Biology subjects

Ross, M. E.

Publications and source records attributed to Ross, M. E..

13 recordsLinked to original sources

bioRxiv2025

Mitochondrial dysfunction precedes neurodegeneration in DRPLA patient-derived neurons, and phenylbutyrate improves survival.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion in the ATN1 gene, which encodes the transcriptional corepressor Atrophin-1. The temporal sequence of molecular mechanisms driving neuronal dysfunction and degeneration in DRPLA is poorly understood, limiting therapeutic development. We generated patient-derived induced pluripotent stem cells and differentiated them into cortical excitatory glutamatergic neurons to model early pathogenic processes. Early pathological features of DRPLA patient-derived neurons included mitochondrial dysfunction and oxidative stress. These alterations occurred before overt neuronal loss, highlighting bioenergetic stress as a key early driver of disease progression toward neurodegeneration. Pharmacological treatment with phenylbutyrate significantly improved neuronal survival and reduced mitochondrial reactive oxygen species production, demonstrating the therapeutic potential of targeting mitochondrial dysfunction and oxidative stress. These findings challenge the conventional aggregation-centric model of polyglutamine disease pathogenesis and position mitochondrial stress as a central and early promoter of neuronal degeneration in DRPLA. By providing mechanistic insight into early-stage disease processes, our study lays the foundation for therapeutic strategies targeting mitochondrial dysfunction in DRPLA and related polyglutamine disorders.

neuroscience↗

bioRxiv2025

Occludin Acts as a Dynein Adaptor Regulating Permeability and Collateral Angiogenesis

Previous studies of the tight junction protein occludin (OCLN) suggest that multiple phosphorylation sites on the carboxy-terminal domain contribute a regulatory role in vascular barrier properties. However, gene deletion studies failed to identify a clear functional role for OCLN, despite multiple phenotypic alterations. Importantly, previous studies targeting exon 3 allowed expression of a splice variant starting at exon 4, (isoform 4) that expresses the full carboxy-terminal tail. Here we show that the OCLN carboxy-terminus forms a complex with the light intermediate chain (LIC) of dynein to link tight junction cargo to the minus end directed motor protein. Mutational analysis revealed S471 phosphorylation is required for binding to the LIC while S490 phosphorylation is required for trafficking. Expressing OCLN S490A mutant prevented endothelial cell proliferation and collateral angiogenesis. OCLN gene deletion targeting exon 5, preventing full length and isoform 4 expression, resulted in embryonic lethality. In summary, OCLN links tight junction cargo to the dynein motor, regulating trafficking in a phosphorylation dependent manner and contributing to both VEGF-induced vascular permeability and collateral angiogenesis.

cell biology↗

bioRxiv2025

A spatial long-read approach at near-single-cell resolution reveals developmental regulation of splicing and polyadenylation sites in distinct cortical layers and cell types.

Genome-wide single-cell and spatial long-read approaches have gained traction, but mostly lack single-cell resolution - and yield limited read lengths. Here, we introduce spatial ISOform sequencing (Spl-ISO-Seq), which reveals exons and polyadenylation sites from long reads with near-single-cell resolution. Spl-ISO-Seq selects long cDNAs and doubles to triples read lengths compared to standard preparations. Adding a highly specific software tool (Spl-ISOquant) and comparing human post-mortem pre-puberty samples of the visual cortex (8-11 years) to post-puberty samples (16-19 years), we find that cortical layers harbor stronger splicing and poly(A)-site regulation than the adjacent white matter, with enrichment of multiple protein-domain types. For oligodendrocytes however, developmental splicing changes are stronger in white matter. Among cortical layers, layer 4 has the most developmental changes in alternative-exon inclusion in excitatory neurons and in poly(A) sites. We also find many repeat elements, especially ERV1 long terminal repeats downstream of developmentally-regulated layer 4 exons. Overall, alternative splicing changes are linked to synapses - specifically at the post-synapse. Age-linked splicing changes in layers 1-3 and 4 are associated with autism spectrum disorder but not with schizophrenia, amyotrophic lateral sclerosis and Alzheimers disease. These results root developmental splicing changes during puberty and the resulting protein changes in specific layers and cell types. More generally, our new technologies enable new observations for any complex tissue.

genomics↗

bioRxiv2025

Cryopreservation of Human Cortical Organoids Using Vitrification.

