Biology subjects

Halford, J.

Publications and source records attributed to Halford, J..

3 recordsLinked to original sources

bioRxiv2025

GFAP Proteolysis in TBI: Linking Novel Modified Cleavage Products to Astrocyte Pathology and Patient Outcomes

Glial fibrillary acidic protein (GFAP) is a significant clinical biomarker of traumatic brain injury (TBI), yet understanding the nature, timing, and impact of its degraded and modified products would inform clinical utility. We report novel GFAP breakdown products (BDPs) and post-translational modifications (PTMs) that are unique to TBI including fragment- and patient-specific citrullination signatures that destabilize GFAP filaments. GFAP and its fragments were sequenced by mass spectrometry (MS) from severe TBI patients cerebrospinal fluid (CSF) and sera, identifying two distinct TBI-specific jointly generated product sets within the rod-domain covering coil1 (20-26kDa) and coil2 (15-19kDa). Their endings differed from GFAP fragments described in Alzheimers and Alexander disease. Label-free quantification showed biofluid co-product abundance differences with coil1-BDPs enriched over coil2-BDPs. Coil imbalance was independently confirmed by immunoblot densitometry in twenty-three TBI patients. Measurements over ten days showed injury day peaks of full-length and end-clipped GFAP fragments, while proteolytic 37/39kDa and small fragments remained elevated. Six-month outcome (Extended Glasgow Outcome Scale) correlated with GFAP fragments, whereas levels of uncleaved and end-clipped GFAP did not. A human astrocyte culture trauma model provided mechanistic insights linking fluid GFAP levels, subcellular localization, and injury-induced astrocytopathy. A new cleavage site between the two coils was identified through selective epitope loss. Coil1-BDPs were fluid-released post-injury, while coil2-BDPs remained intracellular. Their cellular retention was explained by non-filamentous aggregation of citrulline-modified coil2-BDPs in pathological astrocytes. Trauma-triggered proteolysis involved calpains and caspases in specific astrocyte injury states using protease inhibitors and live-dye-reporter imaging. These novel data link TBI biofluid GFAP fragments with assembly-defective GFAP aggregation in pathological astrocytes. Clinical TBI outcome correlated with GFAP degradation rather than with overall GFAP release. These translational findings indicate that TBI biomarker GFAP undergoes degradation and modification alongside astrocyte pathology, providing a new conceptual framework for investigating biomarker-associated astrocyte proteinopathy potentially linked to post-traumatic neurodegeneration.

neuroscience↗

bioRxiv2024

TMEM63A, associated with hypomyelinating leukodystrophies, is an evolutionarily conserved regulator of myelination

Infantile hypomyelinating leukodystrophy 19 (HLD19) is a rare genetic disorder where patients exhibit reduced myelin in central nervous system (CNS) white matter tracts and present with varied neurological symptoms. The causative gene TMEM63A encodes a mechanosensitive ion channel whose role in myelination has not been explored. Our study shows that TMEM63A is a major regulator of OL-driven myelination in the CNS. In mouse and zebrafish, Tmem63a inactivation led to early deficits in myelination, recapitulating the HLD19 phenotype. OL-specific conditional mouse knockouts of Tmem63a exhibited transient reductions in myelin, indicating that TMEM63A regulates myelination cell-autonomously. We show that TMEM63A is present at plasma membrane and on lysosomes and modulates myelin/myelin-associated protein production. Intriguingly, HLD19-associated TMEM63A variants from patients blocked trafficking to cell membrane. Together, our results reveal an ancient role for TMEM63A in fundamental aspects of myelination in vivo and highlight two exciting new models for the development of treatments for devastating hypomyelinating leukodystrophies.

neuroscience↗

bioRxiv2022

Control of stereocilia length during development of hair bundles

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering of mouse apical inner hair cells to measure stereocilia dimensions during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later--in early stage IV--and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (row 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transductions role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8, and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

cell biology↗