**7. Conclusions**

Understanding the mechanisms leading to aberrant immune response and the severe neuro-inflammation in MS plays a relevant role in the development of new treatments directed to decrease pro-inflammatory cytokine production and to ameliorate the clinical symptoms, slowing the disease outcome and delaying or arresting the onset of the disabilities. In this review we have presented recent data in EAE mice and in MS patients, supporting the role of ACh in MS. The dysregulated ACh homeostasis in MS, sugges<sup>t</sup> how its altered functions might represent an additional pathogenetic mechanism negatively influencing the cytokines production with consequent disease outcome.

Interestingly, using the EAE mice model, it has also been described as the alteration of ACh synthesis or degradation may impact on neuron function, contributing to the motor and cognitive disabilities characteristics of MS patients, also influencing the glia network, the differentiation and survival of the oligodendrocytes, and triggering the neuro-inflammation.

The relevant role played by ACh in this context, is supported by the evidence that modulating the cholinergic system activity or cholinergic receptor functions, the clinical disabilities, at least in mice, are significantly reduced. In fact, the altered balance of ACh also in cholinergic area of the brain or spinal cord of EAE mice (i.e., basal ganglia or spinal motoneuros), suggested that the decreased ACh levels may influence the MS motor disabilities both reducing the levels of the neurotransmitter in the cholinergic neurons and increasing the neuro-inflammation with consequent impact on myelin disruption and axonal conduction [63,76,77].

This aspect appears supported by additional studies performed with conventional drugs used in the treatment of MS, as INFβ or dimethyl fumarate [36,88], that demonstrate a significant amelioration of the cholinergic activity with a possible consequent reduction of the clinical symptoms-MS associated. Therefore, further studies will be necessary to evaluate the impact of different immune modulators on the cholinergic system activity both in the immune system and in the brain.

In conclusion, the data described in this review highlight as ACh and its receptors may be implicated in MS pathogenesis. However, further investigation on the genetic aspects and epigenetic modulation of cholinergic markers gene expression could significantly improve the knowledge about MS onset and progression as well as to contribute to identifying new possible therapeutic strategies.

**Author Contributions:** Conceptualization A.M.T., Original draft preparation, writing—review and editing V.G., G.M., M.R., A.M.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** Supported by FISM—Fondazione Italiana Sclerosi Multipla (Project number 2009-R-29; 2013-R-25).

**Conflicts of Interest:** The authors declare there are no conflicts of interest.
