SLC19A1 Genetic Variation Leads to Altered Thiamine Diphosphate Transport: Implications for the Risk of Developing Wernicke-Korsakoff's Syndrome.

AIMS: Wernicke-Korsakoff syndrome (WKS) is commonly associated with chronic alcohol misuse, a condition known to have multiple detrimental effects on thiamine metabolism. This study was conducted to identify genetic variants that may contribute to the development of WKS in individuals with alcohol dependence syndrome through alteration of thiamine transport into cells. METHODS: Exome sequencing data from a panel of genes related to alcohol metabolism and thiamine pathways were analysed in a discovery cohort of 29 individuals with WKS to identify possible genetic risk variants associated with its development. Variant frequencies in this discovery cohort were compared with European frequencies in the Genome Aggregation Database browser, and those present at significantly higher frequencies were genotyped in an additional cohort of 87 alcohol-dependent cases with WKS and 197 alcohol-dependent cognitively intact controls. RESULTS: Thirty non-synonymous variants were identified in the discovery cohort and, after filtering, 23 were taken forward and genotyped in the case-control cohort. Of these SLC19A1:rs1051266:G was nominally associated with WKS. SLC19A1 encodes the reduced folate carrier, a major transporter for physiological folate in plasma; rs1051266 is reported to impact folate transport. Thiamine pyrophosphate (TPP) efflux was significantly decreased in HEK293 cells, stably transfected with rs1051266:G, under thiamine deficient conditions when compared with the efflux from cells transfected with rs1051266:A (P = 5.7 × 10-11). CONCLUSION: This study provides evidence for the role of genetic variation in the SLC19A1 gene, which may contribute to the development of WKS in vivo through modulation of TPP transport in cells.

Mouse maternal protein restriction during preimplantation alone permanently alters brain neuron proportion and adult short-term memory.

Maternal protein malnutrition throughout pregnancy and lactation compromises brain development in late gestation and after birth, affecting structural, biochemical, and pathway dynamics with lasting consequences for motor and cognitive function. However, the importance of nutrition during the preimplantation period for brain development is unknown. We have previously shown that maternal low-protein diet (LPD) confined to the preimplantation period (Emb-LPD) in mice, with normal nutrition thereafter, is sufficient to induce cardiometabolic and locomotory behavioral abnormalities in adult offspring. Here, using a range of in vivo and in vitro techniques, we report that Emb-LPD and sustained LPD reduce neural stem cell (NSC) and progenitor cell numbers at E12.5, E14.5, and E17.5 through suppressed proliferation rates in both ganglionic eminences and cortex of the fetal brain. Moreover, Emb-LPD causes remaining NSCs to up-regulate the neuronal differentiation rate beyond control levels, whereas in LPD, apoptosis increases to possibly temper neuron formation. Furthermore, Emb-LPD adult offspring maintain the increase in neuron proportion in the cortex, display increased cortex thickness, and exhibit short-term memory deficit analyzed by the novel-object recognition assay. Last, we identify altered expression of fragile X family genes as a potential molecular mechanism for adverse programming of brain development. Collectively, these data demonstrate that poor maternal nutrition from conception is sufficient to cause abnormal brain development and adult memory loss.

Evolution of a maternal immune activation (mIA) model in rats: Early developmental effects.

Maternal immune activation (mIA) in rodents is rapidly emerging as a key model for neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Here, we optimise a mIA model in rats, aiming to address certain limitations of current work in this field. Specifically, the lack of clear evidence for methodology chosen, identification of successful induction of mIA in the dams and investigation of male offspring only. We focus on gestational and early juvenile changes in offspring following mIA, as detailed information on these critical early developmental time points is sparse. Following strain (Wistar, Lister Hooded, Sprague Dawley) comparison and selection, and polyriboinosinic-polyribocytidylic acid (poly I:C) dose selection (2.5-15 mg/kg single or once daily for 5 days), mIA was induced in pregnant Wistar rats with 10 mg/kg poly I:C i.p. on gestational day (GD) 15. Early morphometric analysis was conducted in male and female offspring at GD21 and postnatal day (PD) 21, eight dams for each treatment at each time point were used, 32 in total. Subsequent microglia analysis was conducted at PD21 in a small group of offspring. Poly I:C at 10 mg/kg i.p. induced a robust, but variable, plasma IL-6 response 3 h post-injection and reduced body weight at 6 h and 24 h post-injection in two separate cohorts of Wistar rats at GD15. Plasma IL-6 was not elevated at PD21 in offspring or dams. Poly I:C-induced mIA did not affect litter numbers, but resulted in PD21 pup, and GD21 placenta growth restriction. Poly I:C significantly increased microglial activation at PD21 in male hippocampi. We have identified 10 mg/kg poly I:C i.p on GD15 as a robust experimental approach for inducing mIA in Wistar rats and used this to identify early neurodevelopmental changes. This work provides a framework to study the developmental trajectory of disease-relevant, sex-specific phenotypic changes in rats.

