Parkinson's Disease Mutations Research Articles

Mechanism of phospho-Ubls' specificity and conformational changes that regulate Parkin activity.

PINK1 and Parkin mutations lead to the early onset of Parkinson's disease. PINK1-mediated phosphorylation of ubiquitin (Ub), ubiquitin-like protein (NEDD8), and ubiquitin-like (Ubl) domain of Parkin activate autoinhibited Parkin E3 ligase. The mechanism of various phospho-Ubls' specificity and conformational changes leading to Parkin activation remain elusive. Herein, we show that compared to Ub, NEDD8 is a more robust binder and activator of Parkin. Structures and biophysical/biochemical data reveal specific recognition and underlying mechanisms of pUb/pNEDD8 and pUbl domain binding to the RING1 and RING0 domains, respectively. Also, pUb/pNEDD8 binding in the RING1 pocket promotes allosteric conformational changes in Parkin's catalytic domain (RING2), leading to Parkin activation. Furthermore, Parkinson's disease mutation K211N in the RING0 domain was believed to perturb Parkin activation due to loss of pUb binding. However, our data reveal allosteric conformational changes due to N211 that lock RING2 with RING0 to inhibit Parkin activity without disrupting pNEDD8/pUb binding.

Structurally targeted mutagenesis identifies key residues supporting -synuclein misfolding in multiple system atrophy.

Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded -synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in -synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in -synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo, providing key evidence to support the hypothesis that -synuclein adopts unique strains in patients. Here, to further interrogate -synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate -synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of -synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support -synuclein misfolding. Overall, our studies use a structure-based approach to investigate -synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk.

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

The Molecular Impact of Glucosylceramidase Beta 1 (Gba1) in Parkinson's Disease: a New Genetic State of the Art.

Parkinson's disease (PD) is a neurodegenerative disorder affecting 2-3% of those aged over 65, characterized by motor symptoms like slow movement, tremors, and muscle rigidity, along with non-motor symptoms such as anxiety and dementia. Lewy bodies, clumps of misfolded proteins, contribute to neuron loss in PD. Mutations in the GBA1 gene are considered the primary genetic risk factor of PD. GBA1 mutations result in decreased activity of the lysosomal enzyme glucocerebrosidase (GCase) resulting in α-synuclein accumulation. We know that α-synuclein aggregation, lysosomal dysfunction, and endoplasmic reticulum disturbance are recognized factors to PD susceptibility; however, the molecular mechanisms connecting GBA1 gene mutations to increased PD risk remain partly unknown. Thus, in this narrative review conducted according to a systematic review method, we aimed to present the main contributions arising from the molecular impact of the GBA1 gene to the pathogenesis of PD providing new insights into potential impacts for advances in the clinical care of people with PD, a neurological disorder that has contributed to the substantial increase in the global burden of disease accentuated by the aging population. In summary, this narrative review highlights the multifaceted impact of GBA1 mutations in PD, exploring their role in clinical manifestations, genetic predispositions, and molecular mechanisms. The review emphasizes the importance of GBA1 mutations in both motor and non-motor symptoms of PD, suggesting broader therapeutic and management strategies. It also discusses the potential of CRISPR/Cas9 technology in advancing PD treatment and the need for future research to integrate these diverse aspects for improved diagnostics and therapies.

Fibrillation of α-synuclein triggered by bacterial endotoxin and lipid vesicles is modulated by N-terminal acetylation and familial Parkinson's disease mutations.

