I have briefly reviewed three major pathways in neuronal excitation-transcription coupling in Section 1.3.2. Here I am going to focus on the pathway mediated by the transcriptional factor CREB and the underlying mechanisms.
1.6.1 Transcriptional factor CREB and memory formation
CREB (cAMP responsive element binding protein) is a member of a transcriptional factor family that share similar structures and functions (Silva et al., 1998). The transcriptional activity of CREB is critically regulated by the phosphorylation of Ser133 in the kinase inducible domain. As its name suggests, increased level of cytosolic cAMP can lead to CREB activation. This is achieved through dis-inhibition of the catalytic subunit of Protein Kinase A (PKA) and its subsequent nuclear translocation and phosphorylation of CREB Ser133 site (Bacskai et al., 1993). Phosphorylation of CREB Ser133 then recruits CREB-binding protein (CBP), a key regulator of RNA polymerase II-mediated transcription (Chrivia et al., 1993; Kalkhoven, 2004). In addition to PKA, CREB can also be phosphorylated by other kinases, including CaMKII (see Section 1.5.2), CaMKIV, PKC, and casein kinases (Gonzalez et al., 1989; Wu and McMurray, 2001). I am going to focus here and later in Chapter II on the pathway that involves phosphorylation of CREB Ser133 by CaMKIV.
It has been well established that new protein synthesis is required for long-term memory formation (Davis and Squire, 1984; Flexner et al., 1963). The role of CREB in memory was first shown by Kandel’s group (Dash et al., 1990). They injected the cAMP- responsive element (CRE, the DNA fragment that CREB binds to) to the nucleus of
Aplysia sensory neurons to block CREB binding. They found that disruption of CREB binding to its DNA targets selectively blocked serotonin-induced long-term facilitation but not short term facilitation. The importance of CREB in memory retention was further demonstrated in mammals by Silva’s lab, where they found that CREB mutant mice showed normal short-term memory but were deficient in long-term memory in cued or contextual conditioning and Morris water maze text (Bourtchuladze et al., 1994)).
1.6.2 Mechanism of L-type Ca2+ channel-mediated CREB phosphorylation
L-type Ca2+ channels activation can induce global increases of neuronal Ca2+
concentrations. However, at least under some conditions, the initiation of L-type Ca2+
channels signaling to trigger nuclear CREB phosphorylation appears to require increased Ca2+ concentrations only within a nanodomain in the immediate vicinity of the channel. In this paradigm, Ca2+ binds to the ubiquitous Ca2+ sensor, calmodulin, within the L-type Ca2+ channels nanodomain, and Ca2+/calmodulin then translocates to the nucleus to activate CaMKK (Ca2+/calmodulin-dependent protein kinase kinase). CaMKK then phosphorylates a nuclear localized kinase CaMKIV, which in turn phosphorylate CREB at Ser133 (Deisseroth et al., 1998; Ma et al., 2013).
Using this stimulation paradigm, CaMKII is specifically recruited to L-type Ca2+ channels (Li et al., 2016; Wheeler et al., 2008). In fact, recent studies indicate that this form of excitation-transcription coupling requires precisely coordinated recruitment and activation of two CaMKII holoenzymes within the L-type Ca2+ channel nanodomain (Li et al., 2016; Ma et al., 2014). It was proposed that CaMKII serves as a nuclear shuttle for Ca2+/calmodulin, which activates nuclear CaMKIV. In this model, CaMKII achieves the
shuttle function through several steps: 1) Ca2+ influx through L-type Ca2+ channels recruits CaMKII/ and CaMKII to the vicinity of the channel; 2) phosphorylation at Thr287 of CaMKII (presumably by CaMKII/) traps calmodulin; 3) dephosphorylation of Ser334 exposes the unique nuclear localization signal of CaMKII by a Ca2+- dependent phosphatase calcineurin; 4) CaMKII then shuttles to the nuclear and delivers calmodulin that is needed for CaMKK, CaMKIV, and CREB activation.
Chapter II will focus on the first step of this process. CaMKII has been reported to directly interact with multiple proteins within LTCC complexes, including the pore- forming 1 (CaV1.2 or CaV1.3) subunits, auxiliary 1 or 2 subunits, and associated scaffolding proteins such as densin (Abiria and Colbran, 2010; Grueter et al., 2008;
Hudmon et al., 2005; Jenkins et al., 2010; Simms et al., 2014). CaMKII interactions with
2 and densin play a role in modulating Ca2+-dependent facilitation of CaV1.2 and CaV1.3 LTCCs, respectively (Grueter et al., 2008; Jenkins et al., 2010; Koval et al., 2010). However, the roles, if any, of these interactions in E-T coupling are unclear.
Chapter II will present data showing that activated CaMKII directly interacts with the N- terminal domain of neuronal L-type Ca2+ channels, and that this helps to CaMKII to initiate downstream nuclear signaling.
1.6.3 L-type Ca2+ channel-mediated E-T coupling in disease and behavior
Mutations of L-type Ca2+ channels have been linked to multiple neurological and psychological diseases. For example, mutations of CaV1.3 are associated with Autism Spectrum Disorder (ASD) (De Rubeis et al., 2014; Pinggera et al., 2014; Pinggera and Striessnig, 2016). In addition, a mutation of CaV1.2 (G402S) that causes autistic
symptoms in Timothy syndrome disrupts L-type Ca2+ channel-mediate excitation transcription coupling (Li et al., 2016). A de novo mutation of CaMKII (E183V) found in an ASD patient was shown to disrupt CaMKII interactions with Ca2+ channel subunit, which may serve as one of the docking sites in L-type Ca2+ channel-mediated E-T coupling (Stephenson et al., 2017).
A study from Rajadhyaksha’s group showed that CaV1.3, but not CaV1.2, specifically mediates CREB Ser133 phosphorylation in naïve mice that were challenged with acute amphetamine or cocaine in the nucleus accumbens (Giordano et al., 2010).
FIGURE 1.3 L-type Ca2+ channel-mediated excitation-transcription coupling. Ca2+ influx through L-type Ca2+ channels recruits CaMKII and CaMKII to the vicinity of the channel. It is suggested that CaMKII can phosphorylate CaMKII Thr287 in a trans- holoenzyme manner. Thr287 phosphorylation enhances calmodulin binding by ~1000 fold, a phenomenon known as “calmodulin trapping”. In the meantime, Ca2+-dependent phosphatase calcineurin dephosphorylates CaMKII at the Ser334 site, exposing a functional nuclear localization signal that is masked by Ser334 phosphorylation.
CaMKII then translocates to the nucleus with trapped calmodulin. In the nucleus, calmodulin activates Ca2+/calmodulin-dependent kinase kinase (CaMKK), which phosphorylates and activates CaMKIV. CaMKIV in turn phosphorylates Ser133 of the CREB transcription factor, which recruits CREB-binding protein (CPB) and initiates transcription of downstream genes. More recently, it was found that the voltage-induced conformational change of the channel is also required for E-T coupling, and that NMDA receptors somehow are functional linked to L-type Ca2+ channels.