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Alzheimer's disease (AD) is associated with memory and cognitive impairment caused by progressive degeneration of neurons. The events leading to neuronal death are associated with the accumulation of aggregating proteins in neurons and glia of the affected brain regions, in particular extracellular deposition of amyloid plaques and intracellular formation of tau neurofibrillary tangles. Moreover, the accumulation of pathological tau proteoforms in the brain concurring with disease progression is a key feature of multiple neurodegenerative diseases, called tauopathies, like frontotemporal dementia (FTD) where autosomal dominant mutations in the tau encoding MAPT gene provide clear evidence of a causal role for tau dysfunction. Observations from disease models, post-mortem histology, and clinical evidence have demonstrated that pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization, changes in solubility, mislocalization, and intercellular spreading. Despite extensive research, there are few disease-modifying or preventative therapeutics for AD and none for other tauopathies. Challenges faced in tauopathy drug development include an insufficient understanding of pathogenic mechanisms of tau proteoforms, limited specificity of agents tested, and inadequate levels of brain exposure, altogether underscoring the need for innovative therapeutic modalities. In recent years, the development of experimental therapeutic modalities, such as targeted protein degradation (TPD) strategies, has shown significant and promising potential to promote the degradation of disease-causing proteins, thereby reducing accumulation and aggregation. Here, we review all modalities of TPD that have been developed to target tau in the context of AD and FTD, as well as other approaches that with innovation could be adapted for tau-specific TPD., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M. Catarina Silva reports a relationship with Proximity Therapeutics that includes: consulting or advisory. M. Catarina Silva reports a relationship with Casma Therapeutics that includes: funding grants. Stephen J. Haggarty reports a relationship with Proximity Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with Psy Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with Birdwood Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with Frequency Therapeutics Inc that includes: board membership. Stephen J. Haggarty reports a relationship with Souvien Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with Sensorium Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with 4 ​M Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with Ilios Therapeutics that includes: board membership. Stephen J. Haggarty reports a relationship with Entheos Labs that includes: board membership. Stephen J. Haggarty reports a relationship with Amgen that includes: consulting or advisory and speaking and lecture fees. Stephen J. Haggarty reports a relationship with AstraZeneca that includes: consulting or advisory, funding grants, and speaking and lecture fees. Stephen J. Haggarty reports a relationship with Biogen that includes: consulting or advisory and speaking and lecture fees. Stephen J. Haggarty reports a relationship with Merck that includes: consulting or advisory and speaking and lecture fees. Stephen J. Haggarty reports a relationship with Regenacy Pharmaceuticals that includes: consulting or advisory and speaking and lecture fees. Stephen J. Haggarty reports a relationship with Syros Pharmaceuticals that includes: consulting or advisory and speaking and lecture fees. Stephen J. Haggarty reports a relationship with Juvenescence Life that includes: consulting or advisory and speaking and lecture fees. Stephen J. Haggarty reports a relationship with JW Pharmaceuticals that includes: funding grants. Stephen J. Haggarty reports a relationship with Lexicon Pharmaceuticals that includes: funding grants. Stephen J. Haggarty reports a relationship with Vesigen Therapeutics that includes: funding grants. Stephen J. Haggarty reports a relationship with Compass Pathways that includes: funding grants. Stephen J. Haggarty reports a relationship with Atai Life Sciences that includes: funding grants. Stephen J. Haggarty reports a relationship with Stealth Biotherapeutics that includes: funding grants. Stephen J. Haggarty, M. Catarina Silva has patent Compounds for Tau Protein Degradation licensed to Proximity Therapeutics. Stephen J. Haggarty, M. Catarina Silva has patent #Targeted Degrader of Aberrant Tau Based on the PET Tracer PBB3 pending to General Hospital Corp, Dana Farber Cancer Institute Inc. Stephen J. Haggarty, M. Catarina Silva has patent #Inhibitors of TTBK1 pending to General Hospital Corp, Dana Farber Cancer Institute Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we present an approach to neuropharmacological research by integrating few-shot meta-learning algorithms with brain activity mapping (BAMing) to enhance the discovery of central nervous system (CNS) therapeutics. By utilizing patterns from previously validated CNS drugs, our approach facilitates the rapid identification and prediction of potential drug candidates from limited datasets, thereby accelerating the drug discovery process. The application of few-shot meta-learning algorithms allows us to adeptly navigate the challenges of limited sample sizes prevalent in neuropharmacology. The study reveals that our meta-learning-based convolutional neural network ( Meta -CNN) models demonstrate enhanced stability and improved prediction accuracy over traditional machine-learning methods. Moreover, our BAM library proves instrumental in classifying CNS drugs and aiding in pharmaceutical repurposing and repositioning. Overall, this research not only demonstrates the effectiveness in overcoming data limitations but also highlights the significant potential of combining BAM with advanced meta-learning techniques in CNS drug discovery., Competing Interests: P.S. is listed as an inventor on a patent (US 9,897,593) filed by the City University of Hong Kong describing the system for automated handling of larval zebrafish. S.J.H. has served or serves on the advisory board of Proximity Therapeutics, Psy Therapeutics, Frequency Therapeutics, Souvien Therapeutics, Sensorium Therapeutics, 4M Therapeutics, Ilios Therapeutics, Entheos Labs, the Alzheimer’s Disease Drug Discovery Foundation, and the Kissick Family Foundation FTD grant program, none of whom were involved in the present study. S.J.H. has also received speaking or consulting fees from Amgen, AstraZeneca, Biogen, Merck, Regenacy Pharmaceuticals, Syros Pharmaceuticals, and Juvenescence Life, as well as sponsored research or gift funding from AstraZeneca, JW Pharmaceuticals, Lexicon Pharmaceuticals, Vesigen Therapeutics, Compass Pathways, Atai Life Sciences, and Stealth Biotherapeutics. The funders had no role in the design or content of this article or the decision to submit this review for publication., (© 2024 The Author(s).)

