PROJECTS SUPPORTED FOR 2012

INSERM, Centre de Recherche en Neurosciences – Lyon

Anti-NMDA encephalitis: model of AD synaptopathy (Pilote)

SUMMARY OF PROPOSED RESEARCH

In Alzheimer’s disease, Aβ oligomers lead to neuronal dysfunction by different mechanisms ^[1]. Among them, an early alteration of the glutamatergic transmission affects synaptic plasticity. They lead to a decrease of NMDA receptor (NMDAr) expression by ephrin B2 receptor (EphB2) depletion ^[2]. In encephalitis with NMDAr auto-antibodies (NMDAr-Ab), patients’ antibodies specifically alter NMDAr trafficking by reversible disruption of EphB2/NMDAr interactions ^[3]. Thus, NMDAr-Ab of patients could be used as specific and potentially reversible blockers to study effects on synaptic functions of NMDAr/EphB2 interactions.

Using NMDAr-Ab as tools to specifically block the NMDAr/EphB2 interactions, we aim to unravel consequences of this blockade at the synaptic, cellular and circuitry levels. We would like to describe the effect of a long term exposure to NMDAr-Ab on synapse plasticity, morphology and on the distribution of synaptic and extrasynaptic NMDAr subtypes.

Purified patients NMDAr-Ab from CSF and serum samples, collected by the French Reference Center of paraneoplastic neurological syndromes located in Lyon, are available. It has been clearly established that in vitro NMDAr-Ab differentially alter the dynamic trafficking of the 2 NMDAr subtypes (NR2A/NR2B) ^[3]. In vivo, decrease of total NMDAr density has been demonstrated after hippocampal injection of patients NMDAr-Ab in mice. When this exposure is acute, it early depresses long-term potentiation (LTP) ^[4] and its effect could be reversible by concomitant injection of ephrin ligand ^[3]. In this context, we aim at studying the in vivo effects of chronic exposure to patients NMDAr-Ab by infusing purified NMDAr-Ab by Alzet osmotic pumps in hippocampus of C57/Bl6 mice during 1, 7 or 14 days. We will sacrifice animals at each time points and perform electrophysiological and histological studies. At Schaffer collateral-CA1 synapses in acute hippocampal slices, LTP will be induced to study synaptic plasticity by electrophysiology. Phenotype of targeted neurons and NR2A/NR2B distribution will be determined by immunohistochemistry and confocal microscopy using double staining with well-established synaptic proteins. Dendritic spines changes will be studied by Golgi method. On organotypic hippocampal cultures, we will further study the effects of NMDAr-Ab chronic exposure on receptor endocytosis and activation of extrasynaptic receptors. We will use the same methods to evaluate the protective effects of ephrin ligand (full and truncated synthetized peptids) on synaptic plasticity and morphology.

[1] Walsh et al. (2002). Biochel Soc Trans. 30(4):552-7.
[2] Cisse et al., (2011). Nature 469(7328): 47-52.
[3] Mikasova et al. (2012). Brain. 135(5) : 1606-1621.
[4] Zhang et al. (2011) Neurobiol. Dis. 45: 610-5.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

In Alzheimer’s disease, Aβ synaptotoxicity involve complex mechanisms taking decades in the human pathology and months in animal models to set up. NMDAr-Ab of patients with limbic encephalitis offer a reliable experimental model to study early and potentially reversible synaptic alterations. Investigating synaptic NMDAr-Ab effects would most likely lead to identify potential new therapeutic strategies impacting Alzheimer’s disease, such as synaptic NMDAr rescue by ephrin ligand for instance. Indeed, the therapeutic perspective of this molecular and cellular project is to select an ephrin-derived peptide which could reverse NMDAr dysfunction consequences, with limited side-effects and unspecific interactions.

November 1st, 2012 – October 31th, 2014 (2 years)

