Edith Hamel

Distinct choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) bipolar neurons project to local blood vessels in the rat cerebral cortex

Innervation of rat intracortical cerebral blood vessels by acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) remains largely unexplored and it is not known if the cells of origin are intra- or extracortical nor if perivascular fibers colocalize ACh and VIP. Cortical cholinergic innervation arises primarily from the basal forebrain and to a small extent from intrinsic bipolar ACh neurons thought to be the sole source of cortical VIP. In order to evaluate if intracortical perivascular ACh terminals could be distinguished from those of the basal forebrain by their colocalization with VIP, we performed a double immunofluorescence study and determined the percentage of colocalization of choline acetyltransferase (ChAT) and VIP in cortical neurons, as well as in terminal fields associated with intracortical blood vessels. From a total of 2103 cells examined in all cortical areas, VIP neurons accounted for the largest population (58.3%) followed by ChAT-positive cells (28.2%) with only 13.5% of cells being double-labelled for VIP and ChAT. Of the cortical ChAT-immunostained cells (n = 878), 32.3% colocalized VIP whereas only 18.8% of VIP neurons (n = 1509) also contained ChAT. In various cortical areas, ChAT cell bodies were seen to be contacted by VIP terminals which surrounded closely their cell soma and proximal dendrites. Perivascular fibers studied by double immunofluorescence and confocal microscopy were of three categories including cholinergic, VIPergic with a smaller population of fibers which costained for both ChAT and VIP. These results show that cortical VIP neurons are much more numerous than those containing ChAT, and that a majority of VIP neurons do not colocalize with ChAT. This observation indicates that ACh and VIP are primarily located in distinct neuronal populations and that VIP cannot be used as a marker of intracortical ACh neurons and terminals. Our results further suggest that intracortical blood vessels are primarily under the influence of distinct ChAT and VIP perivascular fibers. Also, the presence of a subset of VIP and ChAT/VIP fibers in association with intracortical blood vessels strongly suggests a role for cortical bipolar neurons in local cerebrovascular regulation. The origin of the perivascular ChAT fibers which do not colocalize VIP, however, remains unknown.

Angiotensin II type 1 receptor blocker losartan prevents and rescues cerebrovascular, neuropathological and cognitive deficits in an Alzheimer's disease model.

Angiotensin II (AngII) receptor blockers that bind selectively AngII type 1 (AT1) receptors may protect from Alzheimers disease (AD). We studied the ability of the AT1 receptor antagonist losartan to cure or prevent AD hallmarks in aged (~18months at endpoint, 3months treatment) or adult (~12months at endpoint, 10months treatment) human amyloid precursor protein (APP) transgenic mice. We tested learning and memory with the Morris water maze, and evaluated neurometabolic and neurovascular coupling using [(18)F]fluoro-2-deoxy-D-glucose-PET and laser Doppler flowmetry responses to whisker stimulation. Cerebrovascular reactivity was assessed with on-line videomicroscopy. We measured protein levels of oxidative stress enzymes (superoxide dismutases SOD1, SOD2 and NADPH oxidase subunit p67phox), and quantified soluble and deposited amyloid-β (Aβ) peptide, glial fibrillary acidic protein (GFAP), AngII receptors AT1 and AT2, angiotensin IV receptor AT4, and cortical cholinergic innervation. In aged APP mice, losartan did not improve learning but it consolidated memory acquisition and recall, and rescued neurovascular and neurometabolic coupling and cerebrovascular dilatory capacity. Losartan normalized cerebrovascular p67phox and SOD2 protein levels and up-regulated those of SOD1. Losartan attenuated astrogliosis, normalized AT1 and AT4 receptor levels, but failed to rescue the cholinergic deficit and the Aβ pathology. Given preventively, losartan protected cognitive function, cerebrovascular reactivity, and AT4 receptor levels. Like in aged APP mice, these benefits occurred without a decrease in soluble Aβ species or plaque load. We conclude that losartan exerts potent preventive and restorative effects on AD hallmarks, possibly by mitigating AT1-initiated oxidative stress and normalizing memory-related AT4 receptors.

