Activating transcription factor 4 (ATF4) modulates post-synaptic development and dendritic spine morphology

Abstract

The ubiquitously expressed activating transcription factor 4 (ATF4) has been variably reported to either promote or inhibit neuronal plasticity and memory. However, the potential cellular bases for these and other actions of ATF4 in brain are not well-defined. In this report, we focus on ATF4's role in post-synaptic synapse development and dendritic spine morphology. shRNA-mediated silencing of ATF4 significantly reduces the densities of PSD-95 and GluR1 puncta (presumed markers of excitatory synapses) in long-term cultures of cortical and hippocampal neurons. ATF4 knockdown also decreases the density of mushroom spines and increases formation of abnormally-long dendritic filopodia in such cultures. In vivo knockdown of ATF4 in adult mouse hippocampal neurons also reduces mushroom spine density. In contrast, ATF4 over-expression does not affect the densities of PSD-95 puncta or mushrooom spines. Regulation of synaptic puncta and spine densities by ATF4 requires its transcriptional activity and is mediated at least in part by indirectly controlling the stability and expression of the total and active forms of the actin regulatory protein Cdc42. In support of such a mechanism, ATF4 silencing decreases the half-life of Cdc42 in cultured cortical neurons from 31.5 to 18.5 h while knockdown of Cdc42, like ATF4 knockdown, reduces the densities of mushroom spines and PSD-95 puncta. Thus, ATF4 appears to participate in neuronal development and plasticity by regulating the post-synaptic development of synapses and dendritic mushroom spines via a mechanism that includes regulation of Cdc42 levels.

Specific Downregulation of Hippocampal ATF4 Reveals a Necessary Role in Synaptic Plasticity and Memory

“…The infection efficiency was ~90%. We also observed that an shRNA targeted to another sequence of rat ATF4 had no effect on ATF4 expression (Figure 1A) and used this as a control (shCTRL2) along with another control (shCTRL1) in which 5 bases of shATF4 were changed (Liu et al, 2014). To verify infection efficiency and spreading of the lentiviral particles in vivo , we injected adult mouse hippocampi with an empty lenti-GFP vector, sacrificed them 1 month later and assessed GFP expression.…”

“…Both the amplitude and frequency of mEPSCs were significantly reduced (20% and 40%, respectively) in ATF4 down-regulated cultured hippocampal neurons compared to controls (Figures 4A,B,C). To confirm that these results were not due to off-target effects, we performed a rescue experiment in which the neurons were co-infected with lentiviruses expressing shATF4 and an ATF4 transcript (ATF4add) conservatively mutated to render it unresponsive to shATF4 (Liu et al, 2014). This results in knockdown of endogenous and over-expression of exogenous ATF4 (Figure 4D).…”

“…These shRNAs efficiently reduce ATF4 protein levels in cultured neurons and their actions on dendritic spines and post-synaptic markers are rescued by shRNA-resistant ATF4 expression constructs (Liu et al, 2014). We confirmed effective ATF4 knockdown by shATF4 in cultured hippocampal neurons at 2 weeks after infection (Figure 1A).…”

“…ATF4 knockdown in cultured hippocampal neurons did not alter total protein levels of AMPA receptor subunits GluR1 and GluR2 (Figures S3A,B) or of NMDA receptors subunits NMDAR1, R2A, and R2B (Figures S3C,D,E). We have reported that shATF4 reduces the density of post-synaptic markers (GluR1 and PSD95 puncta) and of dendritic mushroom spines in cultured neurons and mushroom spine density in mouse hippocampus (Liu et al, 2014). These changes could result in decreased glutamatergic synapse function.…”

“…Where possible, we also used ATF4 null mice. We recently reported that direct ATF4 down-regulation reduces the density of dendritic mushroom spines in vitro and in vivo accompanied by a decrease in post-synaptic markers for excitatory glutamatergic synapses (Liu et al, 2014). These effects were partially mediated by reduction in levels of total and active Cdc42, a small Rho family GTPase involved in regulation of the actin cytoskeleton.…”