In vivo evidence that truncated trkB.T1 participates in nociception

Brain-Derived Neurotrophic Factor (BDNF) is a central nervous system modulator of nociception. In animal models of chronic pain, BDNF exerts its effects on nociceptive processing by binding to the full-length receptor tropomyosin-related kinase B (trkB.FL) and transducing intracellular signaling to produce nocifensive behaviors. In addition to trkB.FL, the trkB locus also produces a widely-expressed alternatively-spliced truncated isoform, trkB.T1. TrkB.T1 binds BDNF with high affinity; however the unique 11 amino acid intracellular cytoplasmic tail lacks the kinase domain of trkB.FL. Recently, trkB.T1 was shown to be specifically up-regulated in a model of HIV-associated neuropathic pain, potentially implicating trkB.T1 as a modulator of nociception. Here, we report that trkB.T1 mRNA and protein is up-regulated in the spinal dorsal horn at times following antiretroviral drug treatment and hind paw inflammation in which nocifensive behaviors develop. While genetic depletion of trkB.T1 did not affect baseline mechanical and thermal thresholds, the absence of trkB.T1 resulted in significant attenuation of inflammation- and antiretroviral-induced nocifensive behaviors. Our results suggest that trkB.T1 up-regulation following antiretroviral treatment and tissue inflammation participates in the development and maintenance of nocifensive behavior and may represent a novel therapeutic target for pain treatment

ZRANB3 is an African-specific type 2 diabetes locus associated with beta-cell mass and insulin response.

Genome analysis of diverse human populations has contributed to the identification of novel genomic loci for diseases of major clinical and public health impact. Here, we report a genome-wide analysis of type 2 diabetes (T2D) in sub-Saharan Africans, an understudied ancestral group. We analyze ~18 million autosomal SNPs in 5,231 individuals from Nigeria, Ghana and Kenya. We identify a previously-unreported genome-wide significant locus: ZRANB3 (Zinc Finger RANBP2-Type Containing 3, lead SNP p = 2.831 × 10-9). Knockdown or genomic knockout of the zebrafish ortholog results in reduction in pancreatic β-cell number which we demonstrate to be due to increased apoptosis in islets. siRNA transfection of murine Zranb3 in MIN6 β-cells results in impaired insulin secretion in response to high glucose, implicating Zranb3 in β-cell functional response to high glucose conditions. We also show transferability in our study of 32 established T2D loci. Our findings advance understanding of the genetics of T2D in non-European ancestry populations

ZRANB3 is an African-specific type 2 diabetes locus associated with beta-cell mass and insulin response

Abstract: Genome analysis of diverse human populations has contributed to the identification of novel genomic loci for diseases of major clinical and public health impact. Here, we report a genome-wide analysis of type 2 diabetes (T2D) in sub-Saharan Africans, an understudied ancestral group. We analyze ~18 million autosomal SNPs in 5,231 individuals from Nigeria, Ghana and Kenya. We identify a previously-unreported genome-wide significant locus: ZRANB3 (Zinc Finger RANBP2-Type Containing 3, lead SNP p = 2.831 × 10−9). Knockdown or genomic knockout of the zebrafish ortholog results in reduction in pancreatic β-cell number which we demonstrate to be due to increased apoptosis in islets. siRNA transfection of murine Zranb3 in MIN6 β-cells results in impaired insulin secretion in response to high glucose, implicating Zranb3 in β-cell functional response to high glucose conditions. We also show transferability in our study of 32 established T2D loci. Our findings advance understanding of the genetics of T2D in non-European ancestry populations

An Essential Role for DYF-11/MIP-T3 in Assembling Functional Intraflagellar Transport Complexes

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

BBS4 is necessary for ciliary localization of TrkB receptor and activation by BDNF.

Primary cilia regulate an expanding list of signaling pathways in many different cell types. It is likely that identification of the full catalog of pathways associated with cilia will be necessary to fully understand their role in regulation of signaling and the implications for diseases associated with their dysfunction, ciliopathies. Bardet-Biedl Syndrome (BBS) is one such ciliopathy which is characterized by a spectrum of phenotypes. These include neural defects such as impaired cognitive development, centrally mediated hyperphagia and peripheral sensory defects. Here we investigate potential defects in a signaling pathway associated with neuronal function, brain derived neurotrophic factor (BDNF) signaling. Upon loss of BBS4 expression in cultured cells, we observed decreased phosphorylation and activation by BDNF of its target receptor, TrkB. Assessment of ciliary localization revealed that, TrkB localized to the axonemes or basal bodies of cilia only in the presence of BDNF. Axonemal localization, specifically, was abrogated with loss of BBS4. Finally, we present evidence that loss of the ciliary axoneme through depletion of KIF3A impedes activation of TrkB. Taken together, these data suggest the possibility of a previously uninvestigated pathway associated with perturbation of ciliary proteins

pTRKB in the ciliary axoneme is lost with depletion of <i>BBS4</i> expression.

