Molecular analysis of learning and memory 06/05

Question 1

What is the overall mechanism of CAMKII in LTP?

Answer 1

After activation of NMDA receptors in the PSD, Ca2+ enters the cell.
Ca++ binds to Ca++/Calmodulin, which activates CAMKII.
CAMKII phosophorylates itself.
CAMKII promotes movement of AMPAR receptors (e.g. by phosphorylation of TARPγ-8) to the PSD via RAS-ERK signalling.
CAMKII translocates the PSD binding NMDA receptors.
It phosporylates stargazin (AMPAR associated protein), allowing AMPAR to be anchored to PSD-95 and immobilising AMPAR in the PSD.
It phosphorylates GluRI AMPAR receptors, increasing channel conductance.

Question 2

What is the structure of CAMKII?

Answer 2

Holoenzyme consisting of 12 subunits - 2 stacks of 6 units.

Each subunit has an association domain, which binds subunits together.

Each has a catalytic domain, which phosphorylates substrates.

Each has a regulatory domain, containing an inhibitory domain - this can fold onto the catalytic domain, inactivating the enzyme and switching it ‘off’.

The regulatory domain also contains the Ca++/calmodulin binding domain. When Ca++/calmodulin binds to CAMKII, there is a structural change that removes the inhibition of the catalytic domain.

The inhibitory regulatory domain also contains a phosphorylation site, which is autophosphorylates by other subunits within enzyme (t268). When this site is phosphorylated, the activity of the enzyme becomes independent of Ca++/calmodulin.

The ca++/calmodulin regulatory domain also contains two phosphorylation sites (t305/6), which regulate the binding of ca++/calmodulin. When phosphorylated, they inhibit binding, essentially inhibiting the enzyme.

Sequencing of the protein and it’s structure can provide insight into potential biological function of molecules.

Question 3

What is the autophosphorylation switch of CAMKII?

Answer 3

At low levels of Ca++, the inhibitory domain is folded onto the regulatory domain and the enzyme is inhibited.
When calcium increases, it binds to Ca++/calmodulin, which then binds to Ca++/calmodulin regulatory domain of CAMKII, unfolding the subunit and allowing the catalytic domain to be exposed/active.
When Ca++ levels drop, Ca++/calmodulin unbinds and the subunit refolds, becoming inactive.
If Ca++ levels are high for sustained periods of time, then catalytic subunits phosphorylate one another at a threonine residue in the inhibitory domain, leading to Ca++/calmodulin indepedent activity - i.e., active even if Ca++ drops.

Question 4

What is a holoenzyme?

Answer 4

a biochemically active compound formed by the combination of an enzyme with a coenzyme.

CAMKII is a holoenzyme because until the autophosphorylation, it is dependent on Ca++/calmodulin as a co-enzyme.

Question 5

What is the most specific CAMKII inhibitor?

Answer 5

peptide inhibitor CN21. It is derived from endogenous inhibitory proteins for CAMKII, mimicking their inhibitory domain.

Others such as KN–63 or KN-93 are often cited to be specific, but they inhibit many CAMKs and ion channels. This creates a confound in any experiment using these molecules.

Question 6

What are the effects of CN21?

Answer 6

If injected to the hippocampus prior to contextual fear conditioning, it impairs freezing response (impaired learning).
If injected after contextual fear conditioning, it has no effect.

This implies that activation of CAMKII is involved in fear contextual learning. It is a correlatory study however, with no insight into mechanisms or necessity.

Question 7

What is the effect of knocking out alpha-CAMKII?

Answer 7

An early study in 1992 first provided evidence for CAMKII role in learning in memory. Genetic KO mice (alpha-CAMKII ablated in embryonic stem cells) show slower time to learn where morris water maze is. When the platform is removed, they spend equal time searching in all quadrants. This suggests a memory impairment in spatial memory and learning. This is correlative, but it suggests that CAMKII activation is involved in some process of learning in memory.

They also found that LTP was impaired. They removed hippocampal slices, and measured EPSPs after tetanic stimulation in CA1. They found that normal mice showed LTP, which was impaired in most mutants (panel B) but not all mutants/brain slices (panel C). This shows that CAMKII is involved in LTP, but its role as a required molecule for LTP unclear.

