Kallikrein-kinin system evaluation in neurogenesis, proliferation and differentiation of neural progenitor cells from amyotrophic lateral sclerosis patients to mature motor neurons

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized primarily by a loss of upper and lower motor neurons (MNs) leading to progressive paralysis, disability and death. ALS affects people in the fourth to sixth decade of life, with a survival of less than 5 years after diagnosis. Numerous mechanisms that may contribute to the progression of the disease have been proposed such as axonal transport defects, glutamate excitotoxicity, impaired neurotrophic support, mitochondrial dysfunction and oxidative damage. Several mutations in a heterogeneous pull of genes have been associated with ALS, among them: C9ORF72, FUS/TLS, SOD1, TARDBP, UBQLN2 and VAPB. Mutations in VAPB, a protein involved in the Endoplasmic Reticulum-Golgi-mediated vesicle transport, produces an atypical autosomal dominant form of ALS, known as ALS8, initially identified in a large Brazilian family. Mutant VAPB must interfere in essential functions of the protein such as neuronal maturation, neuroprotection against glutamate excitotoxicity and calcium homeostasis. As ALS is characterized by an irreplaceable loss of motor neurons, the hypothesis about an alteration at the neurogenesis level emerged. There have been described modifications in neurogenesis in other neurological diseases such as Alzheimer's disease. Human brain post-mortem samples from ALS patients as well as animal models (rats and mice SOD1G93A) of the disease corroborate the existence of these alterations in the generation of new neurons. In this context Bradykinin (BK), a biological active peptide that belongs to the Kallikrein-kinin system (KKS), has been proved to promote neurogenesis, increase migration, determines the neural phenotype in several cell lines and induces neuroprotection against excitotoxicity produced by NDMA, by activating the B2Bk receptor (B2BKR). Besides the activation of the B2BKR promotes a signaling pathway which is essential for initial stages of development. Blocking of B2BKR signaling promotes a reduction in the cell survival. Disregulation of B2BKR function or expression has been suggested to be involved in the etiology of neurodegeneration in several brain diseases as well. Taking into account the severity of the ALS disease and the lack of any treatment, the development of compounds to that could increase the cell survival or neurogenesis rate are urgent. As in ALS there is an irreplaceable loss of MN and as it has been proved that BK promotes neurogenesis, we propose the study of the KKS in the neurogenesis and differentiation of neural progenitor cells obtained from induced pluripotent stem cells (iPSC) of ALS8 patients, to MN. We expect to see a significant increment in neurogenesis in samples treated with BK. Possible alterations in the KKS of ALS8 patients that could be responsible for the lack of new neurons regeneration will be determined. The NPC obtaining and iPSC and the NPC differentiation to MN, are very difficult and sensitive procedures that involved a big investment in time, money and requires specific manipulation skills. Prof. Alysson R. Muotri's laboratory, located in San Diego (USA) has been working with iPSC and neurological disorders for many years. His team is composed by professional post-doctoral researchers and Ph.D. students who have a wide background in iPSC handling and researching. Prof. Muotri's laboratory has the entire infrastructure to iPSC researching and they have developed optimized protocols to the differentiation of ALS8-NPC to MN. This scenario makes Prof. Muotri's laboratory the best place not only to learn who to work with iPSC/NPC but also how to differentiate them into MN. These procedures will be applied back in São Paulo, in Prof. Ulrich's laboratory where we are implementing this technique. The results obtained during this stage will be determinant to the writing and finishing of this doctoral thesis project. (AU)