Amyotrophic lateral sclerosis (ALS) is an inexorable neurodegenerative disorder characterized by concomitant degeneration of upper and lower motor neurons [1]. Typically, ALS initially manifests with focal signs and symptoms that progressively deteriorate and expand to involve additional body regions, but generalized weakness and muscle wasting may be evident from its onset [2]. Cognitive and behavioral disorders may coexist; however, they are not uniformly present [3]. Despite its low worldwide prevalence (about 4–5 cases per 100,000 people) conservative projections predict a forthcoming “growth spurt” that will accompany the constant ageing of the world population [4, 5]. Therefore, ALS devastating nature, along with its ominous epidemiological trajectory increases the urgency for a better understanding of its pathogenesis that will pave the way for advances in its management.

To date, the primary event that instigates the pathophysiological cascade leading to progressive and incessant degeneration of motor neurons remains elusive [6]. Regardless, multiple pathophysiological mechanisms have been implicated in the neurodegenerative processes and are considered to be set in motion by complex interactions among numerous important genetic and environmental factors [7]. In brief, glutamate-mediated excitotoxicity, oxidative (reactive oxygen species) and metabolic (mitochondrial dysfunction) stress, protein misfolding, neurofilament accumulation, formation of intracellular aggregates, disruption of axonal transport and activation of microglia are considered to underlie neurodegenerative processes in ALS [8]. Whether these mechanisms are uniformly implicated in all patients or specific mechanisms, tend to assume predominant roles in distinct subgroups of ALS cases remains to be determined [6, 7].

The cluster of differentiation 33 (CD33) is a transmembrane sialic-acid-binding immunoglobulin-like lectin (siglec) which is principally found on leucocyte subsets and is suspected to regulate immune and inflammatory responses [9, 10]. The CD33 gene is located in chromosome 19p13.33 [11]. CD33 rs3865444 polymorphism constitutes a sequence variant situated within a regulatory region of the CD33 gene that is considered to alternate the expression of CD33, ultimately controlling microglial activation [12]. Its pivotal role in neurodegeneration has been primarily demonstrated by previous studies suggesting a protective effect of the minor allele against the development of Alzheimer’s disease (AD) [13, 14]. Lately, limited research has provided evidence on the protective role of rs3865444 against Parkinson’s disease (PD), as well as its implication in the pathogenesis of multiple sclerosis (MS) [15, 16, 17]. Considering the extensive pathophysiologic and genetic overlap across the spectrum of neurodegeneration, rs3865444 could assume a role in ALS related neurodegeneration, as well [18].

To date, no published article has focused on the relationship between CD33 rs3865444 and ALS. Therefore, the current analysis was undertaken to look into this association. For this purpose, a genetic association study involving individuals diagnosed with ALS and healthy controls of Greek ancestry was performed. The associations between (1) rs3865444 and ALS and (2) rs3865444 and age of disease onset were separately investigated.

The current report was the first to focus on CD33 rs3865444 in patients with ALS and did not establish any associations between the two. Although there is no direct evidence to assess the reproducibility of our results, there is indirect congruent evidence originating from genome-wide association studies (GWASs), which have not revealed a link between rs3865444 and ALS [27, 28]. In rare entities such as ALS, however, the sample size which is required by GWASs to identify SNPs conferring small-sized risks and low-frequency risk-variants may not be feasible to achieve [29]. Of note, although heritability is estimated to explain as much as 50% of the variability of sporadic ALS, the ensemble of risk-loci identified by GWASs occurs in $\le$10% of patients with sporadic ALS [28]. Therefore, the absence of GWAS-based evidence on an association between rs3865444 and ALS is not definitive. In any case, despite the negative results of the present article (and the indirect concurring findings of previous GWASs), additional research is warranted since linkage disequilibrium may vary among ethnic groups, and our findings may not be applicable across individuals with different racial backgrounds.

CD33 rs3865444 polymorphism is considered to alternate the expression of CD33, ultimately controlling microglial activation [12]. To date, most evidence of its implication in neurodegeneration stems from AD research [13, 14]. Relevant studies have shown that individuals with rs3865444 A/A alleles exhibit decreased expression of CD33 in peripheral monocytes and brain tissue compared to those with wild type alleles [30]. Reduced micro-glia expression of CD33 appears to lead to increased phagocytosis of amyloid beta (A$\beta$) peptide since CD33-lacking microglia phagocytize considerably greater quantities of A$\beta$ peptide [31]. Thus, individuals with minor alleles probably carry a protective effect against the accumulation of A$\beta$ peptide. Although the pathophysiological underpinnings of ALS are heterogeneous from those of AD, microglial activation and neuroinflammation, as well as the balance between homeostatic and deleterious inflammatory processes, appear to be of pivotal importance in the pathogenesis of the disease. The presumed mechanism of CD33 rs3865444 action is presented at Fig. 1.

