The discovery and reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue- such as the inability to biopsy or test tissue from the central nervous system directly. RNAs originating from hard to access tissues, such as neurons within the brain and spinal cord, have the potential to get to the periphery where they can be detected non-invasively. The formation and extracellular release of microvesicles and RNA binding proteins have been found to carry RNA from cells of the central nervous system to the periphery and protect the RNA from degradation. Extracellular miRNAs detectable in peripheral circulation can provide information about cellular changes associated with human health and disease. In order to associate miRNA signals present in cell-free peripheral biofluids with neurodegenerative disease status of patients with Alzheimer's and Parkinson's diseases, we assessed the miRNA content in cerebrospinal fluid and serum from postmortem subjects with full neuropathology evaluations. We profiled the miRNA content from 69 patients with Alzheimer's disease, 67 with Parkinson's disease and 78 neurologically normal controls using next generation small RNA sequencing (NGS). We report the average abundance of each detected miRNA in cerebrospinal fluid and in serum and describe 13 novel miRNAs that were identified. We correlated changes in miRNA expression with aspects of disease severity such as Braak stage, dementia status, plaque and tangle densities, and the presence and severity of Lewy body pathology. Many of the differentially expressed miRNAs detected in peripheral cell-free cerebrospinal fluid and serum were previously reported in the literature to be deregulated in brain tissue from patients with neurodegenerative disease. These data indicate that extracellular miRNAs detectable in the cerebrospinal fluid and serum are reflective of cell-based changes in pathology and can be used to assess disease progression and therapeutic efficacy.

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.

The developing retinotectal system of the Xenopus laevis tadpole is a model of choice for studying visual experience-dependent circuit maturation in the intact animal. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to play a critical role in the formation of sensory circuits in this preparation, however a comprehensive neuroanatomical study of GABAergic cell distribution in the developing tadpole has not been conducted. We report a detailed description of the spatial expression of GABA immunoreactivity in the Xenopus laevis tadpole brain at two key developmental stages: stage 40/42 around the onset of retinotectal innervation and stage 47 when the retinotectal circuit supports visually-guided behavior. During this period, GABAergic neurons within specific brain structures appeared to redistribute from clusters of neuronal somata to a sparser, more uniform distribution. Furthermore, we found that GABA levels were regulated by recent sensory experience. Both ELISA measurements of GABA concentration and quantitative analysis of GABA immunoreactivity in tissue sections from the optic tectum show that GABA increased in response to a 4 hr period of enhanced visual stimulation in stage 47 tadpoles. These observations reveal a remarkable degree of adaptability of GABAergic neurons in the developing brain, consistent with their key contributions to circuit development and function.