Emerging role of galectin 3 in neuroinflammation and neurodegeneration

Brian M.Lozinski, Khanh Ta, Yifei Dong

Abstract Neuroinflammation and neurodegeneration are key processes that mediate the development and progression of neurological diseases.However, the mechanisms modulating these processes in different diseases remain incompletely understood.Advances in single cell based multi-omic analyses have helped to identify distinct molecular signatures such as Lgals3 that is associated with neuroinflammation and neurodegeneration in the central nervous system (CNS).Lgals3 encodes galectin-3 (Gal3), a β-galactoside and glycan binding glycoprotein that is frequently upregulated by reactive microglia/macrophages in the CNS during various neurological diseases.While Gal3 has previously been associated with non-CNS inflammatory and fibrotic diseases, recent studies highlight Gal3 as a prominent regulator of inflammation and neuroaxonal damage in the CNS during diseases such as multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease.In this review, we summarize the pleiotropic functions of Gal3 and discuss evidence that demonstrates its detrimental role in neuroinflammation and neurodegeneration during different neurological diseases.We also consider the challenges of translating preclinical observations into targeting Gal3 in the human CNS.

Key Words: Alzheimer’s disease; Galectin 3; microglia; multiple sclerosis; neurodegeneration;neuroinflammation; Parkinson’s disease; therapeutics

Introduction

Neuroinflammation and neurodegeneration facilitate the pathology of central nervous system (CNS) diseases, including multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD).Preclinical studies investigating the pathological functions of various cellular and molecular signaling pathways in the CNS during these diseases have uncovered numerous anti-degenerative or pro-reparative targets (Friese et al., 2014; Gan et al., 2018).However,modern therapeutics are largely ineffective against disease progression in human neurological disorders because we do not fully understand the complex interplay between different cells and extracellular molecules that permeate the tissue milieu during these diseases (Wilson et al., 2023b).As neuroinflammation is commonly associated with axonal damage and neuronal loss in the CNS (Chitnis and Weiner, 2017), understanding the extracellular regulators of deleterious neuroinflammation may reveal novel treatments for chronic neurodegenerative diseases.Advances in single cell and spatial transcriptomic and proteomic analyses have generated unprecedented amounts of information to interrogate the heterogeneous signatures associated with various neurodegenerative diseases (Kaufmann et al., 2022; Vijayaragavan et al., 2022; Piwecka et al.,2023).Notably, these types of studies can also provide critical insights into commonly dysregulated molecules found in different diseases and potentially uncover new therapeutic targets that may be broadly effective in multiple neurological disorders.For example, several independent reports have highlighted thatLgals3overexpression by microglia/macrophages in the CNS is associated with the disease associated microglia phenotype in the context of AD (Krasemann et al., 2017; Boza-Serrano et al., 2019, 2022), PD (Boza-Serrano et al., 2014; Garcia-Revilla et al., 2023), MS (Plemel et al., 2020; Xue et al., 2023), retinal degeneration (Margeta et al., 2022; Zhou et al., 2023),and aging (Hammond et al., 2019; Pluvinage et al., 2019; Safaiyan et al., 2021;Dong et al., 2022).These observations suggest thatLgals3upregulation is part of a conserved response by microglia/macrophage to the loss of homeostasis and subsequent damage in the CNS.

Lgals3encodes for galectin-3 (Gal3), a β-galactoside-binding lectin potentially involved in the signaling of pattern recognition receptors such as toll-like receptor 4 (TLR4) (Burguillos et al., 2015; Liu et al., 2022) and scavenger receptors including triggering receptor expressed on myeloid cells 2 (TREM2)(Boza-Serrano et al., 2019) in reactive microglia/macrophages.Gal3 has been implicated in systemic diseases.For example, elevated Gal3 is associated with an increased risk of atrial fibrillation in chronic heart disease (Fashanu et al.,2017).Similarly, Gal3 is upregulated in obese individuals, and it is a marker of cardiac inflammation and fibrosis associated with heart failure (Florido et al., 2022).Gal3 elevation is also a sign of altered lung volume, gas exchange,and lung fibrosis (Ho et al., 2016).More recently, it has been indicated as a biomarker for the severity of coronavirus disease 2019 (Cervantes-Alvarez et al., 2022).Despite that these observations and other clinical evidence link Gal3 to inflammation and fibrosis in systemic diseases (reviewed in Bouffette et al., 2023), less is known about its intracellular functions or its functions after being secreted into the extracellular microenvironment, particularly in neurological diseases.Furthermore, few studies have investigated how microglia/macrophage-derived Gal3 acts on other CNS cells.Given that Gal3 is consistently upregulated by microglia/macrophages in response to neuroinflammation and neurodegeneration, it may be worthwhile to explore Gal3 inhibition as a therapeutic target in neurological diseases.In this review,we will provide an overview of the structure of Gal3 as well as its homeostatic versus inflammatory functions in the CNS.We then summarize the evidence for the role of Gal3 in neurodegeneration and discuss Gal3 as a potential therapeutic target in neurological diseases.

