Role of Heat Shock Protein 25 (Hsp25) in Stress and Regeneration in the Brain.

The molecular mechanisms of response of the central nervous system (CNS) to stress involve the induction of expression of a variety of genes, including transcription factors, neurotrophins, cytokines, and cellular stress proteins.  Expression of cellular stress proteins or heat shock proteins (hsp) has been found to be a universal and evolutionarily conserved response of all cells and organisms to different types of environmental stress, essential for maintenance of cellular homeostasis and development of stress tolerance (for review see Lindquist, 1986; Welch, 1992; Morimoto, 1993).  The expression of cellular stress proteins has been extensively studied in the CNS (for review see Brown, 1994; Nowak et al., 1994).  In the brain, hsp were showed to be induced in response to a variety of stimulations including tissue injury (Gonzales et al., 1989), ischemia/hypoxia (Vass et al., 1988; Nowak, 1990; Ferriero et al., 1990; Kawagoe et al., 1992), epileptic attack (Vass et al., 1989), and hyperthermia (Masing and Brown, 1989; Blake et al., 1990).

The hsp are divided into three major families, hsp90, hsp70 and hsp20, according to their molecular weight and degree of structural homology.  While the mechanisms of action and expression patterns of members of the hsp90 and hsp70 gene families in the CNS have been partially characterized, the role of small heat shock proteins is far less studied.  Hsp25 has been characterized as a molecular chaperone required for protection of protein machinery under conditions of stress (Jakob et al., 1993; Carver et al., 1995) and acquisition of stress tolerance (Landry et al., 1989).  Hsp25 has been found to be expressed during mouse embryogenesis, accumulating in the neurons of the spinal cord and the Purkinje cells of the cerebellum, as well as in some peripheral tissues (Gernold et al., 1993).  Constitutive expression of hsp25 has been detected in the adult rat brain by immunoblot analysis, while immunohistochemistry performed on the cortex and cerebellum failed to detect this protein (Wilkinson and Pollard, 1993).  Different stimuli including injury, ischemia, and kainic acid treatment were found to induce mRNA and protein expression of the rat hsp27 gene, a homologue of the mouse hsp25 gene, in the affected brain areas (Higashi et al., 1994; Kato et al., 1994; Plumier et al., 1996;1997).  Nevertheless, in spite of accumulation of the data of constitutive as well as induced expression of hsp25 in the mammalian CNS, the exact role of hsp25 in the CNS remains far from clear.

Three recent observations (Plumier et al., 1997; Costigan et al., 1998; Murashov et al., 1998a) have shed more light on the possible function of Hsp25 in the CNS.  The expression of Hsp25 was localized to specific neuronal populations of brainstem and the spinal cord under normal physiological conditions and was strongly upregulated at a time of the stress, injury and regeneration (Costigan et al., 1998; Murashov et al., 1998a,b).  In the adult mouse brainstem, the constitutive expression of Hsp25 was limited to neuronal populations of facial, trigeminal, ambiguus, and hypoglossal motor nuclei (Murashov et al., 1998a).  Significant increase in the levels of Hsp25 in these nuclei and along their axonal fibers was observed after experimentally induced hyperthermia and hypoxia treatment.   The rapid induction of Hsp25 after stress in axonal fibers of the facial and trigeminal nerve tracts indicated that Hsp25 was possibly transported by fast axonal transport.  p38 has been shown to be specifically involved in the phosphorylation cascade leading to activation of Hsp25 in vivo, with subsequent stabilization of actin filaments (Guay et al., 1997).  The upstream regulators of phosphorylation cascade activating p38 include p21 (Ras), Raf, and MAP kinase kinase 3 (MEK-3).  P38 in turn regulates MAP kinase-activated protein kinase-2 (MAPKAPK-2), which phosphorylates Hsp25/27 (Saklatvala et al., 1996).  Inhibition of p38 kinase activity with the inhibitor SB203580 specifically blocked Hsp25/27 phosphorylation and prevented stabilization of actin filaments in vivo (Huot et al., 1996).  Furthermore, recent observations showed that during stress, Hsp25 may form complex with Akt/PKB kinase (Matsuzaki et al., 1996; Konishi et al., 1997), a member of PI-3 kinase pathway which prevents neuronal cell death (Dudek et al., 1997; Philpott et al., 1997; Eves et al., 1998).  Therefore, activation of p38/Hsp25/Akt signaling may both promote survival of the injured neurons and contribute to alterations in actin cytoskeleton associated with axonal regeneration.  

Our preliminary studies were focused on regeneration in the spinal cord of adult mice after peripheral nerve injury.  On the level of spinal cord the constitutive expression of Hsp25 was observed predominantly in motor neurons.  After sciatic nerve axotomy we observed induction of Hsp25 in lumbar motor neurons, interneurons of dorsal horn, and along their fibers on the side of the injury which strongly correlated with the pattern of axonal regeneration.  The expression of Hsp25 appeared on the third day postaxotomy, reached maximum on the forth day and sustained at steady level up to twenty days postaxotomy (Murashov et al., 1998b) (Fig.2). 

The pattern of induction of Hsp25 in the spinal cord corresponds to published data (Costigan et al., 1998) and correlates with the pattern of axonal regeneration described in mice and rats after sciatic nerve injury (Hoffman and Lasek, 1980).  The matching pattern of expression after sciatic nerve injury we observed for p38, MAPKAPK-2 and Akt kinases, which indicate their involvement in mechanisms of posttraumatic regeneration in the spinal cord (Fig.3). 

We were able to downregulate expression of Hsp25 by administering p38 kinase inhibitor SB203580, and upregulate Hsp25 expression by administering of d-tubocurarine (Fig.4).  

This suggests that activation of p38 kinase may be necessary for upregulation of Hsp25 expression during regeneration, possibly through phosphorylation of Hsp25 transcription factor.  D-tubocurarine a powerful antagonist of nicotinic receptors has induced expression of Hsp25 along axonal fibers, which may indicate its therapeutic potential for treatment of SCI.  The expression of Hsp25 was not limited to central part of axon.  Cultured neurons of dorsal root ganglion expressed Hsp25 throughout their neurites including the growth cones (Costigan et al., 1998).  We observed concentration of Hsp25 in cultured sympathetic and motor neurons at the growth cones where Hsp25 colocalized with F-actin (Murashov et al., in preparation) (Fig.5). 

Therefore the upregulation of Hsp25 after injury may both promote survival of the injured neurons and contribute to alterations in actin cytoskeleton associated with axonal regeneration.

This project was funded in part by the Department of Naval Research (N00014-96-1-056300).