Silencing the SPCA1 (Secretory Pathway Ca²⁺-ATPase Isoform 1) Impairs Ca²⁺Homeostasis in the Golgi and Disturbs Neural Polarity

Abstract

Neural cell differentiation involves a complex regulatory signal transduction network in which Ca2ϩ ions and the secretory pathway play pivotal roles. The secretory pathway Ca 2ϩ -ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca 2ϩ or Mn 2ϩ from the cytosol to the Golgi lumen. Its expression during brain development in different types of neurons has been documented recently, which raises the possibility that SPCA1 contributes to neuronal differentiation. In the present study, we investigated the potential impact of SPCA1 on neuronal polarization both in a cell line and in primary neuronal culture. In N2a neuroblastoma cells, SPCA1 was immunocytochemically localized in the juxtanuclear Golgi. Knockdown of SPCA1 by RNA interference markedly delayed the differentiation in these cells. The cells retarded in differentiation showed increased numbers of neurites of reduced length compared with control cells. Ca 2ϩ imaging assays showed that the lack of SPCA1 impaired Golgi Ca 2ϩ homeostasis and resulted in disturbed trafficking of different classes of proteins including normally Golgi-localized cameleon GT-YC3.3, bearing a Golgi-specific galactosyltransferase N terminus, and a normally plasma membrane-targeted, glycosyl phosphatidyl inositol-anchored cyan fluorescent protein construct. Also in hippocampal primary neurons, which showed a differential distribution of SPCA1 expression in Golgi stacks depending on differentiation stage, partial silencing of SPCA1 resulted in delayed differentiation, whereas total suppression drastically affected the cell survival. The disturbed overall cellular Ca 2ϩ homeostasis and/or the altered targeting of organellar proteins under conditions of SPCA1 knockdown highlight the importance of SPCA1 function for normal neural differentiation.

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

“…The present data provide additional information on the functional role of TG Ca 2+ . In particular, recent evidence suggests that SPCA1 down-regulation (in cell culture or in knockout animals) affects a number of cellular and GA-specific functions, from GA morphology to apoptosis and from neuronal differentiation to protein trafficking (13,(36)(37)(38)(39). Here we have further expanded this analysis by acutely down-regulating SPCA1 expression using the RNAi technique.…”

Organellar Calcium Buffers

“…ATPase responsible for maintenance of Golgi Ca 2þ gradients (Hu et al 2000;Sudbrak et al 2000). Furthermore, reduced SPCA1 expression in a mouse model caused impaired neural polarity (Sepulveda et al 2009). …”

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

“…Chelator selectivities indicate that Ca 2ϩ acts either through a short pulseor else a continuous gradient of leaking luminal Ca 2ϩ very near the transport machinery (22,23). In vivo evidence for a requirement for luminal Ca 2ϩ in Golgi trafficking came with the recognition that mutations of the Golgi Ca 2ϩ pump SPCA (secretory pathway Ca 2ϩ -ATPase) cause Hailey-Hailey disease (24) and that depletion of SPCA from cells arrests secretion at the Golgi (25,26). In this case, the luminal Ca 2ϩ may act in the lumen; SPCA-dependent luminal Ca 2ϩ appears to be required in the TGN for sorting of a subset of secretory proteins into exocytic carriers via the luminal Ca 2ϩ -binding protein Cab45 (27,28).…”