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
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
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
in vivo.
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.
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 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.
Background
A major hurdle in developing effective treatments for Alzheimer’s Disease (AD) is the fact that pathological hallmarks, such as amyloid beta (Aβ) plaques, develop decades before the first ...clinical symptoms emerge. Moreover, the risk of developing AD is strongly influenced by genetic background. In addition, the role of microglia in the disease pathway is undeniable, albeit dual and complex. We therefore aim to determine the influence of patient genetic background and immunophenotype on the microglial response to AD pathology.
Method
Subject material was sequenced and underwent deep immunophenotyping. Based on their scores patient iPSCs were divided in a high risk and low risk group of developing AD. This microglial response is determined by (i) their phagocytic capacity and (ii) their (change in) morphology. iPSC cells were differentiated into microglia (Mancuso et al., 2019), followed by RT‐qPCR to verify their identity. When microglia are incubated with pHrodo beads, these are phagocytosed and transported to the acidic lysosome, yielding a pH dependent increase in fluorescence, measured with an Operetta confocal microscope. Microglial morphology was recorded in an Incucyte and tracked and scored (e.g. amoeboid, rod‐like, size, motility …) automatically by AI based software. The change in morphology was assessed after addition of stressors such as LPS or Aβ1‐42.
Result
Phagocytotic capacity of a reference microglia strain, measured by pHrodo intensity, reached a maximum level approximately 2 hours after addition of the pHrodo beads. When cytochalasin D, an Actin polymerization inhibitor, was added to the medium, the phagocytosis process is inhibited and baseline pHrodo intensity levels are observed. In addition, the addition of LPS seemed to change the morphology of microglia to a more elongated rod‐like shape with less motility.
Conclusion
The phagocytic capacity of microglia could be dependent on the genetic background and immunophenotype of the iPSC strain. Similarly, the effect on morphology (e.g. more rod‐like shape) as an answer to stressors (e.g. LPS) could be dependent on the microglia strain. The differential microglial response based on genetic background and immunophenotype might yield insight into the further segmentation of different types of Alzheimer’s Disease and personalized therapeutic approaches.
Background
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder with more than half of the patients developing dementia about 10 years later, and is characterized by the ...accumulation of alpha‐synuclein protein aggregates in the PD brain. These inclusions, also called Lewy bodies, accompanied with the degeneration of dopaminergic neurons are the neuropathological hallmark of this devastating incurable disease. Currently a lot of effort is put in studying the involvement of different forms of alpha‐synuclein, i.e. monomeric, oligomeric, fibrillar and aggregated forms or different post translational modifications of the protein, and their contribution to disease progression. Targeting alpha‐synuclein might thus be of potential therapeutic value. Since the research field lacks good and consistent animal models for preclinical research, we’ve set up a mouse model based on the striatal administration of sonicated preformed fibrils (PFF).
Method
In brief, we performed unilateral stereotactical injections in the dorsal striatum of young wild type mice with sonicated PFF’s (n = 8/group). PFF injected mice were sacrificed after 5, 9 and 13 weeks, a control group of vehicle injected mice (n = 7) was sacrificed after 9 weeks. Phosphorylated alpha‐synuclein (pSer129) seed and spread and dopaminergic neurodegeneration was visualized by IHC in different brain regions both ipsilateral and contralateral.
Result
Phosphorylated alpha‐synuclein (pSer129) positive inclusions were shown to be present in Striatum, substantia nigra (SN) and amygdala in the ipsilateral hemisphere already after 5 weeks, indicating clear spreading of pathology. Interestingly, in the contralateral hemisphere we were able to detect clear pSer129 alpha‐synuclein in the amygdala and striatum 5 and 9 weeks after injection respectively, demonstrating the progressive nature of this model. To assess whether the observed pathology also affected the dopaminergic circuitry we quantified the loss of synaptic tyrosine hydroxylase positive terminals in the striatum and found a significant decrease already 5 weeks after PFF administration.
