and a mice model of Diabetes mellitus (DM). DM has been seen plaque deposition in postmortem AD brains (Yamazaki to increase the risk of cognitive dysfunction, and in these et al., 2019). Interestingly, APOE is as many other proteins mice impaired learning and memory is seen together with modified by glycosylation. O-glycosylation of APOE was obesity and hyperglycemia. These findings correlate with first identified at Thr194, and newly secreted APOE was decreased O-GlcNAcylation and increased Tau phosphorylation, found to be highly sialylated (Wernette-Hammond et al., whereas hypoglycemic therapy improved these phenotypes, 1989). More recently, a number of O-glycosylation sites causing increased levels of O-GlcNAc transferase (Huang R. have been identified, and the glycan profile has been seen et al., 2020). Transgenic mice models of AD show the same to differ in the lipid-binding domain of APOE in CSF and trend, with an upregulation of Tau phosphorylation and plasma, indicating tissue specific glycoforms. Sialylated reduced O-GlcNAcylation in the hippocampus, supporting glycans are more abundant on the lipid-binding domain of the findings of an imbalance between O-GlcNAcylation and CSF APOE, and could indicate that glycosylation plays a Tau phosphorylation (Gatta et al., 2016). This imbalance role in the flexibility of lipoprotein-binding (Flowers et al., could potentially also affect the localization of Tau proteins 2020). APOE is known to interact with Ab, and it has been (Lefebvre et al., 2003), and an overall affect on both function suggested that the sialic moiety of APOE affects this interaction, of Tau and formation of neurofibrillary tangles seen in AD thus being an important contributor in AD development patients (Shane Arnold et al., 1996). These findings highlight the (Sugano et al., 2008). Altered glycosylation profile of ApoE potential of O-GlcNAcase inhibitors in treatment of AD, with has been observed in a mouse model of Niemann-Pick the aim to prevent the pathological hyperphosphorylation of Type C (NPC), a cholesterol-storage disorder that causes Tau (Yuzwa et al., 2014). However, increased insight into the neurodegeneration, which shares some of the pathological specific mechanisms of O-GlcNAcylation of Tau is required to mechanisms seen in AD, including Ab deposition. In this elucidate if the hyperphosphorylation in AD is in fact a cause or study, they identified a potential link between changes in ApoE a consequence of decreased O-GlcNAcylation. glycosylation and increased level of the toxic Ab42 peptide The altered glycosylation of the main AD-related molecules (Chua et al., 2010). described above provides an evident implication of glycans in Although the precise mechanisms of how APOE glycosylation AD. Furthermore, organelle pathology such as ER stress and contributes to AD pathogenesis have yet to be elucidated, Golgi fragmentation have been observed in AD conditions, and there is clear indication that this process is implicated in could be a very interesting link to the glycan alterations seen the disease. Although the APOE +4 allele is known as the in AD. A plausible hypothesis could be that the excessive Ab strongest genetic risk factor for developing sAD, genome accumulation and Tau phosphorylation cause a fragmentation wide association studies (GWAS) have identified a number of the Golgi, by causing inactivation of major Golgi proteins of genes that confers an increased risk of sAD. These are and cytoskeleton disruption, respectively (Joshi et al., 2015). especially genes linked to the innate immune system, such Golgi fragmentation will most likely affect glycosylation, as as Triggering receptor expressed on myeloid cells-2 (TREM2), this is the location for a majority of these processes, and highlighting the role of microglia and neuroinflammation in AD could thus contribute to the glycan alterations seen in AD (Efthymiou and Goate, 2017). Altered glycosylation pattern of patients. Consequently, as the altered glycosylation can affect TREM2 has been identified in AD, and provides an interesting APP processing and Ab load, together with Tau phosphorylation, link between glycosylation and neuroinflammation. This link it is not unlikely that it becomes a vicious cycle, enhancing is further emphasized by the fact that glycosylation plays each of these phenotypes. Elucidating the mechanisms behind an important role in cell-to-cell, as well as cell-environment this pathway would therefore be an interesting approach in the interactions, which the immune system is highly dependent search for an AD cure. on. Neuroinflammation and glycosylation will therefore be In addition to the molecules