Programmed cell death-2 isoform1 is ubiquitinated by parkin and increased in the substantia nigra of patients with autosomal recessive Parkinson’s disease

AbstractMutations in parkin gene are responsible for autosomal recessive Parkinson’s disease (ARPD) and its loss-of-function is assumed to affect parkin ubiquitin ligase activity. Accumulation of its substrate may induce dopaminergic neurodegeneration in the substantia nigra (SN) of ARPD. Here, we show that parkin interacts with programmed cell death-2 isoform 1 (PDCD2-1) and promotes its ubiquitination. Furthermore, accumulation of PDCD2-1 was found in the SN of ARPD as well as in sporadic PD, suggesting that common failure of the ubiquitin–proteasome system is associated with neuronal death in both ARPD and sporadic PD.Structured summary:MINT-6805975, MINT-6806032, MINT-6806051, MINT-6806070:PDCD2 (uniprotkb:Q16342) physically interacts (MI:0218) with Parkin (uniprotkb:O60260) by anti tag coimmunoprecipitation (MI:0007)MINT-6805947:Parkin (uniprotkb:O60260) physically interacts (MI:0218) with PDCD2 (uniprotkb:Q16342) by two hybrid (MI:0018)MINT-6806000: PDCD2 (uniprotkb:Q16342) physically interacts (MI:0218) with ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)

PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy

Defective mitochondrial quality control is shown to be a mechanism for neurodegeneration in some forms of Parkinson's disease

Parkinson’s disease-associated iPLA2-VIA/PLA2G6 regulates neuronal functions and α-synuclein stability through membrane remodeling

Mutations in the iPLA2-VIA/PLA2G6 gene are responsible for PARK14-linked Parkinson’s disease (PD) with α-synucleinopathy. However, it is unclear how iPLA2-VIA mutations lead to α-synuclein (α-Syn) aggregation and dopaminergic (DA) neurodegeneration. Here, we report that iPLA2-VIA–deficient Drosophila exhibits defects in neurotransmission during early developmental stages and progressive cell loss throughout the brain, including degeneration of the DA neurons. Lipid analysis of brain tissues reveals that the acyl-chain length of phospholipids is shortened by iPLA2-VIA loss, which causes endoplasmic reticulum (ER) stress through membrane lipid disequilibrium. The introduction of wild-type human iPLA2-VIA or the mitochondria–ER contact site-resident protein C19orf12 in iPLA2-VIA–deficient flies rescues the phenotypes associated with altered lipid composition, ER stress, and DA neurodegeneration, whereas the introduction of a disease-associated missense mutant, iPLA2-VIA A80T, fails to suppress these phenotypes. The acceleration of α-Syn aggregation by iPLA2-VIA loss is suppressed by the administration of linoleic acid, correcting the brain lipid composition. Our findings suggest that membrane remodeling by iPLA2-VIA is required for the survival of DA neurons and α-Syn stability

Twin CHCH Proteins, CHCHD2, and CHCHD10: Key Molecules of Parkinson’s Disease, Amyotrophic Lateral Sclerosis, and Frontotemporal Dementia

Mutations of coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) have been found to be linked to Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and/or frontotemporal lobe dementia (FTD). CHCHD2 and CHCHD10 proteins, which are homologous proteins with 54% identity in amino acid sequence, belong to the mitochondrial coiled-coil-helix-coiled-coil-helix (CHCH) domain protein family. A series of studies reveals that these twin proteins form a multimodal complex, producing a variety of pathophysiology by the disease-causing variants of these proteins. In this review, we summarize the present knowledge about the physiological and pathological roles of twin proteins, CHCHD2 and CHCHD10, in neurodegenerative diseases

PINK1-Mediated Phosphorylation of Parkin Boosts Parkin Activity in <i>Drosophila</i>

<div><p>Two genes linked to early onset Parkinson's disease, <i>PINK1</i> and <i>Parkin</i>, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons <i>in vivo</i>. Here, we examined the effects of Parkin phosphorylation in <i>Drosophila</i>, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in <i>Parkin</i>-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons <i>in vivo</i>, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.</p></div

Phosphorylation of the Parkin Ubl domain regulates mitochondrial morphology.

