Bioenergetic consequences of PINK1 mutations in Parkinson disease

Background

Mutations of the gene for PTEN-induced kinase 1 (PINK1) are a cause of familial Parkinson''s disease (PD). PINK1 protein has been localised to mitochondria and PINK1 gene knockout models exhibit abnormal mitochondrial function. The purpose of this study was to determine whether cells derived from PD patients with a range of PINK1 mutations demonstrate similar defects of mitochondrial function, whether the nature and severity of the abnormalities vary between mutations and correlate with clinical features.

Methodology

We investigated mitochondrial bioenergetics in live fibroblasts from PINK1 mutation patients using single cell techniques. We found that fibroblasts from PINK1 mutation patients had significant defects of bioenergetics including reduced mitochondrial membrane potential, altered redox state, a respiratory deficiency that was determined by substrate availability, and enhanced sensitivity to calcium stimulation and associated mitochondrial permeability pore opening. There was an increase in the basal rate of free radical production in the mutant cells. The pattern and severity of abnormality varied between different mutations, and the less severe defects in these cells were associated with later age of onset of PD.

Conclusions

The results provide insight into the molecular pathology of PINK1 mutations in PD and also confirm the critical role of substrate availability in determining the biochemical phenotype – thereby offering the potential for novel therapeutic strategies to circumvent these abnormalities.     

Copy number variation in familial Parkinson disease

Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility.     

Role of EIF4G1 network in non-small cell lung cancers (NSCLC) cell survival and disease progression

Although the Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) has been found overexpressed in a variety of cancers, its role in non–small cell lung cancers (NSCLC) pathogenesis especially in immunoregulatory functions, its clinical relevance and therapeutic potential remain largely unknown. By using cancer patients tissue assays, the results indicate that EIF4G1 expressional levels are much higher in NSCLC tissues than in adjacent or normal lung tissues, which are also associated with NSCLC patient survival. By using an RNA-Sequencing based pipeline, the data show that EIF4G1 has a significant association with immune checkpoint molecules such as PD-1/PD-L1 in NSCLC. EIF4G1 small-molecule inhibitors effectively repress NSCLC growth in cell culture and xenograft animal models. Protein array results identify the signature of proteins controlled by EIF4G1 in NSCLC cells, in which new candidates such as MUC1 and NRG1 are required for NSCLC survival and tumorigenesis with clinical relevance. Taken together, these results have for the first time demonstrated the immunoregulatory functions, clinical relevance and therapeutic potential of the EIF4G1 network in NSCLC, which may represent a promising and novel target to improve lung cancer treatment.     

Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.     

Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.     

Genomewide association study for susceptibility genes contributing to familial Parkinson disease

Five genes have been identified that contribute to Mendelian forms of Parkinson disease (PD); however, mutations have been found in fewer than 5% of patients, suggesting that additional genes contribute to disease risk. Unlike previous studies that focused primarily on sporadic PD, we have performed the first genomewide association study (GWAS) in familial PD. Genotyping was performed with the Illumina HumanCNV370Duo array in 857 familial PD cases and 867 controls. A logistic model was employed to test for association under additive and recessive modes of inheritance after adjusting for gender and age. No result met genomewide significance based on a conservative Bonferroni correction. The strongest association result was with SNPs in the GAK/DGKQ region on chromosome 4 (additive model: p = 3.4 × 10⁻⁶; OR = 1.69). Consistent evidence of association was also observed to the chromosomal regions containing SNCA (additive model: p = 5.5 × 10⁻⁵; OR = 1.35) and MAPT (recessive model: p = 2.0 × 10⁻⁵; OR = 0.56). Both of these genes have been implicated previously in PD susceptibility; however, neither was identified in previous GWAS studies of PD. Meta-analysis was performed using data from a previous case–control GWAS, and yielded improved p values for several regions, including GAK/DGKQ (additive model: p = 2.5 × 10⁻⁷) and the MAPT region (recessive model: p = 9.8 × 10⁻⁶; additive model: p = 4.8 × 10⁻⁵). These data suggest the identification of new susceptibility alleles for PD in the GAK/DGKQ region, and also provide further support for the role of SNCA and MAPT in PD susceptibility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. Pankratz and J. B. Wilk are joint first authors.     

Functional impairment in RHOT1/Miro1 degradation and mitophagy is a shared feature in familial and sporadic Parkinson disease

Mitophagy is a conserved and highly regulated process of selective degradation crucial in maintaining normal cellular physiology. Genetic defects and cellular aberrations affecting mitophagy have been associated with the development of Parkinson disease. In their recently published article (highlighted in a punctum in this issue of the journal) Hsieh et al. present a putative mitophagy marker, which serves as a mechanistic link between sporadic and familial Parkinson disease.     

Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2)

Only a few cases of exclusive translation initiation at non-AUG codons have been reported. We recently demonstrated that mammalian NAT1 mRNA, encoded by EIF4G2, uses GUG as its only translation initiation codon. In this study, we identified NAT1 orthologs from chicken, Xenopus, and zebrafish and found that in all species, the GUG codon also serves as the initiation codon. In all species, the GUG codon fulfilled the reported requirements for non-AUG initiation: an optimal Kozak motif and a downstream hairpin structure. Site-directed mutagenesis showed that nucleotides at positions -3 and +4 are critical for the GUG-mediated translation initiation in vitro. We found that NAT1 orthologs in Drosophila melanogaster and Halocynthia roretzi also use non-AUG start codons, demonstrating evolutionary conservation of the noncanonical translation initiation.     

Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy

The knowledge gap separating the molecular and cellular underpinnings of Parkinson disease (PD) and its pathology hinders treatment innovation. Adding to this difficulty is the lack of a reliable biomarker for PD. Our previous studies identify a link of 2 PD proteins, PINK1/PRKN Parkin to a mitochondrial motor adaptor RHOT1/Miro-1, which mediates mitochondrial motility and mitophagy. Here we review our recent paper showing that a third PD protein, LRRK2, also targets RHOT1 and regulates mitophagy, and pathogenic LRRK2 disrupts this function. Notably, we discover impairments in RHOT1 and mitophagy in sporadic PD patients with no known genetic backgrounds, pointing to RHOT1-mediated mitophagy as a convergent pathway in PD. This novelty opens new doors in PD research toward RHOT1-based therapy and biomarker development.     

The role of iron in Parkinson disease and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity

Parkinson disease (PD) involves the specific degeneration of dopaminergic neurons of the pars compacta of the substantia nigra. Although the cause of the degeneration of nigrostriatal dopaminergic neurons in PD is unknown, there is significant evidence to suggest that oxidative stress may be involved in this process. This review specifically examines the current status of evidence suggesting iron may contribute to oxidative damage associated with PD.     

Assignment of two of the translation initiation factor-4E (EIF4EL1 and EIF4EL2) genes to human chromosomes 4 and 20

The eukaryotic translation initiation factor (eIF-4E) has recently been cloned from human, mouse, and yeast. This polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. We have designed oligonucleotide primers to the 3' untranslated region of the gene encoding eIF-4E and specifically amplified the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this method, one of the human eIF-4E genes (EIF4EL1, eukaryotic translation initiation factor 4E-like 1) has been mapped to human chromosome 4qter-4p15. In addition, we have localized a second eIF-4E gene (EIF4EL2, eukaryotic translation initiation factor 4E-like 2) to human chromosome 20 by Southern blot analysis of mapping panels established from human/rodent somatic cell hybrids.     

Staurosporine-induced activation of caspase-3 is potentiated by presenilin 1 familial Alzheimer's disease mutations in human neuroglioma cells

Deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, has been known to cause an inhibition of DNA synthesis and cell growth, and to induce apoptosis in nonneuronal cells. To investigate whether this is also the case in neurons, we examined the effect of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor on the viability of neuronal cultures prepared from fetal rat brains. Treatment with compactin, a competitive inhibitor of HMG-CoA reductase, induced neuronal death in a dose-dependent manner. Concurrent treatment with cholesterol, beta-migrating very low density lipoprotein, mevalonate, or squalene substantially inhibited the induction of neuronal death by compactin. Cell death was also induced by treatment with squalestatin, which specifically inhibits cholesterol biosynthesis at a site downstream from the generation of nonsterol metabolites. Furthermore, squalestatin-induced neuronal death was inhibited by concurrent incubation with squalene but not mevalonate. In contrast, cell growth of proliferating cells such as NIH 3T3 and PC12 cells was exclusively dependent on the level of nonsterol isoprenoid products and not that of cholesterol. The results of this study clearly indicate that the viability of neurons, different from that of nonneuronal cells, depends on the intracellular cholesterol content and not on the intermediate nonsterol isoprenoid products.     

Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: three questions

Mutations in the gene encoding LRRK2 (leucine-rich repeat kinase 2) were first identified in 2004 and have since been shown to be the single most common cause of inherited Parkinson’s disease. The protein is a large GTP-regulated serine/threonine kinase that additionally contains several protein–protein interaction domains. In the present review, we discuss three important, but unresolved, questions concerning LRRK2. We first ask: what is the normal function of LRRK2? Related to this, we discuss the evidence of LRRK2 activity as a GTPase and as a kinase and the available data on protein–protein interactions. Next we raise the question of how mutations affect LRRK2 function, focusing on some slightly controversial results related to the kinase activity of the protein in a variety of in vitro systems. Finally, we discuss what the possible mechanisms are for LRRK2-mediated neurotoxicity, in the context of known activities of the protein.     

Apathy in Parkinson disease

Apathy is one of the least investigated symptom of Parkinson disease （PD）. In the article there are data of frequency, diagnostic features, pathophysiology and treatment of apathy in PD. The aim of the investigation was to evaluate the frequency of apathy in PD without dementia, evaluate the relationship with other neuropsychiatric and motor disorders, influence on the life quality. 115 patients （age-63.84±0.6 years, stage 2.6±0.3） with PD without dementia were included in the investigation. There were used the following scales： scale of evaluation stages of PD by Hoehn-Yahr, UPDRS （part 〈〈activity of daily living〉〉, 〈〈motor functions 〉〉）; Beck Depression Inventory, Spielberger State Trait Anxiety Inventory, Parkinson Disease Sleep Scale- PDSS, Epworth Sleepiness Scale, Parkinson Fatigue Scale-PFS- 16, SCOPA-Cog, Lilli Apathy Rating Scale LARS and Apathy Scale AS. Apathy was found in 25% of patients. The frequency and severity of apathy does not depend on stage and duration of PD. It was found positive correlation of apathy and hypokinesia. In different stages of PD there was variability of relationships of apathy with depression, executive functions and sleep disorders. We suppose the heterogeneity of apathy in PD because of the variability of the association with other neuropsychiatric （affective, cognitive, sleep） disorders. It was found the negative influence of apathy on daily activity, emotional and social aspects of life quality.