40 000€

CNRS, Centre de Neurosciences Paris-Sud – Paris

Akt signalling links Diabetes to Alzheimer Disease

SUMMARY OF PROPOSED RESEARCH

Despite the enormous effort, time and financial support that has gone into to research for the development of therapeutic strategies to combat Alzheimer’s Disease (AD), there is still no efficient treatment available. More recently, strategies for eliminating amyloid load in the brain, currently the one that held the most promise has met with failure. More importantly however one study has shown that removal of Aβ does not result in rescue of dementia or prolongation of life. This, coupled with growing evidence suggesting the disease process is starting much earlier than was originally thought, as early as middle age; suggests early interventive strategies would be more propitious. This gives a potential causal role of risk factors for Alzheimer’s Disease, such as Diabetes Mellitus Type 2 (DMT2) a more central focus. The amyloid cascade hypothesis postulates that amyloid or species of Aβ is the driving force behind triggering the disease process; however it is not known what and how Aβ is provoked to start acting in a dysfunctional manner to trigger the disease process and induce dementia. Our hypothesis is that DMT2, largely induced by obesity in mid life, creates a dysfunctional neuronal milieu over a number of years; that will favour dysregulation of Aβ. Disruption of a number of cellular functions common to both diseases, are in part, regulated by the PI3K-Akt signalling cascade; a signalling pathway that responds directly to environmental input. Therefore the project is designed to use 2 different models of DMT2, an in utero model based on epigenetic reprogramming and a pharmaco-obesogenic model based on environmental and life style inputs. These rats will be challenged with intracerebral soluble oligomeric Aβ, that alone has little or no lasting effect; to determine whether it induces a pathological and behavioural phenotype associated with Alzheimer’s Disease. We will then use environmental (exercise, caloric restriction) and pharmacological (Insulin growth factor II, Sirtuin 1 activator, SRT1720) manipulation to positively regulate the PI3K-Akt signalling pathway and determine the importance of these mechanisms in triggering early stage Alzheimer’s Disease. The project will not only open up potential novel therapeutic strategies, but also advance our understanding of how manipulation of the environment may modify cellular function such as energy metabolism, nutrient sensing, apoptosis in DMT2 and Alzheimer’s Disease and the memory deficits associated with these diseases.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

November 1st, 2012 – October 31th, 2014 (2 years)

80 000€

INSERM, Faculté de Médecine Pitié-Salpêtrière – Paris

A new Alzheimer’s Disease treatment strategy using a synthetic TrkB agonist
Programme franco-néerlandais avec le Dr. Gunter Kenis (Maastricht) financé par l’association ISAO

SUMMARY OF PROPOSED RESEARCH

In this cross-border project, the laboratory for Cellular Neuroscience (Maastricht University, Maastricht, the Netherlands) and the Center for Psychiatry and Neuroscience (INSERM, Paris, France) join their expertise to evaluate the therapeutic potential of MIM L4-11, a newly developed, specific agonist of tropomyosin-related kinase receptor B (TrkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF). As neurotrophic support is severely compromised in Alzheimer’s disease (AD) brains and is related to memory dysfunction, we hypothesize that MIM L4-11, having advantageous pharmacological properties as compared to the BDNF protein itself or to non-specific agonists, can enhance neuronal resilience and restore memory formation.

To this end, MIM L4-11 will be chronically administered to transgenic APPswe/PS1dE9 mice at the age of 9 months, an age at which the brains of these mice show significant plaque load and memory impairment. Memory improvement will be monitored with the object location task and with both the spatial and working memory paradigm of the Y-maze test. Plaque load and synapse integrity are determined using immunohistochemical staining procedures. Changes in TrkB related signaling, including receptor and signaling pathway activation and expression of TrkB target genes, is measured with western blotting and quantitative real-time PCR. Further, the neuroprotective properties of
MIM L4-11 against amyloid-β toxicity are assessed in cell culture models.

The Maastricht center has extensive experience in the behavioral and neurochemical analysis of animal models of Alzheimer’s Disease, while the Paris center plays a vital role in the pharmacological validation of MIM L4-11 and has a broad expertise on cell culture models and molecular techniques to measure TrkB-related signaling.

The specific objectives are:

1. To evaluate the effect of MIM L4-11 on cognitive performance in wild-type and transgenic APPswe/PS1dE9 mice
2. To examine the effects of MIM L4-11 on plaque load, synapse integrity, TrkB signaling and expression of TrkB target genes in wild-type and transgenic APPswe/PS1dE9 mice
3. To assess the potential neuroprotective properties against amyloid-β-related toxicity in primary neuronal cultures of mice, and in human cell culture models

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

November 1st, 2012 – October 31th, 2014 (2 years)

