Novel brain-penetrant inhibitor of G9a methylase blocks Alzheimer’s disease proteopathology for precision medication

Current amyloid beta-targeting approaches for Alzheimer’s disease (AD) therapeutics only slow cognitive decline for small numbers of patients. This limited efficacy exists because AD is a multifactorial disease whose pathological mechanism(s) and diagnostic biomarkers are largely unknown. Here we report a new mechanism of AD pathogenesis in which the histone methyltransferase G9a noncanonically regulates translation of a hippocampal proteome that defines the proteopathic nature of AD. Accordingly, we developed a novel brain-penetrant inhibitor of G9a, MS1262, across the blood-brain barrier to block this G9a-regulated, proteopathologic mechanism. Intermittent MS1262 treatment of multiple AD mouse models consistently restored both cognitive and noncognitive functions to healthy levels. Comparison of proteomic/phosphoproteomic analyses of MS1262-treated AD mice with human AD patient data identified multiple pathological brain pathways that elaborate amyloid beta and neurofibrillary tangles as well as blood coagulation, from which biomarkers of early stage of AD including SMOC1 were found to be affected by MS1262 treatment. Notably, these results indicated that MS1262 treatment may reduce or avoid the risk of blood clot burst for brain bleeding or a stroke. This mouse-to-human conservation of G9a-translated AD proteopathology suggests that the global, multifaceted effects of MS1262 in mice could extend to relieve all symptoms of AD patients with minimum side effect. In addition, our mechanistically derived biomarkers can be used for stage-specific AD diagnosis and companion diagnosis of individualized drug effects.


INTRODUCTION
Alzheimer's disease (AD), the most common form of dementia in older adults, is a neurodegenerative disorder characterized by progressive decline in cognition, memory, and emotional states 1,2 .The heterogeneity and multifaceted nature of AD prevents clear mechanistic understanding of disease pathogenesis, which hinders precision diagnosis of AD at a stage when intervention could be effective.
The amyloid hypothesis has guided current approaches to AD therapeutics.That is, investigators have developed monoclonal antibody drugs that target amyloid beta oligomers (AβO), such as aducanumab (Aduhelm; Biogen), lecanemab, and donanemab, to reduce Aβ plaques that were postulated to contribute to neuropathologic processes in patient brains 3 .However, these AβO-targeting drugs showed limited speci city and e cacy toward AD in the clinic because (i) the primary drug effect is slowing cognitive decline at the earliest AD stages, but there are no biomarkers to conclusively diagnose early-stage AD, (ii) drug effects were determined primarily based on dose-dependent reductions in plaques measured by positron emission tomography (PET), yet large amounts of amyloid plaques are apparently present in healthy, non-demented individuals 4 , and (iii) no biomarkers other than Aβ plaque size are available to measure effects of these drugs on AD progression, and drug effects on AD biomarkers downstream of Aβ were mixed.Most importantly, multiple pathological brain alterations other than formation of Aβ plaque and neuro brillary tangles (NFTs) occur during AD pathogenesis, most of which are not readily measurable due to lack of corresponding biomarkers 5 .Because AD patients have highly complicated cognitive and noncognitive symptoms, new mechanism-based drugs are urgently needed for both precision medication of AD and for derivation of new biomarkers to precisely assess drug effects.
Despite the identi cation of a few AD genetic risk factors 6 , the exact etiology of AD pathogenesis is obscure.Epigenetic mechanisms have been implicated in causing AD pathogenicity because mutations in major histone-modifying enzymes or regulators were characterized in association with AD-related neurodegenerative processes such as impaired memory formation 7 .The histone methyltransferases G9a (EHMT2) and G9a-like protein GLP (hereafter G9a will represent both proteins in their functional dimerized form) 8, 9 were among the histone-modifying enzymes associated with behavioral abnormalities 10 .Elevated activity of G9a existed in post-mortem brain tissues from AD patients and the familial AD (5xFAD) mice at the AD stage 8 , which implicated G9a activity in AD pathogenesis.In AD, G9a functioned as an epigenetic (transcriptional) suppressor by catalyzing the dimethylation of lysine 9 of histone 3 at speci c genes associated with synaptic transmission such as glutamate receptor genes 8 .
Recently, Johnson et al. revealed that AD has a proteopathic nature or is a proteomic disease 11 , i.e., AD pathology and cognitive decline strongly correlated with altered expression of a broad spectrum of proteins.However, the canonical, gene-speci c transcriptional silencing function of G9a did not explain how expression or modi cation of speci c proteins is regulated in AD conditions in which G9a was constitutively active.That is, the G9a-regulated mechanism responsible for AD-related proteomic alterations ('proteopathology') remained to be established.
To determine the function(s) of G9a directly associated with AD pathogenesis, we used our chromatin activity-based chemoproteomics (ChaC)-mass spectrometry (MS) 12 approach with UNC0965, a biotinylated version of a G9a inhibitor 13 , to dissect G9a pathways in AD.The a nity-tagged inhibitor, which binds only the constitutively active form of G9a, captured proteins that interacted with G9a in AD, and the identities of these proteins were used to deduce G9a-associated pathways.Unexpectedly, ChaC-MS identi ed a set of translational or post-translational (proteostasis) regulatory proteins that had enhanced interaction with active G9a in AD-related samples.Importantly, the same set of G9a interactors was identi ed in all AD-related samples including the hippocampus of 5xFAD mice and cerebral organoids derived from AD patients.Particularly, we detected increased interaction between G9a and several regulators of N6-methyladenosine (m 6 A) modi cation, including METTL3, an RNA methyltransferase that catalyzes m 6 A modi cation of RNA to promote oncogene translation 14 ; altered expression of METTL3 and m 6 A mRNA were implicated in AD 15,16,17 .Recently, our mechanistic study of in ammation control revealed that constitutively active G9a is the upstream regulator of METTL3/m 6 Amediated translation in chronically in amed macrophages that mimic AD-related neuroin ammation 18 .In agreement with ndings that translational regulation was broadly perturbed in various neurodegenerative diseases, 19,20,21 and loss of proteostasis is an AD hallmark, 22,23 our in vivo ChaC-MS identi cation of G9a interactions with translation regulators such as METTL3 revealed a new, noncanonical function of G9a in the translational or post-translational regulation of AD pathogenesis.Because translational mechanisms ultimately determine function-related protein abundance or modi cation states 24 , we developed a new mechanism-based therapeutic for AD, i.e., MS1262, a brainpenetrant inhibitor of G9a, to block G9a-mediated translation of AD-related proteins.
The blood-brain barrier (BBB) prevents many drugs from entering the brain; thus, the BBB presents challenges for effective AD therapeutic development 25 .Our new G9a inhibitor MS1262 freely enters the brain.In addition, MS1262 has greater potency than any existing G9a inhibitors (e.g., BIX-01294, UNC0638, UNC0642).We studied the MS1262 effects on two AD mouse models, the 5XFAD and the APP NLGF knock-in (KI) 26 mice, which are clinically relevant mouse models of AD that comprehensively recapitulate major pathological features of AD patients.Consistently, we found that intermittent MS1262 treatment of both types of AD mice not only fully restored cognitive functions to healthy levels but also reduced anxiety-and depressive-like behavior, typical noncognitive (affective) symptoms of AD patients.
