The O-GlcNAc modification protects against protein misfolding and

Subscriber access provided by CAL STATE UNIV BAKERSFIELD

Review

The O-GlcNAc modification protects against protein misfolding and aggregation in neurodegenerative disease Philip Ryan, Mingming Xu, Andrew K Davey, Jonathan J. Danon, George D Mellick, Michael Kassiou, and Santosh RUDRAWAR ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00143 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

The O-GlcNAc modification protects against protein misfolding and aggregation in neurodegenerative disease Philip Ryan1,2,3, Mingming Xu4, Andrew K. Davey1,2,3, Jonathan J. Danon5, George D. Mellick3, Michael Kassiou5*, Santosh Rudrawar1,2,3,5*† 1Menzies

Health Institute Queensland, Griffith University, Gold Coast 4222, Australia of Pharmacy and Pharmacology, Griffith University, Gold Coast, 4222, Australia 3Quality Use of Medicines Network, Griffith University, Gold Coast, 4222, Australia 4Griffith Institute for Drug Discovery, Griffith University, Nathan, 4111, Australia 5School of Chemistry, The University of Sydney, NSW 2006, Australia 2School

KEYWORDS: Amyloid, tau, synuclein, Alzheimer’s disease, Parkinson’s disease, neurodegenerative disease, peptides, glycopeptides, OGT, GlcNAc, glycosylation. ABSTRACT: Post-translational modifications (PTM) of proteins are becoming the focus of intense research due to their implications in a broad spectrum of neurodegenerative diseases. Various PTMs have been identified to alter the toxic profiles of proteins which play critical roles in disease etiology. In Alzheimer’s disease (AD), dysregulated phosphorylation is reported to promote pathogenic processing of the microtubuleassociated tau protein. Among the PTMs, the enzymatic addition of N-acetyl-D-glucosamine (GlcNAc) residues to Ser/Thr residues is reported to deliver protective effects against the pathogenic processing of both amyloid precursor protein (APP) and tau. Modification of tau with as few as one single O-GlcNAc residue inhibits its toxic self-assembly. This modification also has the same effect on the assembly of the Parkinson’s disease (PD) associated α-synuclein (ASyn) protein. In fact, O-GlcNAcylation (O-linked GlcNAc modification) affects the processing of numerous proteins implicated in AD, PD, amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD) in a similar manner. As such, manipulation of a protein’s O-GlcNAcylation status has been proposed to offer therapeutic routes toward addressing multiple neurodegenerative pathologies. Here we review the various effects that OGlcNAc modification, and its modulated expression, have on pathogenically significant proteins involved in neurodegenerative disease.

INTRODUCTION Each year, more than 9.9 million people are diagnosed with dementia.1 Today, there are more than 47 million people living with dementia, though this number is expected to triple by 2050. The total global cost of dementia exceeds USD 1 trillion, and yet the large majority of people living with dementia remain undiagnosed, and unable to access care and treatment. This is due, in part, to the difficulties associated with diagnosis. Though dementia symptoms arise from distinct neural abnormalities, autopsy often reveals cases of mixed dementia, complicating the process.2 There is no cure available for dementia, nor the majority of neurodegenerative disorders, which share some pathological similarities. Since Alzheimer’s disease (AD) pathology is present in most dementia cases, a majority of empirical research has been focused on understanding its cause and driving mechanisms. Likewise, AD chief pathological hallmarks are studied in the pursuit of promising therapeutic avenues.

represent the late-stage consequences of protein misfolding and self-assembly and do not necessarily correlate with pathogenic toxicity. Rather findings are suggesting that the misprocessing of smaller monomeric and oligomeric species are implicated.3 Similar observations are being made across the field, with researchers shifting focus from aggregate structures like neurofilament tangles and protein inclusions characteristic of other neurodegenerative diseases (NDs) such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) or Huntington’s disease (HD), to more closely examine the smaller soluble protein species (Figure 1).

The chief pathological hallmarks of AD include extracellular senile plaques and intracellular neurofibrillary tangles (NFTs). These aggregate structures 1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1: O-GlcNAc modification of proteins implicated in NDs is identified to play a protective role. Green indicates effects are due to direct O-GlcNAc modification whereas Red indicates effects are due to O-GlcNAc modification of upstream mediators. The post-translational modification (PTM) of the proteins implicated in NDs are also gaining increased research interest, as various modifications have been identified to alter toxicity profiles. As such, manipulation of a neurotoxic protein’s modified status, or related upstream factors, has been proposed to offer therapeutic routes toward addressing dementia’s underlying pathologies. In AD, a number of PTMs of APP and tau protein are suggested to play critical roles in the disease etiology. Among the PTMs, the enzymatic addition of N-acetyl-Dglucosamine (GlcNAc) residues to serine and threonine residues of proteins is reported to confer protective effects against their pathogenic processing. Furthermore, OGlcNAcylation (O-linked GlcNAc modification) is reported to affect the proteins implicated in ALS, PD and HD in similar ways. Here, in continuation of our interest in NDtargeted therapeutics,4-7 we aim to summarize the evidence that exists for a potential, protective role assumed by the O-GlcNAc modification common throughout neurodegeneration. O-GlcNAcylation in NDs Originally discovered within lymphocytes,8 the O-GlcNAc modification has now been identified on a diverse array of proteins localized within the cytoplasm, nucleus and mitochondria. In fact, more than 4000 proteins have been identified as O-GlcNAcylation candidates. These proteins are involved in almost all aspects of cellular metabolism9-11 and so naturally, O-GlcNAcylation has become the focus of a broad scope of research. The addition and removal of OGlcNAc onto protein substrates is governed entirely by two ubiquitously expressed enzymes: O-GlcNAc transferase [EC 2.4.1.255] (OGT) and O-GlcNAc hydrolase [EC 3.2.1.169] (OGA) respectively (see Figure 2).10 OGT employs the universal donor substrate uridine 5´-diphospho-Nacetylglucosamine (UDP-GlcNAc) to catalyse the transfer of GlcNAc onto serine and threonine residues. Meanwhile, OGA specifically recognises the O-GlcNAc unit occupying its substrate proteins,12 actively catalysing its hydrolytic cleavage.13 For a detailed description of OGT and OGA substrate recognition, please refer to Walker and coworkers’ comprehensive review.14 Between 2-3% of cellular

Page 2 of 16

glucose is biosynthetically transformed into UDP-GlcNAc following the serial processing of five enzymes constituting the hexosamine biosynthetic pathway (HBP). OGTmediated transfer of O-GlcNAc onto proteins is therefore closely tied to cellular glucose concentrations, though notably glucosamine and GlcNAc may also be recycled via salvage pathways.15 In fact, it has been found that a decreased availability of glucose invokes a considerable reduction in the level of O-GlcNAc present in the mouse brain.16 Furthermore, hyperglycaemic conditions yield increased levels of O-GlcNAc.17 Enzymatic cycling of GlcNAc on a protein can take place several times during its lifetime and appears to be a completely conserved cellular mechanism within eukaryotes.18

Figure 2: The cycling of O-GlcNAc onto hydroxyl amino acids is governed exclusively by the OGT and OGA enzymes.10 OGT and OGA are ubiquitously expressed, though the highest levels of expression of both occurs within the brain.13, 19 In particular, the cerebellar cortex and hippocampus exhibit high levels of OGT expression and OGlcNAc modified proteins.20-21 OGT is expressed as three predominant isoforms arising from alternative splicing, namely short OGT (sOGT), mitochondrial OGT (mOGT) and nucleocytoplasmic OGT (ncOGT). These isoforms are differentiated by alterations in their tetratricopeptide repeat (TPR) domains, regions involved in substrate recognition. The ncOGT isoform is the primary product of the ogt gene and is the most-studied to date. It catalyses the transfer of O-GlcNAc via an ordered, sequential bi-bi mechanism.22 Specifically, OGTs carboxy-terminal domain hosts the donor substrate in a catalytic pocket, stabilised by pi-pi interactions and hydrogen-bonding,23 while nearby TPR units arrange a cleft within which the acceptor substrate is invited.22 The cleft is postulated to shift between open and closed states owing to a latch also fabricated by the TPR units, revoking access to the binding pocket upon association of the acceptor substrate. The availability of the crystal structure of OGT and its complex with a catalytically incompetent substrate has led to an influx of potent, selective inhibitors.24-26 These tools are being designed with the goal of probing OGT’s intracellular mechanisms. Only very recently has an inhibitor which is potent, selective, and membranepermeable been described,26 and so potential therapeutic benefits derived from attenuating increased O-GlcNAc levels via OGT inhibition within living models are yet to be fully explored. Effective pharmacological inhibition of OGT’s counterpart enzyme OGA has been studied in greater detail.

2


Page 3 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


OGA is expressed as two unique isoforms. The larger variant (OGA-L) is localised in nuclei and cytosol while the shorter OGA-S, is limited to nuclear localization.27 Regarding O-GlcNAc hydrolysis, OGA-S is relatively less active than OGA-L28 and is only expressed during embryo development.29 Knockout studies in mice suggest OGA plays a critical role in brain development.30-31 Unlike for OGT, a number of effective OGA inhibitors have been identified. Though highly potent, PUGNAc (1, Scheme 1) was found to also effectively inhibit human lysosomal hexosaminidase, limiting its utility. Selectivity over lysosomal hexosaminidases was achieved following the introduction of a fused thiazoline ring carrying a pendant alkyl chain to produce NButGT (2). Rational modification of NButGT finally yielded Thiamet-G (3), differing only by one amino group. Thiamet-G is able to permeate the blood-brain barrier and is highly selective for human OGA. As such, it is being frequently used in cell-based experiments and in vivo. Most recently, a novel PET ligand for OGA, denoted 18F-LSN3316612 (4), has facilitated the imaging and quantification of OGA in mice and rhesus monkeys.32

