Abstract
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.
Highlights
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder, which is characterised by the selective death of motor neurons in the spinal cord, motor cortex and brain stem [1]
Mitochondrial damage leads to phosphorylation of mitochondrial Rho GTPase 1 (Miro1) by Phosphatase and Tensin homolog (PTEN)-induced putative kinase 1 (PINK1) which targets Miro1 for Parkin-dependent proteasomal degradation and causes an irreversible detachment of molecular motors from the mitochondrial surface [23, 26, 27]
To test if this defect could be recapitulated by expressing ALS mutant superoxide dismutase 1 (SOD1) in primary cultures of embryonic rat motor neurons we delivered EGFP, EGFP-SOD1 wild type (WT), A4V, G37R or G93A to DIV3 rat motor neurons by lentiviral transduction and analysed axonal transport of MitoTracker Red CMXRos-labelled mitochondria from time-lapse recordings and corresponding kymographs as described by us before (Fig. 1A, a) [9, 28, 29]
Summary
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder, which is characterised by the selective death of motor neurons in the spinal cord, motor cortex and brain stem [1]. 185 different mutations in the enzyme Cu/Zn superoxide dismutase-1 (SOD1) have been associated with ALS [3] and it is generally accepted that these mutations cause ALS by toxic-gain-of-function mechanisms. In case of SOD1-related ALS we have shown that ALS mutant SOD1 selectively reduced anterograde transport of mitochondria in primary cortical and motor neuron cultures [9]. Mitochondrial damage leads to phosphorylation of Miro by Phosphatase and Tensin homolog (PTEN)-induced putative kinase 1 (PINK1) which targets Miro for Parkin-dependent proteasomal degradation and causes an irreversible detachment of molecular motors from the mitochondrial surface [23, 26, 27]. We report that ALS mutant SOD1 reduces anterograde mitochondrial transport by inducing PINK1/Parkin-dependent degradation of Miro
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