The Vps33a gene regulates behavior and cerebellar Purkinje cell number

Abstract

A mutation in the Vps33a gene causes Hermansky-Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13-14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (approximately 30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may also be a clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function.

Fig. 1

SHIRPA behavior tests. (A) Young (8–13 weeks) bf and C57/BL6 controls were tested (12 mice in each group). (B) Old (13–14 months) bf mice and their heterozygous controls were tested (12 mice each). The defect in startle response was found only in old bf mice. Results are presented only for those SHIRPA tests in which significances were noted. Twenty five other SHIRPA tests in which no significant differences were found are not presented. ^$P≤0.01; ^@P≤0.03; *P≤0.001.

Fig. 2

Analysis of motion and postural symmetry. Video capture of body movements reveals asymmetry (or curving) behavior of bf (lower panel) compared with movement of wild-type C57BL6 mice (top panel). Two animals were videotaped, and subtle body movements and motion analysis were processed as described in methods.

Fig. 3

(A) Effect of the Vps33a mutation on the cerebellum. Immunocytochemical stain (calbindin) of brain sections from young and old bf mice. Specific calbindin stain highlighting cerebellar regions indicates a smaller cerebellum in old bf mutants (panels 2 and 3) compared with heterozygous controls. There is no difference in the size of cerebellum in young bf mice (panel 1). Hematoxylin and eosin was used as counter stain. The pictures are representative of three different animals. Scale bar, 5 μm. (B) Measurement of total brain and cerebellum size by Image J software: There is a nominal 12.5% reduction in the total brain size in bf mutants (92.9 mm²) but this is not significantly different (P=0.11) from bf/+ controls (106 mm²) (a). The cerebellum size (bf/+ 10.8 mm², bf 7.40 mm²) in bf mutants is significantly (P=0.02) reduced (31.4%) compared to their controls (b). 3 wild-type and 3 mutant mice were used for determining sizes. Similar results (i.e. a selective reduction in size of mutant cerebella) were obtained when sections were stained with cresyl violet (not shown).

Fig. 4

MRI analysis illustrates abnormal cerebellar volume in bf. A representative, central slice of MR imaging (left) and 3D renderings (right) show decrease in cerebellum volume (CB, green) and accumulation of more cerebral spinal fluid (CSF, yellow) in the bf mouse (panel B) as compared to control (panel A).

Fig. 5

Purkinje cell loss in old bf. (A) Brain sections from old (13–14 months) bf/bf and bf/+ controls were stained with calbindin to clearly show the cerebellum. Scale bar 2 μm. (B) Higher magnification specifically highlights Purkinje cells. Arrowheads indicate Purkinje cells in the cerebellum. Three different animals were used for the study, and from each animal at least 2 different brain sections were used. Scale bar 40 μm.

Fig. 6

Up-regulation of GFAP in bf. (A) Brain sections of old (13–14 months) bf and controls were immunostained with GFAP antibody. GFAP over expression was observed in cerebellum in bf mice compared to their controls in the regions indicated as arrow head (molecular layer), arrow (deep nuclei) and asterisk (Brain stem). Pictures are representative of three different animals. Scale bar, 100 μm. (B) Western blot analysis of old bf brain cerebellum extracts. The up-regulation of GFAP is apparent as a more intense signal with bf mutant compared to age-matched control. α-tubulin was used as loading control.