Cryopreservation at ultra-low temperatures is a valuable tool for preserving cells and tissues used in research. However, few protocols exist for the preservation of brain organoid models. Current methods for preserving human cortical organoids (hCOs) rely on conventional slow cooling approaches with organoids suspended in a medium containing a cocktail of cryoprotectants. In contrast, we have optimized a vitrification technique previously used to cryopreserve human embryos and oocytes for application to hCOs. We have successfully cryopreserved hCOs that were generated by two different protocols. The vitrified organoids demonstrate a growth rate, cytoarchitecture, cell type composition and electrical activity comparable to non-vitrified controls. Our hCO cryopreservation method provides a useful alternative approach for bio-banking and cross-institutional collaboration using cortical organoids as their model system. Highlights- Human pluripotent stem cell (hPSC) derived hCOs can be cryopreserved using vitrification. - Vitrified hCOs develop similarly to non-vitrified controls. - Vitrified hCOs can be stored long-term and shipped to other institutions. MotivationMany methods of cryopreservation have been developed for the maintenance of cell lines, organoid models and tissues. Most organoid models have been successfully preserved using conventional slow-cooling methods; however, human cortical organoids have been difficult to preserve. Our findings demonstrate that the vitrification of cortical organoids preserves their structure, cell type diversity and function upon rewarming and continued culture.

neuroscience↗

bioRxiv2023

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

genetics↗

bioRxiv2022

Foxp1 acts upstream of Vegfa, suppresses cortical angiogenesis, and promotes hypoxia in radial glia.

Radial glia progenitors within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions at later times, yet the mechanisms controlling this transition remain unclear. Previous work has shown that the autism-linked transcription factor Foxp1 is endogenously expressed by early but not late radial glia, and both loss and gain of Foxp1 can alter their neural progenitor activities and fate choices. Here, we show that premature loss of Foxp1 leads to an increase in transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with an elevation in Vegfa expression in radial glia and precocious vascular network development. Thus, the endogenous decline in Foxp1 expression appears to orchestrate changes in the tissue environment adjacent to radial glia that influence their metabolic state which in turn can alter their self-renewal and neurogenic capacities.

developmental biology↗

bioRxiv2022

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. We focus here on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ has the competence to generate predominantly ipsilaterally-projecting RGCs, but this competence is altered in the albino visual system due to CyclinD2 downregulation and disturbed temporal control of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit a diminished ipsilateral retinogeniculate projection and compromised depth perception. Pharmacological stimulation of calcium channels in albino mice, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs, and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit. HighlightsO_LIThe mouse ventral CMZ produces predominantly ipsilateral RGCs. C_LIO_LIIn the albino visual system, CyclinD2 downregulation leads to delayed G1/S transition toward mitotic exit of CMZ progenitors. C_LIO_LIPerturbations in the temporal control of cell cycle by CyclinD2 lead to reduced Zic2+ RGCs and consequently, a diminished ipsilateral retinogeniculate projection and compromised depth perception. C_LIO_LICalcium channel modulation during embryogenesis normalizes the levels of CyclinD2 and restores binocular vision in albino mice. C_LI

developmental biology↗

bioRxiv2021

Single-nuclei isoform RNA sequencing reveals combination patterns of transcript elements across human brain cell types

Single-nuclei RNA-Seq is being widely employed to investigate cell types, especially of human brain and other frozen samples. In contrast to single-cell approaches, however, the majority of single-nuclei RNA counts originate from partially processed RNA leading to intronic cDNAs, thus hindering the investigation of complete isoforms. Here, using microfluidics, PCR-based artifact removal, target enrichment, and long-read sequencing, we developed single-nuclei isoform RNA-sequencing ( SnISOr-Seq), and applied it to the analysis of human adult frontal cortex samples. We found that exons associated with autism exhibit coordinated and more cell-type specific inclusion than exons associated with schizophrenia or ALS. We discovered two distinct modes of combination patterns: first, those distinguishing cell types in the human brain. These are enriched in combinations of TSS-exon, exon-polyA site, and distant (non-adjacent) exon pairs. Second, those with all isoform combinations found within one neural cell type, which are enriched in adjacent exon pairs. Furthermore, adjacent exon pairs are predominantly mutually associated, while distant pairs are frequently mutually exclusive. Finally, we observed that human-specific exons are as tightly coordinated as conserved exons, pointing to an efficient evolutionary mechanism underpinning coordination. SnISOr-Seq opens the door to single-nuclei long-read isoform analysis in the human brain, and in any frozen, archived or hard-to-dissociate sample.