Meanwhile, to discover biomarkers that can be used to 1) precisely diagnose AD patients at early stage, 2) evaluate individual patient response to MS1262 treatment, and 3) stratify appropriate patients for MS1262 treatment with optimized response, we conducted quantitative proteomic and phosphoproteomic experiments 27 on the hippocampus from the same 5xFAD and APP NLGF KI mouse cohorts that exhibited behavioral improvement after intermittent MS1262 treatment.These AD-correlated proteomic analyses have unique strengths in systematic elucidation of G9a-mediated mechanisms of AD pathogenesis and MS1262 drug action, including drug-affected pathways from which we can derive new AD-speci c biomarkers of drug effects.Technically, these unique strengths arise by the genome-wide linking of AD-disturbed, MS1262-recovered protein and phosphorylation levels to speci c biological processes/pathways and annotated functional outcomes.As results, our proteomic data indicated that MS1262 treatment reversed or recovered the G9a-regulated, pathogenic expression or phosphorylation of speci c proteins that represented AD-disturbed pathways related to learning and memory, cognition, neuronal functions, and non-cognitive behavior.Importantly, these pathway results systematically revealed that AD-activated G9a broadly de nes the proteopathic nature of AD.
Presently, an absence of clinically validated protein makers for AD diagnosis prevents validation of preclinical results (e.g., drug effects) from animal studies.To overcome this challenge, we systematically determined the clinicopathologic accuracy of MS1262 therapy of AD mice by comparing the AD mouse proteome with AD patient proteomes of statistical signi cance (> 1000 biospecimen) 11 This proteomewide cross-species validation is obviously superior to immunoblotting one protein at a time.Importantly, numerous AD mouse proteins that exhibited G9a/AD-coregulated, MS1262-reversed expression or phosphorylation were components of protein co-expression modules in AD patients 11 that strongly correlated with human AD pathology and cognitive decline.Speci cally, MS1262 treatment reversed the expression and phosphorylation of SMOC1 in the Aβ-associated matrisome module; SMOC1 was found elevated in AD CSF nearly 30 years before the onset of symptoms 28 .Thus, our AD-correlated proteomics identi ed new biomarkers of early-stage AD and companion diagnosis of individual patient response to MS1262 treatment.In addition, in the clinic, the MS1262 reversal of this mouse-to-human conserved AD proteopathology suggests a G9a-target therapeutic effect for AD patients.Importantly, at the molecular level these proteomic-identi ed biomarkers revealed that MS1262 treatment broadly alleviated not only cognitive impairments (cognition decline, memory loss) but also lessened noncognitive symptoms associated with AD such as mood disturbances (depression, anxiety).Meanwhile, MS1262 suppressed AD-characteristic expression/phosphorylation of proteins associated with blood coagulation, which may avoid the side effect of brain bleeding or a stroke that were reported for a few AD patients treated by Aduhelm.
In sum, our mechanistic ndings show that G9a regulates translation or post-translational modi cations (phosphorylation) of a broad range of proteins that are the primary AD executors.Thus, in contrast to existing AD therapies that only slow one symptom (e.g., cognitive decline) targeting AD-activated G9a by the brain-penetrant drug MS1262 showed multifaceted effects that relieve all AD symptoms with minimum side effect.In addition, our AD pathology-correlated proteomic data mechanistically derived multiple biomarkers of early-stage AD and companion diagnosis for individualized, precision medication of AD; some of these biomarkers exist in body uids accessible for non-invasive diagnosis, which will greatly reduce the burden of patient screening.

RESULTS
In our overall design (Fig. 1A), in vivo ChaC-MS dissection of AD brain tissues revealed a previously unknown G9a-mediated translational mechanism amenable to directly limit or reverse AD progression or pathogenesis.The 5xFAD or APP NLGF KI mice that were treated with the brain-penetrant inhibitor MS1262 were used to correlate behavior/synaptic function with analyses of the hippocampus proteome and phosphoproteome.In addition, we compared the proteomic analyses of MS1262-treated AD mice with human AD patient data of statistical signi cance to identify an AD-characteristic mouse-to-human conserved proteome or phosphoproteome that showed MS1262-reversed expression or phosphorylation.The identi cation of this AD-correlated proteome/phosphoproteome enabled us to ascertain both G9a translational regulatory pathways associated with AD pathogenesis and the mechanism of MS1262 action on AD pathogenesis.Consequently, we derived new protein biomarkers to evaluate the drug effects on individual patients for precision medication of AD.
Constitutively active G9a regulates the translational mechanism of AD pathogenesis.We used the biotintagged G9a inhibitor UNC0965 with label-free quantitation (LFQ) 27,29 ChaC-MS 13 to identify G9ainteracting proteins in the hippocampus of the 5xFAD mice and cerebral organoids derived from an AD iPSC line F033K with the amyloid precursor protein (APP) V717I mutation 30 .The samples from 5xFAD and normal mice were collected at different ages.The organoid groups at 88 and 102 days represented early AD (MCI) pathology.Based on LFQ ratios (log 2 > 1, t-test: -log p value > 1.3) that are proportional to the relative binding of individual proteins to G9a, UNC0965 ChaC identi ed ~ 560 proteins that had enhanced interaction with G9a in the hippocampus of AD mice or MCI/AD organoids.Principal component analysis identi ed a few clusters of G9a interactors, including clusters of AD/MCI-phenotypic G9a interactors, which were well separated from G9a interactors in age-matched, healthy mice or organoids ( g. S1 and Data S1A & S1B).These results indicated the high speci city of UNC0965 ChaC-MS to dissect AD heterogeneity with mixed cell types, i.e., UNC0965 captured G9a interactors speci cally from AD-related cells that had aberrant G9a activity.
Unexpectedly, the predominant functional networks (mapped by STRING) 31 overrepresented by the aforementioned AD-phenotypic G9a interactors were primarily associated with major translational or post-translational processes such as alternative splicing, RNA modi cation and processing, translation initiation and elongation, ribosome biogenesis, and protein degradation (proteostasis 22 ) (Fig. 1B).
Notably, this characteristic pro le of G9a-interacting translational regulators was shared between the hippocampus of 5xFAD mice and human AD organoids.Particularly, UNC0965 ChaC-MS identi ed most known cofactors of METTL3, including HNRNPA2B1, which showed enhanced interaction with G9a in ADrelated samples.Jiang et al. reported that progression of tauopathy was mediated by interaction of tau with HNRNPA2B1 and m 6 A RNA 32 .Similarly, the ChaC-identi ed interactions of G9a with the METTL3-HNRNPA2B1 translation machinery 14 implicated a function of G9a in translation associated with tauopathy or AD pathology.More broadly, combined ChaC-MS data predicted that, via AD-phenotypic interactions with key translation regulators such as HNRNPA2B1 and other regulators of ribosomal biogenesis, G9a has a noncanonical (nonepigenetic) function in translational and post-translational regulation of AD pathogenesis.Also, to determine the clinicopathological relevance of these ChaC ndings, we used our multiomics approach (iC-MAP, interaction Correlated Multi-omic Aberration Patterning) 13 to retrospectively analyze the NIH Gene Expression Omnibus databases of AD patient blood (Accession: GSE63060) 33 .We identi ed 26 and 47 G9a interactor mRNAs that had interaction-correlated overexpression patterns 34 in the blood of 80 MCI and 145 AD patients, respectively, compared with a healthy population (n = 104) ( g. S2).Interestingly, 20 interactor genes had similarly elevated mRNAs in the peripheral blood cells of both MCI and AD patients 35 36 .This nding revealed a mouse-to-human conservation of AD-related G9ainteracting pathways.
