Galectin‐3 is upregulated in frontotemporal dementia patients with subtype specificity

Abstract

INTRODUCTION
Neuroinflammation is a major contributor to the progression of frontotemporal dementia (FTD). Galectin‐3 (Gal‐3), a microglial activation regulator, holds promise as a therapeutic target and potential biomarker. Our study aimed to investigate Gal‐3 levels in patients with FTD and assess its diagnostic potential.

METHODS
We examined Gal‐3 levels in brain, serum, and cerebrospinal fluid (CSF) samples of patients with FTD and controls. Multiple linear regressions between Gal‐3 levels and other FTD markers were explored.

RESULTS
Gal‐3 levels were increased significantly in patients with FTD, mainly across brain tissue and CSF, compared to controls. Remarkably, Gal‐3 levels were higher in cases with tau pathology than TAR‐DNA Binding Protein 43 (TDP‐43) pathology. Only MAPT mutation carriers displayed increased Gal‐3 levels in CSF samples, which correlated with total tau and 14‐3‐3.

DISCUSSION
Our findings underscore the potential of Gal‐3 as a diagnostic marker for FTD, particularly in MAPT cases, and highlights the relation of Gal‐3 with neuronal injury markers.

Full text

Received: 26 June 2023 Revised: 4 October 2023 Accepted: 11 October 2023
DOI: 10.1002/alz.13536
RESEARCH ARTICLE
Galectin-3 is upregulated in frontotemporal dementia patients
with subtype specificity
Sergi Borrego–Écija1Agnès Pérez-Millan1,2Anna Antonell1Laura Fort-Aznar1
Elif Kaya-Tilki3Alberto León-Halcón4,3Albert Lladó1,2Laura Molina-Porcel1
Mircea Balasa1Jordi Juncà-Parella1Javier Vitorica4,3,5Jose Luis Venero4,3
Tomas Deierborg6Antonio Boza-Serrano1,3,4Raquel Sánchez-Valle1,2
1Alzheimer’s disease and other cognitive disorders Unit. Service of Neurology,Fundació Recerca Clínic Barcelona-IDIBAPS, Hospital Clínic de Barcelona, Barcelona,
Spain
2Institut of Neurosciences. Faculty of Medicine and Medical Sciences, University of Barcelona, Barcelona, Spain
3Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla,Sevilla, Spain, Sevilla, Spain
4Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla,Spain
5Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),M adrid, Spain
6Department of Experimental Medical Sciences, Experimental Neuroinflammatory Lab, Lund University, Lund, Sweden
Correspondence
Antonio Boza-Serrano and Raquel
Sánchez-Valle, Alzheimer’s disease and other
cognitive disorders Unit. Service of Neurology,
Fundació Recerca Clínic Barcelona-IDIBAPS,
Hospital Clínic de Barcelona, Barcelona, Spain.
Email: aboza@us.es and rsanchez@clinic.cat
Funding information
Instituto de Salud Carlos III, Grant/Award
Numbers: PI20/0448, PI18/01556,
PI21/00914; Una manera de hacer Europa,
Grant/AwardNumber: PI19/00449;
Generalitat de Catalunya, Grant/Award
Number: SGR 2021-01126; Vetenskapsrådet,
Grant/AwardNumber: 2019-0633; Kungliga
Fysiografiska Sällskapet i Lund, Grant/Award
Numbers: 20191114ABS, 20211129ABS;
Greta och Johan Kocks stiftelser, Grant/Award
Number: 20201201ABS; Juan de la Cierva
Incorporación, Grant/AwardNumber:
IJC2019-040731-I; Spanish Ministerio de
Ciencia e Innovación /FEDER/UE,
Grant/AwardNumber:
PID2021-124096OB-I00; Swedish
Demensfonden; Swedish Brain Foundation;
Crafoord Foundation; Swedish Dementia
Abstract
INTRODUCTION: Neuroinflammation is a major contributor to the progression of
frontotemporal dementia (FTD). Galectin-3 (Gal-3), a microglial activation regulator,
holds promise as a therapeutic target and potential biomarker. Our study aimed to
investigate Gal-3 levels in patients with FTD and assess its diagnostic potential.
METHODS: We examined Gal-3 levels in brain, serum, and cerebrospinal fluid (CSF)
samples of patients with FTD and controls. Multiple linear regressions between Gal-3
levels and other FTD markers were explored.
RESULTS: Gal-3 levels were increased significantly in patients with FTD, mainly across
brain tissue and CSF, compared to controls. Remarkably, Gal-3 levels were higher
in cases with tau pathology than TAR-DNA Binding Protein 43 (TDP-43) pathology.
Only MAPT mutation carriers displayed increased Gal-3 levels in CSF samples, which
correlated with total tau and 14-3-3.
DISCUSSION: Our findings underscore the potential of Gal-3 as a diagnostic marker
for FTD, particularly in MAPT cases, and highlights the relation of Gal-3 with neuronal
injury markers.
KEYWORDS
C9orf72, CSF, frontotemporal dementia, galectin-3, GRN, MAPT, microglia, neuroinflammation
Sergi Borrego–Écija, Agnès Pérez-Millan, Antonio Boza-Serrano, and Raquel Sánchez-Valle contributed equally to this work.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. Alzheimer’s & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer’s Association.
Alzheimer’s Dement. 2024;20:1515–1526. wileyonlinelibrary.com/journal/alz 1515