Heterozygous loss-of-function mutations in the GRN gene are a major cause of hereditary frontotemporal dementia. The mechanisms linking frontotemporal dementia pathogenesis to progranulin deficiency are not well understood, and there is currently no treatment. Our strategy to prevent the onset and progression of frontotemporal dementia in patients with GRN mutations is to utilize small molecule positive regulators of GRN expression to boost progranulin levels from the remaining functional GRN allele, thus restoring progranulin levels back to normal within the brain. This work describes a series of blood-brain-barrier-penetrant small molecules which significantly increase progranulin protein levels in human cellular models, correct progranulin protein deficiency in Grn +/- mouse brains, and reverse lysosomal proteome aberrations, a phenotypic hallmark of frontotemporal dementia, more efficiently than the previously described small molecule suberoylanilide hydroxamic acid. These molecules will allow further elucidation of the cellular functions of progranulin and its role in frontotemporal dementia and will also serve as lead structures for further drug development., (© 2024. The Author(s).)

Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) is a fatal neurodegenerative disorder with only a limited number of risk loci identified. We report our comprehensive genome-wide association study as part of the International FTLD-TDP Whole-Genome Sequencing Consortium, including 985 cases and 3,153 controls, and meta-analysis with the Dementia-seq cohort, compiled from 26 institutions/brain banks in the United States, Europe and Australia. We confirm UNC13A as the strongest overall FTLD-TDP risk factor and identify TNIP1 as a novel FTLD-TDP risk factor. In subgroup analyses, we further identify for the first time genome-wide significant loci specific to each of the three main FTLD-TDP pathological subtypes (A, B and C), as well as enrichment of risk loci in distinct tissues, brain regions, and neuronal subtypes, suggesting distinct disease aetiologies in each of the subtypes. Rare variant analysis confirmed TBK1 and identified VIPR1 , RBPJL , and L3MBTL1 as novel subtype specific FTLD-TDP risk genes, further highlighting the role of innate and adaptive immunity and notch signalling pathway in FTLD-TDP, with potential diagnostic and novel therapeutic implications.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification., Competing Interests: Competing interests statement:S.J.H. has or currently serves on the SAB of Proximity Therapeutics, Psy Therapeutics, Frequency Therapeutics, Souvien Therapeutics, Sensorium Therapeutics, 4M Therapeutics, Ilios Therapeutics, Entheos Labs, and the Kissick Family Foundation FTD Grant Program, none of whom were involved in the present study. S.J.H. has also received speaking or consulting fees from Amgen, AstraZeneca, Biogen, Merck, Regenacy Pharmaceuticals, Syros Pharmaceuticals, Juvenescence Life, Neumora Therapeutics, and Biohaven Pharmaceuticals, as well as sponsored research or gift funding from AstraZeneca, JW Pharmaceuticals, Lexicon Pharmaceuticals, Vesigen Therapeutics, Compass Pathways, Atai Life Sciences, and Stealth Biotherapeutics. The funders had no role in the design or content of this article, or the decision to submit this review for publication. S.C.P., B.E.G., F.A.H., C.M.J., J.A.P., J.J, Z.G.L., G.Q.F., and S.W. declare no competing interests.

Frontotemporal dementia (FTD) is a debilitating neurodegenerative disorder with currently no disease-modifying treatment options available. Mutations in GRN are one of the most common genetic causes of FTD, near ubiquitously resulting in progranulin (PGRN) haploinsufficiency. Small molecules that can restore PGRN protein to healthy levels in individuals bearing a heterozygous GRN mutation may thus have therapeutic value. Here, we show that epigenetic modulation through bromodomain and extra-terminal domain (BET) inhibitors (BETi) potently enhance PGRN protein levels, both intracellularly and secreted forms, in human central nervous system (CNS)-relevant cell types, including in microglia-like cells. In terms of potential for disease modification, we show BETi treatment effectively restores PGRN levels in neural cells with a GRN mutation known to cause PGRN haploinsufficiency and FTD. We demonstrate that BETi can rapidly and durably enhance PGRN in neural progenitor cells (NPCs) in a manner dependent upon BET protein expression, suggesting a gain-of-function mechanism. We further describe a CNS-optimized BETi chemotype that potently engages endogenous BRD4 and enhances PGRN expression in neuronal cells. Our results reveal a new epigenetic target for treating PGRN-deficient forms of FTD and provide mechanistic insight to aid in translating this discovery into therapeutics., (© 2024. The Author(s).)

