Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.     

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.     

Annexins in cardiac tissue: cellular localization and effect on phospholipase activity

Stimulation of cardiac phospholipid metabolism has diverse biological effects, ranging from subtle changes in cellular function to severe cellular damage. Accordingly, knowledge of the factors governing the activity of cardiac phospholi pases is of great biological importance. A possible role of annexins, intracellular proteins that bind to membranes in a calcium dependent manner, as modulators of phospholipase activity has been proposed. In this study we investigated the cell type specific distribution of Annexin V and VIII in the heart. Recombinant Annexin V was used to examine the effect of this type of Annexin on cardiac phospholipase activity. Western blot analysis shows that annexin V is abundantly present in the heart. Using isolated myocytes and cultured cardiac endothelial and fibroblast-like cells, it is demonstrated that the localization of Annexin V is confined to non-myocytes. No positive bands matching the Mw of recombinant Annexin VIII are found in any of the cell types examined.In vitro studies demonstrate that recombinant Annexin V potently inhibits the activity of cardiac membrane-bound phospholipases, acting on their natural sur rounding substrate, in a calcium dependent manner. Interestingly, annexin V also inhibits triacylglycerol hydrolysis. In conclusion, the expression of annexins is cell-type specific and suggests a cell-type specific function of these proteins in the heart. The absence of Annexin V in cardiac myocytes dismisses involvement of this annexin in cardiomyocyte phospholipid metabolism. The presence of Annexin V in cardiac endothelial and fibroblasts suggests a regulating role in the phospholipid homeostasis of non-myocyte cell types in the heart. (Mol Cell Biochem116: 95–101, 1992)     

Long-lasting alterations in neuronal calcium homeostasis in an in vitro model of stroke-induced epilepsy

Altered calcium homeostatic mechanisms have been implicated in the development of acquired epilepsy in numerous models. Stroke is one of the leading brain injuries that cause acquired epilepsy, yet little is known concerning the molecular mechanisms underlying stroke-induced epileptogenesis. Recently an in vitro model of stroke-induced epilepsy was developed and characterized as a powerful tool to study the pathophysiology of injury and stroke-induced epileptogenesis. Using this glutamate injury-induced epileptogenesis model, we have investigated the role of altered calcium homeostatic mechanisms in the development and maintenance of stroke-induced epilepsy. Epileptic neurons manifested elevated intracellular calcium levels compared to control neurons independent of neuronal activity and seizure discharge for the remainder of the life of the neurons in culture. In addition, epileptic neurons were found to have alterations in the ability to reduce intracellular calcium levels following a calcium load. These long-term epileptic changes in calcium homeostasis were dependent on calcium during the initial glutamate injury. The data demonstrate that significant alterations in calcium homeostatic mechanisms occur in association with stroke-induced epilepsy and suggest that these changes may play a role in both the induction and maintenance of the epileptic phenotype in this model.     

Overexpression of the Neurotrophic Cytokine S100β in Human Temporal Lobe Epilepsy

Abstract: Neuritic sprouting and disturbances of calcium homeostasis are well described in epilepsy. S100β is an astrocyte-derived cytokine that promotes neurite growth and induces increases in levels of intracellular calcium in neurons. In sections of neocortex of surgically resected temporal lobe tissue from patients with intractable epilepsy, we found that the number of S100β-immunoreactive astrocytes was approximately threefold higher than that found in control patients ( p < 0.001). These astrocytes were activated, i.e., enlarged, and had prominent processes. Temporal lobe tissue levels of S100β were shown by ELISA to be fivefold higher in 21 epileptics than in 12 controls ( p < 0.001). The expression of the astrocyte intermediate filament protein, glial fibrillary acidic protein, was not significantly elevated in epileptics, suggesting a selective up-regulation of S100β expression. Our findings, together with established functions of S100β, suggest that this neurotrophic cytokine may be involved in the pathophysiology of epilepsy.     