Question 8

What is the difference between loss of function point mutations in CAMKII and complete KO on learning and memory?

Answer 8

Point mutation of T305D mimicks a constant inhibitory phosphorylation of ca++/calmodulin regulatory zone and blocks ca++/calmodulin from activating CAMKII.

Point mutatation of T305D results in full loss of spatial learning and memory in MWM, whereas KO mice have some learning and memory if they recieve more training. This shows requirement of actiation of CAMKII by ca++/calmodulin and also suggests compensation in KO.

These mice show greater impairment of LTP than the full KO.

Fractionation of the PSD and analysis of b-CAMKII. In point mutation animals, there is the same level of b-CAMKII in the PSD as normal. In full KO mice, there is a 200% increase in b-CAMKII.

This suggests that full KO mice have a compensatory mechanism by b-CAMKII, but does not give evidence for how the mechanism works, what it is, or whether it is required.

Question 9

What is cre-recombinase technology?

Answer 9

It is an inducible gene KO system.
Cre-recombinase is a recombinase from bacteriophages, that recognised loxP sequences in DNA, which are usually exogenously engineered to allow you to target a specific gene for deletions, insertions, translocations and inversions at specific sites in the DNA of cells.

This technology is very useful as it is specific and creates a mutation under then normal promoter region of the targeted gene. This means that expression patterns and levels will be physiological, unlike transgenes.

Question 10

What are the limitations of overexpression models?

Answer 10

Overexpression models have been shown to create many artefacts - such as dysregulations in other genes important for the same processes. For example, high expressions of autophoshorylated CAMKII results in an dsyregulation of many other genes involved in neurotransmission, plasticity and potentially LTP. It important to carry out microassays etc to monitor the confounding effects that your manipulations may be having on other systems.

Question 11

What are three important steps in experimental design for investigating molecules involved in learning and memory?

Answer 11

1. Identifying molecules of study.
   This could be done by selecting a molecule involved in a specific plasticity process such as LTP. It could also be done using an unbiased screening method during learning and memory, such as proteomic analysis to identify a protein whose expression changings during learning/memory events.
2. Showing that a particular event related to this moleucle occurs during learning and memory. Such as a phosphorylation of CAMKII.
3. Showing that this event is required for the process of learning and memory that you are studying. This can be done using knock out experiments, etc - studies should not just be behavioural but also cellular/molecular etc. There are many confounds for methods of blocking molecular processes, which must be considered - such as artefacts, developmental models, compensatory mechanismsm, how your manipulation effects the function of the protein (gain or loss) etc.

Question 12

How has autophosphorylation of a-CAMKII been shown to be important for learning and memory?

Answer 12

Inducible transgene experiments using doxycline have been used to increase expression of mimicked autophosphorylation switch (T286D dominant transgene), which is reversed by doxycycline - this may suggest there is no developmental confound for the T286D mutation. Other experiments showed that high expression of T286D caused loss of LTP due to gene dosing effect where high levels of expression cause compensatory mechanism by other genes to suppress excess LTP. Low expression of mimicked autophosphorylated subunits actually caused enhanced LTP.

Transgene experiments do not necessarily replicate the normal physiological expression of genes due to their use of other promoter regions - this is a considerable confounding factor.

Experiments completely blocking autophosphorylation mechanism but allowing calcium dependent activation (using a knock-in T286A mutation) inhibit the induction LTP in the CA1 in vitro and in vivo. It also reduces the length of time that a place cell retains its place field - suggesting it is important for LTP in spatial episodic memory. (correlative). Other experiments have shown that autonomous regulation lasts for 1 minute after LTP induction - however, it is not know precisely what role it plays.

Knock in mutations follow the normal temporal and spatial expression levels of the physiological gene - they use a loxp cre-recombinase b.p transfer. However, this mutation is present from birth which poses a developmental confound.

Question 13

What is the role of beta-CAMKII in LTP?

Answer 13

Point mutations that block the activity of beta-CAMKII do not result in impairment of LTP, but complete KO do.

beta-CAMKII is likely to play a structural role rather than a kinase role in LTP. It binds to f-actin. In heteroenzymes, it is bound to f-actin, and it dissociates upon phosphorylation.

Question 14

Novel downstream target for CAMKII in LTP?

