Mefenamic acid shows neuroprotective effects and improves cognitive impairment in in vitro and in vivo Alzheimer's disease models

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert anti-inflammatory, analgesic, and antipyretic activities and suppress prostaglandin synthesis by inhibiting cyclooxygenase, an enzyme that catalyzes the formation of prostaglandin precursors from arachidonic acid. Epidemiological observations indicate that the long-term treatment of patients suffering from rheumatoid arthritis with NSAIDs results in reduced risk and delayed onset of Alzheimer's disease. In this study, we investigated the therapeutic potential for Alzheimer's disease of mefenamic acid, a commonly used NSAID that is a cyclooxygenase-1 and 2 inhibitor with only moderate anti-inflammatory properties. We found that mefenamic acid attenuates the neurotoxicities induced by amyloid beta peptide (Abeta)(1-42) treatment and the expression of a Swedish double mutation (KM595/596NL) of amyloid precursor protein (Swe-APP) or the C-terminal fragments of APP (APP-CTs) in neuronal cells. We also show that mefenamic acid decreases the production of the free radical nitric oxide and reduces cytochrome c release from mitochondria induced by Abeta(1-42), Swe-APP, or APP-CTs in neuronal cells. In addition, mefenamic acid up-regulates expression of the antiapoptotic protein Bcl-X(L). Moreover, our study demonstrates for the first time that mefenamic acid improves learning and memory impairment in an Abeta(1-42)-infused Alzheimer's disease rat model. Taking these in vitro and in vivo results together, our study suggests that mefenamic acid could be used as a therapeutic agent in Alzheimer's disease.     

Minocycline attenuates neuronal cell death and improves cognitive impairment in Alzheimer's disease models.

Minocycline is a semi-synthetic tetracycline antibiotic that effectively crosses the blood-brain barrier. Minocycline has been reported to have significant neuroprotective effects in models of cerebral ischemia, traumatic brain injury, amyotrophic lateral sclerosis, and Huntington's and Parkinson's diseases. In this study, we demonstrate that minocycline has neuroprotective effects in in vitro and in vivo Alzheimer's disease models. Minocycline was found to attenuate the increases in the phosphorylation of double-stranded RNA-dependent serine/threonine protein kinase, eukaryotic translation initiation factor-2 alpha and caspase 12 activation induced by amyloid beta peptide1-42 treatment in NGF-differentiated PC 12 cells. In addition, increases in the phosphorylation of eukaryotic translation initiation factor-2 alpha were attenuated by administration of minocycline in Tg2576 mice, which harbor mutated human APP695 gene including the Swedish double mutation and amyloid beta peptide(1-42)-infused rats. We found that minocycline administration attenuated deficits in learning and memory in amyloid beta peptide(1-42)-infused rats. Increased phosphorylated state of eukaryotic translation initiation factor-2 alpha is observed in Alzheimer's disease patients' brains and may result in impairment of cognitive functions in Alzheimer's disease patients by decreasing the efficacy of de novo protein synthesis required for synaptic plasticity. On the basis of these results, minocycline may prove to be a good candidate as an effective therapeutic agent for Alzheimer's disease.     

Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage

Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ(1-42), increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain.     

Involvement of heme oxygenase-1 in neuroprotection by sanguinarine against glutamate-triggered apoptosis in HT22 neuronal cells

Sanguinarine is a natural compound isolated from the roots of Macleaya cordata and M. microcarpa, has been reported to possess several biological activities such as anti-inflammatory and anti-oxidant effects. In the present study, we demonstrated that sanguinarine markedly induces the expression of HO-1 which leads to a neuroprotective response in mouse hippocampus-derived neuronal HT22 cells from apoptotic cell death induced by glutamate. Sanguinarine significantly attenuated the loss of mitochondrial function and membrane integrity associated with glutamate-induced neurotoxicity. Sanguinarine protected against glutamate-induced neurotoxicity through inhibition of HT22 cell apoptosis. JC-1 staining, which is a well-established measure of mitochondrial damage, was decreased after treatment with sanguinarine in glutamate-challenged HT22cells. In addition, sanguinarine diminished the intracellular accumulation of ROS and Ca²⁺. Sanguinarine also induced HO-1, NQO-1 expression via activation of Nrf2. Additionally, we found that si RNA mediated knock-down of Nrf2 or HO-1 significantly inhibited sanguinarine-induced neuroprotective response. These findings revealed the therapeutic potential of sanguinarine in preventing the neurodegenerative diseases.     

