Title of the project: Transferring Non autonomous cell degeneration models between EU and USA for development of effective therapies for Motor Neuron Diseases (MND)

The project “Transferring Non autonomous cell degeneration models between EU and USA for development of effective therapies for Motor Neuron Diseases (MND)” (NO-MND) is funded by the Marie Curie International Research Staff Exchange Scheme with the aim to develop an effective therapies for MND disease transferring non autonomous cell degeneration models between EU and USA.

PROJECT SUMMARY

Amyotrophic lateral sclerosis (ALS) and Spinal Muscular Atrophy (SMA) are fatal diseases characterized by selective degeneration of motor neurons without any effective treatment. Non-neuronal cells like astrocytes play a key role in the pathogenesis of ALS and SMA. Understanding the mechanisms of cell death caused by non-neuronal cells is a major research target and it has been proposed that transplantation of non-neuronal cells can represent a new therapeutic strategy for these disorders. The proposed NO-MND project addresses scientific issues related to “non autonomous cell death” in motor neuron disorders, by strengthening research partnerships through staff exchanges and networking activities between 2 European research organizations from Italy, Greece and 1 organization from the USA. The program is focused on: 1) development of in vitro disease models to investigate molecular interactions between non-neuronal cells and motor neurons using cell reprogramming technology; 2) investigation of potential therapeutic efficacy of non-neuronal cells transplantation in motor neuron disease models. The project is based on a 4 years coordinated joint program of exchange of researchers. The three groups have a long standing experience in motor neuron disease field, in cellular and in vivo models, non cell autonomous diseases mechanism and cell replacement therapy. Their ongoing research is supported by multiple grants. Four topics have been identified as most relevant for all partners involved in the joint program: astrocytes reprogrammed from fibroblasts of ALS and SMA patients in a cellular model to study the pathogeneses of these diseases and their use in therapeutic approaches for ALS and SMA using in vivo models. The IRSES scheme provides a unique opportunity to integrate past collaboration activities into a coherent program addressing an issue of high priority for public health agendas of the EU and USA.

Partners

• University of Milan, Neural Stem Cell Lab

The Neural Stem Cells Lab is one of the research facilities of the Centro Dino Ferrari, University of Milan, and is located in the Foundation Ca ‘Granda Ospedale Maggiore Policlinico (http://www.centrodinoferrari.com/laboratori/laboratorio-di-cellule-staminali-neurali/?lang=en).

Our research group field is in the development of cellular and molecular therapeutic

strategies for neurodegenerative diseases (particularly childhood-onset motor neuron disease such as spinal muscular atrophy and spinal muscular atrophy with respiratory distress type 1 or adult as Amyotrophic Lateral Sclerosis) and hereditary neuropathies (Charcot-Marie-Tooth type 2A 2).

The transplantation of stem cells may represent potential therapeutic approaches for neurodegenerative diseases, and in particular for motor neuron diseases. The mechanisms by which the transplantation acts include (in addition to cell replacement), the release of neuroprotective factors produced by transplanted cells. In the perspective of developing new therapeutic approaches for these diseases, our laboratory is focused on the latest techniques of cellular reprogramming. These techniques allow the obtainment of induced pluripotent stem cells (iPSCs) from human patients and healthy subjects. These patient-specific cells are useful as model of disease, cell sources for cell-mediated therapy and are strategies for the development of new therapies.

Our laboratory has demonstrated that the transplantation of neural stem cells (NSCs) and motor neuron precursors exert a positive effect on the disease phenotype in mouse models of motor neuron disease, (Corti et al., 2006-2014), supporting the potential cell-mediated therapeutic approach for these diseases. Moreover, the research team is focused also on another promising therapeutic approach for these neurodegenerative disorders: the use of antisense oligonucleotides (ASO) or morpholino (MO) with the aim to knock down gene expression, modify RNA splicing or inhibit miRNA activity and maturation in order to correct the defect of the disease at the molecular level. They are also working on gene therapy approaches based on the traditional introduction of a healthy copy of the gene using adenoviral vectors (AAV), for the autosomal recessive disorders.

This research is the  combined contribution of a  collaboration between researchers, medical doctors, trainees, students of Biology, Biotechnology, and Medicine, and researchers of national and international laboratories. The teaching activity and the seminars enable the dissemination of the knowledge acquired by our group at national and international level.

