Decarboxylases

Mouse monoclonal anti-His antibody was purchased from Invitrogen

Mouse monoclonal anti-His antibody was purchased from Invitrogen. dynamic to facilitate tau clearance from the brain and rescue LTP; however, when this property is compromised, Hsp27 may actually facilitate accumulation of soluble tau intermediates. Introduction Aberrant protein production is a common feature of many diseases of aging. The Arimoclomol maleate majority of these disease-associated proteins are also clients of the chaperone network. These abnormal clients are prone to aggregation, forming pathologic inclusion bodies in neurodegenerative diseases. One family of chaperones that may help offset the toxicity of misfolded substrates is the small heat shock protein (Hsp) family. The primary function of small Hsps is to protect unfolded proteins in the cytosol from entering an aggregation pathway. This function has been defined primarily by investigating one particular small Hsp: Hsp27 (Renkawek et al., 1999; Wyttenbach et al., 2002; Shimura et al., 2004; Sanbe et al., 2007). Hsp27 regulates many disease-related proteins that are prone to aggregation; however, validation of these findings in mammalian systems has been slow to follow, in large Arimoclomol maleate part because of several unique properties that distinguish Hsp27 from more classical chaperones such as Hsp70 and Hsp90 (Perrin et al., 2007). Hsp27 facilitates degradation and prevents aggregation of aberrant substrates independent of ATP or ubiquitination (Jakob et al., 1993; Shimura et al., 2004). Thus, measuring its activity is difficult because it essentially has no measurable enzymatic function. The most well characterized modification of Hsp27 is its capacity to be phosphorylated by stress-activated kinases at serine residues 15, 78, and 82 (Stokoe et al., 1992). The known consequence Arimoclomol maleate of this phosphorylation is disassembly of large (200C800 kDa) dormant Hsp27 multimers into smaller oligomeric and monomeric species (Huot et al., 1991; Landry et al., 1992). These structures are not static, and deciphering the role that cycling between phosphorylated and dephosphorylated states has on chaperoning activity will be a critical step forward in understanding the mechanisms used by Hsp27 to process substrates (Haley et al., 2000; Lelj-Garolla and Mauk, 2005, 2006). Our previous work has shown that regulation of the microtubule associated protein tau, which accumulates in 70% of all neurodegenerative diseases, is tightly coupled Arimoclomol maleate to the chaperone machinery. Although a role for Hsp27 in tau regulation has been proposed (Shimura et al., 2004), its involvement with tau is unknown. Neurons normally have low levels of Hsp27 expression, but this can be induced in response to proteotoxic stress. In fact, increases in Hsp27 correlate with pathological accumulation of aberrant proteins in neurodegenerative diseases, both in neurons and in glia (Renkawek et al., 1994; Koren et al., 2009). We hypothesized that dynamic regulation of Hsp27 was a requirement for its ability to remove aberrant tau from the brain. Here, we have investigated whether functionally dynamic and static Hsp27 species alter tau aggregation and clearance in the brain. These studies show that, although these Hsp27 variants act similarly at the molecular level, they act quite differently at the organismal level. Moreover, our studies suggest that tau aggregation can be decoupled from neuronal dysfunction. Materials and Methods Antibodies. Anti-Hsp27 and human Tau-150 rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-human tau was purchased from Dako. Rabbit polyclonal anti-pS199/S202 tau was purchased from Anaspec. Neuronal nuclei (NeuN) antibody was purchased from Millipore. Mouse monoclonal anti-His antibody was purchased from Invitrogen. Secondary antibodies conjugated to HRP and anti-goat secondary antibody conjugated to a fluorophore (594 nm) were purchased from Southern Biotechnology. Alexa Fluor (Invitrogen) were optimized and diluted accordingly as described below. Virus. Adeno-associated virus serotype 9 (AAV9)-expressing wild-type (wt) Hsp27 was a gift KDM3A antibody from T.E.G. AAV9Cenhanced green fluorescent protein (GFP) was also a gift from Dr. Kevin Nash (University of South Florida Health Byrd Alzheimer’s Institute, Tampa, FL). AAV9C3S/D Hsp27 was a gift from Dr. Grant Mauk (University of.