Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the gene encoding dystrophin, a protein critical for proper cytoskeletal scaffolding, which is expressed in muscle but also in the brain. Neuropsychiatric comorbidities are present in up to 30% of DMD patients, and in mdx mice (the mouse model of the disease), but the underlying mechanisms are not yet clear. In the general population, many of the co-morbid disorders are associated with neuroinflammation, which remains poorly investigated in relation to DMD. In the context of dystrophy, neuro-inflammatory processes could be due to both functional changes in the brain and secondary to the massive systemic inflammation that accompanies the disease. In the dystrophic brain inhibitory transmission is dampened, while possible abnormalities in astrocytes can boost the presence of glutamate, leading to excitotoxic damage. Peripherally, immune cells, stimulated by ongoing muscle damage, release cytokines that can reach the brain, also because the blood¿brain barrier is leaky in mdx mice Within the brain, proinflammatory cytokines activate microglia, which also produce cytokines. Thus, chronic systemic inflammation can extend to the brain. Aim of the present project is to examine the presence of inflammation in the brain of mdx mice, examining microglia phenotype, astrogliosis and concentration of inflammatory cytokines, as compared to wt animals. We will then examine whether blockade of Ca2+-activated K+ channels can reprogram microglia towards an anti-inflammatory phenotype, as in other disease, setting the stage for future studies on animal behaviour.
With the proposed experiments, we will:
a) get further data on the expression of dystrophin in astrocytes and microglia;
b) characterize the functional status of glial cells in dystrophic brains;
c) ascertain the presence of neuroinflammation and possibly define approaches to reduce it.
Based on the literature data described in the preceding sections and on our own preliminary data, the possibility that we will not detect differences in astrocytes and microglia of wt and mdx brains is quite unlikely. However, even in this case, we will have conclusive data on the presence (or absence) of neuro-inflammation in the dystrophic brain. Moreover, we will document the effects of KCa3.1 channels blockade on inflammation of dystrophic brain.
These data will provide the building blocks of future research aimed at studying the role of inflammation on behavioural alterations of mdx mice. Once established in a preclinical setting, studies on the role of neuroinflammation, accompanied by glia functional alterations, in many of the neurological comorbidities of DMD patients will be possible.
Corticosteroid use is also associated with increased risk of behavioural problems in DMD patients. Of course, in the frame of DMD, muscle protection is the principal focus, but within this project we propose to evaluate the effects of the inhibition of KCa3.1 channels, known to revert neuroinflammation in a variety of conditions. Of note a blocker of these channels, Senicapoc, is an orphan drug licensed for human use, which might enhance the translational value of positive findings within this project.