Development of Blood-Brain Barrier Permeable Nanoparticles as Potential Carriers for Salvianolic Acid B to CNS.

The blood-brain barrier hinders the passage of systemically delivered therapeutics and the brain extracellular matrix limits the distribution and durability of locally delivered agents. Drug-loaded nanocarriers represent a promising strategy to overcome these barriers and address specific drug delivery challenges due to their small size and versatile design. We synthetized [fluorescent poly(ethyl-cyanoacrylate) nanoparticles coated with Tween 80 by an emulsion polymerization method to target and reach the brain after intravenous and intraperitoneal administration. Nanoparticles were characterized in terms of dimensional analysis, polydispersity and zeta potential (ζ-potential), morphology, encapsulation efficacy, and loading capacity. After intracerebral injection in healthy rats, nanoparticles were distributed within the injected hemisphere and mainly interacted with microglial cells, presumably involved in their clearance by phagocytosis. Furthermore, nanoparticles were able to pass the blood-brain barrier after systemic administration in rats, and the lack of toxicity in C57/B6 mice chronically administered was highlighted. The data obtained helped to clarify the nanoparticles distribution, accumulation, fate, and toxicity into the brain. The selected nanoparticles may represent a biocompatible promising carrier to be further investigated as brain delivery systems. Salvianolic acid B from Salvia miltiorrhiza is a promising molecule in the protection of degeneration in several animal models by various biological mechanisms, but its poor chemical stability and low bioavailability limits its clinical application for central nervous system neuronal injury and degeneration. Nanoparticles were loaded with salvianolic acid B obtaining an encapsulation efficacy and loading capacities of 98.70 % ± 0.45 and 53.3 % ± 0.24, respectively. They were suitable for parental administration because their mean diameter was smaller than 300 nm, with a polydispersity of 0.04 ± 0.03, and a ζ-potential of - 8.38 mV ± 3.87. The in vitro release of salvianolic acid B from the nanoparticles was sustained and prolonged during 8 h, suitable for a promising clinical application.

Oleuropein aglycone and polyphenols from olive mill waste water ameliorate cognitive deficits and neuropathology.

AIM: In TgCRND8 (Tg) mice we checked the dose-response effect of diet supplementation with oleuropein aglycone (OLE) at 12.5 or 0.5 mg kg-1 of diet. We also studied the effects of dietary intake of the mix of polyphenols present in olive mill waste water administered at a total dose as high as the highest dose of OLE (50 mg kg-1 of diet) previously investigated. METHODS: Four month-old Tg mice were equally divided into four groups and treated for 8 weeks with a modified low fat (5.0%) AIN-76 A diet (10 g day-1  per mouse) as such, supplemented with OLE (12.5 or 0.5 mg kg-1 of diet) or with a mix of polyphenols (50 mg kg-1 of diet) found in olive mill waste water. Behavioural performance was evaluated by the step down inhibitory avoidance and object recognition tests. Neuropathology was analyzed by immunohistochemistry. RESULTS: OLE supplementation at 12.5 mg kg-1 of diet and the mix of polyphenols was found to improve significantly cognitive functions of Tg mice (P < 0.0001). Aß42 and pE-3Aß plaque area and number were significantly reduced in the cortex by OLE and in the cortex and hippocampus by the mix of polyphenols (P < 0.01, P < 0.001 and P < 0.0001). Similar autophagy induction was found in the brain cortex of differently treated mice. CONCLUSION: Our results extend previous data showing that the effects of OLE on behavioural performance and neuropathology are dose-dependent and not closely related to OLE by itself. In fact, diet supplementation with the same dose of a mix of polyphenols found in olive mill waste water resulted in comparable neuroprotection.

The Polyphenol Oleuropein Aglycone Modulates the PARP1-SIRT1 Interplay: An In Vitro and In Vivo Study.

