Environmental bacteria as triggers to brain disease: Possible mechanisms of toxicity and associated human risk.

AIMS: Brain disease, in its many forms, has recently demonstrated a great socio-economic impact and represents one of the hardest challenges of present research. Although each pathology of this highly heterogenous group is characterized by individual features, there is an increasing number of common toxicological mechanisms that have been evidenced. This review aims to summarize the state-of-art knowledge concerning the role of environmental bacteria in brain diseases focusing on different mechanisms of action that could be interacting in an additive or synergistic way. MATERIALS AND METHODS: For this wide-range subject, we focused on two emerging types of bacterial-derived brain exposure and damage and specifically treated representative examples: i) environmental bacterial-derived compounds in the form of the cyanobacterial product BMAA (ß-N-methylamino-L-alanine) toxin and its isomers DAB (2,4-diaminobutyric acid) and AEG (N-(2-aminoethyl)glycine) and ii) toxicity related to bacterial infections in the form of the emerging Lyme neuroborreliosis (LNB), determined by Borrelia burgdorferi (Bb). KEY FINDINGS: Defined as pleiotropic contaminants, BMAA and Bb act through multiple toxicological pathways including inflammation, oxidative stress and excitotoxicity. Multiple investigations in in vitro and in vivo models have underlined the involved mechanisms of action but further investigations are needed to clarify the role of possible cocktail effects and underline possible new targets of intervention. SIGNIFICANCE: Environmental bacteria represent emerging risk factors because of environmental changes, anthropogenic activities and human lifestyle evolutions. Future directions and research ambitions are here discussed in order to evaluate human risk and possible ways of intervention and prevention.

Peripheral Routes to Neurodegeneration: Passing Through the Blood-Brain Barrier.

A bidirectional crosstalk between peripheral players of immunity and the central nervous system (CNS) exists. Hence, blood-brain barrier (BBB) breakdown is emerging as a participant mechanism of dysregulated peripheral-CNS interplay, promoting diseases. Here, we examine the implication of BBB damage in neurodegeneration, linking it to peripheral brain-directed autoantibodies and gut-brain axis mechanisms. As BBB breakdown is a factor contributing to, or even anticipating, neuronal dysfunction(s), we here identify contemporary pharmacological strategies that could be exploited to repair the BBB in disease conditions. Developing neurovascular, add on, therapeutic strategies may lead to a more efficacious pre-clinical to clinical transition with the goal of curbing the progression of neurodegeneration.

Donecopride, a Swiss army knife with potential against Alzheimer's disease.

BACKGROUND AND PURPOSE: We recently identified donecopride as a pleiotropic compound able to inhibit AChE and to activate 5-HT4 receptors. Here, we have assessed the potential therapeutic effects of donecopride in treating Alzheimer's disease (AD). EXPERIMENTAL APPROACH: We used two in vivo animal models of AD, transgenic 5XFAD mice and mice exposed to soluble amyloid-ß peptides and, in vitro, primary cultures of rat hippocampal neurons. Pro-cognitive and anti-amnesic effects were evaluated with novel object recognition, Y-maze, and Morris water maze tests. Amyloid load in mouse brain was measured ex vivo and effects of soluble amyloid-ß peptides on neuronal survival and neurite formation determined in vitro. KEY RESULTS: In vivo, chronic (3 months) administration of donecopride displayed potent anti-amnesic properties in the two mouse models of AD, preserving learning capacities, including working and long-term spatial memories. These behavioural effects were accompanied by decreased amyloid aggregation in the brain of 5XFAD mice and, in cultures of rat hippocampal neurons, reduced tau hyperphosphorylation. In vitro, donecopride increased survival in neuronal cultures exposed to soluble amyloid-ß peptides, improved the neurite network and provided neurotrophic benefits, expressed as the formation of new synapses. CONCLUSIONS AND IMPLICATIONS: Donecopride acts like a Swiss army knife, exhibiting a range of sustainable symptomatic therapeutic effects and potential disease-modifying effects in models of AD. Clinical trials with this promising drug candidate will soon be undertaken to confirm its therapeutic potential in humans.

