Neuronal nuclear calcium signaling suppression of microglial reactivity is mediated by osteoprotegerin after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is characterized by massive changes in neuronal excitation, from acute excitotoxicity to chronic hyper- or hypoexcitability. Nuclear calcium signaling pathways are involved in translating changes in synaptic inputs and neuronal activity into discrete transcriptional programs which not only affect neuronal survival and synaptic integrity, but also the crosstalk between neurons and glial cells. Here, we report the effects of blunting neuronal nuclear calcium signals in the context of TBI. METHODS: We used AAV vectors to express the genetically encoded and nuclear-targeted calcium buffer parvalbumin (PV.NLS.mCherry) or the calcium/calmodulin buffer CaMBP4.mCherry in neurons only. Upon TBI, the extent of neuroinflammation, neuronal death and synaptic loss were assessed by immunohistochemistry and targeted transcriptome analysis. Modulation of the overall level of neuronal activity was achieved by PSAM/PSEM chemogenetics targeted to parvalbumin interneurons. The functional impact of neuronal nuclear calcium buffering in TBI was assessed by quantification of spontaneous whisking. RESULTS: Buffering neuronal nuclear calcium unexpectedly resulted in a massive and long-lasting increase in the recruitment of reactive microglia to the injury site, which was characterized by a disease-associated and phagocytic phenotype. This effect was accompanied by a substantial surge in synaptic loss and significantly reduced whisking activity. Transcriptome analysis revealed a complex effect of TBI in the context of neuronal nuclear calcium buffering, with upregulation of complement factors, chemokines and interferon-response genes, as well as the downregulation of synaptic genes and epigenetic regulators compared to control conditions. Notably, nuclear calcium buffering led to a substantial loss in neuronal osteoprotegerin (OPG), whereas stimulation of neuronal firing induced OPG expression. Viral re-expression of OPG resulted in decreased microglial recruitment and synaptic loss. OPG upregulation was also observed in the CSF of human TBI patients, underscoring its translational value. CONCLUSION: Neuronal nuclear calcium signals regulate the degree of microglial recruitment and reactivity upon TBI via, among others, osteoprotegerin signals. Our findings support a model whereby neuronal activity altered after TBI exerts a powerful impact on the neuroinflammatory cascade, which in turn contributes to the overall loss of synapses and functional impairment.

Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition.

Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.

Organic anion transporter 1 is an HDAC4-regulated mediator of nociceptive hypersensitivity in mice.

Persistent pain is sustained by maladaptive changes in gene transcription resulting in altered function of the relevant circuits; therapies are still unsatisfactory. The epigenetic mechanisms and affected genes linking nociceptive activity to transcriptional changes and pathological sensitivity are unclear. Here, we found that, among several histone deacetylases (HDACs), synaptic activity specifically affects HDAC4 in murine spinal cord dorsal horn neurons. Noxious stimuli that induce long-lasting inflammatory hypersensitivity cause nuclear export and inactivation of HDAC4. The development of inflammation-associated mechanical hypersensitivity, but neither acute nor basal sensitivity, is impaired by the expression of a constitutively nuclear localized HDAC4 mutant. Next generation RNA-sequencing revealed an HDAC4-regulated gene program comprising mediators of sensitization including the organic anion transporter OAT1, known for its renal transport function. Using pharmacological and molecular tools to modulate OAT1 activity or expression, we causally link OAT1 to persistent inflammatory hypersensitivity in mice. Thus, HDAC4 is a key epigenetic regulator that translates nociceptive activity into sensitization by regulating OAT1, which is a potential target for pain-relieving therapies.

Coupling of NMDA receptors and TRPM4 guides discovery of unconventional neuroprotectants.

Excitotoxicity induced by NMDA receptors (NMDARs) is thought to be intimately linked to high intracellular calcium load. Unexpectedly, NMDAR-mediated toxicity can be eliminated without affecting NMDAR-induced calcium signals. Instead, excitotoxicity requires physical coupling of NMDARs to TRPM4. This interaction is mediated by intracellular domains located in the near-membrane portions of the receptors. Structure-based computational drug screening using the interaction interface of TRPM4 in complex with NMDARs identified small molecules that spare NMDAR-induced calcium signaling but disrupt the NMDAR/TRPM4 complex. These interaction interface inhibitors strongly reduce NMDA-triggered toxicity and mitochondrial dysfunction, abolish cyclic adenosine monophosphate-responsive element-binding protein (CREB) shutoff, boost gene induction, and reduce neuronal loss in mouse models of stroke and retinal degeneration. Recombinant or small-molecule NMDAR/TRPM4 interface inhibitors may mitigate currently untreatable human neurodegenerative diseases.