Search Strategy

PubMed and Google Scholar were searched for journal articles in this narrative review.In general, Gal3 orLgals3was searched together with key words such as: multiple sclerosis, Alzheimer’s disease, Parkinson’s disease,structure, biochemistry, neurodegeneration, neuroinflammation, microglia,neurological disease, therapeutics, and immune response.Articles from reputable journals preferably within the last 6 years, or topically important articles were selected for citation.

Structure and Biochemical Properties of Gal3

Gal3 is a member of the lectin family of glycoproteins.It has binding specificity to β-galactoside sugars and other galactose containing glycans (Farhadi et al., 2021).Unlike most other galectins that are either dimeric consisting of two identical galectin subunits or tandem consisting of two carbohydraterecognition domain (CRD), Gal3 has an unique chimeric structure consisting of a single C-terminal CRD fused to a long non-lectin N-terminal domain,which enables it to bind to both glycans and peptide motifs (Ippel et al.,2016).In monomeric form, X-ray crystallography and reflectivity experiments indicates the CRD of Gal3 is in a β-sandwich arrangement composed of the carbohydrate-binding 6-stranded S face and the opposing 5-stranded F face (Seetharaman et al., 1998; Vander Zanden et al., 2023).The Gal3 CRD is linked to an N-terminal tail composed of the N-terminal segment (NTS)arranged in a double-stranded anti-parallel β-sheet containing two serine phosphorylation sites as well as nine proline rich triple-helical collagenlike repeats (Flores-Ibarra et al., 2018; Zhao et al., 2021) which are cleavage sites for metalloproteases (Ochieng et al., 1994; Bülck et al., 2023).While the N-terminal is not integral to the folded structure nor to the CRD’s carbohydrate binding ability (Ippel et al., 2016; Vander Zanden et al., 2023),the interactions of the proline rich domains in the N-terminals with the CRD mediate Gal3 liquid-liquid phase separation which promotes oligomerization and facilitates cell signaling functions (Chiu et al., 2020; Zhao et al., 2021).Indeed, Gal3 oligomerization is critical for regulating its extracellular signaling activities as the potency of Gal3 to bind to carbohydrates and to induce apoptosis via caspase activation increases with the number of oligomerized subunits (Farhadi et al., 2021).Further detailed discussion on the structure and biochemistry of Gal3 can be found in additional reviews (Dumic et al.,2006; Modenuttiet al., 2019).

