Human SLC1A2 blocking peptide

ObjectiveMesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS. MethodsmiRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay. ResultsmiRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204. SignificancemiR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS.

Genetic polymorphisms in Solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2) have been linked with essential tremor. SLC1A2 encodes excitatory amino acid transporter type 2 (EAAT2), which clears glutamate from the synaptic cleft. One postulated mechanism for essential tremor is the over-excitation of glutamatergic olivo-cerebellar climbing fibers, leading to excitotoxic death of Purkinje cells. Other glutamatergic excitatory signals are transmitted to Purkinje cells via parallel fibers of cerebellar granule neurons. Therefore, the expression level of glutamate transporters could be important in essential tremor pathogenesis. Using Western blotting, we compared the expression levels of the two main glutamate transporters in the cerebellar cortex, EAAT1 and EAAT2, in postmortem tissue from 16 essential tremor cases and 13 age-matched controls. We also studied the localization of EAAT1 and EAAT2 using immunohistochemistry in 10 essential tremor cases and 12 controls. EAAT1 protein levels were similar in cases and controls (1.12 +/- 0.83 vs. 1.01 +/- 0.69, p = 0.71) whereas EAAT2 protein levels in essential tremor cases were only 1/3 of that in controls (0.35 +/- 0.23 vs. 1.00 +/- 0.62, p < 0.01). Interestingly, EAAT2, but not EAAT1, was expressed in astrocytic processes surrounding the Purkinje cell axon initial segment, a region of previously observed pathological changes in essential tremor. Our main finding, a significant reduction in cerebellar cortical EAAT2 protein levels in essential tremor, suggests that Purkinje cells in essential tremor might be more vulnerable to excitotoxic damage than those of controls.