Neuropeptide Y Protects Rat Cortical Neurons against β-Amyloid Toxicity and Re-establishes Synthesis and Release of Nerve Growth Factor

Neuropeptide Y (NPY) is a 36 amino acid peptide, widely distributed within central nervous system neurons. More recently, it has been shown that NPY is involved in Alzheimer's disease (AD), a disorder characterized by accumulation of amyloid β-peptide (Aβ) in neurons. In a previous study, we investigated the effect of NPY on neuronal damage by exposing SH-SY5Y cells (an established human derived neuroblastoma cell line) to Aβ's pathogenic fragment 25-35 (Aβ(25-35)). We found a NPY-neuroprotective action associated with changes in intracellular production of nerve growth factor (NGF), a member of the neurotrophin family. Since our results were encouraging, we decided to replicate our data using primary cortical neurons cultured in presence of Aβ(25-35), and investigated whether NPY had similar neuroprotective action. Moreover, since cortical neurons are able to produce and release NGF, we investigated whether the synthesis and release of NGF were modified in such experimental conditions. Our results showed that a preincubation with NPY counteracted the toxic effect of Aβ, as measured by increased cell viability. Moreover, NPY pretreatment had an effect on NGF since its intracellular synthesis was increased, release was normalized, and mRNA expression was downregulated. Notably, these effects on NGF were in the opposite direction of those produced by incubating the cells with Aβ alone. This study in primary cortical neurons supports the hypothesis that NPY may be a neuroprotective agent against β-amyloid neurotoxicity. These data also suggest that NPY may influence the synthesis and the release of NGF by cortical neurons.     

BODIPY-Based Molecular Probe for Imaging of Cerebral β-Amyloid Plaques

We designed and synthesized a BODIPY-based probe (BAP-1) for the imaging of β-amyloid plaques in the brain. In binding experiments in vitro, BAP-1 showed excellent affinity for synthetic Aβ aggregates. β-Amyloid plaques in Tg2576 mouse brain were clearly visualized with BAP-1. In addition, the labeling of β-amyloid plaques was demonstrated in vivo in Tg2576 mice. These results suggest BAP-1 to be a useful fluorescent probe for the optical imaging of cerebral β-amyloid plaques in patients with Alzheimer's disease.     

Selective acetylcholinesterase inhibitor activated by acetylcholinesterase releases an active chelator with neurorescuing and anti-amyloid activities

The finding that acetylcholinesterase (AChE) colocalizes with β-amyloid (Aβ) and promotes and accelerates Aβ aggregation has renewed an intense interest in developing new multifunctional AChE inhibitors as potential disease-modifying drugs for Alzheimer's therapy. To this end, we have developed a new class of selective AChE inhibitors with site-activated chelating activity. The identified lead, HLA20A, exhibits little affinity for metal (Fe, Cu, and Zn) ions but can be activated following inhibition of AChE to liberate an active chelator, HLA20. HLA20 has been shown to possess neuroprotective and neurorescuing activities in vitro and in vivo with the ability to lower amyloid precursor holoprotein (APP) expression and Aβ generation and inhibit Aβ aggregation induced by metal (Fe, Cu, and Zn) ion. HLA20A inhibited AChE in a time and concentration dependent manner with an HLA20A-AChE complex constant (K(i)) of 9.66 × 10(-6) M, a carbamylation rate (k(+2)) of 0.14 min(-1), and a second-order rate (k(i)) of 1.45 × 10 (4) M(-1) min(-1), comparable to those of rivastigmine. HLA20A showed little iron-binding capacity and activity against iron-induced lipid peroxidation (LPO) at concentrations of 1-50 μM, while HLA20 exhibited high potency in iron-binding and in inhibiting iron-induced LPO. At a concentration of 10 μM, HLA20A showed some activity against monoamine oxidase (MAO)-A and -B when tested in rat brain homogenates. Defined restrictively by Lipinski's rules, both HLA20A and HLA20 satisfied drug-like criteria and possible oral and brain permeability, but HLA20A was more lipophilic and considerably less toxic in human SHSY5Y neuroblastoma cells at high concentrations (25 or 50 μM). Together our data suggest that HLA20A may represent a promising lead for further development for Alzheimer's disease therapy.     