genomics↗

bioRxiv2021

Astrocytes derived from ASD patients alter behavior and destabilize neuronal activity through aberrant Ca2+ signaling

The cellular mechanisms of Autism Spectrum Disorder (ASD) are poorly understood. Cumulative evidence suggests that abnormal synapse function underlies many features of this disease. Astrocytes play in several key neuronal processes, including the formation of synapses and the modulation of synaptic plasticity. Astrocyte abnormalities have also been identified in the postmortem brain tissue of ASD patients. However, it remains unclear whether astrocyte pathology plays a mechanistic role in ASD, as opposed to a compensatory response. To address this, we strategically combined stem cell culturing with transplantation techniques to determine disease specific properties inherent to patient derived astrocytes. We demonstrate that ASD astrocytes induce repetitive behavior as well as impair memory and long-term potentiation when transplanted into the healthy mouse brain. These in vivo phenotypes were accompanied by reduced neuronal network activity and spine density caused by ASD astrocytes in hippocampal neurons in vitro. Transplanted ASD astrocytes also exhibit exaggerated Ca2+ fluctuations in chimeric brains. Genetic modulation of evoked Ca2+ responses in ASD astrocytes modulates behavior and neuronal activity deficits. Thus, we determine that ASD patient astrocytes are sufficient to induce repetitive behavior as well as cognitive deficit, suggesting a previously unrecognized primary role for astrocytes in ASD.

neuroscience↗

bioRxiv2021

Systems Biology Analysis of Human Genomes Points to Key Pathways Conferring Spina Bifida Risk

Spina bifida (SB) is a debilitating birth defect caused by multiple gene and environment interactions. Though SB shows non-Mendelian inheritance, genetic factors contribute to an estimated 70% of cases. Nevertheless, identifying human mutations conferring SB risk is challenging due to its relative rarity, genetic heterogeneity, incomplete penetrance and environmental influences that hamper GWAS approaches to untargeted discovery. Thus, SB genetic studies may suffer from population substructure and/or selection bias introduced by typical candidate gene searches. We report a population based, ancestry-matched whole-genome sequence analysis of SB genetic predisposition using a systems biology strategy to interrogate 298 case-control subject genomes (149 pairs). Genes that were enriched in likely gene disrupting (LGD), rare protein-coding variants were subjected to machine learning analysis to identify genes in which LGD variants occur with a different frequency in cases vs. controls and so discriminate between these groups. Those genes with high discriminatory potential for SB significantly enriched pathways pertaining to carbon metabolism, inflammation, innate immunity, cytoskeletal regulation and essential transcriptional regulation, indicating their impact on the pathogenesis of human SB. Additionally, interrogation of conserved non-coding sequences identified robust variant enrichment in regulatory regions of several transcription factors critical to embryonic development. This genome-wide perspective offers an effective approach to interrogation of coding and non-coding sequence variant contributions to rare complex genetic disorders.

genomics↗

bioRxiv2020

Cell-type, single-cell, and spatial signatures of brain-region specific splicing in postnatal development

Alternative RNA splicing varies across brain regions, but the single-cell resolution of such regional variation is unknown. Here we present the first single-cell investigation of differential isoform expression (DIE) between brain regions, by performing single cell long-read transcriptome sequencing in the mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 (www.isoformAtlas.com). Using isoform tests for brain-region specific DIE, which outperform exon-based tests, we detect hundreds of brain-region specific DIE events traceable to specific cell-types. Many DIE events correspond to functionally distinct protein isoforms, some with just a 6-nucleotide exon variant. In most instances, one cell type is responsible for brain-region specific DIE. Cell types indigenous to only one anatomic structure display distinctive DIE, where for example, the choroid plexus epithelium manifest unique transcription start sites. However, for some genes, multiple cell-types are responsible for DIE in bulk data, indicating that regional identity can, although less frequently, override cell-type specificity. We validated our findings with spatial transcriptomics and long-read sequencing, yielding the first spatially resolved splicing map in the postnatal mouse brain (www.isoformAtlas.com). Our methods are highly generalizable. They provide a robust means of quantifying isoform expression with cell-type and spatial resolution, and reveal how the brain integrates molecular and cellular complexity to serve function.

