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Galectin-3 (Gal-3) is a beta-galactosidase binding protein involved in microglial activation in the central nervous system (CNS). We previously demonstrated the crucial deleterious role of Gal-3 in microglial activation in Alzheimer's disease (AD). Under AD conditions, Gal-3 is primarily expressed by microglial cells clustered around Aβ plaques in both human and mouse brain, and knocking out Gal-3 reduces AD pathology in AD-model mice. To further unravel the importance of Gal-3-associated inflammation in AD, we aimed to investigate the Gal-3 inflammatory response in the AD continuum. First, we measured Gal-3 levels in neocortical and hippocampal tissue from early-onset AD patients, including genetic and sporadic cases. We found that Gal-3 levels were significantly higher in both cortex and hippocampus in AD subjects. Immunohistochemistry revealed that Gal-3+ microglial cells were associated with amyloid plaques of a larger size and more irregular shape and with neurons containing tau-inclusions. We then analyzed the levels of Gal-3 in cerebrospinal fluid (CSF) from AD patients (n = 119) compared to control individuals (n = 36). CSF Gal-3 levels were elevated in AD patients compared to controls and more strongly correlated with tau (p-Tau181 and t-tau) and synaptic markers (GAP-43 and neurogranin) than with amyloid-β. Lastly, principal component analysis (PCA) of AD biomarkers revealed that CSF Gal-3 clustered and associated with other CSF neuroinflammatory markers, including sTREM-2, GFAP, and YKL-40. This neuroinflammatory component was more highly expressed in the CSF from amyloid-β positive (A+), CSF p-Tau181 positive (T+), and biomarker neurodegeneration positive/negative (N+/-) (A + T + N+/-) groups compared to the A + T-N- group. Overall, Gal-3 stands out as a key pathological biomarker of AD pathology that is measurable in CSF and, therefore, a potential target for disease-modifying therapies involving the neuroinflammatory response., Open access funding provided by Lund University. Vetenskapsrådet, 2019-0633, Antonio Boza Serrano, Ministerio de Ciencia, Innovación y Universidades, RTI2018-098645-B-100, Jose Luis Venero, PID2021-124096OB-100, Jose Luis Venero, Instituto de Salud Carlos III, 20/00448, Raquel Sanchez-Valle, Union PI18/01556, Javier Vitorica, PI21/00914, Javier Vitorica, Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, P18-RT-1372, Jose Luis Venero, US-1262734, Javier Vitorica, Kungliga Fysiografiska Sällskapet i Lund, 20191114ABS, Antonio Boza Serrano, 20211129ABS, Antonio Boza Serrano, Greta och Johan Kocks stiftelser, 20201201ABS, Antonio Boza Serrano, Consejería de Economía, Conocimiento, E mpresas y Universidad, Junta de Andalucía, US-1264806, Jose Luis Venero

Parkinson’s Disease (PD) is a neurodegenerative and progressive disorder characterised by intracytoplasmic inclusions called Lewy bodies (LB) and degeneration of dopaminergic neurons in the substantia nigra (SN). Aggregated α-synuclein (αSYN) is known to be the main component of the LB. It has also been reported to interact with several proteins and organelles. Galectin-3 (GAL3) is known to have a detrimental function in neurodegenerative diseases. It is a galactose-binding protein without known catalytic activity and is expressed mainly by activated microglial cells in the central nervous system (CNS). GAL3 has been previously found in the outer layer of the LB in post-mortem brains. However, the role of GAL3 in PD is yet to be elucidated. In post-mortem samples, we identifed an association between GAL3 and LB in all the PD subjects studied. GAL3 was linked to less αSYN in the LB outer layer and other αSYN deposits, including pale bodies. GAL3 was also associated with disrupted lysosomes. In vitro studies demonstrate that exogenous recombinant Gal3 is internalised by neuronal cell lines and primary neurons where it interacts with endogenous αSyn fbrils. In addition, aggregation experiments show that Gal3 afects spatial propagation and the stability of pre-formed αSyn fbrils resulting in short, amorphous toxic strains. To further investigate these observations in vivo, we take advantage of WT and Gal3KO mice subjected to intranigral injection of adenovirus overexpressing human αSyn as a PD model. In line with our in vitro studies, under these conditions, genetic deletion of GAL3 leads to increased intracellular αSyn accumulation within dopaminergic neurons and remarkably preserved dopaminergic integrity and motor function. Overall, our data suggest a prominent role for GAL3 in the aggregation process of αSYN and LB formation, leading to the production of short species to the detriment of larger strains which triggers neuronal degeneration in a mouse model of PD. The Michael J. Fox Foundation for Parkinson's Research ID: 11902 Ministerio de Ciencia e Innovación de España/FEDER/UE/PID2021-124096OB-I00 Junta de Andalucía/FEDER/EU P18-RT-1372 FEDER I + D + i-USE US-1264806 UK Research and Innovation - Future Leaders Fellowship MR/S033947/1 Alzheimer’s Society, UK - Grant 51 Alzheimer’s Research. UK - ARUK-PG2019B-020