Moreover, MS1262 displayed excellent selectivity for G9a and GLP, lacking signi cant inhibition (< 20% at 1 µM) for any of the 21 other methyltransferases tested (Fig. 2D).Similarly, MS1262 effectively reduced histone H3 lysine 9 dimethylation in cells in a concentration-dependent manner, indicating the strong intracellular inhibitory activity of MS1262 (Fig. 2E).Importantly, MS1262 showed good brain penetration in an in vivo mouse pharmacokinetic study.Following a single 5 mg/kg intraperitoneal (i.p.) injection of MS1262 in C57BL/6 mice, MS1262 displayed brain/plasma ratios of 0.64 and 0.73 at 2 and 4 hours postinjection (Fig. 2F).Taken together, these results indicate that MS1262 is a highly potent, selective, brain penetrant G9a/GLP inhibitor suitable for in vivo e cacy studies in AD mouse models.
MS1262 inhibition of G9a activity thoroughly rescues AD-related de cits in behavior and synaptic function.The 5xFAD and APP NLGF mice experience cognitive and affective behavioral de cits that progressively worsen with age 41,42,43,44 .Previously, we showed that 5xFAD mice exhibited a series of hippocampus-dependent cognitive and affective de cits, including impaired spatial memory in the novel place recognition (NPR) test, elevated innate anxiety in the open eld and zero maze tests, and increased depression-like behavior in the forced swim test 45 .Therefore, we assessed these behaviors in response to long-term, intermittent G9a inhibition by MS1262 (Fig. 3, A and B).Locomotion in an open eld was unaltered between wild-type (WT) controls and either 5xFAD or APP NLGF mice in response to MS1262 or vehicle treatment (Fig. 3, C and H), indicating that G9a inhibition did not affect locomotion.For the NPR test to assess spatial memory, animals treated with MS1262 showed a signi cant preference for the object in the novel location measured by the discrimination ratio (5xFAD: p < 0.0001, APP NLGF : p < 0.001), indicative of improved spatial memory (Fig. 3, D and I).Notably, the discrimination ratio of MS1262treated mice was rescued to the level of age-matched WT animals for both AD mouse models (Fig. 3, D  and I).For open eld and zero maze tests to assess anxiety-like behavior, MS1262 treatment showed no effects on the amount of time 5xFAD mice spent in the center of an open eld (Fig. 3E) but MS1262 treatment increased the time APP NLGF mice spent in the center of the open eld as compared with both wildtype and vehicle-treated controls (Fig. 3J).Furthermore, the 5xFAD and APP NLGF mice spent signi cantly less time in open arms of the zero maze compared with wild-type control animals, which was signi cantly increased by MS1262 treatment (Fig. 3, F and K).These results suggest the anxiolytic effects by G9a inhibition in AD mice.For the forced swim test to assess depression-like behavior, MS1262-treated animals demonstrated a signi cantly lower time of immobility compared with vehicletreated animals, suggestive of less depression-like behavior (Fig. 3, G and L).Together, these results suggest that G9a inhibition effectively rescued both cognitive and affective de cits in two complementary AD mouse models, implying broad applicability of effective AD treatment by MS1262.
Additionally, to assess the effect of MS1262 treatment on synaptic transmission and intrinsic properties of hippocampal cells, we recorded evoked action potential and pharmacologically isolated spontaneous excitatory postsynaptic currents (sEPSCs) of hippocampal dentate granule cells (GCs) from vehicle-and MS1262-treated 5xFAD mice.The rationale of selecting dentate gyrus GCs for recording was based upon our previous ndings showing that the hippocampus-dependent behaviors mentioned above were dentate gyrus dependent 46 .No signi cant differences were observed between vehicle and MS1262-treated animals in membrane capacity, input resistance, resting membrane potential, and intrinsic excitability (Fig. 4, A-D).Interestingly, the frequency of sEPSCs recorded from dentate GCs in MS1262-treated 5xFAD mice was signi cantly increased without altering the amplitude of sEPSCs (Fig. 4, F-I).These results suggested that long-term G9a inhibition by MS1262 in 5xFAD mice increased excitatory glutamatergic synaptic transmission onto the dentate GCs.
A G9a translational mechanism de nes the proteopathologic nature of AD.To dissect a G9a-mediated mechanism of AD pathogenesis and the corresponding action mechanism of MS1262 reversal of AD symptoms, we performed ChaC-MS and tandem mass tag (TMT)-based quantitative proteomics/phosphoproteomics experiments 27,29 using microdissected hippocampal samples from the same mouse cohorts used for the aforementioned behavioral studies (Fig. 3 and Fig. 4).These samples included AD mice (5XFAD or APP NLGF KI) and age-matched wild-type mice with or without MS1262 treatment.Because the hippocampus is the primary brain area affected by AD, the MS1262-induced changes in G9a binding or changes in the hippocampus proteome/phosphoproteome revealed ADrelated, G9a-associated pathways and re ected the inhibitor effects on AD pathogenesis at the molecular level.First, ChaC-MS analysis revealed that MS1262 treatment reversed G9a binding to most regulators of translation, RNA processing, and ribosome biogenesis (correlation coe cient = -0.483,g.S3 and Data S1B).Speci cally, MS1262 reversed AD-related binding of G9a to major translation regulators, which con rmed the dependence of G9a translational function on its activity in AD.In addition, in the same G9ainteracting complex in AD, MS1262 inhibition also reversed AD-characteristic G9a interactions with proteins involved in postsynaptic neurotransmitter receptor internalization such as Rabphilin-3A (Rph3A), a synaptic vesicle protein 47 , methyl CpG binding protein 2 (Mecp2) 48,49 , and clathrin-dependent endocytosis such as the hetero-tetrameric assembly protein complex 2 alpha adaptins (AP2A1 and AP2A2) 50 .Thus, combined ChaC results extended G9a function to global translational regulation of ADrelated neuropathologic processes.
In parallel, we performed AD-correlated proteomic/phosphoproteomic experiments using micro-dissected hippocampus tissue from a large cohort of 5xFAD mice exhibiting mid/late-stage AD (C57BL/6J; n = 4) with or without MS1262 treatment, along with age matched wild type controls (C57BL/6J; n = 3), resulting in identi cation of 7,899 proteins and 14,788 phosphorylated sites (in 4,262 protein groups) in total.
Similar experiments in a second mouse model of AD, i.e., wild type (C57BL/6J-A w-J /J; n = 3) mice versus APP NLGF KI (C57BL/6J-A w-J /J; n = 4) mice with or without MS1262 treatment identi ed 6,576 proteins and 13,6687 phosphorylated sites (in 3,854 protein groups) across all hippocampal samples (Fig. 5A and Data S1C).Principal component analysis showed not only good separation of wild-type, AD, and MS1262-treated AD 5xFAD/APP-NLFG samples but also showed high quantitative reproducibility between biological replicates (Fig. S4A).Interestingly, increased m 6 A writer (METTL3, RBM15) and decreased m 6 A eraser (FTO, ALKBH5) expression along with an overall increase in m 6 A modi cation with age/AD-progression in the hippocampus and cortex of various mouse models (APP/PS1, APPNL-G-F/MAPTP301S) and AD patients has been reported previously 51,52,53,54 .Similarly, oligomeric tau was shown to complex with m 6 A-modi ed transcripts through HNRNPA2B1, a m 6 A reader, to regulate translation and promote neurodegeneration 52 .Interestingly, we recently showed that G9a promotes methyltransferase activity of METTL3 to co-upregulate translation of a subset of m 6 A modi ed transcripts 18 , leading us to ask if MS1262 treatment affects m 6 A machinery in AD mouse model.In line with previous reports, we observed increased expression of m 6 A writers (e.g., METTL3, RBM15/15B, WTAP), reduction in m 6 A erasers (e.g., FTO) and dysregulation of several m 6 A readers (e.g., HNRNPA2B1, RBM27, PCIF1, YTHDF2, YTHDC2, HNRNPC/D, etc.) in the hippocampus of 5xFAD and/or APP-NLGF mice, compared to age matched controls; with MS1262 treatment reversing AD-dysregulated expression of several m 6 A regulators (Fig. S4B).In addition, approximately 47% (5xFAD mice) and 42% (APP-NLGF mice) of proteins that showed AD-characteristic dysregulation that was reversed following MS1262 treatment, classi ed as 'AD/G9a-coregulated' proteins, were translated from m 6 A-tagged RNAs (Fig. S4C) further con rming the function of enzymatically active G9a in regulating translation of AD-related proteins.
