The CDKL5 protein ......
What is CDKL5 ?
CDKL5 stands for Cyclin-Dependent Kinase-Like 5. A kinase is a protein that energises other proteins into action. In doing so, a kinase can also regulate cell function. There are about 500 kinases in the body. One particular group of kinases only become active when linked to another protein called Cyclin. They are therefore known as a Cyclin-Dependent Kinases (CDK) and they have an important role in the cell cycle whereby a single parent cell divides into 2 daughter cells.
The CDKL5 gene codes for a protein that is "Like" a Cyclin-Dependent Kinase in that it contains similar sequences of certain amino acids. There are 5 known Cyclin-Dependent Kinase-Like proteins. CDKL1-4 are also involved with brain development in some way although less seems to be known about them.
Studies have identified the presence of the CDKL5 protein in many different parts of the body. For children affected with a CDKL5 disorder, the role of CDKL5 in the developing brain is obviously important, although in time, it may turn out that the function of CDKL5 in other areas may also be reflected in the clinical features....who knows.
An interesting fact about the CDKL5 gene is that its coding sequence is highly preserved and appears in many other species. This suggests that the protein has an important functional role that has been maintained through evolution.
The structure of the CDKL5 protein
A protein consists of a chain of amino acids . There are 1030 amino acids in the CDKL5 protein which is divided into 2 regions, the catalytic or kinase domain, and the C-terminal domain.
Biochemical factors cause the protein chain to convolute into a 3-D structure. The function of the protein depends on its 3-D structure, which in turn depends on the correct sequencing of amino acids. Furthermore, although there is only one CDKL5 gene on the X-chromosome, there are at least 5 different forms of the protein that can result. One in particular that was previously identified, is thought to be the significant form in relation to CDKL5 disorders.
CDKL5 - the worker protein...
The Kinase domain is the active part of the protein in that, this is the part that energises other proteins into action. The kinase domain could be thought of as the "hands" of the protein that are doing the work.
The C-terminal, in contrast, can be thought of as the "legs" of the protein, in that research suggests that this part is involved in transporting the protein to the right area of the neuron. It may also have a role in regulating the function of the protein ... a sort of downing tools...!
In terms of mutations, those that specifically affect the kinase domain will predominantly affect the kinase (hands) function of the protein so that it can get to work but just can't do its job properly or even do it at all. In contrast, a protein with a mutation affecting the C-terminal (legs) may still potentially be able to do its job but can't get to work to do it. There are some mutations of the kinase domain that will also produce a frameshift (deletions and insertions can do this) such that both the kinase and C-terminal domains are affected - so you end up with a protein with no hands or legs...
It is thought that CDKL5 is important both in the nucleus, where it probably interacts with MeCP2 - hence the association of CDKL5 with Rett Syndrome, and in the cytoplasm of the cell. To understand how mutations in CDKL5 might affect brain function it is worth knowing something about the development and structure of the brain.
The structure of a neuron
The human brain contains about 86 billion neurons which are the main signal carrying cells. There are also other supporting cells called glial cells, such as astrocytes and oligodendrocytes, which actually constitute the majority of cells within the brain.
The structure of a neuron is dramatically different to a typical cell that you often see illustrated. The main cell body of a neuron has a network of branches (dendrites) that act like antennae, gathering information from other surrounding neurons.
Nerve cells generally function by carrying electrical signals. In fact, a “resting” neuron actually has a negative charge inside it. When stimulated by other neurons its negative charge can momentarily flip and become positive – an event called depolarisation. This positive charge can ultimately be carried down the axon of the neuron as an “action potential” and this forms the basis of the signal that the neuron then transmits to other nerve cells.
Types of neuron
In the brain there are actually a whole variety of different types of neuron with very different appearances. However,broadly speaking they can be divided into 3 types according to their function, namely motor neurons that transmit motor information, sensory neurons for sensory information and interneurons which convey information between different types of neurons. They can also be classified as being excitatory (they cause their target neuron to fire off a signal) or inhibitory (inhibit neuron firing)
Alternatively, they can be classified according to the chemicals (neurotransmitters) that are used to transmit signals across synapses from one neuron to another. These include cholinergic neurons, dopaminergic neurons, GABAergic neurons, glutamatergic neurons and serotonergic neurons.
Glutamatergic neurons are commonly excitatory while GABAergic neurons are commonly inhibitory. This is relevant to CDKL5 as the anti-epileptic drug Vigabatrin (Sabril) acts by increasing the levels of GABA in the brain. Furthermore, studies on knockout mouse models have shown that behavioural changes can be mapped to GABAergic neurons in the forebrain in which the Cdkl5 protein has been removed.
Dendrites and spines
The numerous dendrites emanating from the cell body allow information to be gathered from far and wide. Signals are then passed down the axon to the axon terminal where information is passed on to other neurons across synapses (junctions between neurons).
The neurons are densely packed together within various layers of the brain, so you can start to imagine how this arrangement produces the hugely complex information gathering and processing network that is the brain.
(Illustration © 2012 Alzheimer's Association. www.alz.org All rights reserved. Illustration by Stacy Janis.)
Dendrites themselves have small projections - known as spines. These are fairly "dynamic" structures, particularly in the developing brain, in that they can be seen to appear and disappear over relatively short periods of time. They are also able to change their morphology (shape or appearance) often within seconds.
This "plasticity" is a property largely determined, it seems, by active interactions from surrounding neurons - signals that are generated in the brain through the sensory input of our experiences. It is therefore thought that dendritic spines, their function and interactions, may also provide the basis of memory.
Research suggests that CDKL5 has an important role in their development and function. Lack of CDKL5 is associated with a reduction in dendritic arborization.
Early brain development
Previous studies on the normal development of the brain both before and after birth have shown how the morphology of neurons becomes increasingly complex. There is an increase in the size of neurons, and in the complexity of their dendrites (a process called arborization) which allows ever more connections to be made with other neurons around them.
This process is known as cell differentiation, and there are now a number studies investigating the role of CDKL5 in this process. Furthermore, as more studies are published, the complexity of the controlling pathways, as you might expect, are becoming more evident.
Modified from Amendola et al. "Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder." PloS one 9.5 (2014): e91613.
So, what happens in CDKL5?
As studies on mouse models become available, we are learning more about what happens to developing neurons when the CDKL5 protein is deficient. In the absence of the CDKL5 protein, neurons appear to be underdeveloped compared to normal neuron development.
The adjacent figure shows a developing neuron in a normal mouse - usually referred to as a wild-type (WT) mouse in scientific publications - compared to that in a CDKL5 knockout (KO) mouse, in which the CDKL5 protein is absent. The WT mouse neuron is developing a normal complex pattern of dendrite branching (arborization) whereas the KO mouse neuron is much less branched and developed.
Remembering that dendrites and spines gather signals (information) from surrounding neurons so that they can be passed on to other neurons, you can start to understand how the function of these underdeveloped neurons will be impaired. The consequential clinical features and symptoms that are produced will obviously depend on where in the brain these affected neurons are.
CDKL5 in the brain
The CDKL5 protein is probably distributed throughout all the neurons of the brain. However, there is evidence for the activity of the CDKL5 protein in specific areas of the brain. We know that children with CDKL5 often have visual cortical impairment, suggesting specific involvement of the visual cortex.
Most children with CDKL5 do not walk - Ellie doesn’t although she has normal power in her legs and arms - I know, I've been given a good kick... or a brilliant "hand off" on many an occasion...! However, she also has relatively poor balance and co-ordination which might be related to her poor vision or possibly to involvement of her cerebellum - a particular part of the brain responsible for co-ordination.
Studies on the activity of CDKL5 have also focused on an area of the brain called the hippocampus - involved in learning and memory. This is because the neurons here only really start to develop after birth and so the influence of the CDKL5 protein can more readily be studied.
So far, I have tried to provide an overview of the CDKL5 protein in terms of its structure and the effect that CDKL5 mutations have on the brain. That is, we are starting to understand the effects of CDKL5 mutations on the basic structure of neurons. However, to really understand the effects that CDKL5 mutations have on function, it is necessary to determine, study and understand the various molecular pathways in which the CDKL5 protein is involved ...... and this is where it really starts to get technical .....!
The CDKL5 Jigsaw Puzzle - the pieces so far......
CDKL5 - Cyclin-dependent kinase-like 5 is an enzyme that acts like a kinase. Enzymes are molecules that act as catalysts in chemical reactions, and a kinase does this by transferring phosphate groups (clusters of phosphorus and oxygen atoms) which are a source of biological energy. A study from 2005 suggested that CDKL5 belonged to same molecular pathway as MeCP2, the gene responsible for Rett syndrome. A study from 2008 suggested that the levels of CDKL5 in the brain normally start to rise immediately after birth to a maximum at about 14 days of life. There appears to be a correlation between the rise in levels of CDKL5 and neuronal maturation, at least suggesting a role for CDKL5 in neuronal differentiation and arborization. These observed morphological changes occur as a response to the sudden massive increase in sensory information that the brain receives from birth onwards and a further study in 2010 suggested a critical role for CDKL5 in this process. The CDKL5 protein appears to regulate the development of dendrites through a mechanism within the cytoplasm of the cell, by forming a complex with something called Rac1.
Rac1- is a protein that regulates cellular processes, and it would appear that it mediates the function of CDKL5 in neuronal morphogenesis. This process probably also involves another protein, called Brain-derived neurotrophic factor (BDNF) which is also known to be involved in the modulation of dendritic growth and morphology.
BDNF- Brain-derived neurotrophic factor is a protein that acts as a growth factor. It is involved in supporting the survival of existing neurons, and encouraging the growth and differentiation of new neurons and synapses. It's function in this process involves the activation of Rac1 proteins, a process in which CDKL5 has also been implicated.
MYCN gene- is a transcriptional factor that regulates the expression of many other genes. This gene may regulate the role of CDKL5 in cell differentiation and proliferation.
CAM’s- Cell Adhesion Molecules are proteins that are located on the surface of cells and allow cells to bind to each other and to their surroundings. Synapses are the junctions between neurons, and CAM’s appear to regulate various aspects of synaptic development and maturation.
NMDA receptors- N-Methyl-D-aspartate receptors are located in neuronal cell membranes at synaptic and extrasynaptic locations. They are thought to mediate various physiological and pathological processes, including the control of synaptic plasticity and memory function. Extrasynaptic NMDA receptor stimulationhas also been implicated in the regulation of CDKL5 activity.
PSD95- post synaptic density protein 95 is a protein associated with the synaptic NMDA receptor.
Netrins- are proteins involved in the growth and elongation of axons during development, a process known as axon guidance. One particular subgroup of netrins, NGL-1, which also appears to be a CAM, regulates the formation of certain synapses through interacting with PSD95, a process that is reinforced by the kinase action of CDKL5.
Amph 1 - amphiphysin 1 is a protein coded by a gene on chromosome 7. It is associated with the cytoplasmic surface of synaptic vesicles and is thought to have important roles in neuronal transmission and development. It has been identified as a potential important substrate of CDKL5.
Shootin 1 - You will recall that neurons are odd shaped cells that have a cell body and dendrites at one end that collect information, and then a long axon with a terminal end that transmits information to other neurons. Well, shootin 1 is a protein involved in determining neuronal polarisation, that is, which end is which.
MeCP2- Methyl CpG binding protein 2 is a protein involved in regulating the expression of various genes and may act on mRNA. In particular, it regulates those genes involved in brain function and the maintenance of synapse function. It is involved in the regulation of CDKL5 whilst research has also shown that CDKL5 in turn binds and phosphorylates DNA methyltransferase 1- a process that might be involved in the pathological processes of Rett syndrome, which is caused by mutations in the MECP2 gene in most cases.