<p>(<b>A</b>) Parkin is phosphorylated by PINK1 in insect cells. S2 cells transfected with the indicated plasmids with or without <i>Drosophila</i> PINK1 were treated with or without 30 µM carbonyl cyanide <i>m</i>-chlorophenylhydrazone (CCCP) for 1 h. The cell lysate was subsequently separated on a Phos-tag gel, followed by western blotting with anti-<i>Drosophila</i> Parkin. (<b>B</b>) The phosphorylation status of Parkin affects the mitochondrial length in muscle tissue. Fluorescent and TEM images of the indirect flight muscle in the indicated genotypes of 14-day-old adult flies are shown. To visualize the mitochondria, the mitoGFP (green) transgene was co-expressed, and the muscle tissue was counterstained with phalloidin (magenta). Mitochondria in the TEM images are outlined with broken lines to highlight their morphology. Scale bars = 10 µm in the fluorescent images and 2 µm in the TEM images. (<b>C</b>) Mitochondrial morphology of 14-day-old <i>PINK1</i> mutant flies expressing mock, WT Parkin and phospho-mutants. The inset shows a high-magnification TEM image of <i>PINK1^-/-; Parkin SE</i> with intact mitochondrial matrices. Scale bars = 10 µm in the fluorescent images and 2 µm in the TEM images. (<b>D</b>) The length of the long axis of the muscle mitochondria was calculated. The data represent the mean ± SE from three flies (<i>n</i> = 25 in each). ** <i>p</i><0.01 <i>vs</i>. all other genotypes, # <i>p</i><0.01 <i>vs</i>. mock with <i>PINK1^+/+</i>, § <i>p</i><0.01 <i>vs</i>. <i>PINK1^RNAi; WT</i> or <i>SA Parkin</i>, * <i>p</i><0.05, N.S., not significant. (<b>E</b>) Mitochondrial morphology of 14-day-old <i>Parkin</i> mutant flies expressing mock, WT or SA Parkin. Scale bars = 10 µm in all fluorescent images and 2 µm for <i>Parkin^-/-</i> and <i>WT Parkin; Parkin^-/-</i> and 5 µm for <i>SA Parkin; Parkin^-/-</i> in the TEM images. Arrowheads indicate large mitochondrial aggregates. (<b>F</b>) The length of the long axis of the muscle mitochondria was calculated. The data represent the mean ± SE from three flies (<i>n</i> = 25 in each). * <i>p</i><0.05. The genotypes are as follows: (<b>B</b>) <i>UAS-mitoGFP/+</i>; <i>MHC-GAL4/+</i> (control), <i>UAS-mitoGFP/UAS-Parkin</i>; <i>MHC-GAL4</i> (<i>WT, SA</i> and <i>SE Parkin</i>). (<b>C</b>) <i>PINK1^B9/Y; UAS-LacZ</i>; <i>MHC-GAL4</i> (<i>PINK1^-/-</i>), <i>PINK1^B9/Y; UAS-Parkin</i>; <i>MHC-GAL4</i> (<i>PINK1^-/-; WT, SA</i> or <i>SE Parkin</i>). <i>UAS-mitoGFP; MHC-GAL4, UAS-PINK1 RNAi</i> crosses were used rather than <i>PINK1^B9</i> crosses for fluorescent images. (<b>E</b>) <i>UAS-mitoGFP/UAS-LacZ</i>; <i>Da-GAL4, Parkin^Δ21/Parkin¹</i> (<i>Parkin^-/-</i>), <i>UAS-mitoGFP/UAS-Parkin</i>; <i>Da-GAL4, Parkin^Δ21/Parkin¹</i> (<i>Parkin^-/-; WT</i> or <i>SA Parkin</i>).</p

Parkin phosphorylation regulates mitochondrial morphology and the distribution of DA neurons.

<p>Mock (<b>A, A′, E, E′</b>), WT Parkin (<b>B, B′, F, F′</b>), SA Parkin (<b>C, C′, G, G′</b>) or SE Parkin (<b>D, D′, H, H′</b>) were expressed together with mitoGFP in DA neurons of <i>PINK1^+/+</i> (<b>A–D′</b>) or <i>PINK1^-/-</i> (<b>E–H′</b>) flies using the <i>TH</i> driver. Mitochondria and cell bodies of the PPM1/2 cluster DA neurons were visualized using mitoGFP (green in <b>A–H</b> and white in <b>A′–H′</b>) and anti-TH staining (magenta in <b>A–H</b>), respectively. Images (<b>A–H</b>) are higher magnifications of the boxes shown in (<b>A′–H′</b>). Scale bars = 5 µm in (<b>A–H</b>) and 10 µm in (<b>A′–H′</b>). (<b>i</b>) Graph showing the total lengths of mitochondria observed in the cell bodies. # <i>p</i><0.05 <i>vs</i>. mock in <i>PINK1^+/+</i>, § <i>p</i><0.05 <i>vs</i>. WT or SE in <i>PINK1^-/-</i>, ¶ <i>p</i><0.05 <i>vs</i>. all other genotypes in <i>PINK1^-/-</i>, ** <i>p</i><0.01, N.S., not significant. (<b>j</b>) Graph showing the mean areas of aggregated mitochondria greater than 3 µm in each genotype. # <i>p</i><0.05 <i>vs</i>. all other genotypes in <i>PINK1^+/+</i> or <i>PINK1^-/-</i>, § <i>p</i><0.05 <i>vs</i>. mock in <i>PINK1^+/+</i>. ** <i>p</i><0.001, * <i>p</i><0.05 (Student's <i>t</i>-test).</p

Parkin phosphorylation modulates dopaminergic function and survivability of DA neurons.

