Christian J. Pike

Structure-activity analyses of β-amyloid peptides : contributions of the β25-35 region to aggregation and neurotoxicity

Abstract: The neurodegeneration of Alzheimers disease has been theorized to be mediated, at least in part, by insoluble aggregates of β‐amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β‐amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure‐activity analyses of a series of peptides derived from both the proposed active fragment of β‐amyloid, β25–35, and the full‐length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β‐amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β‐amyloid‐induced neurotoxicity. Further, both N‐ and C‐terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β‐sheet secondary structure in aggregating, toxic β‐amyloid peptides but not in nonaggregating, nontoxic β‐amyloid peptides. Together, these data further define the primary and secondary structures of β‐amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimers disease.

Protective actions of sex steroid hormones in Alzheimer’s disease

Risk for Alzheimers disease (AD) is associated with age-related loss of sex steroid hormones in both women and men. In post-menopausal women, the precipitous depletion of estrogens and progestogens is hypothesized to increase susceptibility to AD pathogenesis, a concept largely supported by epidemiological evidence but refuted by some clinical findings. Experimental evidence suggests that estrogens have numerous neuroprotective actions relevant to prevention of AD, in particular promotion of neuron viability and reduction of beta-amyloid accumulation, a critical factor in the initiation and progression of AD. Recent findings suggest neural responsiveness to estrogen can diminish with age, reducing neuroprotective actions of estrogen and, consequently, potentially limiting the utility of hormone therapies in aged women. In addition, estrogen neuroprotective actions are also modulated by progestogens. Specifically, continuous progestogen exposure is associated with inhibition of estrogen actions whereas cyclic delivery of progestogens may enhance neural benefits of estrogen. In recent years, emerging literature has begun to elucidate a parallel relationship of sex steroid hormones and AD risk in men. Normal age-related testosterone loss in men is associated with increased risk to several diseases including AD. Like estrogen, testosterone has been established as an endogenous neuroprotective factor that not only increases neuronal resilience against AD-related insults, but also reduces beta-amyloid accumulation. Androgen neuroprotective effects are mediated both directly by activation of androgen pathways and indirectly by aromatization to estradiol and initiation of protective estrogen signaling mechanisms. The successful use of hormone therapies in aging men and women to delay, prevent, and or treat AD will require additional research to optimize key parameters of hormone therapy and may benefit from the continuing development of selective estrogen and androgen receptor modulators.

Progesterone and Estrogen Regulate Alzheimer-Like Neuropathology in Female 3xTg-AD Mice

Estrogen depletion in postmenopausal women is a significant risk factor for the development of Alzheimers disease (AD), and estrogen-based hormone therapy may reduce this risk. However, the effects of progesterone both alone and in combination with estrogen on AD neuropathology remain unknown. In this study, we used the triple transgenic mouse model of AD (3xTg-AD) to investigate the individual and combined effects of estrogen and progesterone on β-amyloid (Aβ) accumulation, tau hyperphosphorylation, and hippocampal-dependent behavioral impairments. In gonadally intact female 3xTg-AD mice, AD-like neuropathology was apparent by 3 months of age and progressively increased through age 12 months, a time course that was paralleled by behavioral impairment. Ovariectomy-induced depletion of sex steroid hormones in adult female 3xTg-AD mice significantly increased Aβ accumulation and worsened memory performance. Treatment of ovariectomized 3xTg-AD mice with estrogen, but not progesterone, prevented these effects. When estrogen and progesterone were administered in combination, progesterone blocked the beneficial effect of estrogen on Aβ accumulation but not on behavioral performance. Interestingly, progesterone significantly reduced tau hyperphosphorylation when administered both alone and in combination with estrogen. These results demonstrate that estrogen and progesterone independently and interactively regulate AD-like neuropathology and suggest that an optimized hormone therapy may be useful in reducing the risk of AD in postmenopausal women.

Testosterone attenuates β-amyloid toxicity in cultured hippocampal neurons

Abstract Accumulating evidence suggests that testosterone has neurotrophic and perhaps neuroprotective actions. Thus, age-related depletion of testosterone may increase the brain’s vulnerability to Alzheimer’s disease and related disorders. To begin investigating this issue, cultured neurons were exposed to the Alzheimer-related insult β-amyloid in the presence of testosterone. β-Amyloid neurotoxicity was significantly reduced by testosterone via a rapid, estrogen-independent mechanism. These data may provide additional insight into the treatment of age-related neurodegenerative disorders.

