Cyclin E belongs to the core cell cycle machinery, which has been conserved from yeast to humans. The function of this machinery is to drive cell cycle progression. Indeed, many phosphorylation substrates of yeast and mammalian cyclin-Cdk enzymes represent proteins involved in cell division (Hwang and Clurman, 2005
; Sherr and Roberts, 2004
; Ubersax et al., 2003
). Consistent with this notion, analyses of knockout mice lacking particular cyclins or Cdks revealed distinct proliferative deficits in specific compartments (reviewed in Sherr and Roberts, 2004
). The relatively narrow, tissue-specific phenotypes of most cyclin- or Cdk-deficient strains underscored the overlapping, redundant functions of individual cyclins and Cdks in driving cell proliferation.
In clear distinction to these proliferative phenotypes, the work presented here revealed very unexpectedly that mammalian cyclin E, a cancer-related protein that normally serves to drive cell division, plays a cell cycle-independent and rate-limiting function in terminally differentiated neurons by controlling synapse formation and function. We found that acute ablation of cyclin E in postmitotic neurons led to a reduced number of synapses and dendritic spines, and resulted in decreased synaptic transmission. To ablate cyclin E in vivo
, we used conditional cyclin E knockout - Nestin-Cre mice. In these animals, deletion of cyclin E occurred during embryonic development, which might have impacted our analyses of adult cyclin E-deficient brains. However, we verified that ablation of cyclin E did not affect proliferation rates of embryonic brains and did not disturb brain development, as was the case in most other compartments of cyclin E-null mice (Geng et al., 2003
). Moreover analyses of brain-specific cyclin E knockout mice revealed very similar defects to the ones observed upon acute cyclin E shutdown in postmitotic neurons. In fact, the phenotypes seen in conditional cyclin E knockout – Nestin Cre mice were milder than those encountered upon acute cyclin E shutdown, a phenomenon seen in other knockouts and ascribed to developmental compensatory mechanisms (Lin et al., 2008
We propose that cyclin E affects synapse function and memory formation by sequestering Cdk5 into catalytically inactive complexes, thereby affecting the phosphorylation status and function of synaptic proteins (). Hence, cyclin E acts in this setting as a Cdk antagonist, in distinction to the well-described function of cyclins as Cdk activators. Consistent with this model, overexpression of Cdk5 phenocopied acute cyclin E loss, while knock-down of Cdk5 restored normal numbers of synapses and dendritic spines in cyclin E-null neurons. Moreover, we observed hyperphosphorylation of synaptic Cdk5 substrates on Cdk5-specific sites upon acute cyclin E shutdown in vitro
, and in the brains of cyclin E knockout animals. Hyperactivation of Cdk5 is also expected to indirectly affect other events relevant for learning and memory, such as NR1 subunit localization shown here, but also phosphorylation of CREB on Ser133 by PKA (Sindreu et al., 2007
). These abnormalities likely collectively contribute to decreased synaptic function and to memory deficits, observed in cyclin E-knockout mice. It should be noted that in addition to its inhibitory function, Cdk5 was also shown to play positive roles in synaptogenesis and synapse function, for example by phosphorylating the CASK protein and by regulating the interaction between CASK and liprin-α (Samuels et al., 2007
). It remains to be seen how these steps proceed in the absence of cyclin E.
Further studies will be needed to determine whether ablation of cyclin E results in decreased formation of new synapses and dendritic spines, reduced maintenance of the existing ones, or both. Importantly, active learning has been associated both with formation and maintenance of new dendritic spines in the brains (Xu et al., 2009
; Yang et al., 2009
). Given the influence of cyclin E in this process, it will be interesting to determine whether the levels of cyclin E in the brain change locally during learning and memory formation. A related question is what pathways control the abundance of cyclin E in terminally differentiated brain cells. In proliferating cells, the initial induction of E-type cyclins is driven at the transcriptional level by E2F transcription factors, while proteolytic degradation of cyclin E in late S phase is brought about by SCFFbw7
ubiquitin ligase (Hwang and Clurman, 2005
). In postmitotic cells the E2Fs are thought to be held in an inactive state by the pRB and pRB-like “pocket” proteins, therefore it is likely that the proteolytic machinery would be responsible for modulating cyclin E levels.
Of note, the ability of cyclin E to interact with Cdk5 in neurons was also reported by Miyajima et al. (1995)
, however our current work reveals the functional relationship between these two proteins. To the best of our knowledge, this is the first demonstration of a function for a cyclin protein in the formation of neuronal synaptic circuits and memories.
Cdk5 is considered as an essential regulator of neuronal differentiation, and it acts in a complex with its non-cyclin activators p35 or p39. Cdk5-p35/p39 complexes phosphorylate several neuronal proteins, and control a wide range of functions, including synapse formation and plasticity, neuronal migration, axon guidance, membrane transport and neurite outgrowth (Dhavan and Tsai, 2001
). It remains to be seen whether cyclin E affects all these Cdk5 functions, or it only serves to control Cdk5 in its synaptic role.
Another question raised by this work is whether abnormal levels or activity of cyclin E underlie human learning disabilities, cognitive disorders and other pathological conditions. Indeed, cyclin E is a major target of PARK2, an E3 ubiquitin-ligase, mutations in which are the most common cause of early-onset Parkinson's disease. (Staropoli et al, 2003
; Lüking et al., 2000
). Moreover, Cdk5 has been implicated in several human neurological and neurodegenerative diseases, such as Alzheimer's disease (Cruz and Tsai, 2004
; Dhavan and Tsai, 2001
), making cyclin E a potential player in these conditions. Interestingly, recent work indicates that reduction of Cdk5 levels can decrease pathological changes and reduce neuronal loss in an Alzheimer's disease mouse model (Piedrahita et al., 2010
). These observations suggest an exciting possibility that cyclin E, a protein we demonstrate to bind and inhibit Cdk5, might offer a therapeutic tool for ameliorating this devastating disease.