Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive deterioration in cognition, functional ability and behavior, but its underlying causes are to date unclear and a disease-modifying cure is still unavailable. Dysfunction in the clearance of Amyloid β-protein (Aβ) and neuroinflammation are two functionally linked pathological aspects of key importance in the development and progression of the disease (Heppner et al., 2015).

Homotaurine is an aminosulfonate compound naturally found in red algae, which has been demonstrated to have neuroprotective effects in rats systemically administered with kainic acid (Fariello et al., 1982) or following ischemic stroke (Wu et al., 2014). As a possible therapeutic agent for AD treatment, homotaurine reduces Aβ levels in CSF of patients with mild to-moderate disease (Aisen et al., 2006), slows brain atrophy (Gauthier et al., 2009) and exerts positive effect on cognitive impairment (Saumier et al., 2009). In a phase III AD clinical trial, homotaurine does not satisfy pre-fixed primary outcomes failing to demonstrate changes in cognitive function compared to placebo, but this result is possibly confounded by high statistical variability of data and is paralleled by post hoc analyses in a subgroup of patients, revealing some protective effects on hippocampal volume loss (Aisen et al., 2011). Thus, although safe and well tolerated, homotaurine is not authorized as a new AD drug, but it is currently used as a nutraceutical for memory protection and its use in treatment of cognitive decline symptoms is still considered promising. According with its potential favorable effects, we recently demonstrated that homotaurine supplementation has a positive consequence on hippocampus atrophy and short-term episodic memory loss in individuals at the earliest clinical state of AD, namely subjects suffering from amnestic mild cognitive impairment (aMCI; Spalletta et al., 2016). Regardless the favorable disease-modifying activities of homotaurine, its therapeutic efficacy and mechanism of action have yet to be fully elucidated.

Intriguingly, the protective activity of homotaurine appears to be especially evident in AD patients carrying the apolipoprotein E (APOE) ε4 alleles (Caltagirone et al., 2012), suggesting that its effects might be influenced by APOE ε4 genotype, the most powerful genetic risk factor of AD. Since APOE proteins appear to modulate Aβ clearance (Kim et al., 2009) and from in vitro and preclinical studies homotaurine reduces soluble levels of Aβ, inhibits its aggregation and decreases its toxic effects on neurons (Gervais et al., 2007), it is tempting to speculate that homotaurine may act, at least in part, in an APOE-dependent way. Furthermore, since Aβ clearance defect might also be both cause and consequence of the chronically activated neuroinflammatory pathways, which in turn concur to cause neuronal death, we addressed this study to evaluate the ability of homotaurine supplementation in modulating the inflammatory response in treated aMCI patients. In fact, several studies indicate that cerebral Aβ deposits elicit a chronic, disseminated inflammatory response producing neurodegeneration in AD (Akiyama et al., 2000) and, more recently, a skewed immune response both in brain and periphery has been blamed for a defective Aβ clearance leading to AD development (Heneka et al., 2015; Marsh et al., 2016; Ransohoff, 2016). In this regard, accumulation of reactive (and possibly functionally flawed) microglia in damaged brain regions and increased cerebral/peripheral expression of pro-inflammatory cytokines have been broadly described in AD patients. Of note, in response to a peripheral inflammatory stimulus, pro-inflammatory cytokine production is higher with APOE ε4 genotype, compared to the other APOE allele, and recent observations suggest a role for APOE in modulating Aβ-induced neuroinflammation (Tai et al., 2015), supporting the relevance of APOE genotype-specific homotaurine therapeutic potential.

By a mechanistic point of view, Aβ may trigger an innate response through the activation of NALP3 inflammasome (Halle et al., 2008), a multi-protein innate immune pathogen-sensing complex, which is essential for the release of specific inflammatory mediators, determining the cytokine milieu in the brain. Indeed, the two inflammasome-dependent molecules Interleukin (IL)-1β and IL-18 have been shown to be crucial regulators in AD pathology (Shaftel et al., 2008; Bossù et al., 2010). These two cytokines may exert their inflammatory action at both brain and peripheral level. While circulating IL-1β is generally low in normal conditions, with serum concentration often below level of detection and very small visible effects in the periphery, serum IL-18 is constitutively present in high amounts but is regulated by a highly specific natural inhibitor, named IL-18 binding protein (IL-18BP).

Therefore, with the aim to evaluate the potential immunomodulating effects of homotaurine in the serum of APOE genotyped aMCI patients, in this study we measured a panel of pro- and anti-inflammatory cytokines, including other than IL-1β, Tumor necrosis factor-alpha (TNFα), IL-6 and Transforming growth factor-beta (TGFβ), also IL-18BP and IL-18. The latter was evaluated in both its forms, i.e., the total form, including the cytokine bound and unbound to its inhibitor, and the free, unbound and biologically active form. Because of previous evidence that the compound is effective on patients carrying the APOEε4 allele, we hypothesized that homotaurine has an effect on memory deficit and immunomodulation in this subgroup only.

The main outcome of this study reveals that in aMCI patients, who carry the APOE ε4 allele, a 12-month supplementation with homotaurine is specifically associated with a decrease in the serum levels of the pro-inflammatory cytokine IL-18, in turn related to improved episodic memory performances. This finding corroborates previous data showing that the Aβ aggregation-preventing compound homotaurine exerts protective effects on AD in conditions of APOE ε4 genotype (Caltagirone et al., 2012) and in the early stages of the disease, as holding a positive effect on hippocampus atrophy and short-term episodic memory loss in amnestic MCI patients (Spalletta et al., 2016). Of importance, the present work offers novel evidence on homotaurine anti-inflammatory potential in the very precocious clinical phases of aMCI.