HO HO

OH O AcHN

N

HO HO

H N

O

OH O N S

O 1

2

OH O

HO HO

HN 3

N

O

N S 18F

N

O

S N

N H

4

Scheme 1: The most frequently investigated OGA inhibitors in order of their discovery, PUGNAc (1), NButGT (2), Thiamet-G (3) and the recently identified PET ligand, LSN3316612 (4). Given the vast range of O-GlcNAcylation substrates, increasingly sophisticated methods are proving invaluable for the identification and prioritization of disease-relevant modification sites.33-34 O-GlcNAcylation has been found to interplay with other PTMs, most notably drawing parallels to O-phosphorylation.35 This is due to the fact that, like Ophosphorylation, O-GlcNAcylation is dynamic, modifies Ser/Thr residues, and can occur multiple times in a substrate proteins lifetime.18 Site mapping studies have found that many O-GlcNAc sites are also phosphorylation sites, if not neighbours.36-37 Congruent findings suggest OGlcNAcylation and O-phosphorylation competitively modify common substrates, thus reciprocally modulating one another’s functioning.38-39 Furthermore, increased levels of O-GlcNAc via pharmacological OGA inhibition

have frequently led to alterations in protein phosphorylation levels.40-42 A proteomics study conducted by Hart and co-workers found that globally elevated OGlcNAcylation levels lowered phosphorylation at 280 sites while actually increasing it at 148 other sites.43 Recent studies have reported similar observations.38 Moreover, in a number of cases where acute OGA inhibitor administration has decreased phosphorylation levels, longterm treatment has had no significant influence.42, 44-45 Perhaps not surprisingly, in numerous cases OGT and OGA participate in regulating signalling cascades otherwise governed by phosphorylation.46-47 Protein aggregation in NDs The protein aggregation characteristic of NDs typically proceeds via a sigmoidal growth curve that can be subdivided into three principal growth phases. Firstly, a slow-paced initial lag-phase, representative of primary nucleation events such as protein misfolding, establishment of aberrant hydrophobic interactions during pairing, and the arrival of discrete intermediate structures such as soluble oligomers and protofibrils.48 Upon arrival of these intermediate structures, a rapid extension phase is observed, resulting in the appearance of dense, insoluble aggregate structures. Aggregation is also aggressively accelerated by secondary nucleation events catalysed at the surface of these intermediate and mature protein templates. The process is presumably initiated as a result of altered protein homeostasis, however a number of the implicated proteins have been reported to propagate between cells, spreading pathogenesis in a prion-like manner. For more insight on protein aggregation in NDs, please refer to the comprehensive review by Soto & Pritzkow.49 Notably, many of the self-assembling proteins associated with NDs possess intrinsically disordered domains,50 regions rapidly interconverting between collapsed and extended structures. These segments often host known sites of PTM, including O-GlcNAcylation.39, 51 In fact, O-GlcNAc is most abundantly found on such disordered regions. Disordered proteins may adopt structural conformations that promote their self-assembly which, in turn, alters their interactions with chaperones or processing enzymes, triggering these rapid aggregation cascades. Reports suggest O-GlcNAcylation at specific sites inhibits protein misfolding and aggregation, potentially through increasing steric hindrance or protein solubility.5253 Indeed, simple O-GlcNAc addition abrogates the amyloidogenesis of a coiled-coil-based model peptide.54 Though N-linked, the GlcNAc modification of even the human prion protein is reported to significantly inhibit its aggregation and cytotoxicity.55 α-Synuclein PD is characterised by the progressive degeneration of dopamine-producing neurons in the substantia nigra, resulting in impaired motor function. This impairment manifests compromised postural reflexes, muscular rigidity and bradykinesia in patients. Despite extensive research, the exact mechanism of the degeneration of dopaminergic neurons is yet to be uncovered. Close inspection of the substantia nigra led to the identification 3



of Lewy bodies and Lewy neurites. These are proteinaceous aggregates located within neural cells, the primary structural component of which is fibrillar α-synuclein (ASyn). Point mutations in the ASyn gene (SNCA) underlie familial disorders with parkinsonian characteristics, suggesting a pathogenic role for ASyn accumulation. The current consensus is that ASyn self-assembly plays a critical role in PD, whether it be a cause or symptom of the complex pathological cascade. Though present in low concentrations throughout most of the body, ASyn is found predominantly in the presynaptic terminals of neurons, where it interacts with phospholipids and proteins.56 Reports suggest ASyn has a role in regulating supply and management of synaptic vesicles,57 while also possibly regulating neuronal release of dopamine, a neurotransmitter involved in controlling voluntary and involuntary movement. ASyn binds simultaneously to plasma membrane phospholipids (via its N-terminal domain) and to synaptobrevin-2 (via its Cterminal domain) to promote SNARE-complex assembly.58 SNARE-complex assembly and disassembly is critical for repeated neurotransmitter release at presynaptic terminals. The structural conformational of ASyn is highly dependent upon its environment and is subject to modulation following a change in conditions.59 Like a prion, ASyn misfolds and self-assembles. Its interaction with membranes modulates its rate of assembly, as is the case with other amyloid proteins like islet amyloid polypeptide (IAPP) and amyloid-β (Aβ). ASyn most commonly exists in an intrinsically disordered state, though the presence of partial structures and structured oligomeric states have been recently reported.60 Alternative splicing yields three isoforms of ASyn, though full-length 140 amino acid ASyn is the major form. Its primary structure is composed of three distinct domains: i) the N-terminal region spanning residues 1-60, an amphipathic stretch dominated by four 11-residue repeats, the region responsible for modulating ASyn interactions with membranes; ii) the central hydrophobic region spanning residues 61-95, host of the non-amyloid-β component (NAC), the stretch implicated in self-assembly; and iii) the C-terminal region spanning residues 96-140, a proline-rich, highly acidic region able to interact with small molecules, other proteins and metals. Yet to be determined is how far upstream in the pathogenic cascade ASyn self-assembly is positioned, and whether the triggering of other specific molecular events is compulsory. Since aging is the greatest risk factor, normal aging could even be considered an upstream event in PD pathogenesis. It has been suggested that misfolded ASyn may be effectively cleared in young neurons via proteostatic mechanisms compromised in aged, yet otherwise equivalent, cells.61 Also debated is whether it is the oligomeric species or the actual assembly process that is responsible for the pathogenic toxicity. It has been suggested that large ASyn aggregates actually work to sequester other harmful conformers, thus acting in a neuroprotective manner. Currently, the neurotoxicity is thought to be either characteristic of the oligomeric ASyn

Page 4 of 16

species, or that it is somehow triggered by the supraphysiological levels of monomeric ASyn.62-64 Recent findings suggest aggregation prone species propagate between neurons following release into the extracellular space.65-66 Upon arrival, they may initiate secondary nucleation events with endogenous ASyn, accelerating spread of the pathology and disease progress.67 A number of PTMs of ASyn supposedly contribute to Lewy pathology. Notably, cleavages at the ASyn C-terminus typically increase the resultant truncated protein’s aggregation, as has been demonstrated in vitro and in overexpression experiments.68 Specifically calpain, a cysteine protease, cleaves soluble ASyn at a number of sites.69 Calpain also cleaves ASyn fibres to generate Ctruncated forms which promote seeded aggregation.70 Calpain activity correlates with disease progression in PD mouse models.71 An estimated 90% of the proteins constituting ASyn aggregates bear phosphate groups at Ser129, suggesting that ASyn phosphorylation, or at least elevated levels of phosphorylated ASyn, contributes to PD pathogenesis. Disputing this, modified forms of ASyn unable to undergo phosphorylation yielded conflicting results.72 Working competitively against phosphorylation, O-GlcNAcylation of ASyn is speculated to be a protective mechanism against PD pathogenesis. To date, nine different sites of O-GlcNAcylation have been identified in vivo (Figure 3).73-76 The O-GlcNAc modification has been shown to slow the aggregation rate of ASyn relative to the wild-type. Perhaps not coincidently, five of the nine identified O-GlcNAcylation sites reside within the NAC region hosting the pathogenic selfrecognition interactions. As with other NDs, reports are suggesting that O-GlcNAcylation of ASyn plays a protective role in PD.

44 59 72 81 33 54 64 75 87 1

NAC (61-95)

140

-synuclein Figure 3: Known sites of O-GlcNAcylation include 33, 44, 54, 59, 64, T72, 75, 81 and 87, known sites of Ophosphorylation include S87, Y125, S129, Y133 and Y126. Research to date has focused mostly around native sites of O-GlcNAcylation situated within the NAC. Initially it was found that semisynthetic ASyn bearing an O-GlcNAc unit at Thr72 (ASyngT72) displayed a reduced self-assembling propensity relative to the wild-type during thioflavin T (ThT) fluorescence assay.77 In fact, ASyngT72 did not form aggregates even after 7 days of incubation. Similarly, modifications at any of Thr75 (ASyngT75), Thr81 (ASyngT81) and Ser87 (ASyngS87) afford comparable effects.78 Coincubation of ASyngT72 with the wild-type saw only a slight inhibition of the wild-type ASyn aggregation, with results indicating that ASyngT72 was incorporated into the aggregate structures at a reduced efficiency. TEM found semisynthetic ASyngS87 forms shorter fibrils than the wildtype, while ASyngT72 formed only small, broken fibrils.79 4


Page 5 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASyngT75 and ASyngT81 formed rare, highly irregular aggregates, and the peptide tri-O-GlcNAcylated at all of Th72/Thr81/Ser87 did not aggregate at all. In the presence of preformed fibers, self-assembly kinetics of ASyn is heavily dependent upon the site of O-GlcNAcylation. Similarly, treatment of cells infected with preformed fibrils with O-GlcNAcylated ASyn gives varying results dependent upon its site of modification. Interestingly, OGlcNAc modification at any of the listed sites does not induce any significant secondary structure as compared to wild-type ASyn.78 Nor did the modifications have any large effect on biologically relevant activities of the protein, suggesting O-GlcNAcylation might not disrupt ASyn’s endogenous functioning. Interestingly, ASyngT72 was unable to be phosphorylated at Ser129, a pathologically relevant site, by any of CK1, PLK3, or GRK5 kinases.77 Also reported was the incapacity of a triply O-GlcNAc modified, pathologically-relevant A53T mutant to aggregate.78 Looking closer at the self-recognition motif, Pratt and coworkers investigated the aggregative properties of a truncated NAC analogue, ASyn68-77 (GAVVTGVTAV) functionalised with GlcNAc at the central Thr residue, corresponding to the native Thr72.80 As with ASyngT72, this synthetic glycopeptide exhibited a reduced self-assembling propensity. An unmodified control, ASyn68-77, significantly accelerated wild-type aggregation while the GlcNAcylated analogue did not. Their finding supports the mounting evidence that the O-GlcNAc modification disqualifies ASyn from participating in pathogenic aggregation. The O-GlcNAc modification is also reported to inhibit calpain-mediated ASyn proteolysis.81 As mentioned previously, calpain activity correlates well with disease progression in PD mouse models, catalysing the truncation of ASyn into aggregation-prone species. Both the synthetic ASyngT72 and ASyngS87 peptides exhibit strong resistances to cleavage from recombinant calpain, where unmodified ASyn is degraded rapidly. Because a single O-GlcNAc unit was able to inhibit cleavage at fairly distant sites, it was proposed that the saccharide may have been, besides introducing steric bulk, increasing the solubility of the peptide and subtly affecting its natively unfolded structure. The authors speculated that O-GlcNAc may prevent nonspecific interactions with other enzymes through similar means. Though there is a strong case forming for the O-GlcNAc modification qualifying as a protective modification, there also exists contrarian evidence. Li and co-workers studied the effects of O-GlcNAc on aggregation by generating enzymatically O-GlcNAcylated ASyn via its co-expression with sOGT in E. coli.82 During ThT fluorescence studies, their OGT-processed ASyn did not aggregate into fibrils, however subsequent Western blot analyses identified SDSresistant oligomers forming that were absent from the wild-type sample. The authors suggested that OGlcNAcylation as studied in their case, had promoted oligomer-formation underlying the reduced aggregation. They proposed that O-GlcNAcylation of ASyn may therefore escalate pathogenesis by promoting oligomer formation.82

