Biology subjects

Brennand, K.

Publications and source records attributed to Brennand, K..

17 recordsLinked to original sources

bioRxiv2026

Spared corticospinal neurons activate an endogenous plasticity program after partial CNS injury

Functional recovery after incomplete spinal cord injury depends substantially on the capacity of anatomically spared neurons to remodel their connections, yet the molecular programs underlying this endogenous plasticity remain poorly understood. To identify and validate these mechanisms, we combined retrograde labeling, unilateral corticospinal tract injury, spatial transcriptomics, and human stem cell-derived neurons. Injured corticospinal neurons exhibited widespread downregulation with remaining activated pathways dominated by stress, cell death, and degenerative programs. In contrast, spared corticospinal neurons activated a coordinated pro-plasticity program characterized by metabolic, immune, and cytoskeletal remodeling together with selective suppression of growth-inhibitory signaling. Network and drug perturbation analyses identified ARHGEF12 suppression and vorinostat treatment as complementary target- and state-based strategies to enhance neurite regeneration in human neurons. Together, these findings define key components of endogenous plasticity and provide a framework for discovering therapeutic targets for neural repair.

neuroscience↗

bioRxiv2026

Reverse genetics in humanized mice reveals CARD8-mediated pyroptosis causing pancytopenia in human DPP9 deficiency

Loss of function mutation in the human DPP9 gene causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia and patients require bone marrow transplantation, but the mechanism of cell loss is unclear since Dpp9 mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to little transcriptional changes suggesting post-transcriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to the activation of the CARD8 inflammasome resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.

immunology↗

bioRxiv2025

BRD1 haploinsufficiency alters early neuronal programming and disrupts maturation in human induced glutamatergic neurons

BRD1 is an epigenetic regulator implicated in neurodevelopmental and psychiatric disorders, yet its role in human neuronal differentiation, maturation, and function remains poorly understood. Here we show that BRD1 haploinsufficiency disrupts early neuronal programming, resulting in accelerated maturation and altered neurodevelopmental trajectories in human induced glutamatergic neurons. Transcriptomic profiling reveals an early shift toward neuronal identity, characterized by downregulation of pluripotency markers and persistent upregulation of genes involved in synapse assembly and organization, including GRIA3. Despite this, BRD1+/- neurons form significantly smaller synapses and display increased neuronal activity. Our findings highlight BRD1 as a key regulator of neurodevelopmental timing and synaptic maturation, and network activity reinforcing growing evidence that disruptions in chromatin-mediated control of differentiation and synaptic organization contribute to neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Mechanical stretch disrupts calcium dynamics and redistributes Piezo1 in human astrocytes

Astrocytes regulate the activity of nearby neurons so disruption of astrocyte calcium dynamics by traumatic brain injury (TBI) could have profound consequences for neural network activity in the brain. In this study, human induced pluripotent stem cell (hiPSC)-derived astrocytes were used in a two-dimensional (2D) in vitro stretch injury model to evaluate the effect of trauma on calcium dynamics, mitochondrial function, and the mechanosensitive ion channel Piezo1. Outcomes were assessed using live imaging, immunostaining, and RNA sequencing. Cell viability, mitochondrial membrane potential, and spontaneous calcium transients declined as injury severity increased. At moderate injury severity, the decreases in mitochondrial membrane potential and calcium dynamics were temporary. The spatial distribution of Piezo1 also changed temporarily after injury. RNA sequencing identified 196 genes that changed expression after injury, including downregulation of mitochondrial and oxidative metabolic processes and upregulation of cortical thinning pathways. These findings establish this model as a platform for investigating the cellular mechanisms of TBI and its influence on neurodegeneration.

neuroscience↗

bioRxiv2024

Decoding the transcriptomic signatures of psychological trauma in human cortex and amygdala

