The H50Q Mutation Enhances α-Synuclein Aggregation,Secretion, and Toxicity

Over the last two decades, the identification of missense mutations in the α-synuclein (α-Syn) gene SNCA in families with inherited Parkinson disease (PD) has reinforced the central role of α-Syn in PD pathogenesis. Recently, a new missense mutation (H50Q) in α-Syn was described in patients with a familial form of PD and dementia. Here we investigated the effects of this novel mutation on the biophysical properties of α-Syn and the consequences for its cellular function. We found that the H50Q mutation affected neither the structure of free or membrane-bound α-Syn monomer, its interaction with metals, nor its capacity to be phosphorylated in vitro. However, compared with the wild-type (WT) protein, the H50Q mutation accelerated α-Syn fibrillization in vitro. In cell-based models, H50Q mutation did not affect α-Syn subcellular localization or its ability to be phosphorylated by PLK2 and GRK6. Interestingly, H50Q increased α-Syn secretion from SHSY5Y cells into culture medium and induced more mitochondrial fragmentation in hippocampal neurons. Although the transient overexpression of WT or H50Q did not induce toxicity, both species induced significant cell death when added to the culture medium of hippocampal neurons. Strikingly, H50Q exhibited more toxicity, suggesting that the H50Q-related enhancement of α-Syn aggregation and secretion may play a role in the extracellular toxicity of this mutant. Together, our results provide novel insight into the mechanism by which this newly described PD-associated mutation may contribute to the pathogenesis of PD and related disorders.     

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

Background

The pathology of Parkinson''s disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated α-synuclein are demonstrated in the signature inclusions of Parkinson''s disease. However, whether the nitration of α-synuclein is relevant to the pathogenesis of PD is unknown.

Methodology/Principal Findings

In this study, effect of nitrated α-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-α-synuclein intracellular. We provide evidence to show that the nitrated α-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated α-synuclein. Moreover, we show that administration of nitrated α-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D₂R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated α-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified α-synuclein had significantly less severe behavioral effects.

Conclusions/Significance

Our results provide evidence that α-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD.     

Computational investigation on the effects of H50Q and G51D mutations on the α-Synuclein aggregation propensity

The aggregation of α-synuclein is linked directly to the histopathology of Parkinson’s disease (PD). However, several missense mutations present in the α-synuclein gene (SNCA) have been known to be associated with PD. Several studies have highlighted the effect of SNCA mutations on the α-synuclein aggregation, but their pathological roles are not completely established. In this study, we have focused on the effects of the recently discovered α-synuclein missense mutants (H50Q and G51D) on the aggregation using computational approaches. We performed all atom molecular dynamics (MD) simulation on these mutants and compared their conformational dynamics with Wild-Type (WT) α-synuclein. We noticed the solvent accessible surface area (SASA), radius of gyration, atomic fluctuations, and beta strand content to be higher in H50Q than G51D and WT. Using PDBSum online server; we analyzed the inter-molecular interactions that drive the association of monomeric units of H50Q, WT, and G51D in forming the respective homo-dimer. We noticed the interface area, number of interacting residues and binding free energy to be higher for H50Q homo-dimer than the WT and G51D homo-dimers. Our findings in this study suggest that in comparison to WT and G51D, H50Q mutation to have a positive effect on increasing the α-synuclein aggregation propensity. Hence, we see that H50Q and G51D mutation show conflicting effect on the aggregation propensity of α-synuclein.     

Quantitative Analysis of α-Synuclein Solubility in Living Cells Using Split GFP Complementation

Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 60 years of age. The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions (Lewy bodies) in surviving neurons. Lewy bodies are mainly composed of the pre-synaptic protein alpha-synuclein (αsyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble αsyn and monitoring αsyn aggregation in living cells is fundamental to study the molecular mechanisms of αsyn-induced cytotoxicity and develop therapeutic strategies to prevent αsyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify αsyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed αsyn variants and showed that this method can be used to study how αsyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on αsyn solubility. The results presented underscore the utility of the split GFP assay to quantify αsyn solubility in living cells.     

HbS-Savaria: The Anti-polymerization Effect of a Single Mutation in Human α-chains

Recombinant α-Savaria globin (α^S49R) was assembled with β^S chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (α₂^S49Rβ₂^E6V) that exhibited normal O₂ affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O₂ affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 ± 0.025 vs. 0.46 ± 0.03, respectively). The C_SAT of HbS increased from 16.7 ± 0.8 to 27.0 ± 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at α-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory α-chains and gene therapy of sickle cell anemia.     

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (βE6V/αH20R) and rHb (βE6V/αH20Q)) or lateral (rHb (βE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The ¹H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the β-heme pockets. All mutants have similar cooperativity (n₅₀), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P₅₀) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (βE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (βE6V/αH20Q), rHb (βE6V/αH50Q), rHb (βE6V/αH20R/αH50Q), and rHb (βE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (βE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).     