Overall, 9764 entities (global = 1346; phosphosites = 8418; log2FC 0.2 and P < 0.05) were differentially regulated in our proteomic and phosphoproteomics datasets from 5xFAD and APP-NLGF mice (Fig. 5B), with two major clusters of proteins whose expression or phosphorylation levels were either up-or downregulated in 5xFAD and/or APP-NLGF mice compared to age matched controls; a pattern that was reversed following MS1262 treatment (Fig. S4E, and Data S1C).Because MS1262 speci cally inhibited AD-activated G9a, proteins with MS1262-reversed expression or phosphorylation were classi ed as 'AD/G9a-coregulated'. Notably, these AD/G9a-coregulated proteins showed more pronounced phosphorylation changes than their expression changes, which con rmed that G9a activity broadly regulates AD pathogenesis at the translational or post-translational (i.e., phosphorylation) levels.Further, we estimated pathway activity scores to nd AD-dysregulated (i.e., AD versus wild type) and MS1262reversed (i.e., treated AD versus AD) biological processes/pathways in the hippocampus of AD mice (Figs.5C, Fig. S5 and Data S1D).Notably, most AD/G9a coregulated proteins that were conserved in both 5xFAD and APP NLGF KI mice overrepresented the pathways closely associated with AD pathology or dysregulated neurogenesis.Also, these pathways were associated with indicated functions and diseases (Fig. 5D, g.S5B & S5E and Data S1D).These results con rmed that MS1262 is a proper probe for proteomic discovery of G9a activity-regulated pathways.

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Because neuro brillary tangles composed of abnormally hyperphosphorylated Tau are a hallmark of AD, 61 this result demonstrated the speci city of MS1262 in targeting AD tauopathy.
G9a-associated pathways mechanistically contribute to both cognitive and non-cognitive symptoms of AD.Broadly on the basis of MS1262-induced phosphorylation changes, we observed that MS1262 reactivated the interactive pathways involved in calcium signaling, CREB (cAMP-response elementbinding protein) signaling in neurons, and synaptogenesis signaling (Fig. 5E), which are suppressed during AD pathogenesis 62,63,64 .Speci cally, SHANK3 is a large scaffolding protein in the postsynaptic density; phosphorylated forms of SHANK3 have different synaptic properties 65,66 .We found that MS1262 reversed CaMKII (Calcium/CaM-dependent kinase)-mediated phosphorylation of SHANK3 66 and another synaptic protein GluN2A-subunit-containing NMDAR 67 ( g. S5C) in correlation with improved cognition and rescued synaptic function of MS1262-treated AD mice (Figs. 3 and 4).Other ADsuppressed signaling pathways (Fig. 5C) were reactivated by MS1262 as described below: The CDK5 pathway has critical functions in neuronal migration and differentiation, synaptic functions, and memory consolidation.Dysregulated CDK5 activity in AD contributes to formation of senile plaques and neuro brillary tangles, synaptic damage, mitochondrial impairment of cell cycle reactivation, and neuronal cell apoptosis 68 .In agreement with the observation that silencing CDK5 by RNAi reduced neuro brillary tangles in the hippocampi of triple-transgenic mice (3xTg-AD mice) 69 .MS1262 treatment protected 5xFAD mice from memory decline and loss of neuronal function presumably by reversing the phosphorylation states of CDK5 pathway proteins (Fig. S5D).Dopamine-and cAMP-regulated phosphoprotein (DARPP-32) is a regionally distributed neuronal phosphoprotein that robustly integrates dopamine and glutamate signals in the mammalian brain.cAMPmediated signaling and cAMP-dependent protein kinase A pathways are crucial in synaptic plasticity and long-term memory 70 .In AD brains, DARPP-32 was cleaved at Thr153 by activated calpain to reduce CREB phosphorylation via loss of its inhibitory function on PP1 71 .MS1262 reversed the phosphorylationdependent disturbances in CREB function that cause memory de cits in AD patients and animal models 72 .(Figs. 5C & S5D) Dysfunction of the opioid system (opioid receptors and opioid peptides) is implicated in the pathogenesis of AD because of the regulatory functions of opioid receptors in Aβ production, hyperphosphorylated tau, and neuroin ammation 73,74 .In agreement with the observation that activation of opioid receptors limited the production of Aβ 75,76 , MS1262 treatment activated the AD-suppressed opioid system by altering the phosphorylation or expression of major components of opioid signaling pathways (Fig. S5D).Also, MS1262 reprogramed the regulation of actin-based motility that is associated with cytoskeletal abnormalities and synaptic loss 77,78 .AD suppression of pathways involving the SNARE complex, oxytocin in spinal neurons, and endocannabinoid system contributes to neurodegeneration and associated impairments in learning, memory, and cognition.The SNARE complex is crucial for vesicle fusion or recycling and neurotransmitter release; inhibition of SNARE complex formation led to neurodegeneration 79 .In correlation with the observation that Injection of oxytocin into the hippocampus reversed some of the damage caused by amyloid plaques in the learning and memory center in an AD animal model 80 , MS1262 treatment reactivated oxytocin in spinal neurons or brain signaling pathways by reversing the phosphorylation states of proteins related to synaptic plasticity and memory formation such as CAMKK1and CAMKK2 81 .Also, MS1262 treatment upregulated protein components of brain pyrimidine biosynthesis and pyrimidine salvage pathways that are essential for neuronal membrane generation and maintenance and synapses production 82 , and which were downregulated in AD (Figs. 5C,  5E & S5).
Other proteins abnormally expressed in AD and restored to normal levels by MS1262 were gonadotropin releasing hormone, a neuropeptide central regulator of neurogenic and neuroprotective functions and an activator of cAMP-mediated signaling 83,84 , and G-protein coupled receptors involved in numerous key neurotransmitter systems in the brain that were disrupted in AD 85, 86 (Figs.5C & S5).These proteomic/phosphoproteomic results identi ed multiple signaling pathways that synergistically contribute to synaptic plasticity or/and synaptic transmission and whose de cits in mice were rescued by MS1262.
In parallel with MS1262-reduced noncognitive AD-like neuropsychiatric behaviors such as depression and anxiety (Fig. 3), we identi ed numerous phosphoproteins with MS1262-reversed phosphorylation involved in Relaxin signaling that contains markers of depression in AD 94 (Fig. 5C & S5D).Moreover, MS1262 reinstated gustation pathways whose dysregulation was indicative of impaired sensory systems (e.g., olfactory, visual, auditory, somatosensory, gustatory) and poorer memory in AD 95,96 because auditory and visual measures were used to detect prodromal AD 97 (Fig. S5D).In sum, the proteins that showed AD-regulated, MS1262-reversed expression or phosphorylation were identi ed in AD-related, interconnected pathways.These ndings demonstrated that G9a is an upstream translation regulator of proteins that de ne AD proteopathology and associated AD symptoms.
