Diane Houitte

Alteration of sensory-evoked metabolic and oscillatory activities in the olfactory bulb of GLAST-deficient mice

Astrocytes are key cellular elements in both the tripartite synapse and the neurovascular unit. To fulfill this dual role in synaptic activity and metabolism, they express a panel of receptors and transporters that sense glutamate. Among them, the GLT-1 and GLAST transporters are known to regulate extracellular glutamate concentrations at excitatory synapses and consequently modulate glutamate receptor signaling. These major uptake systems are also involved in energy supply to neurons. However, the functional role of GLAST in concurrent regulation of metabolic and neuronal activity is currently unknown. We took advantage of the attractive structural and functional features of the main olfactory bulb to explore the impact of GLAST on sensory information processing while probing both glutamate uptake and neuronal activity in glomeruli and deeper cellular layers, respectively. Using odor-evoked 2-deoxyglucose imaging and local field potential recordings in GLAST knockout mice, we show in vivo that deletion of GLAST alters both glucose uptake and neuronal oscillations in olfactory bulb networks.

A role of mitochondrial complex II defects in genetic models of Huntington's disease expressing N-terminal fragments of mutant huntingtin

Huntingtons disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron–Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.

The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimers disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntingtons disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.

Gadolinium-staining reveals amyloid plaques in the brain of Alzheimer's transgenic mice

Detection of amyloid plaques in the brain by in vivo neuroimaging is a very promising biomarker approach for early diagnosis of Alzheimers disease (AD) and evaluation of therapeutic efficacy. Here we describe a new method to detect amyloid plaques by in vivo magnetic resonance imaging (MRI) based on the intracerebroventricular injection of a nontargeted gadolinium (Gd)-based contrast agent, which rapidly diffuses throughout the brain and increases the signal and contrast of magnetic resonance (MR) images by shortening the T1 relaxation time. This gain in image sensitivity after in vitro and in vivo Gd staining significantly improves the detection and resolution of individual amyloid plaques in the cortex and hippocampus of AD transgenic mice. The improved image resolution is sensitive enough to demonstrate an age-dependent increase of amyloid plaque load and a good correlation between the amyloid load measured by μMRI and histology. These results provide the first demonstration that nontargeted Gd staining can enhance the detection of amyloid plaques to follow the progression of AD and to evaluate the activity of amyloid-lowering therapeutic strategies in longitudinal studies.

Restricted Transgene Expression in the Brain with Cell-Type Specific Neuronal Promoters

Tissue-targeted expression is of major interest for studying the contribution of cellular subpopulations to neurodegenerative diseases. However, in vivo methods to investigate this issue are limited. Here, we report an analysis of the cell specificity of expression of fluorescent reporter genes driven by six neuronal promoters, with the ubiquitous phosphoglycerate kinase 1 (PGK) promoter used as a reference. Quantitative analysis of AcGFPnuc expression in the striatum and hippocampus of rodents showed that all lentiviral vectors (LV) exhibited a neuronal tropism; however, there was substantial diversity of transcriptional activity and cell-type specificity of expression. The promoters with the highest activity were those of the 67 kDa glutamic acid decarboxylase (GAD67), homeobox Dlx5/6, glutamate receptor 1 (GluR1), and preprotachykinin 1 (Tac1) genes. Neuron-specific enolase (NSE) and dopaminergic receptor 1 (Drd1a) promoters showed weak activity, but the integration of an amplification system into the LV overcame this limitation. In the striatum, the expression profiles of Tac1 and Drd1a were not limited to the striatonigral pathway, whereas in the hippocampus, Drd1a and Dlx5/6 showed the expected restricted pattern of expression. Regulation of the Dlx5/6 promoter was observed in a disease condition, whereas Tac1 activity was unaffected. These vectors provide safe tools that are more selective than others available, for the administration of therapeutic molecules in the central nervous system (CNS). Nevertheless, additional characterization of regulatory elements in neuronal promoters is still required.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease

The mechanisms underlying preferential atrophy of the striatum in Huntingtons disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3′-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

The neuroprotective agent CNTF decreases neuronal metabolites in the rat striatum: an in vivo multimodal magnetic resonance imaging study.