It has been hypothesized that --Parkinson's disease (PD) may be initiated in the gastrointestinal tract, before manifesting in the central nervous system. In this respect, it was demonstrated that lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, accelerates the in vitro formation of α-synuclein (aSyn) fibrils, whose intracellular deposits is a histological hallmark of the degeneration of dopaminergic neurons in PD. Herein, N-terminal acetylation and missense mutations of aSyn (A30P, A53T, E46K, H50Q and G51D) linked to rare, early-onset forms of familial PD were investigated regarding their effect on aSyn aggregation stimulated by either LPS or small unilamellar lipid vesicles (SUVs). Our findings indicated that LPS as well as SUVs induce the fibrillation of N-terminally acetylated wild-type aSyn (Ac-aSyn-WT) more remarkably than the non-acetylated protein, while the LPS-free protein alone did not undergo fibrillation under our assay conditions. In addition, with the exception of A30P, PD mutations increased the fibrillation of Ac-aSyn in the presence of LPS compared with Ac-aSyn-WT. The most pronounced effect of LPS was noticed for A53T, as observed when either Thioflavin-T or JC-1 were used as fluorescent probes for fibrils. Overall, our results suggest for the first time the existence of a synergy between LPS and PD mutations/N-terminal acetylation toward aSyn fibrillation.

Breaking down and building up alpha-synuclein: An insight on its N-terminal domain.

Alpha-synuclein (αSyn) is a small presynaptic protein (14 kDa) that is involved in synucleinopathies including Parkinson's disease (PD). In its native state, the αSyn monomer exists in an unfolded state, and its folding is highly dependent on variations of environmental conditions, mutations and interactions with endogenous and/or exogenous molecules. Recently, there is increasing evidence for a direct interplay between αSyn and microtubules (MTs), whose defects are linked to neurodegenerative diseases, such as PD. Understanding the correlation between αSyn and MTs could be fundamental for the correct comprehension of the undergoing mechanisms of PD. Hence, we chemically synthesized a library of peptides, deriving from both native and PD mutated sequences of the N-terminal domain of αSyn. Their secondary structure was characterized by circular dichroism and Fourier transform infrared (FTIR) experiments, in order to evaluate the effect of PD mutations. Finally, the kinetics of polymerizing tubulin in vitro in the presence of the peptides was evaluated.

Unraveling Autonomic Dysfunction in GBA-Related Parkinson's Disease.

Patients with Parkinson's disease (PD) and GBA gene mutations (GBA-PD) develop nonmotor complications more frequently than noncarriers. However, an objective characterization of both cardiovascular and sudomotor autonomic dysfunction using extensive clinical and instrumental measures has never been provided so far. Survival is reduced in GBA-PD regardless of age and dementia, suggesting that other hitherto unrecognized factors are involved. To provide instrumental measures of pattern and severity of autonomic dysfunction in GBA-PD and explore their correlation with other non-motor symptoms and implications for clinical practice. In this cross-sectional study, 21 GBA-PD and 24 matched PD noncarriers underwent extensive assessment of motor and non-motor features, including neuropsychological testing. Cardiovascular autonomic function was explored through a comprehensive battery of indexes, including power spectral analysis of the R-R intervals and blood pressure short-term variability during resting state and active maneuvers. Dynamic Sweat Test was used to assess post-ganglionic sudomotor dysfunction. Despite minimal or absent clinical correlates, cardiovagal and sympathetic indexes, heart rate variability parameters and sudomotor postganglionic function were more severely impaired in GBA-PD than noncarriers (overcoming relatively preserved compensatory peripheral sympathetic function), suggesting more prominent cardiac sympatho-vagal demodulation, efferent baroreflex failure and peripheral sympathetic dysfunction in GBA-PD. Cardiovascular dysautonomia showed marginal correlations with cognitive impairment. Compared to PD noncarriers, GBA-PD display more severe instrumental autonomic abnormalities, which may be underestimated by purely clinical measures, despite their relevance on morbidity and mortality. This supports the necessity of implementing instrumental autonomic assessment in all GBA-PD, regardless of clinically overt symptoms.

Protective Role of AMPK against PINK1B9 Flies' Neurodegeneration with Improved Mitochondrial Function.

