Astrocyte-targeting RNA interference against mutated superoxide dismutase 1 induces motoneuron plasticity and protects fast-fatigable motor units in a mouse model of amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS) caused by SOD1 gene mutations, both cell-autonomous and noncell-autonomous mechanisms lead to the selective degeneration of motoneurons (MN). Here, we evaluate the therapeutic potential of gene therapy targeting mutated SOD1 in mature astrocytes using mice expressing the mutated SOD1G93A protein. An AAV-gfaABC1 D vector encoding an artificial microRNA is used to deliver RNA interference against mutated SOD1 selectively in astrocytes. The treatment leads to the progressive rescue of neuromuscular junction occupancy, to the recovery of the compound muscle action potential in the gastrocnemius muscle, and significantly improves neuromuscular function. In the spinal cord, gene therapy targeting astrocytes protects a small pool of the most vulnerable fast-fatigable MN until disease end stage. In the gastrocnemius muscle of the treated SOD1G93A mice, the fast-twitch type IIB muscle fibers are preserved from atrophy. Axon collateral sprouting is observed together with muscle fiber type grouping indicative of denervation/reinnervation events. The transcriptome profiling of spinal cord MN shows changes in the expression levels of factors regulating the dynamics of microtubules. Gene therapy delivering RNA interference against mutated SOD1 in astrocytes protects fast-fatigable motor units and thereby improves neuromuscular function in ALS mice.

Altered interplay between endoplasmic reticulum and mitochondria in Charcot-Marie-Tooth type 2A neuropathy.

Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

Tmc gene therapy restores auditory function in deaf mice.

Genetic hearing loss accounts for up to 50% of prelingual deafness worldwide, yet there are no biologic treatments currently available. To investigate gene therapy as a potential biologic strategy for restoration of auditory function in patients with genetic hearing loss, we tested a gene augmentation approach in mouse models of genetic deafness. We focused on DFNB7/11 and DFNA36, which are autosomal recessive and dominant deafnesses, respectively, caused by mutations in transmembrane channel-like 1 (TMC1). Mice that carry targeted deletion of Tmc1 or a dominant Tmc1 point mutation, known as Beethoven, are good models for human DFNB7/11 and DFNA36. We screened several adeno-associated viral (AAV) serotypes and promoters and identified AAV2/1 and the chicken ß-actin (Cba) promoter as an efficient combination for driving the expression of exogenous Tmc1 in inner hair cells in vivo. Exogenous Tmc1 or its closely related ortholog, Tmc2, were capable of restoring sensory transduction, auditory brainstem responses, and acoustic startle reflexes in otherwise deaf mice, suggesting that gene augmentation with Tmc1 or Tmc2 is well suited for further development as a strategy for restoration of auditory function in deaf patients who carry TMC1 mutations.

SOD1 silencing in motoneurons or glia rescues neuromuscular function in ALS mice.

OBJECTIVE: Amyotrophic lateral sclerosis is an incurable disorder mainly characterized by motoneuron degeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for 20% of familial forms of the disease. Mutant SOD1 exerts multiple pathogenic effects through the gain of toxic properties in both neurons and glial cells. Here, we compare AAV-based gene therapy suppressing expression of mutant SOD1 in either motoneurons or astrocytes. METHODS: AAV vectors encoding microRNA against human SOD1 were administered to (G93) (A)SOD1 mice either by intracerebroventricular injections in pups or by lumbar intrathecal injections in adults. Vector systems were designed to suppress SOD1 expression predominantly in either spinal motoneurons or astrocytes. Electrophysiological and behavioral tests were performed on treated animals to evaluate disease progression. RESULTS: Following vector injection in (G93) (A)SOD1 pups, efficient silencing of SOD1 expression was achieved in motoneurons and/or astrocytes. Most complete protection of motor units was obtained when targeting human SOD1 predominantly in motoneurons. Suppressing SOD1 mainly in astrocytes led to preserved muscle innervation despite only partial protection of spinal motoneurons. In both cases, injection in pups led to full recovery of neuromuscular function and significantly prolonged survival. Vector injections in adult mice also achieved significant protection of neuromuscular function, which was highest when motoneurons were targeted. INTERPRETATION: These results suggest that AAV-mediated SOD1 silencing is an effective approach to prevent motoneuron degeneration caused by SOD1 mutation. AAV vectors suppressing SOD1 in motoneurons delay disease onset and show effective neuroprotection. On the other hand, AAV-based SOD1 silencing in astrocytes rescues neuromuscular function following initial denervation.

Molecular characterization of the processing of arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme-1/site-1 protease.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242.

The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment leads to virus extinction.