As OGA inhibitor therapies gain increased clinical attention for the treatment of tauopathies, new evidence is suggesting excessive O-GlcNAcylation may actually promote ASyn accrual via activation of alternative cellular pathways. Zhang and co-workers found that OGAinhibition via acute Thiamet-G treatment promoted phosphorylation of mTOR and AKT, leading to decreased autophagic flux and subsequent accretion of dysfunctional ASyn.83 Thiamet-G treatment increased levels of monomeric ASyn after 7 days by 50% relative to the control. Subsequent re-activation of autophagy via rapamycin, an mTOR inhibitor, attenuated the increase in ASyn accumulation also. The authors suggested that administering OGA inhibitors to abate AD-related tau phosphorylation may simultaneously impact ASyn homeostasis, especially since ASyn build-up occurs in ~60% of AD cases. Meanwhile, Vocadlo and co-workers reported that pharmacological inhibition of OGA, in each of neuroblastoma N2a cells, primary rat neurons, and a mouse brain, actually stimulates autophagy via an mTORindependent pathway. They outlined the viability of OGA inhibition as a strategy for addressing ND progression.84 Tau: The intraneuronal, microtubule-associated tau protein has been identified as a prime therapeutic target in addressing AD pathology. A hallmark of the disease, the neurofibrillary tangle arises from the aggregation of paired helical filaments (PHFs), fibrous deposits of self-assembled tau and its truncated derivatives. Like with other NDs, the tau cascade hypothesis attributes the toxicity characteristic of AD to the assembly of the smaller monomeric and oligomeric units rather than the larger aggregate structures which act as templates for secondary nucleation events to take place. Tau is present in neurons where it interacts with tubulin, promoting its assembly into microtubules while stabilising pre-established microtubule structures. In the human brain, tau is expressed in six molecular isoforms as a result of alternative pre-mRNA splicing. The tau isoforms are composed of a combination of either zero (0N), one (1N), or two (2N) 29-residue amino-terminal inserts and either three (3R) or four (4R) microtubule-binding repeats (MTBRs). Within the human brain, a approximate equal ratio of 3R and 4R isoforms are expressed. The largest isoform, tau-441 (2N4R), contains 80 serine and threonine residues and only limited hydrophobic character. Tau is typically unstructured and elastic; CD studies indicate limited secondary structure.85 Prior to aggregation, tau is suggested to adopt a compressed “paperclip” conformation.86 Tau is subject to an array of post-translational modifications, however its phosphorylation on Ser/Thr residues and truncation has been most closely tied to AD pathogenesis. Phosphorylation of tau regulates its modulatory functions by impairing its ability to bind microtubules. While normal tau has been found containing between 2-3 mol phosphate/mol protein, tau from the autopsied AD brain has been found to contain a 3- to 4-fold increase in phosphate groups.87 The longest 5



isoform, tau-441, contains more than 80 phosphorylation sites potentially modified in AD though only very few are well characterised.88 Phosphorylation of tau residue Ser202/Thr205 is well established and specific antibodies are developed as tool labelling phosphorylated Ser202/Thr205 for staging of AD. Abnormal hyperphosphorylation of tau (P-tau) results in its detachment from microtubules, suppressing its activity and secondarily promoting its self-assembly into oligomers, ultimately giving rise to the NFTs present in the AD brain. Tau aggregation patterns and the quantities of NFTs both show clear correlation with AD cognitive decline.89 Prevention of tau phosphorylation did not prevent NFT formation in transgenic (tg) tau mice, however it did block motor impairment and presumptive neuron loss.90 Tau phosphorylation is also not a requirement for fibril propagation,91 rather inhibiting tautau binding in some cases.92 Tau-associated toxicity is thought to be derived from the smaller oligomers and their assembly. PHFs are composed mostly of truncated tau, restricted to its repeat domain units.93-94 Like some other misfolding proteins, low molecular weight aggregate species can transfer between cells, acting as seeds that promulgate further tau misfolding and self-assembly.95-96 The most promising tau aggregation inhibitors are also able to disaggregate preformed oligomers, assaulting the seeding process and promoting release of monomeric tau susceptible to proteolytic processing. Two key segments in tau sequences, PHF6 (306VQIVYK311) and PHF6* (275VQIINK280), located within the MTBR domain are suggested to be the principal sites of oligomer nucleation. Peptides retaining one or both segments exhibit a propensity to stack in parallel β-sheets, form fibrils and ‘steric zippers’97 stabilised by highly ordered H-bonding networks, complementary electrostatic interactions and strong pi interactions.97-98 These segments have been the targets of rationally designed inhibitors which earn utility by mimicking the native binding interactions.

123 191 185 1

N1-N2 (45-103)

409? 413 400 412

R1-R4 (244-372)

441

tau-441, (2N4R) Figure 4: Known sites of tau O-GlcNAcylation include S123, S185, S191, S400, S412 and S413.99-100 Modification at S409 is yet to be confirmed.99 Pathological tau hyperphosphorylation was found to be caused by downregulation of protein phosphatase 2A (PP2A),101-102 a major tau phosphatase governing tau phosphorylation and dephosphorylation. It was later found that tau phosphorylation was inversely regulated by OGlcNAc addition in vitro, in tissue culture cells, and ex vivo in rat brain slices (Figure 4).40 Furthermore, downregulation of OGT leads to increased tau phosphorylation and onset of AD pathology.103 In fact, deletion of the OGT-encoding gene in mice leads to increased tau phosphorylation, besides the obvious global

Page 6 of 16

decrease in O-GlcNAc levels.104 Findings from a recent systematic examination suggest that the reciprocal regulation is the result of indirect mechanisms.100 Wilson and co-workers produced forebrain-specific OGT conditional knockout (OGT cKO) mice which reportedly developed signs of severe, progressive neurodegeneration characterised by neuron death, gliosis, elevated levels of hyperphosphorylated tau and Aβ peptides, neuroinflammation and memory deficits.105 Twelve-month old triple-tg mice (3xTg-Ad) displayed reduced levels of tau O-GlcNAcylation consistent with tau hyperphosphorylation in the hippocampus. The reduction of O-GlcNAcylation was region specific and was not associated with changes in expression or activity of OGlcNAcylation regulatory enzymes.106 In fact, the expression of OGT, OGA and GFAT, the HBP rate-limiting enzyme, remained unaffected in the hippocampus, hinting that the reduction in O-GlcNAcylation may be due to defective glucose metabolism. Gong and co-workers identified that impaired brain glucose metabolism actually leads to neurofibrillary degeneration via downregulation of O-GlcNAcylation.107 Finally, AD patient cortical brain tissues reportedly have significantly reduced levels of OGT expression compared to aged and post-mortem delay matched brain tissue.105 Beyond inhibiting its phosphorylation, the O-GlcNAc modification hinders tau oligomerization.44 Vocadlo and co-workers demonstrated that a recombinantly produced O-GlcNAc-modified tau construct (spanning residues 244441) formed oligomers at a slower rate, and to a lesser extent than the unmodified, recombinant control sequence during ThS fluorescence assays. Modification of recombinant full-length tau-441 also facilitated its resistance to aggregation in vitro.108-109 Indeed, O-GlcNAc modification of tau-441 appeared to slow tau’s extension, perturbing the equilibrium position between oligomeric and free tau. Results suggest O-GlcNAc modification at specifically Ser400 plays a critical role in hindering aggregation.109 Closer interrogation revealed the Ser400 modification had no observable impact on the global structure of tau. NMR analyses of truncated tau353-408 bearing O-GlcNAc at a residue corresponding to Ser400 found that O-GlcNAc, at most, caused only small, localised structural or dynamic changes. Importantly, O-GlcNAc on full-length tau did not impact its ability to bind and stabilise microtubules. As a result of the mounting evidence, specifically that impairment of glucose metabolism and GlcNAc regulation and their downstream consequences constitute early hallmarks of Alzheimer’s disease,107 upregulation of OGlcNAcylation via pharmacological OGA inhibition has become a prime therapeutic objective.44 As mentioned previously, Thiamet-G is the most effective OGA inhibitor developed to date (Ki = 21 nM) and is orally bioavailable. Acute treatment with Thiamet-G effectively decreased phosphorylation of tau at pathologically relevant sites, Thr231 and Ser396, in PC-12 cells.42 Thiamet-G was able to decrease effectively aggregation in tau-BiFC cells, even after treatment with BZX2, an OGT inhibitor reported to 6


Page 7 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


downregulate O-GlcNAcylation and promote phosphorylation and aggregation.110 Thiamet-G remains effective in vivo, efficiently reducing phosphorylation at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus. Long-term treatment of JNPL3 tau tg mice with ThiametG increased levels of O-GlcNAc modified tau while decreasing levels of tau aggregates and neuron death.44 Interestingly, tau phosphorylation was not significantly altered in this case, suggesting the decrease in aggregation may have been due to the O-GlcNAc unit. Acute, sub-acute and chronic treatment of Tau.P301L tg mice led to the increased O-GlcNAcylation of many brain proteins though only marginally affected tau phosphorylation.111 Nonetheless, Thiamet-G exerted therapeutic effects, increasing the mouse survival rate by three-fold. Interestingly, even acute inhibition of OGA was considered innocuous in vivo.111 In rTg4510 mice, acute treatment led to increased tau O-GlcNAcylation, reduced tau phosphorylation at epitopes S202/S205, S626, S356 and S396 (notably excluding S400), besides reducing levels of pathological tau aggregation.45 Moreover, early intervention with Thiamet-G prevents the mice’s characteristic behavioural changes.112 Chronic treatment strongly increased tau O-GlcNAcylation without impacting levels of phosphorylation of non-pathological tau.45 Subsequent studies on rTg4510 mice found their chronic treatment reduced pathological tau in the brain besides total tau in the CSF also.41 As mentioned previously, the tau PHF6 stretch is considered a primary self-recognition site of tau, causative to its self-assembling nature. It establishes strong intermolecular interactions with equivalent segments in other tau and its truncated analogue self-assembles rapidly in vitro.98 PHF6 has been widely studied and is often used to model pathological tau aggregation. Notably, acetylated PHF6 peptide significantly accelerates Aβ40 and Aβ42 fibrillogenesis in vitro.113 Gazit, Segal and co-workers reported that their synthetic, O-GlcNAcylated PHF6 analogue, besides possessing reduced self-assembly propensity, seemed to inhibit unmodified PHF6 fibril formation upon co-incubation.114 They also investigated the effects from two other derivative glycosides on the sequence, finding that each glycopeptides inhibitory effects were strongly dependent on the nature of the glycoside. Their work is the first evidence that the associative properties of amyloidogenic sequences may be exploited in the design of amyloid-targeted glycopeptide inhibitors. Further modification yielded ‘11aa-GlcNAc’ (AcPGGGS(β-GlcNAc)VQIVYK-NH2), which decreased PHF6 aggregation with increased effectiveness. The researchers deduced that the extent to which GlcNAc affected the selfassembly of the native counterpart depended heavily on the sequence to which they were conjugated.115 Amyloid- and Amyloid Precursor Protein: As the chief constituent of AD’s hallmark senile plaques, Aβ has long been suspected to play a major role in AD pathogenesis. Indeed, it was anticipated that clearance of the plaques would halt nerve cell death and consequent