Poly(ADP-ribose) polymerase-1 (PARP1) activation contributes to the cascade of events initiated by amyloid-ß (Aß) peptide eventually leading to cell death in Alzheimer's disease brain. A significant accumulation of PAR polymers and increase of PARP1 expression were detected in the cortex at the early (3.5 months) and intermediate (6 months) stage of Aß deposition in the TgCRND8 mouse model. Our previous data highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the olive oil, against neurodegeneration both in cultured cells and in model organisms. Here we found that 8-week OLE treatment (50 mg/kg of diet) to 6-month-old TgCRND8 mice rescued to control values PARP1 activation and the levels of its product, PAR. In N2a neuroblastoma cells, PARP1 activation and PAR formation upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were abolished by pretreatment for 24 h with either OLE (100µM) or PARP inhibitors. A significant reduction of the NAD+ content, compared to controls, was found in N2a cells exposed to MNNG (100µM) for 90 min; the latter was slightly attenuated by cell treatment for 24 h with PJ-34 or with OLE. In vitro and in vivo, the OLE-induced reduction of PARP1 activation was paralleled by the overexpression of Sirtuin1 (SIRT1), and, in vivo, by a decrease of NF-κB and the pro-apoptotic marker p53. In N2a cells, we also found that OLE potentiates the MNNG-induced increase of Beclin1 levels. In conclusion, our data show that OLE treatment counteracts neuronal damage through modulation of the PARP1-SIRT1 interplay.

The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis.

Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai(-/-) mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai(+/+) mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis.

Albumin Nanoparticles for Brain Delivery: A Comparison of Chemical versus Thermal Methods and in†vivo Behavior.

Human serum albumin nanoparticles (NPs) have gained considerable attention owing to their high loading capacity for various drugs and the fact that they are well tolerated. The aim of this work was to investigate two different methods to produce NPs without the use of organic solvents and to obtain useful drug-delivery systems to cross the blood-brain barrier. NPs were obtained by coacervation, using both chemical and thermal cross-linking processes. They were developed and optimized to target brain tissues after parenteral administration in healthy rats. Furthermore, their distribution, cellular uptake, and fate were investigated in vivo after intracerebral injection in healthy rats. The toxicity of the developed carriers was estimated by behavioral tests. All NPs were chemically and physically characterized by dynamic light scattering, transmission electron microscopy, and high-performance liquid chromatography coupled with diode array and fluorimetric detectors. Their distribution and fate in the brain were evaluated by fluorescence microscopy. NPs were observed to be located in different brain tissues depending on the mode of injection, and did not induce an inflammatory response. Behavioral tests demonstrated no locomotor, explorative, or cognitive function impairment induced by the NPs.

Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight.

The healthy effects of plant polyphenols, some of which characterize the so-called Mediterranean diet, have been shown to arise from epigenetic and biological modifications resulting, among others, in autophagy stimulation. Our previous work highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the extra virgin olive oil, against neurodegeneration both in cultured cells and in model organisms, focusing, in particular, autophagy activation. In this study we investigated more in depth the molecular and cellular mechanisms of autophagy induction by OLE using cultured neuroblastoma cells and an OLE-fed mouse model of amylod beta (Aß) deposition. We found that OLE triggers autophagy in cultured cells through the Ca2+-CAMKKß-AMPK axis. In particular, in these cells OLE induces a rapid release of Ca2+ from the SR stores which, in turn, activates CAMKKß, with subsequent phosphorylation and activation of AMPK. The link between AMPK activation and mTOR inhibition was shown in the OLE-fed animal model in which we found that decreased phospho-mTOR immunoreactivity and phosphorylated mTOR substrate p70 S6K levels match enhanced phospho-AMPK levels, supporting the idea that autophagy activation by OLE proceeds through mTOR inhibition. Our results agree with those reported for other plant polyphenols, suggesting a shared molecular mechanism underlying the healthy effects of these substances against ageing, neurodegeneration, cancer, diabetes and other diseases implying autophagy dysfunction.

Oleuropein Aglycone: A Possible Drug against Degenerative Conditions. In Vivo Evidence of its Effectiveness against Alzheimer's Disease.

