MoMoThe

Motor neuron disease Modeling and Therapy

Background Information and Research Hypothesis

In the aging populations of Western countries one out of four persons is at risk for a neurodegenerative disease. Among neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) is a particularly devastating and always fatal disorder which does not benefit from effective therapies. ALS affects motor neurons in cerebral cortex, brainstem and spinal cord leading to progressive muscle atrophy and paralysis. While most forms of ALS appear sporadically, about 10-20 % are familial and caused by mutations in genes encoding proteins as diverse as superoxide dismutase 1/SOD1 (ALS1), Alsin (ALS2), VAP-B (ALS8), TDP-43, FUS/TLS, CHMP2B or Optineurin. While the molecular mechanisms of ALS remain controversial, a consensus emerges on its natural disease history. According to studies in mutant SOD1 mice, motor neuron degeneration first manifests far distally at the neuromuscular synapse with loss of synaptic vesicles and axon retraction but is initiated in the cell body and modulated by neighboring astrocytes and microglia. Our team hypothesizes that the Golgi apparatus may link peripheral manifestation, central initiation and neuron/glia-interactions in ALS. This novel and potentially unifying hypothesis is corroborated by the following observations: • The Golgi apparatus plays a key role in protein secretion to the extracellular space, vesicle routing to the neuromuscular synapse and is increasingly recognized as a sensor and transducer of cell death signals. • Ultrastructural, molecular and functional Golgi abnormalities have been observed in motor neurons from human patients with various forms of ALS and corresponding mouse models. • An increasing number of ALS genes encode proteins normally involved in the function of the Golgi apparatus and related organelles. In order to test the hypothesis, the team pursues the following specific aims: • To investigate how mutations in ALS genes trigger Golgi pathology, endosome dysfunction and synaptic vesicle loss in motor neurons. • To dissect out the mechanisms converging from Golgi dysfunction to cell death and axon degeneration. • To identify such pathological changes in motor neurons from human ALS patients. • To use this knowledge for the development of new experimental therapies.

Technical approaches Past cell biological studies in ALS were hampered by the low yield and purity of motor neurons isolated from model mice and the impossibility to obtain motor neurons from human patients. To overcome these limitations, the team has developed fluorescence-activated cell sorting (FACS) and cellular reprogramming techniques. Using a cell sorter with an optimized flow chamber motor neuron subtypes innervating limb, axial, tongue, oculomotor and other muscles are now isolated with unprecedented yield and purity. In culture, the FACS-sorted motor neuron subtypes develop well and exhibit striking morphological differences. The molecular bases for these differences are established by whole genome transcriptomic profiling and their functional relevance tested by lentivirus-mediated gene overexpression or silencing. Cellular reprogramming techniques are developed in the frame of the European ERANET network IPSoALS. The IPS cells are generated from human patient skin fibroblasts at Institut Pasteur (team D Bohl) and at Hadassah University (team B Reubinoff) and subsequently differentiated into several neural cell types. Motor neurons are then cultured alone or together with astrocytes and analyzed for phenotypic changes in survival, axon morphology and organelle structure. These techniques are also used in collaboration with other teams of the INT, for instance in a screening of pharmacological compounds capable to up-regulate the ion transporter KCC2, a promising target in spinal cord injury (collaboration with P3M team).

Research Projects

Project 1: ALS1 triggered by SOD1 mutants Degenerating ALS motor neurons show Golgi fragmentation and atrophy at an early disease stage and secrete ALS-linked SOD1 mutants in a Golgi-dependent manner. Our studies showed that embryonic motor neurons from transgenic mice carrying ALS-linked SOD1 mutations have an increased susceptibility to the Fas/No cell death pathway. This pathway involved activation of Daxx, Akt, p38 kinase and neuronal nitric oxide (NO) synthase and NO production. We further demonstrated that the pathway comprises a feed forward amplification mechanism and is activated in mutant mice at presymptomatic stage. Interestingly, the Golgi apparatus represents a hidden reservoir of Fas receptor and caspase 2 suggesting that aberrant Fas signalling might cause or contribute to pathological Golgi fragmentation.

Project 2: Juvenile ALS2 linked to mutant Alsin Alsin/ALS2 encodes a 184 kD protein with guanylate nucleotide exchange factor domains (GEFs) for the small GTPases Rac1 and Rab5. Recessive Alsin mutations have been identified in juvenile ALS2 and related disorders but their precise mechanism(s) remained unclear since Alsin knockout mice do not develop disease. Using RNAi-mediated Alsin knockdown the team demonstrated that Alsin controls endocytosis, axon growth and survival of spinal motor neurons by activating Rac1. The team further showed that Alsin-knockdown-mediated cell death and defective axon growth were completely inhibited by co-cultured astrocytes through release of a diffusible factor. The current objectives are to identify the astrocytic rescue factor, to characterize its mode of action and to evaluate its neuroprotective potential using patient-derived iPS-cells as model system.

Project 3. Progressive motor neuronopathy linked to mutant TBCE Recessive mutations in the tubulin chaperone TBCE are responsible for mouse progressive motor neuronopathy and human Sanjad/Sakati syndrom. The team contributed to the identification of the pmn mutation as a Trp534->Gly substitution and subsequently identified TBCE as the first tubulin chaperone localizing to the Golgi apparatus. Subsequent studies showed that TBCE normally controls axonal tubulin routing and that the pmn mutation compromises axonal tubulin routing and causes retrograde loss of axonal microtubules. More recently the team identified severe and progressive Golgi pathology in pmn motor neurons such as membrane fragmentation and atrophy and demonstrated defects in Golgi-derived microtubules as their molecular origin. The objective is now to investigate the relationship between Golgi pathology and synaptic vesicle loss in degenerating motor neurons. These studies bear therapeutic relevance since neurotrophic factors, which remain among the most potent therapeutic candidates for ALS, seem to exert some of their beneficial effects through retrograde axonal transport to the Golgi apparatus.

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