Neuroscience

Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome.

MicroRNA (miRNA) are short sequences of RNA that function as post-transcriptional regulators by binding to target mRNA transcripts resulting in translational repression. A number of recent studies have identified miRNA as being involved in neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. However, the role of miRNA in multiple system atrophy (MSA), a progressive neurodegenerative disorder characterized by oligodendroglial accumulation of alpha-synuclein remains unexamined. In this context, this study examined miRNA profiles in MSA cases compared with controls and in transgenic (tg) models of MSA compared with non-tg mice. The results demonstrate a widespread dysregulation of miRNA in MSA cases, which is recapitulated in the murine models. The study employed a cross-disease, cross-species approach to identify miRNA that were either specifically dysregulated in MSA or were commonly dysregulated in neurodegenerative conditions such as Alzheimer’s disease, dementia with Lewy bodies, progressive supranuclear palsy and corticobasal degeneration or the tg mouse model equivalents of these disorders. Using this approach we identified a number of miRNA that were commonly dysregulated between disorders and those that were disease-specific. Moreover, we identified miR-96 as being up-regulated in MSA. Consistent with the up-regulation of miR-96, mRNA and protein levels of members of the solute carrier protein family SLC1A1 and SLC6A6, miR-96 target genes, were down-regulated in MSA cases and a tg model of MSA. These results suggest that miR-96 dysregulation may play a role in MSA and its target genes may be involved in the pathogenesis of MSA.

Premature infants often experience chronic hypoxia, resulting in cognitive & motor neurodevelopmental handicaps. These sometimes devastating handicaps are thought to be caused by compromised neural precursor cell (NPC) repair/recovery resulting in variable central nervous system (CNS) repair/recovery. We have identified differential responses of two mouse strains (C57BL/6 & CD1) to chronic hypoxia that span the range of responsiveness noted in the premature human population. We previously correlated several CNS tissue and cellular behaviors with the different behavioral parameters manifested by these two strains. In this report, we use unbiased array technology to interrogate the transcriptome of the subventricular zone (SVZ) in these strains. Our results illustrate differences in mRNA expression in the SVZ of both C57BL/6 and CD1 mice following hypoxia as well as differences between C57BL/6 and CD1 SVZ under both normoxic and hypoxic conditions. Differences in expression were found in gene sets associated with Sox10-mediated neural functions that explain, in part, the differential cognitive and motor responsiveness to hypoxic insult. This may shed additional light on our understanding of the variable responses noted in the human premature infant population and facilitate early intervention approaches. Further interrogation of the differentially expressed gene sets will provide a more complete understanding of the differential responses to, and recovery from, hypoxic insult allowing for more informed modeling of the ranges of disease severity observed in the very premature human population.

EZH2 plays an important role in stem cell renewal and maintenance by inducing gene silencing via its histone methyltransferase activity. Previously, we showed that EZH2 downregulation enhances neuron differentiation of human mesenchymal stem cells (hMSCs); however, the underlying mechanisms of EZH2- regulated neuron differentiation are still unclear. Here, we identify Smurf2 as the E3 ubiquitin ligase responsible for the polyubiquitination and proteasomemediated degradation of EZH2, which is required for neuron differentiation. A ChIP-on-chip screen combined with gene microarray analysis revealed that PPARg was the only gene involved in neuron differentiation with significant changes in both its modification and expression status during differentiation. Moreover, knocking down PPARg prevented cells from undergoing efficient neuron differentiation. In animal model, rats implanted with intracerebral EZH2-knocked-down hMSCs or hMSCs plus treatment with PPARg agonist (rosiglitazone) showed better improvement than those without EZH2 knockdown or rosiglitazone treatment after a stroke. Together, our results support Smurf2 as a regulator of EZH2 turnover to facilitate PPARg expression, which is specifically required for neuron differentiation, providing a molecular mechanism for clinical applications in the neurodegenerative diseases.

Luteolin, a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR- 132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.

