Epigenetic modulation through BET bromodomain inhibitors as a novel therapeutic strategy for progranulin-deficient frontotemporal dementia.

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.

Small molecule regulators of microRNAs identified by high-throughput screen coupled with high-throughput sequencing.

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.

Bipolar disorder-iPSC derived neural progenitor cells exhibit dysregulation of store-operated Ca2+ entry and accelerated differentiation.

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 (Ca2+) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca2+ entry (SOCE) is not well understood. Here, we report Ca2+ 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 Ca2+ 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.

Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations.

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.

A Cdk5-derived peptide inhibits Cdk5/p25 activity and improves neurodegenerative phenotypes.

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.

A prolyl oligopeptidase inhibitor reduces tau pathology in cellular models and in mice with tauopathy.

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.

Small Molecule Regulators of microRNAs Identified by High-Throughput Screen Coupled with High-Throughput Sequencing.

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 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 provide a valuable resource for further miRNA-based drug discovery.

Cholesterol Dysregulation Drives Seed-Dependent Tau Aggregation in Patient Stem Cell-Derived Models of Tauopathy.

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.

Chemically targeting the redox switch in AP1 transcription factor ΔFOSB.

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.

Differentiation of Human Induced Pluripotent Stem Cells into Cortical Neurons to Advance Precision Medicine.

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.

Brain-specific deletion of GIT1 impairs cognition and alters phosphorylation of synaptic protein networks implicated in schizophrenia susceptibility.

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.

Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy.

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.

A bidirectional competitive interaction between circHomer1 and Homer1b within the orbitofrontal cortex regulates reversal learning.

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.

Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis.

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.

Zinc-dependent histone deacetylases drive neutrophil extracellular trap formation and potentiate local and systemic inflammation.

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.

Design, Synthesis, and Evaluation of Thienodiazepine Derivatives as Positron Emission Tomography Imaging Probes for Bromodomain and Extra-Terminal Domain Family Proteins.

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.

High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy.

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.

Brigatinib causes tumor shrinkage in both NF2-deficient meningioma and schwannoma through inhibition of multiple tyrosine kinases but not ALK.

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.

ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids.

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.

Phosphorylation-dependent control of Activity-regulated cytoskeleton-associated protein (Arc) protein by TNIK.

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.