The nuclear receptor PPARγ as a therapeutic target for cerebrovascular and brain dysfunction in Alzheimer's disease

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that regulate peripheral lipid and glucose metabolism. Three subtypes make up the PPAR family (α, γ, β/δ), and synthetic ligands for PPARα (fibrates) and PPARγ (Thiazolidinediones, TZDs) are currently prescribed for the respective management of dyslipidemia and type 2 diabetes. In contrast to the well characterized action of PPARs in the periphery, little was known about the presence or function of these receptors in the brain and cerebral vasculature until fairly recently. Indeed, research in the last decade has uncovered these receptors in most brain cell types, and has shown that their activation, particularly that of PPARγ, is implicated in normal brain and cerebrovascular physiology, and confers protection under pathological conditions. Notably, accumulating evidence has highlighted the therapeutic potential of PPARγ ligands in the treatment of brain disorders such as Alzheimers disease (AD), leading to the testing of the TZDs pioglitazone and rosiglitazone in AD clinical trials. This review will focus on the benefits of PPARγ agonists for vascular, neuronal and glial networks, and assess the value of these compounds as future AD therapeutics in light of evidence from transgenic mouse models and recent clinical trials.

Schizophrenia-Like Features in Transgenic Mice Overexpressing Human HO-1 in the Astrocytic Compartment

Delineation of key molecules that act epigenetically to transduce diverse stressors into established patterns of disease would facilitate the advent of preventive and disease-modifying therapeutics for a host of neurological disorders. Herein, we demonstrate that selective overexpression of the stress protein heme oxygenase-1 (HO-1) in astrocytes of novel GFAP.HMOX1 transgenic mice results in subcortical oxidative stress and mitochondrial damage/autophagy; diminished neuronal reelin content (males); induction of Nurr1 and Pitx3 with attendant suppression of their targeting miRNAs, 145 and 133b; increased tyrosine hydroxylase and α-synuclein expression with downregulation of the targeting miR-7b of the latter; augmented dopamine and serotonin levels in basal ganglia; reduced D1 receptor binding in nucleus accumbens; axodendritic pathology and altered hippocampal cytoarchitectonics; impaired neurovascular coupling; attenuated prepulse inhibition (males); and hyperkinetic behavior. The GFAP.HMOX1 neurophenotype bears resemblances to human schizophrenia and other neurodevelopmental conditions and implicates glial HO-1 as a prime transducer of inimical (endogenous and environmental) influences on the development of monoaminergic circuitry. Containment of the glial HO-1 response to noxious stimuli at strategic points of the life cycle may afford novel opportunities for the effective management of human neurodevelopmental and neurodegenerative conditions.

Transgenic Mice Overexpressing APP and Transforming Growth Factor-β1 Feature Cognitive and Vascular Hallmarks of Alzheimer's Disease

High brain levels of amyloid-β (Aβ) and transforming growth factor-β1 (TGF-β1) have been implicated in the cognitive and cerebrovascular alterations of Alzheimers disease (AD). We sought to investigate the impact of combined increases in Aβ and TGF-β1 on cerebrovascular, neuronal, and mnemonic function using transgenic mice overproducing these peptides (A/T mice). In particular, we measured cerebrovascular reactivity, evoked cerebral blood flow and glucose uptake during brain activation, cholinergic status, and spatial memory, along with cerebrovascular fibrosis, amyloidosis, and astrogliosis, and their evolution with age. An assessment of perfusion and metabolic responses was considered timely, given ongoing efforts for their validation as AD biomarkers. Relative to wild-type littermates, A/T mice displayed an early progressive decline in cerebrovascular dilatory ability, preserved contractility, and reduction in constitutive nitric oxide synthesis that establishes resting vessel tone. Altered levels of vasodilator-synthesizing enzymes and fibrotic proteins, resistance to antioxidant treatment, and unchanged levels of the antioxidant enzyme, superoxide dismutase-2, accompanied these impairments. A/T mice featured deficient neurovascular and neurometabolic coupling to whisker stimulation, cholinergic denervation, cerebral and cerebrovascular Aβ deposition, astrocyte activation, and impaired Morris water maze performance, which gained severity with age. The combined Aβ- and TGF-β1-driven pathology recapitulates salient cerebrovascular, neuronal, and cognitive AD landmarks and yields a versatile model toward highly anticipated diagnostic and therapeutic tools for patients featuring Aβ and TGF-β1 increments.

Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor

Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y2 receptor described here for the first time. More than 70% of the hippocampal neurones were endowed with [125I]‐[Leu31,Pro34]PYY Y1‐like receptor silver grain accumulations and Y1 receptor immunostaining. These radio‐ and immuno‐labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal‐like neuronal cells, as confirmed by neurone‐specific enolase and MAP‐2 staining. Competition binding profiles revealed that specific [125I]‐[Leu31,Pro34]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y1 receptor sub‐type with [Leu31,Pro34]substituted NPY/PYY analogues>>C‐terminal fragments=pancreatic polypeptides, with the non‐peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [125I]‐[Leu31,Pro34]PYY of the newly cloned Y4, Y5 and Y6 receptors. The expression of the genuine Y1 receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y2‐like/[125I]‐PYY3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y1 and the Y2 receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero. Taken together, these results suggest that the Y1 receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

Neurovascular and Cognitive failure in Alzheimer's Disease: Benefits of Cardiovascular Therapy.

Alzheimer’s disease (AD) is a multifactorial and multifaceted disease for which we currently have very little to offer since there is no curative therapy, with only limited disease-modifying drugs. Recent studies in AD mouse models that recapitulate the amyloid-β (Aβ) pathology converge to demonstrate that it is possible to salvage cerebrovascular function with a variety of drugs and, particularly, therapies used to treat cardiovascular diseases such as hypercholesterolemia and hypertension. These drugs can reestablish dilatory function mediated by various endothelial and smooth muscle ion channels as well as nitric oxide availability, benefits that result in normalized brain perfusion. These cerebrovascular benefits would favor brain perfusion, which may help maintain neuronal function and, possibly, delay cognitive failure. However, restoring cerebrovascular function in AD mouse models was not necessarily accompanied by rescue of cognitive deficits related to spatial learning and memory. The results with cardiovascular therapies rather suggest that drugs originally designed to treat cardiovascular diseases that concurrently restore cerebrovascular and cognitive function do so through their pleiotropic effects. Specifically, recent findings suggest that these drugs act directly on brain cells and neuronal pathways involved in memory formation, hence, working simultaneously albeit independently on neuronal and vascular targets. These findings may help select medications for patients with cardiovascular diseases at risk of developing AD with increasing age. Further, they may identify molecular targets for recovering memory pathways that bear potential for new therapeutic avenues.

Selective in vivo antagonism of endothelin receptors in transforming growth factor-β1 transgenic mice that mimic the vascular pathology of Alzheimer’s diseaseThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Increased levels of transforming growth factor-beta1 (TGF-beta1) induce a vascular pathology that shares similarities with that seen in Alzheimers disease, and which possibly contributes to the cognitive decline. In aged transgenic mice that overexpress TGF-beta1 (TGF mice), we previously found reduced dilatory function and selectively impaired endothelin-1 (ET-1)-induced contraction. Here we studied the effects of chronic treatments with selective ETA (ABT-627) or ETB (A-192621) receptor antagonist on cerebrovascular reactivity, cerebral perfusion, or memory performance. The dilatory deficit of TGF mice was not improved by either treatment, but both ET-1 contraction and basal nitric oxide (NO) production were distinctly altered. Although ABT-627 was devoid of any effect in TGF mice, it virtually abolished the ET-1-induced contraction and NO release in wild-type (WT) littermates. In contrast, A-192621 only acted upon TGF mice with full recovery of ET-1 contraction and baseline NO synthesis. TGF mice, treated or not, had no cognitive deficit in the Morris water maze, nor did ABT-627-treated WT controls despite severely impaired vasoreactivity. These findings confirm that ETA receptors primarily mediate the ET-1-induced contraction. Further, they suggest that ETB receptors play a detrimental role in conditions of increased TGF-beta1 and that vascular dysfunction does not inevitably lead to cognitive deficit.

