Simvastatin therapy prevents brain trauma‐induced increases in β‐amyloid peptide levels

Elevations in β‐amyloid peptide (Aβ) levels after traumatic brain injury (TBI) may confer risk for developing Alzheimer's disease in head trauma patients. We investigated the effects of simvastatin, a 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor, on hippocampal Aβ burden in a clinically relevant head injury/intervention model using mice expressing human Aβ. Simvastatin therapy blunted TBI‐induced increases in Aβ, reduced hippocampal tissue damage and microglial activation, and improved behavioral outcome. The ability of statins to reduce post‐injury Aβ load and ameliorate pathological sequelae of brain injury makes them potentially effective in reducing the risk of developing Alzheimer's disease in TBI patients. Ann Neurol 2009;66:407–414     

Human tau increases amyloid β plaque size but not amyloid β‐mediated synapse loss in a novel mouse model of Alzheimer's disease

Alzheimer's disease is characterized by the presence of aggregates of amyloid beta (Aβ) in senile plaques and tau in neurofibrillary tangles, as well as marked neuron and synapse loss. Of these pathological changes, synapse loss correlates most strongly with cognitive decline. Synapse loss occurs prominently around plaques due to accumulations of oligomeric Aβ. Recent evidence suggests that tau may also play a role in synapse loss but the interactions of Aβ and tau in synapse loss remain to be determined. In this study, we generated a novel transgenic mouse line, the APP/PS1/rTg21221 line, by crossing APP/PS1 mice, which develop Aβ‐plaques and synapse loss, with rTg21221 mice, which overexpress wild‐type human tau. When compared to the APP/PS1 mice without human tau, the cross‐sectional area of ThioS+ dense core plaques was increased by ~50%. Along with increased plaque size, we observed an increase in plaque‐associated dystrophic neurites containing misfolded tau, but there was no exacerbation of neurite curvature or local neuron loss around plaques. Array tomography analysis similarly revealed no worsening of synapse loss around plaques, and no change in the accumulation of Aβ at synapses. Together, these results indicate that adding human wild‐type tau exacerbates plaque pathology and neurite deformation but does not exacerbate plaque‐associated synapse loss.     

CSF levels of β‐amyloid 1‐42, tau and phosphorylated tau protein in CADASIL

Background and purpose: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) can be considered a useful model of pure subcortical vascular dementia (SVD) because it occurs in young adults, unlikely to have concomitant age‐ and Alzheimer’s disease (AD)‐related pathology. In patients with CADASIL we evaluated the cerebrospinal fluid (CSF) levels of β‐amyloid 1‐42 (Aβ42), total tau protein (t‐tau) and phosphorylated tau protein (p‐tau), which are well‐accepted biomarkers of AD. Methods: The CSF Aβ42, t‐tau and p‐tau levels were determined with Innotest β‐amyloid 1‐42, Innotest hTAU‐Ag and Innotest Phospho‐tau 181p sandwich enzyme‐linked immunoassay, in 10 CADASIL patients and 17 healthy age‐matched subjects. A case–control statistical analysis was carried out. Results: CSF Aβ42 levels were significantly lower in CADASIL patients than in controls, whereas CSF t‐tau and p‐tau levels did not differ between the two groups. Conclusions: The pattern found in CADASIL patients is similar to that reported in those with sporadic SVD, suggesting that decreased CSF Aβ42 might be related to the subcortical vascular lesions in the white matter.     

Effects of antiparkinsonian agents on β‐amyloid and α‐synuclein oligomer formation in vitro

The aggregation of β‐amyloid protein (Aβ) and α‐synuclein (αS) are hypothesized to be the key pathogenic event in Alzheimer's disease (AD) and Lewy body diseases (LBD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD and LBD. Here, we examined the effects of antiparkinsonian agents (dopamine, levodopa, trihexyphenidyl, selegiline, zonisamide, bromocriptine, peroxide, ropinirole, pramipexole, and entacapone) on the in vitro oligomer formation of Aβ40, Aβ42, and αS using a method of photo‐induced cross‐linking of unmodified proteins (PICUP), electron microscopy, and atomic force microscopy. The antiparkinsonian agents except for trihexyphenidyl inhibited both Aβ and αS oligomer formations, and, among them, dopamine, levodopa, pramipexole, and entacapone had the stronger in vitro activity. Circular dichroism and thioflavin T(S) assays showed that secondary structures of Aβ and αS assemblies inhibited by antiparkinsonian agents were statistical coil state and that their seeding activities had disappeared. The antiparkinsonian agents could be potential therapeutic agents to prevent or delay AD and LBD progression. © 2013 Wiley Periodicals, Inc.     

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

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABA_A modulation for Alzheimer's disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABA_A antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABA_A receptors. © 2013 Wiley Periodicals, Inc.     

