A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects.

Using induced pluripotent stem cells (iPSCs) to understand the mechanisms of neurological disease holds great promise; however, there is a lack of well-curated lines from a large array of participants. Answer ALS has generated over 1,000 iPSC lines from control and amyotrophic lateral sclerosis (ALS) patients along with clinical and whole-genome sequencing data. The current report summarizes cell marker and gene expression in motor neuron cultures derived from 92 healthy control and 341 ALS participants using a 32-day differentiation protocol. This is the largest set of iPSCs to be differentiated into motor neurons, and characterization suggests that cell composition and sex are significant sources of variability that need to be carefully controlled for in future studies. These data are reported as a resource for the scientific community that will utilize Answer ALS data for disease modeling using a wider array of omics being made available for these samples.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Huntington's disease iPSC models-using human patient cells to understand the pathology caused by expanded CAG repeats.

A major advance in the study of Huntington's disease (HD) has been the development of human disease models employing induced pluripotent stem cells (iPSCs) derived from patients with HD. Because iPSCs provide an unlimited source of cells and can be obtained from large numbers of HD patients, they are a uniquely valuable tool for investigating disease mechanisms and for discovering potential disease-modifying therapeutics. Here, we summarize some of the important findings in HD pathophysiology that have emerged from studies of patient-derived iPSC lines. Because they retain the genome and actual disease mutations of the patient, they provide a cell source to investigate genetic contributions to the disease. iPSCs provide advantages over other disease models. While iPSC-based technology erases some epigenetic marks, newly developed transdifferentiation methods now let us investigate epigenetic factors that control expression of mutant huntingtin (mHTT). Human HD iPSC lines allow us to investigate how endogenous levels of mHTT affect cell health, in contrast to other models that often rely on overexpressing the protein. iPSCs can be differentiated into neurons and other disease-related cells such as astrocytes from different brain regions to study brain regional differences in the disease process, as well as the cell-cell dependencies involved in HD-associated neurodegeneration. They also serve as a tissue source to investigate factors that impact CAG repeat instability, which is involved in regional differences in neurodegeneration in the HD brain. Human iPSC models can serve as a powerful model system to identify genetic modifiers that may impact disease onset, progression, and symptomatology, providing novel molecular targets for drug discovery.

Answer ALS, a large-scale resource for sporadic and familial ALS combining clinical and multi-omics data from induced pluripotent cell lines.

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical-molecular-biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics.

Enrichment of SARM1 alleles encoding variants with constitutively hyperactive NADase in patients with ALS and other motor nerve disorders.

SARM1, a protein with critical NADase activity, is a central executioner in a conserved programme of axon degeneration. We report seven rare missense or in-frame microdeletion human SARM1 variant alleles in patients with amyotrophic lateral sclerosis (ALS) or other motor nerve disorders that alter the SARM1 auto-inhibitory ARM domain and constitutively hyperactivate SARM1 NADase activity. The constitutive NADase activity of these seven variants is similar to that of SARM1 lacking the entire ARM domain and greatly exceeds the activity of wild-type SARM1, even in the presence of nicotinamide mononucleotide (NMN), its physiological activator. This rise in constitutive activity alone is enough to promote neuronal degeneration in response to otherwise non-harmful, mild stress. Importantly, these strong gain-of-function alleles are completely patient-specific in the cohorts studied and show a highly significant association with disease at the single gene level. These findings of disease-associated coding variants that alter SARM1 function build on previously reported genome-wide significant association with ALS for a neighbouring, more common SARM1 intragenic single nucleotide polymorphism (SNP) to support a contributory role of SARM1 in these disorders. A broad phenotypic heterogeneity and variable age-of-onset of disease among patients with these alleles also raises intriguing questions about the pathogenic mechanism of hyperactive SARM1 variants.

An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients.

Neurodegenerative diseases are challenging for systems biology because of the lack of reliable animal models or patient samples at early disease stages. Induced pluripotent stem cells (iPSCs) could address these challenges. We investigated DNA, RNA, epigenetics, and proteins in iPSC-derived motor neurons from patients with ALS carrying hexanucleotide expansions in C9ORF72. Using integrative computational methods combining all omics datasets, we identified novel and known dysregulated pathways. We used a C9ORF72 Drosophila model to distinguish pathways contributing to disease phenotypes from compensatory ones and confirmed alterations in some pathways in postmortem spinal cord tissue of patients with ALS. A different differentiation protocol was used to derive a separate set of C9ORF72 and control motor neurons. Many individual -omics differed by protocol, but some core dysregulated pathways were consistent. This strategy of analyzing patient-specific neurons provides disease-related outcomes with small numbers of heterogeneous lines and reduces variation from single-omics to elucidate network-based signatures.