Gal3 Is a Multifunctional Protein

Immune cells such as monocytes and macrophages secrete Gal3 following inflammatory activation (Sato and Hughes, 1994; van Stijn et al., 2009),likely though a nonclassical secretory pathway (Zhang et al., 2020).In the extracellular milieu, Gal3 binds to glycoconjugates in the extracellular matrix(Gordon-Alonso et al., 2017) and on cell surfaces (Xiao et al., 2018).The functional outcome of Gal3 interacting with the extracellular matrix is not fully understood, however a recent study indicates it can limit cytokine diffusion by binding to glycosylated cytokines such as interferon (IFN)-γ and thus indirectly regulate immune cell infiltration (Gordon-Alonso et al., 2017).More is known about Gal3 activities in the context of cellular function.For example, Gal3 interacts with adhesion molecules such as vascular cell adhesion molecule 1 function to support immune cell trafficking (Sano et al., 2000; Rao et al., 2007;Ko et al., 2018).Interestingly, oligomerized Gal3 crosslinks with cell surface receptors to regulate receptor clustering and lattice formation (Nieminen et al., 2007) and this suggests that Gal3 controls various cell signaling pathways and membrane functions.Indeed, loss of function studies also show Gal3 binding to glycosylated proteins such as CD44 on cells is important for biogenesis of endocytic structures during clathrin-independent endocytosis(Lakshminarayan et al., 2014) and for the maintenance of lipid rafts (Hsu et al., 2009).Similarly, Gal3 promotes macrophage phagocytosis (Sano et al.,2003) at least in part through the scavenger receptor MerTK (Nomura et al., 2017; Lew et al., 2022).Cell culture studies also demonstrate Gal3 may promote inflammatory cytokine production by activating nuclear factor-κB through integrin-linked kinase (Zhao et al., 2018) or by activating JAK-STAT signaling via IFN-γ receptor 1 (Jeon et al., 2010).In the context of tissue injury or cancer, Gal3 potentially signals through Akt (Hsieh et al., 2015), Ras (Song et al., 2012), Notch-Jag1 (Zhou et al., 2023), or insulin-like growth factor 1(Lalancette-Hébert et al., 2012) to regulate cell proliferation.Conversely, Gal3 augments inflammasome and caspase activation (Li et al., 2008; Fermino et al., 2011; Lo et al., 2021), suggesting it can also promote apoptosis in cells,especially as oligomers (Farhadi et al., 2021).The involvement of Gal3 in inflammasome and caspase activation indicates that Gal3 also has intracellular signaling functions.Indeed, Chen et al.(2022b) recently demonstrated that Gal3 is an intracellular sensor of lipopolysaccharide (LPS) and Gal3 binding to LPS in the cytoplasm promotes cellular glycolytic reprogramming through Rag GTPases and Ragulator activation of mTORC1.Collectively, these observations,although derived from different cell types or disease models, highlight Gal3 has pleotropic functions (Figure 1).Whether and how Gal3 participates in all these functions in the context of CNS homeostasis and neurodegenerative diseases will be discussed in greater detail in the subsequent sections.

Gal3 and CNS Homeostasis

Figure 1 ｜Galectin-3 (Gal3) and its pleiotropic functions.(A) Gal3 is composed of a carbohydrate-binding domain that binds to β-galactoside sugars, and an N-terminal tail that is a site for phosphorylation and sites of cleavage for metalloproteases.(B) Gal3 can be released into the extracellular space by macrophages and monocytes where it can interact with interferon (IFN)-γ to immune cell infiltration.(C) Pentameric oligomerization of Gal3 crosslinks with cell surface receptors to promote membrane protein reorganizations and receptor lattice formation.(D) Through binding to CD44 and MerTK, Gal3 modulates endocytic activities and phagocytosis.(E) Gal3 mediates inflammatory cytokine production by activating nuclear factor-kB pathway through integrin-linked kinase (ILK) or by activating JAK-STAT signaling via IFN-γ receptor 1 (IFNGR1).(F) Gal3 facilitates cell proliferation through Akt-mTORC1, RAS, Notch-Jag1,or insulin-growth factor 1 (IGF1) signaling.(G) Intracellular Gal3 binding to liposaccharide(LPS) promotes inflammasome and caspase activation, as well as (H) mTORC1 activation via Rag GTPases and Ragulato.Created with BioRender.com.LPS: Lipopolysaccharide;MMP: matrix metalloproteinase; NF-κB: nuclear factor κB.