Psychological trauma has profound effects on brain function and precipitates psychiatric disorders in vulnerable individuals, however, the molecular mechanisms linking trauma with psychiatric risk remain incompletely understood. Using RNA-seq data postmortem brain tissue of a cohort of 304 donors (N=136 with trauma exposure), we investigated transcriptional signatures of trauma exposures in two cortical regions (dorsolateral prefrontal cortex, and dorsal anterior cingulate cortex) and two amygdala regions (medial amygdala and basolateral amygdala) associated with stress processing and regulation. We focused on dissecting heterogeneity of traumatic experiences in these transcriptional signatures by investigating exposure to several trauma types (childhood, adulthood, complex, single acute, combat, and interpersonal traumas) and interactions with sex. Overall, amygdala regions were more vulnerable to childhood traumas, whereas cortical regions were more vulnerable to adulthood trauma (regardless of childhood experience). Using cell-type-specific expression imputation, we identified a strong transcriptional response of medial amygdala excitatory neurons to childhood trauma, which coincided with dysregulation observed in a human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons exposed to hydrocortisone. We resolved multiscale coexpression networks for each brain region and identified modules enriched in trauma signatures and whose connectivity was altered with trauma. Trauma-associated coexpression modules provide insight into coordinated functional dysregulation with different traumas and point to potential gene targets for further dissection. Together, these data provide a characterization of the long-lasting human encoding of traumatic experiences in corticolimbic regions of human brain.

neuroscience↗

bioRxiv2024

Dynamic convergence of autism disorder risk genes across neurodevelopment

Over three hundred and seventy-three risk genes, broadly enriched for roles in neuronal communication and gene expression regulation, underlie risk for autism spectrum disorder (ASD) and developmental delay (DD). Functional genomic studies of subsets of these genes consistently indicate a convergent role in neurogenesis, but how these diverse risk genes converge on a smaller number of biological pathways in mature neurons is unclear. To uncover shared downstream impacts between neurodevelopmental disorder (NDD) risk genes, here we apply a pooled CRISPR approach to contrast the transcriptomic impacts of targeting 29 NDD loss-of-function genes across human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Points of convergence vary between the cell types of the brain and are greatest in mature glutamatergic neurons, where they broadly target not just synaptic and epigenetic, but unexpectedly, mitochondrial biology. The strongest convergent networks occur between NDD genes with common co-expression patterns in the post-mortem brain, biological annotations, and clinical associations, suggesting that convergence may one-day inform patient stratification and treatment. Towards this, ten out of eleven drugs tested that were predicted to reverse convergent signatures in human cells and/or arousal and sensory processing behaviors in zebrafish ameliorated at least one behavioral phenotype in vivo. Altogether, robust convergence in post-mitotic neurons represents a clinically actionable therapeutic window.

genetics↗

bioRxiv2024

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

More than 60 human disorders have been linked to unstable expansion of short tandem repeat (STR) tracts. STR length and the extent of DNA methylation is linked to disease pathology and can be mosaic in a cell type-specific manner in several repeat expansion disorders. Mosaic phenomenon have been difficult to study to date due to technical bias intrinsic to repeat sequences and the need for multi-modal measurements at single-allele resolution. Nanopore long-read sequencing accurately measures STR length and DNA methylation in the same single molecule but is cost prohibitive for studies assessing a target locus across multiple experimental conditions or patient samples. Here, we describe MASTR-seq, Multiplexed Analysis of Short Tandem Repeats, for cost-effective, high-throughput, accurate, multi-modal measurements of DNA methylation and STR genotype at single-allele resolution. MASTR-seq couples long-read sequencing, Cas9-mediated target enrichment, and PCR-free multiplexed barcoding to achieve a >ten-fold increase in on-target read mapping for 8-12 pooled samples in a single MinION flow cell. We provide a detailed experimental protocol and computational tools and present evidence that MASTR-seq quantifies tract length and DNA methylation status for CGG and CAG STR loci in normal-length and mutation-length human cell lines. The MASTR-seq protocol takes approximately eight days for experiments and one additional day for data processing and analyses. Key pointsO_LIWe provide a protocol for MASTR-seq: Multiplexed Analysis of Short Tandem Repeats using Cas9-mediated target enrichment and PCR-free, multiplexed nanopore sequencing. C_LIO_LIMASTR-seq achieves a >10-fold increase in on-target read proportion for highly repetitive, technically inaccessible regions of the genome relevant for human health and disease. C_LIO_LIMASTR-seq allows for high-throughput, efficient, accurate, and cost-effective measurement of STR length and DNA methylation in the same single allele for up to 8-12 samples in parallel in one Nanopore MinION flow cell. C_LI

