Despite the characterization of many molecules involved in the development, function, and plasticity of excitatory synapses, a striking deficit remains in knowledge of the endogenous mechanisms controlling the coordinated expression of these molecules. Few transcription factors have been implicated in dendritic spine and excitatory synapse formation and the relationship between pathways regulating initial synapse development and adult synaptic plasticity is also unclear. This study demonstrates that a subunit of the NF-κB transcription factor, p65, regulates dendritic spine morphology and spine and excitatory synapse density both during developmental synaptogenesis and during increased demand for new synapses in mature neurons.
NF-κB activity is highly regulated during development with activity peaking before and during rapid synaptogenesis and dropping to low levels in mature neurons. Developmental changes in NF-κB activity do not appear to result from changes in the expression of NF-κB components, and are instead attributed to the post-translational activation pathway characteristic of this transcription factor. During developmental synaptogenesis, NF-κB activation can be blocked by the acute inhibition of glutamate receptors, indicating a dependence on excitatory neurotransmission.
In mature neurons, basal NF-κB activity is low and loss of p65 no longer attenuates basal spine density. Instead, stimuli such as estrogen and short-term bicuculline that induce demand for new synapses, both activate NF-κB and require NF-κB in order to upregulate spine density in mature neurons. Collectively, this data reveals that NF-κB may fulfill a unique function in the regulation of excitatory synapse number and function. Other transcription factors, such as Cux1 and Cux2, have been reported to regulate the basal density of spine and excitatory synapses in the mature nervous system (Cubelos et al.). In contrast, NF-κB does not appear to affect the maintenance of basal spine density in mature neurons and is instead specifically required for stimulus-induced upregulation of dendritic spine density during this period. This role is consistent with the requirement for NF-κB in behavioral learning paradigms and in vitro assays of plasticity, as well as the absence of reported brain structural defects in mice lacking subunits of the NF-κB transcription factor.
Structural plasticity of dendritic spines and synapses is increasingly appreciated as a candidate for an enduring memory trace or engram (Hubener and Bonhoeffer). The magnitude of changes in spine density induced by p65-deficiency, near 30 % loss both in vitro
and in vivo
, closely approximates structural plasticity associated with both learning and regulation by estrogen. Neurons from control animals have been reported to have between 10 – 40 % fewer spines than neurons from animals undergoing learning paradigms or estrogen exposure (Gould et al., 1990
; Moser et al., 1994
; Restivo et al., 2009
). Structural plasticity also encompasses changes in dendritic spine morphology, where spine head size is highly responsive to activity. In p65-deficient neurons, we observe a reduction in spine head volume as well as reduced mEPSC amplitudes carried by AMPA receptors. These findings are consistent with previous reports that larger spine heads and larger synapses correlate with increased numbers of AMPA receptors and increased AMPA receptor-mediated currents (Nusser et al., 1998
; Matsuzaki et al., 2001
). NF-κB specifically regulates the density of excitatory, but not inhibitory, synapses and influences spine head volume and, to a lesser extent, spine length (,).
Remarkably, the p65 subunit of NF-κB is enriched in the dendritic spines whose structural plasticity it regulates. Our data clearly demonstrate, however, that p65-mediated regulation of spine density absolutely depends on the ability of p65 to bind DNA and activate transcription of target genes rather than being mediated by local protein-protein interactions. Point mutation of the DNA-binding domain of p65 as well as deletion of the transactivation domain both eliminate NF-κB transcriptional activity and regulation of dendritic spines, while leaving spine enrichment unaffected. The importance of spine enrichment for initial NF-κB activation or other cellular functions is not precluded by these data. In fact, other studies indicate that NF-κB can be activated locally at the synapse and that retrograde motor transport can mediate NF-κB-dependent gene expression in response to synaptic stimulation (Wellmann et al., 2001
; Meffert et al., 2003
; Mikenberg et al., 2007
; Shrum et al., 2009
). The p65 subunit thus appears to be both localized in spines and able to provide feedback to control spine density and morphology and the recruitment of presynaptic elements in a cell autonomous manner.
