longitudinal-vivo-imaging-cerebrovasculature-relevance-to-cns

Longitudinal <em>In Vivo</em> 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 ...

Institute of Functional Genomics

UMR-5203

Université de Montpellier

Cannabinoids exert complex actions on neurotransmitter systems involved in cognition, locomotion, appetite, but no information was available so far on the interactions between the endocannabinoid system and histaminergic neurons that command several, similar behavioural states and memory. In this study, we investigated the effect of cannabimimetic compounds on histamine release using the microdialysis technique in the brain of freely moving rats. We found that systemic administration of the cannabinoid receptors 1 (CB1-r) agonist arachidonyl-2'chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA; 3 mg/kg) increased histamine release from the posterior hypothalamus, where the histaminergic tuberomamillary nuclei (TMN) are located. Local infusions of ACEA (150 nm) or R(+)-methanandamide (mAEA; 1 microm), another CB1-r agonist, in the TMN augmented histamine release from the TMN, as well as from two histaminergic projection areas, the nucleus basalis magnocellularis and the dorsal striatum. When the endocannabinoid uptake inhibitor AM404 was infused into the TMN, however, increased histamine release was observed only in the TMN. The cannabinoid-induced effects on histamine release were blocked by co-administrations with the CB1-r antagonist AM251. Using double-immunofluorescence labelling and confocal laser-scanning microscopy, CB1-r immunostaining was found in the hypothalamus, but was not localized onto histaminergic cells. The modulatory effect of cannabimimetic compounds on histamine release apparently did not involve inhibition of gamma-aminobutyric acid (GABA)ergic neurotransmission, which provides the main inhibitory input to the histaminergic neurons in the hypothalamus, as local infusions of ACEA did not modify GABA release from the TMN. These profound effects of cannabinoids on histaminergic neurotransmission may partially underlie some of the behavioural changes observed following exposure to cannabinoid-based drugs.

Differential effect of cannabinoid agonists and endocannabinoids on histamine release from distinct regions of the rat brain.

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.

Histamine in the brain: beyond sleep and memory.

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.

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

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.

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.

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-β (Aβ) 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 Aβ peptides. Therefore, Amyloid Precursor Protein (APP) synthesis, trafficking and metabolism producing either the toxic Aβ 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 Aβ 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.

Alzheimer culprits: cellular crossroads and interplay.

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.

Serotonin type 4 receptor dimers.

Amyloid β (Aβ) accumulation is considered the main culprit in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that decreasing Aβ 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 Aβ formation. Using the 5XFAD mouse model of AD that shows accelerated Aβ 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 Aβ species. Reduction of Aβ 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.

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.

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-β 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.

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

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.

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.

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.

Serotonin: A New Hope in Alzheimer's Disease?

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 (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, 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 Aβ 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 Aβ 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 Aβ-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.

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.

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 Aβ 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 Aβ 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 (PDGFRβ) 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 Aβ 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 PDGFRβ(+) pericytes was significantly decreased at M4 in the fronto-parietal cortices, with a trend decrease observed in the other structures. At M9-M12, PDGFRβ(+) 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.

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

Amyloid β (Aβ) 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 Aβ 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 Aβ influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aβ 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 Aβ1-40 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 Aβ 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 Aβ efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aβ. 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.

Article	Year
Differential effect of cannabinoid agonists and endocannabinoids on histamine release from distinct regions of the rat brain. The European journal of neuroscience\| PubMed ID: 17004927	2006
Histamine in the brain: beyond sleep and memory. Biochemical pharmacology\| PubMed ID: 17241615	2007
Heterogeneity of histaminergic neurons in the tuberomammillary nucleus of the rat. The European journal of neuroscience\| PubMed ID: 19490084	2009
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 (GSK189... The Journal of pharmacology and experimental therapeutics\| PubMed ID: 19815811	2010
Alzheimer culprits: cellular crossroads and interplay. Cellular signalling\| PubMed ID: 22627093	2012
Serotonin type 4 receptor dimers. Methods in cell biology\| PubMed ID: 24143975	2013
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. Frontiers in aging neuroscience\| PubMed ID: 24399967	2013
Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25157130	2014
Novel multitarget-directed ligands (MTDLs) with acetylcholinesterase (AChE) inhibitory and serotonergic subtype 4 receptor (5-HT4R) agonist activities as potential agents against Alzheimer's... Journal of medicinal chemistry\| PubMed ID: 25793650	2015
Serotonin: A New Hope in Alzheimer's Disease? ACS chemical neuroscience\| PubMed ID: 26011650	2015
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 br... Neurobiology of disease\| PubMed ID: 26581638	2016
Cerebrovascular pathology during the progression of experimental Alzheimer's disease. Neurobiology of disease\| PubMed ID: 26774030	2016
In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Aβ protein. Frontiers in aging neuroscience\| PubMed ID: 27729857	2016

Patrizia Giannoni

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