The nuclear calcium signaling target, activating transcription factor 3 (ATF3), protects against dendrotoxicity and facilitates the recovery of synaptic transmission after an excitotoxic insult.

The focal swellings of dendrites ("dendritic beading") are an early morphological hallmark of neuronal injury and dendrotoxicity. They are associated with a variety of pathological conditions, including brain ischemia, and cause an acute disruption of synaptic transmission and neuronal network function, which contribute to subsequent neuronal death. Here, we show that increased synaptic activity prior to excitotoxic injury protects, in a transcription-dependent manner, against dendritic beading. Expression of activating transcription factor 3 (ATF3), a nuclear calcium-regulated gene and member of the core gene program for acquired neuroprotection, can protect against dendritic beading. Conversely, knockdown of ATF3 exacerbates dendritic beading. Assessment of neuronal network functions using microelectrode array recordings revealed that hippocampal neurons expressing ATF3 were able to regain their ability for functional synaptic transmission and to participate in coherent neuronal network activity within 48 h after exposure to toxic concentrations of NMDA. Thus, in addition to attenuating cell death, synaptic activity and expression of ATF3 render hippocampal neurons more resistant to acute dendrotoxicity and loss of synapses. Dendroprotection can enhance recovery of neuronal network functions after excitotoxic insults.

Nuclear calcium signaling regulates nuclear export of a subset of class IIa histone deacetylases following synaptic activity.

In neurons, dynamic changes in the subcellular localization of histone deacetylases (HDACs) are thought to contribute to signal-regulated gene expression. Here we show that in mouse hippocampal neurons, synaptic activity-dependent nucleo-cytoplasmic shuttling is a common feature of all members of class IIa HDACs, which distinguishes them from other classes of HDACs. Nuclear calcium, a key regulator in neuronal gene expression, is required for the nuclear export of a subset of class IIa HDACs. We found that inhibition of nuclear calcium signaling using CaMBP4 or increasing the nuclear calcium buffering capacity by means of expression of a nuclear targeted version of parvalbumin (PV.NLS-mC) led to a build-up of HDAC4 and HDAC5 in the cell nucleus, which in the case of PV.NLS-mC can be reversed by nuclear calcium transients triggered by bursts of action potential firing. A similar nuclear accumulation of HDAC4 and HDAC5 was observed in vivo in the mouse hippocampus following stereotaxic delivery of recombinant adeno-associated viruses expressing either CaMBP4 or PV.NLS-mC. The modulation of HDAC4 activity either by RNA interference-mediated reduction of HDAC4 protein levels or by expression of a constitutively nuclear localized mutant of HDAC4 leads to changes in the mRNA levels of several nuclear calcium-regulated genes with known functions in acquired neuroprotection (atf3, serpinb2), memory consolidation (homer1, arc), and the development of chronic pain (ptgs2, c1qc). These results identify nuclear calcium as a regulator of nuclear export of HDAC4 and HDAC5. The reduction of nuclear localized HDACs represents a novel transcription-promoting pathway stimulated by nuclear calcium.

Nuclear calcium sensors reveal that repetition of trains of synaptic stimuli boosts nuclear calcium signaling in CA1 pyramidal neurons.

Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo.

Nuclear calcium signaling in spinal neurons drives a genomic program required for persistent inflammatory pain.

Persistent pain induced by noxious stimuli is characterized by the transition from normosensitivity to hypersensitivity. Underlying mechanisms are not well understood, although gene expression is considered important. Here, we show that persistent nociceptive-like activity triggers calcium transients in neuronal nuclei within the superficial spinal dorsal horn, and that nuclear calcium is necessary for the development of long-term inflammatory hypersensitivity. Using a nucleus-specific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics, and functional analyses, we discovered a pain-associated, nuclear calcium-regulated gene program in spinal excitatory neurons. This includes C1q, a modulator of synaptic spine morphogenesis, which we found to contribute to activity-dependent spine remodelling on spinal neurons in a manner functionally associated with inflammatory hypersensitivity. Thus, nuclear calcium integrates synapse-to-nucleus communication following noxious stimulation and controls a spinal genomic response that mediates the transition between acute and long-term nociceptive sensitization by modulating functional and structural plasticity.

Nuclear calcium-VEGFD signaling controls maintenance of dendrite arborization necessary for memory formation.

The role of neuronal dendrites is to receive and process synaptic inputs. The geometry of the dendritic arbor can undergo neuronal activity-dependent changes that may impact the cognitive abilities of the organism. Here we show that vascular endothelial growth factor D (VEGFD), commonly known as an angiogenic mitogen, controls the total length and complexity of dendrites both in cultured hippocampal neurons and in the adult mouse hippocampus. VEGFD expression is dependent upon basal neuronal activity and requires nuclear calcium-calmodulin-dependent protein kinase IV (CaMKIV) signaling. Suppression of VEGFD expression in the mouse hippocampus by RNA interference causes memory impairments. Thus, nuclear calcium-VEGFD signaling mediates the effect of neuronal activity on the maintenance of dendritic arbors in the adult hippocampus and is required for cognitive functioning. These results suggest that caution be employed in the clinical use of blockers of VEGFD signaling for antiangiogenic cancer therapy.