The percentage of band density of CAST single tg and non-tg was calculated based on these three blots as well as additional blots

The percentage of band density of CAST single tg and non-tg was calculated based on these three blots as well as additional blots. by CAST transgene expression reduces A pathology in APP23 mice, with our findings further suggesting that APP metabolism is altered by CAST overexpression as the mice develop -amyloid pathology. Our results indicate that this calpain system in neurons is usually more responsive to CAST inhibition under conditions of -amyloid pathology, suggesting that in the disease state neurons may be more sensitive to ASP3026 the therapeutic use of calpain inhibitors. Keywords:calpain, calpastatin, APP, A, Alzheimers disease == Introduction == Disruption of multiple proteolytic systems contributes to Alzheimers disease (AD) pathobiology, including alterations in the enzymes responsible for -amyloid (A) generation and clearance and dysfunction of the lysosomal system (Mathews, et al., 2002a,Nixon, et al., 2000). Calpains have also shown evidence of hyperactivity in human AD tissue (Grynspan, et al., 1997,Liu, et al., 2005,Nixon, 2003,Saito, et al., 1993). The calpain family is usually a group of Ca2+-activated, cytosolic, neutral pH, cysteine proteases (Huang and Wang, 2001) which modulate, probably indirectly, the localization and proteolytic processing of the amyloid precursor protein (APP) in cultured cells (Mathews, et al., 2002b). The most abundantly expressed calpains are m-calpain and -calpain, which are distinguished by their different affinities for Ca2+, each of which forms a functional heterodimer with the shared regulatory calpain small subunit 1 (Capn4) (Croall and DeMartino, 1991,Sorimachi, et al., 1997). In addition to their Ca2+-dependence, calpains are regulated ASP3026 by cytosol-to-membrane translocation and an endogenous inhibitor, calpastatin (CAST). The CAST protein consists of four calpain-inhibitory domains that are subjected to calpain cleavage and terminal inactivation by caspase (Croall and DeMartino, 1991,Goll, et al., 2003,Maki, et al., 1991,Sorimachi, et al., 1997,Wang, et al., 1998). Activation of calpains in AD is usually evidenced by increased levels of activated -calpain (Saito, et al., 1993) and m-calpain (Grynspan, et al., 1997) in neurons. The role of the calpain system in normal brain function and in pathological conditions has also been examined in various mouse models with altered calpain and CAST expression. While genetic deletion of either the m-calpain large subunit or the single calpain small subunit is usually lethal (Arthur, et al., 2000,Takano, et al., 2005), deletion of the -calpain large subunit does not result in an apparent gross phenotype (Azam, et al., 2001,Grammer, et al., 2005). Mice lacking CAST, while showing decreased locomotor activity and a decreased acoustic startle response, have no switch in hippocampal-dependent memory function (Nakajima, TSHR et al., 2008). CAST overexpressing mice similarly do not have gross memory deficits (Higuchi, et al., 2005,Takano, et al., 2005), arguing that CAST deletion or overexpression produces, in general, moderate effects in the normal mouse brain (Rao, et al., 2008). The role of the calpain system in AD pathobiology ASP3026 has been recently explored in -amyloid depositing mouse models. In Tg2576 (Vaisid, et al., 2007) and in APP/PS1 mice (Liang, et al., 2010), calpain appears to be activated and CAST diminished, consistent with reports in human AD tissue (Grynspan, et al., 1997,Liu, et al., 2005,Nixon, 2003,Saito, et al., 1993). In a mouse overexpressing wild-type APP that does not develop -amyloid pathology, calpains also appear to be activated in neurons (Kuwako, et al., 2002). In APP/PS1 mice, chronic calpain inhibition has been shown to reduce amyloid plaque burden and improve memory and synaptic transmission (Liang, et al., 2010,Trinchese, et al., 2008). This is in contrast to previous results from multiple laboratories showing that the acute pharmacological inhibition of calpains in cell culture systems dramatically increases A42 generation (Klafki, et al., 1996,Mathews, et al., 2002b,Yamazaki, et al., 1997,Zhang, et al., 1999), which is usually thought to be a more pathological A species. Here, we examined brain APP metabolite levels in mice overexpressing CAST in neurons compared to wild-type mice, as well as CAST overexpressing mice crossed to the -amyloid depositing APP23.