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

Abstract

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.

Keywords: Calcium Signaling; Cell Signaling; Gene Expression; Glutamate Receptors Ionotropic (AMPA, NMDA); Neurobiology; Neurodegeneration; Neuroprotection; Neurotransmitters; Signal Transduction.

FIGURE 1.

Dendritic beading, cell death, and neuronal network dysfunction induced by excitotoxic stimuli. A–C, dissociated mouse hippocampal cultures at DIV14–15 were challenged with 2.5, 5, 10, 20, or 30 μm NMDA for 10 min and fixed for morphological evaluation after a recovery period of 0, 2, or 4 h (dendritic morphology for beading) or 24 h (nuclear morphology for cell death). Neuronal cultures were transfected with an EGFP expression construct for individual cell visualization, and nuclei were counterstained with Hoechst 33258 for cell death analysis. Cell death and dendritic beading were evaluated based on morphological alterations (for details see “Experimental Procedures”). A, dose-response analysis of NMDA-induced dendritic beading presented as a percentage of EGFP-positive neurons. Statistically significant differences compared with 0 μm NMDA at 0 h are indicated with asterisks as follows: *, p < 0.05; ****, p < 0.0001. Columns represent mean + S.E. B, dose-response analysis of NMDA-induced cell death as a percentage of whole cell population. Representative images are presented of hippocampal neurons expressing EGFP before and 10 min after 20 μm NMDA bath application. C, scale bars, 20 μm. D and E, MEA analysis of network activity before and after application to 10–20 μm NMDA. Traces shown are representative of an acute response to a 10-min NMDA bath application (D). E, synaptic transmission measured 0.5 to 24 h after a 10-min exposure to 10 or 20 μm NMDA or to a control solution.

FIGURE 2.

Rapid structural changes in hippocampal neurons in response to an excitotoxic insult. A–C, confocal live time-lapse imaging of hippocampal neurons expressing EGFP and mitochondrially targeted mCherry. The protocol used for live imaging is shown (A). Images are single plane projections of z-stacks at the indicated times before, during, and after washout of a 10-min NMDA (20 μm) application. Scale bars, 5 μm (B). C, data are the same as shown in B except that the NMDAR open-channel blocker MK-801 (20 μm) was included. D and E, TEM analysis of cultured hippocampal neurons; representative images are shown in D. Left panel, dendritic segment of a control neuron. Right panel, a swollen dendritic segment of a hippocampal neuron treated for 10 min with 20 μm NMDA (D, dendrite; M, mitochondrion; T, pre-synaptic terminal; small arrows indicate microtubules, large arrows indicate endoplasmic reticulum). Scale bars, 0.5 μm. E, quantitative assessment of mitochondrial rounding using TEM images. A large form factor indicates an elongated morphology, whereas a form factor close to 1 indicates a rounded morphology (see “Experimental Procedures” for details). Graph illustrates individual numbers and mean for each group; n = 31 mitochondria (control) and n = 53 mitochondria (NMDA) from n = 2 experiments. Statistical significance was determined by unpaired two-tailed t test based on n and is indicated with asterisks; ***, p < 0.001.

FIGURE 3.

Synaptic activity protects against dendrotoxicity. A, representative confocal images of dendrites with spines of EGFP-transfected neurons subjected to 6 or 16 h of AP bursting induced by bicuculline (Bic) (50 μm). Scale bars, 5 μm. B and C, histograms show the quantification of the percentage of EGFP-positive neurons displaying dendritic beading after various treatments. B, 16 h of AP bursting induced by Bic reduces dendritic beading induced by a subsequent exposure to 20 μm NMDA for 10 min. Analysis of the role of nuclear calcium signaling for AP bursting mediated protection against NMDA-induced dendritic beading. Nuclear calcium signaling was blocked by infecting hippocampal neurons at DIV7 with rAAV-mCherry-CaMBP4 or, as control, with rAAV-mCherry-NLS. Cells were subsequently transfected with EGFP for visual assessment of dendritic beading (B). Analysis of the importance of transcription for AP bursting mediated protection against NMDA-induced dendritic beading. Transcription was blocked by actinomycin D (10 μg/ml) incubation prior to the induction of AP bursting (C). Tetrodotoxin (1 μm) was included in experiments with CaMBP4 and actinomycin D (B and C) during NMDA application to prevent secondary AP-mediated glutamate release. Statistically significant differences are indicated with asterisks as follows: *, p < 0.05; **, p < 0.01, ns = nonsignificant. Columns represent mean + S.E.

FIGURE 4.

ATF3 is dendroprotective. A and B, confocal images (z-stacks projected into one plane) of cultured hippocampal neurons expressing FLAG-tagged ATF3 and EGFP at DIV11–12. A representative example is shown. Scale bar, 20 μm (A). Confocal images showing dendritic beading of an ATF3-overexpressing and a control neuron at the indicated times before and after a 10-min application of 20 μm NMDA. Scale bars, 1 μm (B). C, quantification of dendritic area of ATF3-overexpressing and control neurons at the indicated time points after NMDA treatment. Statistically significant differences are indicated with asterisks as follows: ***, p < 0.001. Data are presented as mean + S.E.

FIGURE 5.

ATF3 loss-of-function renders neurons more susceptible to dendrotoxicity. A and B, confocal images (z-stacks projected into one plane) of cultured hippocampal neurons transfected with scrambled control shRNA (shSCR) (A) or ATF3 targeting shRNA (shATF3) (B). Transfected neurons are indicated with arrows, and untransfected neurons are indicated with arrowheads. The expression of endogenous ATF3 was detected immunocytochemically. Scale bar, 20 μm. C, quantification of dendritic area of ATF3 knockdown and control neurons at the indicated time points after NMDA treatment. Statistically significant differences are indicated with asterisks as follows: *, p < 0.05; **, p < 0.01. Data are presented as mean + S.E.

FIGURE 6.

ATF3-mediated dendroprotection enhances recovery of neuronal network activity after an excitotoxic insult. A and B, for MEA analysis, hippocampal neurons were infected at DIV7 with either rAAV-ATF3-FLAG or with rAAV-mCherry-NLS. Representative images of rAAV-ATF3-FLAG-infected hippocampal neurons labeled with anti-FLAG antibody (upper panels) and rAAV-NLS-mCherry infected hippocampal neurons (lower panels) plated on MEAs. Scale bars, 20 μm (A). Firing rates were normalized to their respective rates at DIV13 of uninfected hippocampal neurons and hippocampal neurons infected with rAAV-ATF3-FLAG or rAAV-mCherry-NLS at the indicated time points before and after a 10-min application of 20 μm NMDA (B). Statistically significant differences are indicated with asterisks as follows: ***, p < 0.001. Data are presented as mean + S.E.