described above, glycosylation elaborated in the following section. has been observed to affect APOE, identified as a risk factor for developing sAD. NEUROINFLAMMATION AND APOE and Sporadic AD GLYCOSYLATION ApoE is a cholesterol carrier important in transport of lipids and injury repair in the brain, and genetic variants Until recently, AD research has been focused on neural within the APOE gene have been identified as the strongest pathology. However, increasing evidence support an important genetic determinants of sAD risk (Liu et al., 2013). Three role of glial cells, such as microglia and astrocytes in disease polymorphic alleles have been identified, with the +4 allele pathogenesis. Neuroinflammation is a common characteristic being known to cause increased risk of developing AD. seen in AD brains, and is now considered a highly interesting On the other hand, the +2 allele has a protective role, topic within the field of neurodegenerative disorders (Fakhoury, whereas the most common +3 variant has a neutral effect 2017). Especially the role of microglia has become evident, and (Chartier-Harlin et al., 1994). Studies have shown a clear link the cells and genes related to the innate immune systems can be between APOE genotype and Ab, and although uncertainties affected by glycans. Frontiers in Neuroscience | www.frontiersin.org 9 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 10 Haukedal and Freude Implications of Glycosylation in AD explaining the late-onset in AD versus the early-onset seen in Microglia and Glycosylation Alterations NHD (Li and Zhang, 2018). Recently, studies have shown that Microglia are the resident macrophages of the brain, responsible TREM2 activity can be modulated by interactions with TMEM59. for the innate immune system in the central nervous system TMEM59 regulates complex glycosylation, secretion and cell (CNS). Although microglia only accounts for about 5% of the surface expression of APP, with overexpression causing defects in glial cell population in the cerebral cortex (Lawson et al., 1990), glycan maturation (Ullrich et al., 2010). Furthermore, TMEM59 they play a vital role in terms of brain homeostasis and protection could play an important role in immunity, as downregulation against potential threats (Mandrekar-Colucci and Landreth, has resulted in anti-inflammatory effects, potentially mediating 2012). In AD however, microglia have been observed to be microglia activity, functioning as a self-defense in microglia. associated with Ab plaques, presenting with a proinflammatory TREM2 have been observed to interact with TMEM59, and phenotype. As they are unable to clear such plaques through potentially mediating the degradation of the protein. TREM2 phagocytosis, microglia in the condition of AD enters a state deficiency cause impaired microglial survival and phagocytic of chronic activation. These microglia release proinflammatory activities in TREM2 knock-out mice, together with elevated levels factors such as IL-1b, IL-6 and TNFa, which can be detrimental to of TMEM59. Downregulation of TMEM59 in these knock-out surrounding neurons, further enhancing the neurodegenerative mice however, reversed such impairments, indicating a role of progression of the disease. Neuroinflammation can therefore elevated TMEM59 in microglial defects. The fact that TMEM59 be of large impact in AD pathogenesis (Mandrekar-Colucci have an effect on both glycosylation and neuroinflammation in and Landreth, 2012; Sarlus and Heneka, 2017). GWAS studies AD provides yet another potential connection between the two have revealed a number of genes related to the innate immune mechanisms (Liu et al., 2020). system as risk factors for developing AD, and lot of these genes are either highly or exclusively expressed by microglia, SIGLECs and Galectins indicating an important microglial role in AD (Verheijen Lectins are carbohydrate binding proteins that can interact and Sleegers, 2018). Among the identified risk factors are specifically with selected sugar structures, and which is a highly the genes TREM2 and CD33 (Karch et al., 2012; Guerreiro exploited feature used in glycoanalysis, as discussed in the et al., 2013). Altered glycosylation profile have been detected in following section (Van Damme, 2011). SIGLECs are lectins that TREM2 variants associated with sAD, providing a potential link play an important role in regulation of the immune response, between microglial neuroinflammation and glycosylation in AD and an important member of the SIGLEC family is CD33, also progression. Furthermore, sialylation, which is altered in AD, known as Siglec-3 (Crocker and Varki, 2001). Together with have been suggested to play an important role in microglia- the evidence of altered sialylation in AD, such as decreased ST mediated neuroinflammation. CD33 is a sialic acid-binding levels in these patients, desialylation of the microglial surface has receptor, within the sialic-acid-binding immunoglobulin-type been shown to be induced by activating stimuli such as LPS, Ab lectin (SIGLEC) family, known to bind sialylated glycans, and and Tau, which can in turn enhance the complement receptor 3 plays an important role in microglia activation (Estus et al., (CR3) mediated phagocytosis of neurons (Allendorf et al., 2020). 