dementia. Yet although plaque clearance may yield restorative effects in AD mouse models,116 the strategy does not translate well in the human AD brain.117 Contemporary theories instead speculate that neurotoxicity may be inflicted regardless of the arrival of the plaques.118-119 Again, the smaller, soluble monomeric and oligomeric precursors and their accumulation are being held responsible instead. Paradoxically, the appearance and accrual of Aβ and its correlation with symptoms are temporally inconsistent. Furthermore, discrepancies between the locations most affected by NFT formation and neuron death and the locations hosting the highest levels of amyloid deposition remain unaccounted for.120 Finally, levels of Aβ have been shown not to discriminate between AD and healthy brains.121 Despite such controversy, Aβ and its role in AD remains the central focus of a large body of ongoing research. Prior to its truncation, APP functions as a type 1 transmembrane glycoprotein with the majority of its length situated in the exoplasmic space. It is expressed as one of three main isoforms, though the 695-residue long variant is the predominant form expressed within neurons. Though its biological function is poorly understood, it is proposed to play a role in the formation and repair of synapses. It undergoes post-translational processing which, when catalysed by α-secretase, releases free nonpathogenic sAPPα. The sAPPα peptide has a suggested role in neuronal excitability, synaptic plasticity, learning and memory - mechanisms and processes all compromised in the AD patient brain.122-123 Alternatively, APP may be processed by beta-site APP cleavage enzyme 1 (BACE1) to produce sAPPβ which, when subsequently processed by γsecretase, is released as the 39-42 residue-long Aβ. Though the function of Aβ remains undefined, animal studies have shown that its absence does not lead to obvious loss of physiological function. As such, its clearance represents a therapeutic strategy free from mechanism-based toxicity. The majority of Aβ in the human brain exists as the 40-residue variant, Aβ40, however it is the excess Aβ42 generated that predominantly aggregates.124 The production rate of the peptides does not differ in the AD brain, and so impaired efflux mechanisms are speculated to be the cause of increased levels of Aβ42. Aggregation of either variant induces cytotoxicity, however Aβ42 boasts an increased self-assembling propensity, often demonstrating more severe neurotoxic consequences than the Aβ40. Even minor increases in the ratio of Aβ42:Aβ40 reportedly stabilize oligomeric Aβ species and promote neurotoxicity.125 The Aβ sequence is composed of a hydrophilic N-terminal segment, derived from a soluble extracellular domain of APP, and a relatively hydrophobic stretch toward the Cterminal, where it would have occupied a transmembrane helix of APP. In aqueous mixtures, Aβ42 rapidly adopts βsheet structure, contrasted by the Aβ40 peptides random coil.126 Aβ aggregation follows a sigmoidal growth curve, consisting initially of a lag phase prior to rapid aggregation. Oligomerization is catalyzed by dimeric interactions taking place between residues Leu17 to Phe20, the stretch 7



known as the central hydrophobic domain (CHD). Monomer hairpins are united at this site, orienting into antiparallel β-sheet conformation. Seeding and secondary nucleation events significantly accelerate the aggregation process, as with other ND-implicated proteins. Intermolecular and intramolecular forces catalyse the association of partially folded units which elongate to form long fibrils and then further stabilized, bent β-hairpin structures. A number of therapeutic Aβ-targeted strategies are being explored currently. These include blocking βsheet formation, preventing fibrillation, destabilising Aβ oligomers and promoting accumulation via alternative, non-toxic pathways.

292 291

576

1

A 42 (597-638)

695

APP-695 Figure 5: Known sites of APP-695 O-GlcNAcylation include T291, T292, and T576.127 O-GlcNAcylation is also proposed to occur at other sites along the proteins cytoplasmic tail and extracellular domain.75, 128 As with the tau protein, APP is O-GlcNAcylated naturally.128 Like tau, only very few sites of OGlcNAcylation have been identified, and only very recently (See figure 5).127 As such, the effects of APP OGlcNAcylation are currently unknown. Recently, insight into how O-GlcNAcylation affects upstream APP processing has become available. Treatment of SH-SY5Y neuroblastoma with PUGNAc, an OGA inhibitor, has increased levels of O-GlcNAc modified APP and secretion of sAPPα while decreasing secretion of Aβ40. An inverse correlation between O-GlcNAcylation and Aβ formation was reported.129 Treatment of the 5xFAD Aβ mouse model with NButGT, another studied OGA inhibitor, impaired Aβ production and attenuated activated gliosis.130 Clear effects on γ-secretase activity were observed, independent of α- or β-secretase activity. Specifically, NButGT promoted the OGlcNAcylation of γ-secretases essential nicastrin (NCT) domain at the Ser708 residue, possibly impairing its recognition of APP, or at least its ability to initiate its proteolysis. Enhanced levels of NCT increase γ-secretase’s proteolytic activity causing significant APP-CTF accumulation. Interestingly, NButGT treatment did not significantly affect NCT phosphorylation, suggesting phosphorylation and GlcNAcylation may not competitively modify the Ser708 site. Treatment of HeLa cells transfected with the Swedish mutant form of APP also led to increases in levels of sAPPα while decreasing βsecretase products. PUGNAc treatment increased the rate of APP trafficking to the plasma membrane from the transGolgi network and decreased the rate of APP endocytosis. Subsequent work indicated that O-GlcNAcylation of APP promotes its plasma membrane localization, facilitating its non-amyloidogenic processing.131 Thiamet-G treatment of tg TAPP (tau/APP) mice blocked cognitive decline paralleling decreased Aβ and plaque

Page 8 of 16

levels while not affecting tau phosphorylation.132 The researchers suggested that APP processing was not being directly regulated, but that O-GlcNAc was acting at multiple different points along the amyloid cascade. Finally, treatment with prostaglandin J2 (PGJ2), an endogenous mediator of inflammation that promotes cleavage of secreted APP, is reported to increase overall levels of protein O-GlcNAcylation.133 Though the direct effects of PGJ2 on APP O-GlcNAcylation could not be determined, findings suggest that PGJ2-induced OGlcNAcylation of numerous proteins may stimulate the increase of downstream proteolysis of APP. Other neurodegenerative diseases: Huntington disease is characterised by the formation of intracellular protein inclusions comprised primarily of mutant huntingtin (mHtt) protein.134 Normal huntingtin plays a role in vesicular transport, transcription of neuronal genes and production of brain-derived neurotrophic factor (BDNF), besides potentially also possessing some anti-apoptotic function.135 The mutant variant associated with HD is unable to adopt its wild-type folding state, rather it tends to aggregate, inducing cytotoxicity.134 After aggregating, disruption of synaptic transmission and the transport of axonal mitochondria occurs,136-137 before finally transcription factors are sequestered, suffering loss of function.138 Regarding HD, OGlcNAcylation does not necessarily play a protective role. Instead, decreases in global O-GlcNAcylation levels is reported to be protective against huntingtin aggregation and cytotoxicity in neuroblastoma cells and fly models.139 Decreasing O-GlcNAcylation enhances autophagosomelysosome function, which leads to increased basal autophagy flux and clearance of toxic huntingtin aggregates. Hanover and co-workers reported similar results following the expression of a toxic, huntingtin model protein in C. elegans.140 Inactivation of OGA dramatically promoted HD model toxicity while removal of OGT worked to alleviate toxicity. On the other hand, treatment of primary cultured neurons overexpressing mutant huntingtin with Thiamet-G has also been found to improve cell viability, proposedly by alleviating mutant gene-induced disruption of nucleocytoplasmic transport.141 It is reasonable to assume that dysregulated O-GlcNAc cycling is somehow implicated in HD pathogenesis, however determining the precise outcomes of pharmacological modulation of O-GlcNAcylation has proven a complex endeavour. ALS is characterised by neuron death, primarily of motor neurons, in the CNS. Accumulation of axonal spheroids, composed of essential neurofilament (NF) proteins, constitutes a notable pathological feature of ALS.142 As with tau, NFs are naturally O-GlcNAcylated143 and phosphorylated in a mutually reciprocal manner.144 Reports suggest ALS in humans and mice is linked to dysregulation of kinase function, leading to increased levels of phosphorylated products.145-146 It has also been suggested that phosphorylation of NFs precedes their accumulation, and that O-GlcNAcylation may play a protective role, as with other diseases.147 Brandt and co8


Page 9 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


workers reported that decreases of O-GlcNAcylation in the spinal cords of an ALS tg rat model were associated with ALS pathogenesis and that pharmacological OGA inhibition via PUGNAc treatment attenuated pathogenic tangle formation.148 Mice carrying a mutation in superoxide dismutase 1 (SOD1) also exhibit decreased OGlcNAc levels in the spinal cord.149 Familial SOD1 mutations are responsible for 20% of familial ALS cases in humans.150 Treatment of even the mSOD mice with an OGA inhibitor, NButGT, increased levels of O-GlcNAc in the spinal cord.149 These results indicated that O-GlcNAc levels maybe modulated in vivo to increase OGlcNAcylation of NFs, even in familial ALS cases. As such, upregulation of O-GlcNAcylation is emerging as a promising therapeutic strategy toward addressing ALS pathology.

druggable targets. Indeed, enriching our understanding of these cellular events will certainly aid development of novel therapeutics that work to address NDs underlying pathologies. Rational design of O-GlcNAc-modified peptidomimetics may yield selective protein-targeted disruptors of self-assembly and induced neurotoxicity.152 Future findings may facilitate the adjustment of specific proteins’ glycosylation status via alternative means. Regardless, details on the molecular consequences of pharmacologically modulating O-GlcNAcylation are essential if we are to earn a precise understanding of OGlcNAcs definitive and context-specific role.