There is limited knowledge on the role of Gal3 during neurodevelopment and CNS homeostasis (Figure 2A).While Gal3–/–mice are typically healthy without major physiological or behavioral defects (Colnot et al., 1998), there are subtle observations suggesting that Gal3 is required for optimal tissue development and homeostasis.In the healthy CNS of rodents, Gal3 is expressed at low levels in most glial cells (Xue et al., 2023) but can be highly expressed in neurons in brain regions including the cerebral cortex and various subcortical nuclei in the hypothalamus and brainstem (Yoo et al., 2017), suggesting Gal3 expression in neurons may have non-redundant functions.Indeed, while Gal3–/–mice have normal neural progenitor cells expansion and survival in the subventricular zone (SVZ) during development, the migration rate of neural progenitor cells from the SVZ to the olfactory bulb is reduced, indicating that Gal3 regulates neural progenitor cell motility (Comte et al., 2011).Al-Dalahmah et al.recently demonstrated that Gal3 deficiency decreases the number of astrocytes and oligodendrocyte lineage cells in the SVZ during postnatal development and that Gal3 enhances bone morphogenetic protein receptor 1 alpha signaling (Al-Dalahmah et al., 2020b) but lowers β-catenin-Wnt signaling (Al-Dalahmah et al., 2020a) during gliogenesis.Thus, neural progenitor cell differentiation may also be regulated by Gal3.Consistent with this hypothesis, the reduced frequencies of myelinated axons, myelin lamellae and g-ratio in the brain white matter of Gal3–/–mice compared to wildtype mice suggest Gal3 promotes oligodendrocyte differentiation (Pasquini et al., 2011).Together, these studies have demonstrated a limited role of Gal3 in neurodevelopment and CNS homeostasis.Since neurons in certain anatomical brain regions express high levels of Gal3 at steady state (Yoo et al.,2017), the intracellular functions of Gal3 in these neurons and whether they secrete Gal3 upon stress are of interests for future studies.Moreover, agingassociated upregulation of Gal3 in microglia/macrophages (Hammond et al., 2019; Pluvinage et al., 2019; Safaiyan et al., 2021; Dong et al., 2022; Xue et al., 2023) highlights the need to better understand how its function may change in the aging CNS.

CNS Microglia and Macrophages Upregulate Gal3 during Disease

Single-cell RNA sequencing studies highlight that theLgals3gene is upregulated by microglia/macrophage in the CNS across multiple models of neurological disorders and aging (Hammond et al., 2019; Plemel et al., 2020;Safaiyan et al., 2021; Boza-Serrano et al., 2022; Dong et al., 2022; Xue et al.,2023; Zhou et al., 2023), which suggests its overexpression is a feature of the disease associated microglia phenotype and a common biomarker of neuroinflammation.Interestingly, Gal3 expression is higher in non-microglial phagocytes compared with microglia (Xue et al., 2023) and thus may be a useful indicator of peripheral monocyte-derived macrophage infiltration in the CNS (Hohsfield et al., 2022).Consistent with this observation, Gal3 expressing monocyte derived macrophages, distinct from microglia, aggregate at sites of vascular injury and promote aberrant blood vessel elimination in a cerebral microbleed model in diabetic mice (Mehina et al., 2021).These results do not contradict the observations that microglia upregulate Gal3 in disease, as while microglia responding to oxidized phosphatidylcholine(OxPC) lesions upregulate Gal3, their overall expression levels remain lower than monocytic cells (Xue et al., 2023).Whether and how other glial cells or neurons alter their Gal3 expression during CNS diseases is not fully understood.For example, Gal3 immunofluorescence did not increase in astrocytes in mouse models of MS (Xue et al., 2023), but it is increased in astrocytes following brain cortical stab wound injury (Sirko et al., 2015).In addition, as neurons express high levels of Gal3 in certain brain regions at steady-state (Yoo et al., 2017), how Gal3 activity changes in stressed neurons may be important for disease pathology.This is demonstratedin vitrowhere the release of extracellular vesicles containing Gal3 by human neurons with lysosomal membrane damage is associated with α-synuclein fibril formation(Burbidge et al., 2022).Collectively, these studies show microglia and other mononuclear phagocytes infiltrating the CNS during disease upregulate Gal3,whereas changes in Gal3 expression by other neuroglial cells need additional investigation.