Cannabinoids prevent the amyloid β‐induced activation of astroglial hemichannels: A neuroprotective mechanism

The mechanisms involved in Alzheimer's disease are not completely understood and how astrocytes and their gliotransmission contribute to this neurodegenerative disease remains to be fully elucidated. Previous studies have shown that amyloid‐β peptide (Aβ) induces neuronal death by a mechanism that involves the excitotoxic release of ATP and glutamate associated to astroglial hemichannel opening. We have demonstrated that synthetic and endogenous cannabinoids (CBs) reduce the opening of astrocyte Cx43 hemichannels evoked by activated microglia or inflammatory mediators. Nevertheless, whether CBs could prevent the astroglial hemichannel‐dependent death of neurons evoked by Aβ is unknown. Astrocytes as well as acute hippocampal slices were treated with the active fragment of Aβ alone or in combination with the following CBs: WIN, 2‐AG, or methanandamide (Meth). Hemichannel activity was monitored by single channel recordings and by time‐lapse ethidium uptake while neuronal death was assessed by Fluoro‐Jade C staining. We report that CBs fully prevented the hemichannel activity and inflammatory profile evoked by Aβ in astrocytes. Moreover, CBs fully abolished the Aβ‐induced release of excitotoxic glutamate and ATP associated to astrocyte Cx43 hemichannel activity, as well as neuronal damage in hippocampal slices exposed to Aβ. Consequently, this work opens novel avenues for alternative treatments that target astrocytes to maintain neuronal function and survival during AD. GLIA 2016 GLIA 2017;65:122–137     

Oligomeric α‐synuclein and β‐amyloid variants as potential biomarkers for Parkinson's and Alzheimer's diseases

Oligomeric forms of α‐synuclein and β‐amyloid are toxic protein variants that are thought to contribute to the onset and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. The detection of toxic variants in human cerebrospinal fluid (CSF) and blood has great promise for facilitating early and accurate diagnoses of these devastating diseases. Two hurdles that have impeded the use of these protein variants as biomarkers are the availability of reagents that can bind the different variants and a sensitive assay to detect their very low concentrations. We previously isolated antibody‐based reagents that selectively bind two different oligomeric variants of α‐synuclein and two of β‐amyloid, and developed a phage‐based capture enzyme‐linked immunosorbent assay (ELISA) with subfemtomolar sensitivity to quantify their presence. Here, we used these reagents to show that these oligomeric α‐synuclein variants are preferentially present in PD brain tissue, CSF and serum, and that the oligomeric β‐amyloid variants are preferentially present in AD brain tissue, CSF, and serum. Some AD samples also had α‐synuclein pathology and some PD samples also had β‐amyloid pathology, and, very intriguingly, these PD cases also had a history of dementia. Detection of different oligomeric α‐synuclein and β‐amyloid species is an effective method for identifying tissue, CSF and sera from PD and AD samples, respectively, and samples that also contained early stages of other protein pathologies, indicating their potential value as blood‐based biomarkers for neurodegenerative diseases.     

β2 Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK‐dependent/protein kinase A‐independent pathway

Activation of the β2 adrenergic receptor (β2AR) on immune cells has been reported to possess anti‐inflammatory properties, however, the pro‐inflammatory properties of β2AR activation remain unclear. In this study, using rat primary mesencephalic neuron‐glia cultures, we report that salmeterol, a long‐acting β2AR agonist, selectively induces dopaminergic (DA) neurotoxicity through its ability to activate microglia. Salmeterol selectively increased the production of reactive oxygen species (ROS) by NADPH oxidase (PHOX), the major superoxide‐producing enzyme in microglia. A key role of PHOX in mediating salmeterol‐induced neurotoxicity was demonstrated by the inhibition of DA neurotoxicity in cultures pretreated with diphenylene‐iodonium (DPI), an inhibitor of PHOX activity. Mechanistic studies revealed the activation of microglia by salmeterol results in the selective phosphorylation of ERK, a signaling pathway required for the translocation of the PHOX cytosolic subunit p47^phox to the cell membrane. Furthermore, we found ERK inhibition, but not protein kinase A (PKA) inhibition, significantly abolished salmeterol‐induced superoxide production, p47^phox translocation, and its ability to mediate neurotoxicity. Together, these findings indicate that β2AR activation induces microglial PHOX activation and DA neurotoxicity through an ERK‐dependent/PKA‐independent pathway. © 2009 Wiley‐Liss, Inc.     

Detection of isolated cerebrovascular β‐amyloid with pittsburgh compound B

Imaging of cerebrovascular β‐amyloid (cerebral amyloid angiopathy) is complicated by the nearly universal overlap of this pathology with Alzheimer's pathology. We performed positron emission tomographic imaging with Pittsburgh Compound B on 42‐year‐old man with early manifestations of Iowa‐type hereditary cerebral amyloid angiopathy, a form of the disorder with little or no plaque deposits of fibrillar β‐amyloid. The results demonstrated increased Pittsburgh Compound B retention selectively in occipital cortex, sparing regions typically labeled in Alzheimer's disease. These results offer compelling evidence that Pittsburgh Compound B positron emission tomography can noninvasively detect isolated cerebral amyloid angiopathy before overt signs of tissue damage such as hemorrhage or white matter lesions. Ann Neurol 2008;64:587–591     

