Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression

Abstract

BackgroundAmyotrophic lateral sclerosis (ALS) is a disease with a strong neuroinflammatory component sustained by activated microglia contributing to motoneuron death. However, how to successfully balance neuroprotective versus neurotoxic actions by the use of antinflammatory agents is still under scrutiny. We have recently shown that the antihistamine clemastine, an FDA-approved drug, can influence the M1/M2 switch occurring in SOD1-G93A ALS microglia.MethodsHere, we have chronically treated female SOD1-G93A mice with clemastine, evaluated disease progression and performed mice lumbar spinal cord analysis at symptomatic and end stage of the disease. Moreover, we have studied the mechanism of action of clemastine in primary adult spinal SOD1-G93A microglia cultures and in NSC-G93A motor neuron-like cells.ResultsWe found that a short treatment with clemastine (50 mg/kg) from asymptomatic (postnatal day 40) to symptomatic phase (postnatal day 120) significantly delayed disease onset and extended the survival of SOD1-G93A mice by about 10 %. Under these conditions, clemastine induced protection of motor neurons, modulation of inflammatory parameters, reduction of SOD1 protein levels and SQSTM1/p62 autophagic marker, when analysed immediately at the end of the treatment (postnatal day 120). A long treatment with clemastine (from asymptomatic until the end stage) instead failed to ameliorate ALS disease progression. At the end stage of the disease, we found that clemastine short treatment decreased microgliosis and SOD1 protein and increased LC3-II autophagic marker, while the long treatment produced opposite effects. Finally, in spinal microglia cultures from symptomatic SOD1-G93A mice clemastine activated inflammatory parameters, stimulated autophagic flux via the mTOR signalling pathway and decreased SOD1 levels. Modulation of autophagy was also demonstrated in NSC34 SOD1-G93A motor neuron-like cells.ConclusionsBy gaining insights into the ameliorating actions of an antihistaminergic compound in ALS disease, our findings might represent an exploitable therapeutic approach for familial forms of ALS.

M1 and M2 Functional Imprinting of Primary Microglia: Role of P2X7 Activation and miR-125b

“…Moreover we have demonstrated that activation of P2X7 receptor in SOD1-G93A microglia surprisingly non only stimulates common M1 markers but also increases the M2 parameter Arg1 [15, 16, 20]. This would confirm the hypothesis that indeed P2X7 receptor might be involved not only in ALS toxic actions, but also in microglia-dependent neuroprotection [17, 78, 79], thus sustaining the contribution of P2X7 during the resolution of inflammation in macrophages and not only its active part in the release of proinflammatory cytokines [78]. Therefore, we do believe that a better understanding of the molecules and pathways, as well as the timing, responsible for this functional imprinting of microglia might help to select more effective microglia-targeted therapies during diseases.…”

Histaminergic transmission slows progression of amyotrophic lateral sclerosis

“…The histidine treatment is particularly encouraging in symptomatic mice and makes the approach potentially more valuable for later translation, as most patients suffering from sporadic forms are diagnosed only at the symptomatic stage of the disease. In a different way, our recent studies exploring the actions of another histaminergic compound, clemastine, proved that also an anti‐histaminergic strategy reversing the inflammatory phenotype of microglia can exert positive outcomes in extending survival of ALS mice, but only when provided before the insurgence of symptoms . This may suggest that the therapeutic potential of central histamine in ALS mice shows a dual time window, as also demonstrated in a model of neuropathic pain .…”

“…Efforts were made to minimize animal suffering and number of animals necessary to produce reliable results. Adult B6.Cg‐Tg (SOD1‐G93A)1Gur/J mice expressing high copy number of mutant hSOD1 with a G93A substitution (SOD1‐G93A) and originally obtained from Jackson Laboratories (USA) were bred in our animal facility and housed as described . Transgenic hemizygous SOD1‐G93A males were crossbred with C57BL/6 females, both maintained on C57BL/6 genetic background.…”

“…Protein lysates were obtained by homogenization of mice lumbar spinal cords or sciatic nerves . Cells undergoing pharmacological treatments were washed with phosphate buffer saline (PBS) and lysed in RIPA buffer.…”

“…Mice spinal cords perfused with PBS were processed as described . Serial 20 μm sections (one every 10 sections) from lumbar spinal cord segments (L3‐L5) were stained with 1% cresyl violet to detect the Nissl substance of neuronal cells.…”