α-Synuclein and Mitochondrial Dysfunction in Parkinson’s Disease

α-Synuclein (SNCA) is a substantive component of Lewy bodies, the pathological hallmark of Parkinson’s disease (PD). The discovery and subsequent derivation of its role in PD has led to a suprising but fruitful convergence of the fields of biochemistry and molecular genetics. In particular, the manipulation of the cell lines of a number of forms of familial PD has implicated SNCA in distinct and diverse biochemical pathways related to its pathogenesis. This current and rapidly evolving concept indicates PD is a disease in which interacting pathways of oxidative stress, mitochondrial dysfunction and impaired regulation of protein turnover interact to cause dopaminergic cell dysfunction and death. SNCA has a central role in these processes and manipulation of its expression, degradation and aggregation appear to be promising neuroprotective therapeutic targets.     

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.     

α-Synuclein and Anti-α-Synuclein Antibodies in Parkinson’s Disease,Atypical Parkinson Syndromes,REM Sleep Behavior Disorder,and Healthy Controls

α-synuclein is thought to play a key role in Parkinson’s disease (PD) because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (n = 14), atypical Parkinson syndromes (n = 11), idiopathic rapid eye movement sleep behavior disorder (n = 10), and healthy controls (n = 9), to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power) of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rho = 0.16). In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.     

Recent Advances in α-Synuclein Functions, Advanced Glycation, and Toxicity: Implications for Parkinson’s Disease

The toxicity of α-synuclein in the neuropathology of Parkinson’s disease which includes its hallmark aggregation has been studied scrupulously in the last decade. Although little is known regarding the normal functions of α-synuclein, its association with membrane phospholipids suggests its potential role in signaling pathways. Following extensive evidences for its nuclear localization, we and others recently demonstrated DNA binding activity of α-synuclein that modulates its conformation as well as aggregation properties. Furthermore, we also underscored the similarities among various amyloidogenic proteins involved in neurodegenerative diseases including amyloid beta peptides and tau. Our more recent studies show that α-synuclein is glycated and glycosylated both in vitro and in neurons, significantly affecting its folding, oligomeric, and DNA binding properties. Glycated α-synuclein causes increased genome damage both via its direct interaction with DNA and by increased generation of reactive oxygen species as glycation byproduct. In this review, we discuss the mechanisms of glycation and other posttranslational modifications of α-synuclein, including phosphorylation and nitration, and their role in neuronal death in Parkinson’s disease.     

Interaction between Aβ Peptide and α Synuclein: Molecular Mechanisms in Overlapping Pathology of Alzheimer’s and Parkinson’s in Dementia with Lewy Body Disease

Amyloidogenic proteins (Aβ peptide) in Alzheimer’s disease (AD) and alpha-synuclein (α-Syn) in Parkinson’s disease (PD) are typically soluble monomeric precursors, which undergo remarkable conformational changes and culminate in the form of aggregates in diseased condition. Overlap of clinical and neuropathological features of both AD and PD are observed in dementia with Lewy body (DLB) disease, the second most common form of dementia after AD. The identification of a 35-amino acid fragment of α-Syn in the amyloid plaques in DLB brain have raised the possibility that Aβ and α-Syn interact with each other. In this report, the molecular interaction of α-Syn with Aβ40 and/or Aβ42 are investigated using multidimensional NMR spectroscopy. NMR data in the membrane mimic environment indicate specific sites of interaction between membrane-bound α-Syn with Aβ peptide and vice versa. These Aβ–α-Syn interactions are demonstrated by reduced amide peak intensity or change in chemical shift of amide proton of the interacting proteins. Based on NMR results, the plausible molecular mechanism of overlapping pathocascade of AD and PD in DLB due to interactions between α-Syn and Aβ is described. To the best of our knowledge, it is the first report using multidimensional NMR spectroscopy that elucidates molecular interactions between Aβ and α-Syn which may lead to onset of DLB. An erratum to this article can be found at     

Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

The nuclear receptor small heterodimer partner SHP was shown recently to translocate to the mitochondria, interact with Bcl2, and induce apoptosis in liver cancer cells. However, the exact mitochondrial localization of SHP remains to be determined. In addition, the detailed interaction domains between SHP and Bcl2 have not been characterized. Using biochemistry and molecular biology approaches, we demonstrate that SHP is localized to the mitochondrial outer membrane. Interestingly, compared with the full-length SHP, the N-terminal deleted protein exhibits increased expression in the mitochondria and decreased expression in the nucleus. GST pull-down assays demonstrate that the interaction domain of SHP shows the strongest interaction with Bcl2. Furthermore, the interaction of Bcl2 with SHP is completely abolished by deletion of the Bcl2 transmembrane domain (TM), whereas deletion of the Bcl2 BH1 domain enhances the interaction. As expected, AHPN, a synthetic SHP ligand, markedly augments the direct protein-protein interaction between Bcl2 and SHP. Ectopic expression of hepatocyte nuclear factor 4 alpha (HNF4α) results in exclusive nuclear translocation of SHP proteins that contain either the full-length or the N-terminal domain, but has a minimal effect on the subcellular distribution of SHP protein containing only the interaction domain or repression domain. These results indicate that the N-terminal domain of SHP is important for itsnuclear translocation via HNF4α. Overall, this study provides novel insights into the domains of SHP that are critical for its shutting between different subcellular compartments.     

PINK1 Defect Causes Mitochondrial Dysfunction,Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson''s disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation.     

Altered Regulation of CD200 Receptor in Monocyte-Derived Macrophages from Individuals with Parkinson’s Disease

Microglia are the representative myeloid cells in the brain, and their over-activation plays an important role in the pathogenesis of Parkinson’s disease (PD). Microglia activation is believed to be regulated by the CD200-CD200R signaling. As the peripheral counterpart of microglia, monocyte-derived macrophages (MDMs) share the same progenitor and antigen markers, and they have similar biological behaviors and mirror microglial function in the brain. Here, we studied CD200R expression and its regulation in MDMs from 32 PD cases, 27 age-matched old controls, and 28 young controls. We found that the basal CD200R expression is similar in MDMs from young control, old control and PD patients. However, the induction of CD200R expression in MDMs under various conditions is impaired in the old groups, especially in PD patients. There was a selective decrease in CD200R expression induced by co-culture with dying PC12 cells in MDMs from PD cases, as compared with MDMs from the age-matched controls. We also found that the inducible CD200R expression correlated inversely with the onset age of PD and to tumor necrosis factor-α (TNF-α) released from MDMs. These results suggest an intrinsic abnormality in the CD200-CD200R signaling in MDMs during aging and, especially, in PD. We speculate that in the PD brain, microglia might undergo abnormalities similar to MDMs.     

Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines,in Yeast,and in Vivo

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.     

Decrease in Plasma Levels of α-Synuclein Is Evident in Patients with Parkinson’s Disease after Elimination of Heterophilic Antibody Interference

There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfered with ELISA measurements of A-syn and are accordingly considered to be an important confounder in A-syn ELISAs. HA produced falsely exaggerated signals in A-syn ELISAs more prominently in plasma samples than in CSF samples. After elimination of HA interference, it was found that hemolysis did not have a significant effect on the signals obtained using our A-syn ELISA. Furthermore, plasma levels of A-syn were significantly lower in the PD group compared with the control group following elimination of HA interference with an HA inhibitor. Our results demonstrate that HA was a major confounder that should be controlled in A-syn ELISAs, and that plasma A-syn could be a useful biomarker for the diagnosis of PD if adequately quantified following elimination of HA interference.     

Anti-Human α-Synuclein N-Terminal Peptide Antibody Protects against Dopaminergic Cell Death and Ameliorates Behavioral Deficits in an AAV-α-Synuclein Rat Model of Parkinson’s Disease

The protein α-synuclein (α-Syn) has a central role in the pathogenesis of Parkinson’s disease (PD) and immunotherapeutic approaches targeting this molecule have shown promising results. In this study, novel antibodies were generated against specific peptides from full length human α-Syn and evaluated for effectiveness in ameliorating α-Syn-induced cell death and behavioral deficits in an AAV-α-Syn expressing rat model of PD. Fisher 344 rats were injected with rAAV vector into the right substantia nigra (SN), while control rats received an AAV vector expressing green fluorescent protein (GFP). Beginning one week after injection of the AAV-α-Syn vectors, rats were treated intraperitoneally with either control IgG or antibodies against the N-terminal (AB1), or central region (AB2) of α-Syn. An unbiased stereological estimation of TH+, NeuN+, and OX6 (MHC-II) immunostaining revealed that the α-Syn peptide antibodies (AB1 and AB2) significantly inhibited α-Syn-induced dopaminergic cell (DA) and NeuN+ cell loss (one-way ANOVA (F (3, 30) = 5.8, p = 0.002 and (F (3, 29) = 7.92, p = 0.002 respectively), as well as decreasing the number of activated microglia in the ipsilateral SN (one-way ANOVA F = 14.09; p = 0.0003). Antibody treated animals also had lower levels of α-Syn in the ipsilateral SN (one-way ANOVA F (7, 37) = 9.786; p = 0.0001) and demonstrated a partial intermediate improvement of the behavioral deficits. Our data suggest that, in particular, an α-Syn peptide antibody against the N-terminal region of the protein can protect against DA neuron loss and, to some extent behavioral deficits. As such, these results may be a potential therapeutic strategy for halting the progression of PD.     

α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP⁺ toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP⁺ toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP⁺-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP⁺ exposure. We demonstrate that MPP⁺ significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.     

Emotion Processing in Parkinson’s Disease: A Three-Level Study on Recognition,Representation, and Regulation

Background

Parkinson’s disease (PD) is characterised by well-known motor symptoms, whereas the presence of cognitive non-motor symptoms, such as emotional disturbances, is still underestimated. One of the major problems in studying emotion deficits in PD is an atomising approach that does not take into account different levels of emotion elaboration. Our study addressed the question of whether people with PD exhibit difficulties in one or more specific dimensions of emotion processing, investigating three different levels of analyses, that is, recognition, representation, and regulation.

Methodology

Thirty-two consecutive medicated patients with PD and 25 healthy controls were enrolled in the study. Participants performed a three-level analysis assessment of emotional processing using quantitative standardised emotional tasks: the Ekman 60-Faces for emotion recognition, the full 36-item version of the Reading the Mind in the Eyes (RME) for emotion representation, and the 20-item Toronto Alexithymia Scale (TAS-20) for emotion regulation.

Principal Findings

Regarding emotion recognition, patients obtained significantly worse scores than controls in the total score of Ekman 60-Faces but not in any other basic emotions. For emotion representation, patients obtained significantly worse scores than controls in the RME experimental score but no in the RME gender control task. Finally, on emotion regulation, PD and controls did not perform differently at TAS-20 and no specific differences were found on TAS-20 subscales. The PD impairments on emotion recognition and representation do not correlate with dopamine therapy, disease severity, or with the duration of illness. These results are independent from other cognitive processes, such as global cognitive status and executive function, or from psychiatric status, such as depression, anxiety or apathy.

Conclusions

These results may contribute to better understanding of the emotional problems that are often seen in patients with PD and the measures used to test these problems, in particular on the use of different versions of the RME task.