In vitro analysis of amyotrophic lateral sclerosis type 8 and genetic study of spastic paraplegia 4

Author(s)

Miguel Mitne Neto

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2011-03-22

Examining board members

Mayana Zatz; Luciana Amaral Haddad; Fernando Kok; Carlos Frederico Martins Menck; Oswaldo Keith Okamoto

• Advisor: Mayana Zatz

Abstract

The motor neuron diseases (MND) show a huge clinical and genetic variability. Amyotrophic Lateral Sclerosis (ALS) is the most common late-onset form of MND. ALSs devastating and incurable manifestation leads to a profound loss of life quality. ALS8 is an autosomal dominant form of ALS caused by mutations in the VAPB gene. The VAPB protein is involved with many cellular processes and our data suggest that P56S mutation in this protein reduces its interaction with two other proteins: Tubulin and GAPDH. Since these proteins were previously related to other types of neurodegenerative diseases they potentially are key points to reveal the processes responsible for ALS8 and other MND. A substantial number of successful drug tests in ALS animal models could not be translated to humans, showing the need of novel ALS systems. iPSC technology made possible cellular reprogramming. The iPSC technology brings new hope in this area since it can be used to model diseases in vitro. Here we present a new ALS model based on ALS8-iPSC. Compared to control samples, this model shows a reduction of VAPB levels. However, we could not identify intracytoplasmic aggregates, which characterize overexpression models. We show for the first time that human embryonic stem cells express VAPB. Combined with results showing a VAPB reduction in ALS8 samples, it suggests that ALS8 patients present diminished protein levels since the beginning of their lives and reveal the importance of modeling ALS in a human background. The search for copy number variations has led to the identification of the first multiexonic duplication (exon10_12dup) in SPG4 gene, expanding the mutation spectrum in this gene. This mutation leads to a premature stop codon, which suggests that the expressed protein is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. These data suggest specific neuromuscular diseases can be modulated by factors related to individual background and gender. In sum, we support the idea that microtubules can be involved with neuromuscular disorders pathogenesis, since both VAPB and Spastin interact with these cytoskeleton components. Additionally, we present a new in vitro model to ALS analysis and we justify the studies in a human background. (AU)