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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 [18F]3a and [18F]6a have good brain uptake (with maximum SUV = 1.7 and 2, respectively) and binding specificity in mice brains. These results show that [18F]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 [18F]6a are still needed in order to create optimal PET imaging probes of BET family members.

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 and provides protection against genotoxic stress. We show that exifone acts as a mixed, non-essential 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 HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5 that have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, 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.Graphical Abstract

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., Disruption of autophagy function in cellular and animal models of tauopathy increases tau aggregation. Here, the authors describe a small-molecule screen to identify compounds that promote autophagy clearance of tau and rescue disease-relevant phenotypes in tauopathy patient-derived neurons.

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 [11C]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 [11C]PB003 in vivo. Results In our in vitro evaluation, PB003 showed a high BET binding affinity for BRDs (Kd = 2 nM, 1.2 nM, and 1.2 nM for BRD2, BRD3, and BRD4, respectively). In vivo PET/CT imaging demonstrated that [11C]PB003 has favorable uptake with appropriate kinetics and distributions in main peripheral organs. Besides, the blockade of [11C]PB003 binding was found in our blocking study which indicated the specificity of [11C]PB003. However, the BBB penetration and brain uptake of [11C]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 [11C]PB003 binds to BET with high selectivity and specificity and has favorable uptake in peripheral organs. However, the low brain uptake of [11C]PB003 limits the visualization of brain regions indicating the efforts are still needed to discover the new BET imaging probes for brain visualization.

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.

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.

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.

Summary 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., Graphical Abstract, Highlights • A collection of fibroblasts from 140 MAPT mutation carriers, PSP, CBD, and controls • 31 iPSC lines reprogrammed from MAPT mutation carriers, PSP patients, and controls • 33 iPSC lines engineered with CRISPR/Cas9 or TALENs • Comprehensive resource for tauopathy modeling and discovery of novel therapeutics, In this article, Karch, Temple and colleagues describe a resource of fibroblasts, patient-derived induced pluripotent stem cells, and genome engineered stem cells with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for primary tauopathies.

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.

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.

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.

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Recent evidence suggest that tau may be involved in ALS pathogenesis. Here, we demonstrated that hyperphosphorylated tau (pTau-S396) is mis-localized to synapses in human post-mortem motor cortex (mCTX) across ALS subtypes. Treatment with ALS synaptoneurosomes (SNs) derived from post-mortem mCTX, enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity in vitro. Furthermore, our findings revealed that pTau-S396 interacts with the pro-fission dynamin-related protein (DRP1), and similar to pTau-S396, DRP1 accumulated in ALS SNs across ALS subtypes. Lastly, reducing tau with a specific bifunctional degrader, QC-01-175, 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.Graphical abstractpTau-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 specific degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

SUMMARYFrontotemporal dementia (FTD) due to 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 due to the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, and glutamatergic signaling pathways and regulators including the RNA-binding protein ELAVL4. 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 of layers affected in patients. Mutant neurons are susceptible to glutamate toxicity which was rescued pharmacologically by treatment with the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede cell death, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

Summary 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.

Bipolar disorder presents one of the most challenging neuropsychiatric disorders to effectively treat and understand the underlying etiopathogenesis due to its complexity at multiple levels: clinical diagnosis, genetic architecture, and variable responsiveness to pharmacotherapy and other treatments. As a CNS disorder, gaining an understanding of the disease has been greatly hindered by the limited accessibility of human brain tissue and challenges to characterizing genetically accurate models. These limitations have meant the dissection of pathogenic mechanisms along with the discovery of disease-relevant biomarkers and novel therapeutic targets for bipolar disorder that has lagged behind other areas of medicine despite its prevalence and burden to individuals, families, and society as a whole. Increasingly though, human-induced pluripotent stem cell (hiPSC) technology provides an unprecedented opportunity to gain insight into the underling molecular and cellular pathology of a wide range of monogenic and polygenic CNS disorders, including bipolar disorder. Here, we will review this progress and the state of these efforts along with progress toward integrating hiPSC studies with studies in animal models and with human neuropathology that collectively hold potential to advance our understanding of the neurobiology of bipolar disorder and targeted therapeutics for its treatment and ideally prevention.