Conclusion
This alpha‐synuclein preformed fibril based model, which shows clear progressive seed and spread pathology and a clear decrease in dopaminergic nerve terminals in the striatum, can be of great importance to the research field to assess in vivo therapeutic interventions based on targeting alpha‐synuclein pathology.
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.
Abstract
Background
To improve clinical translatability of non‐clinical in‐vivo Alzheimer’s disease (AD) models, a humanized APP knock‐in mouse model (APP
SAA
) was recently created (Xia, D. et al., ...2022). This homozygous APP knock‐in model carries a humanized Aβ
1‐42
sequence and 3 disease causing mutations (
S
wedish,
A
rctic, and
A
ustrian). These modifications lead to increased Aβ
42/40
ratios in AD relevant tissues, resulting in an age‐dependent amyloid deposition in the brain. The highest plaque density is found in cortical and hippocampal regions. APP
SAA
mice also display clear neuroinflammation and an increase in fluid biomarkers of neurodegeneration (NF‐L and total Tau in CSF). Here we assess the value of this model as a tool for non‐clinical efficacy studies of experimental drugs with diverse mechanisms of action to facilitate the development of novel AD therapeutics.
Method
APP
SAA
mice and WT controls were aged and sacrificed at various time points. In addition, APP
SAA
mice were treated with drug or vehicle, on a daily basis for 3 months from the age of 3 months onward. The major pathological hallmarks of this model and the treatment effect will be investigated with biochemical and immunohistological assays. Monthly blood sampling will allow the follow up of pathology progression in individual mice by assessing plasma‐based biomarkers. Since APP
SAA
mice lack an obvious behavioral phenotype, synaptic plasticity will be investigated to serve as a functional readout.
Result
The treatment is well tolerated by the mice in study. Body weights were not altered between compound and vehicle dosing during the first 2 months of the treatment. Data on pathological outcomes and synaptic plasticity are not yet available but will be shared during the presentation.
Conclusion
A humanized knock‐in model for AD is hypothesized to be a broadly applicable tool to investigate disease‐modifying drugs with diverse modes of actions.
Background
To improve clinical translatability of non‐clinical in‐vivo Alzheimer’s disease (AD) models, a humanized APP knock‐in mouse model (APPSAA) was recently created (Xia, D. et al., 2022). This ...homozygous APP knock‐in model carries a humanized Aβ1‐42 sequence and 3 disease causing mutations (Swedish, Arctic, and Austrian). These modifications lead to increased Aβ42/40 ratios in AD relevant tissues, resulting in an age‐dependent amyloid deposition in the brain. The highest plaque density is found in cortical and hippocampal regions. APPSAA mice also display clear neuroinflammation and an increase in fluid biomarkers of neurodegeneration (NF‐L and total Tau in CSF). Here we assess the value of this model as a tool for non‐clinical efficacy studies of experimental drugs with diverse mechanisms of action to facilitate the development of novel AD therapeutics.
Method
APPSAA mice and WT controls were aged and sacrificed at various time points. In addition, APPSAA mice were treated with drug or vehicle, on a daily basis for 3 months from the age of 3 months onward. The major pathological hallmarks of this model and the treatment effect will be investigated with biochemical and immunohistological assays. Monthly blood sampling will allow the follow up of pathology progression in individual mice by assessing plasma‐based biomarkers. Since APPSAA mice lack an obvious behavioral phenotype, synaptic plasticity will be investigated to serve as a functional readout.
Result
The treatment is well tolerated by the mice in study. Body weights were not altered between compound and vehicle dosing during the first 2 months of the treatment. Data on pathological outcomes and synaptic plasticity are not yet available but will be shared during the presentation.
Conclusion
A humanized knock‐in model for AD is hypothesized to be a broadly applicable tool to investigate disease‐modifying drugs with diverse modes of actions.
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.
PDF