Huntington's disease mouse models: unraveling the pathology caused by CAG repeat expansion.

Huntington's disease (HD) is a neurodegenerative disease that results in motor and cognitive dysfunction, leading to early death. HD is caused by an expansion of CAG repeats in the huntingtin gene (HTT). Here, we review the mouse models of HD. They have been used extensively to better understand the molecular and cellular basis of disease pathogenesis as well as to provide non-human subjects to test the efficacy of potential therapeutics. The first and best-studied in vivo rodent model of HD is the R6/2 mouse, in which a transgene containing the promoter and exon 1 fragment of human HTT with 150 CAG repeats was inserted into the mouse genome. R6/2 mice express rapid, robust behavioral pathologies and display a number of degenerative abnormalities in neuronal populations most vulnerable in HD. The first conditional full-length mutant huntingtin (mHTT) mouse model of HD was the bacterial artificial chromosome (BAC) transgenic mouse model of HD (BACHD), which expresses human full-length mHTT with a mixture of 97 CAG-CAA repeats under the control of endogenous HTT regulatory machinery. It has been useful in identifying the role of mHTT in specific neuronal populations in degenerative processes. In the knock-in (KI) model of HD, the expanded human CAG repeats and human exon 1 are inserted into the mouse Htt locus, so a chimera of the full-length mouse protein with the N-terminal human portion is expressed. Many of aspects of the pathology and behavioral deficits in the KI model better mimic disease characteristics found in HD patients than other models. Accordingly, some have proposed that these mice may be preferable models of the disease over others. Indeed, as our understanding of HD advances, so will the design of animal models to test and develop HD therapies.

The mifepristone REMS: A needless and unlawful barrier to care✰.

Mifepristone (brand name Mifeprex) is a prescription drug that has been safely used in the United States for twenty years to end early pregnancies and, more recently, to treat early miscarriages. Although the U.S. Food and Drug Administration (FDA) acknowledges that mifepristone's safety and efficacy are "well-established by both research and experience" [1], it imposes a Risk Evaluation and Mitigation Strategy ("REMS") that severely restricts where, how, and from whom this medication can be obtained. Most notably, FDA requires that mifepristone be dispensed at a hospital, clinic, or medical office-not by mail or through a retail or mail-order pharmacy-even though patients can receive all evaluation and counseling via telemedicine and can self-administer the mifepristone tablet, unsupervised, at the location of their choice. Of more than 20,000 FDA-approved drugs [2], mifepristone is the only one FDA requires patients to pick up in a clinical setting even though they do not have to take it under clinical supervision [3]. FDA's singular restrictions on mifepristone are not only clinically unjustified, but unlawful. On behalf of leading medical associations, individual physicians, and reproductive justice advocates, our organization, the American Civil Liberties Union, has taken FDA to court-twice-because the agency's restrictions on mifepristone do not satisfy the strict constraints Congress established for REMS programs and erect profound and unnecessary barriers to care in violation of the U.S. Constitution.

The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein.

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1). Endogenous UPF1 interacted with the ZIKV capsid protein in coimmunoprecipitation experiments, and capsid expression posttranscriptionally downregulated UPF1 protein levels, a process that we confirmed occurs during ZIKV infection. Cellular fractionation studies show that the ZIKV capsid protein specifically targets nuclear UPF1 for degradation via the proteasome. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures, consistent with a model in which NMD restricts ZIKV infection in the fetal brain. We propose that ZIKV, via the capsid protein, has evolved a strategy to lower UPF1 levels and dampen antiviral activities of NMD, which in turn contributes to neuropathology in vivoIMPORTANCE Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.

The Library of Integrated Network-Based Cellular Signatures NIH Program: System-Level Cataloging of Human Cells Response to Perturbations.

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations.

Identification of hepta-histidine as a candidate drug for Huntington's disease by in silico-in vitro- in vivo-integrated screens of chemical libraries.

We identified drug seeds for treating Huntington's disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.

Targeting TEAD/YAP-transcription-dependent necrosis, TRIAD, ameliorates Huntington's disease pathology.

Neuronal cell death in neurodegenerative diseases is not fully understood. Here we report that mutant huntingtin (Htt), a causative gene product of Huntington's diseases (HD) selectively induces a new form of necrotic cell death, in which endoplasmic reticulum (ER) enlarges and cell body asymmetrically balloons and finally ruptures. Pharmacological and genetic analyses revealed that the necrotic cell death is distinct from the RIP1/3 pathway-dependent necroptosis, but mediated by a functional deficiency of TEAD/YAP-dependent transcription. In addition, we revealed that a cell cycle regulator, Plk1, switches the balance between TEAD/YAP-dependent necrosis and p73/YAP-dependent apoptosis by shifting the interaction partner of YAP from TEAD to p73 through YAP phosphorylation at Thr77. In vivo ER imaging with two-photon microscopy detects similar ER enlargement, and viral vector-mediated delivery of YAP as well as chemical inhibitors of the Hippo pathway such as S1P recover the ER instability and necrosis in HD model mice. Intriguingly S1P completely stops the decline of motor function of HD model mice even after the onset of symptom. Collectively, we suggest approaches targeting the signalling pathway of TEAD/YAP-transcription-dependent necrosis (TRIAD) could lead to a therapeutic development against HD.

Sequence-Level Analysis of the Major European Huntington Disease Haplotype.

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

Modeling Huntington's disease with induced pluripotent stem cells.

Huntington's disease (HD) causes severe motor dysfunction, behavioral abnormalities, cognitive impairment and death. Investigations into its molecular pathology have primarily relied on murine tissues; however, the recent discovery of induced pluripotent stem cells (iPSCs) has opened new possibilities to model neurodegenerative disease using cells derived directly from patients, and therefore may provide a human-cell-based platform for unique insights into the pathogenesis of HD. Here, we will examine the practical implementation of iPSCs to study HD, such as approaches to differentiate embryonic stem cells (ESCs) or iPSCs into medium spiny neurons, the cell type most susceptible in HD. We will explore the HD-related phenotypes identified in iPSCs and ESCs and review how brain development and neurogenesis may actually be altered early, before the onset of HD symptoms, which could inform the search for drugs that delay disease onset. Finally, we will speculate on the exciting possibility that ESCs or iPSCs might be used as therapeutics to restore or replace dying neurons in HD brains.

High-throughput screening in primary neurons.

Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss the adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells.

Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation.

To navigate a complex and changing environment, an animal's sensory neurons must continually adapt to persistent cues while remaining responsive to novel stimuli. Long-term exposure to an inherently attractive odor causes Caenorhabditis elegans to ignore that odor, a process termed odor adaptation. Odor adaptation is likely to begin within the sensory neuron, because it requires factors that act within these cells at the time of odor exposure. The process by which an olfactory sensory neuron makes a decisive shift over time from a receptive state to a lasting unresponsive one remains obscure. In C. elegans, adaptation to odors sensed by the AWC pair of olfactory neurons requires the cGMP-dependent protein kinase EGL-4. Using a fully functional, GFP-tagged EGL-4, we show here that prolonged odor exposure sends EGL-4 into the nucleus of the stimulated AWC neuron. This odor-induced nuclear translocation correlates temporally with the stable dampening of chemotaxis that is indicative of long-term adaptation. Long-term adaptation requires cGMP binding residues as well as an active EGL-4 kinase. We show here that EGL-4 nuclear accumulation is both necessary and sufficient to induce long-lasting odor adaptation. After it is in the AWC nucleus, EGL-4 decreases the animal's responsiveness to AWC-sensed odors by acting downstream of the primary sensory transduction. Thus, the EGL-4 protein kinase acts as a sensor that integrates odor signaling over time, and its nuclear translocation is an instructive switch that allows the animal to ignore persistent odors.

A 3'UTR pumilio-binding element directs translational activation in olfactory sensory neurons.

Prolonged stimulation leads to specific and stable changes in an animal's behavior. In interneurons, this plasticity requires spatial and temporal control of neuronal protein synthesis. Whether such translational control occurs in sensory neurons is not known. Adaptation of the AWC olfactory sensory neurons of C. elegans requires the cGMP-dependent protein kinase EGL-4. Here, we show that the RNA-binding PUF protein FBF-1 is required in the adult AWC for adaptation. In the odor-adapted animal, it increases translation via binding to the egl-4 3' UTR. Further, the PUF protein may localize translation near the sensory cilia and cell body. Although the RNA-binding PUF proteins have been shown to promote plasticity in development by temporally and spatially repressing translation, this work reveals that in the adult nervous system, they can work in a different way to promote experience-dependent plasticity by activating translation in response to environmental stimulation.

DNA breaks promote genomic instability by impeding proper chromosome segregation.

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes. Previously, we showed that the ends of broken chromosomes remain associated. Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis. RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase. This association is partially dependent upon Rad50 and Rad52. After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome. When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time. Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus. CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation. Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments. The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.