Olive-Oil-Derived Oleocanthal Enhances β-Amyloid Clearance as a Potential Neuroprotective Mechanism against Alzheimer’s Disease: In Vitro and in Vivo Studies

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Neurovascular function in Alzheimer's disease patients and experimental models

The ability of the brain to locally augment glucose delivery and blood flow during neuronal activation, termed neurometabolic and neurovascular coupling, respectively, is compromised in Alzheimer''s disease (AD). Since perfusion deficits may hasten clinical deterioration and have been correlated with negative treatment outcome, strategies to improve the cerebral circulation should form an integral element of AD therapeutic efforts. These efforts have yielded several experimental models, some of which constitute AD models proper, others which specifically recapture the AD cerebrovascular pathology, characterized by anatomical alterations in brain vessel structure, as well as molecular changes within vascular smooth muscle cells and endothelial cells forming the blood–brain barrier. The following paper will present the elements of AD neurovascular dysfunction and review the in vitro and in vivo model systems that have served to deepen our understanding of it. It will also critically evaluate selected groups of compounds, the FDA-approved cholinesterase inhibitors and thiazolidinediones, for their ability to correct neurovascular dysfunction in AD patients and models. These and several others are emerging as compounds with pleiotropic actions that may positively impact dysfunctional cerebrovascular, glial, and neuronal networks in AD.     

Inhibition of P2X receptor-mediated inward current by protein kinase C in small-diameter dorsal root ganglion neurons of adult rats

Objective

The aim of the present study is to verify the ATP-induced varied responses in isolated dorsal root ganglion (DRG) neurons of the adult rat, and investigate the modulatory effects of specific P2X receptor agonist β, γ-me-ATP and protein kinase C (PKC) on P2X receptor-mediated inward current in DRG neurons.

Methods

Whole cell patch-clamp was employed to record the currents on acutely isolated DRG neurons in the adult rats.

Results

β, γ-me-ATP, similar as ATP, evoked 2 distinct subtypes of P2X receptor-mediated inward currents in a dose-dependent manner in DRG neurons. Activation of PKC by phorbol 12,13-dibutyrate (PDBu) significantly inhibited both subtypes of inward currents mediated by P2X receptors in a dose-dependent manner.

Conclusion

Activation of PKC negatively modulated the P2X receptor-mediated currents in rat DRG neurons, which may be of benefit to preventing the over-excitation of nociceptor under inflammatory or neuropathic conditions.     

Point mutations in aβ induce polymorphic aggregates at liquid/solid interfaces

A pathological hallmark of Alzheimer's disease (AD), a late onset neurodegenerative disease, is the development of neuritic amyloid plaques, composed predominantly of aggregates of the β-amyloid (Aβ) peptide. It has been demonstrated that Aβ can aggregate into a variety of polymorphic aggregate structures under different chemical environments, and a potentially important environmental factor in dictating aggregate structure is the presence of surfaces. There are also several mutations clustered around the central hydrophobic core of Aβ (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology. The goal of this study was to determine how these mutations influence the morphology of Aβ aggregates under free solution conditions and at an anionic surface/liquid interface. While the rate of formation of specific aggregates was altered by mutations in Aβ under free solution conditions, the respective aggregate morphologies were similar. However, aggregation occurring directly on a negatively charged mica surface resulted in distinct aggregate morphologies formed by different mutant forms of Aβ. These studies provide insight into the potential role anionic surfaces play in dictating the formation of Aβ polymorphic aggregate structures.     