Fig. 1.

Microglia, the immune cells of the central nervous system, assume a pivotal role in the dynamic regulation of inflammatory responses. Although microglia-mediated inflammation aims at the protection of the brain parenchyma from various insults, the inflammatory milieu might have detrimental effects on the neurons leading to dysfunction and death. Regulatory processes are pivotal in maintaining the balance between inflammatory and homeostatic processes, promoting the resolution of cerebral inflammation and inducing reparatory processes whenever appropriate.

Of note, microglia are speculated to assume a dual role in the pathophysiology of ALS, exhibiting both neurotoxic and neuroprotective properties [32]. Several researchers have even reported the co-existence of detrimental and protective roles in the same disease models [33, 34]. It has been argued that neuroinflammation and microglia activation may assume different roles in the temporal course of ALS or, more intriguingly, that different subsets of ALS cases could demonstrate heterogeneous underlying pathophysiologic mechanisms leading to the common “net outcome” (degeneration of the upper and lower motor neurons) [35]. Based on the aforementioned considerations, it is clear that different patient subsets could exhibit different associations with CD33 rs3865444, as well as other markers of microglial activation. Therefore, current analytic approaches assessing cases with ALS as a whole may fail to detect any association between the two due to systemic bias.

Our study has several limitations. First, despite achieving a power above 80%, there is an almost 20% chance that our analyses failed to detect non-trivial associations between rs3865444 and ALS. More so, there is an even greater probability that the size of our sample was insufficient to reveal truly significant, smaller-sized associations (compared to the relative risk of 1.42 that was used in our power-estimation statistics). Furthermore, study participants originated from a restricted area, geographically located in Central Greece. The formation of such a geographically restricted sample has introduced several advantages by the formation of two comparable participant groups with respect to several unmeasured exposures to numerous environmental factors that may be implicated in ALS (e.g., soil and water metal and pesticide concentrations [36]) but has probably introduced an overmatching bias in genetics. Moreover, our cohort has not been screened for the main genes that have been linked to ALS, such as superoxide dismutase type 1 (SOD1), chromosome 9 open reading frame 72 (C9orf72), Transactive response DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS) [37, 38]. Finally, the con-duction of larger, cross-ethnic studies is warranted to confirm the extrapolation of our findings in populations with different racial backgrounds.

Additional research is required to extract definitive conclusions about the connection between CD33 rs3865444 and ALS, while multinational studies are required to capture differential associations across different racial groups. The meticulous documentation of additional data, along with the imperative transparent reporting of future articles, will unravel potential associations between rs3865444 and other important clinical parameters, such as treatment-response and prognosis.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

VS: Conceptualization, methodology, software, validation, formal analysis, investigation, data curation, writing—original draft preparation, writing—review and editing, supervision. IL: Methodology, software, validation, formal analysis, investigation, data curatimon, writing—original draft preparation, writing—review and editing. AMA, CB, AN, DP, DPB: Validation, investigation, data curation, writing—review and editing. ZT: Software, validation, investigation, data curation, writing—review and editing. GN, EL, PL: Validation, writing—review and editing. GMH: Conceptualization, methodology, resources, writing—review and editing, supervision, project administration. ED: Conceptualization, methodology, resources, writing—review and editing, supervision, project administration, funding acquisition. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All procedures were approved by the Institutional Ethics Review Board of the University of Thessaly (59295/23-01-2017) approved the study protocol before its initiation. All participants provided written informed consent prior to participation.

Not applicable.

This study was supported in part by a research grant from the Research Committee of the University of Thessaly, Greece (code: 5287).

The authors declare no conflict of interest. Vasileios Siokas, Christos Bakirtzis, Efthimios Dardiotis, and Ioannis Liampas are serving as one of the Guest editors of this journal. We declare that Vasileios Siokas, Christos Bakirtzis, Efthimios Dardiotis, and Ioannis Liampas had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Gernot Riedel.