Alzheimer's disease-associated ubiquilin-1 regulates presenilin-1 accumulation and aggresome formation.

The Alzheimer's disease (AD)-associated ubiquilin-1 regulates proteasomal degradation of proteins, including presenilin (PS). PS-dependent γ-secretase generates ß-amyloid (Aß) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin-1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ-secretase complex components and subsequent γ-secretase function in human embryonic kidney 293, human neuroblastoma SH-SY5Y and mouse primary cortical cells. Full-length ubiquilin-1 TV1 and TV3 that lacks the proteasome-interaction domain increased full-length PS1 levels as well as induced accumulation of high-molecular-weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1- and TV3-expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitin-proteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aß levels, particularly in cells overexpressing TV3. These effects were not related to altered γ-secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin-1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

Acute polyglutamine expression in inducible mouse model unravels ubiquitin/proteasome system impairment and permanent recovery attributable to aggregate formation.

The presence of intracellular ubiquitylated inclusions in neurodegenerative disorders and the role of the ubiquitin/proteasome system (UPS) in degrading abnormal hazardous proteins have given rise to the hypothesis that UPS-impairment underlies neurodegenerative processes. However, this remains controversial for polyglutamine disorders such as Huntington disease (HD). Whereas studies in cellular models have provided evidence in favor of UPS-impairment attributable to expression of the N-terminal fragment of mutant huntingtin (N-mutHtt), similar studies on mouse models failed to do so. Furthermore, we have recently shown that the increase in polyubiquitin conjugates reported in the brain of N-mutHtt mice occurs in the absence of a general UPS-impairment. In the present study we aim to clarify the potential of N-mutHtt to impair UPS function in vivo as well as the mechanisms by which neurons may adapt after prolonged exposure to N-mutHtt in genetic models. By combining UPS reporter mice with an inducible mouse model of HD, we demonstrate for the first time polyglutamine-induced global UPS-impairment in vivo. UPS-impairment occurred transiently after acute N-mutHtt expression and restoration correlated with appearance of inclusion bodies (IBs). Consistently, UPS recovery did not take place when IB formation was prevented through administration of N-mutHtt aggregation-inhibitors in both cellular and animal models. Finally, no UPS-impairment was detected in old mice constitutively expressing N-mutHtt despite the age-associated decrease in brain proteasome activity. Therefore, our data reconcile previous contradictory reports by showing that N-mutHtt can indeed impair UPS function in vivo and that N-mutHtt aggregation leads to long lasting restoration of UPS function.

Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment.

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.

Chronic exposure to high levels of zinc or copper has little effect on brain metal homeostasis or Abeta accumulation in transgenic APP-C100 mice.

Aberrant metal homeostasis may enhance the formation of reactive oxygen species and Abeta oligomerization and may therefore be a contributing factor in Alzheimer's disease. This study investigated the effect of chronic high intake of dietary Zn or Cu on brain metal levels and the accumulation and solubility of Abeta in vivo, using a transgenic mouse model that over expresses the C-terminal containing Abeta fragment of human amyloid precursor protein but does not develop amyloid deposits. Exposure to chronic high Zn or Cu in the drinking water resulted in only slight elevations of the respective metals in the brain. Total Abeta levels were unchanged although soluble Abeta levels were slightly decreased, without visible plaque formation, enhanced gliosis, antioxidant upregulation or neuronal loss. This study indicates that brain metal levels are only marginally altered by long term oral exposure to extremely high Cu or Zn levels, and that this does not induce Abeta-amyloid formation in human Abeta expressing, amyloid-free mice, although this is sufficient to modulate Abeta solubility in vivo.

Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis.

In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic Ub(G76V)-GFP /SOD1G93A mice an increase in Ub(G76V)-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of Ub(G76V)-GFP was due to deficient reporter degradation. In some motor neurons the increase of Ub(G76V)-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.

Gender and genetic background effects on brain metal levels in APP transgenic and normal mice: implications for Alzheimer beta-amyloid pathology.

The incidence of Alzheimer's disease (AD) is greater in women than men at any age, as is the development of amyloid pathology in several transgenic mouse models of AD. Due to the involvement of metals in AD pathogenesis, variations between the sexes in metal metabolism may contribute to the sex difference in AD risk. In this study, we investigated sex differences in brain metal levels across the lifespan in mice of two different background strains, as well as in mice overexpressing the human amyloid precursor protein (APP) and amyloid-beta protein (Abeta). We demonstrate consistently lower Cu and higher Mn levels in females compared with males at any age studied. The sex differences in Cu and Mn levels are independent of APP/Abeta expression. AD brain exhibits decreased Cu and increased Mn levels, as do transgenic mice overexpressing APP or Abeta. The age-dependent elevations of Cu, Fe and Co levels were found to be significantly greater in mice of B6/SJL background compared with B6/DBA. If depleting Cu and/or rising Mn levels contribute to AD pathogenesis, natural sex differences in these brain metal levels may contribute to the increased propensity of females to develop AD.