UVB irradiation-induced dysregulation of plasma membrane calcium ATPase1 and intracellular calcium homeostasis in human lens epithelial cells

Ultraviolet B (UVB) could lead to the apoptosis of human lens epithelial cell and be hypothesized to be one of the important factors of cataractogenesis. In the human lens, Ca²⁺-ATPase is a major determinant of calcium homeostasis. Plasma membrane calcium ATPase1 (PMCA1) is a putative “housekeeping” isoform and is widely expressed in all tissues and cells, which plays an important role in calcium homeostasis. However, the effects of UVB-irradiation on the expression of PMCA1 and the cellular calcium homeostasis are still unclear. In the present study, we cultured human lens epithelial cells (HLE B-3) in vitro and investigated the effects of UVB irradiation on the expression of PMCA1 and the intracellular calcium homeostasis using real-time cell electronic sensing system, flow cytometry, fluo-3/AM probes, real-time quantitative PCR, and enzyme-linked immunosorbent assay techniques. We found that UVB irradiation could induce human lens epithelial cell death, cause intracellular calcium ion (Ca²⁺) elevation, inhibit Ca²⁺-ATPase activity and decrease the expression of PMCA1 at gene and protein levels, suggesting that the downregulation of PMCA1 and the disruption of calcium homeostasis may play important roles in UVB-induced HLE B-3 cell apoptosis.     

Neuronal and Glial Expression of the Multidrug Resistance Gene Product in an Experimental Epilepsy Model

1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy. 2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group. 3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling. 4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures. 5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.     

GALNT3, a gene associated with hyperphosphatemic familial tumoral calcinosis, is transcriptionally regulated by extracellular phosphate and modulates matrix metalloproteinase activity

GALNT3 encodes UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl-transferarase 3 (ppGalNacT3), a glycosyltransferase which has been suggested to prevent proteolysis of FGF23, a potent phosphaturic protein. Accordingly, loss-of-function mutations in GALNT3 cause hyperphosphatemic familial tumoral calcinosis (HFTC), a rare autosomal recessive disorder manifesting with increased kidney reabsorption of phosphate, resulting in severe hyperphosphatemia and widespread ectopic calcifications. Although these findings definitely attribute a role to ppGalNacT3 in the regulation of phosphate homeostasis, little is currently known about the factors regulating GALNT3 expression. In addition, the effect of decreased GALNT3 expression in peripheral tissues has not been explored so far. In the present study, we demonstrate that GALNT3 expression is under the regulation of a number of factors known to be associated with phosphate homeostasis, including inorganic phosphate itself, calcium and 1,25-dihydroxyvitamin D(3). In addition, we show that decreased GALNT3 expression in human skin fibroblasts leads to increased expression of FGF7 and of matrix metalloproteinases, which have been previously implicated in the pathogenesis of ectopic calcification. Thus, the present data suggest that ppGalNacT3 may play a role in peripheral tissues of potential relevance to the pathogenesis of disorders of phosphate metabolism.     

Participation of annexin At1 in plant response to abiotic stress

Annexins are calcium-dependent phospholipid-binding proteins existing both in animal and plant cells. Mammalian and especially human annexins were examined for many years, and their functions in these organisms are already well known, but it is not the case for plant annexins. On the basis of existing literature and experimental evidence, it can be proposed that plant annexins may have a role in stress response. Annexin At1 of Arabidopsis thaliana (AnnAt1) is one of eight proteins of this family in A. thaliana. In its sequence many potentially functional domains are found, owing to that this protein can play an important role in stress response of the organism. Considering literature data and our own experiments one can postulate that AnnAt1 has weak peroxidase activity and form oligomers in hydrogen peroxide-dependent manner. This can be important in response to oxidative stress. Also we found that this protein forms ion channel in pH-dependent manner. This phenomenon may have particular significance in maintaining calcium homeostasis in the cell and calcium signaling, therefore AnnAt1 may play different roles in regulating stress response of plant. This is extremely important because plants during growth and development have to cope different stress factors like drought, deficiency or excess of mineral compounds in the soil, as well as low or high temperatures.     