Background Alzheimer’s disease (AD) is a progressive multifaceted neurodegenerative disorder for which no disease-modifying treatment exists. Neuroinflammation is central to the pathology progression, with evidence suggesting that microglia-released galectin-3 (gal3) plays a pivotal role by amplifying neuroinflammation in AD. However, the possible involvement of gal3 in the disruption of neuronal network oscillations typical of AD remains unknown. Methods Here, we investigated the functional implications of gal3 signaling on experimentally induced gamma oscillations ex vivo (20–80 Hz) by performing electrophysiological recordings in the hippocampal CA3 area of wildtype (WT) mice and of the 5×FAD mouse model of AD. In addition, the recorded slices from WT mice under acute gal3 application were analyzed with RT-qPCR to detect expression of some neuroinflammation-related genes, and amyloid-β (Aβ) plaque load was quantified by immunostaining in the CA3 area of 6-month-old 5×FAD mice with or without Gal3 knockout (KO). Results Gal3 application decreased gamma oscillation power and rhythmicity in an activity-dependent manner, which was accompanied by impairment of cellular dynamics in fast-spiking interneurons (FSNs) and pyramidal cells. We found that the gal3-induced disruption was mediated by the gal3 carbohydrate-recognition domain and prevented by the gal3 inhibitor TD139, which also prevented Aβ42-induced degradation of gamma oscillations. Furthermore, the 5×FAD mice lacking gal3 (5×FAD-Gal3KO) exhibited WT-like gamma network dynamics and decreased Aβ plaque load. Conclusions We report for the first time that gal3 impairs neuronal network dynamics by spike-phase uncoupling of FSNs, inducing a network performance collapse. Moreover, our findings suggest gal3 inhibition as a potential therapeutic strategy to counteract the neuronal network instability typical of AD and other neurological disorders encompassing neuroinflammation and cognitive decline. Ministerio de Ciencia e Innovación España

According to Braak’s hypothesis, it is plausible that Parkinsońs disease (PD) starts in the enteric nervous system (ENS) to spread the brain via the vagus nerve. Thus, we were wondering whether human inflammatory bowel diseases (IBD) can progress with appearance of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. Analysis of human gastrointestinal tract sections from IBD patients demonstrated the presence of pathogenic phosphorylated α-syn in both myenteric (Auerbach’s) and submucosal (Meissner’s) plexuses. Remarkably, PD subjects exhibit α-syn pathology in identical gastrointestinal locations. Analysis of human midbrain sections from IBD subjects revealed a clear displacement of neuromelanin in some nigral neurons from the ventral mesencephalon, which were inherently associated with presence of α-syn aggregates reminiscent of pale bodies. We also used different dextran sodium sulfate (DSS)-based rat models of gut inflammation (subchronic and chronic) to study the appearance of phosphorylated α-syn inclusions in both Auerbach’s and Meissner’s plexuses (gut), and in dopaminergic neuritic processes (brain) along with degeneration of nigral dopaminergic neurons, which are considered classical hallmarks of PD. Vagotomized DSS-treated animals exhibited pathological α-syn in the gut but failed to show dopaminergic cells degeneration and α-syn aggregation in the ventral mesencephalon. Taken together, these results strongly suggest that Braak’s hypothesis is plausible.

Additional file 3: 2-way ANOVA analysis with Tukey’s multiple comparisons between groups.

Additional file 2: Fig. S2. Flow cytometry gating strategy. (A) Lymphoid gating strategy. TOP (from left to right): Exclusion of doublets (FSC-H vs FSC-A), debris (SSC-A vs FSC-A), dead cells (PI +) and selection of white blood cells (CD45 +). MIDDLE (from right to left): Selection of Natural killer (NK) cells, T helper (CD4 +), T cytotoxic (CD8 +) and B lymphocytes (B220 +) according to the sequence marked by the arrows and corresponding markers indicated in Y and X-axis for each plot. BOTTOM: Discrimination of activated T lymphocytes from CD4 or CD8 subsets indicated by arrows and based on the lack of CD62L expression in each case. (B)Myeloid gating strategy. TOP (from left to right): Exclusion of doublets (FSC-H vs FSC-A), debris (SSC-A vs FSC-A), dead cells (PI +) and selection of white blood cells (CD45 +). MIDDLE and BOTTOM: Selection of Dendritic cells (DC), Eosinophils, Neutrophils, Inflammatory Monocytes, and intermediate populations according to the sequence marked by the arrows and corresponding markers indicated in Y and X-axis for each plot.

