The role of Galectin-3 in α-synuclein-induced microglial activation

Parkinson's disease (PD) is a progressive neurodegenerative disorder clinically typified by bradykinesia, rigidity, postural instability and tremor, as well as a wide range of non-motor symptoms including constipation, bladder dysfunction and cognitive impairment [1]. Pathologically, PD is characterized by the formation of α-synuclein aggregates commonly known as Lewy bodies and Lewy neurites [2], glial activation, brain inflammation and progressive dopaminergic cell degeneration [3]. While the majority of cases of PD appear to be sporadic, genetic mutations or multiplications of the α-synuclein gene (SNCA) lead to the onset of familial PD [4],[5].

α-synuclein is a soluble protein composed of 140 amino acids found predominantly in presynaptic terminals where it is thought to play a role in development and plasticity [6]-[9]. In addition, α-synuclein is highly expressed in immune cells, including T-cells, B-cells, natural killer cells and monocytes [10]. Recent studies suggest that α-synuclein can transfer from one cell to another and promote the self-aggregation and thus possibly contributing to disease propagation [7],[11]-[14].

While microglial activation has been suggested to play major role in the neurodegenerative process in PD [15],[16], the signaling pathways that mediate this process are still poorly understood. For instance, Codolo and colleagues have recently demonstrated that α-synuclein monomers and fibrils induce Interleukin 1β (IL-1β) release from monocytes [17] via the Toll-like receptor 2 (TLR2). Moreover, Kim and colleagues have suggested that oligomeric forms of α-synuclein specifically activate TLR2 [18]. However, the TLR4 has also been implicated in α-synuclein-induced inflammation [19]. Moreover, it has been shown that the effects on cell activation and the subsequent inflammatory response can vary with the source/species of α-synuclein (mammalian cell-derived vs recombinant) and/or the type of protein used (wild type or mutant) [20]. Moreover, the molecular state of the protein used (monomeric, oligomeric or fibrillar) can also play a role in the magnitude of the inflammatory response [18]. Indeed, depending on the microenvironment/insult, activated microglia cells can adopt one of two well-characterized profiles, namely a classical (pro-inflammatory, M1) or an alternative (anti-inflammatory, M2) profile [21],[22]. In these two different states, activated microglia release different factors and express different surface proteins that allow them to sense the microenvironment and coordinate the inflammatory response. In the pro-inflammatory (M1) profile, microglial cells release different pro-inflammatory molecules, e.g. Tumor Necrosis Factor-α (TNF-α), IL-1β, Interleukin-12 (IL-12), Interferon-γ (IFN-γ) or Nitric oxide (NO), which decrease neuronal survival [23],[24]. The alternative profile, however, is characterized by release of anti-inflammatory factors (e.g. Interleukin-4 (IL-4), Interleukin-13 (IL-13) or Transforming Growth factor-β (TGF-β)) which reduce microglial activation [25]. While different pathways have been suggested to be involved in α-synuclein-mediated activation including the ERK 1/2, p38 MAPK, inflammasome or the NF-κβ pathway [17],[26], the involvement of galectin-3 and microglial activation remains to be elucidated. Galectin-3, which is identical to the commonly used macrophage marker Mac-2, is an inflammatory mediator known to be highly expressed in some activated inflammatory cells, including microglia. Galectin-3 levels are increased in several conditions including encephalomyelitis, traumatic brain injury, experimental allergic encephalitis (EAE) and ischemic brain injury [27],[28]. However, a possible role for α-synuclein induced galectin-3 activation during the inflammatory process in PD has yet to be elucidated.

Galectin-3 is a member of the β-galactoside-binding lectin family defined by their typical carbohydrate recognition domains (CRDs) [29],[30]. Galectin-3 plays a role in different biological activities, including cell adhesion, proliferation, clearance, apoptosis, cell activation, cell migration, phagocytosis and inflammatory regulation [27],[31]-[37]. Galectin-3 is found both intra- (in cytoplasm and nucleus) and extracellularly in different cell types and is suggested to play both pro-inflammatory and anti-inflammatory roles which depend on the cell type and insult provided [31],[36],[38],[39]. In this study, we investigated whether galectin-3 is involved in microglial activation induced by α-synuclein proteins. Therefore, we exposed BV2 and primary microglia cells to monomeric and aggregated forms of recombinant α-synuclein and specifically studied the inflammatory response. We then determined the effects of microglial activation following down-regulation of galectin-3 using a specific pharmacological inhibitor or genetic down regulation using siRNA. We then monitored the effects of different forms of α-synuclein on galectin-3-null mice primary microglial cultures. Finally, we determined whether α-synuclein injections into the olfactory bulb of wild type mice result in microglia activation and galectin-3 protein expression.

We demonstrate for the first time that galectin-3; a carbohydrate-binding protein is an immune modulator that plays an important role in the α-synuclein-induced activation of microglia. We identified a profound inflammatory inhibition of microglia cells by genetic down-regulation or pharmacological inhibition of galectin-3 or by using galectin-3 knockout primary microglia following activation by α-synuclein aggregates. In agreement with these results, prior work suggests that α-synuclein oligomers are neurotoxic and induce a strong inflammatory response in microglia cells, exceeding that seen after exposure to α-synuclein monomers [18]. Interestingly, Tokuda and colleagues have identified elevated levels of α-synuclein oligomers and an increased oligomers/total-α-synuclein ratio in the cerebrospinal fluid in PD patients, suggesting that α-synuclein oligomers may contribute to the progression of PD [51].