2019), highlighting the potential implication of glycosylation CD33 is expressed on microglia, and activation of this receptor and sialylation in AD and other neurodegenerative disorders. can inhibit the phagocytic activity of microglia. On the other Exploitation of these pathways could potentially serve as new hand, sialylation of neurons can also contribute in inhibiting target sites for therapeutic intervention (Puigdellívol et al., 2020). microglial phagocytosis of these neurons, protecting against neurodegeneration. When microglia are activated, such as in AD, TREM2 they release a sialidase activity that can act by desialylating both TREM2 is expressed by microglia, and plays a role in modulating neurons and microglia, promoting phagocytosis of neurons, and the inflammatory response and phagocytosis (Li and Zhang, thus neurodegeneration. Microglial release of galectin-3 (Gal- 2018). Loss of TREM2 has been shown to reduce phagocytosis 3), another lectin member, binding to galactose residues (Dong (Kawabori et al., 2015), whereas TREM2 overexpression has et al., 2018), further activates microglia by Toll-like receptor 4 resulted in increased phagocytic activity (Takahashi et al., 2005). (TLR4) and TREM2 binding, as well as binding to desialylated Genetic variations of TREM2 have been associated with increased neurons and promoting phagocytosis of such (Puigdellívol et al., risk of AD, and especially the identified missense mutation 2020). These finding, together with the GWAS identification R47H have been linked to increased AD risk. Interestingly, of CD33 as a risk factor for AD, underline the connection studies have shown that the glycosylation pattern in this rare between altered sialylation of glycans and neuroinflammation in disease-associated TREM2 variant differs from the one seen in AD pathogenesis, providing new potential targets in preventing wild-type TREM2, having increased terminal glycosylation with neuroinflammation and degeneration. complex oligosaccharides in the Golgi and decreased solubility, On an interesting note, inflammation in AD can be linked potentially affecting the function and ligand binding of the to the mitochondrial dysfunction observed in the disease, receptor, and in this way contribute to AD pathogenesis (Park by increasing the production of reactive oxygen species et al., 2017). Furthermore, the TREM2 variants Y38C and (ROS), and such oxidative stress has been implicated in T66M, linked with the early-onset disease Nasu-Hakola disease AD pathogenesis (Perry et al., 2002). Although glycosylation (NHD) display differences in the N -glycosylation profile, but of mitochondrial proteins is a rather unexplored field, a varies from the profile seen within the R47H variant, potentially number of such proteins have been suggested as targets for Frontiers in Neuroscience | www.frontiersin.org 10 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 11 Haukedal and Freude Implications of Glycosylation in AD glycan modification, and may affect mitochondrial dysfunction animal models. Given that these enzymes are the main regulators and the oxidative stress response. The connection between of glycans, targeted inhibitors of these could make an interesting mitochondrial dysfunction, glycosylation and inflammation has therapeutic approach to cope with the abnormal glycosylation for example been reviewed in relation to Parkinson’s disease (PD) observed in diseases (Rempel and Withers, 2008; Videira et al., (Videira and Castro-Caldas, 2018). Furthermore, alterations of 2018). Glycoanalysis is however more challenging in terms of the glycosylation pattern of mitochondrial proteins have been O-linked glycans, due to the lack of a common core structure, observed in the cerebral cortex of a rat sAD model, indicating and further optimization of tools for studying these is needed. an implication of such processes also in AD (Yu et al., 2017), a potentially important future research area. Detection of AD Biomarkers and Diagnostic Properties GLYCANS AS BIOMARKERS IN Although there are currently