Senkyunolide H protects against MPP+-induced apoptosis via the ROS-mediated mitogen-activated protein kinase pathway in PC12 cells

Senkyunolide H (SNH) is a phthalide isolated from the rhizome of Ligusticum chuanxiong Hort. that has been reported to have several pharmacological activities, including anti-atherosclerotic, antiproliferative, and cytoprotective effects. In this study, we investigated the neuroprotective effects and potential mechanisms of SNH against 1-methyl-4-phenylpyridinium (MPP⁺)-induced oxidative stress. We demonstrated that SNH pretreatment significantly attenuated MPP⁺-induced neurotoxicity and apoptosis in PC12 cells. In addition, SNH attenuated the effect of MPP⁺ on the expression of the pro-apoptotic factors Bax and caspase-3. Meanwhile, SNH prevented oxidative stress by reducing reactive oxygen species generation, mitochondrial membrane potential loss, cytochrome C release, and malondialdehyde levels while increasing antioxidant enzyme activity (e.g., superoxide dismutase, catalase, and glutathione peroxidase). In addition, SNH inhibited nuclear accumulation of nuclear factor-κB and c-Jun N-terminal kinase and phosphorylation p38 mitogen-activated protein kinases (MAPKs). Overall, this investigation provides novel evidence that SNH exerts neuroprotective effects via the ROS-mediated MAPK pathway and represents a potential preventive or therapeutic agent for neuronal disorders.     

Amyloid Precursor Protein Binding Protein-1 Is Up-regulated in Brains of Tg2576 Mice

Amyloid precursor protein binding protein-1 (APP-BP1) binds to the carboxyl terminus of amyloid precursor protein and serves as a bipartite activation enzyme for the ubiquitin-like protein, NEDD8. Previously, it has been reported that APP-BP1 rescues the cell cycle S-M checkpoint defect in Ts41 hamster cells, that this rescue is dependent on the interaction of APP-BP1 with hUba3. The exogenous expression of APP-BP1 in neurons has been reported to cause DNA synthesis and apoptosis via a signaling pathway that is dependent on APP-BP1 binding to APP. These results suggest that APP-BP1 overexpression contributes to neurodegeneration. In the present study, we explored whether APP-BP1 expression was altered in the brains of Tg2576 mice, which is an animal model of Alzheimer''s disease. APP-BP1 was found to be up-regulated in the hippocampus and cortex of 12 month-old Tg2576 mice compared to age-matched wild-type mice. In addition, APP-BP1 knockdown by siRNA treatment reduced cullin-1 neddylation in fetal neural stem cells, suggesting that APP-BP1 plays a role in cell cycle progression in the cells. Collectively, these results suggest that increased expression of APP-BP1, which has a role in cell cycle progression in neuronal cells, contributes to the pathogenesis of Alzheimer''s disease.     

Reduction of aluminum ion neurotoxicity through a small peptide application – NAP treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia in late life. It is difficult to precisely diagnose AD at early stages, making biomarker search essential for further developments. The objective of this study was to identify protein biomarkers associated with aluminum ions toxicity (AD-like toxicity) in a human neuroblastoma cell model, SH-SY5Y and assess potential prevention by NAP (NAPVSIPQ). Complete proteomic techniques were implemented. Four proteins were identified as up-regulated with aluminum ion treatment, CBP80/20-dependent translation initiation factor (CTIF), Early endosome antigen 1 (EEA1), Leucine-rich repeat neuronal protein 4 (LRRN4) and Phosphatidylinositol 3-kinase regulatory subunit beta (PI3KR2). Of these four proteins, EEA1 and PI3KR2 were down-regulated after NAP-induced neuroprotective activity in neuroblastoma cells. Thus, aluminum ions may increase the risk for neurotoxicity in AD, and the use of NAP is suggested as a treatment to provide additional protection against the effects of aluminum ions, via EEA1 and PI3KR2, associated with sorting and processing of the AD amyloid precursor protein (APP) through the endosomal system.     

