Exploring The Functional Role of SPAK in Central Nervous System:Behavioral Analysis,Dopamine Uptake and Inflammatory Responses.

• Main Authors: Han-Peng Shih, 施漢鵬
• Other Authors: Chuen-Lin Huang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/m332h8

碩士 === 國防醫學院 === 生理學研究所 === 102 === SPAK (Ste20-related Proline Alanine rich Kinase) is widely present in mammalian protein kinase, belongs the MAPK Ste-20 superfamily members. Previous studies have known that SPAK can modulate the activity of Na+/ K+/ Cl- co-transporters, which locate on plasma membrane, to regulate the GABAergic activity. Except SPAK is riched in heart and kidney, it has been reported that expressed in brain. Recent studies have demonstrated that overexpression of SPAK exacerbates experimental colitis while knockout of SPAK attenuates intestinal inflammation in mice. However, the real function of SPAK in the brain is still unknown. To investigate the possible role of SPAK in the brain in this study, we design three directions, respectively, animal behavioral tests, the striatal dopamine transporter (DAT) activity and biochemical analysis as well as brain inflammation by using SPAK knockout (SPAK (-/-)) mice to access these questions. First, results in animal behavior, SPAK (-/-) mice displayed poor performance in the beam walk test and showed poor learning and memory in the Morris water maze test than wild type. In the forced swimming test, SPAK (-/-) mice appeared more immobile time than wild type. However, there is no difference in accelerating rotarod-, locomotion-, tail suspension- and wire hang- test compare with wild type. Secondly, we found that female SPAK (-/-) mice appear lower DAT activity compared with wild type but no difference in male SPAK (-/-) mice. Using biochemical analysis showed the lower amount of DAT in synaptosomal membrane in female SPAK (-/-) mice. SPAK is mainly expressed in the cytosol of microglia and neuron and fewer signals of SPAK also appeared in astrocyte by immunofluorescence staining of brain sections. Finally, we use intraperitoneal injection of lipopolysaccharide (LPS) and paraquat (PQ) to induce brain inflammation. Under PQ and (LPS+PQ) treatment, the survival number of dopamine neurons in the substantia nigra in SPAK (-/-) mice were more than wild type. (LPS+PQ) treatment significantly increased the cell number which NF-B (p65) translocated into the microglia nucleus in wild type mice. Interestingly, under (LPS+PQ) treatment, reduction of the number of NF-B (p65) into the microglia nucleus in the cerebral cortex and striatum of SPAK (-/-) mice were appeared, however, no significant difference occurred in neuron. These results indicate SPAK (-/-) mice present significant defects in balance and motor coordination, impairment of learning ability and spatial memory. SPAK (-/-) mice also showed a tendency of depression. Lower DAT activity was found in female SPAK (-/-) mice suggested that estrogen might be involved in SPAK-regulated dopamine neurotransmission. In addition, (LPS+PQ)-induced NF-B (p65) into the microglia nucleus was markedly attenuated in SPAK (-/-) mice implied that inhibition or reduction of SPAK might play an anti-inflammatory effect in the brain.