Thuy-Vi V. Nguyen

β-Amyloid-Induced Neuronal Apoptosis Involves c-Jun N-Terminal Kinase-Dependent Downregulation of Bcl-w

β-Amyloid protein (Aβ) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimers disease (AD). Aβ directly induces neuronal apoptosis, suggesting an important role of Aβ neurotoxicity in AD neurodegeneration. However, the mechanism(s) of Aβ-induced neuronal apoptosis remain incompletely defined. In this study, we report that Aβ-induced neuronal death is preceded by selective alterations in expression of the Bcl-2 family of apoptosis-related genes. Specifically, we observe that Aβ significantly reduces expression of antiapoptotic Bcl-w and Bcl-xL, mildly affects expression of bim, Bcl-2, and bax, but does not alter expression of bak, bad, bik, bid, or BNIP3.Aβ-induced downregulation of Bcl-w appears to contribute to the mechanism of apoptosis, because Aβ-induced neuronal death was significantly increased by Bcl-w suppression but significantly reduced by Bcl-w overexpression. Downstream of Bcl-w, Aβ-induced neuronal apoptosis is characterized by mitochondrial release of second mitochondrion-derived activator of caspase (Smac), an important precursor event to cell death. We observed that Smac release was potentiated by suppression of Bcl-w and reduced by overexpression of Bcl-w. Next, we investigated the upstream mediator of Aβ-induced Bcl-w downregulation and Smac release. We observed that Aβ rapidly activates c-Jun N-terminal kinase (JNK). Pharmacological inhibition of JNK effectively inhibited all measures of Aβ apoptosis: Bcl-w downregulation, Smac release, and neuronal death. Together, these results suggest that the mechanism of Aβ-induced neuronal apoptosis sequentially involves JNK activation, Bcl-w downregulation, and release of mitochondrial Smac, followed by cell death. Complete elucidation of the mechanism of Aβ-induced apoptosis promises to accelerate development of neuroprotective interventions for the treatment of AD.

Androgens activate mitogen-activated protein kinase signaling: role in neuroprotection.

Recent evidence indicates that testosterone is neuroprotective, however, the underlying mechanism(s) remains to be elucidated. In this study, we investigated the hypothesis that androgens induce mitogen‐activated protein kinase (MAPK) signaling in neurons, which subsequently drives neuroprotection. We observed that testosterone and its non‐aromatizable metabolite dihydrotestosterone (DHT) rapidly and transiently activate MAPK in cultured hippocampal neurons, as evidenced by phosphorylation of extracellular signal‐regulated kinase (ERK)‐1 and ERK‐2. Importantly, pharmacological suppression of MAPK/ERK signaling blocked androgen‐mediated neuroprotection against β‐amyloid toxicity. Androgen activation of MAPK/ERK and neuroprotection also was observed in PC12 cells stably transfected with androgen receptor (AR), but in neither wild‐type nor empty vector‐transfected PC12 cells. Downstream of ERK phosphorylation, we observed that DHT sequentially increases p90 kDa ribosomal S6 kinase (Rsk) phosphorylation and phosphorylation‐dependent inactivation of Bcl‐2‐associated death protein (Bad). Prevention of androgen‐induced phosphorylation of Rsk and Bad blocked androgen neuroprotection. These findings demonstrate AR‐dependent androgen activation of MAPK/ERK signaling in neurons, and specifically identify a neuroprotective pathway involving downstream activation of Rsk and inactivation of Bad. Elucidation of androgen‐mediated neural signaling cascades will provide important insights into the mechanisms of androgen action in brain, and may present a framework for therapeutic intervention of age‐related neurodegenerative disorders.

The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo.

Oligomeric forms of amyloid-β (Aβ) are thought to play a causal role in Alzheimers disease (AD), and the p75 neurotrophin receptor (p75NTR) has been implicated in Aβ-induced neurodegeneration. To further define the functions of p75NTR in AD, we examined the interaction of oligomeric Aβ(1-42) with p75NTR, and the effects of that interaction on neurite integrity in neuron cultures and in a chronic AD mouse model. Atomic force microscopy was used to ascertain the aggregated state of Aβ, and fluorescence resonance energy transfer analysis revealed that Aβ oligomers interact with the extracellular domain of p75NTR. In vitro studies of Aβ-induced death in neuron cultures isolated from wild-type and p75NTR−/− mice, in which the p75NTR extracellular domain is deleted, showed reduced sensitivity of mutant cells to Aβ-induced cell death. Interestingly, Aβ-induced neuritic dystrophy and activation of c-Jun, a known mediator of Aβ-induced deleterious signaling, were completely prevented in p75NTR−/− neuron cultures. Thy1-hAPPLond/Swe × p75NTR−/− mice exhibited significantly diminished hippocampal neuritic dystrophy and complete reversal of basal forebrain cholinergic neurite degeneration relative to those expressing wild-type p75NTR. Aβ levels were not affected, suggesting that removal of p75NTR extracellular domain reduced the ability of excess Aβ to promote neuritic degeneration. These findings indicate that although p75NTR likely does not mediate all Aβ effects, it does play a significant role in enabling Aβ-induced neurodegeneration in vitro and in vivo, establishing p75NTR as an important therapeutic target for AD.

