Neuroinflammation and ALS: Transcriptomic Insights into Molecular Disease Mechanisms and Therapeutic Targets

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the motor nervous system. Despite the mechanism underlying motor neuron death is not yet clarified, multiple pathogenic processes have been proposed to account for ALS. Among these, inflammatory/immune responses have recently gained particular interest, although there are conflicting reports on the role of these processes in ALS pathogenesis and treatment. This apparent discrepancy may be due to the absence of an effective stratification of ALS patients into subgroups with markedly different clinical, biological, and molecular features. Our research group recently described genome-wide characterization of motor cortex samples from sporadic ALS (SALS) patients, revealing the existence of molecular and functional heterogeneity in SALS. Here, we reexamine data coming from our previous work, focusing on transcriptomic changes of inflammatory-related genes, in order to investigate their potential contribution in ALS. A total of 1573 inflammatory genes were identified as differentially expressed between SALS patients and controls, characterizing distinct topological pathways and networks, suggestive of specific inflammatory molecular signatures for different patient subgroups. Besides providing promising insights into the intricate relationship between inflammation and ALS, this paper represents a starting point for the rationale design and development of novel and more effective diagnostic and therapeutic applications.

in ALS (Supplementary Figure 1). In particular, NPEPPS has been recently identified as a major peptidase acting on neurotoxic protein substrates, including SOD1, and its expression was found significantly decreased in motor neurons of both ALS transgenic mice and patients, supporting its role in the disease pathogenesis [5]. Low expression levels of the gene encoding nardilysin convertase (NRD1) in SALS2 patients (Supplementary Figure 1) are in line with the evidence that the loss of this metalloendopeptidase in neurons leads to impaired motor activities and cognitive deficits by altering axonal maturation and myelination in the CNS [6]. Overexpression of IMPAS-1 in SALS patients is supported by several studies that correlate high levels of this protease with the aberrant autophagic activity associated with numerous neurodegenerative diseases [7].
Several molecular chaperones have been already extensively implicated in the pathogenesis of ALS, playing an essential role in the folding and maturation of proteins [8]. Consistent with our results, deregulated levels of HSP70, HSP90 and their interacting protein CHIP were previously detected in the serum of FALS patients, suggesting that deregulation of this protein system may reflect the degeneration of motor neurons in both forms of the disease [9]. Interestingly, recent studies have reported that treatments with arimoclomol, a strong co-inducer of HSPs, significantly delays disease progression in ALS animal models, supporting it as a potentially efficacious therapy for ALS.
Antigen presentation is mediated by the major histocompatibility complex (MHC) class I and class II molecules that are responsible to deliver short peptides to the APC surface for recognition by CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The main difference between these molecules is that the MHC class I pathway is usually fueled by endogenous antigen, while exogenous peptides reach the MHC class II pathway. SALS patients showed deregulated expression of genes encoding MHC class I and II molecules and different components of the peptide-loading complex (TAP1, TAP2, TPSN, PDIA3, CALR, CANX, UGCGL1, BCAP31, B2M, ENPL, COPII, BCAP31, ERAP1) (Supplementary Figure 1). Genes encoding MHC molecules were found to be differentially expressed in SALS patients, supporting previous findings that reported a dual activity of neuronal MHC in ALS-affected tissues [10]. In fact, although MHC seems to play an essential role in preserving the maximal efficiency of motor axon connectivity with target muscles, a marked activation of this molecular complex was associated with enhanced infiltration of immune cells in the CNS of ALS animal models at the onset and during disease progression [11]. Moreover, activated microglia and astrocytes, present in ALS and other neurodegenerative diseases, showed increased expression of MHC-class II molecules, promoting the release of nitric oxide and other soluble factors that enhance inflammatory response [12]. The pharmacological inhibition of MHC I expression by immunomodulatory agents, such as glatiramer acetate, has shown neuroprotective effects in several neurological conditions, including ALS [13].