1516 BORREGO–ÉCIJA ET AL.
Association; G&J Kock Foundation; Olle
Engkvist Foundation; Gamla Tjänarinnor
Foundation; Swedish Medical Research
Council; Swedish Parkinson Foundation;
Swedish Parkinson Research Foundation; A.E.
Berger Foundation
1BACKGROUND
Frontotemporal dementia (FTD) encloses a group of neurodegener-
ative disorders that have in common the neurodegeneration of the
frontal and temporal lobes.1Due to its early onset, with most peo-
ple presenting symptoms around the sixth decade of life, FTD is
the second most common form of early-onset neurodegenerative
dementia, after Alzheimer’s disease (AD).2,3 Clinically, FTD includes
three different syndromes: the behavioral variant of FTD (bvFTD),
the semantic variant of primary progressive aphasia (svPPA), and
the non-fluent/agrammatical variant of primary progressive apha-
sia (nfvPPA).4,5 FTD is a highly heritable disorder with mutations in
Chromosome 9 open reading frame 72 (C9orf72), Granulin (GRN), and
Microtubule-associated protein Tau (MAPT), explaining most genetic
cases.6,7 The neuropathological substrate of FTD is frontotemporal
lobar degeneration (FTLD). FTD can be classified according to the
abnormally deposited protein, which can be the tau protein (FTD-tau),
the transactive response (TAR) DNA-binding protein 43 (FTD-TDP-43),
or the FET family of proteins (FTD-FET).1,8,9
Along with neuronal dysfunction/death and protein accumulation,
central immune system activation is a major factor in the progres-
sion of the pathology in FTD.10 Abnormal protein conformation and
accumulation activate the immune system, leading to neuroinflam-
mation. This response involves glial activation and increased levels
of pro-inflammatory factors.10 Microglial cells are the main pool of
innate immune cells in the brain, and their phenotype is crucial to
understand the neuroinflammatory response. Recently, Malpetti et al.,
demonstrated that microglial activation measured by [11C]PK11195
in the frontal cortex could predict cognitive decline in patients with
FTD.11 Positron emission tomography (PET) studies using transloca-
tor protein (TSPO) have detected abnormal microglial activity and
protein aggregation in familial cases of FTD.12 PET analysis also
revealed that microglial activation seems more prominent in fron-
totemporal regions.12–14 The microglial activation pattern detected
by PET analysis has also been observed in histological postmortem
studies.15,16 Neuropathological studies reveal microglial activation in
cortical areas and more pronounced involvement in white matter than
gray matter.15,16
A key molecule involved in microglial activation in neurodegen-
erative diseases is galectin-3 (Gal-3), a beta-galactosidase binding
protein expressed mainly by microglial cells and associated with
neurodegeneration.17–19 Gal-3 is released into the extracellular
space and acts in an autocrine or paracrine manner by binding to
different membrane receptors, such as Toll Like Receptor 4 (TLR4)
and Triggering Receptor Expressed on Myeloid Cells 2 (TREM2).20,21
We have demonstrated previously that Gal-3 is an important reg-
ulator microglial activity in AD21 and in the aggregation process of
α-synuclein and Lewy body formation in Parkinson’s disease.22,23 Of
interest, Gal-3 was found primarily in microglia clustering around
amyloid beta (Aβ) plaques and using 5xFAD model of AD lacking Gal-3
reduced the Aβburden and improved their cognitive performance.21
Our study also confirmed that Gal-3 acts as an endogenous TREM2
ligand, a central player in the regulation of microglial activation under
disease conditions.21 It is important to note that in AD we found
Gal-3 in cerebrospinal fluid (CSF) to be strongly associated with
neuroinflammation markers, synapse loss, and cognitive decline.24
A recent study analyzing over 2000 human AD brain tissue samples
identified a microglia module as a highly affected process in AD. The
study found that Gal-3 ranked fifth among the top 30 microglial tran-
scripts associated with AD, suggesting its significance in AD pathology
and microglia dysfunction.25 In support of this perspective and with
regard to FTD, transgenic mice carrying FTD-related genes exhibit
strong and even aberrant microgliosis and inflammatory response,
especially evident in mice lacking GRN26,27 and P301S MAPT mice.28
Moreover, a recent study has shown that mutations in GRN mice led
to significant microglial activation, which seems to be influenced by
GPNMB (glycoprotein non-metastatic melanoma protein B) and Gal-3
in mice.29
In the context of traumatic injury models, it has been observed that
TDP-43 induction contributes to the activation of microglial cells, lead-
ing to the upregulation of Gal-3.30 Like in AD, the role of neuroinflam-
mation and immune-mediated mechanisms in the development of FTD
is well established.10 Indeed, a causal role of neuroinflammation has
been proposed, as evidenced by increased microglial activation in the
frontal and temporal cortices, astrogliosis, and abnormal expression of
pro- and anti-inflammatory factors in CSF and blood.10,31,32
Consequently, the present study explores Gal-3 levels and their link
to FTD through brain, CSF, and serum biomarker analysis for FTD and
its subtypes.
2METHODS
2.1 Clinical cohort
A total of 133 participants were recruited at the Alzheimer’s Dis-
ease and Other Cognitive Disorders Unit of the Hospital Clínic de
Barcelona, including 115 patients fulfilling criteria for bvFTD (62
patients), svPPA (28 patients), or nfvPPA (25 patients), and 18 healthy
controls (HCs) (Table 1). The clinical CSF FTD cohort has 6 MAPT

