Validation of MT5-MMP as a new therapeutic target in Alzheimer’s disease and mechanisms of action – MAD5

We use different models of cells in culture: i) HEKswe or SH-SY5Y human neuroblastoma cells, carrying a Swedish family mutation; ii) cultures of primary neurons / astrocytes from 5xFAD transgenic mice and 5xFAD mice deficient in MT5-MMP; Iii) human neural cells derived from induced pluripotent stem cells (iPS).
We use as an in vivo model essentially the 5xFAD transgenic mouse which summarizes the fundamental traits of the disease: progressive accumulation of soluble / oligomeric and fibrillar Aß, reactive gliosis accompanied by an increase in the levels of pro-inflammatory mediators, deficits in cognitive, emotional and motor behaviors, synaptotoxicity, and neuronal death in the cortex and hippocampus.
Biochemical and molecular biology techniques including: gene cloning and constructing cDNA sequences for insertion into expression plasmids and / or lenti / adenoviral vectors, stable or transient transfection in our cellular models of these constructs, gene expression by qPCR, protein content assessment by immunoblot and ELISA, analysis of protein interactions by immunoprecipitation and the VENUS system.
Immunohistochemical and immunocytochemical labeling using antibodies and confocal and / or super-resolution microscopy, followed by image analysis using Imaris (eg, morpho-quantitative analysis of dendritic spines), analysis of the intracellular trafficking with the aid of markers of different components of the endosomal / lysosomal system.
Analysis of neuronal activity on cultures of mouse brain primary neurons and human neuron cultures derived from iPS cells by the patch-clamp technique in whole cell configuration.

- Evidence of the harmful effects of MT5-MMP in vivo in the frontal cortex, an area of the brain also affected in AD, which expanded in the same direction the previous observations in the hippocampus and neocortex. An article has been published (Baranger et al., 2017).
- Transient expression of MT5-MMP in a heterologous system stimulates APP traffic to early endosomes, one of the main loci of amyloidogenesis, and the production of Aß. These results are also part of the article Baranger et al., (2017).
- The absence of MT5-MMP is neuroprotective at an advanced stage of the pathology in Tg mice (12 months) according to our unbiaised stereological analysis. This is accompanied by a smaller accumulation of fibrillar Aß and also of small Aß oligomers at different phases of the pathology.
- The absence of MT5-MMP in neuronal / astrocyte cultures decreases the response to IL-1ß, suggesting a functional link between the cytokine and the proteinase.
- Development of lenti / adenoviral vectors coding for APP fragments generated by MT5-MMP mediated cleavage, to study their potentially cytotoxic activities. At present only the ?-CTF fragment would be cytotoxic.
- Development of iPS cell lines from healthy individuals and Alzheimer patients that we then differentiated into neurons. We also introduced deletions and inactivating mutations for MT5-MMP in these iPS using CRISPR / Cas9.

Continue to study the functional interactions between MT5-MMP and IL-1ß, in particular via the possible interaction between MT5-MMP and ubiquitin ligases activated during IL-1ß signaling. We will study whether the deficiency of MT5-MMP can influence the a priori disruptive effect of the cytokine on synaptic activity, based on our preliminary results. We will use neuronal cultures derived from genetically modified MT5-MMP- / - mice, as well as neurons derived from iPS cells deleted / inactivated for MT5-MMP to determine how these interactions between MT5-MMP and IL-1ß affect the metabolism of APP / Aß, the inflammatory response and synaptic activity. ICV injection of IL-1ß will allow us to evaluate whether MT5-MMP deficiency also interferes with IL-1ß signaling in vivo.
To study which domains of MT5-MMP interact with APP (and other members of the amyloid cascade, eg, nicastrin) and the functional consequences, using the generated lentiviral constructs. We will evaluate the impact on the metabolism and trafficking of APP and the inflammatory response in our murine and human neuronal cultures.
We undertook the design of AAV and lentiviruses expressing the ßAPP fragments obtained by ?-secretase. These vectors will make it possible to examine the influence of fragments related to ?-secretase in vivo after ICV injection, as well as in the primary neurons in culture with regard to their viability and their electrophysiological responses. AAV can be used in the neonatal stage and can monitor intrinsic toxicity in naïve and also transgenic Alzheimer mice, in order to evaluate the putative influence of ? fragments before or after a given lesion (plaques, entanglements, synaptic loss).