genomics↗

bioRxiv2023

Common genetic variation impacts molecular stress response in the brain

To explain why individuals exposed to identical stressors experience divergent clinical outcomes, we determine how molecular encoding of stress modifies genetic risk for brain disorders. Analysis of post-mortem brain (n=304) revealed 8557 stress-interactive expression quantitative trait loci (eQTLs) that dysregulate expression of 915 eGenes in response to stress, and lie in stress-related transcription factor binding sites. Response to stress is robust across experimental paradigms: up to 50% of stress-interactive eGenes validate in glucocorticoid treated hiPSC-derived neurons (n=39 donors). Stress-interactive eGenes show brain region- and cell type-specificity, and, in post-mortem brain, implicate glial and endothelial mechanisms. Stress dysregulates long-term expression of disorder risk genes in a genotype-dependent manner; stress-interactive transcriptomic imputation uncovered 139 novel genes conferring brain disorder risk only in the context of traumatic stress. Molecular stress-encoding explains individualized responses to traumatic stress; incorporating trauma into genomic studies of brain disorders is likely to improve diagnosis, prognosis, and drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/573459v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1e07fa9org.highwire.dtl.DTLVardef@1dd530forg.highwire.dtl.DTLVardef@17822a5org.highwire.dtl.DTLVardef@2a889f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Precise Therapeutic Targeting of Distinct NRXN1+/- Mutations

Given the large number of genes significantly associated with risk for neuropsychiatric disorders, a critical unanswered question is the extent to which diverse mutations --sometimes impacting the same gene--will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1, a pre-synaptic cell adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain, and are differentially impacted by unique (non-recurrent) deletions. We contrast the cell-type-specific impact of patient-specific mutations in NRXN1 using human induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Via distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1+/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Reciprocal isogenic manipulations causally demonstrate that aberrant splicing drives these changes in synaptic activity. For NRXN1 deletions, and perhaps more broadly, precision medicine will require stratifying patients based on whether their gene mutations act through LOF or GOF mechanisms, in order to achieve individualized restoration of NRXN1 isoform repertoires by increasing wildtype, or ablating mutant isoforms. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disorders, our findings add nuance to future considerations of precision medicine.

neuroscience↗

bioRxiv2023

Schizophrenia Risk Mapping and Functional Engineering of the 3D Genome in Three Neuronal Subtypes

Common variants associated with schizophrenia are concentrated in non-coding regulatory sequences, but their precise target genes are context-dependent and impacted by cell-type-specific three-dimensional spatial chromatin organization. Here, we map long-range chromosomal conformations in isogenic human dopaminergic, GABAergic, and glutamatergic neurons to track developmentally programmed shifts in the regulatory activity of schizophrenia risk loci. Massive repressive compartmentalization, concomitant with the emergence of hundreds of neuron-specific multi-valent chromatin architectural stripes, occurs during neuronal differentiation, with genes interconnected to genetic risk loci through these long-range chromatin structures differing in their biological roles from genes more proximal to sequences conferring heritable risk. Chemically induced CRISPR-guided chromosomal loop-engineering for the proximal risk gene SNAP91 and distal risk gene BHLHE22 profoundly alters synaptic development and functional activity. Our findings highlight the large-scale cell-type-specific reorganization of chromosomal conformations at schizophrenia risk loci during neurodevelopment and establish a causal link between risk-associated gene-regulatory loop structures and neuronal function.

genomics↗

bioRxiv2022

Spatial proteomics and iPSC modeling uncover mechanisms of axonal pathology in Alzheimer's disease

Axonal spheroids (dystrophic neurites) are commonly found around amyloid deposits in Alzheimers disease (AD). They impair electrical conduction, disrupt neural circuits, and correlate with AD severity. Despite their significance, the mechanisms underlying spheroid formation remain unknown. To address this, we developed a proximity labeling proteomics approach to uncover the proteome of spheroids in human postmortem and mouse brains. Additionally, we established a human iPSC-derived AD model allowing mechanistic investigation of spheroid pathology and optical electrophysiology. This approach revealed the subcellular molecular architecture of spheroids and identified abnormalities in key biological processes, including protein turnover, cytoskeleton dynamics, and lipid transport. Notably, the PI3K/AKT/mTOR pathway, which regulates these processes, was activated within spheroids. Furthermore, phosphorylated mTOR levels in spheroids strongly correlated with AD severity in humans. Importantly, inhibition of mTOR in iPSC-derived neurons and in mice ameliorated spheroid pathology. Altogether, our study provides a multidisciplinary toolkit for investigating mechanisms and novel targets for axonal pathology in neurodegeneration.

neuroscience↗

bioRxiv2020

Fitness selection of hyperfusogenic measles virus F proteins associated with neuropathogenic phenotypes

