Mexiletine
Astrocytes expressing mutant TDP-43 contribute to ALS pathogenesis and it was also shown that functional TDP-43 was indispensable in a cell-autonomous manner for the proper functioning of mature oligodendrocytes in myelination and cell survival \cite{Wang2018}. It was reported that Nav channel blockers, including mexiletine, prevent motoneuron cell death induced by ACM-SOD1G93A, ACM-SOD1G86R, and ACM-TDP43A315T \cite{Rojas2014}. Mexiletine is a FDA- approved anti-arrhythmic drug and a lidocaine analog that is utilized as a local anesthetic, and it down-modulates persistent sodium ion channel conductance. Neuromuscular hyperexcitability and involuntary fasciculations or transient muscle twitches, overwhelmingly afflict ALS patients and the majority of patients suffer from debilitating muscle cramping. In a study of sixty patients with sALS, mexiletine (300 mg/d or 900 mg/d) treatment for 12 weeks assuaged muscle cramps intensity and frequency, but did not improve the rate of disease progression, ALSFRS-R, and, in terms of tolerability, the dose of 300 mg/d was well tolerated, while the higher dose was not \cite{Weiss2016}. In another double blind crossover trial in 150 mg twice daily with a 1-week screening period, 2-week treatment interval, followed by a 1-week washout of drug and then and second treatment muscle cramps severity and frequency likewise significantly decreased but not fasciculations \cite{Oskarsson2018}. A few limitations of these important studies which show efficacy against muscle cramps were the study duration, small number of enrolled patients. absence of a quality-of-life questionnaires and power to detect survival. In a follow up study, Mexiletine in Sporadic Amyotrophic Lateral Sclerosis (Mexiletine-2) (ClincicalTrials.gov Identifier NCT02781454 ) some of these questions will be further addressed.
Therapeutics that target DNA double strand break signaling and DNA repair mechanisms
Mitra et al. have shown that depletion of TDP-43 is associated with an increase in double strand break (DSB) signaling \cite{Mitra_2018}. TDP-43 serves as a scaffold for DNA DSB factors, and the aberrant mislocalization of this protein feasibly contributes to genomic instability and the pronounced motor-neuron cell death that is a prominent hallmark evidenced in ALS. The authors report that TDP-43 is a part of the nonhomologous end joining DNA double-stranded damage response pathway. TDP-43 is attracted to sites of DNA double strand breaks and serves as a scaffold for other factors that repair the damage. The authors prove this because when they employed shRNA or CRISPR/Cas9 to conditional deplete TDP-43, they observed an increase in double strand breaks and to genomic instability and stress, which are signatures of the genome of sALS patients. Targeting the DNA repair response as an ALS therapeutic is a novel rationale therapeutic approach.
Immunomodulation in the treatment of ALS and the role of TDP-43
Rapamycin: Rapamycin (Sirolimus) inhibitor of the Mechanistic/Mammalian Target of Rapamycin (mTOR) is a clinically-approved drug prescribed in cancer therapeutics \cite{Apontes2011}, and for the prevention of organ rejection through immunomodulatory activity (that is, both stimulating the immune system and suppressing age-related excessive immunity), anti-inflammatory, anti-senescence and playing a role in the activation of autophagy \cite{Blagosklonny_2019}\cite{Mandrioli2018}. Autophagy, an important cell process for protein turnover and degradation of aberrant proteins, is upregulated by rapamycin \cite{Blagosklonny_2019} \cite{Mandrioli2018} . Rapamycin also downregulates protein synthesis, inhibits inflammatory responses, and has demonstrated efficacy in animal models of neurodegenerative disorders. Rapamycin was shown in vitro in N2A and SH-SY5Y cells to induce autophagy, decrease TDP-43 aggregates, and correct the mislocalization of TDP-43 from the cytoplamic aggregation to restore it to the nucleus \cite{Caccamo_2009}. The clearance of TDP-43 by autophagy has been demonstrated in murine and in human stem cell-derived neurons and astrocytes with mutant TDP43 \cite{Barmada_2014}. In addition, it has been reported that rapamycin increases T lymphocytes and that these increases in these cells is associated with slower disease progression in ALS \cite{Mandrioli2018}.
In eight ALS treatment centers in Italy, the rapamycin for ALS trial (RAP-ALS) is being conducted for an 18 week period, with a total follow-up duration of 54 weeks (ClinicalTrials.gov identifier NCT03359538) . It is a phase II randomized, double-blind, placebo-controlled, and multicenter trial with patients randomized to one of three groups of (n=21 per group): (1) 1 mg/m2/d rapamycin + riluzole, (2) 2 mg/m2/d rapamycin + riluzole or (3) placebo + riluzole, in addition to mainstay supportive care in accordance with established ALS guidelines (e.g. nutrition, respiratory, motor function, communication) \cite{Mandrioli_2018}. The primary endpoint is the number of T regulatory cells (Tregs), which are cells that are produced in the thymus and regulate the immune response against self cells and foreign antigens, and, given that rapamycin activates Tregs, quantifying the change between baseline and 18 weeks of treatment will help monitor rapamycin activity. There are a number of secondary endpoints that will be quantified, such as safety and tolerability, minimum dose required for detection of rapamycin in the cerebrospinal fluid, modulation of inflammatory and mTOR markers, ALSFRS-R and quality of life. The treatment of ALS with  rapamycin, considering its importance in modulation of TDP-43 clearance and correction of cytoplasmic mislocalization of TDP-43, is a very interesting therapeutic approach to follow in the years to come.