Biology subjects

Brandon, N. J.

Publications and source records attributed to Brandon, N. J..

14 recordsLinked to original sources

bioRxiv2023

Estradiol regulates local synthesis of synaptic proteome via sex-specific mechanisms.

Estrogens, specifically 17{beta}-estradiol (estradiol), can modulate synaptic function by regulating the expression and localisation of synaptic proteins. However, the mechanisms underlying estradiols regulation of synaptic protein expression, and whether if they occur in a sex specific manner, is not well understood. In this study, using sex-specific hippocampal slice cultures and mixed-sex primary hippocampal neurons, we investigated whether local protein synthesis is required for estradiol- induced synaptic protein expression. Estradiol rapidly increased the rate of protein synthesis and the number of actively translating ribosomes along dendrites and near synapses in both male and female hippocampal neurons. Importantly, these effects occurred independently of gene transcription. Moreover, estradiol also increased the abundance of nascent proteins localised to synapses, independently of gene transcription. Specifically, estradiol increased the synaptic expression of GluN2B- containing N-methyl-D-aspartate receptors and PSD-95 in male and female hippocampus. Mechanistically, mTOR signalling was required for estradiol-induced increases in overall local protein synthesis only in male but not female hippocampus. Consistent with this, mTOR signalling mediated estradiol increases in GluN2B in male, but not female, hippocampus. Conversely, mTOR inhibition, blocked estradiol-induced increased PSD-95 expression in both male and female hippocampus. Collectively, these data suggest that the rapid modulation of local protein synthesis by estradiol is required for changes in the synaptic proteome in male and female hippocampus, and that the requirement of the mTOR signalling pathway in these effects occur in both a sex-specific and protein-dependent manner, with this signalling pathway have a greater role in male compared to female hippocampus.

neuroscience↗

bioRxiv2021

Opposing roles of p38α phosphorylation and arginine methylation in driving TDP-43 proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder typically characterized by insoluble inclusions of hyperphosphorylated TDP-43. The mechanisms underlying toxic TDP-43 accumulation are not understood. Persistent activation of p38 mitogen-activated protein kinase (MAPK) is implicated in ALS. However, it is unclear how p38 MAPK affects TDP-43 proteinopathy. Here, we demonstrate that inhibition of p38 MAPK reduces pathological TDP-43 phosphorylation, aggregation, cytoplasmic mislocalization, and neurotoxicity. We establish that p38 MAPK phosphorylates TDP-43 at pathological serine 409/410 (S409/S410) and serine 292 (S292), which reduces TDP-43 liquid-liquid phase separation (LLPS) but allows pathological TDP-43 aggregation. Moreover, we show that protein arginine methyltransferase 1 methylates TDP-43 at R293. Importantly, S292 phosphorylation reduces R293 methylation, and R293 methylation reduces S409/S410 phosphorylation. R293 methylation permits TDP-43 LLPS and reduces pathological TDP-43 aggregation. Thus, strategies to reduce p38-mediated TDP-43 phosphorylation and promote R293 methylation could have therapeutic utility for ALS and related TDP-43 proteinopathies.

molecular biology↗

bioRxiv2021

Phosphorylation-dependent control of Arc protein by synaptic plasticity regulator 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, 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 Arcs 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.

neuroscience↗

bioRxiv2021

Mapping origins of variation in neural trajectories of human pluripotent stem cells

Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. We identified differences in the early lineage emergence across hPSC lines that mapped on the antero-posterior axis of embryonic development. RNA-seq analysis revealed dynamic transcriptomic patterns that defined the emergence of mesendodermal versus neuroectodermal lineages conserved across hPSC lines and cell line-specific transcriptional signatures that were invariant across differentiation. The stable cell line-specific transcriptomic patterns predicted the retinoic acid (RA) response of the cell lines, resulting in distinct bias towards fore-versus hind-brain fates. Replicate hPSC lines and paired adult donor tissue demonstrated that cells from individual humans expressed unique and long-lasting transcriptomic signatures associated with evolutionarily recent genes. In addition to this genetic contribution, we found that replicate lines from a single donor showed divergent brain regional fates linked to distinct chromatin states, indicating that epigenetic mechanisms also contribute to neural fate differences. This variation in lineage bias and its correlation with RA responsive gene expression was also observed in a large collection of hPSC lines. These results define transcriptomic differences in hPSCs that initiate a critical early step specifying anterior or posterior neural fates.

