Huayan Hou

Green Tea Epigallocatechin-3-Gallate (EGCG) Modulates Amyloid Precursor Protein Cleavage and Reduces Cerebral Amyloidosis in Alzheimer Transgenic Mice

Alzheimers disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of β-amyloid (Aβ) peptides as senile plaques in the brain. Recent studies suggest that green tea flavonoids may be used for the prevention and treatment of a variety of neurodegenerative diseases. Here, we report that (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, reduces Aβ generation in both murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APPsw line 2576). In concert with these observations, we find that EGCG markedly promotes cleavage of the α-C-terminal fragment of APP and elevates the N-terminal APP cleavage product, soluble APP-α. These cleavage events are associated with elevated α-secretase activity and enhanced hydrolysis of tumor necrosis factor α-converting enzyme, a primary candidate α-secretase. As a validation of these findings in vivo, we treated Tg APPsw transgenic mice overproducing Aβ with EGCG and found decreased Aβ levels and plaques associated with promotion of the nonamyloidogenic α-secretase proteolytic pathway. These data raise the possibility that EGCG dietary supplementation may provide effective prophylaxis for AD.

Stimulation of cannabinoid receptor 2 (CB2) suppresses microglial activation.

BackgroundActivated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimers disease (AD), multiple sclerosis (MS), and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2).MethodsIn this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-γ using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA) analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise Aβ1–42 peptide using a phagocytosis assay.ResultsWe found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-γ-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-γ-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-α and nitric oxide production induced either by IFN-γ or Aβ peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of Aβ1–42 peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

ADAM10 Activation Is Required for Green Tea (–)-Epigallocatechin-3-gallate-induced α-Secretase Cleavage of Amyloid Precursor Protein

Recently, we have shown that green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain Aβ levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the α-COOH-terminal fragment (α-CTF) of APP and corresponding elevation of the NH2-terminal APP product, soluble APP-α (sAPP-α). These beneficial effects were associated with increased α-secretase cleavage activity, but no significant alteration in β-or γ-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidateα-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in EGCG-induced non-amyloidogenic APP metabolism. Results show that EGCG treatment of N2a cells stably transfected with “Swedish” mutant human APP (SweAPP N2a cells) leads to markedly elevated active (∼60 kDa mature form) ADAM10 protein. Elevation of active ADAM10 correlates with increased α-CTF cleavage, and elevated sAPP-α. To specifically test the contribution of ADAM10 to non-amyloidogenic APP metabolism, small interfering RNA knockdown of ADAM9, -10, or -17 mRNA was employed. Results show that ADAM10 (but not ADAM9 or -17) is critical for EGCG-mediated α-secretase cleavage activity. In summary, ADAM10 activation is necessary for EGCG promotion of non-amyloidogenic (α-secretase cleavage) APP processing. Thus, ADAM10 represents an important pharmacotherapeutic target for the treatment of cerebral amyloidosis in Alzheimer disease.

Peripherally Administered Human Umbilical Cord Blood Cells Reduce Parenchymal and Vascular β-Amyloid Deposits in Alzheimer Mice

Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimers disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.

Flavonoid-mediated presenilin-1 phosphorylation reduces Alzheimer's disease β-amyloid production

Glycogen synthase kinase 3 (GSK‐3) dysregulation is implicated in the two Alzheimers disease (AD) pathological hallmarks: β‐amyloid plaques and neurofibrillary tangles. GSK‐3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic γ‐secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid‐β (Aβ) peptide generation in both human ‘Swedish’ mutant APP transgene‐bearing neuron‐like cells and primary neurons. We also find that luteolin induces changes consistent with GSK‐3 inhibition that (i) decrease amyloidogenic γ‐secretase APP processing, and (ii) promote presenilin‐1 (PS1) carboxyl‐terminal fragment (CTF) phosphorylation. Importantly, we find GSK‐3α activity is essential for both PS1 CTF phosphorylation and PS1‐APP interaction. As validation of these findings in vivo, we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Aβ levels, reduces GSK‐3 activity, and disrupts PS1‐APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Aβ pathology. We suggest that GSK‐3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation‐dependent regulation of amyloidogenesis.

Transcutaneous β-amyloid immunization reduces cerebral β-amyloid deposits without T cell infiltration and microhemorrhage