The Imbalanced Expression of Adenosine Receptors in an Epilepsy Model Corrected Using Targeted Mesenchymal Stem Cell Transplantation

Adenosine inhibits epileptic episodes by interacting with G-protein-coupled receptors. This study examined the mechanism by which the inhibitory effect of adenosine becomes impaired during epileptogenesis. Dynamic changes in adenosine A1 receptors (A1Rs) and A2a receptors (A2aRs) were investigated in a kindling model of epilepsy. RT-PCR, Western blotting, and immunofluorescence results indicated that expression of A1Rs was increased in the hippocampus 24 h after kindling, but progressively decreased 1 and 6 months after kindling. Opposite changes were seen in the expression of A2aRs. This bidirectional change resulted in an imbalance between A1Rs and A2aRs and dysregulation of the adenosine system. Autologous mesenchymal stem cell (MSC) transplantation was used to correct this disorder and avoid side effects of systematic adenosine therapy. Paramagnetic iron oxide particles were used to mark and track the MSCs in vivo using MRI. The results indicated that the transplanted cells migrated along the callosum and settled at the ependymal layer. The MSCs displayed a relatively long survival time, at least 3 months. The improved AR expression and EEG findings suggested that MSC transplantation was a potentially effective means of treating refractory epilepsy.     

Annexin A1 N-Terminal Derived Peptide Ac2-26 Stimulates Fibroblast Migration in High Glucose Conditions

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG) or high glucose (HG) we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i) Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii) N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.     

Effect of the calcium sensing receptor on rat bone marrow-derived mesenchymal stem cell proliferation through the ERK1/2 pathway

Migration and proliferation of bone marrowderived mesenchymal stem cells (BMSCs) is critical to treatment of ischemic injury. The calcium sensing receptor (CaSR) has an important role in maintaining systemic calcium homeostasis, which is related to cell proliferation, apoptosis and paracrine signaling. We hypothesize that CaSR may enhance BMSC proliferation. Rat BMSCs were incubated with various calcium concentrations for 48 h in vitro to activate CaSR. To investigate potential mechanisms responsible for growth enhancement by calcium, the rat BMSC cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), and induction of apoptosis confirmed by cytofluorimetric analysis using propidium iodide and Annexin V-FITC double staining. Since the mitogen-activated protein kinase (MAPK) signaling pathway was one of the most significantly affected by CaSR, MAPK activation was measured by western blotting. Calcium exposure significantly enhanced rat BMSCs proliferation, as well as the proportion of the population in S phase, in a dose-dependent manner, effects which were abolished by NPS2390 (a CaSR antagonist) and U0126 (a MEK1/2 inhibitor). These results demonstrate that CaSR is involved in rat BMSC proliferation, as seen by an increased proliferation index, decreased apoptosis, and ERK1/2 activation, and provide important insight into the cellular and molecular mechanisms by which CaSR affects cell proliferation. A CaSR agonist may prove useful to enhance BMSC survival during transplantation.     

Role of Annexin A5 in Cisplatin-induced Toxicity in Renal Cells: MOLECULAR MECHANISM OF APOPTOSIS*

Annexin A5 belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of various pathophysiological processes. There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function of this protein has yet to be elucidated. In this study, we aimed to verify the function of annexin A5 in the apoptosis of renal epithelial cells. Real-time PCR and Western blot analysis, together with immunofluorescence analysis, showed that the expression of annexin A5 significantly increased in the presence of cisplatin in both human and rat renal epithelial cells. With regard to the mechanism of cisplatin-induced apoptosis, apoptosis-inducing factor (AIF) release into the cytosol was observed, and the underlying mechanism was identified as voltage-dependent anion channel (VDAC) oligomerization. Mitochondrial membrane potential (Δψ_m) was found to be greatly disrupted in cisplatin-treated cells. Moreover, cisplatin strongly induced translocation of annexin A5 into mitochondria. To understand the functional significance of annexin A5 in renal cell death, we used a siRNA-mediated approach to knock down annexin A5. Annexin A5 depletion by siRNA led to decreased annexin A5 translocation into mitochondria and significantly reduced VDAC oligomerization and AIF release. Annexin A5 siRNA also increased cell viability compared with the control. Moreover, expression of annexin A5 was induced by other nephrotoxicants such as CdCl₂ and bacitracin. Taken together, our data suggest that annexin A5 may play a crucial role in cisplatin-induced toxicity by mediating the mitochondrial apoptotic pathway via the induction and oligomerization of VDAC.     

