19 research outputs found
Adaptation to G93A superoxide dismutase 1 in a motor neuron cell line model of amyotrophic lateral sclerosis. The role of glutathione
No full textMotor neuron degeneration in amyotrophic lateral sclerosis involves oxidative
damage. Glutathione (GSH) is critical as an antioxidant and a redox
modulator. We used a motor neuronal cell line (NSC-34) to investigate
whether wild-type and familial amyotrophic lateral sclerosis-linked G93A
mutant Cu,Zn superoxide dismutase (wt ⁄G93ASOD1) modified the GSH
pool and glutamate cysteine ligase (GCL), the rate-limiting enzyme for
GSH synthesis. We studied the effect of various G93ASOD1 levels and
exposure times. Mutant Cu,Zn superoxide dismutase induced an adaptive
process involving the upregulation of GSH synthesis, even at very low
expression levels. However, cells with a high level of G93ASOD1 cultured
for 10 weeks showed GSH depletion and a decrease in expression of the
modulatory subunit of GCL. These cells also had lower levels of GSH and
GCL activity was not induced after treatment with the pro-oxidant tertbutylhydroquinone.
Cells with a low level of G93ASOD1 maintained
higher GSH levels and GCL activity, showing that the exposure time and
the level of the mutant protein modulate GSH synthesis. We conclude that
failure of the regulation of the GSH pathway caused by G93ASOD1 may
contribute to motor neuron vulnerability and we identify this pathway as a
target for therapeutic intervention
A Schwann Cell–Neuron Coculture System to Study Neuron–Glia Interaction During Axon Degeneration
No full textTransmission electron microscopy and morphometry of the CNS white matter
No full textTransmission electron microscopy of central nervous system white matter has provided unparalleled access to the ultrastructural features of axons, their myelin sheaths, and the major cells of white matter; namely, oligodendrocytes, oligodendrocyte precursors, astrocytes, and microglia. In particular, it has been invaluable in elucidating pathological changes in axons and myelin following experimentally induced injury or genetic alteration, in animal models. While also of value in the examination of human white matter, the tissue is rarely fixed adequately for the types of detailed analyses that can be performed on well-preserved samples from animal models, perfusion fixed at the time of death. In this chapter we describe methods for obtaining, processing, and visualizing white matter samples using transmission electron microscopy of perfusion fixed tissue and for unbiased morphometry of white matter, with particular emphasis on axon and myelin pathology. Several advanced electron microscopy techniques are now available, but this method remains the most expedient and accessible for routine ultrastructural examination and morphometry
A Cutting-Edge Magnetic Immunocapture Method to Isolate Cell-Type-Specific Mitochondria from Complex Neural Tissue
No full textBest Undergraduate Poster (Discovery Category) - OSU CBI Research Day 2025Alterations in neuronal and glial metabolism contribute to numerous neurodegenerative diseases, and the crosstalk between these two cell types (i.e., axoglial metabolic coupling) is at the center of extensive investigation. Metabolic alterations frequently culminate in mitochondrial dysfunction, but so far it has proven challenging to obtain cell-type-specific metabolic data from neuronal or glial mitochondria in mouse models of injury or disease. Magnetic immunocapture of genetically tagged mitochondria has emerged as a powerful strategy, yet existing methods are either incompatible with sensitive downstream multi omic workflows or not yet tested in complex tissues with highly heterogeneous cell populations. Here, I refined the “MITO-Tag” approach, which leverages a Cre dependent 3×HA EGFP OMP25 epitope tag localized to the outer mitochondrial membrane. Following enzymatic and mechanical dissociation, mitochondria were rapidly immunopurified from cortical neurons, oligodendrocyte lineage cells, and—accomplished here for the first time—peripheral nerve axons using anti-HA magnetic beads in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-compatible KPBS buffer. This method yields specific and structurally intact mitochondria, as confirmed by live-organelle imaging and Western blotting for compartment-specific markers (COXIV, VDAC, citrate synthase), with minimal contamination from other organelles. Proteomic analysis of brain-derived immunoprecipitates (IPs) revealed mitochondrial enrichment comparable to existing magnetic immunopurification workflows. By enabling multi-omic mitochondrial profiling from moderate-abundance cell types within complex tissues, this method provides a versatile tool for investigating mitochondrial involvement in neurological disease and injury in vivo. Notably, isolation from peripheral nerve axons now offers the ability to characterize Wallerian degeneration and axoglial metabolic coupling mechanisms at an unprecedented resolution.NIH R01NS123450-01NIH R01NS111024-02OSU College of Engineering Thesis Research Grant2025 CAN-ACN Travel GrantNo embargoAcademic Major: Biomedical Engineerin
SerpinB3 induces dipeptidyl-peptidase IV/CD26 expression and its metabolic effects in hepatocellular carcinoma
No full textAbstract
Aims: In hepatocellular carcinoma (HCC), the regulatory protease Dipeptidyl-peptidase IV (DPPIV/CD26), that possesses pro-apoptotic properties, has been found abnormally regulated. The protease inhibitor SerpinB3, exerting anti-apoptotic activity, has also been described to be upregulated, especially in HCCs with poor prognosis. The aim of this study was to investigate the possible relationship between these two molecules in HCC patients and in experimental models.
Materials and methods: DPPIV/CD26 and SerpinB3 expression was measured in liver specimens of 67 patients with HCC. HepG2 and Huh7 cells, stably transfected to overexpress SerpinB3, and respective control cells were used to assess biological and metabolic modifications of DPPIV/CD26 activity induced by this serpin.
Key findings: DPPIV/CD26 and SerpinB3 were localized in the same tumoral areas and both molecules were correlated with the grade of tumor differentiation, with the highest values detected in GI tumors. Cell lines over-expressing SerpinB3 displayed upregulation of DPPIV/CD26, likely as a feedback mechanism, due to the DPPIV/CD26 protease activity inhibition by SerpinB3, as confirmed by the similar behavior induced by the inhibitor Sitagliptin. Moreover, they exhibited lower glycogen storage and higher lipid accumulation, typical effects of DPPIV/CD26.
Significance: A close connection between SerpinB3 and DPPPIV has been identified, but further studies are required to better understand the mechanism by which these proteins communicate and exert metabolic effects in HCC