Adenosine 5'-monophosphate-activated protein kinase (AMPK)'s effect in PTEN-induced kinase 1 (PINK1) mutant Parkinson's disease (PD) transgenic flies and the related mechanism is seldom studied. The classic MHC-Gal4/UAS PD transgenic flies was utilized to generate the disease characteristics specifically expressed in flies' muscles, and Western blot (WB) was used to measure the expression of the activated form of AMPK to investigate whether activated AMPK alters in PINK1B9 PD flies. MHC-Gal4 was used to drive AMPK overexpression in PINK1B9 flies to demonstrate the crucial role of AMPK in PD pathogenesis. The abnormal wing posture and climbing ability of PINK1B9 PD transgenic flies were recorded. Mitochondrial morphology via transmission electron microscopy (TEM) and ATP and NADH: ubiquinone oxidoreductase core subunit S3 (NDUFS3) protein levels were tested to evaluate the alteration of the mitochondrial function in PINK1B9 PD flies. Phosphorylated AMPKα dropped significantly in PINK1B9 flies compared to controls, and AMPK overexpression rescued PINKB9 flies' abnormal wing posture rate. The elevated dopaminergic neuron number in PPL1 via immunofluorescent staining was observed. Mitochondrial dysfunction in PINK1B9 flies has been ameliorated with increased ATP level, restored mitochondrial morphology in muscle, and increased NDUFS3 protein expression. Conclusively, AMPK overexpression could partially rescue the PD flies via improving PINK1B9 flies' mitochondrial function.

Potential direct role of synuclein in dopamine transport and its implications for Parkinson's disease pathogenesis

Parkinson Disease (PD) is a progressive neurodegenerative disorder that is caused by dysfunction and death of dopaminergic neurons. Mutations in the gene encoding α-synuclein (ASYN) have been linked with familial PD (FPD). Despite important role of ASYN in PD pathology, its normal biological function has not been clarified, although direct action of ASYN in synaptic transmission and dopamine (DA+) release have been proposed. In the present report we propose a novel hypothesis that ASYN functions as DA+/H+ exchanger that can facilitate transport of dopamine across synaptic vesicle (SV) membrane by taking advantage of proton gradient between SV lumen and cytoplasm. According to this hypothesis, normal physiological role of ASYN consists of fine-tuning levels of dopamine in the SVs based on cytosolic concentration of dopamine and intraluminal pH. This hypothesis is based on similarity in domain structure of ASYN and pHILP, a designed peptide developed to mediate loading of lipid nanoparticles with the cargo molecules. We reason that carboxy-terminal acidic loop D2b domain in both ASYN and pHILP binds cargo molecules. By mimicking DA+ association with E/D residues in D2b domain of ASYN using Tyrosine replacement approach (TR) we have been able to estimate that ASYN is able to transfer 8–12 molecules of dopamine across SV membrane on each DA+/H+ exchange cycle. Our results suggest that familial PD mutations (A30P, E46K, H50Q, G51D, A53T and A53E) will interfere with different steps of the exchange cycle, resulting in partial loss of dopamine transport function phenotype. We also predict that similar impairment in ASYN DA+/H+ exchange function also occurs as a result on neuronal aging due to changes in SV lipid composition and size and also dissipation of pH gradient across SV membrane. Proposed novel functional role of ASYN provides novel insights into its biological role and its role in PD pathogenesis.

Recent advances in novel mutation genes of Parkinson's disease.

With increasing life expectancy, a growing number of individuals are being affected by Parkinson's Disease (PD), a Neurodegenerative Disease (ND). Approximately, 5-10% of PD is explained by genetic causes linked to known PD genes. With improvements in genetic testing and high-throughput technologies, more PD-associated susceptibility genes have been reported in recent years. However, a comprehensive review of the pathogenic mechanisms and physiological roles of these genes is still lacking. This article reviews novel genes with putative or confirmed pathogenic mutations in PD reported since 2019, summarizes the physiological functions and potential associations with PD. Newly reported PD-related genes include ANK2, DNAH1, STAB1, NOTCH2NLC, UQCRC1, ATP10B, TFG, CHMP1A, GIPC1, KIF21B, KIF24, SLC25A39, SPTBN1 and TOMM22. However, the evidence for pathogenic effects of many of these genes is inconclusive. A variety of novel PD-associated genes have been identified through clinical cases of PD patients and analysis of Genome-Wide Association Studies (GWAS). However, more evidence is needed in confirm the strong association of novel genes with disease.

Automated algorithm development to assess survival of human neurons using longitudinal single-cell tracking: Application to synucleinopathy.

The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.

LRRK2 Structure-Based Activation Mechanism and Pathogenesis.

Mutations in the multidomain protein Leucine-rich-repeat kinase 2 (LRRK2) have been identified as a genetic risk factor for both sporadic and familial Parkinson's disease (PD). LRRK2 has two enzymatic domains: a RocCOR tandem with GTPase activity and a kinase domain. In addition, LRRK2 has three N-terminal domains: ARM (Armadillo repeat), ANK (Ankyrin repeat), and LRR (Leucine-rich-repeat), and a C-terminal WD40 domain, all of which are involved in mediating protein-protein interactions (PPIs) and regulation of the LRRK2 catalytic core. The PD-related mutations have been found in nearly all LRRK2 domains, and most of them have increased kinase activity and/or decreased GTPase activity. The complex activation mechanism of LRRK2 includes at least intramolecular regulation, dimerization, and membrane recruitment. In this review, we highlight the recent developments in the structural characterization of LRRK2 and discuss these developments from the perspective of the LRRK2 activation mechanism, the pathological role of the PD mutants, and therapeutic targeting.

Hybrid enhanced sampling approaches to gain structural insights into leucine-rich repeat kinase 2 (LRRK2) Parkinson's disease mutation.

Parkinson's disease (PD) is a neurodegenerative disease affecting millions worldwide. A commonly mutated gene in PD is leucine-rich repeat kinase 2 (LRRK2), which contains multiple domains, including but not limited to kinase, ATPase, and GTPase. LRRK2 has been found to increase kinase activity and colocalize with microtubules. LRRK2 protein dynamics are affected by its conformation, regulating microtubule interactions and potentially enhancing intracellular trafficking. Despite recent advances in the structural insights of LRRK2, detailed studies on the effects of single-point mutations on the dynamics and conformations of LRRK2 are limited. Consequently, an all-atomistic molecular dynamics simulation is required to understand the allosteric and dynamical effects of single-point disease mutations in LRRK2, but it would be computationally expensive. We thereby employ enhanced molecular dynamics simulations to study single-point mutations in LRRK2, particularly the G2019S, R1441C, R1441G, R1441H, and Y1699C mutations. Our two-phase approach incorporates implementing Gaussian-accelerated molecular dynamics (GaMD) for enhanced sampling, followed by the weighted ensemble (WE) method. GaMD is an enhanced MD approach where a boost potential is added to the total potential energy of the system to accelerate the conformational search, while the WE method readjusts the weight of the simulation trajectories by frequent resampling to obtain the kinetic properties of interest accurately. Investigating the dynamics of such single-point mutations in LRRK2 could explain why specific mutations result in increased kinase activation and dynamicity of LRRK2.

Understanding the role of the LRRK2 tail in the crosstalk between kinase and the N-terminal domains.

LRRK2 is a multi-domain protein encompassing biology's two most critical molecular switches; a kinase and a GTPase. Mutations in LRRK2 are one of the key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to exploring the physiological role of LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is a common feature in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and can serve as a multi-functional protein: protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and are using peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB region with a combination of Gaussian MD simulations and peptide arrays. This Ct-Motif contains multiple phosphorylation sites including two auto-phosphorylation sites (Thr2083 and Thr2524), a 14-3-3 binding site (Thr2524) and a putative PKA phosphorylation site. These two sites together form a part of a dynamic “CAP” that regulates the N-lobe of the kinase domain. We also explore how the cellular localization of LRRK2RCKW and its docking onto microtubules is altered by mutations in the Ct helix, if at all. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Putative pore structure of amyloid beta and alpha synuclein in lipid bilayers.

Many degenerative diseases are characterized by aberrant protein aggregation. One possible mechanism of toxicity is the perturbation or permeabilization of cell membranes by certain types of oligomers. However, the structure of such membrane-inserted pore-forming complexes is unknown. Here we consider putative membrane-inserted β-barrels of Amyloid β and α-synuclein and evaluate them first in an implicit membrane model and then by multi-microsecond all atom simulation. The 11 amino acid peptide sequence of Aβ25-35 is relatively hydrophobic and readily embeds into synthetic bilayers producing ion channels. We consider octameric and decameric β barrels of different topology, strand orientation, and shear. Two decameric β barrels remain stably embedded in the membrane at the conclusion of these 5-μs all atom simulations: an antiparallel barrel with K28 in the pore lumen and a parallel barrel with the K28 in the membrane interface. Examination of the α-synuclein sequence reveals two regions that could form membrane-embedded β-hairpins: 35-56 and 64-92. We find that a 35-56 barrel remains inserted but dehydrates and collapses if all His50 are neutral. If half of the His50 are doubly protonated, however, the barrel takes an oval shape but remains hydrated and open. The 64-92 barrel remains hydrated for at least 10 µs. Our results for these pore structures may carry implications for familial Parkinson's mutations.

Deficiency of RAB39B Activates ER Stress-Induced Pro-apoptotic Pathway and Causes Mitochondrial Dysfunction and Oxidative Stress in Dopaminergic Neurons by Impairing Autophagy and Upregulating α-Synuclein.

Deletion and missense or nonsense mutation of RAB39B gene cause familial Parkinson's disease (PD). We hypothesized that deletion and mutation of RAB39B gene induce degeneration of dopaminergic neurons by decreasing protein level of functional RAB39B and causing RAB39B deficiency. Cellular model of deletion or mutation of RAB39B gene-induced PD was prepared by knocking down endogenous RAB39B in human SH-SY5Y dopaminergic cells. Transfection of shRNA-induced 90% reduction in RAB39B level significantly decreased viability of SH-SY5Y dopaminergic neurons. Deficiency of RAB39B caused impairment of macroautophagy/autophagy, which led to increased protein levels of α-synuclein and phospho-α-synucleinSer129 within endoplasmic reticulum (ER) and mitochondria. RAB39B deficiency-induced increase of ER α-synuclein and phospho-α-synucleinSer129 caused activation of ER stress, unfolded protein response, and ER stress-induced pro-apoptotic cascade. Deficiency of RAB39B-induced increase of mitochondrial α-synuclein decreased mitochondrial membrane potential and increased mitochondrial superoxide. RAB39B deficiency-induced activation of ER stress pro-apoptotic pathway, mitochondrial dysfunction, and oxidative stress caused apoptotic death of SH-SY5Y dopaminergic cells by activating mitochondrial apoptotic cascade. In contrast to neuroprotective effect of wild-type RAB39B, PD mutant (T168K), (W186X), or (G192R) RAB39B did not prevent tunicamycin- or rotenone-induced increase of neurotoxic α-synuclein and activation of pro-apoptotic pathway. Our results suggest that RAB39B is required for survival and macroautophagy function of dopaminergic neurons and that deletion or PD mutation of RAB39B gene-induced RAB39B deficiency induces apoptotic death of dopaminergic neurons via impairing autophagy function and upregulating α-synuclein.

Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model

IntroductionMitochondrial defects have been implicated in Parkinson's disease (PD) since complex I poisons were found to cause accelerated parkinsonism in young people in the early 1980s. More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. However, the details of how mitochondrial dysfunction might impact or cause PD remain unclear. The aim of our study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila melanogaster (fruit fly) model of PD. MethodsWe have conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2–5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16–20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Generalized Linear Models and similar statistical tests were used to verify significance of the findings. ResultsWe have discovered that mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. ConclusionsOur work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.