HDAC4 - HDAC4 belongs to the family of histone deacetylases which are enzymes that modify histones. Histones are proteins around which DNA is wound and packaged in the nucleus. DNA is normally kept tightly wound up mainly to save space. It has to be unwound when the genes it contains need to be read. Once the gene has been read HDAC's come along and help package the DNA up again.
This is a schematic overview I have put together of recent research into the function of CDKL5. It is not a comprehensive overview but has allowed me to visualise how pieces of the CDKL5 jigsaw are coming together.....
These are the references I have used.
1. CDKL5 is a brain MeCP2 target gene regulated by DNA methylation - Carouge et al
2. Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1 - Kameshita et al
3. ExtrasynapticN-Methyl-D-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its
Proteasomal Degradation - Rusconi et al
4. Palmitoylation-dependent CDKL5–PSD-95 interaction regulates synaptic targeting of CDKL5 and dendritic spine development - Zhu et al
5. CDKL5 ensures excitatory synapse stability by reinforcing NGL-1–PSD95 interaction in the postsynaptic compartment and is impaired in
patient iPSC-derived neurons - Ricciardi et al
6. CDKL5, a Protein Associated with Rett Syndrome, Regulates Neuronal Morphogenesis via Rac1 Signaling - Chen et al
7. CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells - Valli et al
8. Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental
disorder - Sekiguchi et al
9. CDKL5 and Shootin 1 Interact and Concur in Regulating Neuronal Polarisation - Nawaz et al
10. HDAC4: A Key Factor Underlying Brain Developmental Alterations in CDKL5 Disorder - Trazzi et al
As time goes by, further research will demonstrate the involvement of CDKL5 in other molecular pathways. An excellent review of CDKL5-related disorders published in 2011 is available on-line. It is quite technical in parts but covers CDKL5 from clinical description to molecular genetics.
Signalling and substrates
Cell signalling or signalling pathways are the many complex processes that control and co-ordinate the different activities of cells. These are being increasingly investigated in CDKL5.
Signalling pathways can be simple, such as
Cell A -------- secretes substance B -------- that acts on target C
although they are also usually quite complex !!!
The adjacent diagram summarises the pathways in which the siganalling factor Akt is involved. Akt has also been implicated in the function of CDKL5. This is all pretty complex stuff - so we know much more research is still needed to unravel this….
Substrates are the molecules on which a protein - like CDKL5 - act. So far, MeCP2, DNA methyltransferase and amphiphysin 1 have all been identified as substrates of CDKL5. This is a database of "experimentally determined" CDKL5-substrate relationships.
Two particular research tools to study the function of CDKL5 at the molecular level have been developed, namely iPS technology and the development of knockout mouse models. Mouse models also allow the study of some of the clinical aspects. There is also research into protein replacement therapy (PRT).
iPS cells - induced pluripotent stem cells, are cells that have the ability to grow and divide into any specialised cell, like a muscle cell, a bone cell or a nerve cell for the study of CDKL5. The clever bit is that by using technology that was first described in 2006, iPS cells can now be “made” from ordinary cells like skin cells.
By taking a sample of skin cells from a child with a given CDKL5 mutation, it is possible to turn these into iPS cells which then develop into neurons that have the same mutation. These can then be studied and compared to neurons with a normal CDKL5 gene.
Although there are limitations to this technique, it allows for a detailed study of the function of the CDKL5 protein in the neuron, and also for the development of possible drug treatments. The scientists who developed these techniques were awarded the Nobel prize for medicine in 2012.
A knockout mouse is one where a specific gene has been either removed from the mouse DNA or just disrupted. This then allows for the study of many characteristics of the mouse when the specific protein coded by that gene is absent.
A number of knockout mouse models have been established for CDKL5.
You can also have a "knockin mouse" where the mouse has a particular mutation or other specific protein coding sequence inserted into its DNA. I understand that this is currently being developed for the R59X mutation in exon 5 which a number of children with a CDKL5 disorder have