Measles virus (MeV) is resurgent and caused >200,000 deaths in 2019. MeV infection can establish a chronic latent infection of the brain that can recrudesce months to years after recovery from the primary infection. Recrudescent MeV leads to fatal subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MIBE) as the virus spreads across multiple brain regions. Most clinical isolates of SSPE/MIBE strains show mutations in the fusion (F) gene that result in a hyperfusogenic phenotype in vitro and allow for efficient spread in primary human neurons. Wild-type MeV receptor binding protein (RBP) is indispensable for manifesting these mutant F phenotypes, even though neurons lack canonical MeV receptors (CD150/SLAMF1 or Nectin-4). How such hyperfusogenic F mutants are selected for, and whether they confer a fitness advantage for efficient neuronal spread is unresolved. To better understand the fitness landscape that allows for the selection of such hyperfusogenic F mutants, we conducted a screen of [≥]3.1x105 MeV-F point mutants in their genomic context. We rescued and amplified our genomic MeV-F mutant libraries in BSR-T7 cells under conditions where MeV-F-T461I (a known SSPE mutant), but not wild-type MeV can spread. We recovered known SSPE mutants but also characterized at least 15 novel hyperfusogenic F mutations with a SSPE phenotype. Structural mapping of these mutants onto the pre-fusion MeV-F trimer confirm and extend our understanding of the fusion regulatory domains in MeV-F. Our list of hyperfusogenic F mutants is a valuable resource for future studies into MeV neuropathogenesis and the regulation of paramyxovirus fusion. SignificanceMeasles remains a major cause of infant death globally. On rare occasions, measles virus infection of the central nervous system (CNS) leads to a fatal progressive inflammation of the brain many years after the initial infection. MeV isolates from such CNS infections harbor fusion (F) protein mutations that result in a hyperfusogenic phenotype. The small number of hyperfusogenic MeV-F mutants identified thus far limits our ability to understand how these mutations are selected in the context of CNS infections. We performed a saturating mutagenesis screen of MeV-F to identify a large set of mutants that would mimic the hyperfusogenic phenotype of MeV-F in CNS infection. Characterization of these mutants shed light on other paramyxoviruses known to establish chronic CNS infections.

microbiology↗

bioRxiv2020

Cell-type specific gene expression profiling in heterogeneous in vitro cultures using epitope-tagged RPL22

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease, but also challenge our ability to resolve cell-type specific perturbations. Here we report an extension of the RiboTag system, first developed to achieve cell-type restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables efficient depletion of off-target RNA in mixed species primary co-cultures and in hiPSC-derived neural progenitor cells, motor neurons, and GABAergic neurons. Nonetheless, depletion efficiency varies across independent experimental replicates. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

molecular biology↗

bioRxiv2019

Transcriptional signatures of participant-derived neural progenitor cells and neurons implicate altered Wnt signaling in Phelan McDermid syndrome and autism

BackgroundPhelan-McDermid syndrome (PMS) is a rare genetic disorder with high risk of autism spectrum disorder (ASD), intellectual disability and language delay, and is caused by 22q13.3 deletions or mutations in the SHANK3 gene. To date, the molecular and pathway changes resulting from SHANK3 haploinsufficiency in PMS remain poorly understood. Uncovering these mechanisms is critical for understanding pathobiology of PMS and, ultimately, for the development of new therapeutic interventions. MethodsWe developed human induced pluripotent stem cell (hiPSC)-based models of PMS by reprogramming peripheral blood samples from individuals with PMS (n=7) and their unaffected siblings (n=6). For each participant, up to three hiPSC clones were generated and differentiated into induced neural progenitor cells (iNPCs; n=32) and induced forebrain neurons (iNeurons; n=42). Genome-wide RNA-sequencing was applied to explore transcriptional differences between PMS probands and unaffected siblings. ResultsTranscriptome analyses identified 391 differentially expressed genes (DEGs) in iNPCs and 82 DEGs in iNeurons, when comparing cells from PMS probands and unaffected siblings (FDR <5%). Genes under-expressed in PMS were implicated in Wnt signaling, embryonic development and protein translation, while over-expressed genes were enriched for pre- and post-synaptic density genes, regulation of synaptic plasticity, and G-protein-gated potassium channel activity. Gene co-expression network analysis identified two modules in iNeurons that were over-expressed in PMS, implicating postsynaptic signaling and GDP binding, and both modules harbored a significant enrichment of genetic risk loci for developmental delay and intellectual disability. Finally, PMS-associated genes were integrated with other ASD iPSC transcriptome findings and several points of convergence were identified, indicating altered Wnt signaling, extracellular matrix and glutamatergic synapses. LimitationsGiven the rarity of the condition, we could not carry out experimental validation in independent biological samples. In addition, functional and morphological phenotypes caused by loss of SHANK3 were not characterized here. ConclusionsThis is the largest human neural sample analyzed in PMS. Genome-wide RNA-sequencing in hiPSC-derived neural cells from individuals with PMS revealed both shared and distinct transcriptional signatures across iNPCs and iNeurons, including many genes implicated in risk for ASD, as well as specific neurobiological pathways, including the Wnt pathway.

