Astrocytes influence in cellular and molecular characterization in motor neuron degeneration in vitro model of amyotrophic lateral sclerosis using transgenic mice for mutant human SOD1

Author(s)

Juliana Milani Scorisa

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2013-06-26

Examining board members

Gerson Chadi; Dagoberto Callegaro; Debora Rejane Fior Chadi; Jessica Ruivo Maximino; Acary Souza Bulle Oliveira

• Advisor: Gerson Chadi

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neurons death that readly leads patients to death. Transgenic mice expressing human mutant superoxide dismutase 1 (hSOD1) is the most accepted experimental model for the disease studying. The mechanisms that lead to neuronal loss are still poorly understood and there is no effective treatment able to prolong the pacient\'s life. Recent studies indicate that glial cells accelerate the neurodegenerative process, but the molecular mechanisms are not yet established. Astrocytes deserve particular attention, since they have close interaction with neurons, providing structural, metabolic and trophic support. In addition, they also participate actively in the neuronal excitatory modulation and in neuronal transmission, controlling ions and neurotransmitters at extracellular levels. The present study aimed to investigate the possible in vitro effects in astrocytes on the motor neurons death in ALS from newborn (P1) and adult pre symptomatic (P60) spinal cord mice. Thus, we evaluated spinal motor neuron survival and trophism in cultures treated with astrocytes conditioned medium and also in co-culture neuron/astrocyte systems of SOD1G93A (transgenic) and wild type in different cells combinations. Still, we investigated genes expression related to P1 and P60 astrocytes cultures performed by quantitative PCR (qPCR) and the molecules secreted by astrocytes were quantified by Sandwich ELISA. For the neuronal degeneration and trophism study, cells were immunostained with specific markers and neurouns were also visualized by phase contrast. These cells were quantified by stereological method and their analysis showed that treatment with transgenic P1 and P60 astrocytes conditioned medium cause length retractions and death on transgenic motor neuron. But, the neuronal death on conditioned medium and co-cultures experiments showed that transgenic P1 and P60 astrocytes caused wild type neuronal death and only transgenic P60 astrocytes led transgenic neurons death, demonstrating major toxicity of transgenic astrocytes. For the gene expression analysis NKRF, UBE2I and TGFa genes showed differentially expressed in transgenic P1 astrocytes and HIPK3, TGFa and NTF5 genes showed differentially expressed in transgenic P60 astrocytes. The analizes of molecules secreted by conditioned media a larger amount of NGF was found in transgenic P60 astrocytes comparing to wild type astrocytes. The amount of IGF-I in the conditioned medium was reduced in astrocytes transgenic P60 cultures compared to the wild type astrocytes Also, there is a reduction autocrine of TNF-? and IL-6 on transgenic astrocytes P60. The transgenic astrocytes seem to promote motor neuron toxicity in ALS and molecules released by astrocytes appear to be involved in the ALS development (AU)

• FAPESP's process: 09/16995-6 - CELLULAR AND MOLECULAR CHARACTERIZATION OF MUTANT ASTROCYTE INFLUENCE ON MOTOR NEURON SURVIVAL USING IN VITRO MODEL OF TRANSGENIC MICE FOR HUMAN SOD1
• Grantee: Juliana Milani Scorisa Salgado
• Support Opportunities: Scholarships in Brazil - Doctorate