The amyloid plaques and neurofibrillary tangles found in the Alzheimer's disease (AD) brain arise as a result of self-assembly into fibrillar material of amyloid-ß protein (Aß) and hyperphosphorylated tau, respectively, through a pathological process starting with the appearance of aggregation nuclei and neurotoxic oligomers. Accordingly, the search of inhibitors of oligomer nucleation and growth is considered a promising target to prevent amyloid toxicity. In recent years, a number of dietary factors including antioxidants, vitamins, and polyphenols have been characterized for their ability to protect cells stressed by several factors including the presence of amyloid deposits as well as to inhibit amyloid self-assembly and cytotoxicity and some of them are currently in clinical trial. The present review summarizes the findings on the beneficial effects against neurodegeneration and other peripheral inflammatory and degenerative diseases of oleuropein aglycone (OLE), a natural phenol abundant in the extra virgin olive oil. The data presently available suggest that OLE could provide a protective and therapeutic effect against a number of pathologies, including AD as well as obesity, type 2 diabetes, non-alcoholic hepatitis, and other natural or experimentally-induced pathological conditions. Such a protection could result, at least in part, in a remarkable improvement of the pathological signs arising from stress conditions including oxidative stress, an excessive inflammatory response, and the presence of cytotoxic aggregated material. In particular, the recent data on the cellular and molecular correlates of OLE neuroprotection suggest it could also play a therapeutic role against AD.

Oleuropein aglycone protects against pyroglutamylated-3 amyloid-ß toxicity: biochemical, epigenetic and functional correlates.

Amyloid-ß (Aß) fragments, oligomeric Aß aggregates, and pyroglutamylated-Aß peptides, as well as epigenetic mechanisms and autophagy dysfunction all appear to contribute in various ways to Alzheimer's disease progression. We previously showed that dietary supplementation of oleuropein aglycone, a natural phenol abundant in the extra virgin olive oil, can be protective by reducing Aß42 deposits in the brain of young and middle-aged TgCRND8 mice. Here, we extended our study to aged TgCRND8 mice showing increased pE3-Aß in the brain deposits. We report that oleuropein aglycone is active against glutaminylcyclase-catalyzed pE3-Aß generation reducing enzyme expression and interferes both with Aß42 and pE3-Aß aggregation. Moreover, the phenol astonishingly activates neuronal autophagy even in mice at advanced stage of pathology, where it increases histone 3 and 4 acetylation, which matches both a decrease of histone deacetylase 2 expression and a significant improvement of synaptic function. The occurrence of these functional, epigenetic, and histopathologic beneficial effects even at a late stage of the pathology suggests that the phenol could be beneficial at the therapeutic, in addition to the prevention, level.

Employing Alzheimer disease animal models for translational research: focus on dietary components.

BACKGROUND: Translational research needs valid animal models of disease to discover new pathogenetic aspects and treatments. In Alzheimer's disease (AD), transgenic models are of great value for AD research and drug testing. OBJECTIVE: It was the aim of this study to analyze the power of dietary polyphenols against neurodegeneration by investigating the effects of oleuropein aglycone (OLE), the main phenol in the extra virgin olive oil (EVOO), a key component of the Mediterranean diet (MD), in a mouse model of amyloid-ß deposition. METHODS: TgCRND8 mice (3.5 months old), expressing the mutant KM670/671NL+V717F h-ßAPP695 transgene, and wild-type (wt) mice were used to study in vivo the effects of an 8-week dietary supplementation with OLE (50 mg/kg of diet) [Grossi et al: PLoS One 2013;8:e71702], following the European Communities Council Directive 86/609 (DL 116/92) and National Guidelines (permit number: 283/2012-B). RESULTS: OLE administration ameliorates memory dysfunction, raises a significant autophagic response in the cortex and promotes the proliferation of newborn cells in the subgranular zone of the dentate gyrus of the hippocampus. CONCLUSIONS: Our findings support the beneficial effects of EVOO and highlight the possibility that continuous intake of high doses of OLE, both as a nutraceutical or as a food integrator, may prevent/delay the appearance of AD and reduce the severity of its symptoms.

Oleuropein aglycone counteracts Aß42 toxicity in the rat brain.