no validated glycan biomarkers for AD, several candidates are being investigated in ongoing ALZHEIMER’S DISEASE studies. These biomarkers can be studied in various ways and Treatment of AD is challenging, partially because the disease is can be broadly classified as biochemical (CSF-, blood sample), often far progressed when diagnosed. Pathologically, the course neuroanatomical (CT-, MRI scan), metabolic (PET, SPECT scan), of AD starts decades before the clinical onset, further limiting genetic (e.g., APP/PSEN/APOE profile) and neuropsychological the already restricted treatment options, emphasizing the need (e.g., Memory) (Wattamwar and Mathuranath, 2010). In terms for new biomarkers and diagnostic tools. Even though the field of glycan biomarkers, studies are usually performed in samples of biomarkers in AD has advanced over the past few years, most from postmortem brains, CSF or blood (Figure 5). Identifying studies have focused on markers for Ab and Tau pathology, these biomarkers in serum from patients would be a great together with neurodegeneration, inflammation and synaptic advantage in AD diagnostic, making the procedure as less deficiencies, as reviewed in Zetterberg and Bendlin (2020). Given invasive as possible. the observations of aberrant glycome profiles in AD, these could Several studies have investigated the glycan profile in CSF potentially be used as new early stage biomarkers of AD. In this from AD patients compared to healthy controls. One issue with section tools used to identify and study glycans will be discussed, this approach has been the fact that the CSF contains a rather together with potential glycan biomarkers that can be beneficial low concentration of proteins, and a large volume is often in AD diagnosis and treatment strategies. required. However, recent studies have shown the possibility of performing CSF glycoanalysis with a very small amount of Glycoanalysis fluid (Cho et al., 2019). Increased levels of fucosylated and There are several approaches to glycoanalysis (Figure 6), bisecting GlcNAc in AD CSF has been observed in several including investigation of intact glycoproteins and analysis of studies, which has also been confirmed in postmortem brains. glycan structures after cleavage from their respective protein. In Furthermore, the altered bisecting glycan profile correlates with terms of studying intact glycoproteins as well as the localization CSF levels of phosphorylated and total Tau (Schedin-Weiss et al., of such, lectins are commonly used (Zou et al., 2017). Lectins 2020). A study of the CSF N -glycome in both mild cognitive recognizes and bind specific glycans and can be used for impairment (MCI) and AD patients identifying 90 N -glycan purification by methods such as fluorescence microscopy or structures by mass spectrometry revealed both a significant enzyme-linked immunosorbent assay (ELISA) (Belický et al., increase in bisected N -glycans together with a decrease in overall 2016). Another highly exploited method for intact glycoprotein sialylation. Interestingly, the MCI patients that progressed to assessment is mass spectrometry (MS) (Domínguez-Vega et al., AD all showed such abnormalities in glycan profile, indicating 2018), whereas nuclear magnetic resonance spectroscopy (MRS) that glycan alterations might precede the clinical onset of AD, can be used if a large amount of purified glycans is obtained highlighting their potentials as biomarkers for early AD diagnosis (Zhang et al., 2016). (Palmigiano et al., 2016). Many glycome studies have focused on Removal of glycans, to investigate cleaved glycans, can N -glycans. However, the interplay between various glycosylation be performed either enzymatically or chemically, by peptide pathways have also been assessed both in brain tissue and serum. N -glycosidase F (PNGase F) and hydrazinolysis, respectively. A simultaneous analysis of N - and O-glycomes revealed global PNGase F cleaves N -linked glycans between the inner GlcNAc alterations of protein glycosylation in both MCI and AD patients. at the Asn residues, thus releasing the entire glycan structure. Furthermore, the altered glycosylation pattern appeared to be Furthermore, these released glycans can be analyzed using region-specific in the brain, with O-GlcNAcylation observed to methods such as liquid chromatography (LC), porous graphitic be decreased in the frontal lobe of AD brains, whereas it was carbon chromatography (PGC) or capillary electrophoresis. Prior increased in the hippocampus. Similar changes were observed to the LC analysis, glycans are usually fluorescently labeled. Often in the serum of AD patients, which presented a unique glycol- the