Abstract

Maintenance of protein homeostasis is essential for cellular function and depends on the coordinated regulation of protein synthesis and degradation. The major intracellular pathways responsible for protein degradation are the ubiquitin-proteasome system (UPS) and autophagy. Disruption of these processes has been implicated in a broad range of diseases, including neuromuscular disorders. X-linked myopathy with excessive autophagy (XMEA) is a rare vacuolar myopathy caused by mutations in VMA21, which encodes an assembly chaperone required for proper assembly of the vacuolar H⁺-ATPase (V-ATPase), the primary proton pump responsible for acidifying intracellular compartments, including lysosomes. Although impaired lysosomal acidification and autophagic dysregulation have been implicated in XMEA, the mechanisms linking VMA21 deficiency to muscle pathology in vivo remain poorly understood, in part because of the lack of suitable animal models. To address this gap, conditional Vma21 knockout mouse models were generated with simultaneous deletion in skeletal and cardiac muscle, as well as inducible skeletal muscle-specific deletion. Combined deletion resulted in early lethality associated with severe cardiomyopathy and cardiac autophagic dysregulation, whereas skeletal muscle-specific deletion produced progressive muscle weakness and myopathy characterized by centralized nuclei, fiber splitting, and increased fiber size variability. Importantly, VMA21-deficient skeletal muscle recapitulated defining pathological hallmarks of XMEA, including basal lamina reduplication and autophagic vacuoles with sarcolemmal features (AVSFs). VMA21 deficiency in skeletal muscle was associated with autophagic dysregulation, characterized by accumulation of LAMP2, LC3B-II, and ubiquitinated proteins, as well as cytoplasmic vacuolar structures containing undegraded material. Ultrastructural analysis further revealed enrichment of vesicular and vacuolar structures at the fiber periphery, frequently in association with membrane irregularity. Together with increased CD63-positive structures, a marker associated with late endosomes and exosomes, these findings raise the possibility of altered vesicle trafficking in the context of VMA21 deficiency. Notably, CD63-positive structures strongly colocalized with the complement membrane attack complex C5b-9 in VMA21-deficient mouse muscle and in skeletal muscle biopsies from patients with XMEA, supporting CD63 accumulation and its association with complement deposition as previously unrecognized features of XMEA pathology. Together, these studies establish tractable mammalian models that recapitulate key pathological features of XMEA and provide new insight into mechanisms linking VMA21 deficiency to autophagic dysregulation, altered vesicle trafficking, and complement deposition. These findings broaden the current pathogenic framework of XMEA and establish a platform for future mechanistic and therapeutic investigation.

Committee Chair

Conrad Weihl

Committee Members

Albert Davis; Cindy Ly; Heather True; John Cirrito

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-13-2026

Language

English (en)

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