MS1262 reversed protein expressions or phosphorylation that mark early-stage of AD.To determine the clinical relevance of G9a-translational mechanism that regulates AD proteopathology, and to predict MS1262 e cacy for treating AD patients, we compared our proteomic and phosphoproteomic data from non-treated versus MS1262-treated AD mice with the proteomic data of different cohorts of AD patients.
We rst examined the proteomic data of brain autopsy samples 98 from (i) control individuals with low pathology of plaques and tangles, (ii) controls with high Aβ pathology but no detectable cognitive defects, (iii) MCI persons with Aβ pathology and slight but measurable defect in cognition, and (iv) AD patients.As shown in Fig. 6A, the expression of eighteen proteins including MAPT that mark AD patients was reversed in MS1262-treated AD mice.For example, MS1262 treatment reversed expression or phosphorylation of proteins related to neuroin ammation and AD immunity, including interleukin 33 (IL-33), complement C3, and CD109.Reduced IL-33 expression was observed in the brains of AD mice and AD patients.Similar to the effect of IL-33 injection that reversed cognitive de cits in APP/PS1 mice, 99 MS1262 inhibition of G9a upregulated IL-33 expression.In correlation with tauopathy, elevated levels of complement C3 protein were detected in brains and cerebrospinal uid from AD patients. 100CD109 is a negative regulator of transforming growth factor β receptor that showed reduced functionality in AD 101 .These results indicated that the broad function of G9a in regulating AD-related neuroin ammation was alleviated by MS1262 inhibition of G9a.Moreover, SQSTM1 is a multifunctional scaffolding protein that has a major function in autophagy and whose upregulation has been found in several neurodegenerative disorders including AD 102 .PACAP is a protein encoded by the ADCYAP1 gene.The close relationship between PACAP reduction and the severity of AD pathology suggests that downregulation of PACAP may contribute to AD pathogenesis 103 .In addition, ADCYAP1 was identi ed as a diagnostic biomarker of AD with high discriminatory ability (AUC = 0.850) and validated in AD brains (AUC = 0.935).PBXIP1 is a cerebrospinal uid (CSF) marker of AD.Comparison of cortex and serum led to an AD-correlated protein panel of CTHRC1, GFAP and OLFM3. 104The CAMKK2-AMPK kinase pathway mediates the early synaptic toxic effects of Aβ42 oligomers and is a target for AD therapeutics 105 .In addition, we found that MS1262 reversed phosphorylation of the protein products of 33 AD-risk genes 106 (Fig. S5C).
Further, to identify mouse-to-patient conserved biomarkers for precision diagnosis at a stage when MS1262 treatment could be effective, we compared our mouse pro les of G9a/AD-coregulated, MS1262reversed proteins with CSF protein biomarkers of AD progression 28 .Notably, MS1262 treatment reversed AD-characteristic expression of multiple CSF biomarkers of early-stage AD (Fig. 6B).For example, the level of SPARC-related modular calcium-binding protein 1 (SMOC1), an Aβ plaque-associated synaptic protein 23,107 was found elevated in AD CSF nearly 30 years before the onset of symptoms 28 .Other CSF markers 28 showing MS1262 affected/reversed expression included the following (Fig. 6B): Glia maturation factor β (GMFB), a newly characterized G9a-regulated factor in the regulation of neuronal and glial growth and differentiation 108 ; Thy-1 glycoprotein, which was implicated in extensive growth of abnormal neurites in AD 109 ; proton pump inhibitor A (PPIA), which was related to cognitive decline 110 ; and SCGC, which is typically transported in synaptic vesicles as a marker of synaptic loss and neuronal injury/degeneration for prognosis in prodromal AD 111 .We also noticed that two CSF markers showed abundance in patient CSF, namely, MFGE8, which was implicated in Aβ-induced phagoptosis and a potential therapeutic target to prevent neuronal loss in AD 112 and neuronal pentraxin receptor (NPTXR/NPTX2), which was identi ed as a biomarker of AD progression for CSF-based liquid biopsy 111,113 .These discrepancies of AD-related expression were probably due to different sample origins from the hippocampus or CSF, respectively.Nevertheless, simultaneous identi cation of these CSF biomarkers of AD as MS1262/G9a-regulated proteins suggested that these biomarkers together can be used to stratify appropriate patients who may have maximum response to MS1262 treatment and to determine individual effects of MS1262 treatment.
MS1262 reversed multiple AD-speci c brain pathological processes that mediate Aβ plaque and NFT pathology.We identi ed and focused on two major clusters of proteins/phospho-proteins whose expression (n = 96) or phosphorylation (n = 252) levels were either up-or downregulated in 5x-FAD and/or APP-NLGF mice compared to age matched controls; a pattern that was reversed following MS1262 treatment (Fig. 7A, and Data S1C).These 'AD/G9a-coregulated' entities were primarily involved in synaptic signaling, neuronal transmission and nervous system development related pathways (Fig. 7B) with 148 out of 348 members showing Aβ peptide (residues 6-28) correlated expression in the brain of AD patients including CD109, CD44, ANO6, ICAM1 and PLSCR4 (Fig. 7D, Fig. S6A and Data S1E).
In ammation-related CD44 splice variants are overexpressed in AD hippocampus and may protect neurons from AD damage 114 .TMEM16F (also known as ANO6) mediated microglia polarization is involved in progression of AD and its inhibition shows neuroprotective effects in AD models 115 .ICAM-1 improves cognitive behaviors in 5xFAD mice by inhibiting NF-κB signaling 116 while PLSCR4 is a transcriptome biomarker hub gene in 5xFAD mice 117 .Overall, these AD/G9a-corregulated proteins were involved in mental/CNS/neurodevelopmental disorders, memory impairment, mental deterioration, and thrombosis/coagulation, among other diseases (Figs.7C & S6B).These results con rmed that G9a activity is closely associated with AD pathology or dysregulated neurogenesis and MS1262 is a proper probe for proteomic discovery of G9a activity-regulated pathways.
Next, we compared AD/G9a-corregulated proteins identi ed in 5xFAD/APP-NLGF mice with "modules" (M) of co-expressed proteins showing dysregulation in brains of AD patient 11 to identify 204 entities (51 proteins, 204 phosphoproteins) with AD-related, MS1262-reversed expression changes (Fig. 7E).Notably, 15 out of 38 of these AD/G9a-corregulated 'modules' were only dysregulated at the protein level without concomitant change in transcriptomic networks in the AD patient samples (Fig. 7E; highlighted in cyan).This nding, along with the observation that nearly half of the AD/G9a-corregulated proteins are encoded by m 6 A-tagged transcripts (Fig. S4C) and the fact that AD/G9a-coregulated proteins exhibited more pronounced phosphorylation changes than expression changes, further con rmed that G9a activity broadly in uences AD pathogenesis at the translational or post-translational (i.e., phosphorylation) levels.