Other subunits of mammalian NF-κB, including p50 and c-Rel, have also been implicated in synaptic plasticity and may have additional uncharacterized mechanisms in the regulation of synapse structure and function. A role for the drosophila homologs of NF-κB and IκB, Dorsal and Cactus, in regulating postsynaptic glutamate receptor clustering at the neuromuscular junction has been reported (Heckscher et al., 2007
). Another component of the NF-κB pathway, the IκB-kinase (IKK) was recently reported to alter spine number in medium spiny neurons of the nucleus accumbens in a cocaine reward model (Russo et al., 2009
). IKK can activate NF-κB as well as carry out NF-κB-independent functions, including chromatin remodeling (Perkins, 2007
). While a requirement for IKK-induced chromatin remodeling during plasticity has been demonstrated (Lubin and Sweatt, 2007
), downstream NF-κB activation by this kinase could also regulate nucleus accumbens plasticity in response to cocaine. Additional studies will be required to evaluate a generalized role of NF-κB in promoting dendritic spine and excitatory synapse formation in brain regions outside of the hippocampus.
We identify PSD-95 as a transcriptional target that is critical for NF-κB enhancement of dendritic spine density and is regulated in a p65-dependent manner both during neuronal development and in response to synaptic activity in mature neurons (). PSD-95 is a key component of the postsynaptic density with an ability to organize both post- and presynaptic machinery (El-Husseini et al., 2000
; Futai et al., 2007
; de Wit et al., 2009
) that might permit postsynaptic NF-κB activation to recruit excitatory presynaptic components as observed in our data (). Through its many binding partners, PSD-95 also influences glutamate receptor trafficking and synaptic strength (Fitzjohn et al., 2006
), making it a highly relevant target. PSD-95 is reported to regulate spine and synapse density during periods of rapid synaptogenesis (El-Husseini et al., 2000
; Ehrlich et al., 2007
), but not after synaptogenesis has plateaued (Elias et al., 2006
). These studies phenocopy NF-κB manipulations and further support a functional link between NF-κB and PSD-95. Nonetheless, it is likely that complex functions, such as regulating synaptic networks, will be achieved by the coordinated control of multiple gene targets. NF-κB-dependent genes remain incompletely characterized within the mammalian CNS, but several reported target genes that could act in concert with PSD-95 are: GluR1, BDNF, NGF, nNOS, and several adhesion molecules (e.g. NCAM, P-selectin and ephrin-A1) (Deregowski et al., 2002
; Shrum and Meffert, 2008
). The regulation of diffusible targets such as nitric oxide and neurotrophins could be speculated to contribute to the capacity of postsynaptic NF-κB manipulations to influence presynaptic elements.
Given the fundamental role of dendritic spines and associated synapses in information acquisition and retention, understanding the mechanisms responsible for their transcriptional regulation could provide insights to control points during development and learning and potential sources of synaptic pathology in disorders such as Alzheimer’s and Parkinson’s disease where NF-κB activation is dysregulated (Mattson and Meffert, 2006
). The data presented here demonstrate a conserved pathway requiring the p65 subunit of NF-κB to coordinately regulate excitatory synapse formation both during initial synaptogenesis and in mature neurons in response to increased demand for new synapses. This underscores the capacity of neurons to employ the same downstream pathway in the context of both developmental and mature stimulus-dependent upregulation of excitatory synapses. These findings also present a novel pathway regulating PSD-95 expression to mediate structural plasticity of dendritic spines and highlight transcription factor-specific roles in excitatory synapse regulation. NF-κB-dependent regulation of the density, size, and function of dendritic spines and excitatory synapses provides a potential cellular mechanism to underlie the importance of this transcription factor in learning and memory(Shrum and Meffert, 2008