Recent discoveries have also demonstrated that α-synuclein can transfer from one cell to another and seed endogenous protein aggregation within the recipient cell in a prion-like fashion [13]. Besides spreading from neuron to neuron, α-synuclein can also spread from neurons to glial cells as shown previously in vitro and in vivo[52]. Due to the presence of α-synuclein in the extracellular milieu, several novel treatment strategies focusing on reducing the α-synuclein levels have been proposed including immunotherapy [53],[54], delivery of α-synuclein degrading enzymes [55] or altering microglial activity [56]. Indeed, microglial activation has been linked to several neurodegenerative disorders [57] and therefore, a pharmacological intervention on the inflammatory response exerted by microglia may be a promising therapeutic target. In attempts to reduce microglial activity, several different inflammatory pathways have been targeted in earlier studies. For example peroxiredoxin 2, which inhibits the mitogen-activated protein kinase and the transcription factor nuclear factor-κB (NF-kB), have shown to be effective [58]. Additionally, minocycline, one of the most used inhibitors for microglia activation has also been suggested to specifically inhibit the M1 phenotype [59]. Moreover, inhibition of NADPH oxidase 2 (Nox2) has also been shown to reduce microglial activation in α-synuclein-induced inflammation model [60].

In this study, we used a small molecule inhibitor targeting galectin-3 and found that it inhibited microglial activation following challenge with aggregated α-synuclein. Galectin-3 inhibitor has been successfully tested in other pathological conditions with evidence for a rate-limiting role of galectin-3 [46]. For example, in a mouse model of hepatitis, the galectin-3 inhibitor attenuated liver damage and proinflammatory T cell-mediated cytokine release (IFN-γ- and IL-17- and IL-4 producing CD4+ T cells). The same inhibitor also increased the number of T cells producing the anti-inflammatory IL-10 while promoting activation of M2 phenotype in macrophages [45]. Recently, the inhibitor was shown to support the survival of pancreatic beta cells in an apoptotic model induced by proinflammatory cytokines TNF-α + IFN-γ + IL-1β [44]. In our current model system, we observed an up-regulation of both pro and anti-inflammatory cytokines released from primary and BV2 microglial cells. After analysis, we detected a significant up regulation of pro-inflammatory cytokines TNF-α, IL-2 and IL-12. Using either, the galectin-3 inhibitor for 12 h or genetic down-regulation using siRNA we found a significant down-regulation in different pro-inflammatory molecules that include iNOS and TNF-α, molecules involved in the nuclear factor-kappa Beta (NF-κβ) pathway [61]. Using primary microglial cells derived from galectin-3 knockout mice, we identified a significant reduction in IL-12 and IL-1β release compared to wild type microglia. Interestingly, the absence of galectin-3 did not significantly affect the levels of IFN-γ or cytokines related to alternative activation pathway (e.g. IL-4) suggesting that, in response to α-synuclein, galectin-3 plays a specific inflammatory role in microglial activation. Such selective role for galectin-3 is noteworthy as galectin-3 regulates traffic of specific membrane glycoproteins (e.g. receptors) [62]. While the regulatory roles of galectins vary between different cell types, this variation is likely due to the galectin type and/or the type of glycans expressed in a particular cell [63]. Our findings support the notion that the inflammatory modulation exerted by galectin-3 is related to specific inflammatory pathways.

We have identified a robust reduction of IL-12 cytokine level in the primary galectin-3 KO microglia when compared to wild type microglial cells. The IL-12 production is regulated through multiple pathways that include: NF-κβ, p38 mitogen-activated protein (MAP) kinase, cyclic adenosine monophosphate (cyclic AMP)-modulating molecules and nitric oxide (NO) [64]. In line with our findings, several studies have shown a relationship between iNOS inhibition and a down-regulation of IL-12 expression [65]. Our results demonstrate a profound iNOS expression and a pro-inflammatory cytokines reduction upon galectin-3 knockdown, gene deletion or pharmacological inhibition, suggesting that the NF-κβ pathway may indeed be the effector pathway for galectin-3. Moreover, the inflammasome, which generates mature IL-1β by activating caspase-1, has also been shown to be associated with microglial activation [66]-[69]. Indeed, recent findings suggest that this inflammatory signaling pathway is activated by the phagocytosis of α-synuclein [17],[70]. For instance, Freeman and colleagues described a specific interaction between galectin-3 and the phagosomes/lysosomes containing α-synuclein [70]. We observed a remarkable 80% inhibition of α-synuclein-induced phagocytosis by pharmacological inhibition of galectin-3. This suggests that galectin-3 regulates α-synuclein-induced activation of microglia. On the other hand, increased phagocytosis of α-synuclein by microglia within the substantia nigra could potentially reduce the load of toxic α-synuclein species [71].

Indeed, we found galectin-3 immunoreactive microglia 12 h following injection of monomeric or oligomeric α-synuclein proteins. However, we did not detected galectin-3 immunoreactive cells after fibril injections at the same time points suggesting different up-take dynamics or intracellular processing [47]. At later time point however, α-synuclein fibrils and oligomers induced a robust galectin-3 immunoreactivity whereas monomers failed to induce a similar response indicating that monomers may be processed intracellular within 72 h without galectin-3 activation.

We have demonstrated that galectin-3 is an important molecule that contributes to full-blown microglial activity upon exposure to α-synuclein aggregates. Genetic ablation, down-regulating galectin-3, or pharmacologically inhibition of galectin-3, resulted in a profound down-regulation of microglial activation (i.e. reduced levels of iNOS, TNF-α, IL-12, IL-1β and the phagocytic ability of microglia). Following injections of α-synuclein species in the olfactory bulb, we observe an up-regulation of galectin-3 in microglial cells that had taken up the injected α-synuclein, providing further support for the importance of galectin-3 in vivo.