In the networks of patient protein modules (Fig. 7E), SMOC1, whose level in CSF or in postmortem brain was associated with AD pathology, was an MS1262-targeted component of the M42 matrisome module strongly correlated with AD neuropathology and cognition.The Tau family microtubule-associated proteins 118 and STIM2 responsible for neuronal calcium homeostasis impairment 119 were MS1262targeted components of module M7 MAPK signaling and metabolism that is highly associated with the rate of cognitive decline.Plasma glial brillary acidic protein, an astrocyte reactivity biomarkers for AD 120 and nitric oxide synthase 1 for synaptic transmission and neuroplasticity 121 were MS1262-targeted components of modules M5 post-synaptic density or M11 cell-ECM interaction, respectively, again, major modules strongly correlated to AD neuropathology and cognition.In addition, MS1262 reversed the phosphorylation of components of modules M29, glycosylation/ER, and M42, matrisome, that were correlated with AD endophenotypes, and elevated tau microtubule-binding domain peptide levels correlate with the other MS1262-affected components of M42 matrisome and M11 cell-ECM modules.Overall, AD/G9a-corregulated co-expression modules were involved in neuronal signaling (neurotransmitter regulation, axonogenesis, synapse signaling, myelination, postsynaptic density), translation (RNP binding, translation initiation), RNA metabolism (transcription, splicing, binding), protein transport (glycosylation/ER, Golgi, endosome), cellular energetics (sugar metabolism, mitochondria) and immune response (complement/acute phase, MHC complex).
In short, we examined spatial relationships between MS1262-reversed AD patient proteome and Aβ plaques and neuro brillary tangles (NFTs), the hallmarks of AD neuropathologies.In addition to MS1262 reversal of the phosphorylation of SMOC1 in M42 matrisome, MS1262 reversed the phosphorylation of many module components found in Aβ plaques or/and NFTs, including M1 and M4 components enriched in both Aβ plaques and NFTs, M7 components primarily associated with Aβ plaque, and M13 and M29 components uniquely enriched in NFTs.Notably, the levels of mRNAs encoding these module proteins showed little change in AD patients, although AD-upregulated expression or phosphorylation of these proteins was reversed by MS1262 treatment.This lack of correlation between mRNA and protein expression indicated that constitutively active G9a regulates the translation and post-translational modi cation (phosphorylation) of AD-related proteins, and these proteins mediate protein co-localization with Aβ plaques and NFTs.
In addition, we found that these G9a-dependent, AD-related protein co-expression and co-phosphorylation modules were preserved across the mouse and human samples as G9a-translated because MS1262targeted proteomic or phosphoproteomic changes were more closely aligned with AD pathology and ADlike behavior than changes at the mRNA level.In parallel with the observation that MS1262 repaired ADrelated behavior, MS1262 treatment reversed the proteopathologic landscape in AD patients, which was evidenced by the greater number of phosphoproteins in the protein co-expression modules highly correlated with broad AD pathology and cognitive de cits.In the clinic, these proteomic identi cations of AD mouse-to-patient conservation revealed multiple MS1262-affected biomarkers of AD which can be used to evaluate evidence of downstream disease modi cation and to assess the relation between biomarker changes and clinical outcomes.

DISCUSSION
Epigenetic mechanisms involving G9a are thought to regulate AD pathogenesis.However, few pathways strongly correlated with AD pathology were identi ed by transcriptomic analysis of G9a inhibitor (UNC0642)-treated 5XFAD mice. 8, 122Instead, recent multiomics analyses of large cohort samples of AD patients 11 identi ed the proteopathologic nature of AD, i.e., proteomic changes that were not observed at the mRNA level strongly correlated with AD pathology and cognitive decline, which suggested that AD pathogenesis is predominately a translation abnormality.Using chemical biology, animal behavioral analysis, and AD-correlated proteomics, we discovered a noncanonical, translation-regulatory function of aberrantly activated G9a that de nes the proteopathologic nature of AD.Accordingly, we have developed new mechanism-based, brain-penetrant small molecule therapeutics that inhibit the G9a-mediated translational mechanism in AD.
Present evidence is insu cient to support the hypothesis that abnormal Aβ deposits are uniquely causal in AD progression; it is unknown whether a large Aβ plaque could be the cause or a result of AD.Also, the Aβ aggregation cascade is composed of a mix of species; thus, any Aβ-targeting monoclonal antibody has limited speci city/e cacy toward AD.In contrast, our AD therapeutics approach is based on a new translation-regulatory mechanism of AD pathogenesis whereby AD-activated G9a translates select mRNAs into proteins that are widespread in a range of pathological brain processes that mediate Aβ plaque and NFT pathology.In correlation with AD behavior reversal, we employed G9a inhibitors (either biotin-tagged or tag-free forms) as the pathway probes for AD-correlated proteomic dissection of G9aregulated AD pathogenesis and the mechanism of MS1262 drug action.First, ChaC-MS identifed ADcharacteristic, MS1262-reversed G9a interactions with proteins that constitute the 'pathway skeleton' of the translational control of AD pathogenesis.These ChaC-identi ed G9a interactors included regulators of either translational processes, such as alternative splicing, ribosome biogenesis, protein synthesis, and proteostasis, 22,23 or neuropathogenic processes related to neuronal endocytosis and synaptic functions.
These AD-phenotypic G9a-interacting translation regulators include multiple m 6 A RNA regulators such as HNRNPA2B1, YTHDC2, and YTHDF2, all of which were implicated in AD. 32 Our m 6 A RNA-to-protein correlation analysis identi ed G9a/AD-coregulated m 6 A mRNAs whose protein translation was reversed by MS1262 treatment of AD mice, validating G9a regulation of m 6 A-mediated translation in AD.In addition to the m 6 A translation regulatory axis, G9a also interacted with other translation regulators.For example, SRRM2 contributes the tauopathies in AD by mislocalizing to cytosolic tau aggregates in brains of individuals with AD. 123 HNRNPK, a splicing factor, regulates AD-driving proteoforms. 124The ribosomal protein interactors RPS14, RPS17, RPS15A, RPL21, RPS8, RPL37A, RPL14, RPS2, RPL26, FAU, RPS13, RPS24 were signi cantly upregulated in AD patients. 125The NFT-associated AP2A1 and AP2A2 50 were identi ed as G9a interactors that are the central hub for AD pathogenesis-associated clathrin-dependent endocytosis and postsynaptic neurotransmitter receptor internalization.The synaptic protein Rph3A associated with Aβ burden 47 when Mecp2 was implicated in neuronal endocytosis and synaptic plasticity. 48,49 n line with our nding of similar G9a translational function in chronically in amed macrophages, these results of G9a interactions with translation regulators con rmed that G9a activity is crucial for global translation of AD-causative proteins.
Speci cally for AD therapy, our small molecule G9a inhibitor MS1262 showed high brain permeability and stability and much higher potency than existing G9a inhibitors, e.g., > 20-fold more potent than UNC0642 (the older version of G9a inhibitor).As a result, MS1262 inhibition of G9a fully rescued both cognitive and noncognitive behavioral de cits of 5xFAD mice and APP NLGF KI mice (Figs. 3 and 4).In a nonbiased manner, our global proteomic pro ling revealed that MS1262 systematically reversed the expression or phosphorylation of a range of proteins in all major neurodegenerative or AD-related signaling pathways; little MS1262 effect was observed on expression or phosphorylation of non-AD related proteins in 5xFAD mice.In addition, select mRNAs, termed as 'poised' mRNAs, showed little changes but produced speci c proteins associated with AD pathogenesis.In patient samples we identi ed MS1262-regulated, 'poised' mRNAs that are translated into AD-driving proteins.These proteomic data demonstrated that MS1262 crossed the BBB directly and speci cally targeted G9a-regulated proteomic pathways to reverse AD proteopathy.Notably, not all but most AD-related pathways that are overrepresented by G9a/ADcoregulated proteins were identi ed in both mouse models.The discrepancy of other pathways is probably due to differences in the background of mice in the wild-type (C57BL/6J) and 5xFAD (C57BL/6J) experiments and the mice of the wild-type (C57BL/6J-A w−J /J) and APP NLGF (C57BL/6J-A w−J /J).Furthermore, 5.5 months old of 5xFAD mice were considered as late-stage AD 8 , but the sameaged APP NLGF KI mice were at an earlier stage of AD due to the slow progression of AD pathology in the knock-in line 126,127 .