Peer reviewed international publications
1. Baranger et al., 2017. Frontiers in Molecular Neuroscience doi: 10.3389/fnmol.2016.00163.

Communications (Conferences)
S. Rivera. MT-MMPs new actors and potential targets in Alzheimer’s disease. Gordon Research Conference on Matrix Metalloproteinases, University of New England, Biddeford ME, USA. July 9-14, 2017. Conférence invitée.

K Baranger, A Bonnet, S Girard, JM Paumier, L Garcia-Gonzalez, W Elmanaa, D Stephan, A Bernard, C Bauer, E Charrat, K Moschke, S Lichtenthaler, F Roman, F Checler, M Khrestchatisky and S Rivera. MT5-MMP promotes amyloidogenesis, neuroinflammation and cognitive decline related with the frontal cortex in 5xFAD mice. The 13th International Conference on Alzheimer's & Parkinson's diseases. Vienne, Autriche. 2 Avril 2017. Oral communication.

S. Rivera, A. Bonnet, JM Paumier, N Crouzin, L Garcia Gonzalez, A Carrete, A Bernard, M Vignes, S Lichtenthaler, F Checler, M Khrestchatisky, K BarangerMT5-MMP, a new potential therapeutic target in Alzheimer’s disease. 52nd International Conference on Medicinal Chemistry, July 6-8, 2016 | CAEN, Normandy, France. Conférence invitée.

Our global objective is to better understand some of the proteolytic pathways operating in amyloidogenesis and neuroinflammation, which are key processes in neurodegeneration and cognitive decline in Alzheimer’s disease (AD). AD is the most devastating neurodegenerative disorder with a major socio-economic burden that is accentuated by the absence of effective treatments curing or slowing down the progression of the disease. Despite a variety of therapies currently under investigation, the discovery of a cure does not seem to be within reach at this point. Therefore, there is urge in identifying new targets in the triggering/progression of the disease and understanding the underlying molecular mechanisms preceding irreversible cognitive deficits. It is in this context that the MAD5 project seeks to bridge some of the molecular mechanisms underlying amyloidogenesis and neuroinflammation across different stages of AD. To this end, two leading CNRS laboratories from Marseille and Sophia-Antipolis with complementary skills have joined forces to develop the MAD5 project in the context of an ongoing collaboration. Together, they have generated over the last three years consistent preliminary data now submitted for publication 1. Using in cellulo and in vivo models of AD, in particular transgenic mice (Tg) that develop the main symptoms of AD, we demonstrated for the first time that a member of the matrix metalloproteinase (MMP) family of proteinases contributes to the amyloidogenic processing of amyloid precursor protein (APP), as well as the concomitant inflammatory response. This prompts us to hypothesize that this MMP is a new important factor that could contribute to Alzheimer’s pathogenesis and hence a novel potential therapeutic target. In order to further validate these hypotheses and gain insight into the underlying mechanisms of action, our specific aims are to:
1. Assess the impact of the modulation of this MMP on the pathological process and the cognitive decline in transgenic mice that develop some of the symptoms observed in AD patients.
2. Assess the impact of the MMP modulation on neuroinflammation and the asssociated mechanisms of action.
3 Assess the functional interactions of this MMP with APP and other key elements of the amyloidogenic pathway.

The main originality of MAD5 is that it unveils a new important element of the proteolytic puzzle that opens new research avenues to study the mechanisms of AD pathogenesis. These new areas of research should be complementary and/or alternative to those classically studied under the exclusive scope of well-known ß- and ?-secretases.

The impact of MAD5 is expected at different levels: 1) To identify new molecular pathways that contribute to AD pathogenesis, and lead eventually to innovative therapeutic strategies without the side effects observed when inhibiting ß- and/or ?-secretases. 2) To identify APP processing proteinases that should contribute to better understand the amyloidogenic role of APP. 3) To improve our knowledge of the functional interplay between neuroinflammation and amyloidogenesis, which is still a major unsolved question in AD. 4) To identify a new neuroinflammatory signalling pathway that could be crucial for the comprehension of neuroinflammation in AD and other neurodegenerative diseases. 5) To implement and validate molecular tools that could be used as templates for drugs that could modify MMP activity/interactions.
In all respects, and to the best of our knowledge, the MAD5 project has little competition worldwide at this point, and our consortium is ideally placed to lead present and future researches on the validation of a new promising target in AD.