Summary 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., Graphical abstract, Highlights • Zn-dependent lysine deacetylases govern neutrophil extracellular trap (NET) formation • Class I/IIb HDACs deacetylate histone H3 allowing its citrullination by PAD4 • HDAC inhibition with ricolinostat protects against viral and bacterial pneumonia • In a model of endotoxic shock, ricolinostat inhibits NETosis and dampens inflammation, Molecular biology; Immunology; Cell biology; Functional aspects of cell biology

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 HttQ111 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 HttQ111 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.

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.

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., Defects in DNA repair have been linked to brain aging and neurodegenerative disorders. Here the authors reveal a role for HDAC1 in stimulating OGG1 activity to alleviate 8-oxoG lesions with implications in the aging brain and neurodegenerative diseases.

Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This effect is mediated by pathological cleavage of the Cdk5 activator p35 to produce the truncated product p25, exhibiting increased stability and altered substrate specificity. The benefit of blocking p25 production has been demonstrated in various rodent and human neurodegenerative models. However, important Cdk5/p35 functions in the developing and adult brain have made it challenging to selectively target the detrimental effects of Cdk5/p25 while sparing the physiological functions of Cdk5/p35. Here, we report a 12-amino acid-long peptide fragment derived from Cdk5 (the Cdk5 inhibitory (Cdk5i) peptide) that shows a high binding affinity toward the Cdk5/p25 complex and can efficiently and selectively inhibit Cdk5/p25 kinase activity. Using cellular assays, mouse neurodegeneration models and human cerebral organoids generated from patient-derived iPSCs, we demonstrate beneficial effects of the Cdk5i peptide on various pathological phenotypes including gliosis, DNA damage, and Tau hyperphosphorylation.

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.

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. Patients with schizophrenia (SCZ) experience chronic cognitive deficits. Histone deacetylases (HDACs) are enzymes that regulate cognitive circuitry; however, the role of HDACs in cognitive disorders, including SCZ, remains unknown in humans. We previously determined that HDAC2 mRNA levels were lower in dorsolateral prefrontal cortex (DLPFC) tissue from donors with SCZ compared with controls. Here we investigated the relationship between in vivo HDAC expression and cognitive impairment in patients with SCZ and matched healthy controls using [(11)C]Martinostat positron emission tomography (PET). METHODS. In a case-control study, relative [((11))C]Martinostat uptake was compared between 14 patients with SCZ or schizoaffective disorder (SCZ/SAD) and 17 controls using hypothesis-driven region-of-interest analysis and unbiased whole brain voxel-wise approaches. Clinical measures, including the MATRICS consensus cognitive battery, were administered. RESULTS. Relative HDAC expression was lower in the DLPFC of patients with SCZ/SAD compared with controls, and HDAC expression positively correlated with cognitive performance scores across groups. Patients with SCZ/SAD also showed lower relative HDAC expression in the dorsomedial prefrontal cortex and orbitofrontal gyrus, and higher relative HDAC expression in the cerebral white matter, pons, and cerebellum compared with controls. CONCLUSIONS. These findings provide in vivo evidence of HDAC dysregulation in patients with SCZ and suggest that altered HDAC expression may impact cognitive function in humans. FUNDING. National Institute of Mental Health (NIMH), Brain and Behavior Foundation, Massachusetts General Hospital (MGH), Athinoula A. Martinos Center for Biomedical Imaging, National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH Shared Instrumentation Grant Program.

Background Meningiomas are the most common primary brain tumor in adults, and somatic loss of the neurofibromatosis 2 (NF2) tumor suppressor gene is a frequent genetic event. There is no effective treatment for tumors that recur or continue to grow despite surgery and/or radiation. Therefore, targeted therapies that either delay tumor progression or cause tumor shrinkage are much needed. Our earlier work established mammalian target of rapamycin complex mTORC1/mTORC2 activation in NF2-deficient meningiomas. Methods High-throughput kinome analyses were performed in NF2-null human arachnoidal and meningioma cell lines to identify functional kinome changes upon NF2 loss. Immunoblotting confirmed the activation of kinases and demonstrated effectiveness of drugs to block the activation. Drugs, singly and in combination, were screened in cells for their growth inhibitory activity. Antitumor drug efficacy was tested in an orthotopic meningioma model. Results Erythropoietin-producing hepatocellular receptor tyrosine kinases (EPH RTKs), c-KIT, and Src family kinase (SFK) members, which are biological targets of dasatinib, were among the top candidates activated in NF2-null cells. Dasatinib significantly inhibited phospho-EPH receptor A2 (pEPHA2), pEPHB1, c-KIT, and Src/SFK in NF2-null cells, showing no cross-talk with mTORC1/2 signaling. Posttreatment kinome analyses showed minimal adaptive changes. While dasatinib treatment showed some activity, dual mTORC1/2 inhibitor and its combination with dasatinib elicited stronger growth inhibition in meningiomas. Conclusion Co-targeting mTORC1/2 and EPH RTK/SFK pathways could be a novel effective treatment strategy for NF2-deficient meningiomas.

