Pharmacological modulation of septins restores calcium homeostasis and is neuroprotective in models of Alzheimer's disease.

Abnormal calcium signaling is a central pathological component of Alzheimer's disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-ß and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-ß and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.

CHCHD2 harboring Parkinson's disease-linked T61I mutation precipitates inside mitochondria and induces precipitation of wild-type CHCHD2.

The T61I mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a protein residing in the mitochondrial intermembrane space (IMS), causes an autosomal dominant form of Parkinson's disease (PD), but the underlying pathogenic mechanisms are not well understood. Here, we compared the subcellular localization and solubility of wild-type (WT) and T61I mutant CHCHD2 in human cells. We found that mitochondrial targeting of both WT and T61I CHCHD2 depended on the four cysteine residues in the C-terminal coiled-coil-helix-coiled-coil-helix (CHCH) domain but not on the N-terminal predicted mitochondrial targeting sequence. The T61I mutation did not interfere with mitochondrial targeting of the mutant protein but induced its precipitation in the IMS. Moreover, T61I CHCHD2 induced increased mitochondrial production of reactive oxygen species and apoptosis, which was prevented by treatment with anti-oxidants. Retention of T61I CHCHD2 in the cytosol through mutation of the cysteine residues in the CHCH domain prevented its precipitation as well as its apoptosis-inducing effect. Importantly, T61I CHCHD2 potently impaired the solubility of WT CHCHD2. In conclusion, our data show that the T61I mutation renders mutant CHCHD2 insoluble inside mitochondria, suggesting loss of function of the mutant protein. In addition, T61I CHCHD2 exerts a dominant-negative effect on the solubility of WT CHCHD2, explaining the dominant inheritance of this form of PD.

LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10.

Parkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1- and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1- and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.Abbreviations : ACTB: actin beta; ATP5F1B: ATP synthase F1 subunit beta; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; Co-IP: co-immunoprecipitation; EBSS: Earle's balanced salt solution; GFP: green fluorescent protein; HSPD1: heat shock protein family D (Hsp60) member 1; LAMP1: lysosomal associated membrane protein 1; LRRK2: leucine rich repeat kinase 2; IF: immunofluorescence; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; OMM: outer mitochondrial membrane; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT1: ras homolog family member T1; ROS: reactive oxygen species; TBK1: TANK binding kinase 1; WB: western blot.

Imaging and Harnessing Percolation at the Metal-Insulator Transition of NdNiO3 Nanogaps.

Competition between coexisting electronic phases in first-order phase transitions can lead to a sharp change in the resistivity as the material is subjected to small variations in the driving parameter, for example, the temperature. One example of this phenomenon is the metal-insulator transition (MIT) in perovskite rare-earth nickelates. In such systems, reducing the transport measurement area to dimensions comparable to the domain size of insulating and metallic phases around the MIT should strongly influence the shape of the resistance-temperature curve. Here we measure the temperature dependence of the local resistance and the nanoscale domain distribution of NdNiO3 areas between Au contacts gapped by 40-260 nm. We find that a sharp resistance drop appears below the bulk MIT temperature at ∼105 K, with an amplitude inversely scaling with the nanogap width. By using X-ray photoemission electron microscopy, we directly correlate the resistance drop to the emergence and distribution of individual metallic domains at the nanogap. Our observation provides useful insight into percolation at the MIT of rare-earth nickelates.

Imaging mitophagy in the fruit fly.

