Regulation of expression and role of the GDNF family receptors in neuronal development

The aim of this project was to determine the temporal and spatial pattern of expression of GDNF family receptors in the developing embryo, with particular emphasis on expression in the peripheral nervous system, and to investigate how expression of receptor mRNAs is regulated in developing neurons. It was hoped that the data obtained would prove useful in further characterizing the role that the GDNF family of neurotrophic factors play in embryonic development. Semi- quantitative PCR revealed that GFRα-1, GFRα-2, GFRα-4 and ret mRNAs are widely distributed with both complementary and overlapping, though distinct, patterns of expression in the chicken embryo during development. Different populations of PNS neurons display different levels of responsiveness to GDNF and NTN and their sensitivity to these factors change throughout development. Examination of receptor expression by quantitative RT-PCR revealed that neurons that are more sensitive to GDNF express higher levels of GFRα-1 mRNA than GFRα-2 mRNA, and neurons that are more sensitive to NTN express higher levels of GFRα-2 mRNA compared to GFRα-1 mRNA. However, developmental changes in responsiveness of a population of neurons to these factors are not consistently paralleled by changes in the relative levels of GFRα transcripts. Furthermore, all neuronal populations express relatively high levels of ret mRNA. These results indicate the responsiveness of PNS neurons to GDNF and NTN is in part governed by the relative levels of expression of their GPI-linked receptors. To determine how the expression of the GDNF family receptors is regulated, embryonic neurons were cultured under different experimental conditions. I found that GFRα-1, GFRα-2, GFRα-4 and ret mRNAs are not significantly regulated by GDNF and/or NTN. However, depolarizing levels of KC1 cause marked changes in the expression of GFRα mRNAs. The effects of KCl are inhibited by L-type Ca2+ channel antagonists, suggesting that they were mediated by elevation of intracellular free Ca2+. KCl treatment increases the response of neurons to GDNF and decreases their response to NTN. There is no marked effect of depolarization on ret mRNA expression

Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival

The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival

Neurofibromin 1 is a miRNA target in neurons.

Mutations of the neurofibromin 1 gene cause neurofibromatosis type 1, a disease in which learning and behavioral abnormalities are common. The disease is completely penetrant but shows variable phenotypic expression in patients. The repertoire of regulatory interactions utilized by neurons to control neurofibromin 1 expression is poorly understood. Here, we examined the contribution of microRNAs into this regulatory network. Using reporter assays, we provided evidence that miR-128 and to a lesser extent miR-137 and miR-103 reduced neurofibromin 1 reporter levels through specific binding to Nf1 3'-UTR. Mutations in all three predicted binding sites eliminated the reporter response. MiR-128 and miR-137, unlike miR-103 that showed a more ubiquitous expression, were predominantly expressed in brain with a distribution that resembled neurofibromin 1 expression in different tissues as well as during the course of neuronal development. In the nervous system, all three microRNAs showed highest expression in neurons and least in Schwann cells and astrocytes. Overexpression of miR-128 alone or with miR-103 and miR-137 significantly reduced endogenous neurofibromin 1 protein levels, while antisense inhibition of these microRNAs enhanced translation of endogenous neurofibromin 1 and reporter in primary cultures of hippocampal neurons. These findings revealed a significant additional mechanism by which neurofibromin 1 is regulated in neurons and implicated new candidates for the treatment of multifarious neurofibromatosis type 1 cognitive symptoms

MiR-103, miR-128, miR-137 and Nf1 mRNA are co-expressed in the nervous system.

<p>Representative gels of the RT-PCR amplification products of miR-103, miR-128, miR-137, and <i>NF1</i> mRNA levels in: (<b>A</b>) Different murine tissues of embryonic day 18 animals; (<b>B</b>) Hippocampus of different ages; (<b>C</b>) Cortex of different ages; and (<b>D</b>) Different neural cell types. The amount of starting template for each condition was normalized to <i>U6</i> RNA. The number of PCR cycles for each miRNA RT-PCR assay is different between gels. Cycles were falling within the linear range of amplification for each primer pair. SCG, superior cervical ganglion; TG, trigeminal ganglion; E, embryonic day; P, postnatal day.</p

Unraveling the Pathways to Neuronal Homeostasis and Disease: Mechanistic Insights into the Role of RNA-Binding Proteins and Associated Factors

