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Alpha CaMKII autophosphorylation as a mechanism to regulate alcohol consumption

Final Report Summary - ALCO_CAMK (Alpha CaMKII autophosphorylation as a mechanism to regulate alcohol consumption.)

Alcoholism comprises a collection of fundamental personal and socioeconomic problems including major financial burden for the health care and welfare systems, as well as emotional one for the affected individuals and their families. The World Health Organization estimates that there are 140 million people suffering from alcoholism worldwide. Despite multiple efforts to understand the biology of alcohol addiction, little progress has been made so far to develop effective therapies. One of the main reasons for the lack of successful therapy is high molecular complexity of the disease as well as the lack of appropriate animal models capturing all major aspects of addicted state.
In the current project we focused on: 1/ development of animal model of alcohol addiction-related behaviours and 2/ investigation of the role of calcium and calmoduline-dependent kinase II alpha in development of alcohol addiction-related behaviours.
Calcium and calmoduline-dependent kinase II alpha (alpha CaMKII) is a major protein of forebrain glutamatergic neurons with well established function in neuronal plasticity as well as learning and memory. Surprisingly, alpha CaMKII function in addiction, and alcohol addiction in particular, has not been much investigated.
In order to develop animal model of alcohol addiction-related behaviours, we performed a longitudinal study using the IntelliCage automated learning system, which allows the detailed analysis of mouse behavior in social groups. We measured behavioral traits in mice that were previously shown to predispose to alcohol addiction in humans, such as (1) novelty seeking, measured as the number of exploratory visits to the corners of the novel IntelliCage; (2) impulsivity, defined as an inability to withhold a nosepoke response until signaled reward availability; (3) anxiety, defined as the suppression of reward consumption associated with an air-puff punishment; (4) compulsivity, defined as reward seeking even during signaled ‘no-reward’ periods; and (5) motivation for a natural reward, measured in the progressive ratio test. The mice then had extended and unlimited access to alcohol (70 days), followed by the assessment of four addiction-like behaviors that resemble Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria for addiction (American Psychiatric Association AP 2000): (1) high motivation for alcohol reward, measured in a progressive-ratio schedule of reinforcement; (2) persistence of alcohol seeking, even during signaled alcohol non-availability; (3) alcohol drinking, even in the face of punishment, measured as resistance to air-puff punishment during alcohol drinking; as well as (4) intensity of relapse after alcohol withdrawal, measured by alcohol consumption after alcohol withdrawal. Finally, we analyzed the correlation between the measured behavioral traits and addiction-like behavior. Our data suggest that high levels of anxiety-related traits (i.e. low novelty seeking, low resistance to punishment and a high level of compulsive behaviors) and high impulsivity predict addiction-like alcohol drinking in mice.
In order to understand the role of autophosphorylation of CaMKII in alcohol addiction-related behaviors we used CaMKII-T286A mutant mice and their wild-type littermates. The mice were tested in a battery of standard behavioural tests as well as in the IntelliCages-based training. We found that CaMKII autophosphorylation deficient mutants have altered behavioural response to alcohol. The T286A mutants show decreased psychostimulant response to single injection of alcohol as compared with wild-type mice as well as psychostimulant sensitisation after chronic alcohol injections. Furthermore, we found that single alcohol treatment (2.5 g/kg) decreases an expression of postsynaptic marker protein, PSD95 as observed 2 hours after injection, in the brain regions forming so called “reward system”, such as the hippocampus (CA1 field), amygdala (central and basolateral nuclei) and striatum (nucleus accumbens core) of the wild-type mice, indicating the loss of synapses . This was accompanied by the decrease in the levels of polymerised actin in synapses in the hippocampus and striatum and increase in the levels of actin depolimerising protein - cofilin. Our data also indicate that chronic alcohol treatment leads to increased PSD95 expression in the hippocampus (CA1 field), increased levels of polymerised actin in synapses, and decreased activity of cofilin. These phenomena do not occur in alcohol-treated T286A mutants. Thus our data show that alpha-CaMKII autophosphorylation is involved both in the regulation of psychostimulant effects of alcohol, and alcohol-induced changes in the morphology of synaptic contacts in the reward system. These observations suggest that alcohol psychostimulant effects involve remodelling of postsynaptic densities in the hippocampus and this process is regulated by CaMKII autophosphorylation.
The CaMKII-T286A mutant mice were also tested in the IntelliCages in order to specify which alcohol-addiction related behaviour are regulated by CaMKII authophosphorylation. As we have shown previously, chronic alcohol treatment of wild-type mice leads to clear split of the population into two groups with very high and low motivation for alcohol. Interestingly, this phenomenon is not observed in T286A mutants, and the whole population remains in the middle of the addiction scale. This observation suggest that authophosphorylation of CaMKII regulates neuronal plasticity which is a prerequisite for development of addictive behaviour.

In conclusions, in the Alco_CaMK project: (1) we have established a new, IntelliCage-based model of alcohol addiction; (2) we have found that autophosphorylation of CaMKII regulates alcohol-addiction related behaviours as well as (3) structural plasticity of the synapse, including cofilin-dependent remodelling of actin cytoskeleton and postsynaptic density, induced by short- and long-term alcohol treatment. Our finding may result in the future in new molecular strategies in the treatment of alcohol addiction.