In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.

Background: Miro is a mitochondrial protein involved in mitochondrial trafficking.Results: Mitochondrial damage drives rapid Miro ubiquitination in a manner dependent on Ser-65 in Parkin; however, Miro degradation is delayed.Conclusion: Ubiquitination of Miro, rather than its degradation, could act as a signal for mitochondrial arrest and clearance.Significance: Disruption of the mitochondrial transport machinery could be implicated in Parkinson disease.

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria, where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development.

BackgroundThe R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Cav2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain.ResultsUsing in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Cav2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation.ConclusionsThe present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.