Identification of in vivo, conserved, TAF15 RNA binding sites reveals the impact of TAF15 on the neuronal transcriptome.

RNA binding proteins (RBPs) have emerged as major causative agents of amyotrophic lateral sclerosis (ALS). To investigate the function of TAF15, an RBP recently implicated in ALS, we explored its target RNA repertoire in normal human brain and mouse neurons. Coupling high-throughput sequencing of immunoprecipitated and crosslinked RNA with RNA sequencing and TAF15 knockdowns, we identified conserved TAF15 RNA targets and assessed the impact of TAF15 on the neuronal transcriptome. We describe a role of TAF15 in the regulation of splicing for a set of neuronal RNAs encoding proteins with essential roles in synaptic activities. We find that TAF15 is required for a critical alternative splicing event of the zeta-1 subunit of the glutamate N-methyl-D-aspartate receptor (Grin1) that controls the activity and trafficking of NR1. Our study uncovers neuronal RNA networks impacted by TAF15 and sets the stage for investigating the role of TAF15 in ALS pathogenesis.

The use of specific AAV serotypes to stably transduce primary CNS neuron cultures.

Although primary neuronal cell cultures are a valuable source of in vitro insight for many neurobiologists, all current gene expression technologies for these cells have significant drawbacks. Some of these limitations of current gene expression protocols include toxicity, transient expression, a requirement for postnatal neurons, and/or low efficiency. To date, many types of experiments were not possible because of these limitations. Here, we outline a methodology by which primary cultured neurons can be transduced at any age, after plating, with virtually no toxicity and continued gene expression for the lifetime of the culture. This method involves the use of adeno-associated viral vectors, which have the potential to be highly useful for either upregulation or downregulation of single or multiple genes, including neurotrophins, other neuroprotective genes, and neurotoxins.

Transcriptome transfer produces a predictable cellular phenotype.

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.

Knockdown of m-calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity.

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and mu-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain (capn2 ) was found to be 50-fold higher than for the mu-calpain catalytic subunit (capn1) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively. Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro. Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2-4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity.

Most current methods of gene delivery for primary cultured hippocampal neurons are limited by toxicity, transient expression, the use of immature neurons and/or low efficiency. We performed a direct comparison of seven serotypes of adeno-associated virus (AAV) vectors for genetic manipulation of primary cultured neurons in vitro. Serotypes 1, 2, 7, 8 and 9 mediated highly efficient, nontoxic, stable long-term gene expression in cultured cortical and hippocampal neurons aged 0-4 weeks in vitro; serotypes 5 and 6 were associated with toxicity at high doses. AAV1 transduced over 90% of all cells with approximately 80% of the transduced cells being neurons. The method was readily adapted to a high-throughput format to demonstrate neurotrophin-mediated neuroprotection from glutamate toxicity in cultured neurons at 2 weeks in vitro. These vectors should prove highly useful for efficient overexpression or downregulation of genes in primary neuronal cultures at any developmental stage.

Increased GABA(A)-receptor alpha1-subunit expression in hippocampal dentate gyrus after early-life status epilepticus.

PURPOSE: Previous studies in neonatal (postnatal day 10) and adult rats suggest that status epilepticus (SE) induces changes in the alpha1 subunit of the GABA(A) receptor (GABRA1) in dentate granule neurons (DGNs) that are age dependent and vary inversely with the likelihood of epilepsy development. In the present study, we examined GABRA1 expression after SE at postnatal day 20 (P20), an intermediate age when only a subset of SE-exposed animals develop epilepsy. METHODS: SE was induced with lithium-pilocarpine or kainate at P20. Animals were video-EEG monitored after SE to determine the presence or absence of spontaneous seizures. GABRA1 mRNA and protein levels were determined 7 days or 3 months later in SE-exposed and control animals by using a combination of aRNA amplification, Western blotting, and immunohistochemistry techniques. RESULTS: GABRA1 mRNA levels in DGNs of SE-exposed rats that did not become epileptic were higher than those in control rats, but were not different from DGNs in epileptic SE-exposed rats. GABRA1 protein levels in dentate gyrus were significantly increased in both epileptic and nonepileptic SE-exposed rats compared with controls. GABRA1 mRNA changes were region specific and did not occur in CA1 or CA3 areas of hippocampus. GABRA1 alterations were present by 1 week after P20 SE and were similar whether pilocarpine or kainate was used to induced SE. CONCLUSIONS: P20 SE results in persistent increases in GABRA1 levels selectively in dentate gyrus. These changes preceded the onset of epilepsy, were not model specific, and occurred in both epileptic and nonepileptic animals.

Injuring neurons induces neuronal differentiation in a population of hippocampal precursor cells in culture.

A novel population of hippocampal precursor cells (HPCs) that can be induced to differentiate into astrocytes and oligodendrocytes can be derived from hippocampal cultures grown in serum-free media. The HPCs are PDGF-responsive, do not proliferate with bFGF, and grow as sheets of cells rather than gathering into neurospheres. The HPCs share many markers (A2B5, GD3, poly-sialylated neuronal common adhesion molecule (PSA-NCAM), and NG2) with oligodendrocyte precursor cells (OPCs). The HPCs do not express markers for mature neurons, astrocytes, or oligodendrocytes. Like OPCs, the HPCs differentiate into glial fibrillary acidic protein (GFAP)+ astrocytes and GalC+ oligodendrocytes with the addition of bone morphogenetic protein-4 (BMP-4) and triiodothyronine (T3), respectively. They do not differentiate into neurons with the addition or withdrawal of basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), or retinoic acid (RA). These HPCs can be stimulated to differentiate into neuron-like cells by the induction of neuronal injury or cell death in nearby cultured neurons or by conditioned medium from injured neuronal cultures. Under these conditions, HPCs grow larger, develop more extensive dendritic processes, become microtubule-associated protein-2-immunoreactive, express large voltage-dependent sodium currents, and form synaptic connections. The conversion of endogenous pluripotent precursor cells into neurons in response to local brain injury may be an important component of central nervous system homeostasis.

Fate of newborn dentate granule cells after early life status epilepticus.

PURPOSE: To determine the fate of newborn dentate granule cells (DGCs) after lithium-pilocarpine-induced status epilepticus (SE) in an immature rat. METHODS: Postnatal day 20 (P20) rats were injected with lithium and pilocarpine to induce SE, and then with bromodeoxyuridine (BrdU) 4, 6, and 8 days later (P24, 26, and 28), and killed 1 day (P29), 1 week (P34), and 3 weeks (P50) after the last dose of BrdU for cell counts. Immunohistochemistry and TUNEL staining were performed to assess the fate of newborn DGCs. RESULTS: Pilocarpine-treated animals had significantly more BrdU-labeled DGCs than did littermate controls at all times. The day after the final BrdU injection (P29), sixfold more cells were found in pilocarpine-treated animals than in controls, which was reduced to threefold, 3 weeks later. A decrease in the BrdU-labeled cell density was noted from P29 to P50 in the control and pilocarpine-treated animals. Evidence of DGC cell death was seen in pilocarpine and control animals, with threefold more TUNEL-positive cells in the pilocarpine-treated than in the control animals at P29. The surviving newborn DGCs became mature neurons; expressing the neuronal marker NeuN in both control and pilocarpine-treated animals. CONCLUSIONS: These findings suggest that SE during postnatal development increases the birth and death of DGCs. A subset of the newborn DGCs survive and mature into dentate granule neurons, resulting in an increased population of immature DGCs after SE that may affect hippocampal physiology.