In addition, in contrast to the fact that MS1262 inhibition of G9a reversed the widespread expression of proteins that are causative in AD pathology, we found that treatment of AD mice by UNC0642 had little effect on the global expression of AD-related proteins in the hippocampus (data not shown); this limited effect was probably due to the low brain permeability/stability or relatively low potency of this compound.
Compared with Aβ-targeting monoclonal antibodies that only slow cognitive decline of small numbers (e.g., 27%) of patients at the earliest stage, MS1262 displayed high e cacy and speci city/sensitivity toward AD, i.e., MS1262 showed multifaceted, therapeutic effects on AD including cognition restoration, memory improvement, and attenuation of depression and anxiety.Because AD-activated G9a broadly and simultaneously regulated major signaling pathways that mechanistically contribute to both cognitive and non-cognitive symptoms of AD, MS1262 effects are not 'one target (G9a) at a time' but, instead, the drug effect (target) is proteome-wide.As supporting evidence, Pao et al. reported 128 that targeting CDK5 hyperactivity ameliorated neurodegenerative phenotypes.Accordingly, we identi ed CDK5 signaling as a one of many G9a/AD-coregulated pathways that were simultaneously reversed by MS1262 treatment.This identi cation of CDK5 con rmed that G9a is a broad regulator of AD proteopathologic landscape, hence, MS1262 rescued a range of AD-dysregulated pathways that de ne multiple AD pathological hallmarks such as Aβ plaque and NFT pathology.In addition, AD mice treated with MS1262 for six weeks did not show any signs of toxicity; general appearance, activity, and behavior were all normal or improved.Outward normality was consistent with MS1262 reversal of AD proteome and phosphoproteome of the hippocampus.On a systems evaluation of MS1262 side effects, our AD pathology-correlated proteomic/phosphoproteomic results demonstrated that MS1262 speci cally and effectively inhibited G9a that was aberrantly activated in AD-related cells but not non-AD related cells in diseased brain so that little, if any, off-target toxicity was observed.
In parallel, our AD-correlated proteomic analysis dissected G9a-regulated pathogenesis of AD, from which proteins that showed G9a/AD-coregulated, MS1262-reversed expression or phosphorylation were mechanistically derived as new biomarkers for diagnosing AD or assessing drug effects.Particularly, in addition to multiple known AD markers that were affected by MS1262 treatment such as IL-33, complement C3, CD109, ADCYAP1, PBXIP1, CTHRC1, GFAP, OLFM3, and the protein products of 33 ADrisk genes (Fig. 6A and Data S5C), 106 MS1262 treatment reversed the expression or phosphorylation of SMOC1 and other proteins in the matrisome that were recently characterized as CSF biomarkers of early stage AD 28 (Figs.6B & 7E).Interestingly, Bellver-Sanchis et al. 108 showed that G9a inhibition rescued GMFB-dysregulated neuroprotection in AD.However, their transcriptomic analysis suggested that G9a is a transcriptional suppressor of GMFB, which failed to explain how GMFB protein was found upregulated in both AD mouse hippocampus and patient CSF (Fig. 6B).In agreement with our characterized function of G9a methylation of METTL3 in G9a-regulated translation 18 , they also identi ed GMFB as a nonhistone substrate of G9a, which suggested similar regulation of GMFB by G9a at the translational level.Thus, our identi cation of AD/G9a-coupregulated, MS1262-suppressed expression of GMFB protein validated our discovery that G9a-translational mechanism outperforms G9a-suppressed transcription in de ning the proteopathologic nature of AD pathogenesis.
In clinical practice, these MS1262-affected AD biomarkers can be used in combination or in a panel to 1) diagnose patients at early stage or the stages when intervention is effective, 2) evaluate evidence of druginduced downstream disease modi cation, and 3) assess clinical outcomes of MS1262 treatment.Some candidate biomarkers of companion diagnosis for individualized, precision medicine were found in body uids accessible for noninvasive assessment of drug effects.For example, although aducanumab treatment of some patients was associated with serious side effects, including brain bleeding or stroke, our mouse-to-patient conserved proteomic data revealed that MS1262 can reverse AD-characteristic expression/phosphorylation of proteins associated with blood clotting (Fig. 7D).These results suggested that, during therapy, MS1262 treatment may reduce or avoid the risk of blood clot burst for brain bleeding or a stroke.In addition, these Aβ abundance-correlated biomarkers represent multiple pathological brain processes that are the AD mediators of Aβ plaque, and MS1262 affected biomarkers of AD-speci c Aβ pathology.Thus, these biomarkers can be used to evaluate for evidence of downstream disease modi cation and to assess the relationship between biomarker changes and MS1262-induced clinical outcomes.
To precisely evaluate the drug effects or individual responses to the drug, instead of conventional assays with available AD markers (if any) for one-protein-at-a-time validations, our global comparison of AD mice and AD patient proteomes 11 determined the impact of MS1262 inhibition of G9a on AD therapy.Of clinical signi cance, this cross-species proteomic comparison validated the mouse-to-human conserved, G9a-translation mechanism of MS1262 reversal of AD-correlated pathways; this mouse-to-human correlation strongly suggests that MS1262 inhibition of G9a would be an effective therapy for AD patients.
In summary, for the rst time, our results unambiguously indicated that de ning the G9a translational mechanism is superior to characterizing the G9a epigenetic mechanism in determining the stage of AD pathogenesis and the ultimate effects of drug action.These ndings also validated the mouse-to-human conserved, G9a-translational mechanism that broadly de nes AD proteopathology.Accordingly, aberrantly activated G9a in diseased hippocampus regulates speci c pathways at the translational or post-translational (phosphorylation) level that mechanistically contribute to the major symptoms of AD, including cognition impairment, memory loss, depression, and anxiety.Because our discovered G9atranslational mechanism directly and ultimately determined AD-correlated proteomic or phosphoproteomic changes in diseased brain, MS1262 showed high speci city and e cacy for protecting AD patients from cognitive impairment and noncognitive disorders.In addition, candidate markers of companion diagnosis can be selected from the AD-related, MS1262-reversed proteome to predict AD patient responses to MS1262 therapy and to stratify patients for enhanced therapy with high positive response rates.
Experimental animals.All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.The 5xFAD (C57BL/6J) and wild-type (C57BL/6J) littermate controls (16-24-weeks-old) were obtained from the Jackson laboratory.All 5xFAD mice were heterozygous.APP NLGF (C57BL/6J-A w-J /J) and wild-type (C57BL/6J-A w-J /J) mice were obtained from Dr. Mohanish Deshmukh.All APP NLGF mice were homozygous.Both male and female mice were used, and sex was matched in the various groups.No immune de ciencies or other health problems were observed in these lines, and all animals were experimentally and drug-naïve before use.Animals Novel place recognition.On day 3 of behavioral testing, also known as the encoding phase, two identical glass cylinders (height 4 cm, base diameter 1.5 cm) were xed to the chamber oor (to prevent object movement) on the same side of the chamber 20 cm apart from each other.Animals were allowed to freely explore the chamber for 5 minutes and could interact with the objects while being recorded.Most animals showed no preference for a single object and the locations of the objects were randomized across mice.On day 4 of behavioral testing, also known as the retrieval phase, the position of one of the objects was moved to the opposite side of the chamber, and animals were recorded for 5 minutes while freely exploring the chamber and interacting with the objects.Videos were scored manually using a separately trained researcher who was blinded to the treatment group.Time spent with each object in both encoding and retrieval were scored.Any mouse that showed a preference for one object over the other (de ned by interacting with one object for more than double the time of interacting with the other) during the encoding phase was not included in the analysis.Furthermore, mice that did not spend at least 2 seconds of total object interaction time (IT) were not included in the analysis.The discrimination ratio (DR) was calculated in the following way: DR = (IT novel location -IT familiar location )/ (IT novel location + IT familiar location ).
