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Venu Talla, Sacide Ozdemir, Tsung-Han Chou, Vittorio Porciatti, John Guy; Gene Therapy with the Mitochondrial Heat Shock Protein 70 (mtHSP70) Chaperone Supresses Neurodegeneration and Irreversible Visual Loss in Experimental Optic Neurits. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5967.
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© ARVO (1962-2015); The Authors (2016-present)
To rescue visual loss and optic neuropathy in the experimental autoimmune encephalomyelitis (EAE) mouse model using gene therapy with the mtHSP70 chaperone responsible for unfolding and import of most proteins into the mitochondria.
EAE was induced in female DBA/1J (n=20) mice by subdermal injection of 0.1 ml homologous spinal cord emulsion in CFA. Ten mice were rescued by intravitreal injection of ssAAV-mtHSP70-Flag, 10 EAE and 10 unsensitized mice injected with scAAV-Cox8-mCherry served as controls. Visual function was assessed by pattern electroretinograms (PERG). High resolution spectral domain OCT evaluated the thickness of the inner plexiform layer + nerve fiber layers at 1, 3 and 6 months post injection (MPI). All mice were euthanized 6MPI. Retinas and ONs were dissected for histological and ultrastructural evaluation. Expression of mtHSP70Flag in the retina and ONs was evaluated 15d PI by RT-PCR, immunofluorescence (IF) and western blotting (WB).
IF revealed a typical punctate and perinuclear expression of Flag-HSP70 which colocalized with mitochondrial porin and thy1.2 labeled RGCs. RT-PCR and WB confirmed HSP70 expression in the retina and ON. PERG amplitude at 3M and 6MPI showed 42% and 45% reduction in EAE-mCherry compared to control mCherry (p<0.005,) mtHSP70 rescued mice also showed reduced amplitudes (26% and 27% respectively, p<0.05) though it rescued the by 37% and 41% respectively (p>0.05). PERG latency was delayed by 15% and 21% in EAE-mCherry compared to unsensitized mCherry controls (p<0.05), whereas the mtHSP70 injected mice rescued the delay by 100% and 83% respectively at 3M and 6MPI. OCT showed a thinning in EAE-mCherry compared to mCherry control at 3M (16%) and 6MPI (15%) p<0.05, whereas mtHSP70 rescued this thinning by 90% and 100% respectively (p<0.05). Ultrastructural analysis of the EAE ONs demonstrated different levels of degeneration in axons and myelin. Degenerated axons showed varied mitochondrial number, shape and morphology. mtHSP70 ONs showed relatively normal axons and myelin with organized microtubules and mitochondria.
mtHSP70 gene therapy preserves vision by limiting neurodegeneration of the ONs that causes permanent and untreatable disability that is unaltered by commonly used disease modifying drugs that target inflammation in multiple sclerosis.
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