Novel neuroprotective effects of the aqueous extracts from Verbena officinalis Linn

Verbena officinialis Linn. (Verbenaceae) is a perennial plant which has been used as herbal medicine or health supplement in both Western and Eastern countries for centuries. It has been used to treat acute dysentery, enteritis, amenorrhea and depression. In view of its wide array of biological effects, we hypothesized that V. officinalis can exert cytoprotective effects on cells of the central nervous system. Pre-treatment of aqueous extracts of V. officinalis significantly attenuated the toxicity of beta-amyloid (Abeta) peptide and reducing agent dithiothreitol in primary cultures of cortical neurons. As extracellular accumulation of Abeta peptide is an important cytotoxic factor involved in Alzheimer's disease (AD), we have further explored its neuroprotective effect against Abeta. Treatment of V. officinalis attenuated Abeta-triggered DEVD- and VDVAD-cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of both interferon-inducing protein kinase (PKR) and c-Jun N-terminal kinase (JNK) was attenuated in Abeta-treated neurons. Taken together, we have proved our hypothesis by showing the novel neuroprotective effects of V. officinalis. As V. officinalis has long been used for many years to be a folk medicine, our study may provide a lead for its potential to be a neuroprotective agent against neuronal loss in AD.     

Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation

The amyloid precursor protein (APP) has been mainly studied in its role in the production of amyloid β peptides (Aβ), because Aβ deposition is a hallmark of Alzheimer's disease. Although several studies suggest APP has physiological functions, it is still controversial. We previously reported that APP increased glial differentiation of neural progenitor cells (NPCs). In the current study, NPCs transplanted into APP23 transgenic mice primarily differentiated into glial cells. In vitro treatment with secreted APP (sAPP) dose-dependently increased glial fibrillary acidic protein (GFAP) immuno-positive cells in NPCs and over expression of APP caused most NPCs to differentiate into GFAP immuno-positive cells. Treatment with sAPP also dose-dependently increased expression levels of GFAP in NT-2/D1 cells along with the generation of Notch intracellular domain (NICD) and expression of Hairy and enhancer of split 1 (Hes1). Treatment with γ-secretase inhibitor suppressed the generation of NICD and reduced Hes1 and GFAP expressions. Treatment with the N-terminal domain of APP (APP 1–205) was enough to induce up regulation of GFAP and Hes1 expressions, and application of 22 C11 antibodies recognizing N-terminal APP suppressed these changes by sAPP. These results indicate APP induces glial differentiation of NPCs through Notch signaling.     

Reduction of aluminum ion neurotoxicity through a small peptide application – NAP treatment of Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia in late life. It is difficult to precisely diagnose AD at early stages, making biomarker search essential for further developments. The objective of this study was to identify protein biomarkers associated with aluminum ions toxicity (AD-like toxicity) in a human neuroblastoma cell model, SH-SY5Y and assess potential prevention by NAP (NAPVSIPQ). Complete proteomic techniques were implemented. Four proteins were identified as up-regulated with aluminum ion treatment, CBP80/20-dependent translation initiation factor (CTIF), Early endosome antigen 1 (EEA1), Leucine-rich repeat neuronal protein 4 (LRRN4) and Phosphatidylinositol 3-kinase regulatory subunit beta (PI3KR2). Of these four proteins, EEA1 and PI3KR2 were down-regulated after NAP-induced neuroprotective activity in neuroblastoma cells. Thus, aluminum ions may increase the risk for neurotoxicity in AD, and the use of NAP is suggested as a treatment to provide additional protection against the effects of aluminum ions, via EEA1 and PI3KR2, associated with sorting and processing of the AD amyloid precursor protein (APP) through the endosomal system.     

The novel squamosamide derivative FLZ enhances BDNF/TrkB/CREB signaling and inhibits neuronal apoptosis in APP/PS1 mice

Aim:

The aim of this study was to study the effects of compound FLZ, a novel cyclic derivative of squamosamide from Annona glabra, on brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element-binding protein (CREB) signaling and neuronal apoptosis in the hippocampus of the amyloid precursor protein (APP)/presenilin-1 (PS1) double transgenic mice.

Methods:

APP/PS1 mice at the age of 5 months and age-matched wild-type mice (WT) were intragastrically administered FLZ (150 mg/kg) or vehicle [0.05% carboxymethyl cellulose sodium (CMC-Na)] daily for 20 weeks. The levels of BDNF in the hippocampus of WT and APP/PS1 mice were then measured by immunohistochemistry and Western blot analysis. Neuronal apoptosis in mouse hippocampus was detected by Nissl staining. Expression of NGF, NT3, pTrkB (Tyr515)/TrkB, pAkt (Ser473)/Akt, pERK/ERK, pCREB (Ser133)/CREB, Bcl-2/Bax, and active caspase-3 fragment/caspase-3 in the hippocampus of WT and APP/PS1 mice was detected by Western blot analysis.