neuroscience↗

bioRxiv2020

Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanism neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of Kcc2 from the plasma membrane of murine forebrain. We resolved these using Blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS. Purified Kcc2 migrated as distinct molecular species of 300, 600 and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N and C-termini of Kcc2 was obtained. The 300kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. Intriguingly, lower levels of Kcc1 were also found in this species suggesting the existence of "mixed" Kcc2/Kcc1 heterodimers. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed association of Kcc2. These included spectrins, ankyrins, and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5 and Lmtk3. Finally, we used LC-MS/MS on highly purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

neuroscience↗

bioRxiv2020

The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products.

KCC2 plays a critical role in determining the efficacy of synaptic inhibition and deficits in its activity lead to epilepsy and neurodevelopmental delay. Here we use unbiased proteomic analyses to demonstrate that KCC2 forms stable protein complexes in the neuronal plasma membrane with 96 autism and/or epilepsy risk gene (ASD/Epi) products including ANKB, ANKG, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1, and SPTBN1. Many of these proteins are also targets of Fragile-X mental retardation protein (FMRP), the inactivation of which is the leading monogenic cause of autism. Accordingly, the expression of a subset of these KCC2-binding partners was decreased in Fmr1 knockout mice. Fmr1 knockout compromised KCC2 phosphorylation, a key regulatory mechanism for transporter activity and the postnatal development of GABAergic inhibition. Thus, KCC2 is a point of convergence for multiple ASD/Epi risk genes and therapies targeting this transporter may have broad utility in alleviating these heterogeneous disorders and their associated epilepsies.

neuroscience↗

bioRxiv2019

Human cortical neural stem cells generate regional organizer states in vitro before committing to excitatory neuronal fates

Better understanding the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here we used RNA-sequencing, cell imaging and lineage tracing of mouse and human in vitro NSCs to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs developed from pluripotency in vitro, they first transitioned through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines varied in these early patterning states leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analysis of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.

neuroscience↗

bioRxiv2018

The cortico-striatal circuit regulates sensorimotor gating via Disc1/Huntingtin-mediated Bdnf transport

Sensorimotor information processing that underlies normal cognitive and behavioral traits is dysregulated across a subset of neurological and psychiatric disorders. The cross-disease deficit in sensorimotor gating poses a unique opportunity to integrate hierarchical findings at molecular, cellular, through circuitry levels to obtain an in-depth mechanistic understanding of this process that contributes to brain physiology and pathophysiology beyond categorical segmentation of brain disorders. Based on circuitry recording with wild-type mice, we demonstrated that the cortico-striatal projection mediates sensorimotor gating responses during prepulse inhibition (PPI) task. We also found that these circuitry responses were disrupted in Disc1 locus-impairment (LI) mice, a model representing neuropsychiatric conditions. Thus, we hypothesized that Disc1-mediated molecular and cellular machinery along the cortico-striatal circuit may regulate sensorimotor gating. Anatomical and biochemical analyses of Disc1-LI mice revealed attenuated Bdnf transport along the cortico-striatal circuit. Pharmacologically augmenting Bdnf transport by chronic lithium administration, in part via Ser-421 phosphorylation of Huntingtin (Htt) and its integration into the motor machinery, restored the striatal Bdnf levels and PPI deficits in Disc1-LI mice, suggesting that the Bdnf transport attenuation mechanistically underlies the circuitry and behavioral deficits. These results also shed light on a novel mechanism and utility of lithium that is currently used as a major mood stabilizer in clinical settings. Collectively, the present study illustrates integrative biological mechanisms for sensorimotor gating, underscoring the cross-disease nature of this behavioral dimension and translational utility of the findings under the era of precision medicine in brain disorders.

neuroscience↗

bioRxiv2018

L-type voltage-gated calcium channel regulation of in vitro human cortical neuronal networks

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here, we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days, inhibitory GABergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that, although basal neuronal firing rate is unaffected, there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening.

neuroscience↗

bioRxiv2018

Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods

High-throughput sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of the biases associated with small RNA sequencing have focused on adapter ligation bias with limited evaluation of reverse transcription or amplification biases. Furthermore, evaluations of the accuracy of quantifications of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited and no study has yet evaluated differences in the quantification of isomiRs of altered length. In addition, no studies have yet compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

genomics↗

bioRxiv2018

Estradiol reverses excitatory synapse loss in a cellular model of neuropsychiatric disorders

