Lack of huntingtin-associated protein-1 causes neuronal death resembling hypothalamic degeneration in Huntington's disease

Abstract

Huntington's disease (HD) is caused by a polyglutamine expansion in the disease protein huntingtin. The polyglutamine expansion causes huntingtin to interact abnormally with a number of proteins. However, it is unclear whether, and how, huntingtin-associated proteins are involved in the neurodegeneration in HD. Here, we show that huntingtin-associated protein-1 (HAP1), which is involved in intracellular trafficking of epidermal growth factor receptor (EGFR), is highly expressed in the hypothalamus. Mice lacking HAP1 die after birth because of depressed feeding activity. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining and electron microscopic examination revealed the degeneration in hypothalamic regions that control feeding behavior. Hypothalamic degeneration was also observed in HD transgenic mice that have a significant loss of body weight. Inhibition of HAP1 expression decreases EGFR signaling and cell viability, whereas overexpression of HAP1 enhances this signaling activity and inhibits mutant huntingtin-mediated cytotoxicity. These results suggest that the effect of mutant huntingtin on HAP1 and EGFR signaling may contribute to the hypothalamic neurodegeneration and loss of body weight in HD.

Figure 1.

Targeted disruption of the mouse HAP1 gene. A, DNA structures of the wild-type allele, targeted vector, and mutant allele. The targeted vector contains neomycin (neo) and TK markers flanked by 1.6 and 5.8 kb of the mouse HAP1 gene. The first two exons (black boxes) of the HAP1 gene are deleted in the mutant allele. The probe used for Southern blot analysis is indicated, and the primers (arrows) for PCR genotyping are also indicated. K, KpnI; E, EcoRI. B, Western blot analysis of brain cortex extracts of wild-type, HAP1 (+/-), and HAP1 (-/-) mice. The blots were probed with antibodies against HAP1, HAP1-A, or HAP1-B. C, Pups from HAP1(+/+), HAP1(+/-), and HAP1(-/-) mice were weighed at postnatal days 1, 2, and 3. The data (mean ± SEM) were obtained from 54-204 pups for each group. ^**p < 0.01 compared with wild-type mice. D, The percentage of surviving HAP1 (-/-) pups after birth. The majority of HAP1(-/-) pups died at days 3-4 after birth.

Figure 2.

TUNEL staining of mouse brain. A, HAP1-B immunostaining of a sagittal section of adult mouse brain shows abundant expression of HAP1 in the hypothalamus (Hyp). HAP1 is also highly expressed in the accessory olfactory bulb (AOB), superior and inferior colliculi (C), pedunculopontine nucleus (PPN), and brain stem (BS). Olf, Olfactory bulb; Ctx, cerebral cortex; Str, striatum; Hip, hippocampus; Tha, thalamus; Cereb, cerebellum. B-D, Elimination of HAP1 results in TUNEL-positive cells in the hypothalamus. At P1, TUNEL-positive cells are seen in the PVN and the LH in HAP1(-/-) mice (B) but not in HAP1(+/+) mice (C). At P3, more TUNEL-positive cells are seen in the hypothalamic areas including the VMN, whereas the ARC shows fewer TUNEL-positive cells in HAP1(-/-) mice (D). E, In the cortex of the same HAP1(-/-) mouse, no or very few TUNEL-positive cells are seen. The third ventricle is indicated by arrowheads. Scale bar, 100 μm.

Figure 3.

Electron microscopic examination of the brain of HAP1(-/-) mice. A-D, Ultrastructural morphology of HAP1(-/-) or HAP1(+/+) mice at P1. A, Electron microscopy reveals swollen mitochondria with disrupted structures of internal membranes (arrows) in the LH of a HAP1(-/-) mouse. B, Mitochondria in the hypothalamus of a HAP1(+/+) mouse pup at P1 have normal morphology. C, The cortex of a HAP1(-/-) mouse at P1 reveals no apoptotic cells. D, In the hypothalamus of the same HAP1(-/-) pup, fragmented and condensed nuclei form apoptotic bodies (arrowheads). E, F, Apoptotic profiles of the hypothalamic neurons of a HAP1(-/-) mouse at P3 showing nuclear pyknosis, chromatin fragmentation, and darkening structures (arrows). Apoptotic bodies (arrowheads) are surrounded by degenerated mitochondria and lysosome-like structures (E). A normal nucleus (n) is present near apoptotic cells (E). A large apoptotic body (arrowhead) is being engulfed by a glial cell (gn), indicating a late stage of apoptosis (F). Scale bars: A, B, 0.5 μm; C-F, 1 μm.

Figure 4.