advanced LC technique known as ultra-high performance LC fingerprint, that could be exploited to develop a new class (UPLC) is used to enhance the process (Regan et al., 2019). of biomarkers for AD diagnosis, that thus could be obtained To investigate the function of glycoproteins, inhibitors of by a simple blood sample. This profile appears to be specific the regulating enzymes glycosidases and glycosyltransferases are for AD, and differed from other neurodegenerative disorders commonly used, and this technique is often exploited in cell- and (Frenkel-Pinter et al., 2017). Furthermore, blood plasma from Frontiers in Neuroscience | www.frontiersin.org 11 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 12 Haukedal and Freude Implications of Glycosylation in AD FIGURE 6 | Glycoanalysis. Samples from postmortem brains, CSF and blood can all be obtained for glycoanalysis. Blood samples are especially of interest in terms of biomarker identification and AD diagnosis, due to the preferable less invasive procedure. Glycoproteins can be extracted from these samples, and either intact or released glycans can be investigated. Intact glycoproteins is commonly studied by lectin arrays and ELISA or fluorescence microscopy or by mass spectrometry (MS) or nuclear magnetic resonance spectrometry (MRS). To evaluate released glycoproteins, these can be either enzymatically or chemically cleaved, labeled and detected through liquid chromatography (LC), LC-MS, porous graphitic carbon chromatography (PGC) or ultra-high performance LC (UPLC). AD patients has shown alterations in glycosylation of specific such trials have decreased due to the constant failures, pointing immunoglobulins (IgG), providing increased evidence of a toward a shift in treatment strategy, and need for novel targets. correlation between neuroinflammation and glycosylation in AD Aberrant glycosylation can be identified in AD patients (Lundström et al., 2014). Decreased ST activity and altered before the clinical disease onset. Thereby, re-establishing the glycosylation of transferrin, an iron transport mediator, together glycosylation homeostasis could potentially be of interest in drug with clusterin (CLU), an important player in debris clearance development. As previously described, glycosylation is mainly and apoptosis, both being genetically linked to AD, have been regulated by the action of glycosidases and glycotransferases. observed in AD blood samples (Maguire et al., 1994; Van Modulating the function of these enzymes could thus be a Rensburg et al., 2004; Liang et al., 2015), making them potential potential therapeutic strategy (Wang et al., 2019). Furthermore, blood biomarker candidates. An overview of potential glycan the increase in level of bisecting GlcNAc and decreased biomarkers in AD is presented in Table 3. ST activity, together with the abnormal glycosylation linked to neuroinflammation presents potential new targets in AD treatment (Table 4). GLYCANS IN TREATMENT OF Novel Targets ALZHEIMER’S DISEASE Glycosyltransferase Inhibitors and Glycosidase Inhibitors Today, only symptomatic treatment options exist for AD, and five FDA-approved prescription drugs are currently The activity of glycosyltransferases and glycosidases could be available. This includes the cholinesterase inhibitors Aricept modulated directly or indirectly, and several methods can be (donepezil), Exelon (rivastigmine) and Razadyne (galantamine), used. One example is to inhibit the metabolism of glycan and the N -methyl D-aspartate (NMDA) antagonist Namenda precursors or the glycan transport in the Golgi and ER. Other (memantine). Additionally, in moderate to severe cases of types of inhibition include blocking the addition of N -glycans to AD, Namzaric, a combination of donepezil and memantine, is glycoproteins, for instance by using the inhibitor tunicamycin, commonly used. All of these can only manage symptoms of or to use glycosidase or mannosidase inhibitors that prevents AD for a certain period of time, but cannot reverse or stop the the formation of mature, complex glycoproteins. A suggested disease progression, highlighting the need of new drug targets glycosyltransferase target in AD treatment is the GnT-III enzyme, (Yiannopoulou and Papageorgiou, 2013). Several candidates responsible for the formation of bisecting glycans, observed to are being investigated in ongoing clinical trials, and a lot of be upregulated in AD patients. GnT-III have been shown to these are focusing on the amyloid-, tau- and neuroinflammatory reduce the Ab deposition in mice, by acting on BACE1, without hypotheses, as reviewed in Huang L. K. et al. (2020). Although compromising the