1. Introduction Results Conclusions Acknowledgements Alzheimer’s Disease (AD) is the most common chronic degenerative neurological disease, and there are currently no disease-modifying therapies. Methods  microRNA signaling pathways are disrupted in aMCI and AD  Two families of miRNA are downregulated in aMCI and AD: miR- 23a/b and miR-132/miR-212  Both of these families are expected to target the transcription factors Sirt1 and Foxo3a, and Sirt1 in frontal cortex is inversely correlated to miR-23a and miR-212 in aMCI samples  These changes in the frontal cortex may be part of a compensatory adjustment  Taken together, these data suggest that we have uncovered a novel miRNA-mediated neuroprotective pathway activated during prodromal AD.  These results raise the intriguing possibility that, when detected early in the disease process, augmenting protective pathways may be a viable treatment strategy for preventing or delaying onset of AD. Evidence for a neuroprotective microRNA pathway in mild cognitive impairment Rebecca B. Weinberg 1,2 and Scott E. Counts 1,2,3 1 Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 2 Department of Family Medicine, Michigan State University, Grand Rapids, MI 3 Hauenstein Neuroscience Institute, Mercy Health Saint Mary’s Hospital, Grand Rapids, MI Figure 2: q-PCR of miR-212 and mir-23a in the frontal cortext; mutual targets Sirt1 and Foxo3a protein levels are increased in aMCI Figure 4: miR-212 and Sirt1 levels are inversely correlated in frontal but not temporal cortex Figure 5: shRNA -mediated down-regulation of miR-212 and 23a increases Sirt1 protein expression in human neuronal cultures  Midfrontal cortex (Brodmann area 10) and inferior temporal cortex (Brodmann area 20) tissue was harvested postmortem from 32 participants in the Rush Religious Orders Study  miRNA was isolated via mirVana (Life Technologies) and microarray was performed using the Exiqon miRCURY LNA system  RT-PCR was using Applied Biosystems TaqMan assays and concentrations calculated using the ΔΔCt method.  miRNAs were inhibited using LNA based inhibitory siRNAs (Exiqon)  Cell toxicity assay was performed on differentiated NT2 cells, exposed to Aβ 1-42 (10µM) for 48 hours. Sirt1 activity was blocked with the inhibitor EX 527 (Tocris) Figure 6: Neuroprotection by miR-212 and 23a down-regulation is Sirt1 dependent miRNA NCI vs. AD fold changep value hsa-miR-2120.30.002 hsa-miR-1320.40.000907 hsa-miR-23a0.10.0000592 hsa-miR-23b0.50.00548 Figure 1: miR-212/miR-132 and miR-23a/b families are down-regulated in AD Grant support: NIH AG42146, AG14449 http://www.nia.nih.gov/alzheimers/publication/part-2-what-happens-brain-ad/changing-brain-ad The progression of AD takes place over many years, and involves a variety of cellular and molecular changes that predate symptoms. Clinically, a subset of individuals with mild cognitive impairment will progress to AD. Understanding the changes that occur during these stages is necessary to develop preventative interventions that can protect the brain prior to neuron loss. Increasing attention is being given to the regulation of complex neuronal physiology by small noncoding microRNAs (miRNAs), which regulate mRNA stability. miRNAs that target BACE1, a key enzyme in β-amyloid plaque formation, have been previously found to be altered in AD. However, much less is known about miRNAs disruptions early in the disease process, including in aMCI. Figure 3: in situ hybridization shows miR23a localization in frozen human brain tissue No probe negative control 20X U6 small nuclear RNA 100nM positive control 20X Scrambled miRNA 50nM negative control 20X miR-23a 40nM 20X