Presence of molecular chaperones, heat shock cognate (Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynaptic structures of rat brain

Abstract

The synaptic localization of molecular chaperones, heat shock cognate protein 70 (Hsc70) and Hsp40, was investigated immunohistochemically in the normal rat brain. Postsynaptic density (PSD) fractions contained a constitutive form of HSP70, heat shock cognate protein 70 (Hsc70 or p73) but not inducible form of HSP70 (p72). The immunoreactivities of Hsc70 (p73) were distributed throughout the rat brain, in neuronal somata, dendrites and axons. Their immunoreactivity in neurons was localized in the cytoplasmic matrix, dendrites, and spines at the electron microscopic level. Presynaptic terminals, but less frequently than postsynaptic staining, were also reactive. Postsynaptic areas immediately beneath the synaptic contact or PSDs were immunoreactive for Hsc70. The Hsp40 was highly concentrated in PSD fractions. The staining of Hsp40 immunoreactivity was punctate and distributed widely in the brain. Hsp40 immunoreactivity was localized in dendritic spines, especially in the subsynaptic web, with weak staining of PSDs at the electron microscopic level. Double immunofluorescent staining and confocal microscopy revealed that Hsc70 and Hsp40 were co-localized on somata and neuronal processes of cultured cerebral neurons, on which synaptophysin immunoreactive spots were scattered. These results suggest that Hsp40 and Hsc70 are co-localized at postsynaptic sites and postsynaptic chaperone activity may be mediated by these two heat shock proteins.

Introduction

Several polypeptides known as heat- or stress-inducible proteins are expressed in prokaryotes and eukaryotes. They are commonly referred to as heat-shock proteins (Hsps). The Hsps are classified into several groups according to their molecular sizes. The expressions of these proteins are induced by the activation of heat shock elements (HSE) in the promoter region of their genes. HSP70, a homologue of E. coli DnaK protein, has two major members, Hsp70 (p72), an inducible form, and the heat shock cognate protein (Hsc 70, p73), a constitutively expressed form. We refer to the constitutive form of HSP70 as Hsc70 or p73, and the inducible form as p72. We use fully capitalized names to denote the protein family (ex. HSP70) and an initial capital letter for specific family members (ex. Hsc70, Hsp40) according to the recent instruction [14]. The general term HSP70 implies both Hsc70 (p73) and p72. The two proteins are similar with approximately 90% identity in amino acid sequences and mostly indistinguishable biochemical properties except for a slight difference in their electrophoretic mobility [37]. HSP70 is expressed ubiquitously in various tissues, but among adult tissues, only the brain retains high levels of Hsc70 gene expression [13]. The relative levels of Hsp70 mRNA and Hsc70 mRNA are higher in cerebellum than in cerebral cortex and both the transcription and turnover of Hsp70 mRNAs differs between cerebellum and cerebral cortex [30]. The expression of brain HSP70 can be induced by seizure [20]and ischemia 2, 41. Thus, Hsp70 is one of the candidate plasticity-related genes (CPGs) [33].

HSP70 has chaperone activity, which facilitate the repair partially denatured proteins after various stress or assists correct folding of nascent polypeptides [4]. HSP70s in the yeast endoplasmic reticulum and mitochondria are involved in the translocation and targeting of proteins [6]. Furthermore, HSP70 appears to be involved in the transport of cytoplasmic proteins into nucleus, and shuttles between these two compartments 19, 27, 28, 43, 45. HSP70 does not function by itself in vivo and requires assistance by the DnaJ family proteins. HSP70, with the aid of various kinds of DnaJ-like proteins, fulfills various cellular functions 5, 10. All DnaJ-like proteins contain J-domains, by which the proteins interact with HSP70 and stimulate its ATPase activity. Different DnaJ-like proteins have specialized functions, being coupled to HSP70. Thus, the roles of HSP70 and its target molecules are likely to be determined by the types of partner DnaJ homologs [6]or co-compartmentalization of certain types of DnaJ homologs and HSP70s [44]. Ohtsuka et al. [35]have recently identified a novel DnaJ-like protein, Hsp40, in mammalian and avian cells and showed the co-localization of Hsp40 and HSP70 in HeLa cells and various mammalian cells 17, 56.

A detailed localization of HSP70 and its partner chaperones in the brain have not yet been reported. We report here the distribution of Hsc70 and Hsp40 at synaptic sites, and discuss their potential roles in neuronal and synaptic functions.