activity of the enzyme. BACE1 has several many anti-amyloid trials are still being conducted, the number of substrates besides APP, important for neural functions, making Frontiers in Neuroscience | www.frontiersin.org 12 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 13 Haukedal and Freude Implications of Glycosylation in AD TABLE 3 | Potential biomarkers for AD diagnosis. of these mice, protecting against brain atrophy (Wang et al., 2020). Inhibitors of the glycosylation enzymes could in this way Biomarker Alteration in AD Detected in target the pathological effects of AD related molecules, while Bisecting GlcNAc Increased Postmortem brains, CSF maintaining the normal functions, thereby minimizing potential and Serum side effects. However, inhibition of OGA will most likely affect a Fucosylated GlcNAc Increased Postmortem brains, CSF great number of proteins, potentially causing chronic O-GlcNAc and Serum elevation. It would therefore be crucial to understand the exact ST activity Decreased Postmortem brains, CSF mechanisms of such inhibitors and the consequences of elevated and Serum glycan levels, before implementing those in human clinical trials Overall sialylation Decreased Postmortem brains, CSF (Wang et al., 2020). and Serum O-GlcNAcylation Region-specific Postmortem brains Sialylation Modulators: Siglecs and Galectins alteration As previously discussed, protein sialylation is implicated in AD, IgG Decreased Plasma complex and decreased levels of ST has been identified in the serum of AD glycosylation and patients. ST are a large group of enzymes that attach sialic acids to sialylation glycoproteins, whereas the removal of such residues is performed Transferrin Decreased CSF, serum by sialidases. High sialylation has been indicated to protect sialylation against neurodegeneration, and targeting the sialylation enzymes Clusterin Decreased Plasma could therefore be of interest in drug development, either by N-glycosylation increasing the sialylation process, or reducing desialylation. Microglia initiate sialidase activity when activated, that desialylate TABLE 4 | Potential therapeutic targets of glycosylation and drug both microglia and neurons causing increased phagocytosis candidates for AD. of surrounding neurons and in that way could contribute Drug Candidates Mode of Action to neurodegeneration. Targeting sialidases could therefore contribute to prevent the neuroinflammation observed in AD. GnT-III inhibitors Reduce the level of bisecting GlcNAc and Ab deposition Furthermore, lectins have been shown to be implicated in O-GlcNAcase inhibitors Reduce deglycosylation and phosphorylation of Tau AD. Gal-3 is released by activated microglia, and contribute to ST modulators Increase sialylation, potentially preventing phagocytosis of neurons, further promoting Ab aggregation. Gal- neuroinflammation 3 could thus be another potential therapeutic target. The sialic Sialidase modulators Reduce desialylation, potentially preventing acid binding microglial receptor CD33, that has been genetically neuroinflammation linked to late onset AD, is yet another promising drug candidate. Gal-3 modulators Reduce phagocytosis of neurons and Ab Single nucleotide polymorphisms in this gene have been shown to production increase the risk of AD. On the other hand, reduced expression of CD33 modulators Increase uptake and phagocytosis of Ab deposits the CD33 sialic acid-binding domain has been shown to confer GM1 Increase Ab clearance and improve memory protection against AD, introducing the potential beneficial use of CD33 inhibitors in AD treatment. Crystal structures of the direct inhibition of the enzyme challenging and presented with CD33 protein have recently been studied, providing structural a number of side effects and abnormalities in mouse models. insights into the sialic binding sites and ligands that can increase Knocking out the GnT-III gene in such models has however phagocytosis and Ab uptake, pointing out the possibility of not shown the same difficulties, and the mice remain healthy in therapeutic intervention at this binding site to promote Ab these studies, indicating that the pathological effects of BACE1 clearance (Miles et al., 2019). could be selectively regulated by GnT-III glycosylation. GnT-III Gangliosides inhibitors could thus potentially be safer drug targets for AD treatment, circumventing the potential side effects seen with In this review, the focus has been on glycosylation of proteins. BACE1 inhibition (Kizuka and Taniguchi, 2018). Inhibition of However, glycosylation of lipids is another common process. glycosidases on the other hand, have been proven beneficial in Gangliosides are a type