Based on these findings our current data suggest that the loss of PGRN may increase the expression of miR 922, miR 548b 5p and miR 548c 5p through unknown mechanisms, leading to a decrease LDK378 in the levels of BAI3, an essential protein for synapse biology. Conclusions Overall, our studies support a novel role for miRNAs in FTLD TDP due to PGRN dysfunction and emphasize the value of combined miRNA and mRNA analyses. Future experiments in cell and animal models are needed to further evaluate the clinical potential of the miRNAs and gene targets identified in this study. The recent progress in human trials for miRNA based therapeutics in non CNS related disorders offers hope for new alter natives for the treatment of dementias, including FTLD.
Methods Brain samples For the miRNA array experiment, post mortem midfron tal cortex tissue was isolated from a collection of 40 FTLD TDP patients selected from the Mayo Clinic Jack sonville brain bank. All samples were obtained with appropriate informed consent with ethical commit tee approval. FTLD patients included the following pathologic classifications, FTLD TDP type 1 without PGRN mutations, FTLD TDP type 2, FTLD TDP type 3 and FTLD TDP type 1 with PGRN mutations. Total RNA quantification was performed using a NanoDrop ND 1000 spectrophotometer. RNA quality was evaluated by the Agi lent RNA 6000 Nano Kit and only samples with an RNA integrity value greater than 5 were included in this study. Mean RINs in frontal cortex were PGRN, PGRN type 1, FTLD TDP type 2, and FTLD TDP type 3. Mean RINs in cerebellums were PGRN, PGRN type 1, FTLD TDP type 2, and FTLD TDP type 3.
Cerebellar tissue of sufficient quality for miRNA expression analyses was also available for 31 of these FTLD TDP patients. For the miRNA expression analyses in cerebellum, 9 additional FTLD TDP patients were obtained from the MCJ brain bank. Importantly, all PGRN mutations included in this study were clear pathogenic loss of function mutations, leading to haploinsufficiency. Demographic and neuro pathologic information on all patients included in this study are summarized in Table 1. miRNA array analyses For mature miRNA expression profiling, real time RT PCR was performed using TaqMan Human MicroRNA Low Density Arrays Version 2. 0 which contain 667 unique assays specific to human mature miRNAs in a two card format.
Total RNA was isolated from human frontal cortical tissue using the miRVana PARIS kit from Ambion. Total RNA was reverse transcribed to cDNA for mature miRNAs AV-951 using Megaplex RT Primers in 7. 5 uls of final reaction volume. Subse quently, 2. 5 uls of cDNA was pre amplified in a 25 ul final volume with PreAmp Master Mix and Megaplex PreAmp Primers using standard conditions according to manufacturers instructions. Preamplified cDNA was diluted in 0.
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