• Academy of Athens, Biomedical research foundation

The interest of the lab lies in the pathogenesis of neurodegenerative conditions, such as Parkinson’s, Alzheimer’s and Huntington’s Diseases and Amyotrophic Lateral Sclerosis. We are studying potential mechanisms through which such conditions may initiate and propagate within the nervous system, with the hope that, if these mechanisms are identified, they can be potential targets for neuroprotective therapies. The pathophysiological mechanisms that are more closely studied include protein misfolding, protein aggregation, inclusion formation and dissolution, impairment of protein degradation systems, synaptic dysfunction and neuronal cell death. These processes constitute common threads in such neurodegenerative conditions.  The group is more focused in the pathogenesis of Parkinson’s Disease (PD) and, in particular, in deciphering the link between identified genetic defects and the disease. Activities range from the study of biological material from patients afflicted with PD, up to cell culture and animal models. Models are largely based on genetic defects linked to PD, but also include more classical neurotoxin approaches, such as MPTP. Relevant biochemical pathways identified in such models are then examined in patient biological material, while insights from the study of the patients are used to develop new models.

• Columbia University, Motor Neuron Center for Neurobiology and Disease

The Przedboski Lab is located at the Columbia University inside the Motor Neuron Center. Przedboski is professor of Neurology and Pathology and is the co-director of the Motor Neuron Center (http://www.columbiamnc.org/bio.php?id=4).

The research conducted in Przedborski laboratory is geared toward unraveling the molecular basis of neurodegeneration and devising therapeutic strategies to hamper the processes that cause neuronal death, the source of many debilitating disorders. To that end, this laboratory has concentrated its research efforts on the mouse model of amyotrophic lateral sclerosis (ALS) provided by the transgenic expression of the mutant superoxide dismutase-1 (mSOD1). For the past decade Przedborski have extensively evaluated the role of the apoptotic machinery in the demise of motor neurons. It appears that apoptosis, which is a form of programmed cell death, plays a major role in the degeneration of spinal cord motor neurons in ALS. This view is supported by several publications from this laboratory. Through genetic and pharmacological interventions aimed at alleviating these cytotoxic changes, this laboratory was able to prolong survival and attenuate neuronal death in the mSOD1 mouse model of ALS. More recently Przedborski have also begun to explore the role of neuroinflammation as part of a non-cell-autonomous mechanism by which glial cells could hasten motor neuron death and contribute to the selective motor neuronal degeneration seen in this fatal disease.

Description of the project

Work packages

• WP1: Project management and network coordination

Objectives: To keep the project on track with respect to the overall objectives and in time with respect to the approved schedule; Develop solutions for any deviations from the project plan.

Description of work

Task1.1 – Project Management: Coordinate the project and manage the workflow; Ensure short term project goals are met; Handle liaison with the Commission; Manage project risks; Facilitate communication between all members of the consortium; Organize regular project meetings to review scientific progress, discuss difficulties and devise solutions. In the set -up phase, virtual meetings will also be held to facilitate the management of the project, develop the format for work packages, workshops and working papers, arrange visits and establish procedures for recording activities, financial management and the publication of findings. Beside seminars that will be systematically given by researchers during their visits to each site, our transfer of knowledge plan is based on two yearly events that will be held successively in the three partner countries (Milano/Athens/New York/Milano).

Task1.2 – IPR and financial management: Arrange collection of financial returns and cost statements; Ensure that resources are deployed as needed; Manage IPR issues.

Task1.3 – General supervision of staff exchanges: Supervise and keep track of researcher exchanges; Practical arrangements for travel and accommodation. The coordinator will be supported by the other managers ; they will update each other about this specific aspect of the project with skype conferences/emails. Adopt all mechanisms to guarantee that appointed researchers work in a safe environment.

• WP2: Astrocytes reprogrammed from fibroblasts of ALS patients as an in vitro model and for therapy development

Objectives: To establish a humanized in vitro model of ALS to investigate the mechanisms of astrocyte-mediated toxicity on motor neurons and to determine whether the genetic modification of astrocytes or motor neurons can be used as a possible therapeutic strategy, to adapt this model for high-throughput screening of therapeutic drugs.

Description of work

Task 2.1 – Derivation of astrocytes from two sources: Cultured human astrocytes (ASTRO) will be derived from: reprogrammed SALS/FALS patients’ fibroblasts into iPSCs and directly trans-differentiated cells. The implementation of in vitro protocols for the differentiation of ASTRO from iPSC as well as trans-differentiation into ASTRO is fundamental. Moreover, cell culture conditions specifically optimized for the acquisition of the astrocytic phenotype will be developed. The development of a method for generating highly purified ALS ASTRO to be used in co-culture with motor neurons is a central goal of this WP. This task will be the main topic of the first annual 2-day Workshop that will be held in Milano in September 2014. It will have a particular emphasis on”how to generate astrocytes and MNs from iPS/neurospheres/fibroblasts”. The first day, three international experts will give individual lectures on the subject followed by a 3 hour-round table in the afternoon. The next day, the participants will be separated in 4 groups of 15 students to participate in 4 successive wet labs of 90 minutes to actually observe the basics of these different techniques.