Loss of glutamatergic synapses is thought to be a key cellular pathology associated with neuropsychiatric disorders including schizophrenia (SCZ) and major depressive disorder (MDD). Genetic and cellular studies of SCZ and MDD using in vivo and in vitro systems have supported a key role for dysfunction of excitatory synapses in the pathophysiology of these disorders. Recent clinical studies have demonstrated that the estrogen, 17{beta}-estradiol can ameliorate many of the symptoms experienced by patients. Yet, to date, our understanding of how 17{beta}-estradiol exerted these beneficial effects is limited. In this study, we have tested the hypothesis that 17{beta}-estradiol can restore dendritic spine number in a cellular model that recapitulates the loss of synapses associated with SCZ and MDD. Ectopic expression of wildtype, mutant or shRNA-mediated knockdown of Disrupted in Schizophrenia (DISC1) reduced dendritic spine density in primary cortical neurons. Acute or chronic treatment with 17{beta}-estradiol increased spine density to control levels in neurons with altered DISC1 levels. In addition, 17{beta}-estradiol reduced the extent to which ectopic wildtype and mutant DISC1 aggregated. Furthermore, 17{beta}-estradiol also caused the enrichment of synaptic proteins at synapses and increased the number of dendritic spines containing PSD-95 or that overlapped with the pre-synaptic marker bassoon. Taken together, our data indicates that estrogens can restore lost excitatory synapses caused by altered DISC1 expression, potentially through the trafficking of DISC1 and its interacting partners. These data highlight the possibility that estrogens exert their beneficial effects in SCZ and MDD in part by modulating dendritic spine number.

neuroscience↗

bioRxiv2018

Miro ubiquitination is critical for efficient damage-induced PINK1/Parkin-mediated mitophagy

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1 / Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Combined knockout of both Miro1 and Miro2 or block of Miro ubiquitination and subsequent degradation, lead to slowed mitophagy. In cultured neurons, Miro1 knockout also leads to delayed Parkin translocation onto damaged mitochondria and reduced mitochondrial clearance. In vivo, postnatal knockout of Miro1 in hippocampus and cortex disrupts mitophagy and leads to a dramatic age dependent upregulation of the mitofusin mitochondrial fusion machinery. Fluorescence imaging of aged neurons conditionally knocked out for Miro1 and expressing mitoDendra to label mitochondria in vivo, reveals that Mfn1 / Mfn2 upregulation leads to enlarged and hyperfused somatic mitochondria. Our results provide new insights into the role of Miro in PINK1/Parkin dependent mitophagy and further suggest that disruption of this regulation may be implicated in human neurological pathology.

neuroscience↗

bioRxiv2018

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across nine time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells that were validated using electrophysiology. We identified widespread changes in the expression of individual transcript features and their splice variants, gene networks, and global patterns of transcription. We next demonstrated that co-culturing human NPCs with rodent astrocytes resulted in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without potentially disruptive cell sorting. We lastly developed and validated a computational tool to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue, which were maturationally heterogeneous but contained subsets of cells most akin to adult human neurons.

genomics↗

bioRxiv2017

Developmental And Genetic Regulation Of The Human Cortex Transcriptome In Schizophrenia

GWAS have identified 108 loci that confer risk for schizophrenia, but risk mechanisms for individual loci are largely unknown. Using developmental, genetic, and illness-based RNA sequencing expression analysis, we characterized the human brain transcriptome around these loci and found enrichment for developmentally regulated genes with novel examples of shifting isoform usage across pre- and post-natal life. We found widespread expression quantitative trait loci (eQTLs), including many with transcript specificity and previously unannotated sequence that were independently replicated. We leveraged this eQTL database to show that 48.1% of risk variants for schizophrenia associated with nearby expression. Within patients and controls, we implemented a novel algorithm for RNA quality adjustment, and identified 237 genes significantly associated with diagnosis that replicated in an independent case-control dataset. These genes implicated synaptic processes and were strongly regulated in early development (p < 10-20). These data offer new targets for modeling schizophrenia risk in cellular systems.

neuroscience↗