Upregulated P2X3 Receptor Expression in Patients with Intractable Temporal Lobe Epilepsy and in a Rat Model of Epilepsy

Purinergic P2X3 receptors (P2X3Rs) play extensive roles in nerve cells in the central nervous system, particularly in hyperexcitability and calcium (Ca²⁺) influx. However, the role of P2X3Rs in epilepsy has not been previously investigated. To determine the relationship between P2X3Rs and epilepsy, the expression and cellular location of P2X3Rs in patients with intractable temporal lobe epilepsy (TLE) and in a lithium chloride-pilocarpine-induced chronic rat model of epilepsy were assessed. Furthermore, the function of P2X3Rs was assessed in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to evaluate the expression levels of P2X3Rs in brain tissues from TLE patients and an epileptic rat model, whereas immunofluorescence labeling was applied to determine the distribution of target proteins. Whole-cell recording was subsequently performed to identify the influence of P2X3Rs on seizure-like discharges. P2X3Rs were located at the cell bodies and dendrites of neurons with significantly increased expression in the TLE patients and epileptic rat model. In vitro, P2X3R activation accelerated sustained repetitive firing, whereas P2X3R inhibition led to relatively low-frequency discharges. To the best of our knowledge, this is the first study provide evidence that upregulated P2X3R expression exists in both epileptic humans and rats and may aggravate the epileptic state in vitro. Thus, P2X3Rs may represent a novel therapeutic target for antiepileptic drugs.     

Expression of the IL-7 Receptor Alpha-Chain Is Down Regulated on the Surface of CD4 T-Cells by the HIV-1 Tat Protein

HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7) is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127) on both CD4 and CD8 T-cells in viremic HIV+ patients. We have shown previously that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells in a paracrine fashion. The effects of Tat on CD127 expression in CD4 T-cells has yet to be described. To explore this effect, CD4 T-cells were isolated from healthy individuals and expression levels of CD127 were examined on cells incubated in media alone or treated with Tat protein. We show here that, similar to CD8 T-cells, the HIV-1 Tat protein specifically down regulates CD127 on primary human CD4 T-cells and directs the receptor to the proteasome for degradation. Down regulation of CD127 in response to Tat was seen on both memory and naive CD4 T-cell subsets and was blocked using either heparin or anti-Tat antibodies. Tat did not induce apoptosis in cultured primary CD4 T-cells over 72 hours as determined by Annexin V and PI staining. Pre-incubation of CD4 T-cells with HIV-1 Tat protein did however reduce the ability of IL-7 to up regulate Bcl-2 expression. Similar to exogenous Tat, endogenously expressed HIV Tat protein also suppressed CD127 expression on primary CD4 T-cells. In view of the important role IL-7 plays in lymphocyte proliferation, homeostasis and survival, down regulation of CD127 by Tat likely plays a central role in immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new approaches to mitigate the CD4 T-cell loss evident in HIV infection.     

