title: Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule.
abstract: BACKGROUND
  Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases. Neurotrophic factors have been widely tested to counteract neurodegenerative conditions, despite their unspecific neuronal access. The non-toxic C-terminal fragment of the tetanus toxin (TTC) heavy chain has been studied not only as a carrier molecule to the CNS but also as a neuroprotective agent. Because the neurotrophic effects of BDNF have been demonstrated in vitro and in vivo, the question addressed in this work is whether a fusion molecule of BDNF-TTC may have a synergistic effect and enhance the neuroprotective properties of TTC alone in a mouse model of ALS.
  
  
  METHODS
  Recombinant plasmid constructs (pCMV-TTC and pCMV-BDNF-TTC) were injected into the quadriceps femoris and triceps brachialis muscles of SOD1(G93A) transgenic mice at 8 weeks of age. The hanging wire and rotarod tests were performed to assess motor coordination, strength and balance. Electrophysiological tests, morphological assays of spinal cord sections of L2 and L4 segments, and gene and protein expression analyses were performed. The Kaplan-Meier survival analysis test was used for comparisons of survival. Multiple comparisons of data were analyzed using a one-way analysis of variance (ANOVA).
  
  
  RESULTS
  Treatment with the fusion-molecule BDNF-TTC and with TTC alone significantly delayed the onset of symptoms and functional deficits of SOD1(G93A) mice. Muscle innervation was partially preserved with these treatments, and the number of surviving motoneurons in L2 spinal cord segment was increased particularly by the fusion protein induction. Inhibition of pro-apoptotic protein targets (caspase-3 and Bax) and significant phosphorylation of Akt and ERK were also found in the spinal cord of treated mice.
  
  
  CONCLUSIONS
  Significant improvements in behavioral and electrophysiological results, motoneuron survival and anti-apoptotic/survival-activated pathways were observed with BDNF-TTC treatment. However, no synergistic effect was found for this fusion molecule. Although BDNF in the fusion molecule is capable of activating autocrine and neuroprotective pathways, TTC treatment alone yielded similar neuroprotection. Therefore, an accurate study of the neuroprotective effects of TTC fusion molecules should be performed to obtain a better understanding of its effects.
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO-I3A), Facultad de Veterinaria, Instituto Aragonés de Ciencias de la Salud (I+CS), Universidad de Zaragoza, C/Miguel Servet, 177, 50013 Zaragoza, Spain.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Calvo
authors.forename: María
authors.initials: M
authors.lastname: Moreno-Igoa
authors.forename: Renzo
authors.initials: R
authors.lastname: Mancuso
authors.forename: Raquel
authors.initials: R
authors.lastname: Manzano
authors.forename: Sara
authors.initials: S
authors.lastname: Oliván
authors.forename: María J
authors.initials: MJ
authors.lastname: Muñoz
authors.forename: Clara
authors.initials: C
authors.lastname: Penas
authors.forename: Pilar
authors.initials: P
authors.lastname: Zaragoza
authors.forename: Xavier
authors.initials: X
authors.lastname: Navarro
authors.forename: Rosario
authors.initials: R
authors.lastname: Osta
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000188:drug therapy / Q000235:genetics / Q000628:therapy*; D000818:Animals; D001522:Behavior, Animal / Q000502:physiology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D005260:Female; D020869:Gene Expression Profiling; D015316:Genetic Therapy / Q000379:methods*; D006651:Histocytochemistry; D053208:Kaplan-Meier Estimate; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009043:Motor Activity / Q000502:physiology; D009431:Neural Conduction / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D013116:Spinal Cord / Q000473:pathology; D013744:Tetanus Toxin / Q000494:pharmacology*
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin