Slitrk5 Mediates BDNF-Dependent TrkB Receptor Trafficking and Signaling

SummaryRecent studies in humans and in genetic mouse models have identified Slit- and NTRK-like family (Slitrks) as candidate genes for neuropsychiatric disorders. All Slitrk isotypes are highly expressed in the CNS, where they mediate neurite outgrowth, synaptogenesis, and neuronal survival. However, the molecular mechanisms underlying these functions are not known. Here, we report that Slitrk5 modulates brain-derived neurotrophic factor (BDNF)-dependent biological responses through direct interaction with TrkB receptors. Under basal conditions, Slitrk5 interacts primarily with a transsynaptic binding partner, protein tyrosine phosphatase δ (PTPδ); however, upon BDNF stimulation, Slitrk5 shifts to cis-interactions with TrkB. In the absence of Slitrk5, TrkB has a reduced rate of ligand-dependent recycling and altered responsiveness to BDNF treatment. Structured illumination microscopy revealed that Slitrk5 mediates optimal targeting of TrkB receptors to Rab11-positive recycling endosomes through recruitment of a Rab11 effector protein, Rab11-FIP3. Thus, Slitrk5 acts as a TrkB co-receptor that mediates its BDNF-dependent trafficking and signaling

Tie2 activation contributes to hemangiogenic regeneration after myelosuppression

Chemotherapy- or radiation-induced myelosuppression results in apoptosis of cycling hematopoietic cells and induces regression of bone marrow (BM) sinusoidal vessels. Moreover, timely regeneration of BM neovessels is essential for reconstitution of hematopoiesis. However, the identity of angiogenic factors that support reconstitution of BM's vasculature is unknown. Here, we demonstrate that angiopoietin/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) signaling contributes to the assembly and remodeling of BM neovessels after myelosuppression. Using transgenic mice where the Tie2 promoter drives the reporter LacZ gene (Tie2-LacZ), we demonstrate that at steady state, there was minimal expression of Tie2 in the BM vasculature. However, after 5-fluorouracil (5-FU) treatment, there was a rapid increase in plasma vascular endothelial growth factor A (VEGF-A) levels and expansion of Tie2-positive neovessels. Inhibition of Tie2 resulted in impaired neoangiogenesis, leading to a delay in hematopoietic recovery. Conversely, angiopoietin-1 (Ang-1) stimulated hematopoiesis both in wild-type and thrombopoietin-deficient mice. In addition, Ang-1 shortened the duration of chemotherapy-induced neutropenia in wild-type mice. Exogenous VEGF-A and Ang-1 stimulated Tie2 expression in the BM vasculature. These data suggest that VEGF-A–induced up-regulation of Tie2 expression on the regenerating vasculature after BM suppression supports the assembly of sinusoidal endothelial cells, thereby promoting reconstitution of hematopoiesis. Angiopoietins may be clinically useful to accelerate hemangiogenic recovery after myelosuppression

Rare Synaptogenesis-Impairing Mutations in <i>SLITRK5</i> Are Associated with Obsessive Compulsive Disorder

<div><p>Obsessive compulsive disorder (OCD) is substantially heritable, but few molecular genetic risk factors have been identified. Knockout mice lacking SLIT and NTRK-Like Family, Member 5 (SLITRK5) display OCD-like phenotypes including serotonin reuptake inhibitor-sensitive pathologic grooming, and corticostriatal dysfunction. Thus, mutations that impair SLITRK5 function may contribute to the genetic risk for OCD. We re-sequenced the protein-coding sequence of the human SLITRK5 gene (<i>SLITRK5</i>) in three hundred and seventy seven OCD subjects and compared rare non-synonymous mutations (RNMs) in that sample with similar mutations in the 1000 Genomes database. We also performed <i>in silico</i> assessments and <i>in vitro</i> functional synaptogenesis assays on the Slitrk5 mutations identified. We identified four RNM’s among these OCD subjects. There were no significant differences in the prevalence or <i>in silico</i> effects of rare non-synonymous mutations in the OCD sample versus controls. Direct functional testing of recombinant SLITRK5 proteins found that all mutations identified in OCD subjects impaired synaptogenic activity whereas none of the pseudo-matched mutations identified in 1000 Genomes controls had significant effects on SLITRK5 function (Fisher’s exact test P = 0.028). These results demonstrate that rare functional mutations in <i>SLITRK5</i> contribute to the genetic risk for OCD in human populations. They also highlight the importance of biological characterization of allelic effects in understanding genotype-phenotype relationships as there were no statistical differences in overall prevalence or bioinformatically predicted effects of OCD case versus control mutations. Finally, these results converge with others to highlight the role of aberrant synaptic function in corticostriatal neurons in the pathophysiology of OCD.</p></div

PTPδ-Fc binding is impaired by OCD-associated SLITRK5 mutations.

<p>(A) Representative images of HA-SLITRK5 (red) and PTPδ-Fc (green) immuno-fluorescence for control and OCD RNM. HEK-293 cells were transfected with HA-tagged SLITRK5 mutants and incubated with a PTPδ ectodomain-Fc fragment fusion protein. Areas of low SLITRK5-PTPδ binding (OCD mutants) appear orange, higher levels of binding (control mutants) appear yellow. (B) PTPδ binding to Slitrk5 alleles. All four mutations from OCD subjects (data for A851V not shown) displayed significantly reduced PTPδ whereas none of the control mutations did. ΔECD is a negative control SLITRK5 mutant lacking an extracellular domain. 25–30 cells were analyzed per condition per experiment. (C, D) Co-immunoprecipitation of PTPδ to Slitrk5 alleles. All four mutations from OCD subjects displayed significantly reduced co-precipitation with PTPδ, whereas none of the control mutations did. **P<0.01, ***P<0.001, ****P<0.0001</p

Structure of Slitrk5 and location of rare non-synonymous mutations.

<p>(A) Schematic representation of the Slitrk5 protein with extracellular Leucine Rich Repeat (LRR) domains and transmembrane (TM) domain marked. Mutations identified in OCD subjects are labeled in red and pseudo-matched mutations from 1000 Genome Database subjects are labeled in blue. (B) SLITRK5 mutations placed in their primary sequence context. All mutations alter absolutely conserved peptide contexts denoted by asterisks.</p

In Silico effects of rare non-synonymous Pmutations.

<p>Avg (s.e.m.), p values based on Student's T test.</p