Biology subjects

Kotzbauer, P.

Publications and source records attributed to Kotzbauer, P..

3 recordsLinked to original sources

bioRxiv2025

Complementary Structural and Chemical Biology Methods Reveal the Basis for Selective Radioligand Binding to α-Synuclein in MSA Tissue

Fibrillar aggregation of -synuclein (Syn) is a hallmark of Parkinsons disease (PD) and related disorders, including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Despite advances in Syn fibril structural characterization, the relevance of in vitro and ex vivo structures to patient aggregates remains unclear, particularly for developing therapeutic or diagnostic molecules. Cryo-electron microscopy (cryo-EM) studies of Syn fibrils with ligands often reveal binding at multiple sites, likely due to high ligand concentrations. Here, various structural and chemical biology techniques were used to characterize Syn fibrils in the presence of EX-6, a candidate ligand for positron emission tomography (PET) imaging of synucleinopathies. Transmission electron microscopy (TEM) and cryo-EM revealed no significant fibril core changes upon binding. Forster resonance energy transfer (FRET) further demonstrated that the disordered C-terminus was unaltered. Cryo-EM and crosslinking mass spectrometry (XL-MS) identified consistent binding sites, with one (Site 2*) providing a well-defined pocket for high-resolution analysis. Site 2* showed similar residue positioning in MSA patient-derived structures, suggesting MSA selectivity. [3H]-EX-6 binding assays demonstrated a 10-fold preference for MSA over PD tissue, with autoradiography further confirming MSA selectivity. Taken together, the combined use of structural and chemical biology techniques provides a comprehensive understanding of EX-6 binding that would not be possible with any single method. Optimization of ligand-protein and ligand-ligand interactions observed in the cryo-EM structure will enable the development of EX-6 as a PET imaging probe for MSA.

neuroscience↗

bioRxiv2024

Seeding competent TDP-43 persists in human patient and mouse muscle

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease. SummaryTDP-43 aggregate seeds persist in mouse and human skeletal muscle independent of large TDP-43 inclusions.

pathology↗

bioRxiv2021

VCP protects neurons from proteopathic seeding

Uptake and spread of proteopathic seeds, such as S, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of S seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase S seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of S seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to S since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

neuroscience↗