title: Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.
abstract: OBJECTIVE
  Recessive mutations in alsin, a guanine-nucleotide exchange factor for the GTPases Rab5 and Rac1, cause juvenile amyotrophic lateral sclerosis (ALS2) and related motoneuron disorders. Alsin function in motoneurons remained unclear because alsin knock-out mice do not develop overt signs of motoneuron degeneration.
  
  
  METHODS
  To generate an alsin loss-of-function model in an ALS-relevant cell type, we developed a new small interfering RNA electroporation technique that allows efficient knock down of alsin in embryonic rat spinal motoneurons.
  
  
  RESULTS
  After small interfering RNA-mediated alsin knockdown, cultured motoneurons displayed a reduced apparent size of EEA1-labeled early endosomes and an increased intracellular accumulation of transferrin and L1CAM. Alsin knockdown induced cell death in 32 to 48% of motoneurons and significantly inhibited axon growth in the surviving neurons. Both cellular phenotypes were mimicked by expression of a dominant-negative Rac1 mutant and were completely blocked by expression of a constitutively active Rac1 mutant. Expression of dominant-negative or constitutively active forms of Rab5 had no such effects.
  
  
  CONCLUSIONS
  Our data demonstrate that alsin controls the growth and survival of motoneurons in a Rac1-dependant manner. The strategy reported here illustrates how small interfering RNA electroporation can be used to generate cellular models of neurodegenerative disease involving a loss-of-function mechanism.
authors.affiliation: Institut National de la Sante et de la Recherche Médicale, Marseille, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Jacquier
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Buhler
authors.forename: Michael K E
authors.initials: MK
authors.lastname: Schäfer
authors.forename: Delphine
authors.initials: D
authors.lastname: Bohl
authors.forename: Stephane
authors.initials: S
authors.lastname: Blanchard
authors.forename: Christophe
authors.initials: C
authors.lastname: Beclin
authors.forename: Georg
authors.initials: G
authors.lastname: Haase
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology; D019556:COS Cells; D016923:Cell Death / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002522:Chlorocebus aethiops; D018274:Electroporation / Q000379:methods; D011992:Endosomes / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D009046:Motor Neurons / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D034741:RNA, Small Interfering / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D013116:Spinal Cord / Q000166:cytology*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein
chemical_list: C512358:Als2 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D009479:Neuropeptides; D034741:RNA, Small Interfering; C484602:Rac1 protein, mouse; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein