Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation

<div><p>Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from <i>Hap1</i>-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in <i>Hap1</i>-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.</p></div

Co-localization of Bcr and Hap1 in differentiated neuro-2A cells.

<p>(<b>A</b>) Confocal imaging of differentiated neuro-2a cells transfected with GFP-Hap1A or GFP-Hap1B (green) with Bcr-myc (red). Scale bar, 10 μm. (<b>B</b>) Statistical analysis showing the percentage of GFP, GFP-Hap1A and GFP-Hap1B co-localized with Bcr in differentiated cells. Five or more sets of image were analyzed. * p < 0.05; ** p < 0.01; *** p < 0.001.</p

Lack of Hap1 inhibits Bcr signaling in mouse hypothalamus.

<p>(<b>A</b>) Western blotting analysis of Bcr and downstream signaling molecules including p38, ERK1/2, PAK, JNK and their phosphorylated forms from the WT and <i>Hap1</i>-null mouse hypothalamic (left panel) and non-hypothalamic regions (right panel). (<b>B</b>) Quantitative and statistical analysis of the changes of Bcr and its downstream signaling molecules in the hypothalamic and non-hypothalamic regions. The presented value was the ratio of the phosphorylated protein level to the total protein level and normalized with the result in WT mouse hypothalamic or non-hypothalamic region, which was set as 1. Three independent experiments were performed for statistical analysis. * p < 0.05; ** p < 0.01.</p

Association of Bcr with Hap1 on microtubules in mouse brains.

<p>(<b>A</b>) The relative amount of a microtubule subunit (β-tubulin), a small GTPase (RhoA) and a GAP/GEF (Bcr) in wild-type (WT) and <i>Hap1</i>-null newborn mouse brains normalized to isotope-labeled counterparts in wild type adult mouse brain. (<b>B</b>) Western blotting showing Hap1 isoforms, Bcr, RhoA and α-Tubulin in microtubule pellets precipitated by taxol-GTP treatment from <i>Hap1</i>-null and WT mouse brains. Input is the supernatant of brain lysates after centrifugation at 18,000 × g for 20 min (S2). The nonspecific protein species reacting to Hap1 antibody in the inputs (arrow) was not precipitated with microtubules.</p