Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.

PTEN-induced putative kinase 1 (PINK1) and Parkin act in a common pathway to regulate mitochondrial dynamics, the involvement of which in the pathogenesis of Parkinson's disease (PD) is increasingly being appreciated. However, how the PINK1/Parkin pathway influences mitochondrial function is not well understood, and the exact role of this pathway in controlling mitochondrial dynamics remains controversial. Here we used mammalian primary neurons to examine the function of the PINK1/Parkin pathway in regulating mitochondrial dynamics and function. In rat hippocampal neurons, PINK1 or Parkin overexpression resulted in increased mitochondrial number, smaller mitochondrial size and reduced mitochondrial occupancy of neuronal processes, suggesting that the balance of mitochondrial fission/fusion dynamics is tipped toward more fission. Conversely, inactivation of PINK1 resulted in elongated mitochondria, indicating that the balance of mitochondrial fission/fusion dynamics is tipped toward more fusion. Furthermore, overexpression of the fission protein Drp1 (dynamin-related protein 1) or knocking down of the fusion protein OPA1 (optical atrophy 1) suppressed PINK1 RNAi-induced mitochondrial morphological defect, and overexpression of PINK1 or Parkin suppressed the elongated mitochondria phenotype caused by Drp1 RNAi. Functionally, PINK1 knockdown and overexpression had opposite effects on dendritic spine formation and neuronal vulnerability to excitotoxicity. Finally, we found that PINK1/Parkin similarly influenced mitochondrial dynamics in rat midbrain dopaminergic neurons. These results, together with previous findings in Drosophila dopaminergic neurons, indicate that the PINK1/Parkin pathway plays conserved roles in regulating neuronal mitochondrial dynamics and function.

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

We have used RNA interference (RNAi) to knock down the expression of the c2 subunit of the GABA A receptors (GABA A Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the c2 subunit, one targeting the coding region and the other one the 3¢-untranslated region (UTR) of the c2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA A receptor c2 subunit and the clustering of other GABA A R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the c2 shRNAs had no effect on the clustering of postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A c2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3¢-UTR targeted by c2 RNAi, rescued both the postsynaptic clustering of GABA A Rs and the GABAergic innervation. Decreased GABA A R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the c2 shRNAs. The results indicate that the postsynaptic clustering of GABA A Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts. Studies of c2 subunit-deficient mouse mutants have shown that the c2 subunit of the GABA A receptor (GABA A R) is necessary for the postsynaptic clustering of the GABA A Rs and for the maintenance of GABA A R clusters at GABAergic synapses (Essrich et al. 1998;Schweizer et al. 2003). The c2-/-mouse mutant shows a severe deficit in GABAergic synaptic transmission and dies soon after birth (Günther et al. 1995). GABA A Rs play a morphogenic role during embryonic development (Rudolph and Mohler 2004;Vicini and Ortinski 2004). Thus, some of the observed phenotypes in these and other mutant mice might result from developmental alterations, while the absence of phenotype might be because of compensatory mechanisms. RNA interference (RNAi, Dykxhoorn et al. 2003;Huppi et al. 2005) is a simpler alternative to the gene knockout technology that can also overcome some of the limitations inherent to the use of mouse mutants. In this study, we have used c2 RNAi to study GABA A R clustering in loss-of-function experiments, both in neuronal cultures and in the intact brain after in utero electroporation. Our results support the notion that the c2 subunit is necessary for the postsynaptic clustering and maintenance of GABA A Rs and gephyrin (a postsynaptic scaffolding protein that is present at inhibitory GABAergic and glycinergic synapses). More interesting, because we revealed it with RNAi technology but it had not been previously observed with the c2 mouse mutants, is the observation that the disruption of the postsynaptic clustering Abbreviations used: AIS, axon initial segmen...

Summary Disorders arising from impaired assembly of succinate dehydrogenase (SDH) result in a myriad of pathologies, consistent with its unique role in linking the citric acid cycle and electron transport chain. In spite of this critical function, however, only a few factors are known to be required for SDH assembly and function. We show here that two factors, Sdh6 (SDHAF1) and Sdh7 (SDHAF3), mediate maturation of the FeS cluster SDH subunit (Sdh2/SDHB). Yeast and Drosophila lacking SDHAF3 are impaired in SDH activity with reduced levels of Sdh2. Drosophila lacking the Sdh7 ortholog SDHAF3 are hypersensitive to oxidative stress and exhibit muscular and neuronal dysfunction. Yeast studies revealed that Sdh6 and Sdh7 act together to promote Sdh2 maturation by binding to a Sdh1/Sdh2 intermediate, protecting it from the deleterious effects of oxidants. These studies in yeast and Drosophila raise the possibility that SDHAF3 mutations may be associated with idiopathic SDH-associated diseases.