Gal3 Modulates Inflammation in Multiple Neurological Diseases

Since Gal3 upregulation by microglia/macrophages is associated with many neurological disorders, it may be a common mediator of neuroinflammation(Figure 2B).For example, Gal3 expression is increased in association with reactive microglia/macrophages in experimental autoimmune encephalomyelitis (EAE), in oxidized phosphatidylcholine lesions that model MS, as well as in MS brain lesions (Xue et al., 2023).Likewise, microglia/macrophages also upregulate Gal3 during cuprizone mediated model of MS demyelination in mice (Hoyos et al., 2014).The functional role of Gal3 in MS is not fully explored.Results from animal models suggest Gal3 exacerbates neuroinflammation in MS.In support of this hypothesis, EAE in Gal3 deficient mice compared to wildtype mice is less severe with reduced monocyte infiltration, interleukin (IL)-17, and IFN-γ in the CNS (Jiang et al., 2009).Additionally, excess Gal3 increases IL-1β levels in spinal cord white matter (SCWM) lesions induced by OxPC (Xue et al., 2023), which is a neurotoxic product of lipid peroxidation elevated in MS (Dong et al., 2021,2022).This observation is interesting since Gal3 elevation in peripheral blood mononuclear cells of MS patients unresponsive to fingolimod compared to responders is reported as a biomarker of increased NLRP3 activity (Malhotra et al., 2023), which is an inflammasome that control IL-1β secretion (Cullen et al., 2015).However, since recombinant Gal3 alone do not promote inflammation in SCWM (Xue et al., 2023) and Gal3 overexpression in the postnatal SVZ did not induce inflammation in the absence of injury (Al-Dalahmah et al., 2020b), the ability of Gal3 to modulate neuroinflammation may not be through direct stimulation of microglia/macrophages.Alternatively, Gal3 deposition in the CNS may help recruit immune cells due to its chemoattractant properties (Sano et al., 2000) or by altering blood-brain barrier permeability (Nishihara et al., 2017).Indeed, Gal3 deficiency reduced immune cell infiltration into the CNS during EAE (Jiang et al., 2009) and during Theiler’s murine encephalomyelitis virus model of MS (James et al., 2016).Together, these results suggest that Gal3 deposition in MS may detrimentally increase neuroinflammation.Gal3 activity is also associated with other major neurodegenerative diseases.In AD patients, Gal3 elevated in the cerebrospinal fluid (CSF) is associated with higher expression of neuroinflammatory biomarkers including soluble TREM-2, glial fibrillary acidic protein, and chitinase-3-like protein 1 (Boza-Serrano et al., 2022).Additionally, in the 5×FAD mouse model of AD, Gal3 deletion ameliorated β-amyloid deposition and cognitive defects, in part by limiting microglia reactivity associated with TLR and TREM2/DAP12 signaling pathways (Boza-Serrano et al., 2019).This study also shows that the Gal3 CRD can directly interact with TREM2, which is evidence that Gal3 may be an endogenous ligand that promotes microglia reactivity through TREM2 activation.Finally, Gal3 expression may also be involved in AD associated neuropathic pain.Using the TASTPM double transgenic mouse model of AD together with K/BxN serum-transfer to induce inflammatory arthritis, Sideris-Lampretsas et al.(2023) demonstrates inhibition of neuron derived Gal3 with the orally active antagonist GB1107 reduces allodynia mediated by TLR4 expressing microglia.Together, these studies suggest that Gal3 inhibition can reduce neuroinflammatory and pain burden in AD.

In PD brains, Gal3 is associated with α-synuclein, Lewy bodies, and disrupted lysosomes (Garcia-Revilla et al., 2023).In vitro, siRNA knockdown of Gal3 or Gal3 inhibition attenuates iNOS, IL-1β, and IL-12 production by microglia stimulated by α-synuclein (Garcia-Revilla et al., 2023) and Gal3 is involved in the dissemination α-synuclein fibrils following lysosomal damage in human dopaminergic neurons (Burbidge et al., 2022).However, whether Gal3 deposition in PD tissue environment promote neuroinflammation remains to be exploredin vivo.

Gal3 is also upregulated in the brain of patients with frontotemporal dementia (Huang et al., 2020), in the spinal cord and CSF of patients with ALS(Zhou et al., 2010), and in the plasma and brain of patients with HD (Siew et al., 2019).Although Gal3 is associated with microglia in HD and is involved in lysosomal rupture and NLRP3 activation in microglia derived from the R6/2 mouse model of HD (Siew et al., 2019), the current paradigm mostly proposes Gal3 as a novel biomarker, but its neuroinflammatory role in these diseases requires further investigation.