of glycosphingolipids containing one or terms of Tau pathology. Inhibiting the O-GlcNAcase, as shown more sialic acids, and play an important role in development with the use of compound Thiamet-G (Yuzwa et al., 2008), and protection of the CNS. Ganglioside metabolism is reported has reduced Tau phosphorylation, potentially by reducing the to be associated with AD pathology, and changes in ganglioside deglycosylation of the protein. Tau is modified by O-GlcNAc, profile have been observed in AD patients. Especially the GM1 and increasing the glycosylation could potentially compete ganglioside have been Investigated, and show neuroprotective with and thus reduce the pathological hyperphosphorylation of roles, making it a potential therapeutic target in a number of Tau seen in AD (Bojarová and Køen, 2009). A recent study neurodegenerative disorders, and such beneficial effects have further highlights this potential, presenting a promising new and already been described from clinical trials in the case of both selective OGA inhibitor; MK-8719. In vivo studies in a mouse stroke and PD (Magistretti et al., 2019). In AD, GM1 is observed model of tauopathy has shown that this compound can increase to interact with Ab, and in AD mouse models increased GM1 the level of O-GlcNAc and reduce pathologic Tau in the brains showed reduced Ab accumulation and improved neuropathology Frontiers in Neuroscience | www.frontiersin.org 13 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 14 Haukedal and Freude Implications of Glycosylation in AD (Bernardo et al., 2009). Beneficial effects of GM1 administration have also been observed in an AD rat model, improving memory deficits and spatial learning, potentially by mediating oxidative stress and lipid peroxidation (Yang et al., 2013). The protective actions of GM1 has been suggested to be initiated by increasing autophagy and promoting Ab clearance by microglia (Yuyama et al., 2014; Dai et al., 2017). Further studies are however needed to confirm the GM1 mode of action in AD, establishing the therapeutic potential of the candidate in clinical use. Research within neurological disorders can be challenging, due to the lack of sufficient models. Postmortem brains can only give insight into late stages of the diseases, whereas animal models often fall short in mimicking the precise human pathologies. A number of drugs that have proved to be potent in animal models, have failed to show the same efficacy in human trials. This issue is for instance evident with the potential drug target CD33, where differences in human and mouse properties of this protein is apparent (Bhattacherjee et al., 2019). Induced pluripotent stem cells could therefore provide an advantage in both disease modeling and therapeutic testing for glycosylation related disorders. FIGURE 7 | Induced pluripotent stem cells disease modeling. Somatic cells can be obtained from a patient, reprogrammed into induced pluripotent stem cells (iPSC), and further differentiated into the cell type of interest, such as neurons in the case of AD. These cell models could then be used for INDUCED PLURIPOTENT STEM CELL identifying disease mechanisms, drug screening to develop new therapeutic MODELING AND GLYCOSYLATION candidates. The potential of reprogramming somatic cells into induced pluripotent stem cells (iPSC) revolutionized the scientific world, CONCLUDING REMARKS AND FUTURE when introduced by Takahashi and Yamanaka (2006), Takahashi et al. (2007), providing a new platform for disease modeling DIRECTIONS and personalized medicine, whilst circumventing the ethical issues of embryonic stem cells. Since then, iPSC technology Glycosylation affects both lipids and proteins, the latter accounts has been a widely exploited tool for modeling of a number of for the most common post-translational modification. Given diseases and genetic disorders (Figure 7). iPSC allows study the fact that more than 50% of all proteins are thought to of cellular mechanisms within the same genetic background be glycosylated, it is not surprising that this process could be as the patient themselves, and the differentiation potential implicated in a number of diseases. This is highlighted by the of such stem cells provides the possibility of investigating fact that major AD-related molecules such as APP, BACE1 and cell-type specific phenotypes, such as neurons and glial cells Tau are all modified by glycosylation and abnormalities of glycan in AD. The implication of patient genetics can further be pattern have been observed on several levels in AD patients. explored in these cell models using gene editing techniques Furthermore, modulating the key enzymes