<p>(<b>A</b>) Confocal images of DA neurons (magenta) from the brains of 5-day-old flies expressing VMAT-pHluorin (green) together with the control LacZ and WT, SA or SE Parkin (Left). The <i>TH</i> driver was used for transgene expression. VMAT-pHluorin signals are shown as white signals (Right). Scale bar = 100 µm. (<b>B</b>) The expression of SA or SE Parkin causes early impairment of vesicular dopamine release in the nerve terminals of DA neurons. The fluorescence recovery rate of DA neuron terminals in cultured neural tissue post-photobleaching was used to estimate the spontaneous DA release rate. Data are shown as the mean ± SE. * <i>p</i><0.05 <i>vs.</i> LacZ and WT, # <i>p</i><0.01 <i>vs</i>. WT. n = 15–20. (<b>C</b>) The flying activity of 15- and 22-day-old males expressing LacZ (control) and those expressing WT, SA and SE Parkin using the <i>TH</i> driver. Data are shown as the mean ± SE. ** <i>p</i><0.01 <i>vs</i>. control and WT. n = 10. (<b>D</b>) Quantification of the number of TH⁺ DA neurons in the PPM1/2 clusters in 5- and 25-day-old males expressing LacZ (control) and those expressing WT, SA and SE Parkin using the <i>TH</i> driver. Data are shown as the mean ± SE. ** <i>p</i><0.01 <i>vs</i>. control of the same age, # <i>p</i><0.01, N.S., not significant. n = 17–20 for control, WT and SA at 45 days. n = 5–8 for flies at 5 days and for SE flies at 45 days. (<b>E</b>) The same assay shown in (<b>D</b>) was performed in the <i>PINK1</i>-deficient flies. * <i>p</i><0.05, ** <i>p</i><0.01 <i>vs</i>. control of the same age, # <i>p</i><0.01 <i>vs</i>. control and WT at 45 days, § <i>p</i><0.01 <i>vs</i>. SA at 45 days. n = 17–20 at 45 days. n = 6–9 at 5 days. (<b>F</b>) Survival curves of flies post-eclosion. Expression of WT, SA and SE Parkin in muscle tissues using the <i>MHC</i> driver rescued the shortened lifespan of <i>PINK1</i>-deficient flies (<i>p</i><1⁻²⁰ by log-rank test; n = 93–170 male flies); there were no differences in the rescue effect among WT, SA and SE. (<b>G</b>) SA Parkin (<i>P</i><1⁻¹¹ by log-rank test; n = 146 male flies) or SE Parkin (<i>P</i><1⁻²⁰; n = 223) expression in DA neurons using the <i>TH</i> driver shortened the lifespan compared with the control LacZ (n = 320) and WT Parkin (n = 154).</p

Constitutive expression of SE Parkin impairs motor behavior.

<p>(<b>A</b>) LacZ and WT, SA or SE Parkin were expressed under the control of <i>MHC-GAL4</i> in the <i>w-</i> background. WT and SA Parkin improved the motor activity in older flies, whereas SE Parkin compromised motor activity throughout the trials. LacZ served as a control. The values are presented as the mean ± SE from 20 trials. Male flies were used for the assay. **<i>P</i><0.01 <i>vs</i>. <i>LacZ</i>; #<i>P</i><0.05. Flies expressing SE Parkin had decreased climbing ability compared with flies expressing WT or SA Parkin throughout the trial (<i>P</i><0.01). (<b>B</b>) Muscle-specific expression of Parkin SE under control of <i>MHC-GAL4</i> failed to rescue the motor defect of the <i>PINK1^-/-</i> flies. The loss of climbing ability in the <i>PINK1^-/-</i> flies was rescued by expression of WT and SA Parkin. The values represent the mean ± SE from 20 trials. Male flies were used for the assay. **<i>P</i><0.01 <i>vs</i>. <i>LacZ</i>. Flies expressing SE Parkin had decreased climbing ability compared with flies expressing WT or SA Parkin throughout the trial (<i>P</i><0.01).</p

Effects of phospho-mutant forms of Parkin on various phenotypes.

a<p>Compared with <i>PINK1^-/-; LacZ</i>. ^bCompared with <i>Parkin^-/-; mRFP</i>. ^cCompared with <i>LacZ</i> at 28-day-old. ^dCompared with <i>PINK1^-/-; LacZ</i> at 28-day-old. N.D., not determined.</p