Several recent studies highlight a potential new role for Gal3 in glaucoma and retinal degeneration.Like other diseases discussed above, microglia/macrophages upregulate Gal3 and adopt a neurodegenerative phenotype in mice following intraocular pressure and retinal ganglion cell (RGC) loss(Margeta et al., 2022).Neuroinflammation and microglial reactivity following retinal damage are attenuated in Gal3 deficient mice or through Gal3 inhibition using the small molecule inhibitor TD139 (Margeta et al., 2022;Tabel et al., 2022) at least in part due to reduced TLR4/NFκB signaling (Liu et al., 2022).Interestingly, the apolipoprotein E4 allele associated with AD risk is protective against glaucoma and human glaucoma patients with this allele had reduced Gal3 expression (Margeta et al., 2022).These findings indicate that interactions between Gal3 and apolipoprotein may be a novel and targetable mechanism against neuroinflammation.

Mechanisms of Gal3 Mediated Inflammatory Response in Microglia/Macrophages

Figure 2 ｜Functions of galectin-3 (Gal3) in homeostasis and disease conditions.(A) Gal3 plays a role in neural stem cell function.Gal3-deficient mice have reduced migration of neural stem cells from the subventricular zone to the olfactory bulbs.Gal3 also facilitates bone morphogenetic protein (BMP) signaling and inhibits Wnt-βcatenin signaling, and its depletion impairs the differentiation of neural stem cells into astrocytes and oligodendrocytes.Gal3-deficient mice also have impairments in axonal myelination.(B) In the context of diseases, Gal3 stimulates the expression of vascular cell adhesion molecule 1 (VCAM1) in endothelial cells, thereby promoting increased monocyte infiltration.It also acts as a ligand or facilitates binding to cell surface receptors such as TLR4, MerTK, IFNGR, and TREM2.Gal3 signaling promotes inflammation,cell death, phagocytosis, amyloid deposition, and disease associated phenotypes in microglia.Finally, Gal3 contributes to the prorogation of degeneration through lysosomal dysfunction and propagation of α-synuclein fibrils.Created with BioRender.com.iNOS:Inducible nitric oxide synthase; NF-κB: nuclear factor κB.

How Gal3 modulates neuroinflammation and whether this is conserved in multiple neurological disorders are not fully understood.However,in vitrostudies provide some mechanistic insights.Burguillos et al.(2015) found microglia secreted Gal3 binds to TLR4 through both the CRD and N-terminal domains and act as an endogenous TLR4 ligand.Soluble Gal3 upregulation of induced nitric oxide synthase, IL-1β, TNFα, IL-12, IL-10, and IL-4 by BV2 microglia is also TLR4 dependent, which suggests that Gal3 may modulate microglia reactivity and/or apoptosis as both an autocrine and a paracrine signaling molecule.Although this study found loss of Gal3 reduces microglia reactivity and tissue damage after global brain ischemia in mice, another study reported that Gal3 deficiency worsens neuronal damage following transient focal brain ischemia because Gal3 is required for microglia activation and proliferation in response to IGF-1 during ischemic injury (Lalancette-Hébert et al., 2012).Similarly, loss of Gal3 causes greater disease severity after parasite infection because Gal3 is important for neutrophil mediated parasite clearance in the brain (Quenum Zangbede et al., 2018).Thus, Gal3 is likely to have both beneficial and detrimental roles depending on the tissue microenvironment and the nature of the CNS disease.Furthermore, the observation that microglia derived Gal3 directly promotes neuroinflammatory response through TLR4 is less consistent with studies showing that Gal3 need to oligomerize and interact with LPS in order to enhance inflammasome activation (Li et al., 2008; Fermino et al., 2011; Lo et al., 2021), rather than acting as a potent TLR4 ligand on its own.Indeed, while the injection of recombinant Gal3 alone into the spinal cord white matter of mice do not induce significant inflammation or damage, the addition of Gal3 increases total levels of IL-1β and cleaved caspase 3 in OxPC lesions in the mouse spinal cord white matter (Xue et al., 2023).These observations are similar to how Gal3 enhanced LPS induced IL-1β production and caspase 4/11 activation(Lo et al., 2021).Although these studies highlight potential Gal3 mediated mechanism of microglia/macrophage activation, how other neurons or other glial cells respond to Gal3 remain unclear.