Tauopathies are a class of neurodegenerative diseases characterized by the progressive misfolding and accumulation of pathological tau protein in focal regions of the brain, leading to insidious neurodegeneration. Abnormalities in cholesterol metabolism and homeostasis have also been implicated in various neurodegenerative diseases. However, the connection between cholesterol dysregulation and tau pathology remains largely unknown. To model and measure the impact of cholesterol dysregulation on tau, we utilized a combination of in vitro and ex vivo tau aggregation assays using an engineered tau biosensor cell line and human induced pluripotent stem cell (iPSC)-derived neuronal cultures from an individual harboring an autosomal dominant P301L tau mutation and from a healthy control. We demonstrate that excess cholesterol esters lead to an increased rate of tau aggregation in vitro and an increase in seed-dependent insoluble tau aggregates detected in the biosensor line. We observed a strong correlation between cholesterol ester concentration and the presence of high-molecular-weight, oligomeric tau species. Importantly, in tauopathy patient iPSC-derived neurons harboring a P301L tau mutation with endogenous forms of misfolded tau, we show that acute dysregulation of cholesterol homeostasis through acute exposure to human plasma-purified cholesterol esters formed by the linkage of fatty acids to the hydroxyl group of cholesterol leads to the rapid accumulation of phosphorylated tau. Conversely, treatment with the same cholesterol esters pool did not lead to subsequent accumulation of phosphorylated tau in control iPSC-derived neurons. Finally, treatment with a heterobifunctional, small-molecule degrader designed to selectively engage and catalyze the ubiquitination and proteasomal degradation of aberrant tau species prevented cholesterol ester-induced aggregation of tau in the biosensor cell line in a Cereblon E3 ligase-dependent manner. Degrader treatment also restored the resiliency of tauopathy patient-derived neurons towards cholesterol ester-induced tau aggregation phenotypes. Taken together, our study supports a key role of cholesterol dysregulation in tau aggregation. Moreover, it provides further pre-clinical validation of the therapeutic strategy of targeted protein degradation with heterobifunctional tau degraders for blocking tau seeding.

While most of the efforts to uncover mechanisms contributing to bipolar disorder (BD) focused on phenotypes at the mature neuron stage, little research has considered events that may occur during earlier timepoints of neurodevelopment. Further, although aberrant calcium (Ca 2+ ) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca 2+ entry (SOCE) is not well understood. Here, we report Ca 2+ and developmental dysregulations related to SOCE in BD patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells (BD-NPCs) and cortical-like glutamatergic neurons. First, using a Ca 2+ re-addition assay we found that BD-NPCs and neurons had attenuated SOCE. Intrigued by this finding, we then performed RNA-sequencing and uncovered a unique transcriptome profile in BD-NPCs suggesting accelerated neurodifferentiation. Consistent with these results, we measured a slower rate of proliferation, increased neurite outgrowth, and decreased size in neurosphere formations with BD-NPCs. Also, we observed decreased subventricular areas in developing BD cerebral organoids. Finally, BD NPCs demonstrated high expression of the let-7 family while BD neurons had increased miR-34a, both being microRNAs previously implicated in neurodevelopmental deviations and BD etiology. In summary, we present evidence supporting an accelerated transition towards the neuronal stage in BD-NPCs that may be indicative of early pathophysiological features of the disorder., (© 2023. Crown.)

MicroRNAs (miRNAs) regulate fundamental biological processes by silencing mRNA targets and are dysregulated in many diseases. Therefore, miRNA replacement or inhibition can be harnessed as potential therapeutics. However, existing strategies for miRNA modulation using oligonucleotides and gene therapies are challenging, especially for neurological diseases, and none have yet gained clinical approval. We explore a different approach by screening a biodiverse library of small molecule compounds for their ability to modulate hundreds of miRNAs in human induced pluripotent stem cell-derived neurons. We demonstrate the utility of the screen by identifying cardiac glycosides as potent inducers of miR-132, a key neuroprotective miRNA downregulated in Alzheimer's disease and other tauopathies. Coordinately, cardiac glycosides downregulate known miR-132 targets, including Tau, and protect rodent and human neurons against various toxic insults. More generally, our dataset of 1370 drug-like compounds and their effects on the miRNome provides a valuable resource for further miRNA-based drug discovery., (© 2023. The Author(s).)

Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.

Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i's therapeutic potential.

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite tremendous effort, the molecular and cellular basis of cognitive deficits in schizophrenia remain poorly understood. Recent progress in elucidating the genetic architecture of schizophrenia has highlighted the association of multiple loci and rare variants that may impact susceptibility. One key example, given their potential etiopathogenic and therapeutic relevance, is a set of genes that encode proteins that regulate excitatory glutamatergic synapses in brain. A critical next step is to delineate specifically how such genetic variation impacts synaptic plasticity and to determine if and how the encoded proteins interact biochemically with one another to control cognitive function in a convergent manner. Towards this goal, here we study the roles of GPCR-kinase interacting protein 1 (GIT1), a synaptic scaffolding and signaling protein with damaging coding variants found in schizophrenia patients, as well as copy number variants found in patients with neurodevelopmental disorders. We generated conditional neural-selective GIT1 knockout mice and found that these mice have deficits in fear conditioning memory recall and spatial memory, as well as reduced cortical neuron dendritic spine density. Using global quantitative phospho-proteomics, we revealed that GIT1 deletion in brain perturbs specific networks of GIT1-interacting synaptic proteins. Importantly, several schizophrenia and neurodevelopmental disorder risk genes are present within these networks. We propose that GIT1 regulates the phosphorylation of a network of synaptic proteins and other critical regulators of neuroplasticity, and that perturbation of these networks may contribute specifically to cognitive deficits observed in schizophrenia and neurodevelopmental disorders., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Accumulation of misfolded, aggregating proteins concurrent with disease onset and progression is a hallmark of neurodegenerative proteinopathies. An important class of these are tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD), associated with accumulation of aberrant forms of tau protein in the brain. Pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization and changes in solubility, cellular redistribution, and spreading. Development and testing of experimental therapeutics that target these pathological tau conformers requires use of cellular models that recapitulate neuronal endogenous, non-heterologous tau expression under genomic and physiological contexts relevant to disease. In this study, we employed FTD-patient induced pluripotent stem cells (iPSC)-derived neurons, expressing a tau variant or mutation, as primary models for driving a medicinal chemistry campaign around tau targeting degrader series. Our screening goal was to establish structure-activity relationships (SAR) for the different chemical series to identify the molecular composition that most efficiently led to tau degradation in human FTD ex vivo neurons. We describe the identification of the lead compound QC-01-175 and follow-up optimization strategies for this molecule. We present three final lead molecules with tau degradation activity in mutant neurons, which establishes potential disease relevance and will drive future studies on specificity and pharmacological properties., Competing Interests: MS, FF, QC, NG, and SH are co-inventors on a patent covering the molecules disclosed in this publication (WO/2019/014429). MS is a consultant to Casma Therapeutics. KD is a consultant to Kronos Bio. EF is a founder, SAB member, and equity holder in Civetta Therapeutics, Jengu Therapeutics (board member), Neomorph Inc., and Proximity Therapeutics; an equity holder of C4 Therapeutics; and a consultant to Novartis, AbbVie, Sanofi, Deerfield, Avilar Pfizer, and EcoR1. The Fischer lab receives or has received research funding from Novartis, Astellas, Ajax, and Deerfield. NG is a founder, science advisory board member (SAB), and equity holder at Gatekeeper, Syros, Petra, C4, B2S, Voronoi, EoCys, Larkspur (board member), and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Her2llc, Arbella, Deerfield, and Sanofi. FF is a founder and equity holder in Proximity Therapeutics, an SAB member and equity holder in Triana Therapeutics, and a consultant to RA Capital. SH is a member of the SAB and equity holder of Psy Therapeutics, Frequency Therapeutics, Sensorium Therapeutics, 4M Therapeutics, and Proximity Therapeutics and has received speaking fees from AstraZeneca, Amgen, Merck, and Syros. The Haggarty lab has received funding from the Tau Pipeline Enabling (T-PEP) Program (Alzheimer’s Association/Rainwater Foundation), Tau Consortium, F-Prime Biomedical Research Initiative, AstraZeneca, JW Pharmaceuticals, Lexicon Pharmaceuticals, Stealth Biotherapeutics, atai Life Sciences, and Compass Pathways. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Silva, Nandi, Donovan, Cai, Berry, Nowak, Fischer, Gray, Ferguson and Haggarty.)

Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOMER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease., Competing Interests: Declaration of interests A.K.H. and N.M. have a financial interest as co-founders of Circular Genomics Inc. and are inventors of patents related to the use of circRNAs for brain disease diagnostics (N.M.) or therapeutics (N.M. and A.K.H.)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer's disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicine research for both neurological and psychiatric disorders., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

To better understand the role of bromodomain and extra-terminal domain (BET) proteins in epigenetic mechanisms, we developed a series of thienodiazepine-based derivatives and identified two compounds, 3a and 6a , as potent BET inhibitors. Further in vivo pharmacokinetic studies and analysis of in vitro metabolic stability of 6a revealed excellent brain penetration and reasonable metabolic stability. Compounds 3a and 6a were radiolabeled with fluorine-18 in two steps and utilized in positron emission tomography (PET) imaging studies in mice. Preliminary PET imaging results demonstrated that [ 18 F] 3a and [ 18 F] 6a have good brain uptake (with maximum SUV = 1.7 and 2, respectively) and binding specificity in mice brains. These results show that [ 18 F] 6a is a potential PET radiotracer that could be applied to imaging BET proteins in the brain. Further optimization and improvement of the metabolic stability of [ 18 F] 6a are still needed in order to create optimal PET imaging probes of BET family members.

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) driven by viruses or bacteria, as well as in numerous immune-mediated disorders. Histone citrullination by the enzyme peptidylarginine deiminase 4 (PAD4) and the consequent decondensation of chromatin are hallmarks in the induction of NETs. Nevertheless, additional histone modifications that may govern NETosis are largely overlooked. Herein, we show that histone deacetylases (HDACs) play critical roles in driving NET formation in human and mouse neutrophils. HDACs belonging to the zinc-dependent lysine deacetylases family are necessary to deacetylate histone H3, thus allowing the activity of PAD4 and NETosis. Of note, HDAC inhibition in mice protects against microbial-induced pneumonia and septic shock, decreasing NETosis and inflammation. Collectively, our findings illustrate a new fundamental step that governs the release of NETs and points to HDAC inhibitors as therapeutic agents that may be used to protect against ARDS and sepsis., Competing Interests: V.P., V.P-Y.M., R.M., S.J.H., J.M.K., and I.Z. submitted a patent related to the findings of this work. J.M.K. has been a paid consultant and/or equity holder for companies including Stempeutics, Sanofi, Celltex, LifeVaultBio, Camden Partners, Stemgent, Biogen, Pancryos, Element Biosciences, Frequency Therapeutics, and Mesoblast. J.M.K. is also an inventor on a patent that was licensed to Mesoblast. J.M.K. holds equity in Frequency Therapeutics, a company that has licensed IP generated by J.M.K. that may benefit financially if the IP is further validated. J.M.K. has been a paid consultant and or equity holder for multiple companies (listed here https://www.karplab.net/team/jeff-karp). The interests of J.M.K. were reviewed and are subject to a management plan overseen by his institutions in accordance with its conflict-of-interest policies. S.J.H. was or is a member of the scientific advisory board and equity holder in Rodin Therapeutics, Psy Therapeutics, Frequency Therapeutics, and Vesigen Therapeutics and has received speaking or consulting fees from AstraZeneca, Amgen, Merck, Biogen, Syros Pharmaceuticals, Regenacy Pharmaceuticals. S.J.H.’s laboratory has received sponsored research funding from the Tau Consortium, F-Prime Biomedical Research Initiative, 10.13039/100004325AstraZeneca, JW Pharmaceuticals, 10.13039/100009782ATAI, and Stealth BioTherapeutics, although none of these sponsors were involved in the present study. R.M. is a scientific advisory board (SAB) member and equity holder of Regenacy Pharmaceuticals, ERX Pharmaceuticals, and Frequency Therapeutics., (© 2021 The Author(s).)

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077., (© 2021 International Society for Neurochemistry.)

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies., (© 2021. The Author(s).)

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD., Competing Interests: Declaration of interests J.D.L. employee, Amgen. A.M.G.: Scientific Advisory Board (SAB), Denali Therapeutics (2015–2018); Genetic SAB, Pfizer (2019); SAB, Genentech; consultant, GSK, AbbVie, Biogen, and Eisai. S.J.H.: SAB, Rodin Therapeutics, Frequency Therapeutics, Psy Therapeutics, Vesigen Therapeutics, and Souvien Therapeutics; inventor, patent 6,475,723. S.T.: president, StemCultures; cofounder, LUXA Biotech; SAB, Sana Biotechnology, Blue Rock Therapeutics, and Vita Therapeutics; inventor, patent 16/331,063. J.K.I.: cofounder, AcuraStem and Modulo Bio; SAB, Spinogenix. Named companies were not involved in this project., (Copyright © 2021. Published by Elsevier Inc.)

Neurofibromatosis Type 2 (NF2) is an autosomal dominant genetic syndrome caused by mutations in the NF2 tumor suppressor gene resulting in multiple schwannomas and meningiomas. There are no FDA approved therapies for these tumors and their relentless progression results in high rates of morbidity and mortality. Through a combination of high throughput screens, preclinical in vivo modeling, and evaluation of the kinome en masse, we identified actionable drug targets and efficacious experimental therapeutics for the treatment of NF2 related schwannomas and meningiomas. These efforts identified brigatinib (ALUNBRIG®), an FDA-approved inhibitor of multiple tyrosine kinases including ALK, to be a potent inhibitor of tumor growth in established NF2 deficient xenograft meningiomas and a genetically engineered murine model of spontaneous NF2 schwannomas. Surprisingly, neither meningioma nor schwannoma cells express ALK. Instead, we demonstrate that brigatinib inhibited multiple tyrosine kinases, including EphA2, Fer and focal adhesion kinase 1 (FAK1). These data demonstrate the power of the de novo unbiased approach for drug discovery and represents a major step forward in the advancement of therapeutics for the treatment of NF2 related malignancies., Competing Interests: No authors have competing interests.

Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.

Given its chronicity, contribution to disability and morbidity, and prevalence of more than 2%, the effective treatment, and prevention of bipolar disorder represents an area of significant unmet medical need. While more than half a century has passed since the introduction of lithium into widespread use at the birth of modern psychopharmacology, that medication remains a mainstay for the acute treatment and prevention of recurrent mania/hypomania and depression that characterize bipolar disorder. However, the continued limited understanding of how lithium modulates affective behavior and lack of validated cellular and animal models have resulted in obstacles to discovering more effective mood stabilizers with fewer adverse side effects. In particular, while there has been progress in developing new pharmacotherapy for mania, developing effective treatments for acute bipolar depression remain inadequate. Recent large-scale human genetic studies have confirmed the complex, polygenic nature of the risk architecture of bipolar disorder, and its overlap with other major neuropsychiatric disorders. Such discoveries have begun to shed light on the pathophysiology of bipolar disorder. Coupled with broader advances in human neurobiology, neuropharmacology, noninvasive neuromodulation, and clinical trial design, we can envision novel therapeutic strategies informed by defined molecular mechanisms and neural circuits and targeted to the root cause of the pathophysiology. Here, we review recent advances toward the goal of better treatments for bipolar disorder, and we outline major challenges for the field of translational neuroscience that necessitate continued focus on fundamental research and discovery.

Glycogen synthase kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3β binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3β.

Tauopathies are neurodegenerative diseases characterized by the pathological accumulation of microtubule-associated protein tau (MAPT) in the form of neurofibrillary tangles and paired helical filaments in neurons and glia, leading to brain cell death. These diseases include frontotemporal dementia (FTD) and Alzheimer's disease (AD) and can be sporadic or inherited when caused by mutations in the MAPT gene. Despite an incredibly high socio-economic burden worldwide, there are still no effective disease-modifying therapies, and few tau-focused experimental drugs have reached clinical trials. One major hindrance for therapeutic development is the knowledge gap in molecular mechanisms of tau-mediated neuronal toxicity and death. For the promise of precision medicine for brain disorders to be fulfilled, it is necessary to integrate known genetic causes of disease, i.e., MAPT mutations, with an understanding of the dysregulated molecular pathways that constitute potential therapeutic targets. Here, the growing understanding of known and proposed mechanisms of disease etiology will be reviewed, together with promising experimental tau-directed therapeutics, such as recently developed tau degraders. Current challenges faced by the fields of tau research and drug discovery will also be addressed.

