Recessive Juvenile Parkinsonism Research Articles

Enhanced IRE1α Phosphorylation/Oligomerization-Triggered XBP1 Splicing Contributes to Parkin-Mediated Prevention of SH-SY5Y Cell Death under Nitrosative Stress.

Mutations in parkin, a neuroprotective protein, are the predominant cause of autosomal recessive juvenile Parkinson's disease. Neuroinflammation-derived nitrosative stress has been implicated in the etiology of the chronic neurodegeneration. However, the interactions between genetic predisposition and nitrosative stress contributing to the degeneration of dopaminergic (DA) neurons remain incompletely understood. Here, we used the SH-SY5Y neuroblastoma cells to investigate the function of parkin and its pathogenic mutants in relation to cell survival under nitric oxide (NO) exposure. The results showed that overexpression of wild-type parkin protected SH-SY5Y cells from NO-induced apoptosis in a reactive oxygen species-dependent manner. Under nitrosative stress conditions, parkin selectively upregulated the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) signaling axis, an unfolded protein response signal through the sensor IRE1α, which controls the splicing of XBP1 mRNA. Inhibition of XBP1 mRNA splicing either by pharmacologically inhibiting IRE1α endoribonuclease activity or by genetically knocking down XBP1 interfered with the protective activity of parkin. Furthermore, pathogenic parkin mutants with a defective protective capacity showed a lower ability to activate the IRE1α/XBP1 signaling. Finally, we demonstrated that IRE1α activity augmented by parkin was possibly mediated through interacting with IRE1α to regulate its phosphorylation/oligomerization processes, whereas mutant parkin diminished its binding to and activation of IRE1α. Thus, these results support a direct link between the protective activity of parkin and the IRE1α/XBP1 pathway in response to nitrosative stress, and mutant parkin disrupts this function.

A Splice Intervention Therapy for Autosomal Recessive Juvenile Parkinson's Disease Arising from Parkin Mutations.

Parkin-type autosomal recessive juvenile-onset Parkinson’s disease is caused by mutations in the PRKN gene and accounts for 50% of all autosomal recessive Parkinsonism cases. Parkin is a neuroprotective protein that has dual functions as an E3 ligase in the ubiquitin–proteasome system and as a transcriptional repressor of p53. While genomic deletions of PRKN exon 3 disrupt the mRNA reading frame and result in the loss of functional parkin protein, deletions of both exon 3 and 4 maintain the reading frame and are associated with a later onset, milder disease progression, indicating this particular isoform retains some function. Here, we describe in vitro evaluation of antisense oligomers that restore functional parkin expression in cells derived from a Parkinson’s patient carrying a heterozygous PRKN exon 3 deletion, by inducing exon 4 skipping to correct the reading frame. We show that the induced PRKN transcript is translated into a shorter but semi-functional parkin isoform able to be recruited to depolarised mitochondria, and also transcriptionally represses p53 expression. These results support the potential use of antisense oligomers as a disease-modifying treatment for selected pathogenic PRKN mutations.

Pathophysiological mechanisms linking F-box only protein 7 (FBXO7) and Parkinson's disease (PD).

Mutations of F-box only protein 7 (FBXO7) gene are associated with a severe form of autosomal recessive juvenile Parkinson's disease (PD) (PARK15) with clinical features of Parkinsonian-Pyramidal syndrome (PPS). FBXO7 is an adaptor protein in SCFFBXO7 ubiquitin E3 ligase complex that recognizes and mediates degradative or non-degradative ubiquitination of substrates. The FBXO7 protein can regulate cell cycle, proliferation, mitochondrial and proteasome functions via interactions with multiple target proteins. Five PARK15-linked FBXO7 gene mutations and several PD-associated single nucleotide polymorphisms (SNP) have been identified so far. WT FBXO7 proteins possess dual protective and deleterious functions, whereas PARK15-linked FBXO7 mutants are toxic. FBXO7 is a stress response protein and stress challenges can promote translocation of FBXO7 protein from nucleus into mitochondria and even form deleterious protein aggregate in mitochondria. FBXO7 mutants aggravate protein aggregation in mitochondria and inhibit mitophagy. The pathological mechanisms concerning FBXO7-relevant protein aggregation, mitochondria impairment, reactive oxygen species (ROS) generation and mitophagy modulation in PARK15 pathogenesis are highlighted and discussed in the current review.

Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination.

Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca2+-binding capacity; thus, Syt IV plays an inhibitory role in Ca2+-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca2+-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release.

Abstract 122: PARK2 is a negative regulator of Wnt and EGFR pathways in glioma

Abstract PARK2 is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of autosomal recessive juvenile Parkinson's disease and human malignancies. However, its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival in cohorts of both low-grade glioma and glioblastoma multiforme (GBM). Functional studies revealed a tumor-suppressive role of PARK2 in GBM cells. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, while depletion of endogenous PARK2 promoted cell proliferation. cDNA microarray analysis showed that PARK2 expression strongly altered the activity of both the Wnt and EGFR pathways. Immunoblot analysis confirmed that ectopic expression of PARK2 suppressed the intracellular levels of β-catenin, EGFR, as well as their down-stream targets including Cyclin D1, TCF4, c-Myc, p-AKT and p-S6K, etc. Notably, PARK2 physically interacted with both β-catenin and EGFR, and promoted their ubiquitination in an E3-ligase dependent manner. Similar to the ligase-dead PARK2 mutant (C431S), three PARK2 mutants harboring cancer-derived somatic mutations (T173A, T240M and P294S) showed decreased ability to ubiquitinate either β-catenin or EGFR proteins. We further found that PARK2 attenuated the cellular response to both Wnt3a and EGF stimulation, suggesting PARK2 is a negative regulator of both the Wnt and EGFR pathways. Lastly, inspired by these newly identified functions of PARK2, we tested and proved that the combination of small-molecule inhibitors targeting both Wnt-β-catenin and EGFR-AKT pathways synergistically impaired glioma cell viability. In aggregate, our findings uncover novel, cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma. Citation Format: Liang Xu, De-Chen Lin, Ye Chen, Haiyan Yan, Masaharu Hazawa, Ngan Doan, Jonathan W. Said, Ling-Wen Ding, Li-Zhen Liu, Henry Yang, Shi-zhu Yu, Michael Kahn, Dong Yin, Phillip Koeffler. PARK2 is a negative regulator of Wnt and EGFR pathways in glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 122. doi:10.1158/1538-7445.AM2015-122

Regulation of Parkin E3 ubiquitin ligase activity.

Parkin is an E3 ubiquitin ligase mutated in autosomal recessive juvenile Parkinson's disease. In addition, it is a putative tumour suppressor, and has roles outside its enzymatic activity. It is critical for mitochondrial clearance through mitophagy, and is an essential protein in most eukaryotes. As such, it is a tightly controlled protein, regulated through an array of external interactions with multiple proteins, posttranslational modifications including phosphorylation and S-nitrosylation, and self-regulation through internal associations. In this review, we highlight some of the recent studies into Parkin regulation and discuss future challenges for gaining a full molecular understanding of the regulation of Parkin E3 ligase activity.

Diminution of eIF4E activity suppresses parkin mutant phenotypes

Mutations in the human parkin ( PARK2) gene cause autosomal recessive-juvenile Parkinson's disease (AR-JP). In Drosophila melanogaster, mutant parkin alleles display a broad range of phenotypic alterations, including female infertility. Here we report that reducing the level of eukaryotic translation initiation factor 4E ( eIF4E) activity specifically rescues the female sterile phenotypes associated with the parkin P23 mutant allele. Additional defects, including reduction of pupal viability and body size, are also entirely recovered in both male and female flies of the abovementioned genotype. We further show that a null eIF4E-binding protein ( 4E-BP) allele counteracts the in vivo effects produced, in a parkin P23 mutant background, by the reduction of functional eIF4E copy number. Moreover, Parkin and eIF4E interact in vitro and co-localize at the posterior end of developing oocytes. Finally, we show that eIF4E is over-expressed in parkin P23 mutant ovaries as compared to wild-types. Taken together, our data are consistent with the idea that Parkin and eIF4E act in a common pathway, likely modulating cap-dependent translation initiation events.

Disease connections

PARK2, which causes autosomal recessive juvenile Parkinson's disease, is shown to be a haploinsufficient suppressor of colorectal cancer, and the loss of this gene synergises with the loss of APC.

Transcriptional repression of p53 by parkin and impairment by mutations associated with autosomal recessive juvenile Parkinson's disease

Mutations of the ubiquitin ligase parkin account for most autosomal recessive forms of juvenile Parkinson's disease (AR-JP). Several studies have suggested that parkin possesses DNA-binding and transcriptional activity. We report here that parkin is a p53 transcriptional repressor. First, parkin prevented 6-hydroxydopamine-induced caspase-3 activation in a p53-dependent manner. Concomitantly, parkin reduced p53 expression and activity, an effect abrogated by familial parkin mutations known to either abolish or preserve its ligase activity. ChIP experiments indicate that overexpressed and endogenous parkin interact physically with the p53 promoter and that pathogenic mutations abolish DNA binding to and promoter transactivation of p53. Parkin lowered p53 mRNA levels and repressed p53 promoter transactivation through its Ring1 domain. Conversely, parkin depletion enhanced p53 expression and mRNA levels in fibroblasts and mouse brains, and increased cellular p53 activity and promoter transactivation in cells. Finally, familial parkin missense and deletion mutations enhanced p53 expression in human brains affected by AR-JP. This study reveals a ubiquitin ligase-independent function of parkin in the control of transcription and a functional link between parkin and p53 that is altered by AR-JP mutations.

A Disease State Mutation Unfolds the Parkin Ubiquitin-like Domain

E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.

Autosomal recessive juvenile Parkinson's disease with partial Trisomy of chromosome 6q syndrome: A case report

Autosomal recessive juvenile Parkinson's disease with partial Trisomy of chromosome 6q syndrome: A case report

Down-regulation of parkin protein in transient focal cerebral ischemia: A link between stroke and degenerative disease?

Ubiquitylated protein aggregates are characteristic features of neurodegenerative disorders that are also found in acute pathological states of the brain such as stroke. Many of the proteins connected to neurodegenerative diseases play a role in the ubiquitin-proteasomal pathway. Mutation of one of these proteins, the E3 ubiquitin ligase parkin, is the cause of autosomal recessive juvenile Parkinson's disease. Here we show that transient focal cerebral ischemia of 1-h duration induces marked depletion of parkin protein levels, to 60%, 36%, 33%, and 25% of controls after 1, 3, 6, and 24 h of reperfusion, but that ischemia does not cause lower protein levels of E2 ubiquitin-conjugating enzymes Ubc6, Ubc7, or Ubc9. After 3 h of reperfusion, when parkin protein levels were already reduced to <40% of control, ATP levels were almost completely recovered from ischemia and we did not observe DNA fragmentation, suggesting that parkin depletion preceded development of neuronal cell death. Up-regulation of the expression of parkin has been shown to protect cells from injury induced by endoplasmic reticulum (ER) dysfunction, and this form of cellular stress is also triggered by transient cerebral ischemia. However, in contrast to observations in neuroblastoma cells, we saw no up-regulation of parkin expression in primary neuronal cell cultures after induction of ER dysfunction. Our data thus suggest that ischemia-induced depletion of parkin protein may contribute to the pathological process resulting in cell injury by increasing the sensitivity of neurons to ER dysfunction and the aggregation of ubiquitylated proteins during the reperfusion period.