Profiling of microRNA and protein from purified synaptoneurosomes in a neurodegenerative disease model

Abstract

The synaptic connections between neurons become lost at early stages of prion disease, often long before clinical signs of disease are detectable. In addition, microRNAs play an important role in modulating the localized protein synthesis at the synapse by binding to and regulating mRNA transcripts, of which some microRNAs have been attributed to dendritic remodeling. In the method described here, we simultaneously extracted protein and total RNA from purified synaptoneurosomes that were isolated from disease-affected forebrain and hippocampus tissue from mice infected with the RML strain of scrapie, a model of neurodegeneration. The synaptic protein samples contained enough material to perform Western blot analysis for detection of individual proteins of interest and to perform a more global proteome screen using mass spectrometry. For the isolated RNA, we describe how to screen hundreds of unique microRNAs using a RT-qPCR array. The concurrent detection of changes in protein and RNA at synapses during neurodegenerative disease, particularly at early stages where synaptic loss is reversible, is useful to illuminate the synaptic landscape of not only prion diseases but also other neurodegenerative conditions.