• Amyotrophic Lateral Sclerosis
• Microglia
• neuroinflammation
• Serotonin
• SOD1

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that leads to the deterioration of motor neurons in the brain and in spinal cord. This causes muscle weakness, paralysis, and, eventually, death. The specific causes of ALS are unclear. However, research indicates that factors like neuroinflammation play a role. Microglia are the main immune cells in the central nervous system where they are involved in neuroinflammation and neuroprotection. Microglia plays a vital role in the first stages of ALS, however in the later stage microglia damaging role seems to be more prominent.

The neurotransmitter serotonin (5- hydroxytryptamine, 5-HT) has gained interest for its potential involvement in the pathogenesis of ALS. In fact, microglia express the serotonergic receptor 5-HT- 2BR, and published data show that antagonizing or blocking 5-HT2BR modulates microglia behavior; this suggests that interfering with this receptor might allow us to control how microglia respond to a certain stimulus. Microglia are the macrophages of the central nervous system (CNS), and macrophages express the 5-HT2BR receptor which has been suggested to mediate the response to serotonin by promoting an anti-inflammatory phenotype. This suggests that serotonin can help to reduce microglia-mediated inflammation through this receptor. The relationship between serotonin signaling and microglial function is a key research focus, likely providing insights into ALS progression and potential treatments.

The objective of the thesis is to address three principal aims pertaining to the role of serotonin in neuroinflammation and the degeneration of motor neurons within the framework of ALS.

The first aim is to analyze if the presence or absence of CNS serotonin influences the inflammatory response. This is tested in knockout (KO) mice. Inflammation is induced via the administration of lipopolysaccharide (LPS), and the levels of inflammatory cytokines are studied. The data collected will provide insights into serotonin’s potential role in modulating an immune response.

Second, to investigate the expression and distribution of serotonergic neurons within the brain of ALS mice. The network of serotonergic neurons of WT mice is compared to that of ALS mice. A transgenic mice model is created where one allele for the Tph2 gene (Tryptophan hydroxylase 2) codifies a recombinant Tph2 labeled with the green fluorescent protein, GFP. Tph2 is an enzyme necessary to produce serotonin, therefore these transgenic mice behave like WT mice, but they have the serotonergic neurons GFP-positive. These heterozygous mice are then crossed with the SOD1 mouse. This crossbreeding produces a transgenic SOD1 mouse that produces GFP in the serotonergic neurons, allowing easy visualization of the serotonergic neuron morphology.

The final aim is to evaluate the influence of serotonin modulation on ALS by boosting the production of serotonin. This aim will concentrate on discerning whether the increase of serotonin levels can alleviate motor neuron degeneration and microglia activation in the context of ALS. We will use a chemogenetic mouse model tool specifically expressing the human M3 muscarinic receptor (hM3Dq) in serotonergic neurons. hM3Dq receptor allows specific stimulation of the cells expressing it. This strategy allows to selectively boost serotonin levels in SOD1G93A mice by using the hM3Dq agonist clozapine-N-oxide (CNO).