Results:

Compared with vehicle-treated APP/PS1 mice, FLZ (150 mg/kg) significantly increased BDNF and NT3 expression in the hippocampus of APP/PS1 mice. In addition, FLZ promoted BDNF high-affinity receptor TrkB phosphorylation and activated its downstream ERK, thus increasing phosphorylation of CREB at Ser133 in the hippocampus of APP/PS1 mice. Moreover, FLZ showed neuroprotective effects on neuronal apoptosis by increasing the Bcl-2/Bax ratio and decreasing the active caspase-3 fragment/caspase-3 ratio in the hippocampus of APP/PS1 mice.

Conclusion:

FLZ exerted neuroprotection at least partly through enhancing the BDNF/TrkB/CREB pathway and inhibiting neuronal apoptosis in APP/PS1 mice, which suggests that FLZ can be explored as a potential therapeutic agent in long-term Alzheimer''s disease therapy.     

The profile of β-amyloid precursor protein expression of rats induced by aluminum

The environmental agent aluminum has been extensively investigated for a potential relationship with amyloid precursor protein (APP) expression. Despite many investigations, there is at present no definite proof from which to draw a conclusion. Since APP is an integral membrane protein expressed in different tissues and capable of fluxes across the blood-brain barrier (BBB), which may ultimately affect APP level in brain, it is necessary to assess the expression profile among vital body organs. The present study compared aluminum oxide and aluminum chloride injected rats with control rats (saline treated) to observe if aluminum affected APP expression patterns in different organs by immunohistochemistry (IHC). The expression of APP was observed in the brain of aluminum chloride treated rats and in the liver of aluminum oxide injected group. Results of double IHC staining showed that it is Kupffer cells, which are located in liver sinus and expressed APP after aluminum oxide treatment. Oxidative stress is suggested as the potential pathway that aluminum chloride exert effects in brain. These results suggest that different aluminum compounds may impact the expression of APP in brain and liver tissues. The mechanism that aluminum induced liver APP expression still needs further investigation.     

Neuroprotective effects of resveratrol against beta-amyloid-induced neurotoxicity in rat hippocampal neurons: involvement of protein kinase C

1. Resveratrol, an active ingredient of red wine extracts, has been shown to exhibit neuroprotective effects in several experimental models. 2. The present study evaluated the neuroprotective effects of resveratrol against amyloid beta(Abeta)-induced toxicity in cultured rat hippocampal cells and examined the role of the protein kinase C (PKC) pathway in this effect. 3. Pre-, co- and post-treatment with resveratrol significantly attenuated Abeta-induced cell death in a concentration-dependent manner, with a concentration of 25 microm being maximally effective. 4. Pretreatment (1 h) of hippocampal cells with phorbol-12-myristate-13-acetate, a PKC activator, at increasing concentrations (1-100 ng x ml(-1)), resulted in a dose-dependent reduction in Abeta-induced toxicity, whereas the inactive 4alpha-phorbol had no effect. 5. Pretreatment (30 min) of hippocampal cells with GF 109203X (1 microm), a general PKC inhibitor, significantly attenuated the neuroprotective effect of resveratrol against Abeta-induced cell death. 6. Treatment of hippocampal cells with resveratrol (20 microm) also induced the phosphorylation of various isoforms of PKC leading to activation. 7. Taken together, the present results indicate that PKC is involved in the neuroprotective action of resveratrol against Abeta-induced toxicity.     

FLZ attenuates learning and memory deficits via suppressing neuroinflammation induced by LPS in mice

Alzheimer's disease (AD) is the most common neurodegenerative disorder in which neuroinflammation plays an important role. FLZ is a novel synthetic derivative of natural squamosamide. Previous studies demonstrated that FLZ had neuroprotective effects on AD models and showed strong anti-inflammatory property in Parkinson's disease models. However, whether the neuroprotective effects of FLZ on AD are associated with its anti-inflammatory property is still not fully elucidated. In this study, we aimed to investigate the ability of FLZ in modulating inflammation. The results showed that FLZ significantly improved memory deficits and alleviated neuronal damage as well as neuronal loss in the hippocampus of mice intracerebroventricular injected with lipopolysaccharide (LPS). Mechanistic studies revealed that the neuroprotective effects of FLZ were due to the suppression of neuroinflammation induced by LPS, as indicated by inactivation of astrocytes and microglia, reduced production of tumor necrosis factor-α, interleukin-1β, and nitric oxide, as well as decreased expression of cyclooxygenase-2 and inducible nitric oxide synthase. The beneficial effects of FLZ on AD were further supported by the finding that FLZ attenuated β-amyloid production through inhibiting β-amyloid precursor protein cleaving enzyme 1 expression. These results suggested that anti-inflammatory agent could be useful for the treatment of AD.     

