Linda Greensmith from UCL, UK and Bradley Turner from the Florey Institute, Australia chaired one of the final sessions for Saturday on Murine Models. With the number of causative genes increasing over the last 20 years it is critical to understand the disease mechanism for each gene to progress understanding of the disease and more importantly, to identify novel therapeutic targets. As such, there are increasing numbers of murine models of ALS that contribute to our understanding of ALS. The first presentation was by Professor Kiaei (University of Arkansas, USA) who was reporting on a novel mouse model that has been developed in his lab with the profilin1 (PFN1) mutation. PFN1 mutation is a recently discovered causative gene found in a small number of fALS patients. PFN1 is an actin-binding protein that catalyses the polymerisation of globular actin to filamentous actin. The exact role of mutant PFN1s in ALS, however, requires further analysis. Three lines of transgenic mice were developed with the PFN1 mutation with low, medium or high levels of human PFN1G118V mutation. Using PCR and WB, the PFN1G118V mutation was validated in the brain and spinal cord for each of the three mouse lines. Phenotypic analyses shows that the mice developed ALS-like motor impairments with progressive weight loss. After 110 days the animals display a reduced gait, muscular atrophy and impaired movement that progressively deteriorates. The average end stage of these mice is around 180 days with spinal cord motor neurons loss of around 45%. EM analysis revealed abnormal axonal morphology and damage to the outer membrane of the mitochondria. The low expressing PFN1 mutants have a subtle disease phenotype does not progress and significantly different to the high expressing mutants. This newly developed mouse model will be extremely useful in investigating the role of PFN1 mutations in ALS.
The next presentation was by Dr Da Cruz from Don Clevelands lab (UCSD, USA) who discussed their newly developed mouse model of FUS/TLS. Transgenic mice were engineered with ‘floxed’ human genomic FUS/TLS. Initially the protein levels were equal or slightly above that of the non-transgenic mice signifying an autoregulation mechanism of Fus/TLS. Behaviourally and phenotypically, there was not much difference between transgenic mice and wild type mice after 2 months. After 8 months, however, transgenic mice developed a significant reduction in grip strength levels. The motor phenotypes were getting worse with age were also exhibited muscle denervation. By 22 months, the neuromuscular junction was reduced by about 30% and motor neurons death was also observed. Replacing normal FUS/TLS with disease causing mutant FUS/TLS produces motor and cognitive deficits that are accompanied by mutant-dependent RNA alterations.
The next presentation was from Dr Gaub (McGill University, Canada) who discussed a novel mouse model that has a missense mutation (P56S) in the gene encoding for the vesicle-associated membrane protein-associated protein B (VapB), a mutation that causes a familial variant of ALS. VapB is involved with ER-Golgi trafficking, neurite extension and the development of the neuromuscular junction as well as assisting growth cone guidance. This knock-in mouse model was analysed in order to determine if a link exists between VapB and other variants of ALS. This mouse model exhibits the typical hallmark features of ALS including progressive motor deficits. VapB mutants exhibited TDP43 and FUS cytoplasmic mislocalisation and have a disruption of the nuclear membrane. Furthermore, mutant mice also have a mislocalisation of VapB from the ER that co-localises with p62 (a marker of autophagy) and ubiquitin inclusions. This, therefore, implicates ER stress in the VapB disease-causing mutation.
The following presentation was from Dr Gordon from Professor Kevin Talbots group (Oxford, UK) who was describing their newly developed TDP-43 mouse model. Instead of supraphysiological expression of TDP-43, their mouse model expresses mutant human TDP-43 at physiological levels, which therefore, is a more suitable model of the human disease. The TDP-43 M337V mouse model was characterised by the typical progressive neuromuscular degeneration and denervation. The animals display a reduced survival and develop age dependent CNS pathology with the onset at 6 months. After 9 months of age, TDP-43 was absent in motor neuron nuclei and aggregated in the cytoplasm. Animal weight was slightly reduced but does not progress. However, the skeletal muscle becomes progressively weaker from 6-9 months. Furthermore, culturing spinal cord motor neurons from embryonic mice display a reduced survival phenotype characterised by cytoplasmic aggregates and reduced formation of stress granules. This mouse model requires further analysis but is a more suitable mouse model of the mutated human TDP-43 gene.
The last presentation of a very interesting session was by Dr Bryson from Prof. Greensmiths lab (UCL, UK). The lab has generated ES-derived motor neurons (ESC-MNs) that expression channelrhodopsin-2 and GDNF enabling optogenetic control of muscle function. In the wild-type animal, these ESC-MNs were transplanted into a peripheral nerve after ligation and successfully re-innervated distal muscle. In addition, after transplantation muscular contraction was controlled in vivo by optical stimulation. These cells were then implanted in to the SOD1G93A mouse and survived within the peripheral nerve environment and maintained extensive axonal projections to muscular targets until late-stage disease. This approach has the potential to reinnervate paralysed muscles in ALS patients.
Finally, I would like to acknowledge the Motor Neuron Disease of Victoria, Australia for their generous ‘Nina Buscombe’ Travel award that allowed me to travel to Belgium, present my work and be a part of this very important symposium.