Previous data have shown that oleuropein aglycone (OLE), the main secoiridoid phenol present in extra virgin olive oil, counteracts in vitro aggregation of the Aß42 peptide and protects cultured cells and model organisms against aggregates toxicity. In this study we investigated the relative tissue toxicity of Aß42 aggregated in vitro in the presence or in the absence of OLE by injecting the nucleus basalis magnocellularis (NBM) of adult male Wistar rats with a 1.5 µl solution containing OLE (450 µM) or Aß42 (50 µM) aggregated in the absence (oligomers) or in the presence of 450 µM OLE. Control rats were injected with vehicle (1.5 µl). Thirty days after injection, the number of choline acetyltransferase (ChAT)-positive neurons, glia reaction and the Aß peptide levels were detected by immunohistochemistry. An apparent reduction in the amount of soluble A11-positive oligomers was detected in the NBM injected with Aß42 aggregated with OLE, as compared with the NBM injected with Aß42 alone. In the latter case, the number of ChAT-positive neurons was significantly reduced (≈-33%) respect to that recorded in the NBM injected with phosphate buffer, OLE or Aß42 aggregated with OLE. A markedly attenuated Aß-induced astrocytes and microglia reaction was also found in the NBM injected with Aß42 aggregated with OLE. Altogether, these data provide additional support to the anti-aggregation, neuroprotective and anti-inflammatory activities of this natural phenol, confirming its beneficial properties against neurodegeneration.

The polyphenol oleuropein aglycone protects TgCRND8 mice against Aß plaque pathology.

The claimed beneficial effects of the Mediterranean diet include prevention of several age-related dysfunctions including neurodegenerative diseases and Alzheimer-like pathology. These effects have been related to the protection against cognitive decline associated with aging and disease by a number of polyphenols found in red wine and extra virgin olive oil. The double transgenic TgCRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 1.5 and 4, and age-matched wild type control mice were used to examine in vivo the effects of 8 weeks dietary supplementation of oleuropein aglycone (50 mg/kg of diet), the main polyphenol found in extra virgin olive oil. We report here that dietary supplementation of oleuropein aglycone strongly improves the cognitive performance of young/middle-aged TgCRND8 mice, a model of amyloid-ß deposition, respect to age-matched littermates with un-supplemented diet. Immunofluorescence analysis of cerebral tissue in oleuropein aglycone-fed transgenic mice showed remarkably reduced ß-amyloid levels and plaque deposits, which appeared less compact and "fluffy"; moreover, microglia migration to the plaques for phagocytosis and a remarkable reduction of the astrocyte reaction were evident. Finally, oleuropein aglycone-fed mice brain displayed an astonishingly intense autophagic reaction, as shown by the increase of autophagic markers expression and of lysosomal activity. Data obtained with cultured cells confirmed the latter evidence, suggesting mTOR regulation by oleuropein aglycone. Our results support, and provide mechanistic insights into, the beneficial effects against Alzheimer-associated neurodegeneration of a polyphenol enriched in the extra virgin olive oil, a major component of the Mediterranean diet.

Aß plaque-associated glial reaction as a determinant of apoptotic neuronal death and cortical gliogenesis: a study in APP mutant mice.

The purpose of this study was to investigate the microglia-driven apoptosis and the Aß deposits triggered generation of new microglial cells in the neocortex of TgCRND8 mice. Three- and seven-month-old TgCRND8 mice, displaying an early and widespread amyloid deposition, respectively, were used. In 7-month-old TgCRND8 mice the Aß-associated glial reaction was accompanied by an intense immunoreactivity of both TNF-α and inducible nitric oxide synthase, increased immunoreactivity of the pro-apoptotic protein Bax and a decrease in levels of the anti-apoptotic protein Bcl-2.Cortical and hippocampal neurons of TgCRND8 mice displayed higher immunoreactivity and higher nuclear expression of the transcription factor NF-kB than controls. It is possible that such an increase could represent a defence/compensatory response to degeneration. These findings indicate that Aß deposits activate brain-resident microglia population and astrocytes, and induce overproduction of inflammatory mediators that enhance pro- and anti-apoptotic cascades. In both 3- and 7-month-old TgCRND8 mice apparent gliogenesis was present in the vicinity of Aß plaques in the neocortex, indicating that microglia have a high proliferative rate which might play a more complex role than previously acknowledge.