Task 2.2 – Establishment of an ALS in vitro model based on the co-culture of SALS/FALS ASTRO and motor neurons: To investigate whether ASTRO from FALS and SALS patients are toxic to human wild type motor neurons by co-culturing iPSC–derived motor neurons from ALS patients and wild-type (WT) subjects with differentiated astrocytes (both from iPSCs and trans-differentiated fibroblasts) from each subject. To demonstrate that SALS astrocytes exert a non-autonomous cell death towards motor neurons is one of the major aims. Seminars and Lectures regarding this specific task and involving students and Academic staff will be held by the researchers during their visits to each site.

Task 2.3 – Investigation of the mechanisms of non-autonomous induced cell death of ASTRO: To investigate the mechanisms of astrocytes mediated non-autonomous cell death events focusing on two strategies: 1) genome wide expression analysis of ASTRO using next generation sequencing (RNASeq) in order to provide a thorough assessment of the ALS astrocyte transcriptome and to identify potential targets responsible for motor neuron toxicity. Targets will be prioritized based on the knowledge of their potential direct toxicity but also based on their involvement in toxic signaling pathways; 2) Validation of the target expression level changes in diseased ASTRO by quantitative PCR and western blots.

Seminars and Lectures regarding this specific task and involving students and Academic staff will be held by the researchers during their visits to each site.

Task 2.4 – Testing of possible therapeutic strategies in co-culture model of ALS-ASTRO with ALS motor neurons: To investigate the role in ALS pathogenesis of selected differentially expressed genes in ALS-ASTRO (identified in Task 2.3) compared to controls by overexpressing/silencing them before co-culturing with motor neurons. To express target genes in astrocytes in order to evaluate any neuropathological modifications and/or neuroprotective properties on co-cultured motor neurons. To silence specific up-regulated genes in ALS astrocytes and observe whether a neuroprotective effect occurs on co-cultured motor neurons. Results obtained will be presented for critical discussion at the end-of the year workshops.

• WP3: Astrocytes mediated toxicity on motor neurons death in SMA         

Objectives: To investigate the role of non-neuronal cells in SMA-related motor neuron degeneration. To assess whether SMA astrocytes can exert a toxic effect and evaluate the mechanisms of astrocytes-mediated cell death on motor neurons. To investigate whether interfering with these events can be employed as a possible therapeutic strategy. To establish an in vitro model to study SMA molecular pathogenesis and for therapeutic assessing.The second “NO-MND day meeting” will be held in Milano in May 2015. The main thematic will be the death molecular cascade in Spinal Muscular Atrophy and other MNDs.The second annual 2-day Workshop will be held in New York City in September 2015 and it will have a particular emphasis on “How to select and use biaised and unbiaised strategies such as reverse Gene Engineering to elucidate a molecular death cascade

Description of work:

Task 3.1-Derivation of astrocytes from reprogrammed SMA patients’ fibroblasts into iPSC and directly trans-differentiated cells: To develop a method for generating highly pure SMA/wild-type ASTRO to be used in co-culture with motor neurons. A second goal is the generation of iPSC-derived motor neurons derived from the same subjects, to be used in co-culture experiments.

Task 3.2-Definition of an SMA in vitro model based on the co-culture of human SMA ASTRO and motor neurons: To demonstrate that SMA ASTRO exert a non-autonomous cell death effect towards motor neurons by co-culturing iPS cell–derived motor neurons from SMA patients and WT subjects with differentiated ASTRO from patients or control.

Task 3.3- 1) genome wide expression analysis of ASTRO using next generation sequencing (RNASeq) in order to provide a thorough assessment of the ALS astrocyte transcriptome and to identify potential targets responsible for motor neurons’ toxicity; 2) Validation of the target expression level changes in diseased ASTRO by quantitative PCR and western blots.

Task 3.4-Testing possible therapeutic strategies in co-culture model of SMA-ASTRO with SMA motor neurons: To silence specific up-regulated genes in SMA astrocytes and observe whether a positive effect occurs on co-cultured motor neurons. To express target genes in astrocytes in order to evaluate any neuropathological modifications and/or neuroprotection properties on the motor neurons in co-culture.

• WP4: Transplantation of glial precursors (GRP) derived from stem cells (iPS) as a therapeutic strategy for ALS

Objectives: To contribute to the development of a stem cell−based approach to treat ALS. To test therapeutic efficacy and molecular mechanisms linked to iPSC derived-GRPs transplantation into ALS mice model, (SOD1G93A mice). To find novel therapies for treating motor neuron diseases.          The third “NO-MND day meeting” will be held in New York in May 2016, the main thematic will be the promise and challenge of cell replacement therapy in MNDs. The third annual 2-day Workshop will be held in Athens in September 2016 and it will have a particular emphasis on “how to design a good preclinical study in ALS and SMA mice”

Description of work:

Task 4.1- Establishing the in vitro protocol for GRPs isolation from iPSCs. Derived cells will be processedfor immunocytochemical analysis with antibodies against neurons, astrocytes, and oligodendrocytes markers. To analyze morphologic and gene expression patterns. To establish protocols for GRPs purification using positive selection.

