Therapeutic Strategy For Alzheimer's Disease Treatment Research Articles

Anti-autophagic and anti-apoptotic effects of memantine in a SH-SY5Y cell model of Alzheimer's disease via mammalian target of rapamycin-dependent and -independent pathways.

Memantine non-competitively blocks the N-methyl-d-aspartate receptor in order to inhibit beta-amyloid (Aβ) secretion, and has been used to treat moderate-to-severe Alzheimer's disease (AD). However, the mechanisms underlying the role of memantine in the autophagy and apoptosis of neuronal cells in AD, as well as the association between neuronal autophagy and apoptosis have yet to be elucidated. The present study aimed to establish an AD cell model overexpressing the 695-amino-acid Swedish mutant of Aβ precursor protein (APP695swe) in order to observe the effects of memantine on the cell viability, autophagy and apoptosis of SH-SY5Y cells in the AD model, and to investigate the associated underlying mechanisms. A pcDNA3.1-APP695 plasmid was transfected into the SH-SY5Y cells. Reverse transcription-quantitative polymerase chain reaction and western blot analyses demonstrated that the AD cell model was successfully established. MTT assays demonstrated that memantine was able to upregulate neuronal cell survival, and acridine orange staining and flow cytometry demonstrated that memantine (5 µM) was able to inhibit neuronal autophagy and apoptosis. Following neuronal autophagy induction by rapamycin, cell apoptosis rates increased significantly. Further experiments revealed that memantine was able to upregulate the expression of signaling molecules phosphorylated (p)-phosphoinositide 3-kinase, p-Akt and p-mammalian target of rapamycin (mTOR), and also inhibited the phosphorylation of the B-cell lymphoma 2/Beclin-1 complex via mitogen-activated protein kinase 8. In conclusion, the results of the present study demonstrated that in the AD cell model, autophagy was able to promote apoptosis. Memantine exerted anti-autophagic and anti-apoptotic functions, and mTOR-dependent as well as-independent autophagic signaling pathways were involved in this process. Therefore, these results of the present study strongly supported the use of memantine as a potential therapeutic strategy for AD treatment.

Temsirolimus promotes autophagic clearance of amyloid-β and provides protective effects in cellular and animal models of Alzheimer's disease

Accumulation of amyloid-β peptides (Aβ) within brain is a major pathogenic hallmark of Alzheimer's disease (AD). Emerging evidence suggests that autophagy, an important intracellular catabolic process, is involved in Aβ clearance. Here, we investigated whether temsirolimus, a newly developed compound approved by Food and Drug Administration and European Medicines Agency for renal cell carcinoma treatment, would promote autophagic clearance of Aβ and thus provide protective effects in cellular and animal models of AD. HEK293 cells expressing the Swedish mutant of APP695 (HEK293-APP695) were treated with vehicle or 100nM temsirolimus for 24h in the presence or absence of 3-methyladenine (5mM) or Atg5-siRNA, and intracellular Aβ levels as well as autophagy biomarkers were measured. Meanwhile, APP/PS1 mice received intraperitoneal injection of temsirolimus (20mg/kg) every 2 days for 60 days, and brain Aβ burden, autophagy biomarkers, cellular apoptosis in hippocampus, and spatial cognitive functions were assessed. Our results showed that temsirolimus enhanced Aβ clearance in HEK293-APP695 cells and in brain of APP/PS1 mice in an autophagy-dependent manner. Meanwhile, temsirolimus attenuated cellular apoptosis in hippocampus of APP/PS1 mice, which was accompanied by an improvement in spatial learning and memory abilities. In conclusion, our study provides the first evidence that temsirolimus promotes autophagic Aβ clearance and exerts protective effects in cellular and animal models of AD, suggesting that temsirolimus administration may represent a new therapeutic strategy for AD treatment. Meanwhile, these findings emphasize the notion that many therapeutic agents possess pleiotropic actions aside from their main applications.

Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimer's disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT4) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT4 receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT4 receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT4d receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT4 receptor-induced α-secretase activation.

Se/Ru nanoparticles as inhibitors of metal-induced Aβ aggregation in Alzheimer's disease.

Amyloid β (Aβ) aggregates are considered as possible targets for therapy of Alzheimer's disease (AD). Metal ions play an important role in amyloid aggregation and neurotoxicity in the AD pathogenesis. Disruption of the interactions between these metal ions and peptides holds considerable promise as a therapeutic strategy for AD treatment. In this study, l-Cys-modified Se/Ru nanoparticles (NPs) have been designed as Aβ-binding units to inhibit metal-induced Aβ aggregation. l-Cys was used as both the reducing agent and surface modifier in the formation of SeNPs, RuNPs and Se/RuNPs. We found that RuNPs and Se/RuNPs have a strong affinity toward Aβ species and efficiently suppress extracellular Aβ40 self-assembly and Zn2+-induced fibrillization. Also, Se/RuNPs can suppress the Zn2+-Aβ40 mediated generation of reactive oxygen species (ROS) and their corresponding neurotoxicity in PC12 cells. Intriguingly, SeNPs do not have the same ability as Se/RuNPs. In addition, Se/RuNPs also decrease intracellular Aβ40 fibrillization, but this process does not involve the lysosomal pathway. These results suggest that ruthenium significantly enhances the activity of Se/RuNPs binding to Aβ40. This interaction would block the Zn2+ binding to Aβ40 peptides and lower the concentration of the free monomer, thus decreasing fibrillization. Owing to this, Se/RuNPs may represent a new strategy in AD treatment.

P1‐249: Role of BACE1 and BACE2 in the production of endogenous mouse Aß

The imbalance between Aβ production and/or clearance is hypothesized to be the major contributor to pathogenesis in Alzheimer's disease (AD). Inhibiting the sequential proteolytic cleavage of amyloid precursor protein (APP) by β- or γ-secretase blockade has been extensively pursued as a therapeutic strategy for AD treatment. The β-site APP cleavage enzyme (BACE) remains a popular target for small molecule inhibitors and antibodies because of its rate-limiting role in Aβ production. Targeting BACE1, versus its close homolog BACE2, is favored because BACE1 is highly expressed in neurons of the brain, whereas BACE2 is not. Although much is known about the role of BACE1 and BACE2 in processing human APP in in vitro and in vivo over-expression systems, less is known about their role in processing endogenous mouse APP in the brain and periphery. To determine the contribution of BACE1 and BACE2 to endogenous Aβ production in the brain and periphery, we measured brain and plasma Aβ40 levels by ELISA from BACE1ko, BACE2ko, and BACE1/2 double ko mice. Endogenous Aβ40 levels were measured in wild-type or BACE1ko mice to determine the selectivity of anti-BACE1 antibodies and small molecules against BACE1. In this study, we demonstrated that BACE1 contributes to ∼90% of endogenous brain and 50-60% of plasma Aβ40 production, whereas BACE2 does not contribute to either brain or plasma Aβ40 production. APPko mice had no detectable Aβ40 levels, demonstrating the specificity of the assay. Transient handling-induced increases in peripheral Aβ40 are mediated by BACE1, not BACE2. Anti-BACE1 antibodies are selective in inhibiting BACE1 activity, since no further reductions in Aβ40 levels were observed in BACE1 ko mice. BACE1 is the major β-secretase involved in the production of endogenous mouse Aβ in the brain and periphery, whereas BACE2 has little to no role. The remaining peripheral Aβ production in BACE1/2 ko mice suggests that there may be additional β-secretases yet to be identified.

SAPP Enhances the Transdifferentiation of Adult Bone Marrow Progenitor Cells to Neuronal Phenotypes

The remediation of neurodegeneration and cognitive decline in Alzheimer's Disease (AD) remains a challenge to basic scientists and clinicians. It has been suggested that adult bone marrow stem cells can transdifferentiate into different neuronal phenotypes. Here we demonstrate that the alpha-secretase-cleaved fragment of the amyloid precursor protein (sAPPalpha), a potent neurotrophic factor, potentiates the nerve growth factor (NGF)/retinoic acid (RA) induced transdifferentiation of bone marrow-derived adult progenitor cells (MAPCs) into neural progenitor cells and, more specifically, enhances their terminal differentiation into a cholinergic-like neuronal phenotype. The addition of sAPPalpha to NGF/RA-stimulated MAPCs resulted in their conversion to neuronal-like cells as evidenced by the extension of neurites and the appearance of immature synaptic complexes. MAPCs differentiated in the presence of sAPPalpha and NGF/RA exhibited a 40% to as much as 75% increase in neuronal proteins including NeuN, beta-tubulin III, NFM, and synaptophysin, compared to MAPCs differentiated by NGF/RA alone. This process was accompanied by an increase in the levels of choline acetyltransferase, a marker of cholinergic neurons, compared to those of GABAergic and dopaminergic neuronal subtypes. MAPCs immunopositive for sAPPalpha were identified within the septohippocampal system of transgenic PS/APP mice injected intravenously with sAPPalpha-transfected MAPCs and found in close proximity to the cerebral vasculature. Given that in AD cholinergic neurons are severely vulnerable to neurodegeneration and that the levels of sAPPalpha are significantly reduced, these findings suggest the combined use of sAPPalpha and MAPCs offers a new and potentially powerful therapeutic strategy for AD treatment.

Amyloid-beta neurotoxicity is mediated by FISH adapter protein and ADAM12 metalloprotease activity.

Based on a variety of genetic, biochemical, and neuropathological evidence, amyloid-beta peptide (Abeta) has been suggested to be causal in Alzheimer's disease (AD). Abeta has been shown to mediate neurodegenerative and inflammatory changes associated with amyloid plaques, as well as exert direct neurotoxicity through oligomeric forms of Abeta. The mechanism of Abeta toxicity, however, remains largely unknown. In this work, we show that an early event after exposure of postmitotic neurons to Abeta is tyrosine phosphorylation of FISH adapter protein. FISH binds to and potentially regulates certain ADAM family members. We present evidence that FISH and ADAM12 mediate the neurotoxic effect of Abeta. Expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks Abeta-induced neuronal death, and expression of an N-terminal fragment of FISH reduces Abeta toxicity. The C-terminal fragment of FISH containing the ADAMs binding region is found to be sufficient for induction of neuronal death, which is prevented by coexpression of the ADAM12DeltaMP. Abeta treatment, as well as expression of the C-terminal toxic FISH fragment, induces accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease/sheddase activity. ADAM12 protein is reduced in AD brains, pointing to a possible increase in ADAM12 proteolytic activity. These data suggest that Abeta toxicity is mediated by FISH and ADAM12 and may provide insights into therapeutic strategies for AD treatment.