We show herein that cPLA
2α is upregulated in the spinal cord of 6-week-old mice long before the appearance of the disease symptoms, neuronal death, or gliosis and remained elevated during the whole life span of the hmSOD1 mice. Prevention of cPLA
2α upregulation at the pre-symptomatic period (between 6 and 12 weeks) did not affect the course of the disease. However, prevention of cPLA
2α upregulation shortly before the onset of the disease symptoms significantly (
p < 0.001) delayed the loss of motor neuronal function, suggesting that cPLA
2α upregulation in the spinal cord plays a role in the disease pathology. In line with our results, the role of elevated spinal cord cPLA
2α in neuronal damage was reported also in spinal cord injury [
28] and in spinal inflammatory hyperalgesia [
29].
In the present study we used, specific antisense brain infusion in order to determine the specific role of cPLA
2α upregulation in the spinal cord in the pathogenesis of ALS. The advantage of the AS technique over knockout mice is its ability to inhibit the induction of an accelerated protein at the site of inflammation, and enables its expression at basal levels, whereas in knockout mouse models total elimination of a protein may result in dramatic changes of homeostasis and/or compensation by upregulation of other proteins (with similar biological features). The use of AS strategy is reinforced by two recent studies in cPLA
2α knockout mice reporting that total elimination of cPLA
2α resulted with abnormalities in architecture of synapses and cortical neurons [
30] as well as in alteration of brain phospholipid composition [
31]. AS brain infusion to 15-week-old hmSOD1 mice (for 6 weeks) shortly before the development of motor dysfunction resulted in reduction of cPLA
2α elevated protein expression (as detected at 18–19 weeks in the spinal cord) and significantly delayed the development of the disease symptoms. This blunting of cPLA
2α upregulation resulted in inhibition of activation of microglia and astrocytes as well as inhibition of motor neuronal death, as detected by immunofluorescence analysis in the spinal cord of 18–19-week-old hmSOD1 mice.
A generation of mice expressing a conditional deletion of the mutant SOD1 gene showed that each individual cell types in the spinal cord contributes to the development of ALS [
8,
32]. Likewise, replacing the myeloid lineage of mutant SOD1 mice with wild-type microglia slowed disease progression [
6]. Since cPLA
2α upregulation was detected in each cell type in the spinal cord, it probably contributes by different mechanisms to motor neuron dysfunction and cell death: (i) elevated neuronal cPLA
2α can act directly in inducing neuronal death (ii) while elevated glial cPLA
2α can act indirectly by activating microglia and/or astrocytes. In our recent study in primary neuronal cultures, we reported that cPLA
2α activation and upregulation induced apoptotic neuronal death [
18]. Concomitant with our results, cDNA microarray analysis to monitor gene expression in the spinal cord of hmSOD1 mice during neurodegeneration revealed changes in apoptosis-related gene expression [
4]. In addition, the role of cPLA
2α in glutamate excitotoxicity and oxidative stress-induced cell death in primary spinal cord neuron cultures was recently reported [
33]. Taken together the studies in neuronal cultures and the abundant levels of cPLA
2α in motor neurons in the spinal cord, it is suggested that neuronal cPLA
2α plays a direct role in motor neuron cell death. Similar to other neurodegenerative diseases and acute CNS insults, one of the most striking hallmarks of ALS shared by familial and sporadic patients, as well as by rodent models, is neuro-inflammation, characterized by extensive microglial activation and astrogliosis that contribute to disease progression, rather than to its resolution [
2,
34‐
36]. Reduction of cPLA
2α upregulation in the spinal cord of the hmSOD1 mice prevented astrocyte activation detected by GFAP immunostaining that is in line with
in vitro studies [
37] reporting that activation of cPLA
2α led to an increase in oxidative stress in astrocytes. We show here a massive activation of microglia in the spinal cord (detected by immunostaining of Iba-1 and CD40) that preceded the changes in the motor neurons, in accordance with other studies [
6,
38,
39]. This microglia activation was shown to be cPLA
2α dependent coincided with ours and others’ studies in cell cultures demonstrating the specific role of microglial cPLA
2α in the activation and transformation of microglia to M1 phenotype. We have previously reported that cPLA
2α activity regulated the production of superoxides by NOX-2 NADPH oxidase and the induction of COX-2 and iNOS
via nuclear factor kB (NF-kB) in microglia cultures [
17]. Interestingly, microglial NF-kB specifically has been shown to play a major role in the development of the ALS in hmSOD1 mice [
40]. We show here that reduction of cPLA
2α in the spinal cord also decreased iNOS and COX-2 upregulation that produce two major proinflammatory mediators; nitric oxide and PGE
2, respectively. As shown in the present study for cPLA
2α, it was also reported that in both early symptomatic and end-stage transgenic hmSOD1 mice, neurons and to a lesser extent glial cells in the spinal cord exhibit robust COX-2 [
41] and iNOS immunoreactivity [
42]. Likewise, similar to cPLA
2α, COX-2 was dramatically increased in postmortem spinal cord samples from sporadic ALS patients [
41]. Nitric oxide and superoxides both under cPLA
2α regulation [
17] can form the toxic reagent peroxynitrite [
43,
44]. In this context, a recent study reported that inhibition of cPLA
2α activity ameliorated experimental autoimmune encephalomyelitis
via blocking peroxynitrite formation in mouse spinal cord white matter [
45]. These results suggest that glial cPLA
2α is involved by different inflammatory processes in the development of ALS. In accordance with this suggestion, injections of PLA
2 into the spinal cord of mice caused inflammation and oxidative stress [
46].
The role of monocyte recruitment to the spinal cord in ALS is under debate. On one hand, it was reported that inhibition of monocyte recruitment to the spinal cord delayed motor dysfunction and increased the survival of ALS mice [
48]. In contrast, another study [
49] reported that monocytes could not be detected in the spinal cord of SOD1G93A mice and that microglia are not derived from infiltrating monocytes. Likewise, a recent study [
50] reported that in the mutant SOD1G93A mouse model of ALS, the choroid plexus of the brain did not support leukocyte trafficking during disease progression, due to a local reduction in IFN-γ levels. The results of the present study, in agreement with the two later reports, show that the recruitment of monocytes detected by co-immunostaining with CD169 and Iba-1 was evident only at the end stage.