Heterozygous PINK1 p.G411S increases risk of Parkinson's disease via a dominant-negative mechanism.

SEE GANDHI AND PLUN-FAVREAU DOI101093/AWW320 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: It has been postulated that heterozygous mutations in recessive Parkinson's genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson's disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson's disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson's disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.

(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.

Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development.

A fluorescence-based assay for the measurement of S-adenosylhomocysteine hydrolase activity in biological samples.

The methylation of DNA, RNA, and proteins plays crucial roles in numerous biological processes, including epigenetic control, virus replication, and cell differentiation. In mammals, the rate-limiting step of the S-adenosylmethionine-dependent methylation process is exclusively controlled by S-adenosylhomocysteine (S-AdoHcy) hydrolase (SAHH). SAHH hydrolyzes S-AdoHcy to adenosine and homocysteine (Hcy) and is therefore a potential therapeutic target for various diseases, including cancer, malaria, and viral diseases. However, a simple and highly sensitive assay for the evaluation of SAHH activity, particularly for drug discovery, had not yet been developed. Here we present the development of a fluorescence-based assay for the measurement of SAHH activity in biological samples. We combined the advantages of the detection of fluorescent thiol groups in Hcy by ThioGlo1 with the S-AdoHcy-driven enzyme-coupled reaction. Our results confirmed the reliability of the proposed assay for the measurement of the SAHH activity of purified SAHH and showed the potential of this assay for the measurement of the SAHH activity of biological samples. Therefore, the proposed SAHH activity assay may be utilized in clinical laboratories and in high-throughput screenings for the identification of new SAHH inhibitors with potentially beneficial effects on numerous pathologies.

Substitution of PINK1 Gly411 modulates substrate receptivity and turnover.

The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development.Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.

Genetic ablation and chemical inhibition of IP3R1 reduce mutant huntingtin aggregation.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Previously, it has been shown that inhibition of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activity reduced aggregation of pathogenic polyQ proteins. Experimentally, this effect was achieved by modification of the intracellular IP3 levels or by application of IP3R1 inhibitors, such as 2-aminoethyl diphenylborinate (2-APB). Unfortunately, there are certain concerns about the 2-APB specificity and cytotoxicity. Moreover, a direct link between IP3R1 and polyQ aggregation has not been shown yet. In this study we show, that down-regulation of the IP3R1 levels by shRNA reduced the aggregation of mutant htt. We tested 2-APB analogs in an attempt to identify less toxic and more IP3R1-specific compounds and found that the effect of these analogs on the reduction of the mutant htt aggregation did weakly correlate with their inhibitory action toward the IP3-induced Ca(2+) release (IICR). Their effect on aggregation was not correlated with the store-operated Ca(2+) entry (SOCE), which is another target of the 2-APB related compounds. Our findings suggest that besides functional contribution of the IP3R inhibition on the mutant htt aggregation there are additional mechanisms for the anti-aggregation effect of the 2-APB related compounds.

Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy.

Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson's disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

BACKGROUND: Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. METHODS: Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. RESULTS: Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. CONCLUSIONS: Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients' fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development.

Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate.

This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phostag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.

Pro-apoptotic effect of new quinolone 7- ethyl 9-ethyl-6-oxo-6,9-dihydro[1,2,5]selenadiazolo [3,4-h]quinoline-7-carboxylate on cervical cancer cell line HeLa alone/with UVA irradiation.

7- ethyl 9-ethyl-6-oxo-6,9-dihydro[1,2,5]selenadiazolo [3,4-h]quinoline-7-carboxylate (E2h) is a new synthetically prepared quinolone derivative, which in our primary study showed cytotoxic effects towards tumor cells. The aim of the present study was to examine the antiproliferative and apoptosis inducing activities of E2h towards human cervical cancer cell line HeLa with/without the presence of UVA irradiation. Further, the molecular mechanism involved in E2h-induced apoptosis in HeLa cells was investigated. Our results showed that both non-photoactivated and photoactivated E2h caused morphological changes and inhibited the cell growth of HeLa cells in a time- and dose-dependent manner. Irradiation increased the sensitivity of HeLa cells to E2h. Quinolone induced S and G2/M arrest and apoptosis in HeLa cells, as characterized by DNA fragmentation and flow cytometry. In addition, E2h elevated the level of reactive oxygen species and activated caspases 3. In conclusions, E2h alone/in combination with UVA irradiation induced apoptosis in HeLa cells through the ROS-mitochondrial/caspase 3-dependent pathway.

miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1).

