Intraneuronal ␤-amyloid (A␤ i ) accumulates early in Alzheimer's disease (AD) and inclusion body myositis. Several organelles, receptor molecules, homeostatic processes, and signal transduction components have been identified as sensitive to A␤. Although prior studies implicate the insulin-PI3K-Akt signaling cascade, a specific step within this or any essential metabolic or survival pathway has not emerged as a molecular target. We tested the effect of A␤42 on each component of this cascade. In AD brain, the association between PDK and Akt, phospho-Akt levels and its activity were all decreased relative to control. In cell culture, A␤ i expression inhibited both insulin-induced Akt phosphorylation and activity. In vitro experiments identified that ␤-amyloid (A␤), especially oligomer preparations, specifically interrupted the PDK-dependent activation of Akt. A␤ i also blocked the association between PDK and Akt in cell-based and in vitro experiments. Importantly, A␤ did not interrupt Akt or PI3K activities (once stimulated) nor did it affect more proximal signal events. These results offer a novel therapeutic strategy to neutralize A␤-induced energy failure and neuronal death. INTRODUCTIONThere is widening recognition that Alzheimer's disease (AD) is closely linked to a state of relative insulin resistance in the brain, so-called "type III diabetes" (de la Monte et al., 2006). Levels of insulin-like growth factor I (IGF-I), insulin, and/or cognate receptors are dysregulated in AD brain (Messier and Teutenberg, 2005;Moloney et al., 2008). In normal brain, IGF-I and insulin promote glucose utilization, energy metabolism, and neuronal survival (Hoyer, 2004), largely through PI3K/Akt/GSK-3␤ signaling (Bondy and Cheng, 2004). Insulin receptors (IRs) populate neuronal synapses and astrocytes in memory-processing brain regions (Lee et al., 2005). Acute insulin treatment increased memory function in rats on a passive-avoidance task (Park et al., 2000) and in small studies involving normal adults and AD patients (Craft and Watson, 2004), consistent with positive effects on synaptic plasticity (Horwood et al., 2006). How ␤-amyloid, a major culprit neurotoxin in AD, could cause central insulin resistance is unknown.Intraneuronal ␤-amyloid (A␤i) in particular, is a significant factor in the early pathogenesis of AD LaFerla et al., 2007). Inclusion body myositis (IBM), another disorder associated with intracellular ␤-amyloid (A␤) deposits, is a major cause of skeletal muscle inflammation and degeneration in the elderly. Cytoplasmic A␤ induces programmed cell death (apoptosis) in a number of experimental and transgenic models (Magrane et al., 2005;Oakley et al., 2006).Interference with or alteration of the Akt signaling pathway has emerged as an important feature in several neurodegenerative diseases characterized by neuronal attrition including AD and schizophrenia (Emamian et al., 2004;Griffin et al., 2005). In previous studies of the effects of intracellular A␤ on this pathway using primary cortical neurons as well as Tg257...

The significance of intracellular β-amyloid (Aβ 42 ) accumulation is increasingly recognized in Alzheimer's disease (AD) pathogenesis. Aβ removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody-mediated uptake by immune cells. However, the role of the ubiquitin-proteasome system (UPS) in the disposal of cellular Aβ has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinson's disease. We tested whether Parkin has crossfunction to target misfolded proteins in AD for proteasome-dependent clearance in SH-SY5Y and primary neuronal cells. Wild-type Parkin greatly decreased steady-state levels of intracellular Aβ 42 , an action abrogated by proteasome inhibitors. Intracellular Aβ 42 accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Aβ or Parkin did not account for their effects on the proteasome. Parkin knock-down led to accumulation of Aβ. In AD brain, Parkin was found to interact with Aβ and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Aβ through a proteasome-dependent pathway. Keywords amyloid; ubiquitin ligase; Parkin; Alzheimer's; proteasome Parkin is a 465-amino-acid protein containing an N-terminal ubiquitin-like (Ubl) domain linked to a C-terminal RING box (Shimura et al., 2000). Parkin functions as an E3 ubiquitin-protein ligase (Imai et al., 2000;Shimura et al., 2000; Zhang et al., 2000), facilitating the proteasomal degradation of misfolded proteins. Several Parkin gene mutations have been linked to autosomal-recessive Parkinsonism with juvenile onset (Kitada et al., 1998;Lucking et al., 2000).In cell culture systems, Parkin fusion proteins have been shown to interact with several proteins, including the α-synuclein-binding protein synphilin-1 (Chung et al., 2001) (Huynh et al., 2000), and α/β tubulin (Ren et al., 2003). Parkin has also been found to be upregulated during the integrated cellular response to misfolded protein-induced ER stress (Imai et al., 2001). Specific targets of Parkin activity having intrinsic toxic and aggregative properties include Pael-R, the Parkin-associated endothelin-like receptor (Imai et al., 2001), and possibly an O-glycosylated form of α-synuclein (Shimura et al., 2001). Thus, Parkin has been shown to suppress the toxicity of PAEL-R (Imai et al., 2001), mutated α-synuclein A30P (Petrucelli et al., 2002;Lo Bianco et al., 2004), and a poly(Q)-expanded mutant of ataxin-3 (Tsai et al., 2003). Deletions in the Parkin gene result in the accumulation of nonubiquitinated forms of α-synuclein and Pael-R in the brain (Imai et al., 2001;Shimura et al., 2001).The accumulation of neuronal β-amyloid (Aβ) is increasingly recognized as a critical factor in Alzheimer's disease (AD) and related pathologies (Hartmann, 1999;Wilson et al., 1999;Gouras et al., 2005). Solub...

Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the ␤-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked ␤-amyloid precursor protein and for its metabolic byproducts A␤42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular A␤. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed ␤-amyloid. Correspondingly, exogenous Parkin significantly lowered A␤ levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of A␤ levels. The Parkin protein is considered an E33 ubiquitin ligase and when mutated has been linked to the development of autosomal recessive Parkinson disease (1-3). In overexpression (or in cellular) models it functions to modify specific target proteins by ubiquitination, earmarking them for proteasomal degradation (3). Putative targets of Parkin include, among others, Pael-R (Parkin-associated endothelin-like receptor) (4), synphilin-1 (5), a modified form of ␣-synuclein (6), CDCrel1 (7), and p38/JTV-1, an aminoacyl-tRNA synthetase cofactor (8). Previous reports have indicated that the cellular stress response promotes the formation of a complex between Parkin, the chaperone Hsp70, the C terminus of Hsc70 interacting protein CHIP, and Pael-R (9). Recently, Parkin has also been shown to interact with the ␣4 subunit of the 19 S proteasome, although the latter does not appear to serve as a substrate for parkin-directed ubiquitination (10). Substantial accumulations of Parkin are found associated with ␣-synuclein and ubiquitin bearing Lewy bodies in Parkinson disease and dementia with Lewy bodies (11), suggesting a role in the pathologic sequestration of proteins in these disorders. Although parkin mRNA is expressed throughout the central nervous system, it is also prominently expressed in skeletal and cardiac muscle, tissues with sustained levels of protein turnover (1).Although Parkin can be found in both cytosolic and membrane-associated compartments of the cel...