Alzheimers disease (AD) immunotherapy accomplished by vaccination with β-amyloid (Aβ) peptide has proved efficacious in AD mouse models. However, “active” Aβ vaccination strategies for the treatment of cerebral amyloidosis without concurrent induction of detrimental side effects are lacking. We have developed a transcutaneous (t.c.) Aβ vaccination approach and evaluated efficacy and monitored for deleterious side effects, including meningoencephalitis and microhemorrhage, in WT mice and a transgenic mouse model of AD. We demonstrate that t.c. immunization of WT mice with aggregated Aβ1–42 plus the adjuvant cholera toxin (CT) results in high-titer Aβ antibodies (mainly of the Ig G1 class) and Aβ1–42-specific splenocyte immune responses. Confocal microscopy of the t.c. immunization site revealed Langerhans cells in areas of the skin containing the Aβ1–42 immunogen, suggesting that these unique innate immune cells participate in Aβ1–42 antigen processing. To evaluate the efficacy of t.c. immunization in reducing cerebral amyloidosis, transgenic PSAPP (APPsw, PSEN1dE9) mice were immunized with aggregated Aβ1–42 peptide plus CT. Similar to WT mice, PSAPP mice showed high Aβ antibody titers. Most importantly, t.c. immunization with Aβ1–42 plus CT resulted in significant decreases in cerebral Aβ1–40,42 levels coincident with increased circulating levels of Aβ1–40,42, suggesting brain-to-blood efflux of Aβ. Reduction in cerebral amyloidosis was not associated with deleterious side effects, including brain T cell infiltration or cerebral microhemorrhage. Together, these data suggest that t.c. immunization constitutes an effective and potentially safe treatment strategy for AD.

Soluble amyloid precursor protein-α modulates β-secretase activity and amyloid-β generation

In sporadic age-related forms of Alzheimers disease (AD), it is unclear why amyloid-β (Aβ) peptides accumulate. Here we show that soluble amyloid precursor protein-α (sAPP-α) decreases Aβ generation by directly associating with β-site APP-converting enzyme (BACE)1, thereby modulating APP processing. Whereas specifically targeting sAPP-α using antibodies enhances Aβ production; in transgenic mice with AD-like pathology, sAPP-α overexpression decreases β-amyloid plaques and soluble Aβ. In support, immunoneutralization of sAPP-α increases APP amyloidogenic processing in these mice. Given our current findings, and because a number of risk factors for sporadic AD serve to lower levels of sAPP-α in brains of AD patients, inadequate sAPP-α levels may be sufficient to polarize APP processing towards the amyloidogenic, Aβ-producing route. Therefore, restoration of sAPP-α or enhancement of its association with BACE may be viable strategies to ameliorate imbalances in APP processing that can lead to AD pathogenesis.

CD45 Deficiency Drives Amyloid-β Peptide Oligomers and Neuronal Loss in Alzheimer's Disease Mice

Converging lines of evidence indicate dysregulation of the key immunoregulatory molecule CD45 (also known as leukocyte common antigen) in Alzheimers disease (AD). We report that transgenic mice overproducing amyloid-β peptide (Aβ) but deficient in CD45 (PSAPP/CD45−/− mice) faithfully recapitulate AD neuropathology. Specifically, we find increased abundance of cerebral intracellular and extracellular soluble oligomeric and insoluble Aβ, decreased plasma soluble Aβ, increased abundance of microglial neurotoxic cytokines tumor necrosis factor-α and interleukin-1β, and neuronal loss in PSAPP/CD45−/− mice compared with CD45-sufficient PSAPP littermates (bearing mutant human amyloid precursor protein and mutant human presenilin-1 transgenes). After CD45 ablation, in vitro and in vivo studies demonstrate an anti-Aβ phagocytic but proinflammatory microglial phenotype. This form of microglial activation occurs with elevated Aβ oligomers and neural injury and loss as determined by decreased ratio of anti-apoptotic Bcl-xL to proapoptotic Bax, increased activated caspase-3, mitochondrial dysfunction, and loss of cortical neurons in PSAPP/CD45−/− mice. These data show that deficiency in CD45 activity leads to brain accumulation of neurotoxic Aβ oligomers and validate CD45-mediated microglial clearance of oligomeric Aβ as a novel AD therapeutic target.

Multiple low-dose infusions of human umbilical cord blood cells improve cognitive impairments and reduce amyloid-β-associated neuropathology in Alzheimer mice.

Alzheimers disease (AD) is the most common progressive age-related dementia in the elderly and the fourth major cause of disability and mortality in that population. The disease is pathologically characterized by deposition of β-amyloid plaques neurofibrillary tangles in the brain. Current strategies for the treatment of AD are symptomatic only. As such, they are less than efficacious in terms of significantly slowing or halting the underlying pathophysiological progression of the disease. Modulation by cell therapy may be new promising disease-modifying therapy. Recently, we showed reduction in amyloid-β (Aβ) levels/β-amyloid plaques and associated astrocytosis following low-dose infusions of mononuclear human umbilical cord blood cells (HUCBCs). Our current study extended our previous findings by examining cognition via (1) the rotarod test, (2) a 2-day version of the radial-arm water maze test, and (3) a subsequent observation in an open pool platform test to characterize the effects of monthly peripheral HUCBC infusion (1×10(6) cells/μL) into the transgenic PSAPP mouse model of cerebral amyloidosis (bearing mutant human APP and presenilin-1 transgenes) from 6 to 12 months of age. We show that HUCBC therapy correlates with decreased (1) cognitive impairment, (2) Aβ levels/β-amyloid plaques, (3) amyloidogenic APP processing, and (4) reactive microgliosis after a treatment of 6 or 10 months. As such, this report lays the groundwork for an HUCBC therapy as potentially novel alternative to oppose AD at the disease-modifying level.

Baicalein reduces β‐amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer's disease transgenic mouse model

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABAA modulation for Alzheimers disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABAA antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABAA receptors.