Molecular alterations in the cerebellum of the plasma membrane calcium ATPase 2 (PMCA2)-null mouse indicate abnormalities in Purkinje neurons

Abstract

PMCA2, a major calcium pump, is expressed at particularly high levels in Purkinje neurons. Accordingly, PMCA2-null mice exhibit ataxia suggesting cerebellar pathology. It is not yet known how changes in PMCA2 expression or activity affect molecular pathways in Purkinje neurons. We now report that the levels of metabotropic glutamate receptor 1 (mGluR1), which plays essential roles in motor coordination, synaptic plasticity, and associative learning, are reduced in the cerebellum of PMCA2-null mice as compared to wild type littermates. The levels of inositol 1,4,5-triphosphate receptor type 1 (IP3R1), an effector downstream to mGluR1, which mediates intracellular calcium signaling, and the expression of Homer 1b/c and Homer 3, scaffold proteins that couple mGluR1 to IP3R1, are also reduced in somata and dendrites of some Purkinje cell subpopulations. In contrast, no alterations occur in the levels of mGluR1 and its downstream effectors in the hippocampus, indicating that the changes are region specific. The reduction in cerebellar mGluR1, IP3R1 and Homer 3 levels are neither due to a generic decrease in Purkinje proteins nor extensive dendritic loss as immunoreactivity to total and non-phosphorylated neurofilament H (NFH) is increased in Purkinje dendrites and microtubule associated protein 2 (MAP2) staining reveals a dense dendritic network in the molecular layer of the PMCA2-null mouse cerebellum. PMCA2 coimmunoprecipitates with mGluR1, Homer 3 and IP3R1, suggesting that the calcium pump is a constituent of the mGluR1 signaling complex. Our results suggest that the decrease in the expression of mGluR1 and its downstream effectors and perturbations in the mGluR1 signaling complex in the absence of PMCA2 may cumulatively result in aberrant metabotropic glutamate receptor signaling in Purkinje neurons leading to cerebellar deficits in the PMCA2-null mouse.

Fig. 1

Quantification of mGluR1, IP3R1, Homer 1b/c and Homer 3 levels in the cerebellum of PMCA2^+/+ and PMCA2^−/− mice by Western blot analysis. The top panel shows representative Western blots. Lanes 1–3 and 4–6 are samples obtained from the cerebellum of three distinct PMCA2^+/+ and PMCA2^−/− mice, respectively. Antibodies specific for each protein recognized bands of expected molecular weights; mGluR1: ~133 kDa, Homer 1b/c and Homer 3: ~47 kDa, and IP3R1: ~240 kDa. α-Tubulin (~50 kDa), remained unchanged and was used as a control for experimental variations. The bottom panel is the schematic representation of two to three experiments combined (n=6–8). Values in graphs represent mean ± S.E.M. after normalization to α-tubulin. *p<0.03, **p<0.002 and ***p< 0.0004 by Student’s t-test.

Fig. 2

Quantification of mGluR1, IP3R1 and Homer 3 levels in the hippocampus of PMCA2^+/+ and PMCA2^−/− mice by Western analysis. The top panel shows representative Western blots. Lanes 1–3 and 4–6 are samples obtained from the hippocampus of three distinct PMCA2^+/+ and PMCA2^−/− mice, respectively. The bottom panel is the schematic representation of the results obtained by Western analysis (n=4–5). As α-tubulin was variable in the hippocampus of the wild type versus knockout mouse, another housekeeping gene, GADPH (~36 kDa), was used as control for experimental variations. Values in graphs represent mean ± S.E.M. after normalization to GAPDH. No significant changes were observed.

Fig. 3

IP3R1, Homer 3 and MAP2 immunoreactivity in sagittal sections of the PMCA2^+/+ and PMCA2^−/− mouse cerebellum. IP3R1 staining was predominantly localized to the molecular layer (ML) and Purkinje somata (arrows) of the wild type mouse cerebellum (A). No staining was observed in cell bodies of granule neurons. The staining intensity was decreased in Purkinje neuron cell bodies (arrows) and in the ML of the PMCA2^−/− mouse (B). Similarly, Homer 3 antibody revealed diffuse immunoreactivity in the ML and strong staining in Purkinje somata (arrows) of the wild type cerebellum (C). The staining was decreased both in cell bodies (arrows) and ML of the PMCA2-null mouse (D). The decrease in immunoreactivity occurred in clusters of Purkinje cells especially along cerebellar sulci (see figure 4). Immunostaining with an anti-MAP2 antibody revealed the extensive dendritic network both in wild type (E) and knockout mouse (F). n=4, Bar represents 100 μm.

Fig. 4

IP3R1 and Homer 3 immunoreactivity in folial crowns of PMCA2^+/+ and PMCA2^−/− mouse cerebellum. In folial crowns, the intensity of IP3R1 staining in Purkinje neurons (arrows) or the molecular layer (ML) did not appear different in the PMCA2^+/+ (A) and PMCA2^−/− mice (B). However, the staining pattern was different in the wild type versus the knockout mouse. Immunoreactivity was mostly diffuse and punctate in the molecular layer (ML) of the wild type cerebellum and concentrated along the dendritic branches in the knockout mouse. C) The difference in IP3R1 staining in Purkinje cell clusters located along the fissure (white arrowheads) versus those in the folial crowns (black arrowheads) is noticeable in a low magnification picture of a section through the cerebellum of a knockout mouse. Note that the differences are most pronounced in the molecular layer. As in the case of IP3R1, the intensity of Homer 3 staining was strong in the folial crowns of both the wild type (D) and PMCA2-null mouse (E). However, a change in the pattern of the staining could be observed in the (ML). Note that the staining is diffuse in the ML of the wild type cerebellum whereas most immunoreactivity is localized to dendritic branches in the ML of the knockout mouse. The inset in figure E is a higher magnification picture showing the punctate staining in dendritic branches. F) Low magnification picture of a section through the cerebellum of a knockout mouse showing the difference in staining intensity in the ML and somata of Purkinje neurons located along a fissure (white arrowheads) as compared to folial crowns (black arrowheads). In some regions, indicated by the asterix, immunostaining was completely suppressed. GCL: granule cell layer. n=4, Bar represents 60 μm for A, B, D and E and 200 μm for C and F.

Fig. 5

Alterations in the levels of total and non-phosphorylated neurofilament H immunoreactivity in Purkinje neurons of the PMCA2-null mouse. A) Non-phosphorylated NFH is low in somata of Purkinje cells (arrows) and in the molecular layer (ML) containing Purkinje dendrites in wild type mice (arrowheads). B) A dramatic increase in non-phosphorylated NFH immunostaining is observed both in cell bodies (arrows) and apical dendrites (arrowheads) of Purkinje neurons in the cerebellum of PMCA2-null mice. Similarly, total NFH is lower in Purkinje neurons of the wild type mouse (C and D, arrows show cells in two different regions) and is increased in somata (E and F, arrows) and dendrites (arrowheads) of the PMCA2-null mouse. The increase in NFH immunoreactivity occurred throughout the cerebellum. Non-phosphorylated NFH staining is not altered in other brain regions including the substantia nigra of the wild type (G) or knockout (H) mouse. Arrows point at some cells immunopositive for non-phosphorylated NFH. GCL: granule cell layer. n=4, Bar represents 150 μm for A and B, 100 μm for C, E, G and H and 60 μm for D and F.

Fig. 6

Coimmunoprecipitation of PMCA2 with mGluR1, Homer 3 and IP3R1 from mouse cerebellum. The upper panel shows pictures of 3–8% Tris-acetate gels and the lower panel are pictures of 10% Bis-tris gels, which were used to separate lower molecular weight proteins such as Homers from the ~60 kDa (arrowheads) rabbit IgG heavy chain, described previously (Xiao et al., 1998). The cerebellar extracts were immunoprecipitated either with an anti-Homer 3 or anti-PMCA2 antibody. The antibody used for immunoprecipitation is indicated above each lane. The blots were probed with antibodies against PMCA2 (~129 kDa), IP3R1, Homer 3 or mGluR1, which detected a band at the expected molecular weight (arrows). NG: is negative control, normal rabbit serum or an unrelated polyclonal antibody as indicated in Methods. PC: is positive control, cerebellum extracts.