Lithium improves hippocampal neurogenesis, neuropathology and cognitive functions in APP mutant mice.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of cognitive functions, extracellular ß-amyloid (Aß) plaques and intracellular neurofibrillary tangles within neocortex and hippocampus. Adult hippocampal neurogenesis plays an important role in learning and memory processes and its abnormal regulation might account for cognitive impairments associated with AD. METHODOLOGY/PRINCIPAL FINDINGS: The double transgenic (Tg) CRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 2 and 6 months, were used to examine in vivo the effects of 5 weeks lithium treatment. BrdU labelling showed a decreased neurogenesis in the subgranular zone of Tg mice compared to non-Tg mice. The decrease of hippocampal neurogenesis was accompanied by behavioural deficits and worsened with age and pathology severity. The differentiation into neurons and maturation of the proliferating cells were also markedly impaired in the Tg mice. Lithium treatment to 2-month-old Tg mice significantly stimulated the proliferation and neuron fate specification of newborn cells and fully counteracted the transgene-induced impairments of cognitive functions. The drug, by the inhibition of GSK-3ß and subsequent activation of Wnt/ß-catenin signalling promoted hippocampal neurogenesis. Finally, the data show that the lithium's ability to stimulate neurogenesis and cognitive functions was lost in the aged Tg mice, thus indicating that the lithium-induced facilitation of neurogenesis and cognitive functions declines as brain Aß deposition and pathology increases. CONCLUSIONS: Lithium, when given on time, stimulates neurogenesis and counteracts AD-like pathology.

Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease.

To investigate the role of the Wnt inhibitor Dickkopf-1 (DKK-1) in the pathophysiology of neurodegenerative diseases, we analysed DKK-1 expression and localization in transgenic mouse models expressing familial Alzheimer's disease mutations and a frontotemporal dementia mutation. A significant increase of DKK-1 expression was found in the diseased brain areas of all transgenic lines, where it co-localized with hyperphosphorylated tau-bearing neurons. In TgCRND8 mice, DKK-1 immunoreactivity was detected in neurons surrounding amyloid deposits and within the choline acetyltransferase-positive neurons of the basal forebrain. Active glycogen synthase kinase-3 (GSK-3) was found to co-localize with DKK-1 and phospho-tau staining. Downstream to GSK-3, a significant reduction in beta-catenin translocation to the nucleus, indicative of impaired Wnt signaling functions, was found as well. Cumulatively, our findings indicate that DKK-1 expression is associated with events that lead to neuronal death in neurodegenerative diseases and support a role for DKK-1 as a key mediator of neurodegeneration with therapeutic potential.

Clioquinol decreases amyloid-beta burden and reduces working memory impairment in a transgenic mouse model of Alzheimer's disease.

Clioquinol (CQ) is a "metal protein attenuating compound" that crosses the blood-brain barrier and binds, with high affinity, copper(II) and zinc(II), two metal ions critically involved in amyloid-beta aggregation and toxicity. CQ was recently proposed for the treatment of Alzheimer's disease, but controversial data have been reported so far concerning its real therapeutic advantages. We describe here results of chronic CQ treatment in the TgCRND8 mouse model of Alzheimer's disease. Remarkably, based on classical behavioral tests, CQ treatment was found to reverse, to a large extent, the working memory impairments that are characteristic of this mouse model. Pairwise, a significant reduction of amyloid-beta plaque burden, both in the cortex and in the hippocampus, was detected as well as an attenuation of astrogliosis. MALDI Mass Spectrometry Imaging technique revealed a specific localization of CQ in the above mentioned brain areas. Modest but significant effects on the absolute and relative brain concentrations of the three most important biometals (i.e., copper, zinc, and iron) were highlighted following CQ treatment. The pharmacological and mechanistic implications of the above findings are thoroughly discussed.

Combined treatment with atorvastatin and minocycline suppresses severity of EAE.