T-cell-antigen recognition. The presentation of antigen in the context of MHC molecules serves as a signal to trigger T cell activation and initiate an immunogenic cascade that leads to cytolysis of the APCs. A global dysregulation of T-cell functions has been related to an increased disease progression, decreased survival as well as production of pro-inflammatory effectors in experimental ALS [14,15]. Abnormalities in T lymphocytes were also found in the blood of ALS patients, although there are differences among studies that may be explained by the heterogeneity of the ALS cohorts and the limited numbers of patients examined [14,16,17]. Accordingly, we found that a significant number of membrane protein-encoding genes involved in T cell activation and proliferation (CAV1, TRIM1, TOLLIP, DPP4/CD26, CD4, MIC2, CD45, ICOS, ICOS-L) were differentially deregulated in SALS patients (increased in SALS1 and decreased in SALS2) (Supplementary Figure 1). Among these, decreased expression of CD4 is in line with previous evidence demonstrating that the genetic depletion of CD4 or, more generally, a lack of functional T cells accelerates motor neuron degeneration and diminishes the survival of ALS transgenic mice, confirming a neuroprotective role of CD4+ T cells in ALS [15]. Reduced expression of CAV1 was associated with alterations of lymphocyte trafficking and synaptic transmission in the CNS, contributing to increased risk of neurodegenerative and age-related disorders [18,19]. SALS1 patients showed an increased expression of the gene encoding CD99 (MIC2), a leukocyte surface glycoprotein involved in several biological processes, including the regulation of T cell activation and development (Supplementary Figure 1) [20]. Pharmacological studies showed that CD99 blockade in vivo decreases the accumulation of CNS inflammatory infiltrates, supporting the role of this protein as a possible target for controlling neuroinflammatory events [21]. Another potential therapeutic target for CNS inflammation is represented by the CD45 tyrosine phosphatase, whose expression levels were increased in the spinal cord of ALS mice as well as in activated microglial cells of murine models of other neurodegenerative diseases, including Alzheimer's (AD) [22][23][24].
Natural Killer-cell-antigen recognition. Natural Killer (NK) cells are activated by a range of soluble factors, including cytokines and type I interferons, but also by direct cell-to-cell contact between NK cell receptors and target cell ligands. Contrary to T cells, NK cells recognize MHC I molecules using cell inhibitory receptors (i.e., KIRs, KLRs and NKG2A), leading to inhibition of NK cell activities [25][26][27]. Although further investigation is required to understand the role of NK cells and their receptors in ALS, previous studies have demonstrated significant infiltrations of NK cells in the spinal cord of ALS patients and the consequent inhibition of neuroprotective T-cell responses [14,28]. In our study, we found differential expression of some genes encoding NK inhibitory receptors (NKG2A, KIR2DS1, KIR2DS2 and KLRA1) in SALS patients, indicating the dysfunctions in NK cell-mediated functions may be implicated in the immunopathogenesis of various neurodegenerative diseases, including ALS (Supplementary Figure 1) [29,30]. In accordance with our results, increased expression of NKG2A was previously reported in PD patients, suggesting that high levels of this receptor may induce a chronic antigen-driven stimulation and dysregulated cytokine production, contributing to inflammatory and neurodegenerative events [31].
B-cell-antigen recognition. B cells recognize a specific antigen and initiate immune response through the B cell antigen receptor (BCRs) complex consisting of an antigen-binding subunit (the membrane immunoglobulin) and a signaling subunit, which is composed of a disulfidelinked heterodimer of Ig-α (CD79A) and Ig-β (CD79B) proteins [32]. Compelling evidence supports an important role of B cells in the pathogenesis of various neurological conditions, including ALS, not only as precursors of antibody-producing cells, but also as important regulators of the T-cell activation process through their participation in antigen presentation and cytokine production [33,34].
Our results showed differential expression of genes encoding a component of BCR complex (CD79A) and some B cell co-receptors (FcγRIIB, ITGB1, CD45, CRACM1) in SALS2 patients (Supplementary Figure 1). Low expression levels of CD79 complex and various B cell regulators were also detected in PD patients and seem to be related to aberrant protein glycosylation and folding in the endoplasmic reticulum [35]. Decreased expression of FcγRIIB or its non-functional variants was associated with the development of inflammatory autoimmune diseases and FcγRIIdeficient mice showed an impaired development of Purkinje neurons and poor rotarod performance [36][37][38]. In accordance with our results, up-regulated expression of CRACM1 seems to be associated with excessive neuronal Ca 2+ signaling and excitability, thus contributing to the pathogenesis of several neurological diseases, such as epilepsy, AD and Huntington's [39,40].

Immune and inflammatory signaling cascades
Accumulating evidence indicates that many neurodegenerative diseases, including ALS, are characterized by the massive activation and proliferation of microglia and astrocytes as well as the accumulation of infiltrating blood-derived immune cells (i.e., T lymphocytes and NK cells) at the sites of neurodegeneration, playing critical functions during the disease course. Moreover, signs of activation of the innate and humoral immune response were largely described in both ALS transgenic animal models and in the spinal cord and cortex of ALS patients [41]. In accordance with these studies, we observed differential expression of multiple components of intracellular signaling pathways regulating innate and adaptive immune responses in SALS patients (Supplementary Figure 2). Interestingly, the majority of these signaling cascades were increased in SALS1 and reduced in SALS2 patients, suggesting that diverse subgroups of ALS patients may respond differentially to therapies targeting innate and adaptive immune responses (Figure 4b).
Immunoreceptor signaling. Once activated, antigen receptors induce a complex series of signaling events that are fundamental for various immune functions, including cell activation, proliferation, gene transcription, cytokine secretion and clonal deletion, determining the direction of immune responses. Among neuroinflammatory DEGs in SALS patients, we distinguish the altered expression of some components of the Syk and Src family kinases (LCK, LYN, FYN, SYK and ZAP70) and their regulators (SHP1 and SHP2), which represent the first signaling molecules to be activated downstream of immune cell-specific receptors (Supplementary Figure 2). The biological functions of these inflammatory mediators are diverse and include immune cell-receptor signaling, CNS myelination, cell division and adhesion, platelet function, synaptic activity and plasticity. In accordance with our findings, lack of Syk and Src-family kinases has been associated to defects in actin polymerization and remodeling at the immune synapse [42]. In particular, decreased expression of FYN induces a reduction in brain myelination and structural defects in synapses and dendritic spines [43]. Moreover, deregulated expression of FYN and LCK was associated with AD pathology, and several drugs that aimed at maintaining the physiological functions of the Syk and Src family kinases (e.g., saracatinib or AZD0530) are currently under study for the treatment of this disease [43,44]. Also, ZAP70 was increased in AD patients, suggesting that activated ZAP70 may induce neuronal death through calcium-induced lymphocyte apoptosis and/or aberrant immune responses [45]. In SALS2, we also observed decreased expression of SYK together with the altered expression of two protein tyrosine phosphatases, SHP-1 and SHP-2, supporting their involvement in protecting neurons from genotoxic or oxidative insults responsible for neuronal degeneration [46][47][48][49]. Together, protein kinase and phosphatase cascades impinge on multiple downstream signaling pathways which have broad effects on gene transcription (ERK/MAPK), apoptosis regulation (AKT), Ca 2+ mobilization (PLC-γ; PKC) and cytoskeletal function modulation (Vav/Rac/Rho and AKT), representing critical events for immune activation and inflammatory cytokine/cytolytic enzyme production.
MAPK/ERK signaling. Mitogen-activated protein kinases (MAPKs) are serine-threonine kinases that are activated in response to different types of oxidative stress and inflammatory conditions, and mediate intracellular signaling associated with a variety of cellular activities, including cell proliferation, differentiation, survival and death [50]. Signal transduction via this cascade is usually initiated by activating multifunctional intracellular molecules, including guanine nucleotide exchange factors and small G proteins, which induce the sequential activation of numerous protein kinases, leading to the phosphorylation and activation of transcription factors, such as c-Jun, ATF/CREB, and p53.
In our study, we found increased mRNA expression of several components of the MAPK family (MKK4, MEK1/2, MEK3, ERK1/2, p38, and JNK) and differential expression of some of their upstream activators (Grb2, SOS, VAVs, K-RAS, H-RAS, Tiam1, CDC42, Rac1 and RhoA) in both SALS subgroups (Supplementary Figure 2). Consistent with our results, several lines of evidence demonstrated that compromised MAPK signaling pathway plays a critical role in the pathogenesis of diverse human diseases, including cancer and neurodegenerative disorders, such as ALS [51]. In fact, aberrant expression and persistent activation of p38, ERK and JNK1 have been implicated in ALS pathogenesis through various mechanisms, such as the formation of abnormal intracellular inclusions, alterations in axonal transport and cytoskeletal remodeling, and the induction of motor neuron cell death [52][53][54]. Interestingly, the p38 MAPK inhibitor SB203580 protects motor neurons and proximal axons from excitotoxin-induced degeneration, prolonging survival of ALS mice [55].
Dysregulation of MAPK pathway upstream regulators was reported in a variety of neuronal traumas and neurodegenerative diseases, including ALS. Among these, RHOA, a member of the Rho GTPase family, plays an important role in neuronal cell survival and death by transducing degenerating spinal cord motor neurons of patients and animal models of ALS [70][71][72]. In addition, it was shown that TLRs antagonism, as well as restoration of RAGE signals, exert neuroprotective effects in ALS pathology, significantly extending survival and improving motor functions in a mouse model of ALS [73][74][75]. Altogether, these results demonstrate that activation of these signaling pathways may contribute to motor neuron injury in ALS and suggest that their pharmacological inhibition may represent an effective therapeutic strategy to attenuate neurodegenerative processes.
Among various transcription factors that are activated by the TLR signaling cascade, NF-κB plays a role of fundamental importance in various cellular mechanisms, including the immune response, cytokine production, cellular responses to oxidative stress and synaptic plasticity. Decreased expression of NF-κB was found in SALS1, while increased expression of this gene was detected in SALS2 (Supplementary Figure 2). This discrepancy may be explained by the fact that, while low levels of NF-κB have been associated with a decreased neuroprotection, high levels of this protein complex might be responsible for microglial activation occurring during neuroinflammatory responses [76]. Moreover, although preclinical studies reported contrasting results, several NF-κB pharmacological inhibitors have shown neuroprotective effects in ALS, mainly by preventing apoptotic cell death, inflammation and oxidative damage as well as improving mitochondrial function [77].