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"Delayed Purkinje cell death and functional deficits in plasma membrane calcium ATPase 2 (PMCA2)-heterozygous mice via modulation of calcium and synaptic complexes"

Amanda K. Fakira
Integrative Neuroscience Program
B.S. 2003, Stony Brook University

Thesis Advisor: Stella Elkabes, Ph.D.
Associate Professor
Department of Neurological Surgery

Friday, January 21, 2011
Cancer Center, G-1196, 2:00 P.M.


Plasma membrane Ca2+ ATPase isoform 2 (PMCA2) is a neuronal Ca2+ pump that is expressed highly in cerebellar PCs. PMCAs in conjunction with other Ca2+-handling mechanisms, maintain resting intracellular Ca2+ levels at low nanomolar concentrations resulting in a large Ca2+ gradient across the cell membrane. Since Ca2+ is an important signaling molecule modulating a plethora of neuronal functions, it is critical not to disrupt Ca2+ homeostasis. Additionally, PMCA2 associates with synaptic complexes. Previous studies in our laboratory have shown that, in the cerebellum, PMCA2 physically interacts with mGluR1-IP3R1-homer3 signaling complex which is down regulated in the PMCA2-/- cerebellum. PMCA2-/- mice display ataxia indicating that PMCA2 plays a critical role in cerebellar function. Although previous studies have indicated cerebellar abnormalities in PMCA2 mutant mice, the mechanisms by which PMCA2 causes cerebellar dysfunction are not well understood.
The current studies were undertaken in order to investigate whether a partial reduction in PMCA2 causes PC and cerebellar dysfunction and the underlying mechanisms. Using PMCA2-heterozygous (PMCA2+/-) mice as a model, we investigated the effects of reduced PMCA2 expression on PC Ca2+ signaling and the modulation of glutamate receptor-containing protein complexes in the cerebellum. The amplitude and decay time of depolarization-induced Ca2+ transients were higher in cultured PCs from PMCA2+/- mice than in wild type (PMCA2+/+) controls. The larger amplitude of Ca2+ transients was most likely due to changes in Ca2+ influx, since the expression of P/Q type voltage-gated Ca2+ channels (VGCC) was increased in PC cultures and in the cerebellum of PMCA2+/- mice and -agatoxin TK prevented the elevation in amplitude, in vitro. Long-term exposure of enriched cultures to a selective neuronal nitric oxide sythase (nNOS) inhibitor, nitroindazole monosodium salt (7-NINA), reduced the amplitude of Ca2+ transients in PMCA2+/- PCs to PMCA2+/+ levels. The protein and mRNA expression of metabotropic glutamate receptor 1 (mGluR1) and AMPA receptor (AMPAr) subunits GluR2/3, components of PMCA2-contaning complexes, were decreased in the PMCA2+/- cerebellum as compared to the PMCA2+/+, in an age-dependent manner whereas the expression of Homer 3, inositol 1, 4, 5-trisphosphate receptor type 1 (IP3r1) and post-synaptic density 95 (PSD95) were either not modified or were increased. These changes were followed by delayed PC loss and poorer performance on the balance beam test. Our results indicate that decreases in PMCA2 expression cause long-term adaptive changes in Ca2+ and glutamate receptor-mediated signaling ultimately leading to PC loss and functional deficits.

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