Biology subjects

Crocker, S. J.

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

5 recordsLinked to original sources

bioRxiv2025

Spoti-find: A novel, open-source void spot assay image analysis tool

The void spot assay (VSA) is a widely used, non-invasive method for evaluating urinary behavior in rodents, but existing analysis tools are limited in scope, throughput, or accessibility. We developed Spoti-find, a stand-alone, open-source VSA image analysis application that introduces novel, biologically meaningful metrics including void circularity, distance to paper edge, and volume-based binning into primary, micro-, and nanovoids. Designed with usability in mind, Spoti-find features a graphical interface that enables manual or semi-automated spot identification, adjustable thresholds, and streamlined data export without the need for coding expertise. We validated Spoti-find across diverse datasets, showing strong inter-user consistency, sensitivity to known phenotypes in aging and disease models, and accuracy in capturing novel parameters while demonstrating high agreement with existing tools. By capturing behavioral context and spatial morphology in voiding patterns, Spoti-find expands the interpretive power of VSA and provides a flexible, user-friendly platform for phenotyping urinary dysfunction in preclinical studies.

physiology↗

bioRxiv2023

Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. We have previously demonstrated that murine astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. To better understand the basis of this, we performed unbiased proteomic analyses and identified a fibronectin-derived peptide called anastellin that is unique to the murine Timp1KO astrocyte secretome. Anastellin was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Anastellin is known to act upon the sphingosine-1-phosphate receptor 1 (S1PR1), and we determined that anastellin also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Further, administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-EAE ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 in astrocytes (Timp1cKO) had no effect. Analysis of human TIMP1 and fibronectin (FN1) transcripts from healthy and multiple sclerosis (MS) patient brain samples revealed an inverse relationship where lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Lastly, we analyzed proteomic databases of MS samples and identified anastellin peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high versus low disease activity. The prospective role for anastellin generation in association with myelin lesions as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and the innate remyelination potential of the the MS brain. Significance StatementAstrocytic production of TIMP-1 prevents the protein catabolism of fibronectin. In the absence of TIMP-1, fibronectin is further digested leading to a higher abundance of anastellin peptides that can bind to sphingosine-1-phosphate receptor 1. The binding of anastellin with the sphingosine-1-phosphate receptor 1 impairs the differentiation of oligodendrocytes progenitor cells into myelinating oligodendrocytes in vitro, and negates the astrocyte-mediated therapeutic effects of FTY720 in the EAE model of chronic CNS inflammation. These data indicate that TIMP-1 production by astrocytes is important in coordinating astrocytic functions during inflammation. In the absence of astrocyte produced TIMP-1, elevated expression of anastellin may represent a prospective biomarker for FTY720 therapeutic responsiveness.

neuroscience↗

bioRxiv2022

Therapeutic depletion of CD8+ T-cells prevents myelin pathology in Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD) or Krabbes disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function blocking antibody against CD8 effectively prevented disease onset, reduced morbidity and mortality and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD. One-Sentence SummaryCD8 T-cells mediate demyelination and neuroinflammation in a genetic white matter disease.

neuroscience↗

bioRxiv2019

Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on axon loss in MS, far less is known about synaptic changes. Here, in striking similarity to other neurodegenerative diseases, we identify in postmortem human MS tissue and in nonhuman primate and mouse MS models profound synapse loss and microglial synaptic engulfment. These events can occur independently of local demyelination, neuronal degeneration, and peripheral immune cell infiltration, but coincide with gliosis and increased localization of complement component C3, but not C1q, at synapses. Finally, we use AAV9 to overexpress the complement inhibitor Crry at activated C3-bound synapses in mice and demonstrate robust protection of synapses and visual function. These results mechanistically dissect synapse loss as an early pathology in MS. We further provide a novel gene therapy approach to prevent synapse loss by microglia, which may be broadly applicable to other neurodegenerative diseases.

neuroscience↗

bioRxiv2019

TIMP-1 attenuates the development of cutaneous inflammation-induced hypersensitivity

Unresolved inflammation is a significant predictor for developing chronic pain, and targeting the mechanisms underlying inflammation offers opportunities for therapeutic intervention. During inflammation, matrix metalloproteinase (MMP) activity contributes to tissue remodeling and inflammatory signaling through proteolytic maturation of cytokines. MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs) 1-4. TIMP-1 and -2 have known roles in pain, but only in the context of MMP inhibition. However, TIMP-1 also has receptor-mediated cell signaling functions that are not well understood. Here, we examined how TIMP-1-dependent cell signaling impacted inflammatory hypersensitivity and ongoing pain. We found that hindpaw injection of complete Freunds adjuvant (CFA) increased keratinocyte-derived TIMP-1 that peaked 3 days following inflammation, when mechanical hypersensitivity began to emerge in WT mice. These data suggest that TIMP-1 expression inhibits the development of inflammatory hypersensitivity. To examine this possibility, we injected TIMP-1 knockout (T1KO) mice with a diluted CFA mixture to examine how subtle cutaneous inflammation affected behavioral hypersensitivity. T1KO mice exhibited rapid onset thermal and mechanical hypersensitivity at the site of inflammation that was absent or attenuated in WT controls. We also found that T1KO mice exhibited hypersensitivity in adjacent tissues innervated by different sets of afferents, and skin contralateral to the site of inflammation. Replacement of recombinant murine (rm)TIMP-1 alleviated hypersensitivity when administered at the site and time of inflammation. To examine the MMP-dependent and -independent mechanisms of rmTIMP-1, T1KO mice were administered full-length rmTIMP-1, the N-terminal region (TIMP-1(N)) with MMP-inhibitory properties, or the C-terminal region (TIMP-1(C)) that retains receptor signaling function. Each of the peptides prevented inflammatory hypersensitivity, suggesting that rmTIMP-1 acts through mechanisms that also include receptor-mediated cell signaling. We also found that hypersensitivity was neither due to genotype-specific differences in MMP-9 activity or expression, nor to differences in cytokine expression. Finally, to evaluate the potential clinical utility of TIMP-1, we administered rmTIMP-1 to WT mice and found that rmTIMP-1 prevented clonidine-induced conditioned place preference (e.g., ongoing pain) and inflammatory mechanical hypersensitivity. Collectively, our data suggest a novel role for TIMP-1 in the attenuation of inflammatory pain that occurs through previously uncharacterized cell signaling mechanisms.

neuroscience↗