Ciliary neurotrophic factor (CNTF) is neuroprotective against multiple pathologic conditions including metabolic impairment, but the mechanisms are still unclear. To delineate CNTF effects on brain energy homeostasis, we performed a multimodal imaging study, combining in vivo proton magnetic resonance spectroscopy, high-performance liquid chromatography analysis, and in situ glutamate imaging by chemical exchange saturation transfer. Unexpectedly, we found that CNTF expression through lentiviral gene transfer in the rat striatum significantly decreased the levels of neuronal metabolites (N-acetyl-aspartate, N-acetyl-aspartyl-glutamate, and glutamate). This preclinical study shows that CNTF remodels brain metabolism, and suggests that decreased levels of neuronal metabolites may occur in the absence of neuronal dysfunction.

Capucin does not modify the toxicity of a mutant Huntingtin fragment in vivo

Genes selectively expressed in the striatum may be involved in the preferential vulnerability of striatal neurons to Huntingtons disease (HD). Here, we investigated whether perturbations of Capucin expression, which is enriched in the striatum and downregulated in Huntingtons disease models, could modify the neurotoxicity induced by the injection of a lentiviral vector encoding a short N-terminal fragment of mutant Huntingtin (mHtt) into the mouse striatum. Neither constitutive Capucin deficiency in knockout mice nor lentiviral vector-mediated Capucin overexpression in the striatum of adult wild type mice significantly modified vulnerability to the mHtt fragment in vivo, suggesting that Capucin has no impact on mHtt toxicity.

In vivo amyloid plaque detection using contrast-enhanced magnetic resonance microscopy in transgenic mice

regions of interest in dorsal hippocampus (Fig. 1). Images were acquired using rapid acquisition with relaxation enhancement (RARE) and echo-train multi-slice-multi-echo (MSME) sequences, from which T1 and T2 maps were generated, respectively; and a fast imaging with steady-state precession (FISP) protocol, from which both T1 and T2 maps were generated. Data from manually-selected regions of interest were analyzed using software scripts in IDL language (ITTVIS). Oneor two-way analyses of variance (ANOVA) with Fischer’s post-test were used to statistically assess the data. Tissue from the animals was examined for markers of astrogliosis and microglial activation. Results: T1 in hippocampus and cortex (p<0.01, Fig. 2) were significantly increased with age whilst T2 in hippocampus (p<0.05) and cortex (p<0.01, Fig. 3) were significantly decreased with age. Treatment with rosiglitazone significantly attenuated the age-related T1 increase in both regions (p<0.05, Fig. 2) and it increased T2 relaxation time in hippocampus of aged, but not young rats (p < 0.05, Fig. 3). Conclusions: The data demonstrate an age-related increase in T1 in hippocampus and cortex, which is attenuated by rosiglitazone; this correlates with astrocytosis (Fig. 4). We also show a decrease in T2 relaxation time in hippocampus and cortex, which correlates with an increase in microglial activation (Fig. 5), and report that rosiglitazone-treatment of aged rats increases hippocampal T2.

SPECIFIC INHIBITION OF ASTROCYTE REACTIVITY IMPROVES SOME DISEASE OUTCOMES IN ALZHEIMER'S DISEASE MICE

in the lipid microenvironment, which may result from Ab accumulation and neuronal degeneration in Alzheimer’s disease (AD). Rare TREM2 variants that impair lipid/lipoprotein recognition lead to an increased risk of developing AD. In murine models of AD, TREM2 deficiency prevents Iba-1 myeloid cells clustering around Ab deposits. However, the origin of these cells and their impact on Ab accumulation and Ab-associated neurotoxicity are a matter of debate. Methods:Using parabiosis, we determined the contribution of peripheral blood monocytes to the pool of myeloid cells that are associated with Ab deposits in 5XFAD and APPPS1-21 mice. To understand the role ofmicroglia inAb accumulation andAb-associated neurotoxicity, we examined the impact of TREM2 deficiency at the onset of Abrelated pathology in 5XFAD mice. Results:We found that amyloidassociated myeloid cells were derived from expansion of brain-resident microglia rather than from recruitment of peripheral monocytes. TREM2 deficiency impaired the clustering of microglia around amyloid plaques even at the onset of Ab-related pathology. Remarkably, morphological analysis revealed that plaques from TREM2-deficient 5XFAD mice were drastically distinct, appearing more diffuse and less dense than those from 5XFAD mice. Moreover, Ab plaques in TREM2-deficient mice were associated with significantly greater neuritic dystrophy. Conclusions:We concluded that peripheral blood monocytes have aminimumcontribution to the expansion ofmicroglia under normal physiological condition in two AD transgenic models. One mechanism via which TREM2 protects from AD is by enabling the proliferation of amyloid-associated microglia to prevent the physical diffusion of Ab deposits, thereby limiting their toxicity.