Indeed, neuroinflammation is involved early in the pathogenesis of AD neurodegeneration by taking part in a vicious cycle of Aβ deposition, neuronal death and cognitive decline (Eikelenboom et al., 2010; Heppner et al., 2015). Pro-inflammatory cytokines, including those released into the brain as well as in the periphery, like IL-18, are able to modulate the activation of resident microglia and participate in neuronal degeneration through different mechanisms. An increase in circulating pro-inflammatory factors, including IL-18, has been often observed in patients, as compared to healthy controls (Yaffe et al., 2003; Öztürk et al., 2007; Swardfager et al., 2010; Trollor et al., 2010; Brosseron et al., 2014; Saleem et al., 2015). However, several reports have described controversial results on cytokine blood levels in AD or MCI, mainly because of high inter-individual variability, clinical confounders and differences in assay procedures. Thus, if on the one hand, these observations confirm the potential of circulating inflammatory mediators as index of disease, on the other hand they encourage performing further longitudinal studies that may clarify the issue. In fact, despite the fact we did not observe any difference of serum cytokine levels between baseline and 12 months supplementation with homotaurine in our whole group of aMCI, we found the intriguing result when we split the group based on APOE genotype, according with our hypothesis. Such result confirms the need to limit heterogeneity among patients to identify potential cytokine markers of disease.

Noteworthy, the only over time change in aMCI subgroups is IL-18, making this cytokine an inflammatory mediator of special interest not only in association with homotaurine supplementation but, possibly, in the overall MCI context. In addition, since IL-18 herein exemplifies an inflammasome-dependent cytokine (unfortunately serum levels of IL-1β were undetectable), our results suggest that the activation of the inflammasome complex may play an important role in homotaurine action and conceivably in neurodegenerative dementia progression, as indeed recently reported (Saresella et al., 2016).

Furthermore, the observed homotaurine supplementation-dependent reduction of IL-18 concerned the two circulating forms of IL-18: both total and free, IL-18BP-unbound cytokine, with no effects on the inhibitory molecule IL-18BP. Differently, a trend towards IL-18BP increase was observed in treated MCI not carrying the APOE ε4 allele, suggesting that homotaurine plays a different regulation of IL-18, depending on the APOE genotype. The latter is a quite intriguing result, which strongly encourage driving additional research efforts to the comprehension of the interconnected molecular pathways linking APOE and neuroinflammation to the increased risk to develop AD, as well as to the potential effects played by homotaurine in this context. In fact, as previously reported, ApoE isoforms affect inflammatory processes (Vitek et al., 2009), with APOE ε4 carriers having lower brain levels of ApoE protein, enhanced neuroinflammation, and greater accumulation of Aβ. Hence, the AD-linked inflammatory events may be influenced by the function of ApoE proteins, giving rise to an interesting interpretation that Aβ-dependent inflammation may participate in neurodegenerative processes involving ApoE-mediated mechanisms (Tai et al., 2015). In particular, since Aβ-dependent triggering of inflammation involves the activation of both the transcription factor Nuclear Factor kappa B (NF-kB) and NLRP3 inflammasome (Halle et al., 2008; Vallabhapurapu and Karin, 2009), which together lead to IL-18 production, it is possible that homotaurine, by lowering Aβ levels can also negatively modulate Aβ-dependent NF-kB signaling, inflammasome activation and, eventually, IL-18 release.

The second achievement of this study points to an association between the homotaurine-dependent decrease of serum IL-18 and the improvement of short-term episodic memory performances. Although it remains unclear whether IL-18 plays a role (either primary or secondary) in neurodegeneration-associated cognitive decline, the observed decrease of serum IL-18 after homotaurine supplementation and its significant correlation with clinical parameters sustain the pathogenic importance of IL-18 in AD. Our result complements a whole body of literature describing that IL-18 might inhibit the cellular mechanisms underlying learning and memory (Curran and O’Connor, 2001) and participate in different ways to neuronal damage (Alboni et al., 2010), proposing that this pro-inflammatory cytokine and its related molecular pathways are of some relevance in AD pathogenesis (Bossù et al., 2010).

The present piece of work suggests that homotaurine supplementation in aMCI individuals carrying APOE ε4 has a positive consequence on episodic memory loss due, at least in part, to homotaurine anti-inflammatory effects targeting IL-18. While some studies highlighted the potential of homotaurine and its derivatives to benefit patients with MCI (Martorana et al., 2014; Spalletta et al., 2016), and to modulate inflammation in mouse models (Sternberg et al., 2012), this is the first evidence ever provided that homotaurine supplementation holds anti-inflammatory properties associated with memory improvement in patients with cognitive impairment.

This finding sheds a new light on the therapeutic potential of homotaurine, sustaining its beneficial disease-modifying effects, encouraging further confirmatory studies for treating genetically-defined populations with moderate AD (Abushakra et al., 2017), and also inspiring future research aimed at exploring the mechanisms by which this compound might control brain inflammation during progression of neurodegenerative dementias.

PB and GS planned and supervisioned the study. FS, AC, ES and AM designed and performed experiments. NB, FA, MO, CC, WG and GS coordinated and performed clinical evaluations. PB, GS, FS, AC, FA and MO analyzed the data. PB and GS wrote the article. All authors made substantial contributions to the conception of the work and its interpretation of data. All authors critically revised the manuscript and approved its finalversion.

Some reagents used for this study have been provided by FB Health, in complete absence of any influence from FB Health on the submitted work. Consulting Fees GS was funded from FB Health and Novartis for consultancy outside the submitted work. Lecture Fees GS received one lecture fee from FB Health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.