Abstract
Prestigio C, Ferrante D, Marte A, et al. Elife. 2021;10:e69058. doi: 10.7554/eLife.69058. The repressor-element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) controls hundreds of neuron-specific genes. We showed that REST/NRSF downregulates glutamatergic transmission in response to hyperactivity, thus contributing to neuronal homeostasis. However, whether GABAergic transmission is also implicated in the homeostatic action of REST/NRSF is unknown. Here, we show that hyperactivity-induced REST/NRSF activation triggers a homeostatic re-arrangement of GABAergic inhibition, with increased frequency of miniature inhibitory postsynaptic currents (IPSCs) and amplitude of evoked IPSCs in mouse-cultured hippocampal neurons. Notably, this effect is limited to inhibitory-onto-excitatory neuron synapses, whose density increases at a somatic level and decreases in dendritic regions, demonstrating a complex target- and area-selectivity. The upscaling of perisomatic inhibition was occluded by TrkB receptor inhibition and resulted from a coordinated and sequential activation of the Npas4 and BDNF gene programs. On the opposite, the downscaling of dendritic inhibition was REST-dependent, but BDNF-independent. The findings highlight the central role of REST/NRSF in the complex transcriptional responses aimed at rescuing physiological levels of network activity in front of the ever-changing environment.
Commentary
Homeostasis is the ability that living organisms have to maintain conditions essential for survival by constantly adjusting their internal environment in the face of external or internal disturbances. In neurons, homeostatic mechanisms are in place to control for appropriate responses to the myriad of electrochemical signals that ultimately control our behaviors. Activity-dependent regulation of neuronal excitability is achieved by adjusting the strength/weakness and efficacy of synaptic transmission at synaptic sites (synaptic plasticity), and through the regulation of intrinsic neuronal properties such as ion channel expression. 1 Disruptions in homeostatic plasticity have been reported in epilepsy and suggested as potential sources of neuronal instability during epileptogenesis and in already hyperexcitable networks. 2 One candidate regulatory molecule implicated in homeostatic changes associated with seizures and epilepsy is the RE-1 silencing transcription factor (REST), also known as the neuron-restrictive silencer factor (NRSF).2,3
REST/NRSF is a transcriptional repressor that regulates neuronal genes in both neural and non-neural cells. 4 This molecule controls neuronal differentiation and development as well as the expression of a number of ion channels including voltage-gated sodium channels (Nav1.2) and hyperpolarization-activated cyclic-nucleotide-gated channels (HCN1). 2 Under physiological conditions, neuronal REST/NRSF expression is typically low but can increase in response to neuronal stimulation. Seizure activity in animal models of epilepsy as well as neuronal hyperexcitability in in vitro neural cultures can upregulate REST/NRSF expression.2,3 Existing evidence supports that some of the physiological consequences of activity-driven increases in REST/NRSF signaling include the regulation of intrinsic neuronal excitability through the modulation of excitatory glutamatergic synaptic transmission. 2 More recently, work from Prestigio et al. published in eLife showed new evidence supporting that REST/NRSF signaling can also control homeostatic plasticity of inhibitory synaptic transmission following network hyperactivation. 3
The study by Prestigio et al. interrogated the role of REST/NRSF in the homeostatic control of GABAergic inhibition utilizing a series of elegant step-by-step experiments in which REST silencing was performed in cultured neuronal networks exposed to hyperactivity triggered by the convulsant 4-aminopyrodine (4AP), a potassium channel blocker. A combination of multielectrode array and patch clamp recordings were performed along with live-imaging, histological, and biochemical tests to measure the impact of a decoy oligodeoxynucleotide (ODN)-mediated silencing of REST on the physiological properties of both excitatory and inhibitory neurons. First, the study confirmed that hyperactivity induced nuclear translocation of REST in both excitatory neurons and inhibitory neurons, which were differently tagged and identified with GFP, thereby indicating a possible activity-dependent activation of the REST/NRSF-mediated transcriptional machinery in both cell types. In addition, this work confirmed that the homeostatic recovery from 4AP-induced network hyperactivity is dependent on REST signaling, as shown previously by this group and others.2,3
The novel discovery of this study is that the REST-mediated homeostatic recovery of basal network activity following a hyperactivity challenge included the upscaling in GABAergic neurotransmission from inhibitory synapses onto excitatory neurons. The evidence leading to this finding shows that ODN-mediated REST silencing effectively attenuated the 4AP-provoked increases in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) and the amplitude of evoked IPSCs, among other physiological properties of excitatory but not inhibitory neurons. In parallel, the 4AP-mediated increase and decrease in the density of inhibitory synaptic markers (VGAT and Gephyrin) in soma and dendrites, respectively, of excitatory cells was abolished in the ODN-treated groups. ODN treatment also attenuated the AP-4-induced increases in mRNA and protein levels of the GABAergic synaptic molecules, Gad1 & 2, VGAT, and Gad 67. This set of experiments and findings are straightforward, and convincingly support a target-specific homeostatic role for REST in controlling network hyperactivity by mediating GABAergic neurotransmission onto excitatory neurons. However, the extent at which glutamatergic synaptic markers were altered in comparison to the inhibitory ones was not interrogated. Concurrent re-arrangement of both excitatory and inhibitory synaptic proteins may impact not only the level of network excitability but also the timeline of homeostatic recovery. This information would be useful as a premise to further interrogate the role that those homeostatic mechanisms may play in the pathology of seizures and epilepsy.
The authors also investigated whether the mechanisms underlying the REST-mediated homeostatic effects on the modulation of inhibitory transmission involved signaling via brain-derived neurotrophic factor (BDNF) and neuronal PAS domain protein 4 (Npas4). BDNF signaling supports neuronal differentiation, growth, survival, and plasticity, 5 while the transcription factor Npas4 regulates genes responsible for the homeostatic control of excitatory–inhibitory balance. 6 Although the study shows that BDNF sequestration with the scavenger TrkB-fc attenuated the 4AP-induced increase in the amplitude of evoked postsynaptic inhibitory currents (eIPSCs) as well as the changes in the density of perisomatic inhibitory synaptic proteins on excitatory neurons, the link between REST, BDNF, and Npas4 was limited to a temporal correlation in their expression following 4AP-triggered network stimulation. Thus, it is still unclear how these molecules communicate with each other in this context, and how their crosstalk results in the regulation of inhibitory synaptic function.
Overall, this article provides additional support for REST/NRSF as a potential molecular mechanism that can be targeted to control enhanced neuronal activity. Utilizing simpler neuronal culture systems such as those used in this study can certainly help elucidate basic mechanisms controlling neuronal and network activity that can be later investigated in more complex neural networks (e.g., animal models) and in the context of epilepsy. However, the work by Prestigio et al. leads to more questions regarding the implications of homeostatic mechanisms in the pathology of epilepsy. In the context of seizure activity, would the REST-regulated enhancement of inhibitory synaptic transmission onto excitatory cells be a compensatory mechanism to attempt to suppress seizures or epileptiform activity? While in simple neural culture systems this may be the case, in animal models where REST/NRSF expression varies by age, brain region, and cell type, the role of REST/NRSF in the pathology of seizures and epilepsy becomes more complex, as demonstrated in studies where seizure-induced REST activation triggers a reduction in HCN1 channels that is associated with the generation of unprovoked seizures and memory defects in animal models of epilepsy.7-9 This evidence suggests the need for more research into the roles of REST signaling in different models of epilepsy at varying stages of disease (epileptogenesis vs. chronic epilepsy) to determine its contribution to the neuropathology and pathophysiology of epilepsy.