Gene expression data can offer deep, physiological insights beyond the static coding of the genome alone. We believe that realizing this potential requires specialized, high-capacity machine learning methods capable of using underlying biological structure, but the development of such models is hampered by the lack of published benchmark tasks and well characterized baselines. In this work, we establish such benchmarks and baselines by profiling many classifiers against biologically motivated tasks on two curated views of a large, public gene expression dataset (the LINCS corpus) and one privately produced dataset. We provide these two curated views of the public LINCS dataset and our benchmark tasks to enable direct comparisons to future methodological work and help spur deep learning method development on this modality. In addition to profiling a battery of traditional classifiers, including linear models, random forests, decision trees, K nearest neighbor (KNN) classifiers, and feed-forward artificial neural networks (FF-ANNs), we also test a method novel to this data modality: graph convolugtional neural networks (GCNNs), which allow us to incorporate prior biological domain knowledge. We find that GCNNs can be highly performant, with large datasets, whereas FF-ANNs consistently perform well. Non-neural classifiers are dominated by linear models and KNN classifiers.

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

As knowledge of Alzheimer's disease (AD) progression improves, the field has recognized the need to diversify the pipeline, broaden strategies and approaches to therapies, as well as delivery mechanisms. A better understanding of the earliest biological processes of AD/dementia would help inform drug target selection. Currently there are a number of programs exploring these alternate avenues. This meeting will allow experts in the field (academia, industry, government) to provide perspectives and experiences that can help elucidate what the pipeline looks like today and what avenues hold promise in developing new therapies across the stages of AD. The focus here is on Active Immunotherapies and Alternative Therapeutic Modalities. This topic includes active vaccines, antisense oligomers, and cell-based therapy among others, and highlights new clinical developments that utilize these modalities., Competing Interests: Stacie Weninger is employed by FBRI LLC, a wholly owned subsidiary of FMR LLC. FMR LLC and its affiliates invest broadly in many companies, including life sciences and pharmaceutical companies. Robert Alexander is a full‐time employee of Takeda. FMM is an employee and shareholder of Eli Lilly and Co. Stephen J. Haggarty was or is a member of the scientific advisory board of Rodin Therapeutics, Psy Therapeutics, Frequency Therapeutics, and Souvien Therapeutics, none of which were involved in the publication. Stephen J. Haggarty has also received speaking or consulting fees from Amgen, AstraZeneca, Biogen, Merck, and Regenacy Pharmaceuticals, as well as sponsored research or gift funding from AstraZeneca, JW Pharmaceuticals, Juvenescence, and Vesigen unrelated to the content of this manuscript. Stephen J. Haggarty is an inventor on patent applications related to targeted protein degradation for tauopathies and other CNS disorders., (© 2020 The Authors. Alzheimer's & Dementia: Translational Research & Clinical Interventions published by Wiley Periodicals, Inc. on behalf of Alzheimer's Association.)

Somatic expansion of the Huntington's disease (HD) CAG repeat drives the rate of a pathogenic process ultimately resulting in neuronal cell death. Although mechanisms of toxicity are poorly delineated, transcriptional dysregulation is a likely contributor. To identify modifiers that act at the level of CAG expansion and/or downstream pathogenic processes, we tested the impact of genetic knockout, in Htt Q111 mice, of Hdac2 or Hdac3 in medium-spiny striatal neurons that exhibit extensive CAG expansion and exquisite disease vulnerability. Both knockouts moderately attenuated CAG expansion, with Hdac2 knockout decreasing nuclear huntingtin pathology. Hdac2 knockout resulted in a substantial transcriptional response that included modification of transcriptional dysregulation elicited by the Htt Q111 allele, likely via mechanisms unrelated to instability suppression. Our results identify novel modifiers of different aspects of HD pathogenesis in medium-spiny neurons and highlight a complex relationship between the expanded Htt allele and Hdac2 with implications for targeting transcriptional dysregulation in HD., Competing Interests: MK, SE, MA, JS, MS, LH, JN, AS, RM, JW, MT No competing interests declared, DF DMF is a member of the scientific advisory board of Psy Therapeutics. SH SJH is a member of the scientific advisory board of Psy Therapeutics, Frequency Therapeutics and Souvien Therapeutics, and former member of the scientific advisory board of Rodin Therapeutics that is focused on HDAC2 inhibitors, none of whom were involved in the present study. SJH has also received speaking or consulting fees from Amgen, AstraZeneca, Biogen, Merck, Regenacy Pharmaceuticals, as well as sponsored research or gift funding from AstraZeneca, JW Pharmaceuticals, and Vesigen unrelated to the content of this manuscript. His financial interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. VW VCW is a scientific advisory board member of Triplet Therapeutics, a company developing new therapeutic approaches to address triplet repeat disorders such Huntington's disease and Myotonic Dystrophy and of LoQus23 Therapeutics. Her financial interests in Triplet Therapeutics, were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies., (© 2020, Kovalenko et al.)

