Characterization of the frameshift c.515dupC knock-in mouse model of HSPB8-associated myopathy (MFM13) and evaluation of Trehalose as autophagy modulating therapy.

Abstract

Heat shock protein family B member 8 (HSPB8) is a chaperone involved in the chaperone-assisted selective autophagy (CASA) complex. HSPB8 in conjunction with cochaperone Bcl2-associated athanogene 3 (BAG3), promotes autophagy-mediated removal of misfolded proteins associated with various neurodegenerative diseases. Mutations in HSPB8, previously associated with Charcot Marie Tooth disease type 2L, have recently been linked to an autosomal dominant rimmed vacuolar myopathy (MFM13), and is considered a multisystem proteinopathy. Patients have distal and proximal limb girdle myopathy with muscle biopsy showing fatty replacement, endomysial fibrosis, and rimmed vacuoles leading to muscle atrophy and early demise. We have demonstrated reduced expression of HSPB8, altered autophagy, and TAR DNA-binding protein 43 (TDP-43) accumulation in patient fibroblasts. Overexpressed murine Hspb8 frameshift mutant (c.515dupC, fs) displays insolubility and aggregation propensity in Murine Neuroblastoma X Spinal Cord 34 (NSC-34) cells. Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we generated a knock-in Hspb8 mouse model of the c.515dupC hot spot frameshift variant to study disease pathology. Mutant Hspb8 mice developed late onset muscle weakness beginning at 15 months. Muscle biochemical analyses revealed reduced HSPB8 levels, increased TDP 43, and altered autophagy markers, partially recapitulating the human phenotype. Fiber type analysis, neuromuscular junction integrity, and motor neurons show mild myopathy without neurodegeneration. Given the lack of available treatments, we evaluated trehalose, a natural disaccharide that is known to induce HSPB8 and enhance autophagy as a potential therapy. Administration of 2% trehalose in the drinking water restored HSPB8 expression; however, it did not significantly ameliorate autophagic dysfunction or TDP-43 pathology in mutant mice. These findings support the value of our preclinical models for translational studies, and autophagy enhancement as a potential therapeutic strategy for HSPB8-related myopathy.