AUTHOR INFORMATION Corresponding Author

Conclusions and Future Perspectives

* [email protected];

The physiological consequences of manipulating levels of O-GlcNAcylation are gradually being uncovered thanks to many advances made in the field.32, 151 Due to its complex relationships with a vast diversity of proteins and biological processes however, a definitive functional role of O-GlcNAcylation is still yet to be identified. Beyond its common engagement as a nutrient-driven mediator of signalling and transcription, O-GlcNAcylation seemingly plays a key role in the etiology of a smorgasbord of neurological disorders. Regarding PD, O-GlcNAcylation is reported to inhibit the phosphorylation, truncation and self-assembly of ASyn, though OGA inhibition may be associated with its impaired autophagic clearance. OGlcNAcylation of Alzheimer’s disease-associated tau dampens its pathogenic phosphorylation and selfassembly. Furthermore, upregulated O-GlcNAcylation disrupts the pathogenic processing of APP mediated by γsecretase.

* [email protected]

Studies consistently demonstrate that promoting OGlcNAcylation protects against a wide range of cellular stresses. Increasingly it is being suggested that this protective mechanism becomes compromised within the aged brain, possibly due to impaired glucose metabolism. As such, manipulating the posttranslational modifications, particularly the O-GlcNAc status of the various aggregating proteins is emerging as a potential therapeutic strategy for the treatment of various age-related disorders. Because OGlcNAc cycling lies at the interface of so many cellular processes however, OGT- and OGA-targeted interventions are speculated to lead to widespread, cell-biological alterations. Such changes may prove deleterious in nature, if not compensatory.

1. World Alzheimer Report 2016. Alzheimer's Disease International. 2. Schneider, J. A., Arvanitakis, Z., Bang, W., and Bennett, D. A. (2007) Mixed brain pathologies account for most dementia cases in community-dwelling older persons. Neurology 69, 2197. 3. Gadad, B. S., Britton, G. B., Rao, K. S. (2011) Targeting oligomers in neurodegenerative disorders: lessons from αsynuclein, tau and amyloid-β peptide. J. Alzheimers Dis. 24 Suppl. 2, 223-32. 4. Moir, M., Chua, S. W., Reekie, T., Martin, A. D., Ittner, A., Ittner, L. M., and Kassiou, M. (2017) Ring-opened aminothienopyridazines as novel tau aggregation inhibitors. MedChemComm 8, 1275-1282. 5. Chaney, A., Bauer, M., Bochicchio, D., Smigova, A., Kassiou, M., Davies, K. E., Williams, S. R., and Boutin, H. (2018) Longitudinal investigation of neuroinflammation and metabolite profiles in the APPswe×PS1Δe9 transgenic mouse model of Alzheimer's disease. J. Neurochem. 144, 318-335. 6. McLeary, F. A., Rcom-H’cheo-Gauthier, A. N., Kinder, J., Goulding, M., Khoo, T. K., Mellick, G. D., Chung, R. S., and Pountney, D. L. (2018) Dexamethasone inhibits copper-induced alpha-synuclein aggregation by a

As the number of ND cases are expected to increase dramatically, enriching our understanding of the pathophysiological mechanisms underlying the diseases is a priority. Interestingly, a number of these conditions share common pathogenic profiles, characterised by formerly innocuous proteins spontaneously employing prion-like mechanisms. Since in this context O-GlcNAc tends to confer neuroprotective effects, some hypothesise that the specific interactions occurring between proteins that are engineered by O-GlcNAc may be considered

Present Addresses †School of Pharmacy and Pharmacology, Griffith University, Gold Coast 4222, Australia.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources Australian Research Council – Discovery Early Career Research Award (ARC DECRA: DE140101632).

Notes

The authors declare no competing financial interest.

REFERENCES

9



metallothionein-dependent mechanism. Neurotox. Res. 33, 229-238. 7. Chua, S. W., Cornejo, A., van Eersel, J., Stevens, C. H., Vaca, I., Cueto, M., Kassiou, M., Gladbach, A., Macmillan, A., Lewis, L., Whan, R., and Ittner, L. M. (2017) The polyphenol altenusin inhibits in vitro fibrillization of tau and reduces induced tau pathology in primary neurons. ACS Chem. Neurosci. 8, 743-751. 8. Torres, C. R., and Hart, G. W. (1984) Topography and polypeptide distribution of terminal Nacetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J. Biol. Chem. 259, 3308-3317. 9. Wells, L., Vosseller, K., and Hart, G. W. (2001) Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291, 2376-2378. 10. Hart, G. W., Housley, M. P., and Slawson, C. (2007) Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446, 1017-1022. 11. Zachara, N. E., and Hart, G. W. (2006) Cell signaling, the essential role of O-GlcNAc! Biochim. Biophys. Acta 1761, 599-617. 12. Li, S., Wang, J., Zang, L., Zhu, H., Guo, J., Zhang, J., Wen, L., Chen, Y., Li, Y., Chen, X., Wang, P. G., and Li, J. (2019) Production of glycopeptide derivatives for exploring substrate specificity of human OGA toward sugar moiety. Front. Chem. 6, 646. 13. Gao, Y., Wells, L., Comer, F. I., Parker, G. J., and Hart, G. W. (2001) Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain. J. Biol. Chem. 276, 9838-9845. 14. Joiner, C. M., Li, H., Jiang, J., and Walker, S. (2019) Structural characterization of the O-GlcNAc cycling enzymes: insights into substrate recognition and catalytic mechanisms. Curr. Opin. Struct. Biol. 56, 97-106. 15. Hinderlich, S., Berger, M., Schwarzkopf, M., Effertz, K., and Reutter, W. (2000) Molecular cloning and characterization of murine and human Nacetylglucosamine kinase. Eur. J. Biochem. 267, 3301-3308. 16. Li, X., Lu, F., Wang, J. Z., and Gong, C. X. (2006) Concurrent alterations of O-GlcNAcylation and phosphorylation of tau in mouse brains during fasting. Eur. J. Neurosci. 23, 2078-2086. 17. Clark, R. J., McDonough, P. M., Swanson, E., Trost, S. U., Suzuki, M., Fukuda, M., and Dillmann, W. H. (2003) Diabetes and the accompanying hyperglycemia impairs cardiomyocyte calcium cycling through increased nuclear O-GlcNAcylation. J. Biol. Chem. 278, 44230-44237. 18. Roquemore, E. P., Chevrier, M. R., Cotter, R. J., and Hart, G. W. (1996) Dynamic O-GlcNAcylation of the small heat shock protein αB-crystallin. Biochemistry 35, 3578-3586. 19. Okuyama, R., and Marshall, S. (2003) UDP-Nacetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellular distribution of cytosolic and nuclear enzyme. J. Neurochem. 86, 1271-1280. 20. Akimoto, Y., Comer, F. I., Cole, R. N., Kudo, A., Kawakami, H., Hirano, H., and Hart, G. W. (2003) Localization of the O-GlcNAc transferase and O-GlcNAc-

Page 10 of 16

modified proteins in rat cerebellar cortex. Brain Res. 966, 194-205. 21. Liu, K., Paterson, A. J., Zhang, F., McAndrew, J., Fukuchi, K., Wyss, J. M., Peng, L., Hu, Y., and Kudlow, J. E. (2004) Accumulation of protein O-GlcNAc modification inhibits proteasomes in the brain and coincides with neuronal apoptosis in brain areas with high O-GlcNAc metabolism. J. Neurochem. 89, 1044-1055. 22. Lazarus, M. B., Nam, Y., Jiang, J., Sliz, P., and Walker, S. (2011) Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469, 564567. 23. She, N., Zhao, Y., Hao, J., Xie, S., and Wang, C. (2019) Uridine diphosphate release mechanism in O-Nacetylglucosamine (O-GlcNAc) transferase catalysis. Biochimica et Biophysica Acta. General Subjects 1863, 609622. 24. Zhang, H., Tomašič, T., Shi, J., Weiss, M., Ruijtenbeek, R., Anderluh, M., and Pieters, R. J. (2018) Inhibition of O-GlcNAc transferase (OGT) by peptidic hybrids. MedChemComm 9, 883-887. 25. Rafie, K., Gorelik, A., Trapannone, R., Borodkin, V. S., and van Aalten, D. M. F. (2018) Thio-linked UDPpeptide conjugates as O-GlcNAc transferase inhibitors. Bioconjug. Chem. 29, 1834-1840. 26. Martin, S. E. S., Tan, Z.-W., Itkonen, H. M., Duveau, D. Y., Paulo, J. A., Janetzko, J., Boutz, P. L., Törk, L., Moss, F. A., Thomas, C. J., Gygi, S. P., Lazarus, M. B., and Walker, S. (2018) Structure-based evolution of low nanomolar O-GlcNAc transferase inhibitors. J. Am. Chem. Soc. 140, 13542-13545. 27. Wells, L., Gao, Y., Mahoney, J. A., Vosseller, K., Chen, C., Rosen, A., and Hart, G. W. (2002) Dynamic Oglycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic β-Nacetylglucosaminidase, O-GlcNAcase. J. Biol. Chem. 277, 1755-1761. 28. Macauley, M. S., and Vocadlo, D. J. (2009) Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase. Carbohydr. Res. 344, 10791084. 29. Liu, Y., Li, X., Yu, Y., Shi, J., Liang, Z., Run, X., Li, Y., Dai, C. L., Grundke-Iqbal, I., Iqbal, K., Liu, F., and Gong, C. X. (2012) Developmental regulation of protein OGlcNAcylation, O-GlcNAc transferase, and O-GlcNAcase in mammalian brain. PLoS ONE 7, e43724. 30. Olivier-Van Stichelen, S., Wang, P., Comly, M., Love, D. C., and Hanover, J. A. (2017) Nutrient-driven Olinked N-acetylglucosamine (O-GlcNAc) cycling impacts neurodevelopmental timing and metabolism. J. Biol. Chem. 292, 6076-6085. 31. Yang, Y. R., Song, M., Lee, H., Jeon, Y., Choi, E. J., Jang, H. J., Moon, H. Y., Byun, H. Y., Kim, E. K., Kim, D. H., Lee, M. N., Koh, A., Ghim, J., Choi, J. H., Lee-Kwon, W., Kim, K. T., Ryu, S. H., and Suh, P. G. (2012) O-GlcNAcase is essential for embryonic development and maintenance of genomic stability. Aging Cell 11, 439-448. 32. Paul, S., Haskali, M. B., Liow, J. S., Zoghbi, S. S., Barth, V. N., Kolodrubetz, M. C., Bond, M. R., Morse, C. L., Gladding, R. L., Frankland, M. P., Kant, N., Slieker, L., 10


