Analysis of the expression and function of mammalian CSP isoforms

Abstract

Exocytosis, the fusion of intracellular vesicles with the plasma membrane, is fundamental to intercellular communication in multicellular organisms. This pathway facilitates the release or secretion of molecules from the cell. In addition, exocytosis is essential for delivery of resident proteins to the plasma membrane. There are two different pathways of exocytosis, constitutive and regulated exocytosis. Constitutive exocytosis occurs without regulation, e.g. pathways regulating the delivery of lipids and ‘house-keeping’ proteins to the plasma membrane or the secretion of antibodies and extra-cellular matrix components from the cell. In contrast, regulated exocytosis facilitates the controlled release of extra-cellular molecules or insertion of new membrane components only in response to a physiological signal. The most common signal for regulated exocytosis is an increase in intracellular Ca2+ concentration. Several proteins function in exocytosis, and the membrane fusion step is widely believed to result from an interaction between SNARE (SNAP receptor) proteins on the vesicle membrane and plasma membrane. In neuroendocrine cells, these SNARE proteins are VAMP2, which is bound to vesicle membranes and syntaxin1A and SNAP25, which are associated with the plasma membrane. Several proteins have been implicated as SNARE regulators, such as NSF (N-ethylmaleimide-sensitive factor) and its cofactor α-SNAP, Munc18 and synaptotagmin. Another possible SNARE regulator is the cysteine string protein (CSP). CSPα was first identified in Drosophila melanogaster and was later identified in Torpedo as a possible Ca2+-channel regulator. Inactivation of the CSPα gene in Drosophila is lethal at an embryonic stage and in embryos synaptic vesicle exocytosis was decreased by ~50% at 22°C and was abolished at higher temperatures. These results provided strong evidence that CSPα has an important role in presynaptic neurotransmission. However, more recent work on CSPα null mice uncovered an important neuroprotective function for CSPα in brain, but also challenged the proposed function of CSPα in neuronal exocytosis, as no defect in this pathway was evident, at least in young animals. The only reported developmental abnormality of CSPα null mice was bilateral cryptorchidism, a failure of testicular descent during development. Interestingly, two additional CSP isoforms were recently identified in mouse and human testis, CSPβ and CSPγ. One consequence of the identification of CSPβ and CSPγ is that they may complicate analysis of CSPα knockout mice. Here, we have used a combination of techniques, cell systems and human brain samples to examine the function of CSPα in exocytosis, the expression of novel CSPα isoforms in testis, and expression changes of CSPα and its partner proteins in neurological disorders. Furthermore, we have initiated studies to examine how CSPα function is linked to cryptorchidism at the molecular level. My results show that CSPα depletion perturbs regulated exocytosis in neuroendocrine cells, but has no consistent effect on constitutive exocytosis. CSPα has been reported to have an important neuroprotective function; however, no significant changes in CSPα expression were detected in brain samples for schizophrenia, depression and bipolar disorder. Nevertheless the expression of specific CSPα binding partners was found to be significantly changed in some of these disorders. In addition to these studies focussing on CSPα function and expression in neuronal and neuroendocrine cells, studies were undertaken to analyse expression profiles of CSP isoforms in testis. This analysis found that CSPβ and CSPγ are exclusively expressed in testis, and that mRNA transcription of both isoforms is initiated with sexual maturation. Furthermore expression of both isoforms is restricted to germ cells, whereas CSPα is expressed throughout testes. Previous work has shown that the secretory hormone INSL3, which is exclusively expressed in testicular Leydig cells, is involved in the development of cryptorchidism. Confocal microscopic analysis revealed that CSPα and INSL3 colocalise on vesicles in Leydig cells, suggesting the intriguing possibility that CSPα inactivation might cause cryptorchidism due to a loss of INSL3 secretion.