Hypothalamic degeneration in HD mice. Electron microscopic examination of the LH of HD mice at 4 months of age. A, Several condensed and darkening apoptotic bodies (arrowheads) are evident. Some swollen mitochondria are also seen. B, A condensed nucleus with dark chromatin is shown in a degenerating neuron. The nuclear membrane is not intact, and several cytoplasmic condensed structures (arrows) are also seen. C, D, EM48 immunogold labeling of hypothalamic neurons in HD mice. Immunogold particles are diffusely distributed in a neuron in which degenerating mitochondria (arrow) and lysosomal structures (arrowhead) are also present (C). Mutant Htt also forms aggregates (arrow) in a nucleus that is undergoing chromosomal condensation (D). Scale bars: A, C, 0.4 μm; B, D, 0.8 μm.

Figure 5.

Expression of HAP1 and EGFR in HD transgenic mice. A, Immunoprecipitation of HAP1 and Htt from brains of wild-type mice (WT) and R6/2 mice (HD) at 4 weeks of age. Anti-HAP1 or the preimmune serum (lane C) was used for immunoprecipitation, and the precipitates were blotted and probed with mEM48. The bottom panel is the same blot reprobed with anti-HAP1 antibody. Input, 20% of tissue lysates. The bracket indicates the stacking gel in which aggregated Htt is retained. B, Western blot analysis of the expression of HAP1, transgenic huntingtin (htt), and tubulin shows that the expression of HAP1 is lower in the hypothalamic tissue from three different N171-82Q mice (HD-1, -2, -3) than in that from wild-type mice (WT-1, -2, -3). C, HAP1 immunocytochemistry of the LH and VMN of N171-82Q and wild-type (WT) mice at 4 months of age. D, Western blot analysis of brain cortical tissues from HAP1(+/+) and HAP1(-/-) pups at P1 and the hypothalamic tissues from two wild-type mice (WT) or N171-82Q mice (HD) at 4 months of age. The blots were probed with antibodies to EGFR and tubulin (Tub).

Figure 6.

Decreased EGFR signaling activity in PC12 cells expressing antisense HAP1 or mutant Htt. A, Western blot analysis of PC12 cell lines (A2, A3, and A6) that were stably transfected with antisense HAP1 construct. WT, Wild-type PC12 cells. The expression of HAP1 (top), dynactin p150 (middle), and tubulin (bottom) is shown. B, Neurite outgrowth of PC12 cells (wild-type and A2 cell line) after NGF (100 ng/ml) treatment for 48 hr. C, Expression of HAP1, EGFR, phosphorylated ERK (pERK), ERK2, phosphorylated AKT (pAKT), and AKT in wild-type cells, A2 cells, and PC12 cells stably expressing exon1 Htt with a 150-glutamine repeat (150Q) or 20-glutamine repeat (20Q) after EGF (100 ng/ml) treatment for 5 min. D, The viability of wild-type, A2, 20Q, and 150Q PC12 cells after treatment with AG1478 and wortmannin for 5 and 8 hr. The control is untreated cells for each group. Data are expressed as mean ± SD (n = 4). ^*p < 0.05; ^**p < 0.01 compared with WT.

Figure 7.

Protection of HAP1 against Htt toxicity. A, Transfection of HEK293 cells with HAP1-B, N-terminal Htt (1-208 amino acids) containing 23 or 120 glutamines (N208-23Q or -120Q), or HAP1-B with Htt. Transfected cells were treated with EGF (100 ng/ml) for 45 min. The expression of EGFR, phosphoralted ERK (pERK), ERK2, and HAP1 was analyzed by Western blotting. The levels of pERK were quantified and expressed as a ratio (pERK/ERK) for each sample. B, HEK293 cells transfected with HAP1-B, N208-120Q alone, N208-120Q with vector, or HAP1 were treated with wortmannin (500 nm) for 4 hr, followed by caspase-3 activity assay. Control is untreated cells. Data are expressed as mean ± SD (n = 4), and ^*p < 0.05. C, Transfection of primary hypothalamic neurons with HAP1-A (top) or mutant Htt (bottom). Cytoplasmic HAP1 and Htt inclusions are indicated by arrows. Hoechst DNA staining (right) shows nuclear DNA fragmentation (arrowhead) in Htt-transfected neurons. Coexpression of HAP1-B (top) or HAP1-A (bottom) with Htt resulted in diffuse HAP1 (green) and Htt (red) and a colocalization of some HAP1 with Htt aggregates (arrows). Note that doubly transfected cells show intact nuclei (arrowheads in insets). D, The percentage of transfected cells showing nuclear DNA fragmentation. Cultured neurons were singly transfected with vector, HAP1-B (HAP1), N208-23Q, or N208-120Q and HAP1-B together (120Q+HAP1). Data are mean ± SE of three to five transfections. ^**p < 0.01 compared with 120Q transfection.