The Effects Of NBP On The Lifespan And CDK5P35Expression In The Mice Model Of ALS

Objective: Amyotrophic lateral sclerosis (ALS) is a degenerativedisease of nervous system that selectively injurys upper and lower motorneurons, reflecting progressive muscle weaknessand atrophy, more than90%of ALS cases are sporadic and about10%are familial. The mechanism ofmotor neuron degeneration in ALS remains unkonwn oxidative,mitochondriadisfunction,excitotoxicity,neuroinflammatory,calcium overload,abnormalprotein aggregation may be involved in, and so far, there is no effective cure.Cyclin-dependent kinase5(CDK5) is a proline-directed serine threoninekinase, including292amino acid residue. The CDK5protein is a33kDamolecule and it is an unusual molecule that belongs to the large family ofproteins, cyclin-dependent kinase, it has little role to play in cell regulationand is activated by non-cyclin protein, P35and P39. Recent research hasshowed that the abnormaly increase of CDK5activity is involved in thepathogenesis of many neurodegenerative disease, such an Alzheimer's disease,Parkinson's disease and Amyotrophic lateral sclerosis. The mechanism is thatvenenosus factors such as excitatory amino acids toxicity,calcium overloadand free radical fomation can lead to increase CDK5activity, activitingCa~2+-dependent prolease (Calpain), that cleave P35to P25, and prolongedactivation of P25/CDK5complex further phosphorylate its substrates such asneurofilament protein. Neuraxis transportation and cytoskeleton are centerpiece of ALS pathogenesis, however NFs anormaly accumulation isimmediate cause leading to neuraxis transportation disfunction, CDK5overphosphorylate Tau protein and neurofilament protein further lead tocytoskeleton and neuraxis transportation anormaly, participating indevelopment of ALS pathology. Therefore inhibiting CDK5cytoplasm transfer,decreasing P35conversion to P25may slow down disease progress. DL-3n-butyphthalide (NBP) is extract from seeds of the Chinese celery, itwas effective in treating ischemic cerebrovascular disease. Many studies havedemonstrated that NBP possesses neuroprotective by improving mitochondrialfunction, inhibiting calcium overload, reducing neuronal apoptosis andinflammation. Recent research has showed that NBP has also curative effecton the neurodegenerative disease.In the present sytudy, we apply NBP to interfere familial amyotrophiclateral sclerosis mice model (SOD1~G93Atransgenic mice), and observe thechanges of behaviors and lifespain of SOD1~G93Atransgenic mice by RotationTest, investigate the spinal cord anteral horn motor neurons numbers bytoluidine blue staining and CDK5,P35expression in the spinal cord anteriorhorn by IHC and WB in the final stage, aim to study the effects of NBP onSOD1~G93Atransgenic mice.Methods:1Animal and treatmentThe48hSOD1~G93Agene positive mice were screened and genotyped.And then were randomly divided into low middle high dose of NBP groupsand placebo group, the hSOD1~G93Agene negative mice were selectived aslittermates. Each group has12mice with equal numbers of males andfemales. At the age of6weeks, L-NBP was administered by oral gavagedaily at a dose of60,120or180mg/kg/day respectively, placebo groupreceived coequeal (10ml/kg/day) corn oil alone.2Behaviour observation2.1Rotation TestBeginning at10weeks of age,we evaluate motor function of hSOD1~G93Amice, we messured the time a given animal remained on the rotating cylinder(30mm in a diameter) of a rotarod apparatus (YLS-4C Rota-Rod) revolving ata constant speed of30rpm. Each animal was given3trials,and the longestlatency before falling was used, The rotarod analysis was performed weekly,5days of daily adaptability training were received before the test.2.2Life span Assessment The animals were considered as sacrificed when they were unable to rightthemselves within20s after placed on either side, this day was recorded asdeathtime, The life span of the mice was the time from born to death.3PathologyAfter10%chloral hydrate anesthesia, animal tissues were fixated viaheart perfusion by4%paraformaldehyde for20min. The lumbar enlargementswere fixated in4%paraformaldehyde for48hour. Then they were dehydratedby gradient ethanol, transparentized by xylene, embedded by paraffin andmade into conventional histological sections futher (5μm thick).5μm thickparaffin sections were used for1%toluidine blue staining to observe the spinalanterior horn neurons survival condition.4ImmunohistochemistyMice were anesthetized with10%chloral hydrate and perfusedtranscardially with4%paraformaldehyde for20min. The lumbar enlargementswere quickly dissected and fixed in4%pareformaldehyde for48hour. Thetissues were then cut into30μm using a vibratome (LeicaVT1000S). Byimmunohistochemisty we observe CDK5expression and distribution of thespinal anterior horn neurons.5ImmunoblottingMice were anesthetized with10%chloral hydrate intraperitonealinjection, then quickly extract the lumbar spinal cord of mice and put intoliquid nitrogen freezing,-80℃storage.Extract total protein to detect theexpress of CDK5,P35protein in lumbar spinal cord by Western-blotting.6Statistical analysisThe data representation are the Mean±SEM, all experiment data weretreated with SPSS13.0statistical software, Kaplane-Meier survival analysiswas used for survival comparisons, and repeated measures ANOVA test wasused for assessment of rotation test comparisons. Biochemical andpathological results were analyzed using ANOVA or Nonparametric test. p <0.05was considered as statistically significant. Results:1Life spanThe life span of the three NBP-treated groups were increased by about6,2,12days respectively compared with the placebo group. However, only180mg/kg treatment group significantly extended the lifespan of SOD1~G93Atransgenic mice by9.2%, P=0.008.2BehaviourThough rotation test we found that performance of rotarod wassignificantly improved from13weeks to16weeks at the dose of high dose ofNBP-treatment group. The low and middle dose NBP-treatment group couldslightly improve performance of rotarod compared with placebo group, therewas no statistical significance.3Toluidine blue stainingToluidine blue staining results display, in final stage, high dose ofNBP-treatment group reserved more neurons than placebo group,(10±0.687vs4±0.553), compared with placebo group, P=0.000.4ImmunohistochemistyImmunohistochemisty results reveal, the number of CDK5proteinpositive were6.7±1.2,3.0±1.0respectively in NBP-treatment group,placebogroup, and compared with placebo group, P=0.038. An intense cytoplasmicimmunostaining of motor neurons for Cdk5in placebo group, and very lowlevels of cytoplasmic CDK5immunoreactivity was detected in spinal cordsections of NBP-treated group.5Western boltsWestern bolts showed the levels of Cdk5protein expression are similarand had no difference. But P35protein in placebo group was obviously lowerthan negative control group, P=0.021. P35protein levels revealed increase inhingh-dose of NBP group and compared with placebo group, p=0.000.Conclusions:NBP can extend the life span of hSOD1~G93Amice, improve the motordysfunction of hSOD1~G93Amice, reduce the loss of motor neurons, inhibitCDK5cytoplasm transfer, increase membrane P35/CDK5expression.