Evidence of Native α-Synuclein Conformers in the Human Brain

α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.     

Comparative Analysis of MPTP Neurotoxicity in Mice with a Constitutive Knockout of the α-Synuclein Gene

Molecular Biology - Aggregated forms of α-synuclein are core components of pathohistological inclusions known as Lewy bodies in substantia nigra (SN) neurons of patients with Parkinson’s...     

17-AAG Induces Cytoplasmic α-Synuclein Aggregate Clearance by Induction of Autophagy

Background

The accumulation and aggregation of α-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. α-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved.

Methodology/Principal Findings

In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human α-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of α-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH₄Cl the aggregate clearing effects of 17-AAG were abolished and α-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of α-synuclein aggregates in OLN-A53T cells.

Conclusions

Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which α-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.     

A Protofilament-Protofilament Interface in the Structure of Mouse α-Synuclein Fibrils

Fibrillar α-synuclein (AS) is the major component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Using solid-state nuclear magnetic resonance (ssNMR), we previously reported a structural characterization of mouse AS (mAS) fibrils and found that the secondary structure of the mAS fibrils is highly similar to a form of human AS (hAS) fibrils. Recently, a three-dimensional structure of these same hAS fibrils was determined by ssNMR and scanning transmission electron microscopy. Using medium- and long-range distance restraints obtained from ssNMR spectra, we found that the single protofilament structure of mAS fibrils is also similar to that of the hAS fibrils. However, residue-specific water accessibility of mAS fibrils probed by water polarization transfer ssNMR measurements indicates that residues S42–T44 and G84–V95 are largely protected from water even though they are located at the edge of the protofilament. Some of the corresponding resonances also exhibit peak doubling. These observations suggest that these residues may be involved in, to our knowledge, a novel protofilament-protofilament interface. We propose a structural model of mAS fibrils that incorporates this dimer interface.     

The Z Mutation Alters the Global Structural Dynamics of α1-Antitrypsin

α₁-Antitrypsin (α₁AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α₁AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α₁AT compared to the wild type protein, M α₁AT, and so it is unclear why Z α₁AT has an increased polymerization propensity. We speculated that the loss of these interactions would make the native state of Z α₁AT more dynamic than M α₁AT and that this change renders the protein more polymerization prone. We have used hydrogen/deuterium exchange combined with mass spectrometry (HXMS) to determine the structural and dynamic differences between native Z and M α₁AT to reveal the molecular basis of Z α₁AT polymerization. Our HXMS data shows that the Z mutation significantly perturbs the region around the site of mutation. Strikingly the Z mutation also alters the dynamics of regions distant to the mutation such as the B, D and I helices and specific regions of each β-sheet. These changes in global dynamics may lead to an increase in the likelihood of Z α₁AT sampling a polymerogenic structure thereby causing disease.     

New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease

The causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neuroblastoma cell model of PD induced by MPP⁺, the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway, regulating cathD activity and affecting two main biological processes, α-synuclein aggregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology.     

The Novel S527F Mutation in the Integrin ��3 Chain Induces a High Affinity ��IIb��3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor αIIbβ3 in a patient with an ill defined platelet disorder: one in the β3 gene (S527F) and two in the αIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant αIIbβ3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the β3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated αIIbβ3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of αIIbβ3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe⁵²⁷ in β3 and the calf-1 domain in αIIb and by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin receptors that consist of noncovalently linked α/β-heterodimers. They are cell-surface receptors that play a role in cell-cell and cell-matrix interactions. Under resting conditions, integrin receptors adopt the low affinity conformation and do not interact with their ligands. Inside-out signaling turns the receptor into a high affinity conformation capable of ligand binding. Ligand binding itself induces additional conformational changes resulting in exposure of neoantigenic sites called ligand-induced binding sites (LIBS)³ and generates in turn outside-in signaling, which triggers a range of downstream signals (1, 2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In flowing blood under resting conditions, αIIbβ3 does not interact with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere at sites of vascular injury and become activated. As a consequence, αIIbβ3 adopts the high affinity conformation and binds fibrinogen. This results in platelet aggregation and thrombus formation, which eventually will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal ligand-binding head domain (the β-propeller domain in the αIIb chain and the βI domain in the β3 chain) standing on two long legs or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial growth factor-like (I-EGF), and β-tail domains in the β3 chain), followed by transmembrane and cytoplasmic domains (1, 2). X-ray crystal structures of the extracellular domain of non-activated αVβ3 revealed that the legs are severely bent, putting the head domain next to the membrane-proximal portions of the legs (4, 5). The bending occurs between I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1 domains in the α-subunit. This bent conformation represents the low affinity state of the receptor. The high affinity state of the receptor is induced by activation and is associated with a large-scale conformational rearrangement in which the integrin extends with a switchblade-like motion (2). Recently, the crystal structure of the entire extracellular domain of αIIbβ3 in its low affinity conformation was resolved and revealed that this integrin also adopts the bent conformation under resting conditions (6). Structural rearrangements in αIIbβ3 between the bent and extended conformations are similar to what has been reported for other integrins (7).We report here that the S527F mutation in the I-EGF3 region of the β3 polypeptide chain of the αIIbβ3 receptor induces a constitutively active receptor adopting an extended high affinity conformation. This was evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding. These data were further corroborated by modeling the replacement of Ser⁵²⁷ with Phe in the crystal structure of the extracellular domain of αIIbβ3. In this model, the S527F mutation decreases the flexibility of I-EGF3 and appears to prevent movement of the lower β-leg into the cleft between the upper β-leg and the lower α-leg. As a consequence, formation of the bent conformation of the non-activated receptor is hampered.     