Benfotiamine prevents increased β-amyloid production in HEK cells induced by high glucose

Objective To determine whether high glucose enhances β-amyloid (Aβ) production in HEK293 Swedish mutant (APPsw) cells with Aβ precursor protein (APP) overexpression, and whether under this condition benfotiamine reduces the increased Aβ production. Methods HEK293 APPsw cells were cultured with different concentrations of glucose for different times. TheAβ content in the supernatant was determined by ELISA. To investigate the mechanism by which benfotiamine reduced Aβ production, glycogen synthase kinase-3 (GSK-3) activity and expression were measured after the cells were cultured with 5.5 g/L glucose for 12 h. Results With 1.0, 3.0, 4.5, 5.5, 6.5, 7.5, 8.5, or 10.5 g/L glucose, Aβ production by HEK293 APPsw cells was highest in the presence of 5.5 g/L glucose for 6 and 12 h. The difference in Aβ content between 5.5 and 1.0 g/L was most marked after incubation for 12 h. Benfotiamine at 20 and 40 μg/mL significantly reduced Aβ production in cells incubated with 5.5 g/L glucose for 12 h. Moreover, 40 μg/mL benfotiamine significantly enhanced the ratio of phosphorylated GSK-3 to total GSK-3, together with consistent down-regulation of GSK-3 activity. Conclusion High glucose increases Aβ production by HEK293 APPsw cells while benfotiamine prevents this increase. This is correlated with the modulation of GSK-3 activity.     

Mutant ubiquitin-mediated ��-secretase stability via activation of caspase-3 is related to ��-amyloid accumulation in ischemic striatum in rats

Previous studies have demonstrated that ischemic stroke increases β-amyloid (Aβ) production by increasing β-secretase (BACE1) through activation of caspase-3, and stimulates generation of mutant ubiquitin (UBB⁺¹) in rat brains. In this study, we examined whether caspase-3 activation participates in the regulation of UBB⁺¹ generation and UBB⁺¹-mediated BACE1 stability in ischemic injured brains. The results showed that UBB⁺¹ and activated caspase-3-immunopositive-stained cells were time dependently increased in the ipsilateral striatum of rat brains after middle cerebral artery occlusion. UBB⁺¹-immunopositive cells could be co-stained with caspase-3, Aβ (UBB⁺¹–Aβ), and BACE1 (UBB⁺¹–BACE1). BACE1 protein could also be pulled down by immunoprecipitation with UBB⁺¹ antibody. Z-DEVD-FMK (DEVD), a caspase-3 inhibitor, significantly decreased the level of UBB⁺¹ protein and the number of UBB⁺¹–Aβ and UBB⁺¹–BACE1 double-stained cells in the ischemic striatum, as well as the level of UBB⁺¹/BACE1 protein complex. We conclude that activation of caspase-3 might be upstream of UBB⁺¹ formation and that excessive UBB⁺¹ could bind to BACE1 and increase the stability of BACE1, thereby increasing Aβ in ischemic injured brains. These results suggest new biological and pathological effects of caspases and regulation of the ubiquitin–proteasome system in the brain. Our results provide new therapeutic targets to prevent further neurodegeneration in patients after stroke.     

Regulation of β cleavage of amyloid precursor protein

Alzheimer’s disease ranks the first cause for senile dementia. The amyloid cascade is proposed to contribute to the pathogenesis of this disease. In this cascade, amyloid β peptide (Aβ) is produced through a sequential cleavage of amyloid precursor protein (APP) by β and γ secretases, while its cleavage by α secretase precludes Aβ production and generates neurotrophic sAPPα. Thus, enhancing αsecretase activity or suppressing βand γcleavage may reduce A βformation and ameliorate the pathological process of the disease. Several regulatory mechanisms of APP cleavage have been established. The present review mainly summarizes the signaling pathways pertinent to the regulation of APP β cleavage.     