Gal3 Is a Secondary Amplifier of Neurodegeneration

Neurodegeneration as defined by axonal and neuronal loss mediates disease progression and permanent disabilities in neurodegenerative diseases including MS, AD, PD, and ALS (Wilson et al., 2023a).Although the mechanisms underlying neurodegeneration are not completely understood,evidence indicates that aberrant intracellular and extracellular aggregation of proteins such as β-amyloid and tau (Boza-Serrano et al., 2022), or α-synuclein(Burbidge et al., 2022; Garcia-Revilla et al., 2023) contributes significantly to neuroinflammation and neurodegeneration (Colonna and Butovsky, 2017;Wilson et al., 2023a).As Gal3 can oligomerize (Chiu et al., 2020; Farhadi et al.,2021; Zhao et al., 2021), its aggregation inside neurons or in the extracellular compartment may also facilitate neuroaxonal injury.The association of Gal3 with lysosomal dysfunction in neurons may be preliminary evidence for this hypothesis (Burbidge et al., 2022; Garcia-Revilla et al., 2023).In addition, the elevation of Gal3 levels in the serum or CSF of patients with AD (Wang et al.,2013), PD (Yazar et al., 2019), and ALS (Yan et al., 2016) indicates that it is at least a biomarker of neurodegeneration.

While mechanistic evidence for Gal3 as a direct mediator of neuroaxonal damage is lacking, studies indicate Gal3 indirectly facilitates neurodegeneration in various animal models of neurological diseases.For instance, Gal3 deficient 5×FAD mice has reduced β-amyloid burden compared to Gal3 sufficient mice and a single injection of Gal3 with β-amyloid monomers promotes the formation of insoluble β-amyloid in the hippocampus of mice (Boza-Serrano et al., 2019), which suggests Gal3 deposition in an extracellular milieu primed for misfolding protein aggregation or injury may exacerbate the mechanisms that cause neurodegeneration.In support of this hypothesis, co-injection of Gal3 together with OxPC also promoted greater axon degeneration in the SCWM of mice compared to mice injected with OxPC alone (Xue et al., 2023).Similarly, in a PD model where adenovirus induced overexpression of human α-synuclein in CNS of mice, dopaminergic neurons in Gal3 deficient mice are better at retaining α-synuclein within their cytoplasm compared to wildtype mice, which improves their cellular integrity and the overall motor function of the animal (Garcia-Revilla et al., 2023).These results are consistent with the recent observations that Gal3 mediates extracellular release of α-synuclein(Burbidge et al., 2022).Finally, siRNA knockdown of Gal3 in the hippocampus of 23-month-old mice reduces neuroinflammation and neuronal death from LPS induced systemic inflammation (Chen et al., 2022a), suggesting Gal3 upregulation in the aging CNS (Hammond et al., 2019; Pluvinage et al., 2019; Safaiyan et al., 2021; Dong et al., 2022; Xue et al., 2023) may also contribute to aging associated neurodegeneration.Interestingly, treating hippocampal brain slices with Gal3 decreases with gamma oscillation and impairs fast-spiking interneurons and pyramidal cells (Arroyo-García et al.,2023), suggesting Gal3 deposition may interfere with neuronal electrical activity and directly contribute to cognitive decline associated with aging and neurodegeneration.Collectively, these observations indicate that Gal3 contributes to the amplification of neurodegeneration in an abnormal tissue environment.

Conversely, reports also suggest Gal3 may be protective against neurodegeneration in specific context.For example, Gal3 deletion in super oxide dismutase 1 (G93A) mutant mice for ALS exacerbated inflammation,disability, and mortality (Lerman et al., 2012).Additionally, injection of recombinant Gal3 reduces tau hyperphosphorylation in 4-month-old 5×FAD mice (Lim et al., 2020).Moreover, intracerebral injection of Gal3 increases vascular repair, neuron survival, and motor sensor recovery following the induction of middle cerebral artery occlusion injury model of stroke in rats(Wesley et al., 2021).This study also suggests the neuroprotective effects from Gal3 may be associated with Akt signaling, downregulation of proapoptotic genes and upregulation of pro-survival genes.Overall, these results provide evidence that Gal3 has pleiotropic roles in neurodegeneration and the mechanism underlying these protective effects needs additional investigation.