Platelets as a peripheral district where to study pathogenetic mechanisms of alzheimer disease: the case of amyloid precursor protein

Alzheimer disease is a progressive neurodegenerative disease, characterised by a progressive cognitive and memory decline. From a neuropathological point of view, Alzheimer disease is defined by the presence of characteristic lesions, i.e. mature senile plaques, neurofibrillary tangles (NFTs) and amyloid angiopathy. In particular, accumulation of the amyloid beta-peptide in the brain parenchyma and vasculature is an invariant event in the pathogenesis of both sporadic and familial Alzheimer cases. Amyloid beta-peptide originates from a larger precursor, the amyloid precursor protein (APP) ubiquitously expressed. Among the different peripheral cells expressing APP forms, platelets are particularly interesting since they show concentrations of its isoforms equivalent to those found in brain. Moreover, a number of laboratories independently described alterations in APP metabolism/concentration in platelets of Alzheimer patients when compared to control subjects matched for demographic characteristics. These observations defined the frame of our work aimed to investigate if a correlation between levels of platelet APP forms and Alzheimer disease could be detected. We have reported that patients affected by Alzheimer disease show a differential level of platelet APP forms. This observation has several implications: APP processing abnormalities, believed to be a very early change in Alzheimer disease in neuronal compartment, do occur in extraneuronal tissues, such as platelets, thus, suggesting that Alzheimer disease is a systemic disorder; further, our data strongly indicate that a differential level of platelet APP isoforms can be considered a potential peripheral marker of Alzheimer disease allowing for discrimination between Alzheimer and other types of dementia.     

The search for alpha-secretase and its potential as a therapeutic approach to Alzheimer s disease

In the nonamyloidogenic processing pathway the Alzheimer s amyloid precursor protein (APP) is proteolytically cleaved by alpha-secretase. As this cleavage occurs at the Lys16-Leu17 bond within the amyloid beta domain, it prevents deposition of intact amyloidogenic peptide. In addition, the large ectodomain (sAPP(alpha)) released by the action of alpha-secretase has several neuroprotective properties. Studies with a range of hydroxamic acid-based compounds, such as batimastat, indicate that alpha-secretase is a zinc metalloproteinase, and members of the adamalysin family of proteins, TACE, ADAM10 and ADAM9, all fulfil some of the criteria required of alpha-secretase. APP is constitutively cleaved by alpha-secretase in most cell lines. However, on stimulation with muscarinic agonists or activators of protein kinase C, such as phorbol esters, the alpha-secretase cleavage of APP is up-regulated. The constitutive alpha-secretase activity is primarily at the cell surface, while the regulated activity is predominantly located within the Golgi. The beneficial action of cholinesterase inhibitors may in part be due to activation of muscarinic receptors, resulting in an up-regulation of alpha-secretase. Other agents can also increase the nonamyloidogenic cleavage of APP including estrogen, testosterone, various neurotransmitters and growth factors. As the alpha-secretase cleavage of APP both precludes the deposition of the amyloid beta peptide and releases the neuroprotective sAPP(alpha), pharmacological up-regulation of alpha-secretase may provide alternative therapeutic approaches for Alzheimer s disease.     

The role of amyloid-beta in the regulation of memory

In this review there is evidence that amyloid-beta peptide is a memory enhancer at physiological (picomolar) concentrations. Pathological overproduction of amyloid-beta leads to impaired memory, oxidative damage, damage to the blood brain barrier, neurofibrillary tangles and amyloid plaque formation. Antisenses to amyloid precursor protein (APP) can reverse these effects in mice when they lower amyloid-beta protein to physiological levels. Data suggests that overproduction of APP leads to oxidative stress producing a vicious cycle of neuronal damage. For these reasons we have revised the “amyloid cascade hypothesis” removing emphasis from the plaque to amyloid-beta overproduction and suggest that an “amyloid-beta mitochondrial vicious cycle” hypothesis may be a better pathophysiological model for understanding Alzheimer's disease.