Task 4.2- Testing GRPs transplantation in a transgenic mouse model of ALS, the SOD1G93A mouse, defining an optimal number of cells to be transplanted, performing a dose-effect analysis and investigating the occurrence of any side effects. To assess the engraftment and therapeutic efficacy of transplanted cells, evaluating whether GRPs migrate, survive, and integrate into the host in anatomically and physiologically appropriate ways. To determine if GRPs give rise to endogenous neuroectodermal cells, replacing endogenous dysfunctional MNs. To determine whether treated ALS transgenic mice have delayed disease

Task 4.3- Characterization of the mechanisms involved in cell transplantation outcomes: To examine bi-directional cellular and molecular interactions between transplanted stem cells and endogenous MNs and the mechanisms that lead to the modulation of disease pathways and MNs protection, evaluating the capacity of GRPs to exert a neuroprotective effect on ALS MNs in vitro and in vivo. To co-culture GRPs and ALS MNs (from iPSC) to elucidate whether stem cell transplantation may lead to concurrent modulation of the neuronal microenvironment, favoring endogenous MN survival.

Task 4.4 – Generation of GRP from ALS patients for use in vitro and in vivo experiments: To evaluate the in vivo effects of GRPs from ALS patients, generating ALS donor GRPs and transplanting these cells in vivo, in order to create for the first time an in vivo model of ALS-non autonomous cell death.

• WP5: Transplantation of glial precursors derived from stem cells (iPS) as a therapeutic strategy for SMA

Objectives: To explore the therapeutic potential of human GRPs derived from iPSCs in providing neuroprotection in SMA. To transplant GRPs in SMA mice, determining survival, differentiation, and function of GRPs following transplantation. To determine the ability of transplanted cells to protect motor neurons and study GRPs properties e.g. their capacity to produce growth factors before and after transplantation. These experiments will also identify other potential neuroprotective mechanisms. To assess the capacity of wild-type GRPs to protect motor neurons and neuromuscular junctions (NMJs), to increase muscle strength and survival following transplantation into SMA mice.

The fourth annual 2-day Workshop will be held in Milano in May 2017 and it will have a particular emphasis on “The basics of high-throughput screening for neuroprotective drugs”The fourth “NO-MND day meeting” will be held in Athens in September 2017. The main thematic will be “Combined therapeutic strategies for MNDs

Description of work:

Task 5.1- Establishing and optimizing the in vitro protocol for GRPs isolation from iPSCs.

Task 5.2- Testing GRP transplantation in a transgenic mouse model of SMA (SMA ∆7 mice), to define an optimal number of cells to be transplanted, to perform a dose-effect analysis and to investigate the occurrence of any side effects. To assess the engraftment and therapeutic efficacy of transplanted cells. To determine if GRPs contribute to endogenous neuroectodermal cells, replacing endogenous dysfunctional MNs. To determine whether treated SMA transgenic mice have delayed disease onset, delayed progression, improved neuromuscular function, and a prolonged lifespan accompanied by reduced MN loss.

Task 5.3- Characterization of the mechanisms involved in cell transplantation outcomes. To examine bi-directional cellular and molecular interactions between transplanted stem cells and endogenous MNs evaluating the capacity of GRPs to exert a neuroprotective effect on SMA MNs. To co-culture GRPs and SMA MNs (from iPSCs) to elucidate whether stem cells transplantation may lead to concurrent modulation of the neuronal microenvironment, favoring endogenous MN survival.

Task 5.4 – Generation of GRPs from SMA patients for further in vitro and in vivo experiments: To evaluate the in vivo effects of GRPs derived from SMA patients, by transplanting these cells in vivo in wt animals, in order to create for the first time an in vivo model of SMA-non autonomous cell death.

• WP6: Training, practice and dissemination activities

Objectives: To disseminate the documentation produced and the tools developed by the project partners. To provide training videos and documentation, useful to the interested user or researcher to understand the tools at a different level of details.

Description of work

Task 6.1-On-site and remote (online) training sessions for users and researchers: to design and develop training documentation assembling the WPs documentation and adding new material to in vitro and in vivo tools. We will organize conference video lab meeting trough skype every 2 o 3 months, each staff exchange will give a seminar on her/his work to the host institute.