Page 11 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Shcherbinin, S., Nuthall, H. N., Zanotti-Fregonara, P., Hanover, J. A., Jesudason, C., Pike, V. W., and Innis, R. B. (2019) Evaluation of a PET radioligand to image OGlcNAcase in brain and periphery of rhesus monkey and knock-out mouse. J. Nucl. Med. 60, 129-134. 33. Wang, S., Yang, F., Petyuk, V. A., Shukla, A. K., Monroe, M. E., Gritsenko, M. A., Rodland, K. D., Smith, R. D., Qian, W. J., Gong, C. X., and Liu, T. (2017) Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease. J. Pathol. 243, 78-88. 34. Ma, J., Wang, W., Li, Z., Shabanowitz, J., Hunt, D. F., and Hart, G. W. (2019) O-GlcNAc Site mapping by using a combination of chemoenzymatic labeling, copper-free click chemistry, reductive cleavage, and electron-transfer dissociation mass spectrometry. Anal. Chem.91, 2620-2625. 35. van der Laarse, S. A. M., Leney, A. C., and Heck, A. J. R. (2018) Crosstalk between phosphorylation and OGlcNAcylation: friend or foe. FEBS J. 285, 3152-3167. 36. Kamemura, K., Hayes, B. K., Comer, F. I., and Hart, G. W. (2002) Dynamic interplay between Oglycosylation and O-phosphorylation of nucleocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitogens. J. Biol. Chem. 277, 19229-19235. 37. Comer, F. I., and Hart, G. W. (2000) OGlycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate. J. Biol. Chem. 275, 29179-29182. 38. Xu, S. L., Chalkley, R. J., Maynard, J. C., Wang, W., Ni, W., Jiang, X., Shin, K., Cheng, L., Savage, D., Hühmer, A. F., Burlingame, A. L., and Wang, Z. Y. (2017) Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 114, E1536-e1543. 39. Trinidad, J. C., Barkan, D. T., Gulledge, B. F., Thalhammer, A., Sali, A., Schoepfer, R., and Burlingame, A. L. (2012) Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol. Cell. Proteomics 11, 215-229. 40. Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. W., and Gong, C. X. (2004) O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 101, 10804-10809. 41. Hastings, N. B., Wang, X., Song, L., Butts, B. D., Grotz, D., Hargreaves, R., Fred Hess, J., Hong, K. K., Huang, C. R., Hyde, L., Laverty, M., Lee, J., Levitan, D., Lu, S. X., Maguire, M., Mahadomrongkul, V., McEachern, E. J., Ouyang, X., Rosahl, T. W., Selnick, H., Stanton, M., Terracina, G., Vocadlo, D. J., Wang, G., Duffy, J. L., Parker, E. M., and Zhang, L. (2017) Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice. Mol. Neurodegener. 12, 39. 42. Yuzwa, S. A., Macauley, M. S., Heinonen, J. E., Shan, X., Dennis, R. J., He, Y., Whitworth, G. E., Stubbs, K. A., McEachern, E. J., Davies, G. J., and Vocadlo, D. J. (2008) A potent mechanism-inspired O-GlcNAcase inhibitor that

blocks phosphorylation of tau in vivo. Nat. Chem. Biol. 4, 483-490. 43. Wang, Z., Gucek, M., and Hart, G. W. (2008) Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc. Proc. Natl. Acad. Sci. U. S. A. 105, 13793-13798. 44. Yuzwa, S. A., Shan, X., Macauley, M. S., Clark, T., Skorobogatko, Y., Vosseller, K., and Vocadlo, D. J. (2012) Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat. Chem. Biol. 8, 393399. 45. Graham, D. L., Gray, A. J., Joyce, J. A., Yu, D., O'Moore, J., Carlson, G. A., Shearman, M. S., Dellovade, T. L., and Hering, H. (2014) Increased O-GlcNAcylation reduces pathological tau without affecting its normal phosphorylation in a mouse model of tauopathy. Neuropharmacology 79, 307-313. 46. Shi, J., Gu, J. H., Dai, C. L., Gu, J., Jin, X., Sun, J., Iqbal, K., Liu, F., and Gong, C. X. (2015) O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling. Sci Rep 5, 14500. 47. Xu, C., Liu, G. D., Feng, L., Zhang, C. H., and Wang, F. (2018) Identification of O-GlcNAcylation modification in diabetic retinopathy and crosstalk with phosphorylation of STAT3 in retina vascular endothelium cells. Cell. Physiol. Biochem. 49, 1389-1402. 48. Agorogiannis, E. I., Agorogiannis, G. I., Papadimitriou, A., and Hadjigeorgiou, G. M. (2004) Protein misfolding in neurodegenerative diseases. Neuropathol. Appl. Neurobiol. 30, 215-224. 49. Soto, C., and Pritzkow, S. (2018) Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat. Neurosci. 21, 1332-1340. 50. Uversky, V. N. (2015) Intrinsically disordered proteins and their (disordered) proteomes in neurodegenerative disorders. Front. Aging Neurosci. 7, 18. 51. Bond, M. R., and Hanover, J. A. (2015) A little sugar goes a long way: the cell biology of O-GlcNAc. J. Cell Biol. 208, 869-880. 52. Srikanth, B., Vaidya, M. M., and Kalraiya, R. D. (2010) O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18. J. Biol. Chem. 285, 34062-34071. 53. Sohn, K. C., Lee, K. Y., Park, J. E., and Do, S. I. (2004) OGT functions as a catalytic chaperone under heat stress response: a unique defense role of OGT in hyperthermia. Biochem. Biophys. Res. Commun. 322, 10451051. 54. Broncel, M., Falenski, J. A., Wagner, S. C., Hackenberger, C. P., and Koksch, B. (2010) How posttranslational modifications influence amyloid formation: a systematic study of phosphorylation and glycosylation in model peptides. Chem. Eur. J. 16, 7881-7888. 55. Yi, C. W., Wang, L. Q., Huang, J. J., Pan, K., Chen, J., and Liang, Y. (2018) Glycosylation significantly inhibits the aggregation of human prion protein and decreases its cytotoxicity. Sci. Rep. 8, 12603. 56. Chandra, S., Chen, X., Rizo, J., Jahn, R., and 11



Südhof, T. C. (2003) A broken α-helix in folded αsynuclein. J. Biol. Chem. 278, 15313-15318. 57. Diao, J., Burré, J., Vivona, S., Cipriano, D. J., Sharma, M., Kyoung, M., Sudhof, T. C., and Brunger, A. T. (2013) Native α-synuclein induces clustering of synapticvesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. eLife 2, e00592. 58. Burré, J., Sharma, M., Tsetsenis, T., Buchman, V., Etherton, M. R., and Südhof, T. C. (2010) α-Synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329, 1663-1667. 59. Uversky, V. N. (2003) A protein-chameleon: conformational plasticity of α-synuclein, a disordered protein involved in neurodegenerative disorders. J. Biomol. Struct. Dyn. 21, 211-234. 60. Neupane, K., Solanki, A., Sosova, I., Belov, M., and Woodside, M. T. (2014) Diverse metastable structures formed by small oligomers of α-synuclein probed by force spectroscopy. PLoS ONE 9, e86495-e86495. 61. Chu, Y., and Kordower, J. H. (2007) Ageassociated increases of α-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: is this the target for Parkinson's disease? Neurobiol. Dis. 25, 134-149. 62. Wang, C., Zhao, C., Li, D., Tian, Z., Lai, Y., Diao, J., and Liu, C. (2016) Versatile structures of α-synuclein. Front. Mol. Neurosci. 9, 48. 63. Peng, C., Gathagan, R. J., and Lee, V. M. Y. (2018) Distinct α-Synuclein strains and implications for heterogeneity among α-Synucleinopathies. Neurobiol. Dis. 109, 209-218. 64. Melki, R. (2015) Role of different α-synuclein strains in synucleinopathies, similarities with other neurodegenerative diseases. J Parkinsons Dis. 5, 217-227. 65. Falcon, B., Masuda-Suzukake, M., and Goedert, M. (2016) Like prions: the propagation of aggregated tau and α-synuclein in neurodegeneration. Brain 140, 266-278. 66. Guo, Jing L., Covell, Dustin J., Daniels, Joshua P., Iba, M., Stieber, A., Zhang, B., Riddle, Dawn M., Kwong, Linda K., Xu, Y., Trojanowski, John Q., and Lee, Virginia M. Y. (2013) Distinct α-Synuclein strains differentially promote tau inclusions in neurons. Cell 154, 103-117. 67. Chan, D. K. Y., Xu, Y. H., Chan, L. K. M., Braidy, N., and Mellick, G. D. (2017) Mini-review on initiatives to interfere with the propagation and clearance of alphasynuclein in Parkinson's disease. Translational neurodegeneration 6, 33-33. 68. Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752. 69. Mishizen-Eberz, A. J., Guttmann, R. P., Giasson, B. I., Day, G. A., 3rd, Hodara, R., Ischiropoulos, H., Lee, V. M., Trojanowski, J. Q., and Lynch, D. R. (2003) Distinct

Page 12 of 16

cleavage patterns of normal and pathologic forms of αsynuclein by calpain I in vitro. J. Neurochem. 86, 836-847. 70. Mishizen-Eberz, A. J., Norris, E. H., Giasson, B. I., Hodara, R., Ischiropoulos, H., Lee, V. M., Trojanowski, J. Q., and Lynch, D. R. (2005) Cleavage of α-synuclein by calpain: potential role in degradation of fibrillized and nitrated species of α-synuclein. Biochemistry 44, 78187829. 71. Diepenbroek, M., Casadei, N., Esmer, H., Saido, T. C., Takano, J., Kahle, P. J., Nixon, R. A., Rao, M. V., Melki, R., Pieri, L., Helling, S., Marcus, K., Krueger, R., Masliah, E., Riess, O., and Nuber, S. (2014) Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice. Hum. Mol. Genet. 23, 3975-3989. 72. Febbraro, F., Sahin, G., Farran, A., Soares, S., Jensen, P. H., Kirik, D., and Romero-Ramos, M. (2013) Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease. Neurobiol. Dis. 56, 4758. 73. Wang, Z., Udeshi, N. D., O'Malley, M., Shabanowitz, J., Hunt, D. F., and Hart, G. W. (2010) Enrichment and site mapping of O-linked Nacetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 9, 153-160. 74. Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L. C., Saudek, C. D., and Hart, G. W. (2009) Site-specific GlcNAcylation of human erythrocyte proteins: potential biomarker(s) for diabetes. Diabetes 58, 309-317. 75. Alfaro, J. F., Gong, C. X., Monroe, M. E., Aldrich, J. T., Clauss, T. R., Purvine, S. O., Wang, Z., Camp II, D. G., Shabanowitz, J., Stanley, P., Hart, G. W., Hunt, D. F., Yang, F., and Smith, R. D. (2012) Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc. Natl. Acad. Sci. U. S. A. 109, 7280-7285. 76. Morris, M., Knudsen, G. M., Maeda, S., Trinidad, J. C., Ioanoviciu, A., Burlingame, A. L., and Mucke, L. (2015) Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat. Neurosci. 18, 1183. 77. Marotta, N. P., Lin, Y. H., Lewis, Y. E., Ambroso, M. R., Zaro, B. W., Roth, M. T., Arnold, D. B., Langen, R., and Pratt, M. R. (2015) O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease. Nature Chemistry 7, 913-920. 78. Levine, P. M., Galesic, A., Balana, A. T., MahulMellier, A. L., Navarro, M. X., De Leon, C. A., Lashuel, H. A., and Pratt, M. R. (2019) α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson's disease. Proc. Natl. Acad. Sci. U. S. A. 116, 1511-1519. 79. Lewis, Y. E., Galesic, A., Levine, P. M., De Leon, C. A., Lamiri, N., Brennan, C. K., and Pratt, M. R. (2017) OGlcNAcylation of α-synuclein at serine 87 reduces 12


Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aggregation without affecting membrane binding. ACS Chem. Biol. 12, 1020-1027. 80. Marotta, N. P., Cherwien, C. A., Abeywardana, T., and Pratt, M. R. (2012) O-GlcNAc modification prevents peptide-dependent acceleration of α-synuclein aggregation. ChemBioChem 13, 2665-2670. 81. Levine, P. M., De Leon, C. A., Galesic, A., Balana, A., Marotta, N. P., Lewis, Y. E., and Pratt, M. R. (2017) OGlcNAc modification inhibits the calpain-mediated cleavage of α-synuclein. Bioorg. Med. Chem. 25, 4977-4982. 82. Zhang, J., Lei, H., Chen, Y., Ma, Y. T., Jiang, F., Tan, J., Zhang, Y., and Li, J. D. (2017) Enzymatic OGlcNAcylation of α-synuclein reduces aggregation and increases SDS-resistant soluble oligomers. Neurosci. Lett. 655, 90-94. 83. Wani, W. Y., Ouyang, X., Benavides, G. A., Redmann, M., Cofield, S. S., Shacka, J. J., Chatham, J. C., Darley-Usmar, V., and Zhang, J. (2017) O-GlcNAc regulation of autophagy and α-synuclein homeostasis; implications for Parkinson's disease. Mol. Brain 10, 32. 84. Zhu, Y., Shan, X., Safarpour, F., Erro Go, N., Li, N., Shan, A., Huang, M. C., Deen, M., Holicek, V., Ashmus, R., Madden, Z., Gorski, S., Silverman, M. A., and Vocadlo, D. J. (2018) Pharmacological inhibition of O-GlcNAcase enhances autophagy in brain through an mTORindependent pathway. ACS Chem. Neurosci. 9, 1366-1379. 85. Cleveland, D. W., Hwo, S. Y., and Kirschner, M. W. (1977) Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J. Mol. Biol. 116, 227-247. 86. Jeganathan, S., Hascher, A., Chinnathambi, S., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (2008) Proline-directed pseudo-phosphorylation at AT8 and PHF1 epitopes induces a compaction of the paperclip folding of tau and generates a pathological (MC-1) conformation. J. Biol. Chem. 283, 32066-32076. 87. Ksiezak-Reding, H., Liu, W. K. and Yen, S. H. (1992) Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments. Brain. Res. 597, 209-219. 88. Neddens, J., Temmel, M., Flunkert, S., Kerschbaumer, B., Hoeller, C., Loeffler, T., Niederkofler, V., Daum, G., Attems, J. and Hutter-Paier, B. (2018) Phosphorylation of different tau sites during progression of Alzheimer's disease. Acta Neuropathol. Commun., 6, 52. 89. Bierer, L. M., Hof, P. R., Purohit, D. P., Carlin, L., Schmeidler, J., Davis, K. L., and Perl, D. P. (1995) Neocortical neurofibrillary tangles correlate with dementia severity in Alzheimer's disease. Arch. Neurol. 52, 81-88. 90. Le Corre, S., Klafki, H. W., Plesnila, N., Hubinger, G., Obermeier, A., Sahagún, H., Monse, B., Seneci, P., Lewis, J., Eriksen, J., Zehr, C., Yue, M., McGowan, E., Dickson, D. W., Hutton, M., and Roder, H. M. (2006) An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 103, 9673-9678. 91. Morozova, O. A., March, Z. M., Robinson, A. S., and Colby, D. W. (2013) Conformational features of tau fibrils from Alzheimer's disease brain are faithfully

propagated by unmodified recombinant protein. Biochemistry 52, 6960-6967. 92. Schneider, A., Biernat, J., von Bergen, M., Mandelkow, E., and Mandelkow, E. M. (1999) Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments. Biochemistry 38, 3549-3558. 93. Wischik, C. M., Novak, M., Edwards, P. C., Klug, A., Tichelaar, W., and Crowther, R. A. (1988) Structural characterization of the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 85, 4884-4888. 94. Wischik, C. M., Novak, M., Thogersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., Roth, M., and Klug, A. (1988) Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 85, 4506-4510. 95. Kfoury, N., Holmes, B. B., Jiang, H., Holtzman, D. M., and Diamond, M. I. (2012) Trans-cellular propagation of tau aggregation by fibrillar species. J. Biol. Chem. 287, 19440-19451. 96. Frost, B., Jacks, R. L., and Diamond, M. I. (2009) Propagation of tau misfolding from the outside to the inside of a cell. J. Biol. Chem. 284, 12845-12852. 97. Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I., Thompson, M. J., Balbirnie, M., Wiltzius, J. J., McFarlane, H. T., Madsen, A. O., Riekel, C., and Eisenberg, D. (2007) Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature 447, 453-457. 98. Ganguly, P., Do, T. D., Larini, L., LaPointe, N. E., Sercel, A. J., Shade, M. F., Feinstein, S. C., Bowers, M. T., and Shea, J. E. (2015) Tau assembly: the dominant role of PHF6 (VQIVYK) in microtubule binding region repeat R3. J. Phys. Chem. B 119, 4582-4593. 99. Yuzwa, S. A., Yadav, A. K., Skorobogatko, Y., Clark, T., Vosseller, K., and Vocadlo, D. J. (2011) Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody. Amino Acids 40, 857868. 100. Bourré, G., Cantrelle, F. X., Kamah, A., Chambraud, B., Landrieu, I., and Smet-Nocca, C. (2018) Direct crosstalk between O-GlcNAcylation and phosphorylation of tau protein investigated by NMR spectroscopy. Front. Endocrinol. 9, 595. 101. Liu, F., Grundke-Iqbal, I., Iqbal, K., and Gong, C. X. (2005) Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur. J. Neurosci. 22, 1942-1950. 102. Gong, C. X., Singh, T. J., Grundke-Iqbal, I., and Iqbal, K. (1993) Phosphoprotein phosphatase activities in Alzheimer disease brain. J. Neurochem. 61, 921-927. 103. Liu, F., Shi, J., Tanimukai, H., Gu, J., Gu, J., Grundke-Iqbal, I., Iqbal, K., and Gong, C.-X. (2009) Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease. Brain 132, 1820-1832. 13



104. O'Donnell, N., Zachara, N. E., Hart, G. W., and Marth, J. D. (2004) Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Mol. Cell. Biol. 24, 16801690. 105. Wang, A. C., Jensen, E. H., Rexach, J. E., Vinters, H. V., and Hsieh-Wilson, L. C. (2016) Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proc. Natl. Acad. Sci. U. S. A. 113, 15120. 106. Gatta, E., Lefebvre, T., Gaetani, S., Dos Santos, M., Marrocco, J., Mir, A.-M., Cassano, T., Maccari, S., Nicoletti, F., and Mairesse, J. (2016) Evidence for an imbalance between tau O-GlcNAcylation and phosphorylation in the hippocampus of a mouse model of Alzheimer’s disease. Pharmacol. Res. 105, 186-197. 107. Gong, C. X., Liu, F., Grundke-Iqbal, I., and Iqbal, K. (2006) Impaired brain glucose metabolism leads to Alzheimer neurofibrillary degeneration through a decrease in tau O-GlcNAcylation. J. Alzheimers Dis. 9, 1-12. 108. Yuzwa, S. A., and Vocadlo, D. J. (2017) Production of O-GlcNAc modified recombinant tau in E. coli and detection of Ser400 O-GlcNAc tau in vivo. Methods Mol. Biol. 1523, 237-248. 109. Yuzwa, S. A., Cheung, A. H., Okon, M., McIntosh, L. P., and Vocadlo, D. J. (2014) O-GlcNAc modification of tau directly inhibits its aggregation without perturbing the conformational properties of tau monomers. J. Mol. Biol. 426, 1736-1752. 110. Lim, S., Haque, M. M., Nam, G., Ryoo, N., Rhim, H., and Kim, Y. K. (2015) Monitoring of intracellular tau aggregation regulated by OGA/OGT inhibitors. Int. J. Mol. Sci. 16, 20212-20224. 111. Borghgraef, P., Menuet, C., Theunis, C., Louis, J. V., Devijver, H., Maurin, H., Smet-Nocca, C., Lippens, G., Hilaire, G., Gijsen, H., Moechars, D., and Van Leuven, F. (2013) Increasing brain protein O-GlcNAc-ylation mitigates breathing defects and mortality of Tau.P301L mice. PLoS ONE 8, e84442. 112. Wang, X., Smith, K., Pearson, M., Hughes, A., Cosden, M. L., Marcus, J., Hess, J. F., Savage, M. J., Rosahl, T., Smith, S. M., Schachter, J. B., and Uslaner, J. M. (2018) Early intervention of tau pathology prevents behavioral changes in the rTg4510 mouse model of tauopathy. PLoS ONE 13, e0195486. 113. Mohamed, T., Gujral, S. S., and Rao, P. P. N. (2017) Tau derived hexapeptide AcPHF6 promotes beta-amyloid (Aβ) fibrillogenesis. ACS Chem. Neurosci. 9, 773-782. 114. Frenkel-Pinter, M., Richman, M., Belostozky, A., Abu-Mokh, A., Gazit, E., Rahimipour, S., and Segal, D. (2016) Selective inhibition of aggregation and toxicity of a tau-derived peptide using its glycosylated analogues. Chem. Eur. J. 22, 5945-5952. 115. Frenkel-Pinter, M., Richman, M., Belostozky, A., Abu-Mokh, A., Gazit, E., Rahimipour, S., and Segal, D. (2018) Distinct effects of O-GlcNAcylation and phosphorylation of a tau-derived amyloid peptide on aggregation of the native peptide. Chem. Eur. J. 24, 1403914043. 116. Buttini, M., Masliah, E., Barbour, R., Grajeda, H., Motter, R., Johnson-Wood, K., Khan, K., Seubert, P.,