Estrogen Regulates Bcl-w and Bim Expression: Role in Protection against β-Amyloid Peptide-Induced Neuronal Death

Estrogen is neuroprotective against a variety of insults, including β-amyloid peptide (Aβ); however, the underlying mechanism(s) is not fully understood. Here, we report that 17β-estradiol (E2) selectively regulates neuronal expression of the Bcl-2 family (bcl-2, bcl-x, bcl-w, bax, bak, bad, bik, bnip3, bid, and bim). In primary cerebrocortical neuron cultures under basal conditions, we observe that E2 upregulates expression of antiapoptotic Bcl-w and downregulates expression of proapoptotic Bim in an estrogen receptor (ER)-dependent manner. In the presence of toxic levels of Aβ, we observe that E2 attenuates indices of neuronal apoptosis: c-Jun N-terminal kinase (JNK)-dependent downregulation of Bcl-w and upregulation of Bim, mitochondrial release of cytochrome c and Smac, and cell death. These neuroprotective effects of E2 against Aβ-induced apoptosis are mimicked by the JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). In addition, E2 attenuates Aβ-induced JNK phosphorylation in an ER-dependent manner, but does not affect basal levels of JNK phosphorylation. These results suggest that E2 may reduce Aβ-induced neuronal apoptosis at least in part by two complementary pathways: (1) ER-dependent, JNK-independent upregulation of Bcl-w and downregulation of Bim under basal conditions, and (2) ER-dependent inhibition of Aβ-induced JNK activation and subsequent JNK-dependent downregulation of Bcl-w and upregulation of Bim, resulting in mitochondrial release of cytochrome c and Smac and eventual cell death. These data provide new understanding into the mechanisms contributing to estrogen neuroprotection, a neural function with potential therapeutic relevance to Alzheimers disease.

Androgen cell signaling pathways involved in neuroprotective actions

As a normal consequence of aging in men, testosterone levels significantly decline in both serum and brain. Age-related testosterone depletion results in increased risk of dysfunction and disease in androgen-responsive tissues, including brain. Recent evidence indicates that one deleterious effect of age-related testosterone loss in men is increased risk for Alzheimers disease (AD). We discuss recent findings from our laboratory and others that identify androgen actions implicated in protecting the brain against neurodegenerative diseases and begin to define androgen cell signaling pathways that underlie these protective effects. Specifically, we focus on the roles of androgens as (1) endogenous negative regulators of beta-amyloid accumulation, a key event in AD pathogenesis, and (2) neuroprotective factors that utilize rapid non-genomic signaling to inhibit neuronal apoptosis. Continued elucidation of cell signaling pathways that contribute to protective actions of androgens should facilitate the development of targeted therapeutic strategies to combat AD and other age-related neurodegenerative diseases.

B-Lymphocyte-Mediated Delayed Cognitive Impairment following Stroke

Each year, 10 million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have more than twice the risk of subsequently developing dementia compared with people who have never had a stroke. The link between stroke and the later development of dementia is not understood. There are reports of oligoclonal bands in the CSF of stroke patients, suggesting that in some people a B-lymphocyte response to stroke may occur in the CNS. Therefore, we tested the hypothesis that a B-lymphocyte response to stroke could contribute to the onset of dementia. We discovered that, in mouse models, activated B-lymphocytes infiltrate infarcted tissue in the weeks after stroke. B-lymphocytes undergo isotype switching, and IgM, IgG, and IgA antibodies are found in the neuropil adjacent to the lesion. Concurrently, mice develop delayed deficits in LTP and cognition. Genetic deficiency, and the pharmacologic ablation of B-lymphocytes using an anti-CD20 antibody, prevents the appearance of delayed cognitive deficits. Furthermore, immunostaining of human postmortem tissue revealed that a B-lymphocyte response to stroke also occurs in the brain of some people with stroke and dementia. These data suggest that some stroke patients may develop a B-lymphocyte response to stroke that contributes to dementia, and is potentially treatable with FDA-approved drugs that target B cells.

Signal transduction in Alzheimer disease : p21-activated kinase signaling requires C-terminal cleavage of APP at Asp664