Genetic associations between cathepsin D exon 2 C-->T polymorphism and Alzheimer's disease, and pathological correlations with genotype

Genetic variations represent major risk factors for Alzheimer's disease (AD). While familial early onset AD is associated with mutations in the amyloid precursor protein and presenilin genes, only the e4 allele of the apolipoprotein E (APOE) gene has so far been established as a genetic risk factor for late onset familial and sporadic AD. It has been suggested that the C-->T (224Ala-->Val) transition within exon 2 of the cathepsin D gene (CTSD) might represent a risk factor for late onset AD. The objective of this study was to investigate whether possession of the CTSD exon 2 T allele increases the risk of developing AD, and to determine whether this modulates the amyloid pathology of the disease in conjunction with, or independent of, the APOE e4 allele. Blood samples were obtained from 412 patients with possible or probable AD and brain tissues from a further 148 patients with AD confirmed by postmortem examination. CTSD and APOE genotyping were performed by PCR on DNA extracted from blood, or from frontal cortex or cerebellum in the postmortem cases. Pathological measures of amyloid beta protein (Abeta), as plaque Abeta40 and Abeta42(3) load and degree of cerebral amyloid angiopathy were made by image analysis or semiquantitative rating, respectively. CTSD genotype frequencies in AD were not significantly different from those in control subjects, nor did these differ between cases of early or late onset AD or between younger and older controls. There was no gene interaction between the CTSD T and APOE e4 alleles. The amount of plaque Abeta40 was greater in patients carrying the CTSD T allele than in non-carriers, and in patients bearing APOE e4 allele compared with non-carriers. Possession of both these alleles acted synergistically to increase levels of plaque Abeta40, especially in those individuals who were homozygous for the APOE e4 allele. Possession of the CTSD T allele had no effect on plaque Abeta42(3) load or degree of CAA. Possession of the CTSD T allele does not increase the risk of developing AD per se, but has a modulating effect on the pathogenesis of the disorder by increasing, in concert with the APOE e4 allele, the amount of Abeta deposited as senile plaques in the brain in the form of Abeta40.     

Association of platelet-derived soluble glycoprotein VI in plasma with Alzheimer's disease

Accumulating evidence from epidemiological, clinical and experimental studies suggests that vascular risk factors and angiopathic mechanisms are involved in the pathogenesis of Alzheimer’s disease (AD). Platelets could be the missing link between AD and the vasculature.

Soluble glycoprotein VI (sGPVI) and β-thromboglobulin (β-TG) plasma and cerebrospinal fluid (CSF) levels as markers of platelet activity were measured in 30 AD patients and 20 age-matched healthy elderly controls by ELISA. The severity of dementia was assessed by mini-mental state examination (MMSE).

We found in AD patients significantly decreased sGPVI plasma levels (0.55 ± 0.18 ng/ml) as compared to healthy controls (0.75 ± 0.43 ng/ml; p = 0.033). In AD patients, sGPVI levels were positively correlated with β-TG plasma levels (r = 0.244, p = 0.05) and with cognitive status as measured by MMSE score (r = 0.271; p = 0.048). In unconcentrated CSF samples, levels of β-TG and sGPVI were below the detection limit of the assays in AD patients and healthy controls.

Our results suggest an association of sGPVI with the pathogenesis of AD. These findings encourage future research into whether sGPVI plasma levels may reflect or even mediate neuroprotective mechanisms in AD.     

