Amyotrophic lateral sclerosis-associated mutant SOD1 inhibits anterograde axonal transport of mitochondria by reducing Miro1 levels.

Defective axonal transport is an early neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously shown that ALS-associated mutations in Cu/Zn superoxide dismutase 1 (SOD1) impair axonal transport of mitochondria in motor neurons isolated from SOD1 G93A transgenic mice and in ALS mutant SOD1 transfected cortical neurons, but the underlying mechanisms remained unresolved. The outer mitochondrial membrane protein mitochondrial Rho GTPase 1 (Miro1) is a master regulator of mitochondrial axonal transport in response to cytosolic calcium (Ca2+) levels ([Ca2+]c) and mitochondrial damage. Ca2+ binding to Miro1 halts mitochondrial transport by modifying its interaction with kinesin-1 whereas mitochondrial damage induces Phosphatase and Tensin Homolog (PTEN)-induced Putative Kinase 1 (PINK1) and Parkin-dependent degradation of Miro1 and consequently stops transport. To identify the mechanism underlying impaired axonal transport of mitochondria in mutant SOD1-related ALS we investigated [Ca2+]c and Miro1 levels in ALS mutant SOD1 expressing neurons. We found that expression of ALS mutant SOD1 reduced the level of endogenous Miro1 but did not affect [Ca2+]c. ALS mutant SOD1 induced reductions in Miro1 levels were Parkin dependent. Moreover, both overexpression of Miro1 and ablation of PINK1 rescued the mitochondrial axonal transport deficit in ALS mutant SOD1-expressing cortical and motor neurons. Together these results provide evidence that ALS mutant SOD1 inhibits axonal transport of mitochondria by inducing PINK1/Parkin-dependent Miro1 degradation.

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy.

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc-51-like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a-dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62-positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient-derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD-associated p62 pathology.

Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA.

BACKGROUND: The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. FINDINGS: We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. CONCLUSION: The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.

Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson's disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson's disease.