2007b)

2007b). a pathological Rabbit Polyclonal to DARPP-32 hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should T-5224 include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest. Electronic supplementary material The online version of this article (doi:10.1007/s12192-013-0427-x) contains supplementary material, which is available to authorized users. as a risk factor for Alzheimers disease. Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, neurodegenerative disorder characterized by gradual loss of muscle function due to death of motor neurons in T-5224 the cortex, brain stem, and spinal cord. Mutations in several genes have been linked to familial forms, including fALS1 due to dominant mutations in (Rosen et al. 1993). Evidence points to misfolding and altered solubility of the mutant protein as the underlying gain of toxic function leading to disease, with multiple downstream effects including mitochondrial abnormalities, calcium dysregulation, T-5224 and formation of cytosolic protein aggregates (Boillee et al. 2006). Inducing expression of HSPs [stress-inducible Hsp70 (HspA1) and Hsp40 (DNAJ)] is protective in cell culture models, including a primary culture model developed in our laboratory by expressing wild-type or mutant SOD1 in primary motor neurons of dissociated spinal cordCdorsal root ganglion (DRG) cultures (Batulan et al. 2006). In addition to accumulating in the cytoplasm, mutant SOD1 is secreted from cells and thus could exert toxicity through extracellular mechanisms (Turner et al. 2005; Urushitani et al. 2006). We therefore asked what effect expression of mutant SOD1 or treatments known to induce HSPs would have on the expression and secretion of clusterin from spinal cord cells, given that clusterin is a stress protein, cytosolic clusterin is protective, and secreted clusterin can prevent the aggregation of misfolded proteins in the extracellular milieu (Poon et al. 2000). In this study, the distribution of clusterin was examined in motor neurons and astrocytes of long-term (3C6?weeks) spinal cordCDRG cultures using antibodies recognizing either nuclear clusterin or cytoplasmic/secreted clusterin. Each cell type expressed both nuclear and cytoplasmic clusterin, the strong constitutive expression of nuclear clusterin arguing against an apoptotic role. In addition, clusterin was secreted into the culture medium. Our previous studies using this model showed that motor neurons have a high threshold for stress-induced upregulation of HSPs, but astrocytes mount a robust stress response (Batulan et al. 2003), replicating the properties of these cells in vivo (Manzerra and Brown 1996). However, thermal stress sufficient to induce expression of Hsp70 in astrocytes failed to T-5224 increase expression of clusterin in either astrocytes or motor neurons. In contrast to heat shock, treatment of spinal cordCDRG cultures with the Hsp90 inhibitor, geldanamycin, did induce expression of nuclear and cytoplasmic clusterin in both motor neurons and astrocytes, as previously shown for Hsp70 T-5224 and Hsp40 (Batulan et al. 2003, 2006). Thus, clusterin could contribute to the neuroprotective properties of Hsp90 inhibitors. Expression of mutant SOD1 in cultured motor neurons or astrocytes did not alter clusterin expression. In lumbar spinal cord of overtly symptomatic SOD1G93A mice, the major finding was strong immunolabeling of mutant SOD1 inclusions, a hallmark of disease, in common with other proteins associated with protein quality.