Potential of a γ-Glutamyl-Transpeptidase-Stable Glutathione Analogue against Amyloid-β Toxicity

The antioxidant properties of glutathione (GSH) and their relevance to oxidative stress induced pathological states such as Alzheimer's disease is well-established. The utility of GSH itself as a pharmacotherapeutic agent for such disorders is limited because of the former's lability to breakdown through amide cleavage by the ubiquitous enzyme γ-glutamyl transpeptidase (γ-GT). In the present study, a GSH analogue, Ψ-GSH, where the γ-glutamylcysteine amide linkage is replaced with a ureide linkage, was synthesized. Ψ-GSH was found to be stable toward γ-GT mediated breakdown. Ψ-GSH fulfilled four cardinal properties of GSH, namely, traversing across the blood brain barrier (BBB) via the GSH active uptake machinery, replacing GSH in the glyoxalase-I mediated detoxification of methylglyoxal, protecting cells against chemical oxidative insult, and finally lowering the cytotoxicity of amyloid-β peptide. These results validate Ψ-GSH as a viable metabolically stable replacement for GSH and establish it as a potential preclinical candidate for treatment of oxidative stress mediated pathology.     

Increased serum interleukin-1beta and interleukin-6 in elderly, chronic schizophrenic patients on stable antipsychotic medication

In schizophrenia, alterations of proinflammatory cytokine levels have been reported and related to the disease and psychopathology. However, only limited conclusions can be drawn in view of confounding factors such as infection, age, sex, smoking, and antipsychotic medication. Chronic schizophrenic patients with a long-term disease process and medication period have not been investigated so far. We have measured serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)α in 41 elderly, chronic schizophrenic patients and 23 age- and sex-matched controls using enzyme-linked immunosorbent assay (ELISA). We assessed detailed psychopathology and neuropsychological performance and determined serum levels of haloperidol, clozapine, and the two main clozapine metabolites, desmethylclozapine and clozapine metabolite N-oxide, by high-pressure liquid chromatography (HPLC). IL-1β and IL-6 levels were increased in treatment-resistant schizophrenic patients compared with healthy controls, whereas TNFα showed no difference. We did not find statistically significant differences of cytokine levels between medication groups and there was no correlation with serum levels of antipsychotics or psychopathological rating scores. Elevations of IL-1β and IL-6 in elderly chronic schizophrenic patients may be related to an active disease process lasting until old age. Despite missing correlations, long-term treatment effects in treatment-resistant patients may have affected TNFα, leading to control levels. Post-mortem and animal studies should clarify the presence of altered immune function in the brain as well as the effect of cytokine levels in relation to neurodevelopmental disturbances and schizophrenia-associated behavior.     

Structure-Activity Relationships in Peptide Modulators of β-Amyloid Protein Aggregation: Variation in α,α-Disubstitution Results in Altered Aggregate Size and Morphology

Neuronal cytotoxicity observed in Alzheimer's disease (AD) is linked to the aggregation of β-amyloid peptide (Aβ) into toxic forms. Increasing evidence points to oligomeric materials as the neurotoxic species, not Aβ fibrils; disruption or inhibition of Aβ self-assembly into oligomeric or fibrillar forms remains a viable therapeutic strategy to reduce Aβ neurotoxicity. We describe the synthesis and characterization of amyloid aggregation mitigating peptides (AAMPs) whose structure is based on the Aβ "hydrophobic core" Aβ(17-20), with α,α-disubstituted amino acids (ααAAs) added into this core as potential disrupting agents of fibril self-assembly. The number, positional distribution, and side-chain functionality of ααAAs incorporated into the AAMP sequence were found to influence the resultant aggregate morphology as indicated by ex situ experiments using atomic force microscopy (AFM) and transmission electron microscopy (TEM). For instance, AAMP-5, incorporating a sterically hindered ααAA with a diisobutyl side chain in the core sequence, disrupted Aβ(1-40) fibril formation. However, AAMP-6, with a less sterically hindered ααAA with a dipropyl side chain, altered fibril morphology, producing shorter and larger sized fibrils (compared with those of Aβ(1-40)). Remarkably, ααAA-AAMPs caused disassembly of existing Aβ fibrils to produce either spherical aggregates or protofibrillar structures, suggesting the existence of equilibrium between fibrils and prefibrillar structures.     