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements.

BACKGROUND/AIMS: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. METHODS: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. RESULTS: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. CONCLUSION: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques.

Counter-regulation of alpha- and beta-synuclein expression at the transcriptional level.

Alpha-synuclein is a cytosolic protein associated with a range of diseases including Parkinson's disease. In these diseases alpha-synuclein aggregates and this is believed to play a causative role in disease progression. Alpha-synuclein aggregation has been suggested to be caused by increased expression levels and has also been suggested to be countered by increased beta-synuclein expression. In this regard, strategies to counter-regulate the expression of the synucleins by increasing beta-synuclein expression relative to alpha-synuclein may be beneficial in preventing disease progression. We therefore studied the regulation of alpha-synuclein to try to identify pathways that might counter-regulate the synucleins. We identified members of the ZSCAN family of transcription factors as specific repressors of alpha-synuclein. In particular ZSCAN21 was found to both repress alpha-synuclein and increase beta-synuclein expression. These findings support the notion that a single pathway in the cell can counter-regulate the expression of the synucleins. Support for this came from experiments that showed that ZSCAN21 expression decreases alpha-synuclein aggregation in the cells.

Maternal Undernutrition Induces Cell Signalling and Metabolic Dysfunction in Undifferentiated Mouse Embryonic Stem Cells.

Peri-conceptional environment can induce permanent changes in embryo phenotype which alter development and associate with later disease susceptibility. Thus, mouse maternal low protein diet (LPD) fed exclusively during preimplantation is sufficient to lead to cardiovascular, metabolic and neurological dysfunction in adult offspring. Embryonic stem cell (ESC) lines were generated from LPD and control NPD C57BL/6 blastocysts and characterised by transcriptomics, metabolomics, bioinformatics and molecular/cellular studies to assess early potential mechanisms in dietary environmental programming. Previously, we showed these lines retain cellular and epigenetic characteristics of LPD and NPD embryos after several passages. Here, three main changes were identified in LPD ESC lines. First, their derivation capacity was reduced but pluripotency marker expression was similar to controls. Second, LPD lines had impaired Mitogen-activated protein kinase (MAPK) pathway with altered gene expression of several regulators (e.g., Maff, Rassf1, JunD), reduced ERK1/2 signalling capacity and poorer cell survival characteristics which may contribute to reduced derivation. Third, LPD lines had impaired glucose metabolism comprising reduced upstream enzyme expression (e.g., Gpi, Mpi) and accumulation of metabolites (e.g., glucose-6-P, fructose-6-P) above the phosphofructokinase (PFK) gateway with PFK enzyme activity reduced. ESC lines may therefore permit investigation of peri-conceptional programming mechanisms with reduced need for animal experimentation.

The Identification of Human Translational Biomarkers of Neuropathic Pain and Cross-Species Validation Using an Animal Model.

Neuropathic pain is a common chronic condition, which remains poorly understood. Many patients receiving treatment continue to experience severe pain, due to limited diagnostic/treatment management programmes. The development of objective clinical diagnostic/treatment strategies requires identification of robust biomarkers of neuropathic pain. To this end, we looked to identify biomarkers of chronic neuropathic pain by assessing gene expression profiles in an animal model of neuropathic pain, and differential gene expression in patients to determine the potential translatability. We demonstrated cross-species validation of several genes including those identified through bioinformatic analysis by assessing their expression in blood samples from neuropathic pain patients, according to conservative assessments of significance measured using Bonferroni-corrected p-values. These include CASP5 (p = 0.00226), CASP8 (p = 0.00587), CASP9 (p = 2.09 × 10-9), FPR2 (p = 0.00278), SH3BGRL3 (p = 0.00633), and TMEM88 (p = 0.00038). A ROC analysis revealed several combinations of genes to show high levels of discriminatory power in the comparison of neuropathic pain patients and control participants, of which the combination SH3BGRL3, TMEM88, and CASP9 achieved the highest level (AUROC = 0.923). The CASP9 gene was found to be common in five combinations of three genes revealing the highest levels of discriminatory power. In contrast, the gene combination PLAC8, ROMO1, and A3GALT2 showed the highest levels of discriminatory power in the comparison of neuropathic pain and nociceptive pain (AUROC = 0.919), when patients were grouped by S-LANSS scores. Molecules that demonstrate an active role in neuropathic pain have the potential to be developed into a biological measure for objective diagnostic tests, or as novel drug targets for improved pain management.

Astragalus and human mesenchymal stem cells promote wound healing by mediating immunomodulatory effects through paracrine signaling.

Background: Skin regeneration from an injury without a scar is still a challenge. Methods: A murine model of a skin wound was treated with a combination of extract of astragalus and exosomes of mesenchymal stem cells (MSCs). CD11b+ and CD45 macrophages were detected and levels of cytokines were tested. Results: The expression of growth factors VEGF, FGF2 and EGF was elevated after treatment administered to MSCs. The administration of ethanolic extract of astragalus decreased the expression of TNF-α, IL-1ß and IL-6 and simultaneously increased the levels of IL-10. The combination sped up the process of wound healing. A sustained-release gel with both ingredients was developed to enhance restoration from granulation. Conclusion: The extract of astragalus promotes the efficacy of MSC-derived exosomes in skin repair.

Recovery from and regeneration of skin wounds are essential to maintaining epidermal function. Improving restoration and reducing scar tissue effectively need to be explored. Here, the authors investigated the potential role of extracts from the combination of an herbal plant (astragalus) and mesenchymal stem cells in wound healing. The administration of ethanolic extract of astragalus decreased the expression of inflammatory factors, increased the anti-inflammatory factor IL-10 and inhibited the proliferation of fibroblasts. The authors found that the combination treatment reduced the recovery time, with a lighter scar. Finally, the authors developed a slow-release gel with the mixture to prolong the effect and promote wound repair. Ethanolic extract of astragalus could enhance the properties of mesenchymal stem cells by effectively increasing recovery speed and improving prognosis.

The Identification of Blood Biomarkers of Chronic Neuropathic Pain by Comparative Transcriptomics.

In this study, we recruited 50 chronic pain (neuropathic and nociceptive) and 43 pain-free controls to identify specific blood biomarkers of chronic neuropathic pain (CNP). Affymetrix microarray was carried out on a subset of samples selected 10 CNP and 10 pain-free control participants. The most significant genes were cross-validated using the entire dataset by quantitative real-time PCR (qRT-PCR). In comparative analysis of controls and CNP patients, WLS (P = 4.80 × 10-7), CHPT1 (P = 7.74 × 10-7) and CASP5 (P = 2.30 × 10-5) were highly significant, whilst FGFBP2 (P = 0.00162), STAT1 (P = 0.00223), FCRL6 (P = 0.00335), MYC (P = 0.00335), XCL2 (P = 0.0144) and GZMA (P = 0.0168) were significant in all CNP patients. A three-arm comparative analysis was also carried out with control as the reference group and CNP samples differentiated into two groups of high and low S-LANSS score using a cut-off of 12. STAT1, XCL2 and GZMA were not significant but KIR3DL2 (P = 0.00838), SH2D1B (P = 0.00295) and CXCR31 (P = 0.0136) were significant in CNP high S-LANSS group (S-LANSS score > 12), along with WLS (P = 8.40 × 10-5), CHPT1 (P = 7.89 × 10-4), CASP5 (P = 0.00393), FGFBP2 (P = 8.70 × 10-4) and FCRL6 (P = 0.00199), suggesting involvement of immune pathways in CNP mechanisms. None of the genes was significant in CNP samples with low (< 12) S-LANSS score. The area under the receiver operating characteristic (AUROC) analysis showed that combination of MYC, STAT1, TLR4, CASP5 and WLS gene expression could be potentially used as a biomarker signature of CNP (AUROC - 0.852, (0.773, 0.931 95% CI)).

Contribution of individual histidines to prion protein copper binding.

The prion protein is well-established as a copper binding protein. The N-terminus of the protein contains an octameric repeat region with each of the four repeats containing a histidine. The N-terminus has two additional histidines distal to the repeat region that has been commonly known as the fifth site. While binding of copper by the protein has been extensively studied, the contribution of each histidine to copper binding in the full-length protein has not. Here we used a battery of mutants of the recombinant mouse prion protein to assess copper binding with both isothermal titration calorimetry and cyclic voltammetry. The findings indicate that there is extensive cooperativity between different binding sites in the protein. The two highest-affinity binding events occur at the fifth site and at the octameric repeat region. However, the first binding is that to the octameric repeat region. Subsequent binding events after the two initial binding events have lower affinities within the octameric repeat region.

Activation and repression of prion protein expression by key regions of intron 1.

Expression of the prion protein is necessary for infection with prion diseases. Altered expression levels may play an important role in susceptibility to infection. Therefore, understanding the mechanisms that regulate prion protein expression is of great importance. It was previously shown that expression of the prion protein is to some degree regulated by an alternative promoter within intron 1. Studies using GFP and luciferase reporter systems were undertaken to determine key sites for the repression and activation of expression of the prion protein driven by intron 1. We identified a region within intron 1 sufficient to drive prion protein expression. Our findings highlight two potential repressor regions. Both regions have binding sites for the known repressor Hes-1. Hes-1 overexpression caused a dramatic decrease in PrP protein expression. Additionally, we have identified Atox-1 as a transcription factor that upregulates prion protein expression. These findings clearly indicate that intron 1 plays a key role in regulation of prion protein expression levels.

Repurposing of Tranilast for Potential Neuropathic Pain Treatment by Inhibition of Sepiapterin Reductase in the BH4 Pathway.

Tetrahydrobiopterin (BH4) is a cofactor in the production of various signaling molecules including nitric oxide, dopamine, adrenaline, and noradrenaline. BH4 levels are critical for processes associated with cardiovascular function, inflammation, mood, pain, and neurotransmission. Increasing pieces of evidence suggest that BH4 is upregulated in chronic pain. Sepiapterin reductase (SPR) catalyzes both the reversible reduction of sepiapterin to dihydrobiopterin (BH2) and 6-pyruvoyl-tetrahydrobiopterin to BH4 within the BH4 pathway. Therefore, inhibition of SPR by small molecules can be used to control BH4 production and ultimately alleviate chronic pain. Here, we have used various in silico and in vitro experiments to show that tranilast, licensed for use in bronchial asthma, can inhibit sepiapterin reduction by SPR. Docking and molecular dynamics simulations suggest that tranilast can bind to human SPR (hSPR) at the same site as sepiapterin including S157, one of the catalytic triad residues of hSPR. Colorimetric assays revealed that tranilast was nearly twice as potent as the known hSPR inhibitor, N-acetyl serotonin. Tranilast was able to inhibit hSPR activity both intracellularly and extracellularly in live cells. Triple quad mass spectrophotometry of cell lysates showed a proportional decrease of BH4 in cells treated with tranilast. Our results suggest that tranilast can act as a potent hSPR inhibitor and therefore is a valid candidate for drug repurposing in the treatment of chronic pain.

Proteomic analysis of embryonic stem cell-derived neural cells exposed to the antidepressant paroxetine.

Antidepressant drugs can have significant effects on the mood of a patient suffering from major depression or other disorders. The pharmacological actions of these drugs generally affect the uptake or metabolism of the neurotransmitters serotonin, noradrenalin, and, to a lesser extent, dopamine. However, many aspects of antidepressant action are not understood. We conducted a proteomic analysis in a neuronal cell culture model in an attempt to identify molecules important to the operation of pathways functionally relevant to antidepressant action. The model involved generating cultures containing mixed neural and glial cells by controlled differentiation of mouse embryonic stem cells, followed by exposure to 1 microM paroxetine for 14 days. After antidepressant exposure, we observed increased expression or modification of sepiapterin reductase (SPR), heat shock protein 9A, RAS and EF-hand domain containing, and protein disulfide isomerase associated 3 and decreased expression or modification of creatine kinase, actin, prohibitin, a T-cell receptor alpha chain, defensin-related cryptdin 5, and the intermediate filament proteins glial fibrillary acidic protein and vimentin. SPR, the most strongly up-regulated protein observed, controls production of tetrahydrobiopterin, an essential cofactor for the synthesis of many neurotransmitters including serotonin, making it a plausible and intriguing candidate protein for involvement in mood control and antidepressant drug action. SPR and the other proteins identified may represent links to molecular processes of importance to mood dysregulation and control, and their respective genes may be novel candidates for the study of antidepressant pharmacogenetics.