Targeting Gal3 in Neurological Disorders

As discussed above, while most studies support a detrimental role for Gal3 in neuroinflammation and neurodegeneration, there is some evidence demonstrating Gal3 has neuroprotective effects.The opposing observations may arise from differences and/or limitations in the experimental approach to interrogate Gal3 function.Notably, reliance on total deletion of Gal3 in transgenic mice cannot distinguish how different levels of Gal3 expression modulate neuroinflammation and neurodegeneration at different stages of disease progression.Thus, using newer genetic tools such as Gal3-flox mice (Jia et al., 2021) to better elucidate the cell specific and time dependent roles of Gal3 in CNS disease models will provide critical insights to support the development of Gal3 as an effective therapeutic target for neurological diseases.Nevertheless, pharmacological inhibitors of Gal3 may help to ameliorate neuroinflammation and neurodegeneration.Since the Gal3 CRD naturally binds to polysaccharide ligands and located at the surface of the protein, drug development efforts have largely focused on antagonizing the Gal3 CRD (Bouffette et al., 2023).Since most animal studies of neurodegenerative diseases utilize TD139 to target Gal3, the discussion here with focus on this molecule.Details on other Gal3 inhibitors such as modified citrus pectin or belapectin are discussed in additional reviews(Sethi et al., 2021; Bouffette et al., 2023).TD139 is a 3,3′-bis-(4-aryltriazol-1-yl)thiodigalactoside that has high affinity for the human Gal3 CRD (Kumar et al., 2021) and antagonizes Gal3 activity (Delaine et al., 2016).TD139 is demonstrated to be safe and well tolerated in a randomized, double-blind,multicenter, placebo-controlled, phase 1/2a idiopathic pulmonary fibrosis study (Hirani et al., 2021).Notably TD139 inhalation treatment in this trial had a plasma half-life of 8 hours and the reduction of Gal3 in alveolar macrophages associates with decrease in idiopathic pulmonary fibrosis biomarkers.Inin vivomodels of AD or retinal degeneration, TD139 treatment reduces microglia reactivity and neuronal impairment or loss (Liu et al., 2022;Margeta et al., 2022; Tabel et al., 2022; Arroyo-García et al., 2023).Thein vivoeffects of TD139 in models of other neurodegenerative diseases such as MS or PD have not been thoroughly reported.More importantly, clinical trials targeting Gal3 in human neurodegenerative diseases remain lacking, perhaps because it remains unclear whether Gal3 inhibitors such as TD139 can cross the blood-brain barrier to modulate the CNS tissue environment.

Concluding Thoughts

Accumulating evidence has indicated that Gal3 is an important modulator of neuroinflammation and neurodegeneration in multiple human neurological disorders.Mechanistically, the current paradigm suggests that Gal3 is most effective following oligomerization and that Gal3 exerts it effect on cells through the interaction of its CRD with cell surface receptors.Given that Gal3 is involved in multiple cellular processes, the molecular mechanism by which Gal3 interacts with different glial cells or neurons/axons in the CNS is a key unanswered question.In neuroinflammation, Gal3 may be a damageassociated molecular pattern and can increase inflammatory responses by microglia/macrophages through TLR and inflammasome associated signaling pathways.How Gal3 impact the inflammatory function of astrocytes or other types of immune cells that have infiltrated the CNS is another area that needs to be investigated.In addition to its role in neuroinflammation, Gal3 may also exacerbate neurodegeneration by promoting intracellular lysosomal stress in neurons, extracellular aggregation of misfolded proteins, and dysfunctional neuronal activity.A major limitation of most preclinical animal models is the use of transgenic mice with total Gal3 depletion, so how the temporal kinetics and physiological elevation of Gal3 in diseases link to neurodegeneration and neurological impairments remains unclear.Elucidating these details will be informative for the development of appropriate therapeutic approaches and clinical trials.Finally, determining how to deliver Gal3 antagonists into the CNS tissue environment is a major hurdle to overcome for targeting Gal3 as an effective treatment for neurological diseases.

Author contributions:BML, KT, and YD wrote the first draft of the manuscript.BML and YD edited the manuscript.YD conceived the manuscript and supervised the overall manuscript.All authors read and approved the final version of the manuscript.

Conflicts of interest:The authors declare no conflicts of interest.

Data availability statement:Not applicable.

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