The Na+-Ca2+ exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in ischemic brain preconditioning (PC) by hypoxia-inducible factor 1 (HIF-1). Because ncx1 brain promoter (ncx1-Br) has five putative consensus sequences, named Sp1A–E, for the specificity protein (Sp) family of transcription factors (Sp1–4), we investigated the role of this family in regulating ncx1 transcription in rat cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1-Br in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C–E. Furthermore, by transient middle cerebral artery occlusion (tMCAO) in rats, the transcriptional repressors Sp3 and REST colocalized with the two histone-deacetylases (HDACs) HDAC1 and HDAC2 on the ncx1-Br, with a consequent hypoacetylation. Contrarily, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalized with histone acetyltransferase p300 on ncx1-Br with a consequent hyperacetylation. In addition, in neurons silenced with siRNA of NCX1 and subjected to oxygen and glucose deprivation (OGD) (3 h) plus reoxygenation (RX) (24 h), the neuroprotection of Class I HDAC inhibitor MS-275 was counteracted, whereas in neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD/RX), the neurotoxic effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by the Sp3/REST/HDAC1/HDAC2 complex in tMCAO and by the Sp1/HIF-1/p300 complex in PC+tMCAO and that epigenetic intervention, by modulating the acetylation of ncx1-Br, may be a strategy for the development of innovative therapeutic intervention in stroke.
Wednesday, 13 May 2015
Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination
MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3+splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3+ SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS.
ApoE4 and A{beta} Oligomers Reduce BDNF Expression via HDAC Nuclear Translocation
Apolipoprotein E4 (ApoE4) is a major genetic risk factor for several neurodegenerative disorders, including Alzheimer's disease (AD). Epigenetic dysregulation, including aberrations in histone acetylation, is also associated with AD. We show here for the first time that ApoE4 increases nuclear translocation of histone deacetylases (HDACs) in human neurons, thereby reducing BDNF expression, whereas ApoE3 increases histone 3 acetylation and upregulates BDNF expression. Amyloid-β (Aβ) oligomers, which have been implicated in AD, caused effects similar to ApoE4. Blocking low-density lipoprotein receptor-related protein 1 (LRP-1) receptor with receptor-associated protein (RAP) or LRP-1 siRNA abolished the ApoE effects. ApoE3 also induced expression of protein kinase C (PKC) and PKC retained HDACs in the cytosol. PKC activation and ApoE3 supplementation prevented ApoE4-mediated BDNF downregulation. PKC activation also reversed Aβ oligomer- and ApoE4-induced nuclear import of HDACs, preventing the loss in BDNF. ApoE4 induced HDAC6–BDNF promoter IV binding, which reduced BDNF exon IV expression. Nuclear HDAC4 and HDAC6 were more abundant in the hippocampus of ApoE4 transgenic mice than in ApoE3 transgenic mice or wild-type controls. Nuclear translocation of HDA6 was also elevated in the hippocampus of AD patients compared with age-matched controls. These results provide new insight into the cause of synaptic loss that is the most important pathologic correlate of cognitive deficits in AD.
Wednesday, 6 May 2015
Faithful Representation of Tactile Intensity under Different Contexts Emerges from the Distinct Adaptive Properties of the First Somatosensory Relay Stations
Adaptation allows neurons to respond to a wide range of stimulus intensities. However, it also leads to ambiguity as the representation of the external world depends on the context. We recorded neurons from Wistar rats' brainstem nuclei belonging to two major somatosensory pathways (lemniscal and paralemniscal) and explored the way in which they encode noisy stimuli under different contexts. We found that although their unadapted intensity-response curves are very similar, the adapted curves of the two pathways are distinctively different as they are optimized for encoding different intensity ranges. Lemniscal neurons most faithfully encoded stimuli when the background intensity was high, whereas paralemniscal cells best encoded stimuli under low intensity context. Intracellular recordings indicate that these differences emerge already at the synaptic level. We suggest that the two pathways synergistically improve the ability of this system to encode a wide range of intensities during natural stimulation, potentially reducing the inherent ambiguity of adaptive coding.
Layer 4 Pyramidal Neurons Exhibit Robust Dendritic Spine Plasticity In Vivo after Input Deprivation
Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinal in vivo two-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (~50%) increase in the rate of dendritic spine loss, acutely (4–8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1.
Audio-Vocal Interaction in Single Neurons of the Monkey Ventrolateral Prefrontal Cortex
Complex audio-vocal integration systems depend on a strong interconnection between the auditory and the vocal motor system. To gain cognitive control over audio-vocal interaction during vocal motor control, the PFC needs to be involved. Neurons in the ventrolateral PFC (VLPFC) have been shown to separately encode the sensory perceptions and motor production of vocalizations. It is unknown, however, whether single neurons in the PFC reflect audio-vocal interactions. We therefore recorded single-unit activity in the VLPFC of rhesus monkeys (Macaca mulatta) while they produced vocalizations on command or passively listened to monkey calls. We found that 12% of randomly selected neurons in VLPFC modulated their discharge rate in response to acoustic stimulation with species-specific calls. Almost three-fourths of these auditory neurons showed an additional modulation of their discharge rates either before and/or during the monkeys' motor production of vocalization. Based on these audio-vocal interactions, the VLPFC might be well positioned to combine higher order auditory processing with cognitive control of the vocal motor output. Such audio-vocal integration processes in the VLPFC might constitute a precursor for the evolution of complex learned audio-vocal integration systems, ultimately giving rise to human speech.
Distinct Brainstem and Forebrain Circuits Receiving Tracheal Sensory Neuron Inputs Revealed Using a Novel Conditional Anterograde Transsynaptic Viral Tracing System
Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.