Answer 14

Recent paper Park 2016

A modulatory subunit of the AMPA receptor, TARPγ-8.

It has two phosphorylation sites where CAMKII acts, and this phosphorylation is upregulated after chemically induced LTP and inhibited when NMDA is blocked.

If these sites are point mutated to prevent phosphorylation, then LTP is reduced and fear conditioning reduced - but not completely.

It is believed that phosphorylation of TARPγ-8 drives synpatic insertion of AMPAR during LTP.

Question 15

What is the human evidence that CAMKII is involved in learning and memory?

Answer 15

23 de novo mutations have been identified in CAMKII, and they are all linked to intellectual disability. Some of these mutations affect autophosphorylation of alpha and beta CAMKII.

Question 16

What are the two types of CAMKII involved in learning and memory?

Answer 16

Alpha CAMKII - kinase role
Beta CAMKII - structural role

They show different expression levels and spatial patterns during development and adulthood. Alpha is increasingly expressed in hoppocampus and neocortex during rodent development, and beta CAMKII begins in the neocortex but by adulthood it is expressed in the frontal cortex, the hippocampus and the cerebellum.

Question 17

5 take home messages. What insights has CAMKII has given as to important considerations in experimental designs in molecular mechanisms in learning?

Answer 17

1. There are different levels at which memory can be analysed, e.g. behaviour and plasticity mechanisms such as NMDA-dependent LTP. The strongest experimental evidence for implicating a molecule in learning and memory is to show that it occurs and is required for both a learning/memory behavioural paradigm, and that occurs and is required for the mechanism underlying the behavioural change. This may rely on other experiments, for example ones which show that NMDA dependent LTP in the CA1 occurs and is required for learning and memory in the morris water maze test.
2. There are many different behavioural paradigms, different types of memory, different areas of the brain involve and different underlying plasticity mechanisms. It is therefore important to select the appropriate measurements at each level so that results are meaningful to one another, and also important to recognise that one process in learning and memory cannot necessarily be extrapolated to all processes.
3. Knock out experiments carry a considerable confound, in that they may alter the development of the brain. It is therefore better to use inducible systems of gene manipulation, which can be carried out once the brain is fully developed.
4. Genetic manipulations can carry different implications. A point mutation (t305D) in the ca++/calmodulin regulatory region of CAMKII prevents ca++/calmodulin from activating CAMKII - essentially knocking out CAMKII activity. But a point mutation of threonine to Aspartic Acid at position 286 of CAMKII-a mimicks the effect of a constant autophosphorylation of inhibitory subunit, constitutively activating CAMKII independent of ca++/calmodulin - giving a constant activation indepedent of calcium levels. A switch from threonine to alanine prevents this autophosphorylative switch - this actually prevents CAMKII activation in the absense of ca++/calmodulin, so the molecule loses its normal ‘memory’ for previous high calcium levels. It should also be considered that knock-in manipulations are more reliable than trasgene experiments - transgenes are randomly inserted and may also use it’s own promoter. Knock-ins are under the phsyiological promoter of the gene under manipulation, therefore gene expression patterns and levels are normally conserved.
5. Different expression levels can alter the effect that the mutation has. For example a low level of expression of CAMKII mutation threonine to Aspartic Acid at position 286 (constitutively auto-phosphorylated/calcium calmodulin indepedent) leads to increased LTP. High expression decreases LTP. This is due to artefats caused, where the high expression causes compensatory mechanisms of other genes involved in LTP. These confounds need to be carefully considered when designing models and interpreting results.

Question 18

T286A has been questioned for it’s neccesity as a memory mechanism molecule. Why? What may be an alternative explanation.

Answer 18

T286A prevents LTP and memory formation after one training, but if successive rounds of training are given then the level of LTP and memory is normal.

However, it has been shown that T286A mutations may form memories via an alternative plasticity mechanism, as they have been shown to have a transient increase in spine density 2hrs after training, which disappears after 24hrs.

It has also been shown that these mutants have an abnormal spine morphology, where a dendritic spine is innervated by multiple axons. This effect is retained for at least 24hrs.

Question 19

T305D

Answer 19

This point mutation of T305D is in the regulatory domain.

It mimicks an inhibitory phosphorylation of ca++/calmodulin regulatory zone.

This blocks ca++/calmodulin from activating CAMKII.