Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Natural history study and statistical modeling of disease progression in a preclinical model of myotubular myopathy.

Generating reliable preclinical data in animal models of disease is essential in therapy development. Here, we performed statistical analysis and joint longitudinal-survival modeling of the progressive phenotype observed in Mtm1-/y mice, a reliable model for myotubular myopathy. Analysis of historical data was used to generate a model for phenotype progression, which was then confirmed with phenotypic data from a new colony of mice derived via in vitro fertilization in an independent animal house, highlighting the reproducibility of disease phenotype in Mtm1-/y mice. These combined data were used to refine the phenotypic parameters analyzed in these mice and improve the model generated for expected disease progression. The disease progression model was then used to test the therapeutic efficacy of Dnm2 targeting. Dnm2 reduction by antisense oligonucleotides blocked or postponed disease development, and resulted in a significant dose-dependent improvement outside the expected disease progression in untreated Mtm1-/y mice. This provides an example of optimizing disease analysis and testing therapeutic efficacy in a preclinical model, which can be applied by scientists testing therapeutic approaches using neuromuscular disease models in different laboratories. This article has an associated First Person interview with the joint first authors of the paper.

BIN1 modulation in vivo rescues dynamin-related myopathy.

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2RW/+ and Dnm2RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2RW/+ mice and rescued the perinatal lethality and survival of Dnm2RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations.

Myostatin: a Circulating Biomarker Correlating with Disease in Myotubular Myopathy Mice and Patients.

Myotubular myopathy, also called X-linked centronuclear myopathy (XL-CNM), is a severe congenital disease targeted for therapeutic trials. To date, biomarkers to monitor disease progression and therapy efficacy are lacking. The Mtm1 -/y mouse is a faithful model for XL-CNM, due to myotubularin 1 (MTM1) loss-of-function mutations. Using both an unbiased approach (RNA sequencing [RNA-seq]) and a directed approach (qRT-PCR and protein level), we identified decreased Mstn levels in Mtm1 -/y muscle, leading to low levels of myostatin in muscle and plasma. Myostatin (Mstn or growth differentiation factor 8 [Gdf8]) is a protein released by myocytes and inhibiting muscle growth and differentiation. Decreasing Dnm2 by genetic cross with Dnm2 +/- mice or by antisense oligonucleotides blocked or postponed disease progression and resulted in an increase in circulating myostatin. In addition, plasma myostatin levels inversely correlated with disease severity and with Dnm2 mRNA levels in muscles. Altered Mstn levels were associated with a generalized disruption of the myostatin pathway. Importantly, in two different forms of CNMs we identified reduced circulating myostatin levels in plasma from patients. This provides evidence of a blood-based biomarker that may be used to monitor disease state in XL-CNM mice and patients and supports monitoring circulating myostatin during clinical trials for myotubular myopathy.

Different in vivo impacts of dynamin 2 mutations implicated in Charcot-Marie-Tooth neuropathy or centronuclear myopathy.

Dynamin 2 (DNM2) is a ubiquitously expressed GTPase implicated in many cellular functions such as membrane trafficking and cytoskeleton regulation. Dominant mutations in DNM2 result in tissue-specific diseases affecting peripheral nerves (Charcot-Marie-Tooth neuropathy, CMT) or skeletal muscles (centronuclear myopathy, CNM). However, the reason for this tissue specificity is unknown, and it remains unclear if these diseases share a common pathomechanism. To compare the disease pathophysiological mechanisms in skeletal muscle, we exogenously expressed wild-type DNM2 (WT-DNM2), the DNM2-CMT mutation K562E or DNM2-CNM mutations R465W and S619L causing adult and neonatal forms, respectively, by intramuscular adeno-associated virus (AAV) injections. All muscles expressing exogenous WT-DNM2 and CNM or CMT mutations exhibited reduced muscle force. However, only expression of CNM mutations and WT-DNM2 correlated with CNM-like histopathological hallmarks of nuclei centralization and reduced fiber size. The extent of alterations correlated with clinical severity in patients. Ultrastructural and immunofluorescence analyses highlighted defects of the triads, mitochondria and costameres as major causes of the CNM phenotype. Despite the reduction in force upon expression of the DNM2-CMT mutation, muscle histology and ultrastructure were almost normal. However, the neuromuscular junction was affected in all DNM2-injected muscles, with the DNM2-CMT mutation inducing the most severe alterations, potentially explaining the reduction in force observed with this mutant. In conclusion, expression of WT and CNM mutants recreate a CNM-like phenotype, suggesting CNM mutations are gain-of-function. Histological, ultrastructural and molecular analyses pointed to key pathways uncovering the different pathomechanisms involved in centronuclear myopathy or Charcot-Marie-Tooth neuropathy linked to DNM2 mutations.

Amphiphysin 2 modulation rescues myotubular myopathy and prevents focal adhesion defects in mice.

Centronuclear myopathies (CNMs) are severe diseases characterized by muscle weakness and myofiber atrophy. Currently, there are no approved treatments for these disorders. Mutations in the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for X-linked CNM (XLCNM), also called myotubular myopathy, whereas mutations in the membrane remodeling Bin/amphiphysin/Rvs protein amphiphysin 2 [bridging integrator 1 (BIN1)] are responsible for an autosomal form of the disease. Here, we investigated the functional relationship between MTM1 and BIN1 in healthy skeletal muscle and in the physiopathology of CNM. Genetic overexpression of human BIN1 efficiently rescued the muscle weakness and life span in a mouse model of XLCNM. Exogenous human BIN1 expression with adeno-associated virus after birth also prevented the progression of the disease, suggesting that human BIN1 overexpression can compensate for the lack of MTM1 expression in this mouse model. Our results showed that MTM1 controls cell adhesion and integrin localization in mammalian muscle. Alterations in this pathway in Mtm1 -/y mice were associated with defects in myofiber shape and size. BIN1 expression rescued integrin and laminin alterations and restored myofiber integrity, supporting the idea that MTM1 and BIN1 are functionally linked and necessary for focal adhesions in skeletal muscle. The results suggest that BIN1 modulation might be an effective strategy for treating XLCNM.

Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy.

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2-related CNM in a knock-in mouse harboring the p.R465W mutation (Dnm2RW/+) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2RW/+mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2RW/+mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases.

Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice.

Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Here we show that systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. Moreover, systemic ASO injection into severely affected mice leads to reversal of muscle pathology within 2 weeks. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease.