thetheDuring years of research on ALS, numerous models of this disease have been developed. Currently, scientists have many different tools for this purpose. From animals carrying ALS mutations to various cell lines and cultures. Although whole organisms like mutated flies and mice give excellent opportunity for some studies, sometimes scientists are overwhelmed by the vast number of factors which can affect the results in in vivo models. For those cases in vitro modelling is a much better solution. It allows control of much more parameters and factors during procedures. Also, recent advances in techniques used for in vitro modelling simplifies experiment preparation, saving a lot of time and money, which can be dedicated for other purposes. During the 25th International Symposium on ALS/MND we had a wonderful opportunity to acquaint with data provided by in vitro models of ALS. Induced pluripotent stem cells: a step towards personalized treatments
During the first lecture of the session Dr Eggan (Harvard, USA) was putting a lot of emphasis on personalized therapies. This approach can help to diminish adverse effects of drugs and improve effectiveness of many therapies. For example in the case of cancer, material from a biopsy can be used to verify which therapy approach will be the most effective. In the case of diseases affecting neurons like ALS, obtaining a material for such studies is practically impossible. However, it all changed with the development of techniques that allowed us to create induced pluripotent stem cells (iPSC). Currently simplification of the methodology allows to reprogram easily-reachable cells like fibroblasts, into cells from another tissue. For example neurons. Then can be used to evaluate different therapy approaches, study new drugs and in the future to determine the best possible treatment for particular patients. This issue is particularly important in the case of such complicated disease like ALS.
Dr Eggan and his colleagues were already able to use this approach for new drug analysis. After observing changes of the electrical properties in neurons obtained from iPSC from ALS patients, they have tried to reverse those changes with existing epilepsy drug called Retigabine. Their study lead to approval of clinical trials for this drug which will start before the end of 2014.
Induced pluripotent stem cells from fibroblasts were also used by Dr Mutihac (University of Oxford, UK) and her colleagues. In their study they have concentrated on patients with expanded hexanucleotide repeat (GGGGCC)n in the C9ORF72 gene. This mutation is currently known to cause ALS in a vast number of familial cases of this disease. In their research, she revealed many functional deficits of those cells, including calcium dysregulation, increase in pro-apoptotic protein levels like Bak or Bim, increase in apoptotic marker cleaved caspase-3 and reduced levels of anti-apoptotic Bcl-2. They have also detected PABP+ stress granules, which suggests autophagy impairments and protein aggregation in those cells. Their data will not only help to understand the pathology mechanisms behind the development of ALS, but it also shows how valuable iPSC are as a tool in research.
Connecting RBM45 with oxidative stress
Oxidative stress damage is one of the theories behind ALS development. Numerous studies in patients and animal models confirmed that this process is taking place in affected cells. Overproduction of reactive oxygen species or malfunction of protective mechanism can lead to severe damage and cellular death.
There is several RNA-binding proteins involved in ALS. Except TDP-43 and FUS, a new one has recently been described. RBM45 has been found to accumulate together with TDP-43 in inclusion bodies and its increased levels were detected in cerebrospinal fluid of ALS patients. Dr Bakkar (Barrow Neurological Institute, USA) has presented results from investigations of several cell lines and cell cultures in search for the role of this protein in oxidative stress damage. She and her colleagues have found that cellular localization of this molecule is regulated by oxidative stress. They also proved that cytoplasmic RBM45 can directly interfere with the neuroprotective antioxidant response. During oxidative stress RBM45 can bind and stabilise the KEAP, which is inhibiting NRF2 (antioxidant response transcription factor). This further leads to increased cell death. These results provided very nice evidence for direct interaction between inclusion forming RNA-binding protein and the antioxidant pathway.
C9ORF72 and DNA damage
Of all the organelles, the nucleus can be considered as the cellular headquarters. It is very active. During the whole life of the cell their gene expression profile is being modified, hundreds of genes are switched on and off, different transcription and RNA processing is taking place. Those adjustments are necessary to allow cells to accommodate to changing environments and to respond to external signalling. In the case of pathological conditions, cells need to activate protective pathways or die. During stress or DNA damage the organization and composition of the nucleus can undergo dramatic changes.
In the last lecture of the session Dr Farg (La Trobe institute of Molecular Science, Aurstralia) presented her study on C9ORF72. She and her colleagues used neuronal cell lines to investigate the effect of this gene mutation (hexanucleotide expansions) on DNA damage and repair pathways. They have found that the presence of repeat expansions in this gene can lead to formation of intra-nuclear inclusions, activate heterogeneous nuclear ribonucleoproteins (hnRNP A2/B1, hnRNP A1), impair ribosomal biogenesis, induce nucleolar stress, and activate the P53 apoptosis pathway. These are very interesting findings, which may suggest that DNA repair pathways may be one of the therapy targets for the ALS.