A DR of 0 indicated no preference for either object, and a DR of 0.33 indicates spending twice as much time with the object in the novel location compared with the object in the familiar location.
Elevated zero maze.The apparatus was an elevated white plastic ring platform (width of ring 6 cm, outer diameter 60 cm).The entire ring was elevated 60 cm off the ground.Each animal was placed in the closed arm to start the trial and was recorded for 5 minutes.Time spent in the open arm sections was scored by a trained, blinded researcher.After each trial, the apparatus was cleaned with 70% ethanol.
Forced Swim.The apparatus was an acrylic cylinder (diameter 20 cm, height 30 cm) lled with room temperature (23±1 o C) water to a depth of 20 cm.Each mouse was recorded during a 5-minute swimming trial, and the video was later scored manually by a trained, blinded researcher for time spent immobile.
Time spent immobile was de ned as when all four paws of the mouse remained immobile.After each trial, the apparatus was lled with fresh water.
METHOD DETAILS Drug treatment.The G9a inhibitor MS1262 was dissolved in DMSO to 10 mg/mL and aliquoted into single doses and stored at -20°C.Immediately prior to injection, these aliquots were thawed and diluted in 0.9% saline.The nal solution that was injected intraperitoneally consisted of 1% DMSO and equated to a 1 mg MS1262/kg of animal weight.Control animals received 1% DMSO in 0.9% saline at weight matched volumes.Mice were randomly selected for MS1262 or vehicle treatment and received 1 injection every 3.5 days for 6 weeks.
Microdissection of hippocampi.After 6 weeks of intermittent MS1262 or vehicle treatment, animals were anesthetized with a 5% iso urane in oxygen mixture until the animal was no longer responsive to a toe pinch.Animals were then transcardially perfused with ice-cold PBS and the brain was isolated.The brain was sliced bilaterally across the sagittal midline.Each half was then taken, and the hippocampus was carefully dissected.Both hippocampi were placed in a cryogenic tube and ash frozen in liquid nitrogen were group housed and bred in a dedicated husbandry facility with 12/12 hour light-dark cycles with ad libitum food and water.All mice were under veterinary supervision.Behavioral experiments were performed in the light phase.Once drug or vehicle administration began, animals were moved to a satellite housing facility with the same light-dark cycle.BEHAVIORAL TESTSHandling.Mice were handled ve days a week for 6 weeks for 5 min/day leading up to behavioral testing.Open Field and Habituation.On days 1 and 2, mice were placed in an empty open eld environment (45 cm square plastic chamber) for 10 minutes.After each test, the chamber was cleaned with 70% ethanol to eliminate scents from previously tested mice.The rst 5 minutes of the day-1 test was analyzed by Noldus Ethovision XT to monitor animal position and locomotion.A 25 cm square was used to indicate the center of the chamber when analyzing the recordings.Both total locomotion and time spent in the center of the open eld were quanti ed by the Noldus Ethovision XT program.

Figure 3 Treatment
Figure 3

Figure 7 MS1262
Figure 7 mM nal) for 45 min in the dark at ambient temperature.Samples were diluted 4fold with 25 mM Tris-HCl pH 8.0, 1 mM CaCl 2 and digested with trypsin at a ratio of 1:100 (w/w, trypsin : protein) overnight at ambient temperature.There were three wild-type (WT) controls, 4 AD, and 4 ADtreated with MS1262.Peptides were cleaned by homemade C18 stage tips and the concentration was determined (Peptide assay, Thermo 23275).One hundred microgram each was used for labeling with isobaric stable tandem mass tags (TMT11, Thermo Fisher Scienti c, San Jose, CA) following supplier instruction.The mixture of labeled peptides was desalted on Cep-Pak light C18 cartridge (Waters).Phosphopeptides were enriched with High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scienti c).One hundred microgram of peptides was fractionated into 20 fractions on C18 stage tip with 10 mM trimethylammonium bicarbonate (TMAB), pH 8.5 containing 5 to 50% acetonitrile.to100%Bin 1 min and 19-min wash with 100%B, where buffer A was aqueous 0.1% formic acid, and buffer B was 80% acetonitrile and 0.1% formic acid.The ow rate was kept at a 250 nl/min.Mass spectrometry experiments were also conducted in a data-dependent mode with full MS (externally calibrated to a resolution of 60,000 at m/z 200) followed by high energy collision-activated dissociation-MS/MS of the top 10 most intense ions with a resolution of 45,000 at m/z 200.High energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 32 eV in the presence of nitrogen bath gas atoms.Dynamic exclusion was 45 seconds.Leica vibratome (VT1200, Germany).Next, slices were warmed to 34.5°C for 8 minutes.Then, slices were maintained in the holding chamber containing HEPES ACSF (in mM):92NaCl, 30 NaHCO 3 , 25 glucose, 20 HEPES, 5 sodium ascorbate, 3 sodium pyruvate, 2.5 KCl, 2 thiourea, 2 MgSO 4 , 2 CaCl 2 , 1.25 NaH 2 PO 4 (pH 7.3, 10 mOsm) at ambient temperature for at least 1 hour before recording.Electrophysiological recordings were made at 32°C using a heater controller (TC-324C, Warner Instruments) in ACSF containing (in mM): 125 NaCl, 26 NaHCO 3 , 20 glucose, 2.5 KCl, 2 CaCl 2 , 1.3 MgSO 4 , 1.25 NaH 2 PO 4 , (pH 7.3, 310 mOsm).The ow rate was 2 ml/min.MgCl 2 , 4 Na-ATP, 2 NaCl, 0.5 EGTA, 0.4 Na-GTP (pH 7.2, 290 mOsm).Series resistance (Rs) was monitored throughout all experiments, and cells with Rs changes over 20% were discarded.All recordings were made from granule cells located in the middle or outer layer of the dentate gyrus.For spontaneous excitatory postsynaptic currents (sEPSCs) recordings, dentate granule cells were held at -70 mV in voltage-clamp mode and bicuculine (20 μM; GABA A receptor antagonist; Tocris Bioscience) was added to the ACSF.Intrinsic cellular properties were recorded in current-clamp mode.To test neuronal input resistance, hyperpolarizing current pulses (20 pA, 200 ms) were applied to the neurons.Resting MS1262 (1 mg/kg) or vehicle as described previously.Animals were anesthetized with iso urane (5% in O 2 ) and transcardially perfused with ice-cold oxygenated arti cial cerebrospinal uid (ACSF) containing the following items (in mM): 92 NMDG, 30 NaHCO 3 , 25 glucose, 20 HEPES, 10 MgSO 4 , 5 sodium ascorbate, 3 sodium pyruvate, 2.5 KCl, 2 thiourea, 1.25 NaH 2 PO 4 , and 0.5 CaCl 2 (pH 7.3, 310 mOsm).Brains were rapidly removed, and acute transverse hippocampal slices (280 μm) were cut using a