Operational dissection of β‐amyloid cytopathic effects on cultured neurons

Alzheimer disease (AD) affects mainly people over the age of 65 years, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The toxic role of β‐amyloid peptide (Aβ) has now shifted from insoluble Aβ fibrils to smaller, soluble oligomeric Aβ aggregates. The urgent need for efficient new therapies is high; robust models dissecting the physiopathological aspects of the disease are needed. We present here a model allowing study of four cytopathic effects of Aβ oligomers (AβO): oxidative stress, loss of synapses, disorganization of the neurite network, and cellular death. By generating a solution of AβO and playing on the concentration of and time of exposure to AβO, we have shown that it was possible to reproduce early effects (oxidative stress) and the long‐term development of structural alterations (death of neurons). We have shown that 1) all toxic events were linked to AβO according to a specific timing and pathway and 2) AβO were probably the key intermediates in AD pathogenesis. The present model, using Aβ peptide solution containing AβO, reproduced essential neuropathological features of AD; the effects involved were similar whatever the kind of neurons tested (cortical vs. hippocampal). By using a single system, it was possible to embrace all toxic mechanisms at defined times and concentrations, to study each involved pathway, and to study the effects of new molecules on the different neurotoxic pathways responsible for development of AD. © 2013 Wiley Periodicals, Inc.     

Dual pathways mediate β‐amyloid stimulated glutathione release from astrocytes

Oxidative stress plays an important role in the progression of Alzheimer's disease (AD) and other neurodegenerative conditions. Glutathione (GSH), the major antioxidant in the central nervous system, is primarily synthesized and released by astrocytes. We determined if β‐amyloid (Aβ42), crucially involved in Alzheimer's disease, affected GSH release. Monomeric Aβ (mAβ) stimulated GSH release from cultured cortical astrocytes more effectively than oligomeric Aβ (oAβ) or fibrillary Aβ (fAβ). Monomeric Aβ increased the expression of the transporter ABCC1 (also referred to as MRP1) that is the main pathway for GSH release. GSH release from astrocytes, with or without mAβ stimulation, was reduced by pharmacological inhibition of ABCC1. Astrocytes robustly express connexin proteins, especially connexin43 (Cx43), and mAβ also stimulated Cx43 hemichannel‐mediated glutamate and GSH release. Aβ‐stimulation facilitated hemichannel opening in the presence of normal extracellular calcium by reducing astrocyte cholesterol level. Aβ treatment did not alter the intracellular concentration of reduced or oxidized glutathione. Using a mouse model of AD with early onset Aβ deposition (5xFAD), we found that cortical ABCC1 was significantly increased in temporal register with the surge of Aβ levels in these mice. ABCC1 levels remained elevated from 1.5 to 3.5 months of age in 5xFAD mice, before plunging to subcontrol levels when amyloid plaques appeared. Similarly, in cultured astrocytes, prolonged incubation with aggregated Aβ, but not mAβ, reduced induction of ABCC1 expression. These results support the hypothesis that in the early stage of AD pathogenesis, less aggregated Aβ increases GSH release from astrocytes (via ABCC1 transporters and Cx43 hemichannels) providing temporary protection from oxidative stress which promotes AD development. GLIA 2015;63:2208–2219     

In vivo labelling of hippocampal β‐amyloid in triple‐transgenic mice with a fluorescent acetylcholinesterase inhibitor released from nanoparticles

The drastic loss of cholinergic projection neurons in the basal forebrain is a hallmark of Alzheimer’s disease (AD), and drugs most frequently applied for the treatment of dementia include inhibitors of the acetylcholine‐degrading enzyme acetylcholinesterase (AChE). This protein is known to act as a ligand of β‐amyloid (Aβ) in senile plaques, a further neuropathological sign of AD. Recently, we have shown that the fluorescent, heterodimeric AChE inhibitor PE154 allows for the histochemical staining of cortical Aβ plaques in triple‐transgenic (TTG) mice with age‐dependent β‐amyloidosis and tau hyperphosphorylation, an established animal model for aspects of AD. In the present study, we have primarily demonstrated the targeting of Aβ‐immunopositive plaques with PE154 in vivo for 4 h up to 1 week after injection into the hippocampi of 13–20‐month‐old TTG mice. Numerous plaques, double‐stained for PE154 and Aβ‐immunoreactivity, were revealed by confocal laser‐scanning microscopy. Additionally, PE154 targeted hippocampal Aβ deposits in aged TTG mice after injection of carboxylated polyglycidylmethacrylate nanoparticles delivering the fluorescent marker in vivo. Furthermore, biodegradable core‐shell polystyrene/polybutylcyanoacrylate nanoparticles were found to be suitable, alternative vehicles for PE154 as a useful in vivo label of Aβ. Moreover, we were able to demonstrate that PE154 targeted Aβ, but neither phospho‐tau nor reactive astrocytes surrounding the plaques. In conclusion, nanoparticles appear as versatile carriers of AChE inhibitors and other promising drugs for the treatment of AD.