Multiple sclerosis (MS) is the most common inflammatory demyelinating disorder of the central nervous system (CNS). An approach to improve MS treatment is to identify a rational combination of new medications or existing therapies that impact different aspects of the disease process. Statins are effective in the treatment of MS animal models and are promising candidates for future treatment. Minocycline ameliorates clinical severity of experimental autoimmune encephalomyelitis (EAE) and exhibits several anti-inflammatory and neuroprotective activities. In this study, we tested whether the combination of these two drugs could produce beneficial effects in EAE mice immunized with myelin oligodendrocyte protein (MOG). Our findings show that combined treatment, compared to using the medications alone, resulted in a significant reduction in disease severity, in both the acute and chronic phases of the disease, along with attenuation of inflammation, demyelination and axonal loss. Stereological analysis revealed that the combined treatment significantly guarded against neuroinflammation and neurodegeneration. Moreover, a significant suppression of anti-MOG antibody production in animals treated with the two medications was found. In conclusion, our findings prove that this combination of drugs is neuroprotective and suppresses the severity of EAE. Furthermore, this pharmacological approach appears to be promising as a future therapeutic strategy to control MS.

Immunogenicity and therapeutic efficacy of phage-displayed beta-amyloid epitopes.

In vitro and in vivo studies indicate that Alzheimer's Disease (AD) could be prevented or treated by active immunization against self-peptide beta-amyloid. In this study, we compared the immunogenicity of different regions of beta-amyloid, displayed on filamentous phages. We established that a filamentous phage displaying epitope 2-6 (AEFRH) of beta-amyloid at the N-terminus of Major Capside Protein (phage fdAD(2-6)) is more immunogenic than a phage displaying epitope 1-7 (DAEFRHD) that differs only in flanking residues. Monthly injections of fdAD(2-6) trigger a robust anti-beta-amyloid antibody response, and afford a significant reduction of plaque pathology in a mouse model of AD, whereas the same treatment, performed with phage fdAD(1-7), induces a lower anti-beta-amyloid titer and does not protect from amyloid deposition. "Memory" anti-amyloid antibodies induced by a single prime-boost cycle with vaccine fdAD(2-6), that have a lower titer compared to antibodies induced by monthly restimulations, do not prevent plaque pathology. Our data show that optimization of epitope display is essential in vaccine design, and suggest that the titer of the anti-amyloid response is the crucial parameter to obtain therapeutic efficacy in vivo.

Abnormal processing of tau in the brain of aged TgCRND8 mice.

Amyloid plaques and neurofibrillary tangles are the main histopathological hallmarks of Alzheimer's disease (AD). In the neocortex and hippocampus of aged TgCRND8 mice, tau is hyperphosphorylated at different sites recognized by PHF-1, AT100, AT8 and CP13 antibodies. Phospho-SAPK/JNK levels were increased in the tg mouse brain, where activated SAPK/JNK co-localizes with PHF-1-positive cells. Phosphorylated tau-positive cells showed Bielschowsky- and Thioflavine S-positive intraneuronal deposits. PHF-1 and nitrotyrosine immunoreactivity merged within neurons surrounding amyloid deposits in cortical and hippocampal areas and immunoprecipitation studies confirmed that tau is nitrosylated. Our findings, demonstrating the presence of hyperphosphorylated and nitrosylated tau protein as well as of insoluble aggregates after the onset of amyloid deposition in the TgCRND8 mouse brain, indicate that the abnormal processing of tau may occur subsequently to cerebral amyloidosis and that activation of SAPK/JNK and induction of nitrosative stress are the more likely connecting factors between amyloidosis and tauopathy in AD.

Cholinergic dysfunction, neuronal damage and axonal loss in TgCRND8 mice.

In 7-month-old TgCRND8 mice, the extracellular cortical acetylcholine levels in vivo, the number and morphology of cholinergic neurons in the nucleus basalis magnocellularis and the ability to acquire an inhibitory avoidance response in the step-down test were studied. The TgCRND8 mouse brain is characterized by many beta-amyloid plaques, reduced neuronal and axonal staining, white matter demyelination, glia reaction and inducible nitric oxide synthase immunoreactivity. Choline acetyltransferase immunoreactivity in the nucleus basalis magnocellularis was significantly decreased. Basal and potassium-stimulated extracellular acetylcholine levels, investigated by microdialysis, and m2 muscarinic receptor immunoreactivity were reduced in the cortex of TgCRND8 mice, and scopolamine administration increased cortical extracellular acetylcholine levels in control but not in TgCRND8 mice. A cognitive impairment was demonstrated in the step-down test. These findings demonstrate that neuronal damage and cholinergic dysfunction in vivo underlie the impairment in learning and memory functions in this mouse model of Alzheimer's disease.