36 000€

INSERM, Université de Montpellier – Montpellier

Symptomatic and curative multi-targeted drugs

SUMMARY OF PROPOSED RESEARCH

The current treatments for Alzheimer’s disease (AD) have only symptomatic benefits. It is urgent to complete this symptomatic approach by a curative one. However, aiming at reducing the formation of the toxic amyloid-β peptide (Aβ), recent developments of drugs inhibiting β- and g-secretases enzymes have been disappointing and many have been stopped due to adverse effects. In the present project, we have a similar objective: the reduction of the formation of the noxious Aβ peptide and its aggregation but using original molecules synthesized by Partner 2 and characterized by their triple-functional activities. One of these activities consists in increasing the cleavage of APP by a-secretase, allowing the release of soluble and non-amyloidogenic sAPPa, a neuroprotective peptide. To that purpose, we propose to synthesize novel agonists of the serotonin 5-HT₄ receptor (5-HT₄R). This receptor, discovered by Partner 1‘s laboratory, is known to promote the extracellular release of sAPPa by increasing a-secretase activity and also to have promnesic effect in rat and monkey. However, those 5‑HT₄R agonists will be designed to have two others activities. The first one will be the ability to inhibit the acetylcholinesterase (AChE) activity by acting on the catalytic active site (CAS), thus resulting in an increased level of acetylcholine (ACh) known to be beneficial in improving cognitive, behavioural, and functional impairment. The second one will be another disease-modifying activity by inhibiting a less-considered AChE property, the Aβ aggregation. Indeed, a peripheral anionic site (PAS) in AChE is known to form a stable complex with Aβ favouring its aggregation. Preliminary experiments indicated that it is possible to find a drug having both 5-HT₄R agonist profile and AChE inhibitory property. This drug stimulates the release of sAPPa and reduces the senile plaque formation in vivo. A common pharmacophore will be designed, gathering the structural features of both this drug and of donepezil, known to interact with PAS. Novel compounds will be synthesized based on this pharmacophore, selected to have all the aforementioned properties and tested in vivo for their ability to reduce the formation of senile plaques (Partner 1) in transgenic mouse models of AD (5XFAD). Thus, the drugs that we aim to obtain will have dual-symptomatic properties (due to their ability to increase ACh release and cAMP production via 5-HT₄ receptor activation and to increase ACh concentration via their inhibition of ACh hydrolysis) and disease-modifying properties (increase in sAPPa release and inhibition of Aβ aggregation). This experimental work is part of a modern approach that consists in designing Multi-Target-Directed Ligands (MTDLs) for treating complex pathologies.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

November 1st, 2012 – October 31th, 2014 (2 years)

80 000€

INSERM, Centre de Recherche Saint-Antoine – Paris

Targeting Neuronal IGF Signaling in Alzheimer Mice

SUMMARY OF PROPOSED RESEARCH

Alzheimer disease (AD) is a progressive and irreversible neurodegenerative dementia, for which the main risk factor is age. Research into the mechanisms of ageing has provided strong evidence that the pleiotropic hormone insulin-like growth factor 1 (IGF-1) is an important regulator of organismal longevity suggesting that it could also affect age-related AD phenotype. Indeed, a few studies suggest a role for IGF signaling in AD. Intriguingly, both systemic IGF-1 treatment and partial inactivation of its receptor IGF-1R reduces astrogliosis and cognitive deficits in AD mouse models. Clearly, more research is needed to clarify this apparent discrepancy. Our aim is to identify cellular mechanisms and pathophysiology underlying the link between brain IGF-1 signaling during ageing and AD. To test the hypothesis that IGF-1 signaling can affect AD progression, we propose to use a novel AD mouse model, where IGF-1R can be conditionally deleted in adult forebrain neurons upon tamoxifen treatment. In summary, we combined IGF-1R^flox/flox, CaMKIIα-CreER^T2, and APPswePSEN1dE9 mice to create the inducible model CaMKIIα-CreER^T2;APPswePSEN1dE9;IGF-1R^flox/flox, that will enable us to choose the specific time for KO induction during AD progression. Using this versatile in vivo approach, our group is currently examining IGF-1R impact on AD onset. To further explore the potential of therapeutic use, the present project aims at evaluating whether inactivation of IGF-1 signaling can affect established AD phenotype. KO will thus be induced after amyloid plaques formation (symptomatic stage). As a complementary approach and to get insights into cellular mechanisms of neuronal IGF-1R inhibition on CNS response to Aβ oligomers-induced neurotoxicity, we will inject Aβ peptides into the brain of adult inIGF-1R mice. This model will exclude compensatory effects due to early onset of IGF-1R inactivation and will help distinguish between systemic and local effects of IGF-1R deletion. Finally, this model, currently being examined at young age in the lab, is especially relevant to study therapeutic intervention targeting IGF-1. Parameters to be examined include sensorimotor (Rotarod, open field) and cognitive (Barnes maze, Novel Object Recognition) performances, histopathological changes (amyloid plaque/Aβ burden, glial cell activation, neuroinflammation, neurodegeneration) and activation of IGF-1R downstream signaling molecules IRS, PI3K and Akt.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Abnormalities in IGF-1 receptor expression and its downstream signal transducing molecules have been demonstrated in postmortem AD brains. However, little is known about the role of brain IGF-1R during aging and AD. Because a treatment for AD must be effective when applied upon diagnosis late in life, we propose to inactivate neuronal IGF-1 signaling during established AD phenotype using a novel AD model capable of inducible and neuron-specific deletion of IGF-1R upon tamoxifen treatment. This project will assess in vivo the impact of IGF-1 signaling on cognitive performances and pathological changes. Resulting data should allow a better translation from preclinical research to clinical studies by providing further insights into the potential of IGF-1 as therapeutic target in Alzheimer’s Disease.