Metals and amyloid-beta in Alzheimer's disease.

Mounting evidence is demonstrating roles for the amyloid precursor protein (APP) and its proteolytic product Abeta in metal homeostasis. Furthermore, aberrant metal homeostasis is observed in patients with Alzheimer's disease (AD), and this may contribute to AD pathogenesis, by enhancing the formation of reactive oxygen species and toxic Abeta oligomers and facilitating the formation of the hallmark amyloid deposits in AD brain. Indeed, zinc released from synaptic activity has been shown to induce parenchymal and cerebrovascular amyloid in transgenic mice. On the other hand, abnormal metabolism of APP and Abeta may impair brain metal homeostasis as part of the AD pathogenic process. Abeta and APP expression have both been shown to decrease brain copper (Cu) levels, whereas increasing brain Cu availability results in decreased levels of Abeta and amyloid plaque formation in transgenic mice. Lowering Cu concentrations can downregulate the transcription of APP, strengthening the hypothesis that APP and Abeta form part of the Cu homeostatic machinery in the brain. This is a complex pathway, and it appears that when the sensitive metal balance in the brain is sufficiently disrupted, it can lead to the self-perpetuating pathogenesis of AD. Clinical trials are currently studying agents that can remedy abnormal Abeta-metal interactions.

Overexpression of Alzheimer's disease amyloid-beta opposes the age-dependent elevations of brain copper and iron.

Increased brain metal levels have been associated with normal aging and a variety of diseases, including Alzheimer's disease (AD). Copper and iron levels both show marked increases with age and may adversely interact with the amyloid-beta (Abeta) peptide causing its aggregation and the production of neurotoxic hydrogen peroxide (H(2)O(2)), contributing to the pathogenesis of AD. Amyloid precursor protein (APP) possesses copper/zinc binding sites in its amino-terminal domain and in the Abeta domain. Here we demonstrate that overexpression of the carboxyl-terminal fragment of APP, containing Abeta, results in significantly reduced copper and iron levels in transgenic mouse brain, while overexpression of the APP in Tg2576 transgenic mice results in significantly reduced copper, but not iron, levels prior to the appearance of amyloid neuropathology and throughout the lifespan of the mouse. Concomitant increases in brain manganese levels were observed with both transgenic strains. These findings, complemented by our previous findings of elevated copper levels in APP knock-out mice, support roles for APP and Abeta in physiological metal regulation.

Mechanisms of peripheral microvascular dysfunction in transgenic mice overexpressing the Alzheimer's disease amyloid Abeta protein.

Freshly prepared soluble amyloid (Abeta) peptide has been reported to have vascular actions both in vitro and in vivo. This study was designed to examine the in vivo microvascular effects of beta in two skin microvascular model systems that might reflect possible short and long-term vascular effects of this peptide. Short-term vascular effects were examined using freshly prepared soluble Abeta(1-40) peptide superfused over naive rat skin microvasculature for 15 min. Peripheral microvascular functional changes in 9-months-old transgenic (Tg) mice overexpressing soluble beta in the brain, peripheral circulation and other tissues, were also examined. Microvascular responses were monitored using laser Doppler flowmetry from the base of a blister raised on the hind footpad of the animals. Endothelial-dependent and independent vasodilatation responses (VD) were examined using acetylcholine (ACh) and sodium nitroprusside (SNP) respectively. The exposure of naïve rat skin microvasculature to Abeta(1-40) resulted in an immediate vasoconstriction (VC) that prevented ACh but not SNP from inducing a subsequent VD response. The vascular effects of Abeta(1-40) were reversed by antioxidants (superoxide dismutase and catalase) and an endothelin A (ETA) receptor antagonist (BQ-123). Tg mice overexpressing soluble Abeta and C100 showed significant reductions in both endothelial-dependent and endothelial-independent VD that were also reversed by antioxidants and BQ-123. In conclusion, this study provided evidence to support the notion of peripheral vascular effects of Abeta in vivo and present novel evidence for alterations in endothelial and smooth muscle cell function in peripheral skin microvasculature in Tg mice overexpressing Abeta and C100. We suggest that skin microvasculature is a useful model to examine the mechanisms underlying the vascular actions of the Abeta protein.