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

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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.

Introduction

PMCAs are P-type ATPases that play a major role in expelling calcium from cells. Four isoforms, PMCA1-4, encoded by different genes, have been described (Strehler and Zacharias, 2001). PMCA isoform 2 is enriched in the brain and heart and is predominantly expressed in neurons (Stahl et al., 1992, Filoteo et al., 1997, Stauffer et al., 1995, Stauffer et al., 1997, Lehotsky et al., 1999, Burette et al., 2003). PMCA2 is particularly abundant in Purkinje neurons of the cerebellum and is found both in cell bodies and dendrites (Stahl et al., 1992, Stauffer et al., 1997). The pivotal role of PMCA2 in the function of the cerebellum is indicated by the phenotype of the PMCA2-null mouse and the deafwaddler 2J (dfw2J), a mouse with a spontaneous mutation in the PMCA2 gene which causes null pump activity (Kozel et al., 1998, Street et al., 1998, Penheiter et al., 2001). Both PMCA2-null and dfw2J mice manifest motor deficits and ataxia, which may be partially attributable to cerebellar pathology. In fact, an increase in the density of Purkinje neurons and a reduction in the thickness of the molecular layer in the cerebellum of PMCA2-null mice have been documented (Kozel et al., 1998). Additional studies further pinpoint the importance of PMCA2 in the function of other CNS regions (Lehotsky et al., 2002). Loss of motor neurons in the spinal cord of PMCA2-null and dfw2J mice has recently been reported (Kurnellas et al., 2005). Accordingly, PMCA2-null and dfw2J mice manifest hindlimb weakness and loss of grip strength in addition to the aforementioned neural deficits. A decrease in PMCA2 levels in the inflamed spinal cord of rodents affected by experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), has also been documented (Nicot et al., 2003, Nicot et al., 2005).

Our earlier studies, which focused on the proteomic analysis of the PMCA2-null versus wild type mouse cerebellum, indicated a significant decrease in the levels of IP3R1 (Hu et al., 2006). This finding was of particular interest as IP3R1 is an effector downstream to mGluR1, a glutamate receptor subtype, which plays a critical role in important cerebellar functions including motor coordination, synaptic plasticity, associative learning and developmental innervation of Purkinje cells by climbing fibers (Ichise et al., 2000, Kano et al., 1997). Activation of mGluR1 in parallel fiber-Purkinje cell synapses leads to production of IP3, which binds IP3Rs on the endoplasmic reticulum, a key step in the induction of intracellular calcium release and signaling (Knöpfel and Grandes, 2002). Coupling of IP3R1 to mGluR1 is mediated by Homer proteins, which are components of the molecular scaffold at postsynaptic densities of excitatory synapses (Brakeman et al., 1997, Xiao et al., 1998, Tu et al., 1998). This family of small proteins comprises several members including Homer 1a, 1b/c, 2 and 3. Homer 3 is highly expressed in the cerebellum, especially in the dendrites of Purkinje cells, and Homer proteins regulate the localization, expression and function of group I mGluRs (Xiao et al., 1998, Sheng, 1997, Thomas, 2002). Co-localization of IP3R with Homer 1b/c and PMCA in Purkinje cells has been reported, although the antibodies used could not differentiate between different PMCA isoforms (Sandona et al., 2003). Our double-labeling studies also indicated co-localization of mGluR1 with PMCA2 in dendrites of Purkinje neurons (Kurnellas et al., in press). Furthermore, the co-expression of Ania-3/Homer with PMCA2 in soma and dendrites of hippocampal neurons and the interaction of Ania-3/Homer with the b-splice form of all PMCAs via their PDZ binding domain has been documented (Sgambato-Faure et al., 2006).

The present studies were undertaken in order to determine the molecular pathways that are affected in Purkinje neurons of PMCA2-null mice with a special emphasis on the mGluR1 signaling pathway and to define a potential link between PMCA2 and the components of the mGluR1–Homer3–IP3R1 complex in the cerebellum.

Section snippets

Results

Previous studies reported an increase in the density of Purkinje neurons and a small but significant decrease (17%) in the thickness of the molecular layer in the cerebellum of PMCA2-null mice as compared to wild type controls, indicating that the lack of PMCA2 induces morphological changes (Kozel et al., 1998). However, the molecular alterations in the cerebellum of the PMCA2-null mouse have not yet been studied. We undertook novel studies in order to gain an initial insight into this issue.

Discussion

The present study demonstrates specific molecular alterations in Purkinje neurons of the PMCA2-null mouse. The decrease in mGluR1 and IP3R1 in Purkinje somata and dendrites of the PMCA2-null mouse may be particularly important as both receptors play essential roles in cerebellar function including motor coordination, a form of synaptic plasticity called long term depression (LTD), associative learning and synapse elimination during development (Linden et al., 1991, Shigemoto et al., 1994,

Animals

PMCA2+/+ and PMCA2−/− mice have been previously described (Kozel et al., 1998, Kurnellas et al., 2005). PMCA2−/− mice have a normal life span similar to wild type littermates but are smaller in size. All animal protocols were performed according to institutional guidelines.

Immunocytochemistry

Mice were anesthetized by injection of Ketamine/Xylazine and perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were dissected out, postfixed, cryoprotected in 10 and 20%

Acknowledgments

We are grateful to Dr. Paul F. Worley for providing the antibodies against Homer 1b/c and Homer 3. We thank Dr. Jian Cheng Tu and Dr. Tao Zu for advice with some of the techniques. This research was supported by NIH grants NS046363 to SE and NS046593 to HL.

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