We previously identified a number of perfluorooctane sulfonic acid (PFOS)-responsive transcripts in developing rat brains using microarray analysis. However, the underlying mechanisms and functional consequences remain unclear. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA and protein levels, might be responsible for PFOS-induced mRNA changes and consequent neural dysfunctions. We used eight miRNA arrays to profile the expression of brain miRNAs in neonatal rats on postnatal days (PND) 1 and 7 with maternal treatment of 0 (Control) and 3.2 mg/kg of PFOS feed from gestational day 1 to PND 7, and subsequently examined six potentially altered synapse-associated proteins to evaluate presumptive PFOS-responsive functions. Twenty-four brain miRNAs on PND 1 and 17 on PND 7 were significantly altered with PFOS exposure (P < 0.05), with miR-466b, -672, and -297, which are critical in neurodevelopment and synapse transmission, showing a more than 5-fold reduction. Levels of three synapse-involved proteins, NGFR, TrkC, and VGLUT2, were significantly decreased with no protein up-regulated on PND 1 or 7. Perfluorooctane sulfonic acid might affect calcium actions during synapse transmission in the nervous system by interfering with SYNJ1, ITPR1, and CALM1 via their targeting miRNAs. Our results indicated that miRNA had little direct regulatory effect on the expression of mRNAs and synapse-associated proteins tested in the developing rat brain exposed to PFOS, and it seems that the PFOS-induced synaptic dysfunctions and changes in transcripts resulted from a combinatory action of biological controllers and processes, rather than directed by one single factor.

EPAC proteins are the guanine nucleotide exchange factors that act as the intracellular receptors for cyclic AMP. Two variants of EPAC genes including EPAC1 and EPAC2 are cloned and are widely expressed throughout the brain. But, their functions in the brain remain unknown. Here, we genetically delete EPAC1 (EPAC1(-/-)), EPAC2 (EPAC2(-/-)), or both EPAC1 and EPAC2 genes (EPAC(-/-)) in the forebrain of mice. We show that EPAC null mutation impairs long-term potentiation (LTP) and that this impairment is paralleled with the severe deficits in spatial learning and social interactions and is mediated in a direct manner by miR-124 transcription and Zif268 translation. Knockdown of miR-124 restores Zif268 and hence reverses all aspects of the EPAC(-/-) phenotypes, whereas expression of miR-124 or knockdown of Zif268 reproduces the effects of EPAC null mutation. Thus, EPAC proteins control miR-124 transcription in the brain for processing spatial learning and social interactions.

To investigate the mechanism of £m receptor 1 (£mR1) neuroprotection in retinal neurons. Oxidative stress, which is implicated in diabetic retinopathy, was induced in mouse primary ganglion cells (GCs) and RGC-5 cells, and the effect of the £mR1 ligand (+)-pentazocine on pro- and anti-apoptotic and endoplasmic reticulum (ER) stress gene expression was examined. Binding of £mR1 to BiP, an ER chaperone protein, and £mR1 phosphorylation status were examined by immunoprecipitation. Retinas were harvested from Ins2Akita/+ diabetic mice treated with (+)-pentazocine, and the expression of ER stress genes and of the retinal transcriptome was evaluated. Oxidative stress induced the death of primary GCs and RGC-5 cells. The effect was decreased by the application of (+)-pentazocine. Stress increased £mR1 binding to BiP and enhanced £mR1 phosphorylation in RGC-5 cells. BiP binding was prevented, and £mR1 phosphorylation decreased in the presence of (+)-pentazocine. The ER stress proteins PERK, ATF4, ATF6, IRE1£, and CHOP were upregulated in RGC-5 cells during oxidative stress, but decreased in the presence of (+)-pentazocine. A similar phenomenon was observed in retinas of Ins2Akita/+ diabetic mice. Retinal transcriptome analysis of Ins2Akita/+ mice compared with wild-type revealed differential expression of the genes critically involved in oxidative stress, differentiation, and cell death. The expression profile of those genes was reversed when the Ins2Akita/+ mice were treated with (+)-pentazocine. In retinal neurons, the molecular chaperone £mR1 binds BiP under stressful conditions; (+)-pentazocine may exert its effects by dissociating £mR1 from BiP. As stress in retinal cells increases, phosphorylation of £mR1 is increased, which is attenuated when agonists bind to the receptor.

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of approximately 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.