Change in the Oligomeric State of α-Synuclein Variants in Living Cells.

The accumulation of β-sheet-rich α-synuclein (α-Syn) protein in human brain cells is a pathological hallmark of Parkinson's disease (PD). Moreover, it has been reported that familial PD mutations (A30P, E46K, H50Q, G51D, and A53T) accumulate at an accelerated rate both in vivo and in vitro. In addition, accumulations of various C-terminal α-Syn truncations, such as C-terminal-truncated N103 α-synuclein (N103), were found in an aggregated form in the brain tissue of PD patients. Fluorescent protein-tagged wild-type α-Syn, A30P, E46K, H50Q, G51D, A53T, and N103 were transfected into HEK293T and SHSY5Y cells, and their diffusion behaviors were investigated with a custom-built fluorescence microscope system. Based on our experimental results, the oligomerization of α-Syn is a time-dependent process in both HEK293T and SHSY5Y cells, and the oligomer state approaches a plateau after 48 h of transfection. The change in the oligomeric state of E46K, H50Q, and G51D exhibited a similar trend to the wild type at a lower concentration but became intense at a higher concentration. A53T and N103 possess smaller diffusion coefficients than wild-type α-synuclein and other family PD mutations, indicating that these two mutants could form higher oligomeric states or stronger interactions in HEK293T and SHSY5Y cells. In contrast, the smallest oligomer and the lowest intracellular interaction among all investigated α-Syn variants were found for A30P. These phenomena indicated the presence of different pathogeneses among familial PD mutants and C-terminal α-Syn truncations.

ANXA1 and the risk for early-onset Parkinson's disease

Recently, homozygous missense variants in ANXA1 were identified to cause parkinsonism by segregation analysis in a consanguineous family. However, no further replication has been conducted in a wider range of Parkinson's disease (PD) populations. To investigate the involvement of ANXA1 mutations in PD, we analyzed the rare variants in 743 Chinese early-onset PD (EOPD) patients (age at onset <50) using whole exome sequencing. The over-representation of rare variants in patients was examined with Fisher's exact test at allele and gene levels. We did not find the disease-causing variant described in the original study, and no patient carried other homozygous or compound heterozygous variants of ANXA1. Six rare missense mutations in ANXA1 were identified (minor allele frequency <0.01). No significant association was found between ANXA1 variants and PD at allele and gene levels. Genetic screening of ANXA1 mutations suggested rare variants of ANXA1 were rare in EOPD in the Asian ethnic background. Further explorations with larger sample size were warranted to better understand the role of ANXA1 in PD.

Endophilin-B regulates autophagy during synapse development and neurodegeneration

Synapses are critical for neuronal communication and brain function. To maintain neuronal homeostasis, synapses rely on autophagy. Autophagic alterations cause neurodegeneration and synaptic dysfunction is a feature in neurodegenerative diseases. In Parkinson's disease (PD), where the loss of synapses precedes dopaminergic neuron loss, various PD-causative proteins are involved in the regulation of autophagy. So far only a few factors regulating autophagy at the synapse have been identified and the molecular mechanisms underlying autophagy at the synapse is only partially understood. Here, we describe Endophilin-B (EndoB) as a novel player in the regulation of synaptic autophagy in health and disease. We demonstrate that EndoB is required for autophagosome biogenesis at the synapse, whereas the loss of EndoB blocks the autophagy induction promoted by the PD mutation LRRK2G2019S. We show that EndoB is required to prevent neuronal loss. Moreover, loss of EndoB in the Drosophila visual system leads to an increase in synaptic contacts between photoreceptor terminals and their post-synaptic synapses. These data confirm the role of autophagy in synaptic contact formation and neuronal survival.