Imaging Net Retrograde Axonal Transport In Vivo: A Physiological Biomarker

OBJECTIVE: The objective of this study is to develop a novel method for monitoring the integrity of motor neurons in vivo by quantifying net retrograde axonal transport. METHODS: The method uses single photon emission computed tomography to quantify retrograde transport to spinal cord of tetanus toxin fragment C ((125) I-TTC) following intramuscular injection. We characterized the transport profiles in 3 transgenic mouse models carrying amyotrophic lateral sclerosis (ALS)-associated genes, aging mice, and SOD1(G93A) transgenic mice following CRISPR/Cas9 gene editing. Lastly, we studied the effect of prior immunization of tetanus toxoid on the transport profile of TTC. RESULTS: This technique defines a quantitative profile of net retrograde axonal transport of TTC in living mice. The profile is distinctly abnormal in transgenic SOD1(G93A) mice as young as 65 days (presymptomatic) and worsens with disease progression. Moreover, this method detects a distinct therapeutic benefit of gene editing in transgenic SOD1(G93A) mice well before other clinical parameters (eg, grip strength) show improvement. Symptomatic transgenic PFN1(C71G/C71G) ALS mice display gross reductions in net retrograde axonal transport, which is also disturbed in asymptomatic mice harboring a human C9ORF72 transgene with an expanded GGGGCC repeat motif. In wild-type mice, net retrograde axonal transport declines with aging. Lastly, prior immunization with tetanus toxoid does not preclude use of this assay. INTERPRETATION: This assay of net retrograde axonal transport has broad potential clinical applications and should be particularly valuable as a physiological biomarker that permits early detection of benefit from potential therapies for motor neuron diseases

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

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

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

Summary of <i>ERBB4</i> and <i>NRG1</i> variants in GWAS datasets.

<p>Chromosome location, according to NCBI Build 36, of <i>ERBB4</i> and <i>NRG</i>1 are shown in the table. SNPs were pooled after Hardy-Weinberg and minor allele frequency cut-off that are 0.05 and 0.001, respectively.</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

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