Development of effective therapeutics for neurological disorders has historically been challenging partly because of lack of accurate model systems in which to investigate disease etiology and test new therapeutics at the preclinical stage. Human stem cells, particularly patient-derived induced pluripotent stem cells (iPSCs) upon differentiation, have the ability to recapitulate aspects of disease pathophysiology and are increasingly recognized as robust scalable systems for drug discovery. We review advances in deriving cellular models of human central nervous system (CNS) disorders using iPSCs along with strategies for investigating disease-relevant phenotypes, translatable biomarkers, and therapeutic targets. Given their potential to identify novel therapeutic targets and leads, we focus on phenotype-based, small-molecule screens employing human stem cell-derived models. Integrated efforts to assemble patient iPSC-derived cell models with deeply annotated clinicopathological data, along with molecular and drug-response signatures, may aid in the stratification of patients, diagnostics, and clinical trial success, shifting translational science and precision medicine approaches. A number of remaining challenges, including the optimization of cost-effective, large-scale culture of iPSC-derived cell types, incorporation of aging into neuronal models, as well as robustness and automation of phenotypic assays to support quantitative drug efficacy, toxicity, and metabolism testing workflows, are covered. Continued advancement of the field is expected to help fully humanize the process of CNS drug discovery., (© 2019 New York Academy of Sciences.)

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired OGG1 activity, 8-oxoG accumulation at the promoters of genes critical for brain function, and transcriptional repression. Moreover, we observe elevated 8-oxoG along with reduced HDAC1 activity and downregulation of a similar gene set in the 5XFAD mouse model of Alzheimer's disease. Notably, pharmacological activation of HDAC1 alleviates the deleterious effects of 8-oxoG in aged wild-type and 5XFAD mice. Our work uncovers important roles for HDAC1 in 8-oxoG repair and highlights the therapeutic potential of HDAC1 activation to counter functional decline in brain aging and neurodegeneration.

Introduction: Bromodomain and extra-terminal domain (BET) family proteins play a vital role in the epigenetic regulation process by interacting with acetylated lysine (Ac-K) residues in histones. BET inhibitors have become promising candidates to treat various diseases through the inhibition of the interaction between BET bromodomains and Ac-K of histone tails. With a molecular imaging probe, noninvasive imaging such as positron emission tomography (PET) can visualize the distribution and roles of BET family proteins in vivo and enlighten our understanding of BET protein function in both healthy and diseased tissue., Methods: We radiolabeled the potent BET inhibitor INCB054329 by N-methylation to make [ 11 C]PB003 as a BET PET radiotracer. The bioactivity evaluation of unlabeled PB003 in vitro was performed to confirm its binding affinity for BRDs, then the PET/CT imaging in rodents was performed to evaluate the bioactivity of [ 11 C]PB003 in vivo., Results: In our in vitro evaluation, PB003 showed a high BET binding affinity for BRDs (K d  = 2 nM, 1.2 nM, and 1.2 nM for BRD2, BRD3, and BRD4, respectively). In vivo PET/CT imaging demonstrated that [ 11 C]PB003 has favorable uptake with appropriate kinetics and distributions in main peripheral organs. Besides, the blockade of [ 11 C]PB003 binding was found in our blocking study which indicated the specificity of [ 11 C]PB003. However, the BBB penetration and brain uptake of [ 11 C]PB003 was limited, with only a maximum 0.2% injected dose/g at ~2 min post-injection., Conclusion: The imaging results in rodents in vivo demonstrate that [ 11 C]PB003 binds to BET with high selectivity and specificity and has favorable uptake in peripheral organs. However, the low brain uptake of [ 11 C]PB003 limits the visualization of brain regions indicating the efforts are still needed to discover the new BET imaging probes for brain visualization., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Leucine rich repeat kinase 2 (LRRK2) is an enigmatic enzyme and a relevant target for Parkinson's disease (PD). However, despite the significant amount of research done in the past decade, the precise function of LRRK2 remains largely unknown. Moreover, the therapeutic potential of its inhibitors is in its infancy with the first clinical trial having just started. In the present work, the molecular mechanism of LRRK2 in the control of neurogenesis or gliogenesis was investigated. We designed and synthesized novel benzothiazole-based LRRK2 inhibitors and showed that they can modulate the Wnt/β-catenin signaling pathway. Furthermore, compounds 5 and 14 were able to promote neural progenitors proliferation and drive their differentiation toward neuronal and oligodendrocytic cell fates. These results suggest potential new avenues for the application of LRRK2 inhibitors in demyelinating diseases in which oligodendrocyte cell-death is one of the pathological features.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Background: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin., Methods: Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq., Results: Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs., Conclusion: MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD., Competing Interests: Competing interestsSJH is a member of the scientific advisory boards of Frequency Therapeutics, Psy Therapeutics, and Souvien Therapeutics, none of who were involved in this study. SDS is a scientific advisor for Outermost Therapeutics, Inc., which played no part in the present study. The other authors declare no competing interests., (© The Author(s). 2020.)

Alterations in the autophagosomal-lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal-lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts., Competing Interests: U.C., E.B., S.L.C., M.C.K., W.-N.Z., and S.A.R. declare no conflicts of interest. A.P. is a shareholder in Enspire Bio, Inc, which played no role in the study. M.O.R. is currently an employee of Image Computing & Information Technologies (ICIT). This company played no role in the study. S.J.H. is a member of the SAB and equity holder in Rodin Therapeutics, Frequency Therapeutics, Psy Therapeutics and Souvien Therapeutics, as well as having received consulting or speaking fees from Sunovion, Biogen, AstraZeneca, Amgen, and Merck, none of whom were involved in the study. The funders had no role in the design, data collection, interpretation, writing of the manuscript, or in the decision to the publish the study.

Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [ 3 H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [ 11 C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC 50 = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2 T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.