Cerebrolysin decreases amyloid-beta production by regulating amyloid protein precursor maturation in a transgenic model of Alzheimer's disease

Cerebrolysin is a peptide mixture with neurotrophic effects that might reduce the neurodegenerative pathology in Alzheimer's disease (AD). We have previously shown in an amyloid protein precursor (APP) transgenic (tg) mouse model of AD-like neuropathology that Cerebrolysin ameliorates behavioral deficits, is neuroprotective, and decreases amyloid burden; however, the mechanisms involved are not completely clear. Cerebrolysin might reduce amyloid deposition by regulating amyloid-beta (Abeta) degradation or by modulating APP expression, maturation, or processing. To investigate these possibilities, APP tg mice were treated for 6 months with Cerebrolysin and analyzed in the water maze, followed by RNA, immunoblot, and confocal microscopy analysis of full-length (FL) APP and its fragments, beta-secretase (BACE1), and Abeta-degrading enzymes [neprilysin (Nep) and insulin-degrading enzyme (IDE)]. Consistent with previous studies, Cerebrolysin ameliorated the performance deficits in the spatial learning portion of the water maze and reduced the synaptic pathology and amyloid burden in the brains of APP tg mice. These effects were associated with reduced levels of FL APP and APP C-terminal fragments, but levels of BACE1, Notch1, Nep, and IDE were unchanged. In contrast, levels of active cyclin-dependent kinase-5 (CDK5) and glycogen synthase kinase-3beta [GSK-3beta; but not stress-activated protein kinase-1 (SAPK1)], kinases that phosphorylate APP, were reduced. Furthermore, Cerebrolysin reduced the levels of phosphorylated APP and the accumulation of APP in the neuritic processes. Taken together, these results suggest that Cerebrolysin might reduce AD-like pathology in the APP tg mice by regulating APP maturation and transport to sites where Abeta protein is generated. This study clarifies the mechanisms through which Cerebrolysin might reduce Abeta production and deposition in AD and further supports the importance of this compound in the potential treatment of early AD.     

Effect of 17beta-estradiol on olfactory bulbectomy-induced oxidative stress and behavioral changes in rats

The present study evaluated 17beta-estradiol (17betaE(2)) (2.5 mg/kg sc) effects on bilateral OBX-induced behavioral changes and oxidative stress. OBX in male Wistar rats produced an increase in lipid peroxidation products and a decline in reduced glutathione (GSH) content and glutathione peroxidase (GSH-Px) activity, together with an increase in caspase-3 activity. Additionally, OBX triggered changes of behavior such as an enhancement of immobility time in the forced swim test and hyperactivity in the open field test. These changes were reversed by treatment with 17betaE(2) (14 days). Our results reveled that 17betaE(2) has a protective effect against oxidative stress, cell damage and behavioral changes induced by OBX, and present antidepressant and antianxiety properties.     

Bcl-xl-Specific antibody labels activated microglia associated with Alzheimer's disease and other pathological states

This report describes the production of a monoclonal antibody raised against Bcl-xl, and includes an initial study of bcl-xl expression in neuropathology including Alzheimer's disease (AD). Bcl-xl is a potent apoptotic inhibitor and is known to be the predominant Bcl-x isoform in brain. To examine the expression of bcl-xl in aged brain and neurodegenerative disease, we raised a Bcl-xl-specific monoclonal antibody. In aged human brain, the highest bcl-xl expression was observed in cerebellum. By immunohistochemistry, significant bcl-xl expression was detected in reactive microglia of patients with AD and other neurological diseases such as progressive supranuclear palsy. Bcl-xl-positive microglia frequently colocalized with β-amyloid plaques in AD and with activated astrocytes in non-AD and AD brains, suggesting a general role for Bcl-xl in regions of pathology. High levels of Bcl-xl protein might render microglia more resistant to cytotoxic environments such as areas of neurodegeneration and astrogliosis. J. Neurosci. Res. 47:98–108, 1997. © 1997 Wiley-Liss, Inc.