Changes Of Caspase-12Expression In Spinal Cord And Cerebral Motor Cortex Of Familial Amyotrophic Lateral Sclerosis Model HSOD1^G93A Transgeneic Mouse

Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressingneurodegenerative disorder primarily affecting motor neurons of the spinalcord, brainstem, and cortex. The disease is characterised by muscle weaknessleading to paralysis and death usually within several years of diagnosis.Approximately10%of ALS cases are inherited, and up to20%of thesefamilial cases are caused by mutations in the gene encoding superoxidedismutase1(SOD1). G93A-SOD1transgenic mice show progressive andselective loss of motor neurons and mimic clinical manifestations of humanALS and are, therefore, regarded to be the most established ALS animalmodel.The linkage of ALS1to chromosome21q22.1was initially described in1991by Siddique. Two years later, Rosen et al. described elevendisease-associated mutations in the SOD1gene. To date, more than a hundreddifferent point mutations that result in amino acid substitutions or incorrecttranslation termination in the SOD1gene are known to causeautosomal-dominant ALS. The vast majority of mutations are missensesubstitutions distributed throughout the five exons of the gene. Exon that inthe codon4alanine (Ala) mutation of the valine (Val) or A4V mutation is themost common mutations. Initially, it was hypothesized that mutations wouldimpair the enzymatic activity of the protein, thus resulting in increased cellularlevels of reactive oxygen species, oxidative stress, and neuronal death.Additional evidence against a loss-of-function hypothesis comes from animalmodels. Several studies showed that mutant SOD1is prone to missfolding andforms cytoplasmic aggregates. In turn, aggregates may lead to cell death bysequestering other cytoplasmic proteins essential for neuronal survival. At present,we have created a number of kinds of transgenic rodent modelsexpressing human SOD1(hSOD1)mutations, hSOD1G93Atasnsgenic mice is theinternational recognized ALS pathogenesis and pre-clinical drug research inanimal models, and is most widely used.Recent studies with autopsied ALS patients and studies using mutantSOD1(mSOD1) transgenic mice have suggested that endoplasmic reticulum(ER) stress-related toxicity may be a relevant ALS pathomechanism. The ERis an important organelle for the folding and post-translational modification ofmany proteins, and it also acts as a significant intracellular calcium store. Theover-accumulated misfold proteins results in ER stress, which triggerssignaling pathways collectively known as the unfolded protein response(UPR).The UPR is a homeostatic mechanism which is initially protective byupregulation of chaperone proteins. The UPR is mediated by activation ofthree upstreamsensors of ER stress, namely, PKR-like endoplasmicreticulumkinase (PERK), inositol-requiring kinase1(IRE1), and activatingtranscription factor6(ATF6). Prolonged ER stress, as occurs in ALS, causescell death via apoptotic signalling. ER stress-activated apoptosis is mediated inpart by the release of Ca2+from the ER and upregulation of proapoptoticfactors, including CCAAT/enhancer binding protein-(C/EBP-) homologousprotein(CHOP), as well as activation of ER stress-specific caspases.Caspase-12,which a caspase-associated recruit domain (CARD) andis specifically localized on the cytoplasmic side of the ER, is thought to play arole in ER stress-mediated cell death.Recent studies show that endoplasmic reticulum stress and endoplasmicreticulum stress-induced apoptosis exist in hSOD1^G93Amice spinal cord. Thispreliminary study is to find whether caspase-12of endoplasmic reticulumstress induced apoptosis signaling pathway activation exists in the hSOD1^G93Aspinal cord and motor cortex of the brain in mice.Objective：To observe the changes of endoplasmic reticulum apoptosisfactor caspase-12in the spinal cord and cerebral motor cortex of hSOD1^G93Atransgenic mice, to reveals endoplasmic reticulum stress and apoptosis in the pathogenesis of ALS.Methods: PartⅠ hSOD1^G93Atransgenic mice breeding and identifyB6SJL-Tg (SOD1-G93A)1Gur/J hemizygous males and B6SJLF1/J+/+females breeding, extracting tip of its tail to DNA, for PCR analysisidentified. After identifying, hSOD1G93Atransgenic mice as an animal modelof FALS study, wtSOD1mice as an control group.PartⅡ To observe the changes of endoplasmic reticulum apoptosisprotein caspase-12in the spinal cord and motor cortex of hSOD1^G93Atransgenic miceMethods:1．Experimental group: divided into four groups, first: wtSOD1mice the control group3mice (120); second: pre-symptoms hSOD1^G93Atransgenic mice3mice (60days); third: symptoms of hSOD1G93Atransgenicmice3mice (100-120days); fourth: end-stage hSOD1G93Atransgenic mice3mice (120-130days).2．Drawn: drawn with5%of the chloral hydrate anesthetized mice,4%paraformaldehyde perfusion-fixed within the heart, remove the spinal cord andbrain, with a separation of lumbar spinal cord and motor cortex area,immersed in4%paraformaldehyde(at least24houres), then the vibrationslicer Continuous slice, slice thickness30μm.3．Immunohistochemistry: vibration slice0.01MPBS washing three times,each5minutes,3%H2O2at room temperature for15minutes,0.01MTBSwashed three times, each5minutes,10%horse serum closed1hour, add1:100caspase-12polyclonal rabbit antibodies,4℃overnight incubation onshaker; adding1:200biotinylated goat anti-rabbit IgG at room temperature onshaker2h; adding1:200horseradish HRP-streptavidin at room temperature onshaker1h; DAB chromogenic terminate the obvious color, stretched,conventional dehydration, coverslipping.4. Statistical analysis: The results as the mean±standard deviation(x±s), using SPSS13.0software for statistical analysis, comparison betweengroups using analysis of variance and SNK multiple comparison, statisticalresults to P <0.05as significant difference. Results:1.The experimental animals were divided into hSOD1^G93Atransgenic mice and negative mice by PCR.2. In spinal cord, the number ofcleaved caspase-12positive motor neurons which cleaved caspase-12changedinto the nucleus were significantly increased in symptoms early group andend-stage group comparing with control and pre-symptoms group, the numberof cleaved caspase-12positive motor neurons in the symptoms early groupwas increased comparing with the end-stage group, but there was nosignificantly changes between the group of pre-symptoms and control. Mostmotor neurons'morphology changed at the early symptoms and end-stagesuch as becoming smaller, nuclear shrinking and decreasing in number.3. Incerebral motor cortex, we did not find changes of cleaved caspase-12positivecells, and did not find nuclear shirinking but only find the volum of somemotor neurons become slightly smaller.Conclusion:1In spinal cord, we did not find the change of apoptosisfactor caspase-12which is induced by endoplasmic reticulum stress in thepre-symptoms group (60days). In early symptoms of ALS disease caspase-12changed significantly and increased with the disease progressing, the numberof motor neurons at the end stage was decreasing greatly. It is indicating thatapoptosis induced by endoplasmic reticulum stress participate in thepathogenesis of ALS. However, the clear pathogenesis of ALS needs furtherstudy.2. In cerebral motor cortex, we did not find the significant change ofcaspase-12, it is indicating that apoptosis which is induced by endoplamicreticulum stress don't occur in the cerebral cortex, and it's clear pathogensisneeds futher research.