The pericyte-glia interface at the blood-brain barrier.

The cerebrovasculature is a multicellular structure with varying rheological and permeability properties. The outer wall of the brain capillary endothelium is enclosed by pericytes and astrocyte end feet, anatomically assembled to guarantee barrier functions. We, here, focus on the pericyte modifications occurring in disease conditions, reviewing evidence supporting the interplay amongst pericytes, the endothelium, and glial cells in health and pathology. Deconstruction and reactivity of pericytes and glial cells around the capillary endothelium occur in response to traumatic brain injury, epilepsy, and neurodegenerative disorders, impacting vascular permeability and participating in neuroinflammation. As this represents a growing field of research, addressing the multicellular reorganization occurring at the outer wall of the blood-brain barrier (BBB) in response to an acute insult or a chronic disease could disclose novel disease mechanisms and therapeutic targets.

Chronic treatments with a 5-HT4 receptor agonist decrease amyloid pathology in the entorhinal cortex and learning and memory deficits in the 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the main cause of dementia and a major health issue worldwide. The complexity of the pathology continues to challenge its comprehension and the implementation of effective treatments. In the last decade, a number of possible targets of intervention have been pointed out, among which the stimulation of 5-HT4 receptors (5-HT4Rs) seems very promising. 5-HT4R agonists exert pro-cognitive effects, inhibit amyloid-ß peptide (Aß) production and therefore directly and positively impact AD progression. In the present work, we investigated the effects of RS 67333, a partial 5-HT4R agonist, after chronic administration in the 5xFAD mouse model of AD. 5xFAD male mice and their wild type (WT) male littermates received either RS 67333 or vehicle solution i.p., twice a week, for 2 or 4 months. Cognitive performance was evaluated in a hippocampal-dependent behavioral task, the olfactory tubing maze (OTM). Mice were then sacrificed to evaluate the metabolism of the amyloid precursor protein (APP), amyloidosis and neuroinflammatory processes. No beneficial effects of RS 67333 were observed in 5xFAD mice after 2 months of treatment, while 5xFAD mice treated for 4 months showed better cognitive abilities compared to vehicle-treated 5xFAD mice. The beneficial effects of RS 67333 on learning and memory correlated with the decrease in both amyloid plaque load and neuroinflammation, more specifically in the entorhinal cortex. The most significant improvements in learning and memory and reduction of pathology stigmata were observed after the 4-month administration of RS 67333, demonstrating that treatment duration is important to alleviate amyloidosis and glial reactivity, particularly in the entorhinal cortex. These results confirm the 5-HT4R as a promising target for AD pathogenesis and highlight the need for further investigations to characterize fully the underlying mechanisms of action.

In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Aß protein.

Amyloid ß (Aß) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Aß oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Aß influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aß clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Aß1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Aß physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Aß efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aß. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

Cerebrovascular pathology during the progression of experimental Alzheimer's disease.

Clinical and experimental evidence point to a possible role of cerebrovascular dysfunction in Alzheimer's disease (AD). The 5xFAD mouse model of AD expresses human amyloid precursor protein and presenilin genes with mutations found in AD patients. It remains unknown whether amyloid deposition driven by these mutations is associated with cerebrovascular changes. 5xFAD and wild type mice (2 to 12months old; M2 to M12) were used. Thinned skull in vivo 2-photon microscopy was used to determine Aß accumulation on leptomeningeal or superficial cortical vessels over time. Parenchymal microvascular damage was assessed using FITC-microangiography. Collagen-IV and CD31 were used to stain basal lamina and endothelial cells. Methoxy-XO4, Thioflavin-S or 6E10 were used to visualize Aß accumulation in living mice or in fixed brain tissues. Positioning of reactive IBA1 microglia and GFAP astrocytes at the vasculature was rendered using confocal microscopy. Platelet-derived growth factor receptor beta (PDGFRß) staining was used to visualize perivascular pericytes. In vivo 2-photon microscopy revealed Methoxy-XO4(+) amyloid perivascular deposits on leptomeningeal and penetrating cortical vessels in 5xFAD mice, typical of cerebral amyloid angiopathy (CAA). Amyloid deposits were visible in vivo at M3 and aggravated over time. Progressive microvascular damage was concomitant to parenchymal Aß plaque accumulation in 5xFAD mice. Microvascular inflammation in 5xFAD mice presented with sporadic FITC-albumin leakages at M4 becoming more prevalent at M9 and M12. 3D colocalization showed inflammatory IBA1(+) microglia proximal to microvascular FITC-albumin leaks. The number of perivascular PDGFRß(+) pericytes was significantly decreased at M4 in the fronto-parietal cortices, with a trend decrease observed in the other structures. At M9-M12, PDGFRß(+) pericytes displayed hypertrophic perivascular ramifications contiguous to reactive microglia. Cerebral amyloid angiopathy and microvascular inflammation occur in 5xFAD mice concomitantly to parenchymal plaque deposition. The prospect of cerebrovascular pharmacology in AD is discussed.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 µm of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer's disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain.

Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aß) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aß, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aß in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Aß injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Aß-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

Serotonin: A New Hope in Alzheimer's Disease?

Alzheimer's disease (AD) is the most common form of dementia affecting 35 million individuals worldwide. Current AD treatments provide only brief symptomatic relief. It is therefore urgent to replace this symptomatic approach with a curative one. Increasing serotonin signaling as well as developing molecules that enhance serotonin concentration in the synaptic cleft have been debated as possible therapeutic strategies to slow the progression of AD. In this Viewpoint, we discuss exciting new insights regarding the modulation of serotonin signaling for AD prevention and therapy.

Novel multitarget-directed ligands (MTDLs) with acetylcholinesterase (AChE) inhibitory and serotonergic subtype 4 receptor (5-HT4R) agonist activities as potential agents against Alzheimer's disease: the design of donecopride.

In this work, we describe the synthesis and in vitro evaluation of a novel series of multitarget-directed ligands (MTDL) displaying both nanomolar dual-binding site (DBS) acetylcholinesterase inhibitory effects and partial 5-HT4R agonist activity, among which donecopride was selected for further in vivo evaluations in mice. The latter displayed procognitive and antiamnesic effects and enhanced sAPPα release, accounting for a potential symptomatic and disease-modifying therapeutic benefit in the treatment of Alzheimer's disease.

Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment.

RS67333 is a partial serotonin subtype 4 receptor (5-HT4R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-ß peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer's disease (AD). We show herein that RS67333 is also a submicromolar acetylcholinesterase (AChE) inhibitor and therefore, could contribute, through this effect, to the restoration of the cholinergic neurotransmission that becomes altered in AD. We planned to pharmacomodulate RS67333 to enhance its AChE inhibitory activity to take advantage of this pleiotropic pharmacological profile in the design of a novel multitarget-directed ligand that is able to exert not only a symptomatic but also, a disease-modifying effect against AD. These efforts allowed us to select donecopride as a valuable dual (h)5-HT4R partial agonist (Ki = 10.4 nM; 48.3% of control agonist response)/(h)AChEI (IC50 = 16 nM) that further promotes sAPPα release (EC50 = 11.3 nM). Donecopride, as a druggable lead, was assessed for its in vivo procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improvement of memory performances observed at 0.3 and 1 mg/kg on the object recognition test. On the basis of these in vitro and in vivo activities, donecopride seems to be a promising drug candidate for AD treatment.

Serotonin type 4 receptor dimers.

Numerous class A G protein-coupled receptors and especially biogenic amine receptors have been reported to form homodimers. Indeed, the dimerization process might occur for all the metabotropic serotonergic receptors. Moreover, dimerization appears to be essential for the function of serotonin type 2C (5-HT2C) and type 4 (5-HT4) receptors and required to obtain full receptor activity. Several techniques have been developed to analyze dimer formation and properties. Due to our involvement in deciphering 5-HT4R transduction mechanisms, we improved and set up new procedures to study 5-HT4R dimers, by classical methods or modern tools. This chapter presents detailed protocols to detect 5-HT4R dimers by Western blotting and coimmunoprecipitation, including the optimizations that we routinely carry out. We developed an innovative method to achieve functional visualization of 5-HT4R dimers by immunofluorescence, taking advantage of the 5-HT4-RASSL (receptor activated solely by synthetic ligand) mutant that was engineered in the laboratory. Finally, we adapted the powerful time-resolved FRET technology to assess a relative quantification of dimer formation and affinity.

Early administration of RS 67333, a specific 5-HT4 receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5XFAD mouse model of Alzheimer's disease.

Amyloid ß (Aß) accumulation is considered the main culprit in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that decreasing Aß production at very early stages of AD could be a promising strategy to slow down disease progression. Serotonin 5-HT4 receptor activation stimulates α-cleavage of the amyloid precursor protein (APP), leading to the release of the soluble and neurotrophic sAPPα fragment and thus precluding Aß formation. Using the 5XFAD mouse model of AD that shows accelerated Aß deposition, we investigated the effect of chronic treatments (treatment onset at different ages and different durations) with the 5-HT4 receptor agonist RS 67333 during the asymptomatic phase of the disease. Chronic administration of RS 67333 decreased concomitantly the number of amyloid plaques and the level of Aß species. Reduction of Aß levels was accompanied by a striking decrease in hippocampal astrogliosis and microgliosis. RS 67333 also transiently increased sAPPα concentration in the cerebrospinal fluid and brain. Moreover, a specific 5-HT4 receptor antagonist (RS 39604) prevented the RS 67333-mediated reduction of the amyloid pathology. Finally, the novel object recognition test deficits of 5XFAD mice were reversed by chronic treatment with RS 67333. Collectively, these results strongly highlight this 5-HT4 receptor agonist as a promising disease modifying-agent for AD.

Alzheimer culprits: cellular crossroads and interplay.

Alzheimer's disease (AD) is the primary cause of dementia in the elderly and one of the major health problems worldwide. Since its first description by Alois Alzheimer in 1907, noticeable but insufficient scientific comprehension of this complex pathology has been achieved. All the research that has been pursued takes origin from the identification of the pathological hallmarks in the forms of amyloid-ß (Aß) deposits (plaques), and aggregated hyperphosphorylated tau protein filaments (named neurofibrillary tangles). Since this discovery, many hypotheses have been proposed to explain the origin of the pathology. The "amyloid cascade hypothesis" is the most accredited theory. The mechanism suggested to be one of the initial causes of AD is an imbalance between the production and the clearance of Aß peptides. Therefore, Amyloid Precursor Protein (APP) synthesis, trafficking and metabolism producing either the toxic Aß peptide via the amyloidogenic pathway or the sAPPα fragment via the non amyloidogenic pathway have become appealing subjects of study. Being able to reduce the formation of the toxic Aß peptides is obviously an immediate approach in the trial to prevent AD. The following review summarizes the most relevant discoveries in the field of the last decades.

Regional differential effects of the novel histamine H3 receptor antagonist 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) on histamine release in the central nervous system of freely moving rats.

After oral administration, the nonimidazole histamine H(3) receptor antagonist, 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254), increased histamine release from the tuberomammillary nucleus, where all histaminergic somata are localized, and from where their axons project to the entire brain. To further understand functional histaminergic circuitry in the brain, dual-probe microdialysis was used to pharmacologically block H(3) receptors in the tuberomammillary nucleus, and monitor histamine release in projection areas. Perfusion of the tuberomammillary nucleus with GSK189254 increased histamine release from the tuberomammillary nucleus, nucleus basalis magnocellularis, and cortex, but not from the striatum or nucleus accumbens. Cortical acetylcholine (ACh) release was also increased, but striatal dopamine release was not affected. When administered locally, GSK189254 increased histamine release from the nucleus basalis magnocellularis, but not from the striatum. Thus, defined by their sensitivity to GSK189254, histaminergic neurons establish distinct pathways according to their terminal projections, and can differentially modulate neurotransmitter release in a brain region-specific manner. Consistent with its effects on cortical ACh release, systemic administration of GSK189254 antagonized the amnesic effects of scopolamine in the rat object recognition test, a cognition paradigm with important cortical components.