The deficits in Alzheimer disease (AD) stem at least partly from neurotoxic β‐amyloid peptides generated from the amyloid precursor protein (APP). APP may also be cleaved intracellularly at Asp664 to yield a second neurotoxic peptide, C31. Previously, we showed that cleavage of APP at the C‐terminus is required for the impairments seen in APP transgenic mice, by comparing elements of the disease in animals modeling AD, with (platelet‐derived growth factor B‐chain promoter‐driven APP transgenic mice; PDAPP) versus without (PDAPP D664A) a functional Asp664 caspase cleavage site. However, the signaling mechanism(s) by which Asp664 contributes to these deficits remains to be elucidated. In this study, we identify a kinase protein, recently shown to bind APP at the C‐terminus and to contribute to AD, whose activity is modified in PDAPP mice, but normalized in PDAPP D664A mice. Specifically, we observed a significant increase in nuclear p21‐activated kinase (isoforms 1, 2, and or 3; PAK‐1/2/3) activation in hippocampus of 3 month old PDAPP mice compared with non‐transgenic littermates, an effect completely prevented in PDAPP D664A mice. In contrast, 13 month old PDAPP mice displayed a significant decrease in PAK‐1/2/3 activity, which was once again absent in PDAPP D664A mice. Similarly, in hippocampus of early and severe AD subjects, there was a progressive and subcellular‐specific reduction in active PAK‐1/2/3 compared with normal controls. Interestingly, total PAK‐1/2/3 protein was increased in early AD subjects, but declined in moderate AD and declined further, to significantly below that of control levels, in severe AD. These findings are compatible with previous suggestions that PAK may be involved in the pathophysiology of AD, and demonstrate that both early activation and late inactivation in the murine AD model require the cleavage of APP at Asp664.

Androgens, aging, and Alzheimer's disease

Testoterone depletion is a normal consequence of aging in men that is associated with senescent effects in androgen-responsive tissues. We discuss new evidence that one consequence of testosterone depletion in men is an increased risk for the development of Alzheimers disease (AD). Furthermore, we discuss two candidate mechanisms by which test osterone may affect AD pathogenesis. First, testosterone has been identified as an endogenous regulator of β-amyloid, a protein that abnormally accumulates in AD brain and is implicated as a causal factor in the disease. Second, findings from several different paradigms indicate that testosterone has both neurotrophic and neuroprotective functions. These new findings support the clinical evaluation of androgen-based therapies for the prevention and treatment of AD.

Androgens regulate neprilysin expression: role in reducing β‐amyloid levels

Age‐related testosterone depletion in men is a risk factor for Alzheimer’s disease. Prior studies suggest that androgens affect Alzheimer’s disease risk by regulating accumulation of β‐amyloid protein (Aβ) by an undefined mechanism. In this study, we investigated the role of the Aβ‐catabolizing enzyme neprilysin (NEP) in this process. First, we observed that androgens positively regulate neural expression of NEP in adult male rats. Next, we investigated androgen regulatory effects on both NEP expression and Aβ levels using cultured hippocampal neurons and neuronally differentiated rat pheochromocytoma cell 12 with or without androgen receptor (AR). Dihydrotestosterone (DHT) induced a time‐dependent increase in NEP expression. DHT also significantly decreased levels of Aβ in AR‐expressing cells transfected with amyloid precursor protein, but did not affect levels of either full‐length or non‐amyloidogenic, soluble amyloid precursor protein. Importantly, the DHT induced decrease of Aβ was blocked by pharmacological inhibition of NEP. The DHT‐mediated increase in NEP expression and decrease in Aβ levels were (i) not observed in rat pheochromocytoma cell 12 lacking AR and (ii) blocked in AR‐expressing cells by the antagonists, cyproterone acetate and flutamide. Together, these findings suggest that androgen regulation of Aβ involves an AR‐dependent mechanism requiring up‐regulation of the Aβ catabolizing enzyme NEP.

Thy1-hAPPLond/Swe+ mouse model of Alzheimer's disease displays broad behavioral deficits in sensorimotor, cognitive and social function

Alzheimer’s disease (AD), the most common form of dementia, is an age‐dependent progressive neurodegenerative disorder. β‐amyloid, a metabolic product of the amyloid precursor protein (APP), plays an important role in the pathogenesis of AD. The Thy1‐hAPPLond/Swe+ (line 41) transgenic mouse overexpresses human APP751 and contains the London (V717I) and Swedish (K670M/N671L) mutations. Here, we used a battery of behavioral tests to evaluate general activity, cognition, and social behavior in six‐month‐old male Thy1‐hAPPLond/Swe+ mice. We found hyperactivity in a novel environment as well as significant deficits in spontaneous alternation behavior. In fear conditioning (FC), Thy1‐hAPPLond/Swe+ mice did not display deficits in acquisition or in memory retrieval in novel context of tone‐cued FC, but they showed significant memory retrieval impairment during contextual testing in an identical environment. Surprisingly, in a standard hidden platform water maze, no significant deficit was detected in mutant mice. However, a delayed‐matching‐to‐place paradigm revealed a significant deficit in Thy1‐hAPPLond/Swe+ mice. Lastly, in the social novelty session of a three‐chamber test, Thy1‐hAPPLond/Swe+ mice exhibited a significantly decreased interest in a novel versus a familiar stranger compared to control mice. This could possibly be explained by decreased social memory or discrimination and may parallel disturbances in social functioning in human AD patients. In conclusion, the Thy1‐hAPPLond/Swe+ mouse model of AD displayed a behavioral phenotype that resembles, in part, the cognitive and psychiatric symptoms experienced in AD patients.