BACKGROUND: Loss-of-function mutations in PINK1 and PARKIN are the most common causes of autosomal recessive Parkinson's disease (PD). PINK1 is a mitochondrial serine/threonine kinase that plays a critical role in mitophagy, a selective autophagic clearance of damaged mitochondria. Accumulating evidence suggests mitochondrial dysfunction is one of central mechanisms underlying PD pathogenesis. Therefore, identifying regulatory mechanisms of PINK1 expression may provide novel therapeutic opportunities for PD. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, little is known about the regulation mechanism of PINK1 at the transcriptional or translational levels. RESULTS: Here, we demonstrated that microRNA-27a (miR-27a) and miR-27b suppress PINK1 expression at the translational level through directly binding to the 3'-untranslated region (3'UTR) of its mRNA. Importantly, our data demonstrated that translation of PINK1 is critical for its accumulation upon mitochondrial damage. The accumulation of PINK1 upon mitochondrial damage was strongly regulated by expression levels of miR-27a and miR-27b. miR-27a and miR-27b prevent mitophagic influx by suppressing PINK1 expression, as evidenced by the decrease of ubiquitin phosphorylation, Parkin translocation, and LC3-II accumulation in damaged mitochondria. Consequently, miR-27a and miR-27b inhibit lysosomal degradation of the damaged mitochondria, as shown by the decrease of the delivery of damaged mitochondria to lysosome and the degradation of cytochrome c oxidase 2 (COX2), a mitochondrial marker. Furthermore, our data demonstrated that the expression of miR-27a and miR-27b is significantly induced under chronic mitophagic flux, suggesting a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b. CONCLUSIONS: We demonstrated that miR-27a and miR-27b regulate PINK1 expression and autophagic clearance of damaged mitochondria. Our data further support a novel negative regulatory mechanism of PINK1-mediated mitophagy by miR-27a and miR-27b. Therefore, our results considerably advance our understanding of PINK1 expression and mitophagy regulation and suggest that miR-27a and miR-27b may represent potential therapeutic targets for PD.

Properties of the basal calcium influx in human red blood cells.

The basal (45)Ca(2+) influx in human red blood cells (RBC) into intact RBC was measured. (45)Ca(2+) was equilibrated with cells with t(1/2)=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5+/-0.2 micromol/l(packed cells) (n=6) at 37 degrees C. The average value of the Ca(2+) influx rate was 43.2+/-8.9 micromol/l(packed cells) hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 degrees C. The basal Ca(2+) influx was saturable with Ca(2+) up to 5 mmol/l but at higher extracellular Ca(2+) concentrations caused further increase of basal Ca(2+) influx. The (45)Ca(2+) influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3',4',5-tetrachloro-salicylanilide (TCS) 10(-6)-10(-5) mol/l) inhibited in part the Ca(2+) influx. The results show that the basal Ca(2+) influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca(2+) influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca(2+) are coupled via the RBC H(+) homeostasis.

Salvia officinalis L. extract and its new food antioxidant formulations induce apoptosis through mitochondrial/caspase pathway in leukemia L1210 cells.

