Preclinical study testing in-vivo the effect of MSC-derived extracellular vesicles on SOD1G93A mice, as a cell-free therapeutic strategy for amyotrophic lateral sclerosis

BACKGROUND. Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons, leading to paralysis and death. Effective treatments are still lacking, due to the complex pathogenesis involving oxidative stress, neuroinflammation, mitochondrial dysfunction, and excitotoxicity. Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic tool because of their neuroprotective and immunomodulatory properties, mainly mediated through their secretome and extracellular vesicles (EVs). Previous studies from our group demonstrated that MSC-derived EVs can modulate the aberrant reactive phenotype of ALS astrocytes and reduce their neurotoxicity towards co-cultured MNs. As a translational step, the present work aimed to investigate the in vivo therapeutic potential of intranasally administered MSC-derived EVs in the SOD1G93A mouse model of the disease, by performing behavioral tests and ex vivo analyses. We first assessed the effect of acute EVs administration, and after observing limited efficacy, we shifted to a chronic treatment. RESULTS. Fluorescently labelled EVs were detected in the olfactory bulb and cortex following intranasal administration, confirming their ability to reach the central nervous system. Acute EVs intranasal administration did not significantly affect pathological parameters or neuroinflammation- related gene expression, possibly due to limited exposure. Conversely, chronic (5 weeks of treatment) intranasal administration of MSC-derived EVs improved motor functions and extended survival in SOD1G93A mice, particularly in male mice, paralleled by spinal motor neurons preservation, confirming the neuroprotective effect. Astrogliosis was not affected by MSC-derived EVs, while reducing the Iba1 microglia proliferation marker. At the molecular level, chronic MSC-derived EVs treatment did not significantly affect neuroinflammatory markers or NFkB pathway activation, but markedly enhanced antioxidant defenses and reduced oxidative stress. Upregulation of antioxidant defence enzymes (catalase, glutathione reductase, and glutathione peroxidase) along with a reduction in oxidative damage markers (MDA, 4-HNE, NT, 8-OHdG), indicating an increased antioxidant response in both brain and spinal cord. Similar effects were observed in skeletal muscle, where EVs treatment normalized oxidative stress parameters and restored the enzymatic activity of mitochondrial complexes I and III and the ATP/AMP ratio, particularly in males. CONCLUSIONS. We show here that mimicking the systemic administration of MSCs, MSCs- derived EVs intranasal administration exerts beneficial effects in the SOD1G93A mouse model of ALS, primarily through redox regulation rather than direct modulation of neuroinflammation, with more pronounced effects in male mice. This data paves the way for the application of MSCs-derived EVs or EV-mimicking synthetic particles as an innovative and promising cell-free therapeutic strategy for ALS cure.