Adipose mesenchymal stem cells-derived extracellular vesicles exert their preferential action in damaged central sites of SOD1 mice rather than peripherally

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.

Figure 1.

Characterization of ASC-EVs. A) NTA profile for concentration and particle size of the ASC-EVs. B) Representative cryo-EM images of ASC-EVs with single membrane layer. C) Representative cryo-EM images of ASC-EVs with multiple layers; magnification: 25kx, scale bar: 100 nm; magnification: 2000x, scale bar: 2 µm. D) Western blot of typical EVs markers: CD9 (25 kDa), HSP70 (70 kDa) and Alix (96 kDa) on ASC-EVs; GM-130 (130 kDa) was used to exclude the presence of Golgi’s proteins. ASCs lysates (ASCs) were used as a positive control.

Figure 2.

Motor performances and survival of SOD1(G93A) mice treated with ASC-EVs. A) The paw grip endurance (PaGE) test shows a global improvement of motor performance of the ASC-EVs-treated mice as compared with the PBS control group, with significant differences from 1 to 3 weeks after clinical onset; *p<0.05, ***p<0.001. B) The graph shows the percentage of survival of ASC-EVs-treated mice compared with the PBS control group. Data are represented as mean ± SEM.

Figure 3.

Neuroprotective effect of ASC-EVs treatment on lumbar MNs in SOD1(G93A) mice. A) The graph shows a significantly higher MN number (expressed as cell density) for the ASC-EVs injected mice compared to PBS injected controls (**p<0.005) at the end-stage of the disease. Data are represented as mean ± SEM. B) Representative Nissl staining of lumbar spinal cord MNs of PBS and ASC-EVs treated mice. Magnification: 4x, 10x; scale bar: 50 µm.

Figure 4.

ASC-EVs effects on NMJs and muscle fibers. A) The graph shows a significant increase in colocalization of NF-H and αBTx in SOD1(G93A) mice injected with ASC-EVs as compared with PBS-injected control mice (*p<0.05). Data are represented as mean ± SEM. B) Representative images of the NMJ architecture: on the upper panel the colocalization of presynaptic NF-H (green) and αBTx (red); on the lower panel the degeneration of the NMJ; magnification: 40x, scale bar: 25 µm. C) The graph shows the quantitative analysis of the average muscular fiber area in ASC-EVs injected SOD1(G93A) mice and PBS control mice; data are shown as mean ± SEM. D) Representative images of HE staining in SOD1(G93A) mice treated with PBS or ASC-EVs. Magnification: 20x; scale bar: 100 µm.