Galectin-3 (Gal-3) is a beta-galactosidase binding protein involved in microglial activation in the central nervous system (CNS). We previously demonstrated the crucial deleterious role of Gal-3 in microglial activation in Alzheimer’s disease (AD). Under AD conditions, Gal-3 is primarily expressed by microglial cells clustered around Aβ plaques in both human and mouse brain, and knocking out Gal-3 reduces AD pathology in AD-model mice. To further unravel the importance of Gal-3-associated infammation in AD, we aimed to investigate the Gal-3 infammatory response in the AD continuum. First, we measured Gal-3 levels in neocortical and hippocampal tissue from early-onset AD patients, including genetic and sporadic cases. We found that Gal-3 levels were signifcantly higher in both cortex and hippocampus in AD subjects. Immunohistochemistry revealed that Gal-3+microglial cells were associated with amyloid plaques of a larger size and more irregular shape and with neurons containing tau-inclusions. We then analyzed the levels of Gal-3 in cerebrospinal fuid (CSF) from AD patients (n=119) compared to control individuals (n=36). CSF Gal-3 levels were elevated in AD patients compared to controls and more strongly correlated with tau (p-Tau181 and t-tau) and synaptic markers (GAP-43 and neurogranin) than with amyloid-β. Lastly, principal component analysis (PCA) of AD biomarkers revealed that CSF Gal-3 clustered and associated with other CSF neuroinfammatory markers, including sTREM-2, GFAP, and YKL-40. This neuroinfammatory component was more highly expressed in the CSF from amyloid-β positive (A+), CSF p-Tau181 positive (T+), and biomarker neurodegeneration positive/negative (N+/−) (A+T+N+/−) groups compared to the A+T−N− group. Overall, Gal-3 stands out as a key pathological biomarker of AD pathology that is measurable in CSF and, therefore, a potential target for disease-modifying therapies involving the neuroinfammatory response. Ministerio de Ciencia, Innovación y Universidades de España RTI2018-098645-B-100 Ministerio de Ciencia, Innovación y Universidades, RTI2018-098645-B-100 y PID2021-124096OB-100 Instituto de Salud Carlos III de España - 20/00448 Fondos FEDER de la Unión Europea - PI18/01556 y PI21/00914 Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía. España - P18-RT-1372

Additional file 1: Fig. S1. Microglia and Ab plaques are not affected by the MS in the hippocampal dentate gyrus, CA1 and CA3 areas and amygdala at 4 months old. (A) Representative microphotographs of microglia (Iba1: white; DAPI: blue) in a whole-brain section of 4 months old mice. Scale bar: 500 μm. Quantification of Iba1 + area relative to the total area in each section from 2–3 sections/animal in (B) dentate gyrus (DG), (C) CA1, (D) CA3 and (E) amygdala (n = 5–9 animals/group). (F) Representative Congo Red staining in a whole-brain section of 4 months 5xFAD mice (left) and (right) the total positive plaques/mm2 (n = 5–8 animals/group). Data are shown as mean ± SD. *P < 0.05.

Additional file 4: 3-way ANOVA analysis with Tukey’s multiple comparisons between groups.

Background: Alzheimer’s disease (AD) is a progressive multifaceted neurodegenerative disorder for which no disease-modifying treatment exists. Neuroinflammation is central to the pathology progression, with evidence suggesting that microglia-released galectin 3 (gal3) plays a pivotal role by amplifying neuroinflammation in AD. However, possible involvement of gal3 in the disruption of cognition-relevant neuronal network oscillations typical of AD remains unknown. Methods: Here, we investigate the functional implications of gal3 signaling on cognition-relevant gamma oscillations (30-80 Hz) by performing electrophysiological recordings in hippocampal area CA3 of wild-type (WT) and 5xFAD mice in vitro. Results: Gal3 application decreases gamma oscillation power and rhythmicity in an activity-dependent manner and is accompanied by impairment of cellular dynamics in fast-spiking interneurons (FSN) and pyramidal cells (PCs). We found that gal3-induced disruption is mediated by the gal3-carbohydrate-recognition domain and prevented by the gal3 inhibitor TD139, which also prevents Aβ42-induced degradation of gamma oscillations. Furthermore, we demonstrate that 5xFAD mice lacking gal3 (5xFAD-Gal3KO) exhibit WT-like gamma network dynamics.Conclusions: We report for the first time that gal3 impairs cognition-relevant neuronal network dynamics by spike-phase uncoupling of FSN inducing a network performance collapse. Moreover, our findings suggest gal3 inhibition as a potential therapeutic target to counteract the neuronal network instability typical of AD and other neurological disorders encompassing neuroinflammation and cognitive decline.