<p>(A–G) Immunofluorescent staining of hTERT-RPE1 cells transfected with empty vector (EV), sh<i>BBS4</i> or both 3′UTR sh<i>BBS4</i> and <i>BBS4</i> expression construct. Cells were cultured in BDNF-supplemented media and stained using antibody against pTRKB (red) or ciliary markers labeling axoneme (ARL13B, green) or basal body (γ-tubulin, green). Region around cilia denoted by dashed box and magnified inset. Basal bodies highlighted by arrows and axoneme in (A,D) highlighted by arrowheads. Scale bar  = 10 µm. Imaged at 100× magnification. (H) Quantification of ciliary localization of pTRKB in transfected cells calculated as the proportion of either basal bodies or axonemes that co-localize with pTRKB. Error bars represent standard deviation. *significant difference (p<0.01, chi-square test).</p

Reduced TRKB activation with loss of ciliary axoneme.

<p>(A–D) Immunofluorescent staining of empty vector (EV) control or sh<i>KIF3A-</i>treated hTERT-RPE1 cells cultured in BDNF-supplemented media and stained using antibody against pTRKB (red) or ciliary markers labeling axoneme (ARL13B, green) or basal body (γ-tubulin, green). Region around cilia denoted by dashed box and magnified inset. Scale bar  = 10 µm. Imaged at 100× magnification. (E) Quantification of basal body localization of pTRKB and TRKB in control (black) or sh<i>KIF3A</i>-treated (striped) cells calculated as the proportion that co-localize with pTRKB or TRKB with or without BDNF. Error bars represent standard deviation. No significant difference between control and sh<i>KIF3A</i>. (F) Western blot detection for pTRKB and TRKB in hTERT-RPE1 cells treated with or without BDNF and with or without sh<i>KIF3A</i>. (G) Quantification of TRKB activation calculated as the average ratio of pTRKB to TRKB protein, measured by ImageJ densitometry analysis. Error bars depict standard deviation across a minimum of three experiments. *significant change (p<0.01, t-test) from control; **significant change (p<0.01, t-test) from BDNF-treated control cells. (H) Western blot detection in hTERT-RPE1 cells of pTRKB and TRKB, as well as Actin, in the presence or absence of BDNF and the presence or absence of a short hairpin targeting the 3′UTR (sh<i>BBS4</i>) or a vector expressing <i>BBS4</i>. (I) Quantification of the average activation of TRKB in hTERT-RPE1 cells quantified as the amount of pTRKB relative to the amount of TRKB for indicated treatments. *significant change (p<0.01, t-test) from control; **significant change (p<0.05, t-test) from BDNF-treated control.</p

TRKB localizes to the basal body and axoneme of hTERT-RPE1 cells in the presence of BDNF.

<p>(A–M) Immunofluorescent staining of hTERT-RPE1 cells transfected with empty vector (EV), sh<i>BBS4</i> or both 3′UTR sh<i>BBS4</i> and <i>BBS4</i> expression construct (Rescue). Cells were either cultured in BDNF-deficient (BDNF-) or BDNF-supplemented (BDNF+) media and stained using antibody against TRKB (red) or ciliary markers labeling axoneme (ARL13B, green) and basal body (γ-tubulin, green, arrows). Region around cilia denoted by dashed box and magnified inset. Scale bar  = 10 µm. Imaged at 100× magnification. (N) Quantification of ciliary localization of TRKB calculated as the proportion of either basal bodies (black bars) or axonemes (striped bars) that co-localize with TRKB. Error bars represent standard deviation. *significant difference (p<0.01, chi-square test).</p

BDNF-induced phosphorylation of TRKB is reduced in <i>BBS4</i>-deficient cells.

<p>(A) Western blot detection in hTERT-RPE1 cells of phosphorylated TRKB (pTRKB) and unphosphorylated TRKB, as well as Actin as loading control, in the presence or absence of BDNF and the presence or absence of short hairpin targeting expression of <i>BBS4</i> (sh<i>BBS4</i>). (B) Average activation of TRKB in hTERT-RPE1 cells quantified as the amount of pTRKB relative to the amount of TRKB for indicated treatments measured by ImageJ densitometry analysis. Error bars depict standard deviation across a minimum of 3 experiments. *significant change (p<0.01, t-test) from control; **significant change (p<0.01, t-test) from BDNF-treated control cells. (C) Western blot detection in hTERT-RPE1 cells of pTRKB and TRKB, as well as Actin, in the presence or absence of BDNF and the presence or absence of a short hairpin targeting the 3′UTR (sh<i>BBS4</i>) or a vector expressing <i>BBS4</i> (BBS4). (D) Quantification of the average activation of TRKB in hTERT-RPE1 cells quantified as the amount of pTRKB relative to the amount of TRKB for indicated treatments. *significant change (p<0.01, t-test) from control; **significant change (p<0.05, t-test) from BDNF-treated control. (E) Western blot detection SH-SY5Y cells pTRKB and TRKB in the presence of absence of BDNF and the presence or absence of sh<i>BBS4</i>. (F) Average activation of TRKB in SH-SY5 cells quantified as the amount of pTRKB relative to the amount of TRKB for indicated treatments. Error bars depict standard deviation across a minimum of 3 experiments. *significant change (p<0.01, t-test) from control; **significant change (p<0.01, t-test) from BDNF-treated control cells.</p