Increased Oxidative Stress as a Risk Factor in Chronic Idiopathic Axonal Polyneuropathy.

Chronic idiopathic axonal polyneuropathy (CIAP) is a disorder with insidious onset and slow progression, where no etiology is identified despite appropriate investigations. We aimed to investigate the role of oxidative stress as a risk factor for the pathogenesis of CIAP. Sera of patients with CIAP were tested for protein carbonyl (PC) and 8-hydroxydeoxyguanosine (8H). As a control group, we recruited patients with gluten neuropathy. Twenty-one patients with CIAP and 21 controls were recruited. The two groups did not differ significantly regarding demographics or clinical characteristics (i.e., neuropathy type or disease severity). After adjusting for gender, having CIAP was positively correlated with both the 8H titer (standardized beta coefficient 0.349, p = 0.013) and the PC titer (standardized beta coefficient 0.469, p = 0.001). Oxidative stress appears to be increased in CIAP and might have a role in the pathogenesis of the disease.

The Development of Translational Biomarkers as a Tool for Improving the Understanding, Diagnosis and Treatment of Chronic Neuropathic Pain.

Chronic neuropathic pain (CNP) is one of the most significant unmet clinical needs in modern medicine. Alongside the lack of effective treatments, there is a great deficit in the availability of objective diagnostic methods to reliably facilitate an accurate diagnosis. We therefore aimed to determine the feasibility of a simple diagnostic test by analysing differentially expressed genes in the blood of patients diagnosed with CNP of the lower back and compared to healthy human controls. Refinement of microarray expression data was performed using correlation analysis with 3900 human 2-colour microarray experiments. Selected genes were analysed in the dorsal horn of Sprague-Dawley rats after L5 spinal nerve ligation (SNL), using qRT-PCR and ddPCR, to determine possible associations with pathophysiological mechanisms underpinning CNP and whether they represent translational biomarkers of CNP. We found that of the 15 potential biomarkers identified, tissue inhibitor of matrix metalloproteinase-1 (TIMP1) gene expression was upregulated in chronic neuropathic lower back pain (CNBP) (p = 0.0049) which positively correlated (R = 0.68, p = ≤0.05) with increased plasma TIMP1 levels in this group (p = 0.0433). Moreover, plasma TIMP1 was also significantly upregulated in CNBP than chronic inflammatory lower back pain (p = 0.0272). In the SNL model, upregulation of the Timp1 gene was also observed (p = 0.0058) alongside a strong trend for the upregulation of melanocortin 1 receptor (p = 0.0847). Our data therefore highlights several genes that warrant further investigation, and of these, TIMP1 shows the greatest potential as an accessible and translational CNP biomarker.

Nonsynonymous Polymorphism in Guanine Monophosphate Synthetase Is a Risk Factor for Unfavorable Thiopurine Metabolite Ratios in Patients With Inflammatory Bowel Disease.

Background: Up to 20% of patients with inflammatory bowel disease (IBD) who are refractory to thiopurine therapy preferentially produce 6-methylmercaptopurine (6-MMP) at the expense of 6-thioguanine nucleotides (6-TGN), resulting in a high 6-MMP:6-TGN ratio (>20). The objective of this study was to evaluate whether genetic variability in guanine monophosphate synthetase (GMPS) contributes to preferential 6-MMP metabolizer phenotype. Methods: Exome sequencing was performed in a cohort of IBD patients with 6-MMP:6-TGN ratios of >100 to identify nonsynonymous single nucleotide polymorphisms (nsSNPs). In vitro assays were performed to measure GMPS activity associated with these nsSNPs. Frequency of the nsSNPs was measured in a cohort of 530 Caucasian IBD patients. Results: Two nsSNPs in GMPS (rs747629729, rs61750370) were detected in 11 patients with very high 6-MMP:6-TGN ratios. The 2 nsSNPs were predicted to be damaging by in silico analysis. In vitro assays demonstrated that both nsSNPs resulted in a significant reduction in GMPS activity (P < 0.05). The SNP rs61750370 was significantly associated with 6-MMP:6-TGN ratios ≥100 (odds ratio, 5.64; 95% confidence interval, 1.01-25.12; P < 0.031) in a subset of 264 Caucasian IBD patients. Conclusions: The GMPS SNP rs61750370 may be a reliable risk factor for extreme 6MMP preferential metabolism.

Cognitive ability in Down syndrome and its relationship to urinary neopterin, a marker of activated cellular immunity.

BACKGROUND: Neopterin is an unconjugated pteridine that is secreted in large quantities by activated macrophages and can be used as a clinical marker of activated cellular immunity and oxidative stress. We aimed to investigate whether urinary neopterin levels are associated with cognitive function in people with Down syndrome (DS). METHODS: Out of 32 adults with DS who originally participated in a longitudinal study, 25 were followed up at 4 years. Informants rated their adaptive behavior (ABAS) and the adults with DS attempted assessments of language skills and memory at both baseline and follow-up time points (Modified Memory Object Task, MOMT), and receptive vocabulary (British Picture Vocabulary Scale, BPVS). RESULTS: Neopterin/creatinine levels were negatively correlated with change in the MOMT total score (Spearman's Rho=-0.517, p=0.020) and change in the MOMT delayed recall score (Spearman's Rho=-0.577, p=0.008) over time, i.e. higher neopterin/creatinine level was associated with worse performance on a test of cognitive ability over time. CONCLUSION: Urine neopterin may have potential as a biomarker for memory decline in Down syndrome, and could potentially also help to track progression of mild cognitive impairment (MCI) to Alzheimer's disease in other high risk populations.

Pain as a First Manifestation of Paraneoplastic Neuropathies: A Systematic Review and Meta-Analysis.

INTRODUCTION: Paraneoplastic neurological syndromes (PNS) consist of a heterogeneous group of neurological disorders triggered by cancer. The aim of this systematic review is to estimate the reported prevalence of pain in patients with paraneoplastic peripheral neuropathy (PPN). METHODS: A systematic computer-based literature search was conducted on PubMed database. RESULTS: Our search strategy resulted in the identification of 126 articles. After the eligibility assessment, 45 papers met the inclusion criteria. Full clinical and neurophysiological data were further extracted and involved 92 patients with PPN (54.5% males, mean age 60.0 ± 12.2 years). The commonest first manifestation of PPN is sensory loss (67.4%), followed by pain (41.3%), weakness (22.8%), and sensory ataxia (20.7%). In 13.0% of the cases, pain was the sole first manifestation of the PPN. During the course of the PPN, 57.6% of the patients may experience pain secondary to the neuropathy. CONCLUSIONS: Pain is very prevalent within PPN. Pain specialists should be aware of this. Detailed history-taking, full clinical examination, and requesting nerve conduction studies might lead to an earlier diagnosis of an underlying malignancy.

Effects of different isoforms of apoE on aggregation of the α-synuclein protein implicated in Parkinson's disease.

Parkinson's disease is a progressive brain disorder due to the degeneration of dopaminergic neurons in the substantia nigra. The accumulation of aggregated forms of α-synuclein protein into Lewy bodies is one of the characteristic features of this disease although the pathological role of any such protein deposits in causing neurodegeneration remains elusive. Here, the effects of different apolipoprotein E isoforms (apoE2, apoE3, apoE4) on the aggregation of α-synuclein in vitro were examined using thioflavin T assays and also an immunoassay to detect the formation of multimeric forms. Our results revealed that the aggregation of α-synuclein is influenced by apoE concentration. At low concentrations of apoE (<15nM), all of the isoforms were able to increase the aggregation of α-synuclein (50µM), with apoE4 showing the greatest stimulatory effect. This is in contrast to a higher concentration (>15nM) of these isoforms, where a decrease in the aggregation of α-synuclein was noted. The data show that exceptionally low levels of apoE may seed α-syn aggregation, which could potentially lead to the pathogenesis of α-synuclein-induced neurodegeneration. On the other hand, higher levels of apoE could potentially lower the degree of α-synuclein aggregation and confer protection. The differential effects noted with apoE4 could explain why this particular isoform results in an earlier age of onset for Parkinson's disease.

The structure and function of the dopamine transporter and its role in CNS diseases.

In this chapter, we explore the basic science of the dopamine transporter (DAT), an integral component of a system that regulates dopamine homeostasis. Dopamine is a key neurotransmitter for several brain functions including locomotor control and reward systems. The transporter structure, function, mechanism of action, localization, and distribution, in addition to gene regulation, are discussed. Over many years, a wealth of information concerning the DAT has been accrued and has led to increased interest in the role of the DAT in a plethora of central nervous system diseases. These DAT characteristics are explored in relation to a range of neurological and neuropsychiatric diseases, with a particular focus on the genetics of the DAT. In addition, we discuss the pharmacology of the DAT and how this relates to disease and addiction.