November 1st, 2012 – October 31th, 2014 (2 years)

80 000€

INSERM, Collège de France – Paris

Role of glial connexins in Alzheimer’s disease

SUMMARY OF PROPOSED RESEARCH

Our project aims at understanding the contribution of astroglial connexins (Cxs), the membrane proteins forming gap junction channels and hemichannels, to neuronal dysfunction and disease progression in APP_swe/PS1_dE9 mice, a murine model of Alzheimer’s disease (AD). We have previously shown that Cx expression changes occur in astrocytes in the vicinity of amyloid-β (Aβ plaques in brains from AD patients and APP_swe/PS1_dE9 mice. We hypothetize that these changes affect the ability of astrocytes to exchange small molecules (ATP, glutamate, energy metabolites) with the extracellular medium via hemichannels and with other astrocytes via gap junctions, hence contributing to alter the periplaque environment but also more distant sites via long-range diffusion of potentially neurotoxic or neuroprotective signals through Cx-mediated astrocytic networks. Firstly, we propose to analyze whether Cx channel functions are modified in APP_swe/PS1_dE9 mice at the proximity of Aβ plaques. The activity of hemichannel and gap junction channels will be tested in acute brain slices of APP_swe/PS1_dE9 mice, using respectively ethidium bromide uptake quantified in cells identified by GFAP immunostaining and measure of the intercellular diffusion of gap junction permeant dyes injected into one patched astrocyte. Aβ plaques will be visualized by Aβ immunostaining. A battery of pharmacological tools will be used to confirm the specific involvement of Cxs in these functions. We will also investigate whether Cxs working as hemichannels and/or full channels are involved in the propagation of interastrocytic Ca²⁺ waves already described in this mouse; Ca²⁺ imaging will be performed in slices loaded with a fluorescent calcium dye or in vivo using a multiphoton microscope. Secondly, we will generate APP_swe/PS1_dE9 mice deleted in Cxs to analyze the consequences of Cx knocking-out in APP_swe/PS1_dE9 mice on diverse aspects related to the amyloid pathology: the propagation of interastroglial Ca²⁺ waves, the extent and chronology of Aβ deposition evaluated by ELISA and immunohistochemical approaches, the extent of neuronal damage and dysfunction assessed by immunohistochemical stainings and electrophysiological recordings of neuronal activity in slices as well as behavioral performances tested in memory tasks using Y maze task and Morris water maze.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Our project concerns AD in the framework of a neuroprotective strategy. Astrocytes treated with the Aβ peptide release toxic molecules (glutamate and ATP) via activated Cx hemichannels, that affect neuronal survival. Cx-mediated astroglial networks can also propagate neurotoxic or neuroprotective effects at more distant sites. The completion of our project should allow to determine whether astroglial Cxs contribute to the amyloid pathology developed by a murine model of AD, in particular neuronal dysfunction, neurodegeneration and cognitive impairment and to identify which Cx channel function is involved (hemichannel and/or gap junction) in this process. Hence, Cxs should represent new therapeutical targets on which to act to slow down the process of neurodegeneration and/or to improve neuronal functions.

Publication :

Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology and roles. Giaume C., Leybaert L., Naus C.C. and Saez JC. Frontiers in Pharmacology. 2013 Jul 17;4:88.

November 1st, 2012 – October 31th, 2014 (2 years)

80 000€