neuroscience↗

bioRxiv2020

A real-time search strategy for finding urban disease vector infestations

Containing domestic vector infestation requires the ability to swiftly locate and treat infested homes. In urban settings where vectors are heterogeneously distributed throughout a dense housing matrix, the task of locating infestations can be challenging. Here, we present a novel stochastic compartmental model developed to help locate infested homes in urban areas. We designed the model using infestation data for the Chagas disease vector species Triatoma infestans in Arequipa, Peru. Our approach incorporates disease vector counts at each observed house, and the vectors complex spatial dispersal dynamics. We used a Bayesian method to augment the observed data, estimate the insect population growth and dispersal parameters, and determine posterior infestation probabilities of households. We investigated the properties of the model through simulation studies, followed by field testing in Arequipa. Simulation studies showed the model to be accurate in its estimates of two parameters of interest: the growth rate of a domestic triatomine bug colony and the probability of a triatomine bug successfully invading a new home after dispersing from an infested home. When testing the model in the field, data collection using model estimates was hindered by low household participation rates, which severely limited the algorithm and in turn, the models predictive power. While future optimization efforts must improve the models capabilities when household participation is low, our approach is nonetheless an important step toward integrating data with predictive modeling to carry out evidence-based vector surveillance in cities.

bioinformatics↗

bioRxiv2018

Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types.

Full-length isoform sequencing has advanced our knowledge of isoform biology1-11. However, apart from applying full-length isoform sequencing to very few single cells12,13, isoform sequencing has been limited to bulk tissue, cell lines, or sorted cells. Single splicing events have been described for <=200 single cells with great statistical success14,15, but these methods do not describe full-length mRNAs. Single cell short-read 3 sequencing has allowed identification of many cell sub-types16-23, but full-length isoforms for these cell types have not been profiled. Using our new method of single-cell-isoform-RNA-sequencing (ScISOr-Seq) we determine isoform-expression in thousands of individual cells from a heterogeneous bulk tissue (cerebellum), without specific antibody-fluorescence activated cell sorting. We elucidate isoform usage in high-level cell types such as neurons, astrocytes and microglia and finer sub-types, such as Purkinje cells and Granule cells, including the combination patterns of distant splice sites6-9,24,25, which for individual molecules requires long reads. We produce an enhanced genome annotation revealing cell-type specific expression of known and 16,872 novel (with respect to mouse Gencode version 10) isoforms (see isoformatlas.com).\n\nScISOr-Seq describes isoforms from >1,000 single cells from bulk tissue without cell sorting by leveraging two technologies in three steps: In step one, we employ microfluidics to produce amplified full-length cDNAs barcoded for their cell of origin. This cDNA is split into two pools: one pool for 3 sequencing to measure gene expression (step 2) and another pool for long-read sequencing and isoform expression (step 3). In step two, short-read 3-sequencing provides molecular counts for each gene and cell, which allows clustering cells and assigning a cell type using cell-type specific markers. In step three, an aliquot of the same cDNAs (each barcoded for the individual cell of origin) is sequenced using Pacific Biosciences (\"PacBio\")1,2,4,5,26 or Oxford Nanopore3. Since these long reads carry the single-cell barcodes identified in step two, one can determine the individual cell from which each long read originates. Since most single cells are assigned to a named cluster, we can also assign the cells cluster name (e.g. \"Purkinje cell\" or \"astrocyte\") to the long read in question (Fig 1A) - without losing the cell of origin of each long read.\n\nO_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC=\"FIGDIR/small/364950_fig1.gif\" ALT=\"Figure 1\">\nView larger version (66K):\norg.highwire.dtl.DTLVardef@4df0cdorg.highwire.dtl.DTLVardef@fc4beborg.highwire.dtl.DTLVardef@1dc485forg.highwire.dtl.DTLVardef@1138a3e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO (A) Outline of our ScISOr-Seq approach. (B) TSNE-plot depicting cell clusters, marker genes and names given to clusters, including: Bergman glia (BG), External granule cell layer neurons (EGL), Internal granule cell layer and other neurons in the interior of the cerebellum (IGL), two clusters of Purkinje cell layer neurons (PCL), oligodendrocyte progenitor cells (OPCs), Atoh1+ neuronal progenitors, Ptf1a+ neuronal progenitors and other neuronal progenitors (NPCs) (C) In-situ hybridization images from the Allen Brain Atlas depicting expression of marker genes in specific layers. (D) Expression patterns of selected marker genes across cell types.\n\nC_FIG

molecular biology↗