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.

Brain levels of sex steroid hormones in men and women during normal aging and in Alzheimer’s disease

We examined the relationships between normal aging, Alzheimers disease (AD), and brain levels of sex steroid hormones in men and women. In postmortem brain tissue from neuropathologically normal, postmenopausal women, we found no age-related changes in brain levels of either androgens or estrogens. In comparing women with and without AD at different ages, brain levels of estrogens and androgens were lower in AD cases aged 80 years and older but not significantly different in the 60-79 year age range. In male brains, we observed that normal aging was associated with significant decreases in androgens but not estrogens. Further, in men aged 60-79 years, brain levels of testosterone but not estrogens were lower in cases with mild neuropathological changes as well as those with advanced AD neuropathology. In male cases over age 80, brain levels hormones did not significantly vary by neuropathological status. To begin investigating the relationships between hormone levels and indices of AD neuropathology, we measured brain levels of soluble β-amyloid (Aβ). In male cases with mild neuropathological changes, we found an inverse relationship between brain levels of testosterone and soluble Aβ. Collectively, these findings demonstrate sex-specific relationships between normal, age-related depletion of androgens and estrogens in men and women, which may be relevant to development of AD.

β-Amyloid induces neuritic dystrophy in vitro : similarities with Alzheimer pathology

beta-Amyloid protein, the major component of neuritic plaques found in Alzheimers disease, has been implicated as a potential contributor to the diseases progressive neuropathology. We report that within a two day exposure to aggregates of synthetic beta-amyloid peptide, the neurites of cultured rat hippocampal neurons adopt a dystrophic appearance. Observed morphological changes in the neurites include beading, fragmentation, terminal swelling and tortuous growth patterns. The degenerative changes are similar to those observed in neurites associated with neuritic plaques, suggesting that beta-amyloid may induce the neuritic abnormalities of Alzheimer neuropathology.

Gender, sex steroid hormones, and Alzheimer's disease

Age-related loss of sex steroid hormones is a established risk factor for the development of Alzheimers disease (AD) in women and men. While the relationships between the sex steroid hormones and AD are not fully understood, findings from both human and experimental paradigms indicate that depletion of estrogens in women and androgens in men increases vulnerability of the aging brain to AD pathogenesis. We review evidence of a wide range of beneficial neural actions of sex steroid hormones that may contribute to their hypothesized protective roles against AD. Both estrogens and androgens exert general neuroprotective actions relevant to a several neurodegenerative conditions, some in a sex-specific manner, including protection from neuron death and promotion of select aspects of neural plasticity. In addition, estrogens and androgens regulate key processes implicated in AD pathogenesis, in particular the accumulation of β-amyloid protein. We discuss evidence of hormone-specific mechanisms related to the regulation of the production and clearance of β-amyloid as critical protective pathways. Continued elucidation of these pathways promises to yield effective hormone-based strategies to delay development of AD.

Androgens Regulate the Development of Neuropathology in a Triple Transgenic Mouse Model of Alzheimer's Disease

Normal age-related testosterone depletion in men is a recently identified risk factor for Alzheimers disease (AD), but how androgen loss affects the development of AD is unclear. To investigate the relationship between androgen depletion and AD, we compared how androgen status affects the progression of neuropathology in the triple transgenic mouse model of AD (3xTg-AD). Adult male 3xTg-AD mice were sham gonadectomized (GDX) or GDX to deplete endogenous androgens and then exposed for 4 months to either the androgen dihydrotestosterone (DHT) or to placebo. In comparison to gonadally intact 3xTg-AD mice, GDX mice exhibited robust increases in the accumulation of β-amyloid (Aβ), the protein implicated as the primary causal factor in AD pathogenesis, in both hippocampus and amygdala. In parallel to elevated levels of Aβ, GDX mice exhibited significantly impaired spontaneous alternation behavior, indicating deficits in hippocampal function. Importantly, DHT treatment of GDX 3xTg-AD mice attenuated both Aβ accumulation and behavioral deficits. These data demonstrate that androgen depletion accelerates the development of AD-like neuropathology, suggesting that a similar mechanism may underlie the increased risk for AD in men with low testosterone. In addition, our finding that DHT protects against acceleration of AD-like neuropathology predicts that androgen-based hormone therapy may be a useful strategy for the prevention and treatment of AD in aging men.