Loss-of-function mutations in the genes encoding PRKN/parkin and PINK1 cause autosomal recessive Parkinson disease (PD). Seminal work in Drosophila revealed that loss of park/parkin and Pink1 causes prominent mitochondrial pathology in flight muscle and, to a lesser extent, in dopaminergic neurons. Subsequent studies in cultured mammalian cells discovered a crucial role for PRKN/PARK2 and PINK1 in selective macroautophagic removal of mitochondria (mitophagy). However, direct evidence for the existence of a PINK1-PRKN/PARK2-mediated mitophagy pathway in vivo is still scarce. Recently, we engineered Drosophila that express the mitophagy reporter mt-Keima. We demonstrated that mitophagy occurs in flight muscle cells and dopaminergic neurons in vivo and increases with aging. Moreover, this age-dependent rise depends on park and Pink1. Our data also suggested that some aspects of the mitochondrial phenotype of park- and Pink1-deficient flies are independent of the mitophagy defect, and that park and Pink1 may have multiple functions in the regulation of the integrity of these organelles. Here, we discuss implications of these findings as well as possible future applications of the mt-Keima fly model.

Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila.

Mutations in the genes for PINK1 and parkin cause Parkinson's disease. PINK1 and parkin cooperate in the selective autophagic degradation of damaged mitochondria (mitophagy) in cultured cells. However, evidence for their role in mitophagy in vivo is still scarce. Here, we generated a Drosophila model expressing the mitophagy probe mt-Keima. Using live mt-Keima imaging and correlative light and electron microscopy (CLEM), we show that mitophagy occurs in muscle cells and dopaminergic neurons in vivo, even in the absence of exogenous mitochondrial toxins. Mitophagy increases with aging, and this age-dependent rise is abrogated by PINK1 or parkin deficiency. Knockdown of the Drosophila homologues of the deubiquitinases USP15 and, to a lesser extent, USP30, rescues mitophagy in the parkin-deficient flies. These data demonstrate a crucial role for parkin and PINK1 in age-dependent mitophagy in Drosophila in vivo.

Direct Mapping of Phase Separation across the Metal-Insulator Transition of NdNiO3.

Perovskite rare-earth nickelates RNiO3 are prototype correlated oxides displaying a metal-insulator transition (MIT) at a temperature tunable by the ionic radius of the rare-earth R. Although its precise origin remains a debated topic, the MIT can be exploited in various types of applications, notably for resistive switching and neuromorphic computation. So far, the MIT has been mostly studied by macroscopic techniques, and insights into its nanoscale mechanisms were only provided recently by X-ray photoemission electron microscopy through absorption line shifts, used as an indirect proxy to the resistive state. Here, we directly image the local resistance of NdNiO3 thin films across their first-order MIT using conductive-atomic force microscopy. Our resistance maps reveal the nucleation of ∼100-300 nm metallic domains in the insulating state that grow and percolate as temperature increases. We discuss the resistance contrast mechanism, analyze the microscopy and transport data within a percolation model, and propose experiments to harness this mesoscopic electronic texture in devices.

The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy.

Loss-of-function mutations in PARK2, the gene encoding the E3 ubiquitin ligase Parkin, are the most frequent cause of recessive Parkinson's disease (PD). Parkin translocates from the cytosol to depolarized mitochondria, ubiquitinates outer mitochondrial membrane proteins and induces selective autophagy of the damaged mitochondria (mitophagy). Here, we show that ubiquitin-specific protease 15 (USP15), a deubiquitinating enzyme (DUB) widely expressed in brain and other organs, opposes Parkin-mediated mitophagy, while a panel of other DUBs and a catalytically inactive version of USP15 do not. Moreover, knockdown of USP15 rescues the mitophagy defect of PD patient fibroblasts with PARK2 mutations and decreased Parkin levels. USP15 does not affect the ubiquitination status of Parkin or Parkin translocation to mitochondria, but counteracts Parkin-mediated mitochondrial ubiquitination. Knockdown of the DUB CG8334, the closest homolog of USP15 in Drosophila, largely rescues the mitochondrial and behavioral defects of parkin RNAi flies. These data identify USP15 as an antagonist of Parkin and suggest that USP15 inhibition could be a therapeutic strategy for PD cases caused by reduced Parkin levels.

Ambra1: a Parkin-binding protein involved in mitophagy.

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.

Parkin interacts with Ambra1 to induce mitophagy.

Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy.