Cognitive Dysfunction In The SOD1^G93A Transgenic Mouse Model Of Familial Amyotrophic Lateral Sclerosis

Objective: Amyotrophic lateral sclerosis(ALS)is a degenerative disease of nervous system, which leads to the lose of motor neurons in brain and spinal cord, while the pathogenesis is unknown. Generally, we take ALS as a disease that involved pyramidal cells of cerebrum cortex, motor nucleus of brainstem and anterior horn motor neurons of spinal cord selectivity. However,during clinical work, we find some ALS patients have non-motor system symptoms, for example cognitive function impairment, sensory disturbance, ocular motility disorders, autonomic nerves function impairment and so on. Therefore, to explore whether ALS was multi-system involvement, is one of the issues need to focus.Among the non-motor system symptoms of ALS, cognitive function impairment is more common. Autopsy found round or ellipsoidal ubiquitin inclusion body in hippocampus neurons, which in support of ALS involving non-motor systems, and may be a common clinical cognitive impairment related.Ubiquitin-proteasome system (UPS) contains ubiquitin-activating enzyme, ubiquitin conjugating enzyme, ubiquitin-protein ligase and 26S proteasome. It exists in eukaryotic organism generally. More than 80% of misfolding proteins or other mutant proteins within the cells are degradated by it. Also it is subtile and specific protein degradated system and important controlling cell system. Studies suggest that disorders of ubiquitin-proteasome degradation pathway can lead to accumulation of abnormal proteins in cells, and further lead to cell dysfunction and degeneration. Therefore, the abnormal expression of ubiquitin protein in the hippocampus can lead to the substrate protein aggregated abnormally in neurons, and form cytoplasm inclusion, which injury the normal function of hippocampus neurons and lead to cognitive function impairment.In present study, we used familial amyotrophic lateral sclerosis model- SOD1^G93A transgenic mice and observed the changes of mouse'behaviors and hippocampus structure, to explored whether ALS exist cognitive function impairment or not. The study could provide a theoretical basis for clinical research.Methods:1 developed animal modelUsing B6SJL2 BTg (SOD12^G93A) 1Gur /J hemizygote males and B6SJLF1 /J + / + females( Jackson Lab, America) to breed. Raising them in constant temperature(22～27℃), constant humidity(40～50%), depuratory housing, eating sterilizated water and granular pattern food of murids. Clipping tail of ^G93A transgenic mice about 1mm, extracting DNA, using PCR amplification, through 1.5% agarose gel electrophoresis, observing straps under long wave ultraviolet light, to identificate positive or negative mutants.2 grouped laboratory animalwe select 60 mouse that is sensitive to electric shock through Y labyrinth (positive mutants :30 mouse, negative mutants:30 mouse ) (+/–mutants: 30 females and 30 males) and divide them into three group randomly:60 days,90 days and 120 days (onset 3.5). There are 20 mouse in each group.3 SOD1^G93A transgenic mice score standardFor weekly assessment of general condition starting at day 100 we used a behavioural score system based on the score developed by Vercelli etc. from 1 to 5 defined as follows:5: healthy without any symptoms of paralysis,4: slight signs of destabilized gait and paralysis of the hind limbs,3: obvious paralysis and destabilized gait,2: fully developed paralysis of the hind limbs, animals only crawl on the forelimbs,1: fully developed paralysis of the hind limbs, animals predominantly lie on the side and/or are not able to straighten up after turning them on the back or lost more than 20% of their starting weight.4 step down testThe step down test lasts two days. The first day: Putting the mice into the step down equipment to move freely about 3 minutes. Then take it on the copper grille and connect 36V alternating current. To observe in 5 minutes after electric shock, how long it takes to find the refuge area at the first time (as learning action time) and how manytimes it jumps down from refuge area (as learning errors frequency). Take all above as learning training results.The second day:24 hours after learning training,put transgene mice on the refuge area and connect 36 V alternating current.Take notes in the 5 minutes ,how long it take to jump down from refuge area (if overtopping 5 minutes, record it as 5 minutes) and how many times it jumps down from refuge area (as memory errors frequency).Take all above as memory training results.5 pathologyPour 4%paraformaldehyde into left ventricle fast when animals under anesthesia. Take the tissue of brains and fix them with 4% paraformaldehyde about 24 hours. Exposure hippocampus at optic nerve plane, dehydrate in gradient alcohol, then the tissues of brains were embedded in paraffin and sectioned at 5μm thickness. These sections were strained with anti-ubiquitin body to observe the forms of ubiquitin cytoplasm inclusion.Results: Comparing the memory action time of SOD1^G93A transgenic mice to negative controls , it shortens apparently.The memory action time of the 60 days ,90 days , and 120 days SOD1^G93A transgenic mouse are 68.00±47.16s, 55.20±92.99s, 110.10±116.52s. While the the memory action time of the negative controls are 65.60±89.94s, 158.00±88.31s, 169.80±122.96s.At the same time, the memory errors frequency in 5 minutes of SOD1^G93A transgenic mouse are 3.40±2.84 times, 5.20±3.08 times, 1.80±1.32 times, and the negative controls are 3.30±2.16 times, 2.30±1.95 times, 2.00±2.75 times. Through independent-samples T test, each group's memory action time and memory errors frequency in 5 minutes have statistical significance comparing to the negative controls(P<0.05). And the memory errors frequency in 5 minutes increases with duration prolonged of ALS(P<0.05). While the learning action time and learning errors frequency in 5 minutes have no statistical significance(P>0.05).But the memory action time has no correlation with duration of ALS(P>0.05).The pathology finds crescent-or ring-shaped ubiquitin cytoplasm inclusions and granular cytoplasmic staining without a well-defined structure in CA1, CA3 and dentate gyrus of the SOD1^G93A transgenic mouse'hippocampus through immunohistochemistry. The most common type of dentate fascia inclusion had a crescent or ring shape. While generally in the negative controls , ubiquitin expresses in CA1,CA3 and dentate gyrus neurons cytoplasm, showed as tiny granular cytoplasmic staining. And the rate of positive neurons has statistical significance.Conclusions: Comparing to the negative controls, the spatial discrimination memory ability of SOD1^G93A transgenic mouse are injuried at prophase of onset. While learning ability injuring is not notablely. But the memory ability impairment is not complete correlation with duration of ALS. However, immunohistochemistry of SOD1^G93A transgenic mouse finds apparente polyubiquitinated protein aggregated abnormally in neurons cytoplasmic of hippocampus, which maybe correlation with the memory ability impairment.