Biology subjects

Mitchell, J. S.

Publications and source records attributed to Mitchell, J. S..

7 recordsLinked to original sources

bioRxiv2025

Synaptic Dysfunction and Compensation After NMDA Receptor Ablation in the Mouse Medial Prefrontal Cortex

N-methyl-D-aspartate receptors (NMDARs) in the prefrontal cortex (PFC) are critical regulators of neuronal excitability, synaptic plasticity, and cognitive function. NMDAR disruptions, including pharmacological blockade and anti-NMDAR encephalitis, can mimic symptoms of schizophrenia. These observations support the glutamate hypothesis of schizophrenia, which posits that symptoms arise from abnormal corticolimbic glutamatergic signaling. Further evidence for this theory includes abnormal expression of NMDARs and decreased dendritic spine density in the PFC of individuals with schizophrenia, as well as altered spine density and synaptic transmission caused by genetic manipulation of NMDARs. However, it is unknown how progressive loss of NMDAR function in the PFC during adolescence - a developmental time period associated with significant synaptic pruning and symptom onset in schizophrenia - affects excitatory synaptic structure and function. In this study, we used in vivo genome editing to ablate expression of the Grin1 gene, which encodes the obligate GluN1 subunit of NMDARs, in medial PFC neurons of female and male adolescent mice. We assessed synaptic density and function in layer V pyramidal neurons at multiple time points using whole-cell patch-clamp electrophysiology, integrated with confocal imaging of dendritic spine architecture in recorded neurons. NMDAR ablation caused an early decrease in basilar dendritic spine density, followed by a rebound in spine density and corresponding increase in AMPAR-mediated synaptic transmission, suggesting that synaptic compensation maintains an allostatic set point. Our findings demonstrate that NMDAR ablation initially disrupts local PFC networks, followed by recovery via compensatory processes that could be impaired in disease states.

neuroscience↗

bioRxiv2025

A systematic approach to analyze T cell migration: application to mouse melanoma tumors

T cells must assess and choose between surveilling large areas, but also engage efficiently with the target cells. This process is translated into variations in speed and turning angle of T cells. In this study, we propose a generalized algorithm to analyze cell migration data with focus on CD8+ T cells, using clustering technique to identify the number of different migration states and Hidden Markov Model to capture the dynamical switching between them. The algorithm only requires a set of position observations in a series of times, independent of other factors. While this study focuses on CD8+ T cell migration, this approach can potentially be used broadly to study the migration of other cell types as well. For the current analysis, low and high avidity T cells in melanoma tumors were tracked ex vivo using two-photon microscopy. Our findings suggest that CD8+ T cells follow a two-state migration dynamic, with one state being faster, while the other slower and more localized. Moreover, we established a statistical methodology to analyze T cell migration to assess whether there is true variability in cell speeds as distinguished from stochastic fluctuations about a single speed, and it can be applied across different experimental platforms.

immunology↗

bioRxiv2023

RELAXING SELECTIVE PRESSURES ON DEVELOPMENTALLY COMPLEX INTEGUMENTARY STRUCTURES: FEATHER VANE SYMMETRY EVOLVES IN ADDITION TO BODY MASS AND WING LENGTH AFTER FLIGHT LOSS IN RECENT BIRDS

Feathers are complex integumentary structures with high diversity across species and within plumage and have varied functions (e.g., thermoregulation, flight). Flight is lost in many crown lineages, and frequently occurs in island founding or semiaquatic context. Different extant lineages lost flight across at least three orders of magnitude of time ([~]79.58 Ma-15 Ka). Flight losss effect on sensory capacity, brain size, and skeletomusculature have been studied, but less work exists on relations between flightlessness and feathers. To understand how flight loss affects feather anatomy, we measured 11 feather metrics (e.g., barb length, barb angle) from primaries, tertials, rectrices, and contour feathers on skins of 30 flightless taxa and their phylogenetically closest volant taxa, supplemented with broader sampling of primaries across all orders of volant crown birds. Our sample includes 27 independent losses of flight; the sample contains nearly half the extant flightless species count and matches its [~]3:2 terrestrial:semiaquatic ratio. Vane symmetry increases in flightless lineages, and these patterns are strongest in flight feathers and weakest in coverts. Greatest changes in feathers are in the oldest flightless lineages like penguins, which show robust filaments (rachis, barbs, and barbules) on small feathers, and ratites, which show high interspecific diversity with plumulaceous filaments and/or filament loss. Phylogenetic comparative methods show that some of these microscopic feather traits, such as barb/barbule length and rachis width, are not as dramatically modified upon flight loss as are body mass increase and relative wing and tail fan reduction, whereas the effect on vane symmetry is more easily detected. Upon relaxing selection for flight, feathers do not soon significantly modify many of their flight adaptations, although increased vane symmetry is likely the most detectable shift. Feathers of recently flightless lineages are in many ways like those of their volant relatives. Feather microstructure evolution is often subtle in flightless taxa, except when flight loss is ancient, perhaps because developmental constraints act upon feathers and/or selection for novel feather morphologies is not strong. Changes in skeletomusculature of the flight apparatus are likely more evident in recently flightless taxa and may be a more reliable way to detect flight loss in fossils, with increased vane symmetry as potentially a microscopic signal. Finally, we see an intriguing, reversed pattern in feather evolution after flight loss from the pattern proposed in popular developmental models of feathers, with the later stages of feather development (asymmetric displacement of barb loci) being lost more readily, while early stages of development (e.g., differentiated barb ridges on follicle collar) are only lost after many millions of years of flightlessness.

evolutionary biology↗

bioRxiv2023

Insulin B peptide-MHC class II-specific chimeric antigen 1 receptor-Tregs prevent autoimmune diabetes

Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs stably expressed Foxp3 and prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes. Brief SummaryChimeric antigen receptor Tregs specific for an insulin B-chain peptide presented by MHC class II prevent autoimmune diabetes.

immunology↗

bioRxiv2023

Regional vulnerability in a neurodegenerative disease: Delineating SCA1 CNS and muscle therapeutic targets using a conditional mutant ATXN1 mouse

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model (f-ATXN1146Q/2Q) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1146Q/2Q;Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1146Q/2Q;Rgs9-Cre mice. Mice lacking striatal ATXN1146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1146Q/2Q mice. Kyphosis was not improved in f-ATXN1146Q/2Q;Nestin-Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

neuroscience↗

bioRxiv2019

Widespread FUS mislocalization is a molecular hallmark of ALS

Amyotrophic lateral sclerosis (ALS)-causing mutations clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins (RBPs), which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RBPs mislocalize without necessarily becoming constituents of ALS cytoplasmic inclusions themselves remains unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RBP Fused in Sarcoma (FUS), in an unaggregated state, may occur more widely in ALS that previously recognized. To address this hypothesis, we analysed motor neurons (MNs) from an human ALS induced-pluripotent stem cells (iPSC) model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron-retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

neuroscience↗

bioRxiv2018

Chrysalis: A new method for high-throughput histo-cytometry analysis of images and movies.

Advances in imaging have led to the development of powerful multispectral, quantitative imaging techniques, like histo-cytometry. The utility of this approach is limited, however, by the need for time consuming manual image analysis. We therefore developed the software Chrysalis and a group of Imaris Xtensions to automate this process. The resulting automation allowed for high-throughput histo-cytometry analysis of 3D confocal microscopy and two-photon time-lapse images of T cell-dendritic cell interactions in the spleen. It was also applied to epi-fluorescence images to quantify T cell localization within splenic tissue by using a signal absorption strategy that avoids computationally intensive distance measurements. In summary, this image processing and analysis software makes histo-cytometry more useful for immunology applications by automating image analysis.

immunology↗