Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.

Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). Patients with ARSACS, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca2+-evoked responses in Sacs-/- PCs as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca2+ buffer proteins. Alteration of cytoskeletal linkers, which we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/- cerebellum. Based on this pathogenetic cascade, we treated Sacs-/- mice with Ceftriaxone, a repurposed drug that exerts neuroprotection by limiting neuronal glutamatergic stimulation and, thus, Ca2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/- mice, at both pre- and postsymptomatic stages. We correlated this effect to restored Ca2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in preclinical and clinical settings for the treatment of patients with ARSACS.

Assessment of Sacsin Turnover in Patients With ARSACS: Implications for Molecular Diagnosis and Pathogenesis.

BACKGROUND AND OBJECTIVES: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by variations in SACS gene encoding sacsin, a huge multimodular protein of unknown function. More than 200 SACS variations have been described worldwide to date. Because ARSACS presents phenotypic variability, previous empirical studies attempted to correlate the nature and position of SACS variations with the age at onset or with disease severity, although not considering the effect of the various variations on protein stability. In this work, we studied genotype-phenotype correlation in ARSACS at a functional level. METHODS: We analyzed a large set of skin fibroblasts derived from patients with ARSACS, including both new and already published cases, carrying variations of different types affecting diverse domains of the protein. RESULTS: We found that sacsin is almost absent in patients with ARSACS, regardless of the nature of the variation. As expected, we did not detect sacsin in patients with truncating variations. We found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense variations. In this case, we excluded SACS mRNA decay, defective translation, or faster posttranslational degradation as possible causes of protein reduction. Conversely, our results demonstrate that nascent mutant sacsin protein undergoes cotranslational ubiquitination and degradation. DISCUSSION: Our results provide a mechanistic explanation for the lack of genotype-phenotype correlation in ARSACS. We also propose a new and unambiguous criterion for ARSACS diagnosis that is based on the evaluation of sacsin level. Last, we identified preemptive degradation of a mutant protein as a novel cause of a human disease.

Impaired turnover of hyperfused mitochondria in severe axonal neuropathy due to a novel DRP1 mutation.

Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5'-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense mutation in the GTPase domain of DRP1 (NM_012062.3:c.436G>A, NP_036192.2: p.D146N variant in DNM1L). Patient's fibroblasts show hyperfused/balloon-like giant mitochondria, highlighting the importance of D146 residue for DRP1 function. This dramatic mitochondrial rearrangement phenocopies what observed overexpressing DRP1-K38A, a well-known experimental dominant negative version of DRP1. In addition, we demonstrated that p.D146N mutation has great impact on peroxisomal shape and function. The p.D146N mutation compromises the GTPase activity without perturbing DRP1 recruitment or assembly, causing decreased mitochondrial and peroxisomal turnover. In conclusion, our findings highlight the importance of sensory neuropathy in the clinical spectrum of DRP1 variants and, for the first time, the impact of DRP1 mutations on mitochondrial turnover and peroxisomal functionality.