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.

Selective dopaminergic neurotoxicity of three heterocyclic amine subclasses in primary rat midbrain neurons.

Heterocyclic amines (HCAs) are primarily produced during high temperature meat cooking. These compounds have been intensively investigated as mutagens and carcinogens. However, converging data suggest that HCAs may also be neurotoxic and potentially relevant to neurodegenerative diseases such as Parkinson's disease (PD). The identification of new potential etiological factors is important because most PD cases are sporadic. Our group previously showed that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was selectively neurotoxic to dopaminergic neurons. However, PhIP is one of many HCAs, a class of compounds that exhibits wide structural variability. The goal of this study was to determine the neurotoxicity of the most prevalent and best studied HCAs from three subclasses: aminoimidazoaazarenes (AIA), α-carbolines, and ß-carbolines. Using E17 rat primary midbrain cultures, we tested dopaminergic and non-dopaminergic neurotoxicity elicited by the following compounds: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), PhIP, 1-methyl-9H-pyrido[3,4-b]indole (harmane), 9H-pyrido[3,4-b]indole (norharmane) and 2-amino-9H-pyrido[2,3-b]indole (AαC) at concentrations ranging from 100 nM-5 µM. All tested HCAs were selectively neurotoxic, though the dose required to elicit selective loss of dopaminergic neurons or decreases in dopaminergic neurite length was compound specific. Non-dopaminergic neurons were unaffected at all tested doses. The sensitivity (determined by threshold dose required to elicit selective neurotoxicity) appears to be unrelated to published mutagenic potency. Both AIA and α/ß-carbolines produced oxidative damage, which was magnified in dopaminergic neurons vs. non-dopaminergic neurons as further evidence of selective neurotoxicity. These studies are expected to prompt clinical and mechanistic studies on the potential role of HCA exposure in PD.

Alterations in the nigrostriatal dopamine system after acute systemic PhIP exposure.

Heterocyclic amines (HCAs) are primarily formed during cooking of meat at high temperature. HCAs have been extensively studied as mutagens and possible carcinogens. Emerging data suggest that HCAs are neurotoxic and may be relevant to Parkinson's disease (PD) etiology. However, the majority of HCAs have not been evaluated for in vivo neurotoxicity. Here, we investigated acute in vivo neurotoxicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is the most prevalent genotoxin in many types of meats. Adult, male Sprague-Dawley rats were subjected to acute, systemic PhIP at doses and time-points that have been extensively utilized in cancer studies (100 and 200 mg/kg for 8, 24 h) and evaluated for changes in dopaminergic, serotoninergic, GABAergic, and glutamatergic neurotransmission. PhIP exposure resulted in decreased striatal dopamine metabolite levels and dopamine turnover in the absence of changes to vesicular monoamine transporter 2 levels; other neurotransmitter systems were unaffected. Quantification of intracellular nitrotyrosine revealed higher levels of oxidative damage in dopaminergic neurons in the substantia nigra after PhIP exposure, while other neuronal populations were less sensitive. These changes occurred in the absence of an overt lesion to the nigrostriatal dopamine system. Collectively, our study suggests that acute PhIP treatment in vivo targets the nigrostriatal dopaminergic system and that PhIP should be further examined in chronic, low-dose studies for PD relevance.

From the Cover: Harmane-Induced Selective Dopaminergic Neurotoxicity in Caenorhabditis elegans.

Parkinson's disease (PD) is a debilitating neurodegenerative disease. Although numerous exposures have been linked to PD etiology, causative factors for most cases remain largely unknown. Emerging data on the neurotoxicity of heterocyclic amines suggest that this class of compounds should be examined for relevance to PD. Here, using Caenorhabditis elegans as a model system, we tested whether harmane exposure produced selective toxicity to dopamine neurons that is potentially relevant to PD. Harmane is a known tremorigenic ß-carboline (a type of heterocyclic amine) found in cooked meat, roasted coffee beans, and tobacco. Thus, this compound represents a potentially important exposure. In the nematode model, we observed dopaminergic neurons to be selectively vulnerable, showing significant loss in terms of structure and function at lower doses than other neuronal populations. In examining mechanisms of toxicity, we observed significant harmane-induced decreases in mitochondrial viability and increased reactive oxygen species levels. Blocking transport through the dopamine transporter (DAT) was not neuroprotective, suggesting that harmane is unlikely to enter the cell through DAT. However, a mitochondrial complex I activator did partially ameliorate neurodegeneration. Further, mitochondrial complex I activator treatment reduced harmane-induced dopamine depletion, measured by the 1-nonanol assay. In summary, we have shown that harmane exposure in C. elegans produces selective dopaminergic neurotoxicity that may bear relevance to PD, and that neurotoxicity may be mediated through mitochondrial mechanisms.

The distinct genetic pattern of ALS in Turkey and novel mutations.

The frequency of amyotrophic lateral sclerosis (ALS) mutations has been extensively investigated in several populations; however, a systematic analysis in Turkish cases has not been reported so far. In this study, we screened 477 ALS patients for mutations, including 116 familial ALS patients from 82 families and 361 sporadic ALS (sALS) cases. Patients were genotyped for C9orf72 (18.3%), SOD1 (12.2%), FUS (5%), TARDBP (3.7%), and UBQLN2 (2.4%) gene mutations, which together account for approximately 40% of familial ALS in Turkey. No SOD1 mutations were detected in sALS patients; however, C9orf72 (3.1%) and UBQLN2 (0.6%) explained 3.7% of sALS in the population. Exome sequencing revealed mutations in OPTN, SPG11, DJ1, PLEKHG5, SYNE1, TRPM7, and SQSTM1 genes, many of them novel. The spectrum of mutations reflect both the distinct genetic background and the heterogeneous nature of the Turkish ALS population.

Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones.

Genome-wide copy number variation in sporadic amyotrophic lateral sclerosis in the Turkish population: deletion of EPHA3 is a possible protective factor.

The genome-wide presence of copy number variations (CNVs), which was shown to affect the expression and function of genes, has been recently suggested to confer risk for various human disorders, including Amyotrophic Lateral Sclerosis (ALS). We have performed a genome-wide CNV analysis using PennCNV tool and 733K GWAS data of 117 Turkish ALS patients and 109 matched healthy controls. Case-control association analyses have implicated the presence of both common (>5%) and rare (<5%) CNVs in the Turkish population. In the framework of this study, we identified several common and rare loci that may have an impact on ALS pathogenesis. None of the CNVs associated has been implicated in ALS before, but some have been reported in different types of cancers and autism. The most significant associations were shown for 41 kb and 15 kb intergenic heterozygous deletions (Chr11: 50,545,009-50,586,426 and Chr19: 20,860,930-20,875,787) both contributing to increased risk for ALS. CNVs in coding regions of the MAP4K3, HLA-B, EPHA3 and DPYD genes were detected however, after validation by Log R Ratio (LRR) values and TaqMan CNV genotyping, only EPHA3 deletion remained as a potential protective factor for ALS (p = 0.0065024). Based on the knowledge that EPHA4 has been previously shown to rescue SOD1 transgenic mice from ALS phenotype and prolongs survival, EPHA3 may be a promising candidate for therepuetic interventions.

Discovery, validation and characterization of Erbb4 and Nrg1 haplotypes using data from three genome-wide association studies of schizophrenia.

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 NRG1 and ERBB4, 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 NRG1 and ERBB4 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 ERBB4 between chromosome locations 212,156,823 and 212,162,848 in CATIE and GAIN datasets (p = 0.0206 and 0.0095, respectively). In NRG1, 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 ERBB4 and NRG1 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.

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.