The Effect of a ΔK280 Mutation on the Unfolded State of a Microtubule-Binding Repeat in Tau

Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer''s disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW), samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2) in wild-type (WT) tau and a ΔK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and ΔK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of β-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.     

Characterization of Inhibitor-Bound α-Synuclein Dimer: Role of α-Synuclein N-Terminal Region in Dimerization and Inhibitor Binding

α-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other α-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit α-synuclein assembly with low micromolar IC₅₀ values, and that SDS-stable, noncytotoxic soluble α-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound α-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound α-synucleins in detail, we have prepared α-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated α-synuclein monomer and dimer were oxidized at all four methionine residues of α-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of α-synuclein. Ultra-high-field NMR analysis of inhibitor-bound α-synuclein dimers showed that the signals derived from the N-terminal region of α-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric α-synuclein. We propose that the N-terminal region of α-synuclein plays a key role in the formation of α-synuclein assemblies.     

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.     

The Cancer Mutation D83V Induces an α-Helix to β-Strand Conformation Switch in MEF2B

MEF2B is a major target of somatic mutations in non-Hodgkin lymphoma. Most of these mutations are non-synonymous substitutions of surface residues in the MADS-box/MEF2 domain. Among them, D83V is the most frequent mutation found in tumor cells. The link between this hotspot mutation and cancer is not well understood. Here we show that the D83V mutation induces a dramatic α-helix to β-strand switch in the MEF2 domain. Located in an α-helix region rich in β-branched residues, the D83V mutation not only removes the extensive helix stabilization interactions but also introduces an additional β-branched residue that further shifts the conformation equilibrium from α-helix to β-strand. Cross-database analyses of cancer mutations and chameleon sequences revealed a number of well-known cancer targets harboring β-strand favoring mutations in chameleon α-helices, suggesting a commonality of such conformational switch in certain cancers and a new factor to consider when stratifying the rapidly expanding cancer mutation data.     

The role of α-synuclein in the pathophysiology of alcoholism

Alcoholism has complex etiology and there is evidence for both genetic and environmental factors in its pathophysiology. Chronic, long-term alcohol abuse and alcohol dependence are associated with neuronal loss with the prefrontal cortex being particularly susceptible to neurotoxic damage. This brain region is involved in the development and persistence of alcohol addiction and neurotoxic damage is likely to exacerbate the reinforcing effects of alcohol and may hinder treatment. Understanding the mechanism of alcohol’s neurotoxic effects on the brain and the genetic risk factors associated with alcohol abuse are the focus of current research. Because of its well-established role in neurodegenerative and neuropsychological disorders, and its emerging role in the pathophysiology of addiction, here we review the genetic and epigenetic factors involved in regulating α-synuclein expression and its potential role in the pathophysiology of chronic alcohol abuse. Elucidation of the mechanisms of α-synuclein regulation may prove beneficial in understanding the role of this key synaptic protein in disease and its potential for therapeutic modulation in the treatment of substance use disorders as well as other neurodegenerative diseases.     

The Role of N53Q Mutation on the Rat μ-Opioid Receptor Function

Glycosylation of the μ-opioid receptor may play an important role on its function. Using nested PCR, N53Q mutation was prepared in the N-terminal region of the rat μ-opioid receptor cDNA and cloned into the pcDNA3 vector. The plasmids containing the wild-type and mutated receptor cDNA were transfected into the COS-7 cells. Intracellular cAMP was measured in the morphine-treated and untreated transfected cells using an ELISA kit. Plasmid expression was evaluated using X-gal staining. Intracellular concentration of cAMP in the N53Q-mutated cells was not significantly different from the wild-type. The expression of the transfected plasmids was confirmed. Therefore, based on these results, it seems that glycosylation at the N53 site of the rat μ-opioid receptor does not influence the function of this receptor significantly.