Salvia officinalis, L. (Lamiaceae) is one of the most widespread herbal species used in the area of human health and in the food-processing industry. Salvia and its extracts are known to be a rich source of antioxidants. As shown previously, the crude ethanolic extract of salvia (SE) exerts lower anti-oxidative properties in lard compared to the new salvia food formulations No. 1 (SF1; 32% of SE + 68% of the emulsifier Dimodan S-T) and No. 2 (SF2; 32% of SE + 68% of the emulsifier Topcithin 50). The aim of the present study was to investigate and compare the effects of the SE and its food formulations SF1 and SF2 on the toxicity and/or proliferation of L1210 leukemia cells. We found that SE and both SF1 and SF2 demonstrated different concentration- and time-dependent cytotoxic/antiproliferative cellular effects already within the first 24 h of the treatment. However, SE was nearly 10 times more effective than the new salvia food formulations SF1 and SF2. We investigated partially also the molecular mechanisms lying behind the action of SE, SF1 and SF2 induced apoptosis in our cell model. We found an apparent involvement of the mitochondrial/caspase-dependent pathway in the described processes. Nevertheless, further investigation is needed before salvia extract and its new antioxidant formulations can be included among the potential food antioxidants with protective properties against cancer.

ROCK-phosphorylated vimentin modifies mutant huntingtin aggregation via sequestration of IRBIT.

BACKGROUND: Huntington's Disease (HD) is a fatal hereditary neurodegenerative disease caused by the accumulation of mutant huntingtin protein (Htt) containing an expanded polyglutamine (polyQ) tract. Activation of the channel responsible for the inositol-induced Ca²âº release from ensoplasmic reticulum (ER), was found to contribute substantially to neurodegeneration in HD. Importantly, chemical and genetic inhibition of inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) has been shown to reduce mutant Htt aggregation. RESULTS: In this study, we propose a novel regulatory mechanism of IP3R1 activity by type III intermediate filament vimentin which sequesters the negative regulator of IP3R1, IRBIT, into perinuclear inclusions, and reduces its interaction with IP3R1 resulting in promotion of mutant Htt aggregation. Proteasome inhibitor MG132, which causes polyQ proteins accumulation and aggregation, enhanced the sequestration of IRBIT. Furthermore we found that IRBIT sequestration can be prevented by a rho kinase inhibitor, Y-27632. CONCLUSIONS: Our results suggest that vimentin represents a novel and additional target for the therapy of polyQ diseases.

Huntington's disease, calcium, and mitochondria.

Huntington's disease (HD) is caused by a mutation that increases the number of CAG repeats in the gene encoding for the protein Huntingtin (Htt). The mutation results in the pathological expansion of the polyQ stretch that is normally present within the N-terminal region of Htt. Even if Htt is ubiquitously expressed in tissues, the changes in the protein finally result in the clinical manifestation of motor and cognitive impairments observed in HD patients. The molecular ethiology of the disease is obscure: a number of cellular and animal models are used as essential tools in experimental approaches aimed at understanding it. Biochemical changes have been described that correlate with the malfunction of HD neurons (primarily in the striatum): consensus is gradually emerging that the dyshomeostasis of Ca(2+) and/or mitochondria stress are important factors in the linkage of the Htt mutation to the onset and progression of the disease. Here, we present a succint overview of the changes of Htt, of its possible effect on the transcription of critical genes and of its causative role in the disturbance of the neuronal Ca(2+) homeostasis. Particular emphasis will be placed on the role of mitochondria as key player in the molecular pathogenesis of the disease.

Mitochondrial fission and cristae disruption increase the response of cell models of Huntington's disease to apoptotic stimuli.

Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, is accompanied by multiple mitochondrial alterations. Here, we show that mitochondrial fragmentation and cristae alterations characterize cellular models of HD and participate in their increased susceptibility to apoptosis. In HD cells, the increased basal activity of the phosphatase calcineurin dephosphorylates the pro-fission dynamin related protein 1 (Drp1), increasing its mitochondrial translocation and activation, and ultimately leading to fragmentation of the organelle. The fragmented HD mitochondria are characterized by cristae alterations that are aggravated by apoptotic stimulation. A genetic analysis indicates that correction of mitochondrial elongation is not sufficient to rescue the increased cytochrome c release and cell death observed in HD cells. Conversely, the increased apoptosis can be corrected by manoeuvres that prevent fission and cristae remodelling. In conclusion, the cristae remodelling of the fragmented HD mitochondria contributes to their hypersensitivity to apoptosis.

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts.

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.