Up-Regulation of Epithelial Membrane Protein-1 in the Temporal Neocortex of Patients with Intractable Epilepsy

Epithelial membrane protein-1 (EMP-1), called Tumor-associated membrane protein, is the marker of a drug-resistant tumor and take part in the drug-resistant mechanism of tumor, with the relationship of epidermal growth factor receptor (EGFR). Because there are some similarities between the pathogenesis and the drug resistance mechanism of tumor and the drug resistance mechanisms in epilepsy. EMP1 expression may be connected with the drug-resistance mechanism of epilepsy. We detected EMP-1 by gene scanning and immunohistochemistry staining, comparing the IE group and the control group, and we investigated the relationship between EMP-1 and EGFR by double-label immunofluorescence staining in the IE group. We found expression of EMP-1 mRNA was higher in IE per the gene scanning, EMP-1 immunoreactivity was apparent in neurons of IE patients but not in the control group, and the expression of EMP-1 and EGFR occurred in the same neuron. We confirm EMP-1 is abnormally expressed in IE and suggest the interaction of EGFR and EMP-1 plays a role in the mechanism of drug resistance in epilepsy and may be a new gene for drug resistance.     

Plasma membrane Ca2+-ATPase expression during colon cancer cell line differentiation

The differentiation of colon cancer cell lines is associated with changes in calcium homeostasis. Concomitantly there are changes in the expression of some calcium transporters and G-protein-coupled receptors, which are capable of altering cytosolic-free calcium levels. Recent studies associate alterations in calcium transporter expression with tumourigenesis, such as changes in specific isoforms of the plasma membrane calcium ATPase (PMCA) in breast cancer cell lines. In this study, we examined the expression of PMCA isoforms in the HT-29 colon cancer cell line using two methods of differentiation (sodium butyrate-mediated and spontaneous post-confluency induced differentiation). Our studies show that differentiation of HT-29 colon cancer cells is associated with the up-regulation of the PMCA isoform PMCA4 but no significant alteration in PMCA1. These results suggest that PMCA4 may be important and have a specific role in colon cells as well as being significant in colon cancer tumourigenesis.     

运用BioID技术筛选S100A7互作蛋白*

目的: 通过邻近生物素鉴定(BioID)技术筛选S100A7互作蛋白并进行验证。方法: 采用邻近生物素BioID和质谱相结合的方法,筛选S100A7互作蛋白。通过免疫荧光和免疫共沉淀方法,对相互作蛋白进行验证。结果: 与对照组比对,通过BioID技术共获得94个可能与 S100A7 相互作用的候选蛋白质。选取 Annexin A2(AnxA2)蛋白在 HEK293 细胞中进行了验证,结果表明S100A7 与 AnxA2 存在直接的相互作用,且二者共定位于细胞质和细胞膜。结论: 可将BioID技术作为互作蛋白筛选的一项新技术,通过该技术发现S100A7和AnxA2存在相互作用。     

Defective calcium homeostasis in the cerebellum in a mouse model of Niemann-Pick A disease

We recently demonstrated that calcium homeostasis is altered in mouse models of two sphingolipid storage diseases, Gaucher and Sandhoff diseases, owing to modulation of the activities of a calcium-release channel (the ryanodine receptor) and of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) respectively, by the accumulating sphingolipids. We now demonstrate that calcium homeostasis is also altered in a mouse model of Niemann-Pick A disease, the acid sphingomyelinase (A-SMase)-deficient mouse (ASM-/-), with reduced rates of calcium uptake via SERCA in the cerebellum of 6-7-month-old mice. However, the mechanism responsible for defective calcium homeostasis is completely different from that observed in the other two disease models. Thus, levels of SERCA expression are significantly reduced in the ASM-/- cerebellum by 6-7 months of age, immediately before death of the mice, as are levels of the inositol 1,4,5-triphosphate receptor (IP3R), the major calcium-release channel in the cerebellum. Systematic analyses of the time course of loss of SERCA and IP3R expression revealed that loss of the IP3R preceeded that of SERCA, with essentially no IP3R remaining by 4 months of age, whereas SERCA was still present even after 6 months. Expression of zebrin II (aldolase C), a protein found in about half of the Purkinje cells in the adult mouse cerebellum, was essentially unchanged during development. We discuss possible pathological mechanisms related to calcium dysfunction that may cause Purkinje cell degeneration, and as a result, the onset of neuropathology in Niemann-Pick A disease.