The main goal of this study was to assess the effect of a novel therapeutic approach combining a Sigma-1R agonist, PRE-084, and RSV in the SOD1
G93A mouse model of ALS, since separate administration of the two compounds had resulted in significant improvement of disease progression and survival of these mice [
22,
32]. Our results indicate that co-administration of PRE-084 and RSV from 8 weeks of age significantly preserved spinal MNs function and reduced MN degeneration together with a reduction of microglial immunoreactivity in the lumbar spinal cord. This effect was accompanied by improvement in the locomotor performance and significant extension of the animals survival. Western blot analyses revealed that, as we previously described, PRE-084 induced PKC-specific phosphorylation of Ser896 of the NMDA-NR1 subunit, whereas RSV increased the expression and activation of Sirt1 and AMPK in the ventral part of the lumbar spinal cord of SOD1
G93A mice. Unfortunately, the combinatory therapy did not represent a clear improvement compared to RSV-only or PRE-084-only treated animals.
Mechanisms of neuroprotection and possible overlapping effects
As we previously described, PRE-084 promotes potent neuroprotective effects to MNs both
in vitro after excitotoxic insults [
43], and
in vivo after spinal root avulsion [
11] and in the SOD1
G93A mouse model accompanied by a significant extension of animals survival [
22]. Calcium dysregulation and excitotoxicity are two pathophysiological mechanisms contributing to ALS pathology [
44,
45]. In fact, spinal ALS-vulnerable MNs have an endogenous calcium buffering capacity 5–6 times lower than that found in ALS-resistant MNs, increasing their susceptibility to excitotoxic insults [
46]. NMDA receptor plays an important role during excitotoxicity [
45] and Sigma-1R agonists have been shown to suppress calcium influx to the cells by modulating NMDA receptor through PKC activation. Consistent with our previous observations that PRE-084 administration promotes PKC-specific NMDA-NR1 subunit phosphorylation [
22], we have found that PRE + RSV combined treatment also increased Ser896 phosphorylation of the NDMA-NR1 subunit in the ventral part of the spinal cord. Interestingly, increased NMDA-NR1 phosphorylation was only present in the treated group, suggesting that the treatment is activating compensatory protective pathways rather than counteracting a pre-existent pathological event. Although it has been demonstrated that Sigma-1R physically interacts with NMDA-NR1 subunits [
47], it has been also reported that Sigma-1R agonists can modulate ionic flow through calcium, sodium and potassium channels, thus modifying cell excitability properties [
14,
48,
49]. In fact, recent findings by Mavlyutov et al. [
50] indicate that the lack of Sigma-1R is detrimental in SOD1
G93A mice, probably because it acts by reducing the excitability of spinal MNs. Moreover, Sigma-1R is found associated to ER chaperones (such as BiP) and plays a role in clearance of misfolded proteins by the ERAD response [
51,
52]. Sigma-1R is enriched in the so-called mitochondrial-associated ER membrane (MAM) and its activation can also modulate mitochondrial metabolism [
8,
53].
It has been reported that RSV promotes protective effects both in neurodegenerative and traumatic injury models, including Alzheimer’s disease and accelerated aging [
23,
28,
54], multiple sclerosis [
55,
56], Huntington’s disease [
57,
58], Parkinson’s disease [
24,
59], and reducing peripheral axonal degeneration [
60] or promoting functional improvements after spinal cord injury [
25]. We have recently found that RSV administration significantly delays clinical symptoms onset, improves spinal MNs function and survival, and extends SOD1
G93A mice lifespan. We also determined that the therapeutic effect was mediated by the increased expression and activation of both Sirt1 and AMPK, leading to normalization of the autophagic flux and enhanced mitochondrial biogenesis [
32]. Although there is some controversy about the exact molecular mechanisms underlying RSV effect, it has been established that Sirt1 or AMPK activation are upstream of pathways that participate in several cellular processes, including inflammation [
61‐
64], autophagy [
32] and mitochondrial function [
29,
54]. Consistent with our previous findings, PRE + RSV treated animals also presented higher expression and activation of both Sirt1 and AMPK compared to SOD1
G93A untreated mice. As we previously commented regarding increased NMDA-NR1 phosphorylation, Sirt1 and AMPK overactivation was only present in the treated group, suggesting that the treatment activates protective pathways rather than counteracts a pre-existent pathological condition. This fact may increase the interest of these treatments since the potentiation of endogenous protective mechanisms can be translated to other non-SOD1 ALS situations.
Although the absence of summative effect in the PRE + RSV treated group may be due to an insufficient dose of any of the compounds, a possible overlapping in the pathways activated by both compounds may underlie the lack of synergy. As above mentioned, RSV protective effect is likely related to normalization of the autophagic flux and increased mitochondrial biogenesis [
32]. Although PRE-084 main therapeutic effects have been considered associated to modulation of calcium influx and MNs excitability [
14,
48,
49], it also participates on the response to misfolded protein accumulation [
8,
52] and the modulation of mitochondrial metabolism [
53]. Considering this action, RSV effects on autophagy and mitochondrial biogenesis may mask those of PRE-084 administration thus explaining the lack of summative effect of the combined treatment. Alternatively, both compounds may exert opposite effects on some pathways. For example, it has been reported that Sigma-1R stabilize IRE1α and increase cell survival through transcriptional activity of X Box binding Protein 1 (XBP1) which in turn regulates genes responsible for protein folding and degradation during the Unfolded Protein Response (UPR) [
51]. In contrast, RSV has been shown to suppress the transcriptional activity of XBP1 through Sirt1 [
65], therefore promoting an opposite action that could contribute to the lack of synergistic effect of the combinatory treatment assayed.
Another explanation for the lack of summative effect of the combined treatment lies in the pathology state of the animals in the moment we began the drug administration. SOD1
G93A mice at 8 weeks of age present 25-30% loss of CMAP amplitude in proximal muscles (TA and gastrocnemius) [
36,
66], due to early neuromuscular junction retraction and motor axons degeneration [
41]. Thus, although MN cell bodies in the anterior spinal cord are intact, up 30% of them are already under a degenerative process that may be irreversible. Since our treatment is focused on the preservation of remaining functional MNs, it is likely that we are acting on the still functioning population of MNs and thus, the maximum effect that can be reached would be limited.