glycotransferases and such as CRISPR/Cas9, underlining the unique possibilities of glycosidases have been shown to affect the Ab load in AD, iPSC in disease modeling (Hsu et al., 2014). The features of the main pathological hallmark of the disease, indicating that iPSC are being exploited in modeling of both glycosylation targeting the glycan balance could be a beneficial approach disorders as well as neurodegenerative disorders (Berger et al., for AD intervention. In the scope of AD treatment, inhibitors 2016), and can be of great benefit to test the potential drug of the APP processing molecules BACE1 and g-secretase have candidates identified in other model systems, on a human been thoroughly investigated as drug candidates. However, level. Implications of glycosylation that for instance have been such inhibitors will not only affect the pathogenic actions of observed in transgenic mice with mutations in APP and these proteins, but will also implicate normal functions crucial PSEN1 can be further investigated using iPSCs either from for neural development. By targeting the glycan structures patients with the same mutations, or by introducing these attached to AD-related molecules, and not the protein itself, one mutations into healthy cells. This will be a great advantage could potentially inhibit the harmful effects whilst maintaining to potentially validate disease phenotypes and therapeutic the normal function of these proteins. With this approach targets in a human model, which is an important step before one could potentially minimize side effects seen in current translating it into clinical use. These cell models also provide clinical trials. a great test platform for potential drug candidates, which Furthermore, altered glycosylation and sialylation has been will evaluate the safety and potential side effects of drugs observed in relation to the recently identified genetic AD risk in a human matter. factors TREM2 and CD33. These are both linked to microglia Frontiers in Neuroscience | www.frontiersin.org 14 January 2021 | Volume 14 | Article 625348 fnins-14-625348 January 2, 2021 Time: 16:0 # 15 Haukedal and Freude Implications of Glycosylation in AD activity and neuroinflammation, presenting a possible link role of glycosylation in AD pathogenesis, glycans remain as between neuroinflammation and glycosylation in AD. promising new biomarkers for early diagnosis and drug targets Although studies have indicated that glycans could serve in AD treatment. as new and reliable biomarkers for AD, and could be great targets for medical intervention, further research is necessary to confirm these potentials. One challenge is the AUTHOR CONTRIBUTIONS fact that although modulators of the glycosylation process have been beneficial, precise mechanistic insights are still HH wrote the manuscript and prepared the figures. KF wrote lacking, and due to the fact that multiple proteins are being and edited the manuscript. Both authors approved the final modified in a similar matter, it is difficult to predict if the version. Both authors contributed to the article and approved the effects of treatments is caused by one or more proteins. submitted version. Studying the potential link between glycosylation, ER stress and Golgi fragmentation could be an interesting new aspect in future AD research, to potentially elucidate these mechanisms. FUNDING iPSC modeling provides a potential new platform for glycan research in neurodegenerative disorders, and could be a This work was supported by Innovation Fund Denmark great addition accompanying the animal and postmortem (BrainStem and NeuroStem), Alzheimer Foundation Denmark, studies. Whilst more knowledge is needed to confirm the and Novo Nordisk Foundation (GliAD – NNF1818OC0052369). Chua, C.-C., Lim, M.-L., and Wong, B.-S. (2010). 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Conflict of Interest: The authors declare that the research was conducted in the doi: 10.1074/jbc.M114.577213 absence of any commercial or financial relationships that could be construed as a Yuzwa, S. A., Macauley, M. S., Heinonen, J. E., Shan, X., Dennis, R. J., He, Y., potential conflict of interest. et al. (2008). A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat. Chem. Biol. 4, 483–490. doi: 10.1038/ Copyright © 2021 Haukedal and Freude. This is an open-access article distributed nchembio.96 under the terms of the Creative Commons Attribution License (CC BY). The use, Yuzwa, S. A., Shan, X., Jones, B. A., Zhao, G., Woodward, M. L., Li, X., et al. distribution or reproduction in other forums is permitted, provided the original (2014). 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