Epigenetic control of hypersensitivity in chronic inflammatory pain by the de novo DNA methyltransferase Dnmt3a2.

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study, we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant, an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by Complete Freund's Adjuvant injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.

Reliable optical detection of coherent neuronal activity in fast oscillating networks in vitro.

Cognitive and behavioral functions depend on the activation of stable neuronal assemblies, i.e. distributed groups of co-active neurons within neuronal networks. It is therefore crucial to monitor distributed patterns of activity in real time with single-neuron resolution. Microelectrode recordings allow detection of coincidence between discharges of identified units at high temporal resolution, but are not able to reveal the full spatial pattern of activity in multi-cellular assemblies. Therefore, observation of such distributed sets of neurons is a stronghold of optical techniques, but the required resolution, sensitivity, and speed are still challenging current technology. Here, we report a new approach for monitoring neuronal assemblies, using memory-related network oscillations in rodent hippocampal circuits as a model. The cytosolic calcium-sensitive fluorescent protein GCaMP3.NES was expressed using recombinant adeno-associated viral (rAAV)-mediated gene transfer in CA3 pyramidal neurons of cultured mouse hippocampal slices. After 14-21 days in culture, field potential recordings revealed spontaneous occurrence of sharp wave-ripple network events during which a fraction of local neurons is coherently activated. Using a custom-built epi-fluorescence microscope we could monitor a field of view of 410 µm × 410 µm with single-neuron optical resolution (20× objective, 0.4 NA). We developed a highly sensitive and specific wavelet-based method of cell identification allowing simultaneous observation of more than 150 neurons at frame rates of up to 60 Hz. Our recording configuration and image analysis provide a tool to investigate cognition-related activity patterns in the hippocampus and other circuits.

Reversible occlusion (on-off) valves in shunted tumor patients.

The first commercially produced adjustable valve for shunted hydrocephalus patients was introduced by H. Portnoy and R. Schulte in 1973. This valve is still in use and known as reversible occlusion or on-off valve. The reversible occlusion valve is mainly used in conjunction with an existing shunt in patients receiving intraventricular cytostatic therapy. The valve has a simple mechanical lock that is closed by external pressure application with a single finger. The study method is a retrospective clinical series of 15 patients undergoing a total of 16 valve implantations between 2003 and 2010 was carried out, and the valve was tested in vitro. We report a high incidence of accidental occlusions leading to a loss of consciousness in five patients (33.3%). We furthermore demonstrate in vitro that accidental occlusions can occur. The reversible occlusion valve is needed in shunted tumor patients receiving intrathecal administration of cytostatica. The mechanism works as long as no external pressure compresses the valve. However, head positions pose significant risks for unintentional occlusions. We stress the importance of: (1) a position near the midline avoiding the retroauricular or occipital regions, (2) a handling training for nurses and doctors, (3) instruction of patients and relatives, and (4) removal of the device after intrathecal cytostatic treatment.

2-Aminoethoxydiphenyl-borate (2-APB) increases excitability in pyramidal neurons.

Calcium ions (Ca(2+)) released from inositol trisphosphate (IP(3))-sensitive intracellular stores may participate in both the transient and extended regulation of neuronal excitability in neocortical and hippocampal pyramidal neurons. IP(3) receptor (IP(3)R) antagonists represent an important tool for dissociating these consequences of IP(3) generation and IP(3)R-dependent internal Ca(2+) release from the effects of other, concurrently stimulated second messenger signaling cascades and Ca(2+) sources. In this study, we have described the actions of the IP(3)R and store-operated Ca(2+) channel antagonist, 2-aminoethoxydiphenyl-borate (2-APB), on internal Ca(2+) release and plasma membrane excitability in neocortical and hippocampal pyramidal neurons. Specifically, we found that a dose of 2-APB (100 microM) sufficient for attenuating or blocking IP(3)-mediated internal Ca(2+) release also raised pyramidal neuron excitability. The 2-APB-dependent increase in excitability reversed upon washout and was characterized by an increase in input resistance, a decrease in the delay to action potential onset, an increase in the width of action potentials, a decrease in the magnitude of afterhyperpolarizations (AHPs), and an increase in the magnitude of post-spike afterdepolarizations (ADPs). From these observations, we conclude that 2-APB potently and reversibly increases neuronal excitability, likely via the inhibition of voltage- and Ca(2+)-dependent potassium (K(+)) conductances.