The role of Arc in synaptic plasticity and memory consolidation has been investigated for many years with recent evidence that defects in the expression or activity of this immediate-early gene may also contribute to the pathophysiology of brain disorders including schizophrenia and fragile X syndrome. These results bring forward the concept that reversing Arc abnormalities could provide an avenue to improve cognitive or neurological impairments in different disease contexts, but how to achieve this therapeutic objective has remained elusive. Here, we present results from a chemogenomic screen that probed a mechanistically diverse library of small molecules for modulators of BDNF-induced Arc expression in primary cortical neurons. This effort identified compounds with a range of influences on Arc, including promoting its acetylation—a previously uncharacterized post-translational modification of this protein. Together, our data provide insights into the control of Arc that could be targeted to harness neuroplasticity for clinical applications., The activity-regulated cytoskeleton-associated protein (Arc) has been implicated in synaptic plasticity and memory consolidation. Here the authors show that Arc acetylation regulates its stability and identify small molecules that modulate Arc expression.

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KImice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region ofP301Smice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENTAccumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KItransgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

Histone deacetylase 6 (HDAC6) function and dysregulation have been implicated in the etiology of certain cancers and more recently in central nervous system (CNS) disorders including Rett syndrome, Alzheimer’s and Parkinson’s diseases, and major depressive disorder. HDAC6-selective inhibitors have therapeutic potential, but in the CNS drug space the development of highly brain penetrant HDAC inhibitors has been a persistent challenge. Moreover, no tool exists to directly characterize HDAC6 and its related biology in the living human brain. Here, we report a highly brain penetrant HDAC6 inhibitor, Bavarostat, that exhibits excellent HDAC6 selectivity (>80-fold over all other Zn-containing HDAC paralogues), modulates tubulin acetylation selectively over histone acetylation, and has excellent brain penetrance. We further demonstrate that Bavarostat can be radiolabeled with 18F by deoxyfluorination through in situ formation of a ruthenium π-complex of the corresponding phenol precursor: the only method currently suitable for synthesis of [18F]Bavarostat. Finally, by using [18F]Bavarostat in a series of rodent and nonhuman primate imaging experiments, we demonstrate its utility for mapping HDAC6 in the living brain, which sets the stage for first-in-human neurochemical imaging of this important target., Bavarostat, an HDAC6-selective inhibitor with high brain penetrance, was identified. Through innovative ruthenium-mediated radiofluorination PET imaging was enabled and shows promise for translation.

Ketamine, a molecule of many faces, has contributed immeasurably to numerous realms of clinical practice and scientific inquiry. From anesthesia and analgesia to depression and schizophrenia, it continues to shed light on the molecular underpinnings of pain, consciousness, and the pathophysiology of neuropsychiatric disorders. In particular, research on ketamine's mechanism of action is providing new hope in the search for therapies for treatment-resistant depression and affords insights into disorders of glutamatergic dysfunction. In this Review, we will cover aspects of ketamine's synthesis, manufacturing, metabolism, pharmacology, approved and off-label indications, and adverse effects. We will also discuss the captivating history of this molecule, its influence on neuropsychiatry, and its potential to advance the fields of chemical neuroscience and neuropharmacology.

The discovery of haloperidol catalyzed a breakthrough in our understanding of the biochemical basis of schizophrenia, improved the treatment of psychosis, and facilitated deinstitutionalization. In doing so, it solidified the role for chemical neuroscience as a means to elucidate the molecular underpinnings of complex neuropsychiatric disorders. In this Review, we will cover aspects of haloperidol's synthesis, manufacturing, metabolism, pharmacology, approved and off-label indications, and adverse effects. We will also convey the fascinating history of this classic molecule and the influence that it has had on the evolution of neuropsychopharmacology and neuroscience.

Dysregulation of glycogen synthase kinase-3β (GSK-3β) is implicated in the pathogenesis of neurodegenerative and psychiatric disorders. Thus, development of GSK-3β radiotracers for positron emission tomography (PET) imaging is of paramount importance, because such a noninvasive imaging technique would allow better understanding of the link between the activity of GSK-3β and central nervous system disorders in living organisms, and it would enable early detection of the enzyme’s aberrant activity. Herein, we report the synthesis and biological evaluation of a series of fluorine-substituted maleimide derivatives that are high-affinity GSK-3β inhibitors. Radiosynthesis of a potential GSK-3β tracer [18F]10a is achieved. Preliminary in vivo PET imaging studies in rodents show moderate brain uptake, although no saturable binding was observed in the brain. Further refinement of the lead scaffold to develop potent [18F]-labeled GSK-3 radiotracers for PET imaging of the central nervous system is warranted.

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.

63 p.-15 fig.-1 tab.-2 schem., 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 last 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 towards 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., This work was supported by MINECO (grant SAF2016-76693-R to A.M. and SAF2017-85717-R to A.V.M.), CIBERNED (CB18/05/00040 to A.M.), MECD (FPU13-003262 to J.Z.-D.), CEU-Banco Santander (Scholarships for the Research Mobility Program CEU-BANCO SANTANDER to J.Z.-D.), Fundación Alicia Koplowitz (Research Project, 2018 to A.V.M.) and Tau Consortium and Stuart & Suzanne Steele MGH Research Scholar Award to S.J.H

Alterations in the autophagosomal&ndash, 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&ndash, 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.

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.

Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01–175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, to trigger tau ubiquitination and proteasomal degradation. QC-01–175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01–175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.

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.

High-content functional screening of primary patient blood cells reveals repurposed psychiatric drug candidates., 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.

Through epigenetic and other regulatory functions, the histone deacetylase (HDAC) family of enzymes has emerged as a promising therapeutic target for central nervous system and other disorders. Here we report on the synthesis and functional characterization of new HDAC inhibitors based structurally on tianeptine, a drug used primarily to treat major depressive disorder (MDD) that has a poorly understood mechanism of action. Since the chemical structure of tianeptine resembles certain HDAC inhibitors, we profiled the in vitro HDAC inhibitory activity of tianeptine and demonstrated its ability to inhibit the lysine deacetylase activity of a subset of class I HDACs. Consistent with a model of active site Zn2+ chelation by the carboxylic acid present in tianeptine, newly synthesized analogues containing either a hydroxamic acid or ortho-aminoanilide exhibited increased potency and selectivity among the HDAC family. This in vitro potency translated to improved efficacy in a panel of high-content imaging assays designed to assess HDAC target engagement and functional effects on critical pathways involved in neuroplasticity in both primary mouse neurons and, for the first time, human neurons differentiated from pluripotent stem cells. Most notably, tianeptinaline, a class I HDAC-selective analogue of tianeptine, but not tianeptine itself, increased histone acetylation, and enhanced CREB-mediated transcription and the expression of Arc (activity-regulated cytoskeleton-associated protein). Systemic in vivo administration of tianeptinaline to mice confirmed its brain penetration and was found to enhance contextual fear conditioning, a behavioral test of hippocampal-dependent memory. Tianeptinaline and its derivatives provide new pharmacological tools to dissect chromatin-mediated neuroplasticity underlying memory and other epigenetically related processes implicated in health and disease.