neuroscience↗

bioRxiv2019

Molecular Networks and Key Regulators of the Dysregulated Neuronal System in Alzheimer’s Disease

To study the molecular mechanisms driving the pathogenesis and identify novel therapeutic targets of late onset Alzheimers Disease (LOAD), we performed an integrative network analysis of whole-genome DNA and RNA sequencing profiling of four cortical areas, including the parahippocampal gyrus, across 364 donors spanning the full spectrum of LOAD-related cognitive and neuropathological disease severities. Our analyses revealed thousands of molecular changes and uncovered for the first-time multiple neuron specific gene subnetworks most dysregulated in LOAD. ATP6V1A, a critical subunit of vacuolar-type H+-ATPase (v-ATPase), was predicted to be a key regulator of one neuronal subnetwork and its role in disease-related processes was evaluated through CRISPR-based manipulation of human induced pluripotent stem cell derived neurons and RNAi-based knockdown in transgenic Drosophila models. This study advances our understanding of LOAD pathogenesis by providing the global landscape and detailed circuits of complex molecular interactions and regulations in several key brain regions affected by LOAD and the resulting network models provide a blueprint for developing next generation therapeutics against LOAD.

neuroscience↗

bioRxiv2019

Functional annotation of rare structural variation in the human brain

Structural variants (SVs) contribute substantially to risk of many brain related disorders including autism and schizophrenia. However, annotating the potential contribution of SVs to disease remains a major challenge. Here, we integrated high resolution SV calling from genome-sequencing in 755 human post-mortem brains with dorsal lateral prefrontal cortex RNA-sequencing from a subset of 629 samples to quantify the dosage and regulatory effects of SVs. We show that genic (p = 5.44x10-9) and regulatory SVs (enhancer p = 3.22x10-23, CTCF p = 3.86x10-18) are present at significantly lower frequencies than intergenic SVs after correcting for SV length. Copy number variants (CNVs)--deletions and duplications--exhibit a significant quantitative and directional relationship between the proportion of genic and regulatory content altered and gene expression, and the size of the effect is inversely correlated with the loss-of-function intolerance of the gene. We trained a joint linear model that leverages genic and regulatory annotations to predict expression effects of rare CNVs in independent samples (R2 = 0.21-0.41). We further developed a regulatory disruption score for each CNV that aggregates the predicted expression across all affected genes weighted by the genes intolerance score and applied it to an independent set of SVs from 14,891 genome-sequenced individuals. Pathogenic deletions implicated in neurodevelopmental disorders by ClinGen had significantly more extreme regulatory disruption scores than the rest of the SVs. Rank ordering based on the most extreme regulatory disruption scores prioritized pathogenic deletions that would not have been prioritized by frequency or length alone. This work points to the deleteriousness of regulatory SVs, particularly those altering CTCF sites. We further provide a simple approach for functionally annotating the regulatory effects of SVs in the human brain that has potential to be useful in larger SV studies and should improve as more regulatory annotation data is generated.

genomics↗

bioRxiv2017

An Efficient Platform For Astrocyte Differentiation From Human Induced Pluripotent Stem Cells

Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. Using this method, we generated hiPSC-derived astrocyte populations (hiPSC-astrocytes) from 42 NPC lines (derived from 30 individuals) with an average of [~]90% S100{beta}-positive cells. Transcriptomic analysis demonstrated that the hiPSC-astrocytes are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our novel protocol is a reproducible, straightforward (single media) and rapid (<30 days) method to generate homogenous populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.\n\nABBREVIATIONS

neuroscience↗