MSC have emerged as a promise in the treatment of human ALS (10). Indeed, rather recent clinical trials have pointed out potential positive effects of MSC for neurodegenerative diseases (38, 39), including ALS (7, 8, 40, 41), it is still lacking information on putative cellular/molecular mechanisms underlying MSC-induced neuroprotection (12, 14, 42) as well as to counteract motor neuron death in ALS (3, 43). Remarkably, the descriptions of molecules possible involved in MSC effects on neurons have increased experimentally (11, 14) but not as far clinically (42, 44). Considering a well described failure to translate therapeutical targets for ALS from bench to bed side (45, 46), researches that explore in details cellular mechanisms and corresponded molecules related to MSC treatment in ALS disorder are desirable.
This manuscript innovated by combining large omics, specifically Proteomics and Protein Interaction Network, as well as defined criteria for molecular modeling in order to highlight cellular mechanisms and their related molecules in the CSF of ALS subjects 30 days after intrathecal infusion of autologous bone marrow-derived MSC. Nevertheless, our study is in agreement to previous reports that investigated molecular responses in CSF after local deliver of MSC in ALS patients by applying different methodologies (2, 47–49). Moreover, despites investigations have searched molecular responses to MSC in blood serum in clinical ALS (50, 51) as well as striatal muscles in experimental ALS (52, 53), CSF has been considered an important body compartment for molecular investigation due to CSF anatomical proximity to suffering neurons as well as for carrying bio molecular signatures of aberrant biochemical processes related to central nervous system pathophysiology (54, 55). In this context, it seems likely that CSF administration of autologous MSC performed in this trial may be relevant to facilitate cell signals to reach neurodegeneration zones in ALS, as discussed elsewhere (3, 11, 48), contrasting previous clinical design that analyzed molecular responses to MSC after muscular deliver (24, 56).
Furthermore, to our concern, this study is the first one to employ Proteomics by means of mass spectrometry for molecular investigation in CSF of intrathecal autologous MSC-treated ALS subjects, regardless the methodology has been recently employed on biomarker discovery program in CSF of ALS patients (57–59). Moreover, despite a lack of omics investigation on that matter, molecular regulation in CSF of MSC-treated ALS patients has been performed using classical non omics methodology (2, 49). In line to present study, majority of ALS clinical trials on MSC-delivered CSF have employed autologous bone marrow-derived MSC (2, 6, 47, 60, 61), rather than stem cells derived from adipose tissue (48), umbilical cord or other sources that are mainly employed in experimental investigations. The advantage of bone marrow-derived MSC to clinical application, specially in neurodegenerative disorders, has been well described, that is specially related to their ability to interact in an autocrine/paracrine matter to injured tissue (11–14, 62). Indeed, the molecular crosstalk among MSC and nervous tissue might interfere with inflammatory events at wound with the potential to modify the progression of neurodegeneration which is substantially important for progressive neurodegenerative disorders like ALS (63, 64).
Remarkably, proteomic analysis has pointed out 220 deregulated proteins in CSF of ALS subjects thirty days after autologous bone marrow-derived MSC intrathecal delivery. This result represents an important set of molecular responses to MSC presence in ALS indeed a number far way larger than the set of deregulated molecules described by similar clinical trials on ALS that have not applied omics technology (2, 47) in the screening of molecular biomarkers. Among those deregulated proteins, upregulated and downregulated molecules might be able to address mechanisms related to MSC in ALS or even may contribute as biomarkers of MSC effects in ALS in future investigations.
In fact, the present study has worthy contributed to original description of cellular mechanisms and related molecular targets facing intrathecal MSC in ALS by employing enrichment analysis of deregulated molecules (36, 37). The clusterization of deregulated proteins by means of REVIGO has pointed out a set of clusters and superclusters of cellular/molecular mechanisms possibly related to MSC actions thirty days after intrathecal delivery in ALS patients. Remarkably, extracellular matrix and cell adhesion terms were highlighted among superclusters thus representing an important contribution of herein employed methodology. In fact, despites REVIGO clusterization has been largely applied (37), it is an original contribution in the search for mechanism related to MSC in ALS. In fact, the literature analysis of “Extracellular matrix” and “Cell adhesion molecules” MeSHs indicated a huge involvement of such matters in the context of ALS as well as MSC. Our study remarkably highlighted specific Pathways/Categories related to “Extracellular matrix” and “Cell adhesion molecules” MeSHs. The observation that 92% of proteomics deregulated molecules belonged to Pathways/Categories related to “Extracellular matrix” and “Cell adhesion molecules” MeSHs strongly emphasized the possible involvement of Extracellular matrix and Cell adhesion molecules in the putative mechanisms of MSC delivered in CSF of ALS subjects thus representing an important contribution of this study.
It has to be mentioned that we are still not able to address whether Extracellular Matrix/Cell Adhesion Molecules highlighted in this study are related to putative MSC actions after intrathecal delivery in ALS patients, to ongoing ALS motor neuron degeneration or a possible interaction of both, a matter that must be the subject of further investigations. Anyhow, extracellular matrix and cell adhesion have been largely described in the context of ALS motor neuron degeneration (65–68) as well as MSC mechanisms of action (10, 43, 69). Actually, Extracellular Matrix and Cell Adhesion Molecules largely interact in the mechanisms of cell signaling driving autocrine (70) and paracrine (71) cellular mechanisms that indeed have been largely correlated to MSC actions (42) and motor neuron degeneration/protection (39, 72). Those observations remarkably open up the possibility for an integrated mechanism related to Extracellular Matrix and Cell Adhesion Molecules that might be involved in the effects of MSC in the injury sites of ALS thus possibly interfering with motor neuron degeneration in the disorder. All in all, this paper has highlighted for the first time the importance of Extracellular Matrix and Cell Adhesion Molecules in the interactive mechanisms of MSC and motor neuron death in ALS.
The demonstration of highlighted molecules APOA1, APOE, APP, PLG, C4A, C5, FGA, FGB and FGG as possible important proteins related to the presence of MSC in CSF of ALS subjects is an additional contribution of this study. Moreover, it should be emphasized the contribution of Protein Interaction Network evaluation (73, 74) in the criteria herein applied to point out those highlighted molecules. Importantly, it is the first time APOA1, APOE, APP, C4A, C5, FGA, FGB, FGG and PLG have been mentioned in the context of biomarkers of MSC presence in \ CSF of ALS subjects 30 days after intrathecal cell delivery.
Indeed, all nine highlight molecules belong or signalize to elements of Extracellular Matrix and Cell Adhesion Molecules as well as they have been described to interact to stem cells in general or to MSC in particular (75–78). C4A, FGB, FGG and PLG have been described in the context of neuronal degeneration/survival or neurodegenerative disorders (79–83) and APOA1, APOE, APP, C5 and FGA have been investigated in the context of ALS (84–90).
All in all, the highlighted molecules described above have a potential possibility to be involved in the mechanisms of MSC in motor neuron degeneration in human ALS, a matter to be explored in future investigation.