Systemic Copper Disorders Influence the Olfactory Function in Adult Rats: Roles of Altered Adult Neurogenesis and Neurochemical Imbalance

Disrupted systemic copper (Cu) homeostasis underlies neurodegenerative diseases with early symptoms including olfactory dysfunction. This study investigated the impact of Cu dyshomeostasis on olfactory function, adult neurogenesis, and neurochemical balance. Models of Cu deficiency (CuD) and Cu overload (CuO) were established by feeding adult rats with Cu-restricted diets plus ip. injection of a Cu chelator (ammonium tetrathiomolybdate) and excess Cu, respectively. CuD reduced Cu levels in the olfactory bulb (OB), subventricular zone (SVZ), rostral migratory stream (RMS), and striatum, while CuO increased Cu levels in these areas. The buried pellet test revealed both CuD and CuO prolonged the latency to uncover food. CuD increased neural proliferation and stem cells in the SVZ and newly differentiated neurons in the OB, whereas CuO caused opposite alterations, suggesting a “switch”-type function of Cu in regulating adult neurogenesis. CuO increased GABA in the OB, while both CuD and CuO reduced DOPAC, HVA, 5-HT and the DA turnover rate in olfactory-associated brain regions. Altered mRNA expression of Cu transport and storage proteins in tested brain areas were observed under both conditions. Together, results support an association between systemic Cu dyshomeostasis and olfactory dysfunction. Specifically, altered adult neurogenesis along the SVZ-RMS-OB pathway and neurochemical imbalance could be the factors that may contribute to olfactory dysfunction

Phip-Induced Dopaminergic Neurotoxicity: In vitro and in vivo Mechanisms of Action

Parkinson’s disease (PD) is the second most common neurodegenerative disease with ~1% prevalence in the population over 60 years of age. PD patients manifest cardinal motor symptoms including bradykinesia, postural instability, resting tremor and rigidity, while non-motor symptoms arise decades earlier, and can be also debilitating. The neuropathology of the disease is characterized by loss of dopaminergic neurons in substantia nigra and subsequent depletion of dopamine in striatum in the midbrain. The hallmark molecular mechanisms underlying PD are aggregation of the protein alpha synuclein, oxidative stress, mitochondrial inhibition, disruption of dopamine homeostasis and neuroinflammation. Currently, there is no cure for this devastating disease whereas the treatments available only relieve the symptoms temporarily. ~10% of PD patients have been found to carry one or more mutations in the PD-relevant genes such as alpha synuclein and LRRK2. However, the rest is considered as sporadic comprising approximately nine million individuals worldwide. The role of environmental factors has been repeatedly emphasized in sporadic PD cases. Some of the PD-associated neurotoxins include pesticides, heavy metals, and accidental byproducts of illicit opioid drug synthesis. Although association of these compounds with the increased risk of PD has been confirmed in epidemiological studies, as well as cellular and animal models, the exposures only occur in minority of population such as in occupational settings. Thus, the search for neurotoxins that are exposed frequently in higher doses is still ongoing. Dietary factors represent such a group that receives far less attention compared to some of the PD-associated environmental toxins. Micro- and macronutrients (i.e. vitamins, polyphenols, fatty acids), and dietary habits (high calorie intake, Mediterranean diet) have been investigated as causative or protective factors of PD. However, the byproducts xx of cooking processes have not received much attention so far compared to raw dietary factors regarding neurodegeneration. Heterocyclic amines (HCAs) are formed during the cooking of meat at high temperatures, and were identified as potent mutagens three decades ago. Since then, they have been shown to induce tumor formation in several different types of tissues. HCAs have not been considered as dopaminergic neurotoxicants. However, structural similarities HCAs and PD-associated dopaminergic neurotoxins share, and limited number of studies showed their adverse effects in dopamine metabolism suggest their possible roles as contributors to dopaminergic neurotoxicity and PD-relevant mechanisms. Thus, we hypothesized that 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most HCAs found in cooked meat, induces selective PD-relevant dopaminergic neurotoxicity. In this dissertation, three specific aims are represented to test the role of PhIP as a dopaminergic neurotoxicant. First aim was to test PhIP-induced dopaminergic neurotoxicity in vitro using primary midbrain cultures. Second aim involved detailed and more in depth examination of in vitro mechanisms of PhIP-induced toxicity. The final goal in this thesis was to test whether PhIP exposure elicits motor deficits and neuropathology relevant to PD in vivo. Our studies showed that PhIP and its select phase I metabolite N-OH-PhIP cause selective dopaminergic neuronal loss, and neurite shortening in surviving dopaminergic neurons in primary midbrain cultures, while they did not affect other neuronal populations. Cell loss observed was found to be mediated by oxidative stress, and was alleviated in the presence of anti-oxidants such as blueberry extracts. In depth examination of mechanism of PhIP-induced dopaminergic neurotoxicity in vitro revealed that it inhibits proteasome degradation pathway, possibly leading to protein aggregation. Increase in free cytosolic dopamine levels was shown to enhance PhIP toxicity, suggesting dopamine metabolism as a contributor of the toxicity observed. Finally, PhIP induced DNA damage in astrocytes while sparing the neuronal populations. In vivo testing of PhIP neurotoxicity showed that acute PhIP exposure in wild type rats caused oxidative damage in dopaminergic neurons of substantia nigra while other dopaminergic xxi populations and neuronal cells nearby are less sensitive. Acute PhIP exposure also revealed decreases in striatal dopamine metabolites and turnover rate, suggesting possible inhibition of dopamine metabolism in the midbrain. These results suggest that acute PhIP exposure induces early mechanisms of PD without an overt lesion in the midbrain. Later, our studies testing subacute PhIP suggested systemic toxicity observed by decreased weight gain, and also subtle decreases in striatal dopamine turnover and nigral serotonin turnover in the absence of dopaminergic system lesion or motor deficits. Finally, we investigated gene-environment interaction using a transgenic line of rats that lacks an efflux transporter that results in increased accumulation of PhIP in brain. However, these animals did not show any exacerbated motor deficits or neuropathology compared to wild type rats. Overall, the findings presented in this dissertation, suggest the role of PhIP as a dopaminergic neurotoxicant in vitro and its contribution to early PD-relevant mechanisms including oxidative stress and disruption of dopamine metabolism in vivo. We believe the data generated in this thesis will pave the way for understanding how food consumed everyday might lead early neurodegeneration. Further studies are needed to test the chronic exposure in rodent models and to investigate gene-environment interactions considering differential metabolism of PhIP in rodents and humans

ATXN2 and Its Neighbouring Gene SH2B3 Are Associated with Increased ALS Risk in the Turkish Population

Expansions of the polyglutamine (polyQ) domain (>= 34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27-33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1-4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31-32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway

Transcription factors that bind common and significant haplotypes of <i>NRG1</i>.

<p>The frequencies and p-values of transcription factors that bind to the 25-kb haplotype block of <i>ERBB4 NRG1</i> block (chr 8: 32,291,552–32,317,192) in CATIE, GAIN and nonGAIN datasets are illustrated. <b>A.</b> The most common haplotype of 25-kb block in CATIE, GAIN and nonGAIN, and transcription factors that bind to this haplotype are shown. <b>B</b>, <b>C</b> and <b>D</b> depict the significant haplotypes of the same <i>NRG1</i> block in CATIE, GAIN and nonGAIN respectively.</p

Validation of previously identified and identification of novel haplotypes of <i>ERBB4</i> in schizophrenia GWAS datasets.

<p><b>A. </b><i>ERBB4</i> polymorphims in three schizophrenia GWAS datasets are illustrated in GWADview software. SNPs are plotted by their location on the y-axis and by their genomic position on the x-axis. Blue represents CATIE, red GAIN and green nonGAIN. The lower panel shows haplotype blocks of this region in Hapmap CEU population. <b>B.</b> LD plots of <i>ERBB4</i> with the most significant haplotypes in the region from 212,100,000 bp to 212,200,000 bp. Cut-off value for Hardy-Weinberg is 0.05 and for minor allele frequencies 0.001.</p

Transcription factors that bind common and significant haplotypes of <i>ERBB4</i>.

<p>The frequencies and p-values of transcription factors that bind to the 6-kb haplotype block of <i>ERBB4</i> (chr 2: 212,156,823–212,162,848) in CATIE, GAIN and nonGAIN datasets are illustrated. <b>A.</b> The most common haplotype of 6-kb block in CATIE, GAIN and nonGAIN, T-A-G-C, and transcription factors that bind to this haplotype are shown. <b>B</b>, <b>C</b> and <b>D</b> depict the significant haplotypes of the same <i>ERBB4</i> block in CATIE, GAIN and nonGAIN respectively.</p

Identification of gender-specific association of <i>NRG1</i> in schizophrenia GWAS datasets.

<p>LD plots of <i>NRG1</i> with the most significant haplotypes <b>A.</b> in the region from 31,618,950 bp to 31,732,358 bp in females. <b>B.</b> in the region from 32,257,152 bp to 32,288,979 bp in males Cut-off value for Hardy-Weinberg is 0.05 and for minor allele frequencies 0.001.</p

Discovery, Validation and Characterization of <em>Erbb4</em> and <em>Nrg1</em> Haplotypes Using Data from Three Genome-Wide Association Studies of Schizophrenia

<div><p>Schizophrenia is one of the most common and complex neuropsychiatric disorders, which is contributed both by genetic and environmental exposures. Recently, it is shown that NRG1-mediated ErbB4 signalling regulates many important cellular and molecular processes such as cellular growth, differentiation and death, particularly in myelin-producing cells, glia and neurons. Recent association studies have revealed genomic regions of <em>NRG1</em> and <em>ERBB4</em>, which are significantly associated with risk of developing schizophrenia; however, inconsistencies exist in terms of validation of findings between distinct populations. In this study, we aim to validate the previously identified regions and to discover novel haplotypes of <em>NRG1</em> and <em>ERBB4</em> using logistic regression models and Haploview analyses in three independent datasets from GWAS conducted on European subjects, namely, CATIE, GAIN and nonGAIN. We identified a significant 6-kb block in <em>ERBB4</em> between chromosome locations 212,156,823 and 212,162,848 in CATIE and GAIN datasets (p = 0.0206 and 0.0095, respectively). In <em>NRG1</em>, a significant 25-kb block, between 32,291,552 and 32,317,192, was associated with risk of schizophrenia in all CATIE, GAIN, and nonGAIN datasets (p = 0.0005, 0.0589, and 0.0143, respectively). Fine mapping and FastSNP analysis of genetic variation located within significantly associated regions proved the presence of binding sites for several transcription factors such as SRY, SOX5, CEPB, and ETS1. In this study, we have discovered and validated haplotypes of <em>ERBB4</em> and <em>NRG1</em> in three independent European populations. These findings suggest that these haplotypes play an important role in the development of schizophrenia by affecting transcription factor binding affinity.</p> </div

FastSNP analysis summary of SNPs in defined region of <i>ERBB4</i> and <i>NRG1</i>.

<p>The frequencies for common, rare and very rare SNPs are >25%, 10%-1% and <1%, respectively.</p

Validation of previously identified and identification of novel haplotypes of <i>NRG1</i> in schizophrenia GWAS datasets.

<p><b>A. </b><i>NRG1</i> polymorphims in three schizophrenia GWAS datasets are illustrated in GWADview software. SNPs are plotted by their location and genomic position on the y and x-axis respectively. Blue represents CATIE, red GAIN and green nonGAIN. The lower panel shows haplotype blocks of this region in Hapmap CEU population. <b>B.</b> LD plots of <i>NRG1</i> with the most significant haplotypes in the region from 32,250,000 bp to 32,400,000 bp. Cut-off value for Hardy-Weinberg is 0.05 and for minor allele frequencies 0.001.</p