Heterogeneity of histaminergic neurons in the tuberomammillary nucleus of the rat.

Histaminergic neurons of the hypothalamic tuberomammillary nuclei (TMN) send projections to the whole brain. Early anatomical studies described histaminergic neurons as a homogeneous cell group, but recent evidence indicates that histaminergic neurons are heterogeneous and organized into distinct circuits. We addressed this issue using the double-probe microdialysis in freely moving rats to investigate if two compounds acting directly onto histaminergic neurons to augment cell firing [thioperamide and bicuculline, histamine H(3)- and gamma-aminobutyric acid (GABA)(A)-receptor (R) antagonists, respectively] may discriminate groups of histaminergic neurons impinging on different brain regions. Intra-hypothalamic perfusion of either drug increased histamine release from the TMN and cortex, but not from the striatum. Thioperamide, but not bicuculline, increased histamine release from the nucleus basalis magnocellularis (NBM), bicuculline but not thioperamide increased histamine release from the nucleus accumbens (NAcc). Intra-hypothalamic perfusion with thioperamide increased the time spent in wakefulness. To explore the local effects of H(3)-R blockade in the histaminergic projection areas, each rat was implanted with a single probe to simultaneously administer thioperamide and monitor local changes in histamine release. Thioperamide increased histamine release from the NBM and cortex significantly, but not from the NAcc or striatum. The presence of H(3)-Rs on histaminergic neurons was assessed using double-immunofluorescence with anti-histidine decarboxylase antibodies to identify histaminergic cells and anti-H(3)-R antibodies. Confocal analysis revealed that all histaminergic somata were immunopositive for the H(3)-R. This is the first evidence that histaminergic neurons are organized into functionally distinct circuits that influence different brain regions, and display selective control mechanisms.

Histamine in the brain: beyond sleep and memory.

A few decades elapsed between the attribution of unwanted side effects of classic antihistamine compounds to the blockade of central H(1) receptors, and the acceptance of the concept that the histaminergic system commands general states of metabolism and consciousness. In the early 80s, two laboratories discovered independently that histaminergic neurons are located in the posterior hypothalamus and project to the whole CNS [Panula P, Yang HY, Costa E. Histamine-containing neurons in the rat hypothalamus. Proc Natl Acad Sci 1984;81:2572-76, Watanabe T, Taguchi Y, Hayashi H, Tanaka J, Shiosaka S, Tohyama M, Kubota H, Terano Y, Wada H. Evidence for the presence of a histaminergic neuron system in the rat brain: an immunohistochemical analysis. Neurosci Lett 1983;39:249-54], suggesting a global nature of histamine regulatory effects. Recently, functional studies demonstrated that activation of the central histaminergic system alters CNS functions in both behavioral and homeostatic contexts, which include sleep and wakefulness, learning and memory, anxiety, locomotion, feeding and drinking, and neuroendocrine regulation. These actions are achieved through interactions with other neurotransmitter systems, and the interplay between histaminergic neurons and other neurotransmitter systems are becoming clear. Hence, numerous laboratories are pursuing novel compounds targeting the three known histamine receptors found in the brain for various therapeutic indications. Preclinical studies are focusing on three major areas of interest and intense research is mainly oriented towards providing drugs for the treatment of sleep, cognitive and feeding disorders. This commentary is intended to summarize some of the latest findings that suggest functional roles for the interplay between histamine and other neurotransmitter systems, and to propose novel interactions as physiological substrates that may partially underlie some of the behavioral changes observed following manipulation of the histaminergic system.