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In AD, an imbalance between Aβ production and removal drives elevated brain Aβ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing viaα-cleavage at the plasma membrane, amyloidogenicβ- andγ-cleavage within endosomes to generate Aβ, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aβ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adenoassociated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, theα andβ C-terminal APP fragments (CTFs), and in the steady-state Aβ levels in the brain interstitial fluid. In aged mice, totalAβ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and β-CTFs, and attenuatedA β generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD. [ABSTRACT FROM AUTHOR]

In sporadic Alzheimer's disease (AD), impaired Aβ removal contributes to elevated extracellular Aβ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood- brain barrier, and cellular uptake facilitate physiologic Aβ clearance. Astrocytes can take up and degradeAβ, but it remains unclear whether this function is insufficient inADor can be enhanced to accelerate Aβ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles whereinAβ localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote Aβ uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied Aβ42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of Aβ. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced Aβ levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate Aβ removal and counter amyloid plaque pathogenesis in AD. [ABSTRACT FROM AUTHOR]

The bcl-x gene appears to play a critical role in regulating apoptosis in the developing and mature CNS and following CNS injury. Two isoforms of Bcl-x are produced as a result of alternative pre-mRNA splicing: Bcl-xL (the long form) is anti-apoptotic, while Bcl-xs (short form) is pro-apoptotic. Despite the antagonistic activities of these two isoforms, little is known about how regulation of alternative splicing of bcl-x may mediate neural cell apoptosis. Here, we report that apoptotic stimuli (staurosporine or C2-ceramide) reciprocally altered Bcl-x splicing in neural cells, decreasing Bcl-xL while increasing Bcl-xs. Specific knockdown of Bcl-xs attenuated apoptosis. To further define regulatory elements that influenced Bcl-x splicing, a Bcl-x minigene was constructed. Deletional analysis revealed several consensus sequences within intron 2 that altered splicing. We found that the splicing factor, CUG-binding-protein-1 (CUGBP1), bound to a consensus sequence close to the Bcl-xL 5′ splice site, altering the Bcl-xL/Bcl-xs ratio and influencing cell death. In vivo, neonatal hypoxia-ischemia reciprocally altered Bcl-x pre-mRNA splicing, similar to the in vitro studies. Manipulation of the splice isoforms using viral gene transfer of Bcl-xs shRNA into the hippocampus of rats before neonatal hypoxia-ischemia decreased vulnerability to injury. Moreover, alterations in nuclear CUGBP1 preceded Bcl-x splicing changes. These results suggest that alternative pre-mRNA splicing may be an important regulatory mechanism for cell death after acute neurological injury and may potentially provide novel targets for intervention. [ABSTRACT FROM AUTHOR]

Amyloid plaques are primarily composed of extracellular aggregates of amyloid-β (Aβ) peptide and are a pathological signature of Alzheimer's disease. However, the factors that influence the dynamics of amyloid plaque formation and growth in vivo are largely unknown. Using serial intravital multiphoton microscopy through a thinned-skull cranial window in APP/PS1 transgenic mice, we found that amyloid plaques appear and grow over a period of weeks before reaching a mature size. Growth was more prominent early after initial plaque formation: plaques grew faster in 6-month-old compared with 10-month-old mice. Plaque growth rate was also size-related, as smaller plaques exhibited more rapid growth relative to larger plaques. Alterations in interstitialAβ concentrations were associated with changes in plaque growth. Parallel studies using multiphoton microscopy and in vivo microdialysis revealed that pharmacological reduction of soluble extracellular Aβ by as little as 20-25% was associated with a dramatic decrease in plaque formation and growth. Furthermore, this small reduction in Aβ synthesis was sufficient to reduce amyloid plaque load in 6-month-old but not 10-month-old mice, suggesting that treatment early in disease pathogenesis may be more effective than later treatment. In contrast to thinned-skull windows, no significant plaque growth was observed under open-skull windows, which demonstrated extensive microglial and astrocytic activation. Together, these findings indicate that individual amyloid plaque growth in vivo occurs over a period of weeks and may be influenced by interstitial Aβ concentration as well as reactive gliosis. [ABSTRACT FROM AUTHOR]

Methylprednisolone (MP) is used to treat a variety of neurological disorders involving white matter injury, including multiple sclerosis, acute disseminated encephalomyelitis, and spinal cord injury (SCI). Although its mechanism of action has been attributed to antiinflammatory or antioxidant properties, we examined the possibility that MP may have direct neuroprotective activities. Neurons and oligodendrocytes treated with AMPA or staurosporine died within 24 h after treatment. MP attenuated oligodendrocyte death in a dose-dependent manner; however, neurons were not rescued by the same doses of MP. This protective effect was reversed by the glucocorticoid receptor (GR) antagonist (11, 17)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (RU486) and small interfering RNA directed against GR, suggesting a receptor-dependent mechanism. MP reversed AMPA-induced decreases in the expression of anti-apoptotic Bcl-xL, caspase-3 activation, and DNA laddering, suggesting anti-apoptotic activity in oligodendrocytes. To examine whether MP demonstrated this selective protection in vivo, neuronal and oligodendrocyte survival was assessed in rats subjected to spinal cord injury (SCI); groups of rats were treated with or without MP in the presence or absence of RU486. Eight days after SCI, MP significantly increased oligodendrocytes (CC-1-immunoreactive cells) after SCI, but neuronal (neuronal-specific nuclear protein-immunoreactive cells) number remained unchanged; RU486 reversed this protective effect. MP also inhibited SCI induced decreases in Bcl-xL and caspase-3 activation. Consistent with these findings, the volume of demyelination, assessed by Luxol fast blue staining, was attenuated by MP and reversed by RU486. These results suggest that MP selectively inhibits oligodendrocyte but not neuronal cell death via a receptor-mediated action and may be a mechanism for its limited protective effect after SCI. [ABSTRACT FROM AUTHOR]

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-βpeptide (Aβ). Although familial AD appears to be caused by Aβoverproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Aβ. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Aβdegradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Aβdegradation.Wefound that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Aβ, lowering levels and producing several fragments after incubation with synthetic human Aβ1-40 and Aβ1-42. This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Aβ were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001) increased brain interstitial fluid Aβlevels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Aβclearance by promoting Aβcatabolism. [ABSTRACT FROM AUTHOR]

Amyloid-β peptide (Aβ)-induced death in cerebral endothelial cells (CECs) is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. Mitochondria-dependent apoptosis engages Bcl-2 family proteins, especially the BH3-only homologues, which play a key role in initiating the apoptotic cascade. Here, we report that the expression of bim, but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy (CAA), suggesting a pivotal role for Bim in Aβ-induced cerebrovascular degeneration in vivo. A similar expression profile was observed in Aβ-treated CECs. Furthermore, Aβ induction of bim expression involved a pro-apoptotic transcription factor, FKHRL1. FKHRL1 bound to a consensus sequence in the bim promoter region and was activated by Aβ before bim expression. FKHRL1 activity was negatively regulated by phosphorylation catalyzed by Akt, an anti-apoptotic kinase. Akt upregulation by adenoviral gene transfer inhibited Aβ-induced FKHRL1 activation and bim induction. In addition, Aβ increased the activity of protein phosphatase 2A (PP2A), a ceramide-activated protein phosphatase. Suppression of PP2A activity by RNA interference or a specific inhibitor, okadaic acid, effectively suppressed Aβ-induced Akt inactivation and FKHRL1 activation, leading to an attenuation of bim expression and cell death in CECs. Coimmunoprecipitation experiments revealed that Aβ enhanced the binding of the PP2A regulatory subunit PP2ACαβto Akt. These results implicate PP2A as an early regulator of Aβ-induced bim expression and CEC apoptosis via the Akt/FKHRL1 signaling pathway. We raise the possibility that this pathway may play a role in cerebrovascular degeneration in CAA. [ABSTRACT FROM AUTHOR]