Primary tauopathies are characterized neuropathologically by inclusions containing abnormal forms of the microtubule-associated protein tau (MAPT) and clinically by diverse neuropsychiatric, cognitive, and motor impairments. Autosomal dominant mutations in the MAPT gene cause heterogeneous forms of frontotemporal lobar degeneration with tauopathy (FTLD-Tau). Common and rare variants in the MAPT gene increase the risk for sporadic FTLD-Tau, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We generated a collection of fibroblasts from 140 MAPT mutation/risk variant carriers, PSP, CBD, and cognitively normal controls; 31 induced pluripotent stem cell (iPSC) lines from MAPT mutation carriers, non-carrier family members, and autopsy-confirmed PSP patients; 33 genome engineered iPSCs that were corrected or mutagenized; and forebrain neural progenitor cells (NPCs). Here, we present a resource of fibroblasts, iPSCs, and NPCs with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for tauopathies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we report the development of novel PET radiotracer ([11C]CW22) of BET proteins. In vivo imaging results in rodents and nonhuman primates (NHP) demonstrate that [11C]CW22 has excellent brain uptake, good specificity, good selectivity, suitable metabolism, appropriate kinetics and distribution in the brain. Our studies demonstrated that [11C]CW22 exhibits ideal properties as a PET imaging probe of BET proteins for further validation.

A subset of individuals with major depressive disorder (MDD) elects treatment with complementary and alternative medicines (CAMs), including the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Previous studies in rodents suggest that DHA modulates neurodevelopmental processes, including adult neurogenesis and neuroplasticity, but the molecular and cellular mechanisms of DHA's potential therapeutic effect in the context of human neurobiology have not been well established. Here we sought to address this knowledge gap by investigating the effects of DHA using human iPSC-derived neural progenitor cells (NPCs) and post-mitotic neurons using pathway-selective reporter genes, multiplexed mRNA expression profiling, and a panel of metabolism-based viability assays. Finally, real-time, live-cell imaging was employed to monitor neurite outgrowth upon DHA treatment. Overall, these studies showed that DHA treatment (0-50 μM) significantly upregulated both WNT and CREB signaling pathways in human neuronal cells in a dose-dependent manner with 2- to 3-fold increases in pathway activation. Additionally, we observed that DHA treatment enhanced survival of iPSC-derived NPCs and differentiation of post-mitotic neurons with live-cell imaging, revealing increased neurite outgrowth with DHA treatment within 24 h. Taken together, this study provides evidence that DHA treatment activates critical pathways regulating neuroplasticity, which may contribute to enhanced neuronal cell viability and neuronal connectivity. The extent to which these pathways represent molecular mechanisms underlying the potential beneficial effects of omega-3 fatty acids in MDD and other brain disorders merits further investigation., (Copyright © 2019. Published by Elsevier Inc.)

Histone deacetylases (HDACs) are enzymes involved in the epigenetic control of gene expression. A handful of HDAC inhibitors have been approved for the treatment of cancer, and HDAC inhibition has also been proposed as a novel therapeutic strategy for neurodegenerative disorders. These disorders include progranulin (PGRN)-deficient forms of frontotemporal dementia caused by mutations in the GRN gene that lead to haploinsufficiency. Hydroxamic-acid-based inhibitors of HDACs 1-3, reported to have fast-on/fast-off binding kinetics, induce increased expression of PGRN in human neuronal models, while the benzamide class of slow-binding HDAC inhibitors does not produce this effect. These observations indicate that the kinetics of HDAC inhibitor binding can be tuned for optimal induction of human PGRN expression in neurons. Here, we further expand on these findings using human cortical-like, glutamatergic neurons. We provide evidence that two prototypical, potent hydroxamic acid HDAC inhibitors that induce PGRN (panobinostat and trichostatin A) exhibit an initial fast-binding step followed by a second, slower step, referred to as mechanism B of slow binding, rather than simpler fast-on/fast-off binding kinetics. In addition, we show that trapoxin A, a macrocyclic, epoxyketone-containing class I HDAC inhibitor, exhibits slow binding with high, picomolar potency and also induces PGRN expression in human neurons. Finally, we demonstrate induction of PGRN expression by fast-on/fast-off, highly potent, macrocyclic HDAC inhibitors with ethyl ketone or ethyl ester Zn 2+ binding groups. Taken together, these data expand our understanding of HDAC1-3 inhibitor binding kinetics, and further delineate the specific combinations of structural and kinetic features of HDAC inhibitors that are optimal for upregulating PGRN expression in human neurons and thus may have translational relevance in neurodegenerative disease.

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

There is a paucity of efficacious new compounds to treat neuropsychiatric disorders. We present a novel approach to neuropsychiatric drug discovery based on high-content characterization of druggable signaling network responses at the single-cell level in patient-derived lymphocytes ex vivo. Primary T lymphocytes showed functional responses encompassing neuropsychiatric medications and central nervous system ligands at established (e.g., GSK-3β) and emerging (e.g., CrkL) drug targets. Clinical application of the platform to schizophrenia patients over the course of antipsychotic treatment revealed therapeutic targets within the phospholipase Cγ1-calcium signaling pathway. Compound library screening against the target phenotype identified subsets of L-type calcium channel blockers and corticosteroids as novel therapeutically relevant drug classes with corresponding activity in neuronal cells. The screening results were validated by predicting in vivo efficacy in an independent schizophrenia cohort. The approach has the potential to discern new drug targets and accelerate drug discovery and personalized medicine for neuropsychiatric conditions.