The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/− and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/− mouse hippocampus. Live-cell imaging of EB3 movement in primary neurons from Ank3+/− mice revealed impaired elongation of microtubules. Using a CRISPR-dCas9-KRAB transcriptional repressor in mouse neuro-2a cells, we determined that repression of brain-specific Ank3 increased EB3 expression, decreased tubulin acetylation, and increased the soluble:polymerized tubulin ratio, indicating enhanced microtubule dynamics. These changes were rescued by inhibition of glycogen synthase kinase 3 (GSK3) with lithium or CHIR99021, a highly selective GSK3 inhibitor. Brain-specific Ank3 repression in neuro-2a cells increased GSK3 activity (reduced inhibitory phosphorylation) and elevated collapsin response mediator protein 2 (CRMP2) phosphorylation, a known GSK3 substrate and microtubule-binding protein. Pharmacological inhibition of CRMP2 activity attenuated the rescue of EB3 expression and tubulin polymerization in Ank3-repressed cells by lithium or CHIR99021, suggesting microtubule instability induced by Ank3 repression is dependent on CRMP2 activity. Taken together, our data indicate that ANK3 functions in neuronal microtubule dynamics through GSK3 and its downstream substrate CRMP2. These findings reveal cellular and molecular mechanisms underlying brain-specific ANK3 disruption that may be related to its role in psychiatric illness.

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 find that these mice have deficits in fear conditioning learning and spatial memory. 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 to cognitive deficits observed in schizophrenia and neurodevelopmental disorders.

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca(2+)-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation. Interrogation of intracellular Ca(2+) handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in store-operated Ca(2+) uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca(2+) handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared to their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4+ NPCs with a pharmacological inhibitor of glycogen synthase kinase 3 (GSK3), a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD-patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

SUMMARY Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders., In Brief Nehme et al. combine two strong neuralizing factors (transcription factor programming and small molecule patterning) to generate human excitatory neurons from stem cells. They further undertake single-cell and reporter gene approaches to select highly differentiated neurons with increased functionality, augmenting their utility in the modeling of nervous system disorders.

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47–mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.

Schizophrenia (SCZ) and Bipolar Disorder (BD) are heterogeneous psychiatric disorders with severe socioeconomic impacts and unknown pathogenesis, despite the plethora of genomics and genetic studies focused on protein-coding genes. However, the emerging consensus is that protein-coding genes are only the tip of the iceberg of the mammalian transcriptome, given the plethora of actively transcribed non-coding RNAs (ncRNAs). Circular RNAs (circRNAs) are a novel category of ncRNAs that are derived from the back-splicing and covalent joining of exons, yet lack the capacity to become translated into protein. Recent studies have suggested that circRNAs are enriched in the brain, are preferentially generated from brain plasticity-associated genes, and are abundant in dendrites and synapses. However, very little is known about the function of circRNAs in the human brain and their potential involvement in neuropsychiatric disease. Here we carried out systematic profiling of circRNA expression in a large cohort of human postmortem brains from subjects with SCZ and BD disorder and uncovered a subset of differentially expressed circRNAs produced from genes with known links to synaptic plasticity and neuronal excitability. We validated the expression of psychiatric disease-altered circRNAs in human postmortem brain with qRT-PCR and uncovered a subset of circRNAs that were also dysregulated in induced pluripotent stem cell-derived human neuronal progenitor and immature neuronal cultures from subjects with BD and SCZ. Analysis of RNA-Binding proteins (RBPs) that could bind to psychiatric disease-associated circRNAs revealed interactions with RBPs that affect alternative splicing and control of local translation in synapses. Ongoing experiments using circRNA shRNA knockdown in human stem cell-derived neuronal cultures are attempting to elucidate the function of SCZ- and BD-associated circRNAs in human neuronal development and maturation and their interplay with RBPs and disease-related molecular pathways. Collectively, our experiments will shed light into the unexplored role of circRNAs in brain function and disease.

Epigenetic enzymes are now targeted to treat the underlying gene expression dysregulation that contribute to disease pathogenesis. Histone deacetylases (HDACs) have shown broad potential in treatments against cancer and emerging data supports their targeting in the context of cardiovascular disease and central nervous system dysfunction. Development of a molecular agent for non-invasive imaging to elucidate the distribution and functional roles of HDACs in humans will accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and validation of an HDAC imaging agent, [(11)C]6. Our imaging results demonstrate that this probe has high specificity, good selectivity, and appropriate kinetics and distribution for imaging HDACs in the brain, heart, kidney, pancreas, and spleen. Our findings support the translational potential for [(11)C]6 for human epigenetic imaging.