Page 14 of 16

Freedman, S., Schenk, D., and Games, D. (2005) β-amyloid immunotherapy prevents synaptic degeneration in a mouse model of Alzheimer's disease. J. Neurosci. 25, 90969101. 117. Boche, D., Denham, N., Holmes, C., and Nicoll, J. A. (2010) Neuropathology after active Aβ42 immunotherapy: implications for Alzheimer's disease pathogenesis. Acta Neuropathol. 120, 369-384. 118. Holmes, C., Boche, D., Wilkinson, D., Yadegarfar, G., Hopkins, V., Bayer, A., Jones, R. W., Bullock, R., Love, S., Neal, J. W., Zotova, E., and Nicoll, J. A. R. (2008) Longterm effects of Aβ42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet 372, 216-223. 119. Chui, D.-H., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, F., Ueda, O., Suzuki, H., Araki, W., Inoue, H., Shirotani, K., Takahashi, K., Gallyas, F., and Tabira, T. (1999) Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation. Nat. Med. 5, 560-564. 120. Giannakopoulos, P., Herrmann, F. R., Bussière, T., Bouras, C., Kövari, E., Perl, D. P., Morrison, J. H., Gold, G., and Hof, P. R. (2003) Tangle and neuron numbers, but not amyloid load, predict cognitive status in Alzheimer's disease. Neurology 60, 1495-1500. 121. Corrada, M. M., Berlau, D. J., and Kawas, C. H. (2012) A population-based clinicopathological study in the oldest-old: the 90+ study. Curr. Alzheimer Res. 9, 709-717. 122. Furukawa, K., and Mattson, M. P. (1998) Secreted amyloid precursor protein α selectively suppresses Nmethyl-D-aspartate currents in hippocampal neurons: involvement of cyclic GMP. Neuroscience 83, 429-438. 123. Furukawa, K., Barger, S. W., Blalock, E. M., and Mattson, M. P. (1996) Activation of K+ channels and suppression of neuronal activity by secreted β-amyloidprecursor protein. Nature 379, 74-78. 124. Selkoe, D. J. (2001) Alzheimer's disease: genes, proteins, and therapy. Physiol. Rev. 81, 741-766. 125. Kuperstein, I., Broersen, K., Benilova, I., Rozenski, J., Jonckheere, W., Debulpaep, M., Vandersteen, A., SegersNolten, I., Van Der Werf, K., Subramaniam, V., Braeken, D., Callewaert, G., Bartic, C., D'Hooge, R., Martins, I. C., Rousseau, F., Schymkowitz, J., and De Strooper, B. (2010) Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio. EMBO J. 29, 3408-3420. 126. Sgourakis, N. G., Yan, Y., McCallum, S. A., Wang, C., and Garcia, A. E. (2007) The Alzheimer's peptides Aβ40 and 42 adopt distinct conformations in water: a combined MD / NMR study. J. Mol. Biol. 368, 1448-1457. 127. Chun, Y. S., Kwon, O.-H., and Chung, S. (2017) OGlcNAcylation of amyloid-β precursor protein at threonine 576 residue regulates trafficking and processing. Biochem. Biophys. Res. Commun. 490, 486-491. 128. Griffith, L. S., Mathes, M., and Schmitz, B. (1995) β-Amyloid precursor protein is modified with O-linked Nacetylglucosamine. J. Neurosci. Res. 41, 270-278. 129. Jacobsen, K. T., and Iverfeldt, K. (2011) OGlcNAcylation increases non-amyloidogenic processing of 14


Page 15 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the amyloid-β precursor protein (APP). Biochem. Biophys. Res. Commun. 404, 882-886. 130. Kim, C., Nam, D. W., Park, S. Y., Song, H., Hong, H. S., Boo, J. H., Jung, E. S., Kim, Y., Baek, J. Y., Kim, K. S., Cho, J. W., and Mook-Jung, I. (2013) O-linked β-Nacetylglucosaminidase inhibitor attenuates β-amyloid plaque and rescues memory impairment. Neurobiol. Aging 34, 275-285. 131. Chun, Y. S., Park, Y., Oh, H. G., Kim, T. W., Yang, H. O., Park, M. K., and Chung, S. (2015) O-GlcNAcylation promotes non-amyloidogenic processing of amyloid-β protein precursor via inhibition of endocytosis from the plasma membrane. J. Alzheimers Dis. 44, 261-275. 132. Yuzwa, S. A., Shan, X., Jones, B. A., Zhao, G., Woodward, M. L., Li, X., Zhu, Y., McEachern, E. J., Silverman, M. A., Watson, N. V., Gong, C. X., and Vocadlo, D. J. (2014) Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice. Mol. Neurodegener. 9, 42. 133. Jean-Louis, T., Rockwell, P., and FigueiredoPereira, M. E. (2018) Prostaglandin J2 promotes OGlcNAcylation raising APP processing by α- and βsecretases: relevance to Alzheimer's disease. Neurobiol. Aging 62, 130-145. 134. Zheng, Z., and Diamond, M. I. (2012) Huntington disease and the huntingtin protein. Prog. Mol. Biol. Transl. Sci. 107, 189-214. 135. Ho, L. W., Brown, R., Maxwell, M., Wyttenbach, A., and Rubinsztein, D. C. (2001) Wild type Huntingtin reduces the cellular toxicity of mutant Huntingtin in mammalian cell models of Huntington's disease. J. Med. Genet. 38, 450-452. 136. Rozas, J. L., Gómez-Sánchez, L., Tomás-Zapico, C., Lucas, J. J., and Fernández-Chacón, R. (2011) Increased neurotransmitter release at the neuromuscular junction in a mouse model of polyglutamine disease. J. Neurosci. 31, 1106-1113. 137. Shirendeb, U., Reddy, A. P., Manczak, M., Calkins, M. J., Mao, P., Tagle, D. A., and Reddy, P. H. (2011) Abnormal mitochondrial dynamics, mitochondrial loss and mutant huntingtin oligomers in Huntington's disease: implications for selective neuronal damage. Hum. Mol. Genet. 20, 1438-1455. 138. Nucifora, F. C., Jr., Sasaki, M., Peters, M. F., Huang, H., Cooper, J. K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V. L., Dawson, T. M., and Ross, C. A. (2001) Interference by Huntingtin and Atrophin-1 with CBP-mediated transcription leading to cellular toxicity. Science 291, 2423-2428. 139. Kumar, A., Singh, P. K., Parihar, R., Dwivedi, V., Lakhotia, S. C., and Ganesh, S. (2014) Decreased O-linked GlcNAcylation protects from cytotoxicity mediated by huntingtin exon1 protein fragment. J. Biol. Chem. 289, 13543-13553. 140. Wang, P., Lazarus, B. D., Forsythe, M. E., Love, D. C., Krause, M. W., and Hanover, J. A. (2012) O-GlcNAc cycling mutants modulate proteotoxicity in Caenorhabditis elegans models of human neurodegenerative diseases. Proc. Acad. Natl. Sci. U. S. A. 109, 17669.

141. Grima, J. C., Daigle, J. G., Arbez, N., Cunningham, K. C., Zhang, K., Ochaba, J., Geater, C., Morozko, E., Stocksdale, J., Glatzer, J. C., Pham, J. T., Ahmed, I., Peng, Q., Wadhwa, H., Pletnikova, O., Troncoso, J. C., Duan, W., Snyder, S. H., Ranum, L. P. W., Thompson, L. M., Lloyd, T. E., Ross, C. A., and Rothstein, J. D. (2017) Mutant huntingtin disrupts the nuclear pore complex. Neuron 94, 93-107.e106. 142. Corbo, M., and Hays, A. P. (1992) Peripherin and neurofilament protein coexist in spinal spheroids of motor neuron disease. J. Neuropathol. Exp. Neurol. 51, 531-537. 143. Dong, D. L., Xu, Z. S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) Glycosylation of mammalian neurofilaments. Localization of multiple Olinked N-acetylglucosamine moieties on neurofilament polypeptides L and M. J. Biol. Chem. 268, 16679-16687. 144. Deng, Y., Li, B., Liu, F., Iqbal, K., Grundke-Iqbal, I., Brandt, R., and Gong, C. X. (2008) Regulation between O-GlcNAcylation and phosphorylation of neurofilamentM and their dysregulation in Alzheimer disease. FASEB J. 22, 138-145. 145. Hu, J. H., Chernoff, K., Pelech, S., and Krieger, C. (2003) Protein kinase and protein phosphatase expression in the central nervous system of G93A mSOD overexpressing mice. J. Neurochem. 85, 422-431. 146. Hu, J. H., Zhang, H., Wagey, R., Krieger, C., and Pelech, S. L. (2003) Protein kinase and protein phosphatase expression in amyotrophic lateral sclerosis spinal cord. J. Neurochem. 85, 432-442. 147. Gong, C. X., Wang, J. Z., Iqbal, K., and GrundkeIqbal, I. (2003) Inhibition of protein phosphatase 2A induces phosphorylation and accumulation of neurofilaments in metabolically active rat brain slices. Neurosci. Lett. 340, 107-110. 148. Lüdemann, N., Clement, A., Hans, V. H., Leschik, J., Behl, C., and Brandt, R. (2005) O-glycosylation of the tail domain of neurofilament protein M in human neurons and in spinal cord tissue of a rat model of amyotrophic lateral sclerosis (ALS). J. Biol. Chem. 280, 31648-31658. 149. Shan, X., Vocadlo, D. J., and Krieger, C. (2012) Reduced protein O-glycosylation in the nervous system of the mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis. Neurosci. Lett. 516, 296-301. 150. Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J. P., Deng, H. X., Rahmani, Z., Krizus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S. M., Berger, R., Tanzi, R. E., Halperin, J. J., Hertzfeldt, B., Van den Bergh, R., Hung, W.-Y., Bird, T., Deng, G., Mulder, D. W., Smyth, C., Laing, N. G., Soriano, E., Pericak-Vance, M. A., Haines, J., Rouleau, G. A., Gusella, J. S., Horvitz, H. R., and Brown Jr., R. H. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62. 151. Tan, H. Y., Eskandari, R., Shen, D., Zhu, Y., Liu, T. W., Willems, L. I., Alteen, M. G., Madden, Z., and Vocadlo, D. J. (2018) Direct one-step fluorescent labeling of OGlcNAc-modified proteins in live cells using metabolic intermediates. J. Am. Chem. Soc. 140, 15300-15308. 15



Page 16 of 16

152. Ryan, P., Patel, B., Makwana, V., Jadhav, H. R., Kiefel, M., Davey, A., Reekie, T. A., Rudrawar, S., and Kassiou, M. (2018) Peptides, peptidomimetics, and carbohydrate–peptide conjugates as amyloidogenic aggregation anhibitors for Alzheimer’s disease. ACS Chem. Neurosci. 9, 1530-1551.

16


The O-GlcNAc modification protects against protein misfolding and

Recommend Documents