Ipsilateral alterations in tryptophan hydroxylase activity in rat brain after hypothalamic 5,7-di-hydroxytryptamine lesion

The in vivo relationship between the amounts of tryptophan hydroxylase (TPH) protein and its intrinsic synthetic activity, measured by quantifying the amounts of alpha-[3H]methyl-5-hydroxytryptamine (alpha-[3H]M5-HT), is reported in cell body and terminal areas of intact and disturbed serotonergic neurons following a unilateral 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsolateral hypothalamus. Five days after the lesion, the relationships between TPH and its synthetic product 5-HT were evaluated on adjacent brain sections in serotonergic cells bodies of the dorsal raphe nucleus (DRN) and nerve fibres of the medial forebrain bundle (MFB). On the side contralateral to the lesion, TPH and alpha-[3H]M5-HT levels in the intact hemi-DRN exhibited a caudo-rostral distribution and were positively and significantly correlated (p < or = 0.001); the calculated TPH-specific activity was 0.76 nCi of alpha-[3H]M5-HT formed per U TPH. In the MFB, quantitative measurements of TPH and alpha-[3H]M5-HT showed no correlation between enzyme and product and no specific activity for TPH could be determined. On the side ipsilateral to the lesion, the density of TPH-immunoreactive fibers was drastically decreased in the dorsolateral hypothalamus where a significant reduction in TPH content (45.5% of control side, P < 0.001) was found. In the overall ipsilateral hemi-DRN, TPH and alpha-[3H]M5-HT levels, their correlation as well as TPH-specific activity were unaltered by the lesion but a significant increase in alpha-[3H]M5-HT and TPH contents was observed in the lateral wings of the DRN. The lesion also induced a significant increase in alpha-[3H]M5-HT and TPH levels (136% and 93.8%, P < 0.001, respectively) in the ipsilateral MFB, which resulted in a positive and significant correlation between these two markers and yielded a TPH-specific activity of 1.0 nCi of alpha-[3H]M5-HT formed per U TPH. TPH topological area was also significantly increased in the lateral aspect of the ipsilateral MFB 5 days post lesion. These results show that 5-HT synthesis in the intact DRN is proportional to and dependent on TPH activity while in the MFB, 5-HT accumulation appears unrelated to TPH content which is most likely in an inactive enzymatic form. Moreover, the data show that a local disruption of serotonergic terminals in the dorsolateral hypothalamus does not affect 5-HT synthesis in the overall ipsilateral DRN neurons but results in local activation of TPH within the serotonergic projection neurons and the ipsilateral MFB, as evidenced by active de novo synthesis of 5-HT. Altogether the results point to circumscribed activation of compensatory mechanisms in 5-HT synthesis after selective destruction of serotonergic terminals.

IC-P1-013: 3D whole-brain perfusion MRI in APP transgenic mice

typical bvFTD overlap with those of dementia with Lewy bodies (FTDDLB) such that hallucinations and Parkinsonism are also presenting features; MND is not observed clinically. Objective: We compared brain perfusion patterns in 4 bvFTD, 4 FTD-MND and 5 FTD-DLB patients compared to 19 normal controls. Methods: All 13 patients completed a single photon emission computer tomography (SPECT) study using the radioligand 99mTc-Ethyl cysteinate dimer. Individual SPECT studies were spatially warped to a standard SPECT template (MNI SPECT template) using Statistical Parametric Mapping (SPM2). The result was then masked and smoothed by convolution with an isotropic Gaussian Kernel of 16mm. Group analysis was performed by statistically comparing each group’s processed images for significant regions of hypoperfusion against the control group, using SPM. Results were corrected for family-wise error and a cluster extent of 125 contiguous voxels was applied deemed consistent with the minimum resolution of SPECT imaging. The resultant t-statistics data were transformed in the form of 3D statistical maps and were displayed on the standard T1 MRI template provided by SPM. Results: Average time of disease onset to SPECT was: 1.6 typical bvFTD, 1.5 FTD-MND, and 3 years FTD-DLB. In the typical bvFTD group there was bilateral frontal lobe involvement without striatal involvement. In the FTD-MND group, cortical perfusion changes were similar to typical bvFTD, but there was also striatal hypoperfusion changes not seen in typical bvFTD. Those with FTD-DLB, however had predominantly striatal hypoperfusion with moderate frontotempoal involvment. Conclusions: In this study of three different clinical bvFTD subgroups we found three different patters of hypoperfusion on SPECT scan. These findings suggest that varying involvement of frontotemporal cortex and striatal nuclei may be associated with different presenting features of bvFTD. Given our small sample size, further work is needed to confirm FTD-DLB as a subtype of bvFTD, as well as the association of the three phenotypes with the three patterns of hypoperfusion.