The timing, dosage and location of gene expression are fundamental determinants of brain architectural complexity. In neurons, this is, primarily, achieved by specific sets of trans-acting RNA-binding proteins (RBPs) and their associated factors that bind to specific cis elements throughout the RNA sequence to regulate splicing, polyadenylation, stability, transport and localized translation at both axons and dendrites. Not surprisingly, misregulation of RBP expression or disruption of its function due to mutations or sequestration into nuclear or cytoplasmic inclusions have been linked to the pathogenesis of several neuropsychiatric and neurodegenerative disorders such as fragile-X syndrome, autism spectrum disorders, spinal muscular atrophy, amyotrophic lateral sclerosis and frontotemporal dementia. This review discusses the roles of Pumilio, Staufen, IGF2BP, FMRP, Sam68, CPEB, NOVA, ELAVL, SMN, TDP43, FUS, TAF15, and TIA1/TIAR in RNA metabolism by analyzing their specific molecular and cellular function, the neurological symptoms associated with their perturbation, and their axodendritic transport/localization along with their target mRNAs as part of larger macromolecular complexes termed ribonucleoprotein (RNP) granules

MiR-128 and miR-137 synergize to reduce NF1 reporter protein expression.

<p>HEK293 cells were co-transfected with both the reporter gene (0.2 µg/reaction) and variable concentrations of pri-mir-128 and pri-mir-137 expression vectors. Luciferase activity was measured 48 hours later. (<b>A</b>) pri-mir-128 plasmid concentration was kept at 0.4 µg/reaction while pri-mir-137 concentration varied from 0.1–0.4 µg/reaction. (<b>B</b>) pri-mir-137 plasmid concentration was kept at 0.4 µg/reaction while pri-mir-128 concentration varied from 0.1–0.4 µg/reaction. Scramble 2 plasmid was supplemented in some of the reactions to achieve final miRNA plasmid concentration of 0.8 µg/reaction. These assays demonstrated that miR-128 and miR-137 synergistically reduce NF1 reporter expression when levels of the less abundant miRNA in the reaction are 50% or higher of the more abundant miRNA. The average value of two single miRNA expression plasmids (combination of scramble 2 plus pri-mir-9 or pri-mir-181 or pri-mir-218) predicted not to bind <i>Nf1</i> 3′-UTR were used as controls. Data show the mean ± s.e.m from 4 independent transfections (*, P<0.05; **, P<0.01, ***, P<0.001).</p

MiR-103, miR-128 and miR-137 reduce endogenous NF1 protein but not mRNA expression.

<p>(<b>A</b>) Representative gel of the RT-PCR amplification of <i>Nf1</i> mRNA in HEK293 cells transfected with scramble or miR-128 vectors for 48 hours. The amount of starting template for each condition was equilibrated relative to <i>U6</i> RNA. Cycles were falling within the linear range of amplification for each primer pair. (<b>B</b>) Representative Western Blot analysis demonstrating that miR-128, miR-128/103, and miR-128/137 reduce endogenous NF1 protein levels in human cells. HEK293 cells were transfected with miR-128 constructs for 48 hours. 15 µg of whole-cell lysate was then loaded in each lane. B-tubulin was used as an internal control for loading. Data show the mean ± s.e.m from 4 (for RT-PCR) and 8 (for Western Blot) independent experiments (***, P<0.001). B</p

MiR-128 alone or together with miR-103 or miR-137 reduces endogenous NF1 protein levels in neurons.

<p>(<b>A</b>) Representative images of transfected hippocampal neurons (green) stained with NF1 antibody (red). Merged images are shown in the third column of the panel. E16 murine hippocampal neurons were transfected with scramble or pri-mir-128 plasmids immediately after plating by using Lipofectamine 2000. Immunocytochemistry was carried out 40 hours after transfection. (<b>B</b>) Average decrease in NF1 protein levels. 80 neurons were analyzed per experiment. Data show the mean ± s.e.m from 6 independent experiments (*, P<0.05; **, P<0.01). (<b>C</b>) Freshly dissociated hippocampal neurons were transfected with <i>Nf1</i> reporter vector plus antisense 2′-O-methyl inhibitors (miR-128 alone or together with miR-103 or miR-137) and assayed 48 hours later. Data show the mean ± s.e.m from 4 independent experiments (*, P<0.05).</p