BORREGO–ÉCIJA ET AL.1517
symptomatic mutation carriers, 5 presymptomatic MAPT mutation car-
riers, 13 GRN mutation carriers, and 13 C9orf72 expansion carriers. All
the participants underwent a complete clinical and neuropsychologi-
cal examination. The participants included in the study had Gal-3 levels
in CSF (N=133 participants) and/or serum (N=120 participants). All
subjects were Caucasian white and did not present other neurological
diseases. The study was approved by the ethics committee of the Hos-
pital Clínic de Barcelona (HCB 2019/0105). Written informed consent
was obtained from all participants.
2.2 Human brain tissue
Frozen frontal cortices from 10 cognitively healthy controls and 37
FTD cases were obtained from the Neurological Tissue Bank, Biobanc-
Hospital Clínic-IDIBAPS, Barcelona, Spain. All subjects were White.
The control cases were not diagnosed with any neurological condi-
tion (other than migraine or essential tremor) during life, and the
postmortem examination did not disclose findings supporting any neu-
ropathological diagnosis, although minor vascular changes or a low
grade of incidental pathologies were not exclusionary.33 FTD cases
included 18 mutation carriers (4 MAPT,5GRN,and8C9orf72;Table2)
and 19 sporadic FTD (10 tau and 9 TDP-43; Table 2). Written informed
consent for using brain tissue and clinical data for research purpose
was obtained from all patients or their next of kin following the Inter-
national Declaration of Helsinki and Europe’s Code of Conduct for
Brain Banking. The medical ethics committee of the institutional review
board of the Hospital Clínic (Barcelona) has approved the procedures
for brain tissue collection.
2.3 ELISA
Enzyme-linked immunosorbent assay (ELISA) plates from Abcam
(ab269555) were used to measure the levels of Gal-3 (detection range
58.8 to 2000 pg/mL) in tissue homogenates, CSF, and serum samples.
The protocol was carried out according to the manufacturer’s instruc-
tions. A Biotek Synergy 2 was used to read the ELISA Gal-3 assay.
All samples were run in duplicate once. Mean inter-assay Coeficients
of Variances (CV) was 6.23. Samples were distributed in the plates
according to the clinical group in similar proportions to avoid a bias
caused by the plate. The ELISA and the analysis of the raw data were
performed by different persons. CSF samples were not diluted. Tis-
sue homogenates were diluted 1:100. Serum samples were diluted
1:20 or 1:50; the correction factor on diluted samples was applied
when needed for the comparison. Our kit has been used previously to
measure Gal-3 levels in brain, CSF, and serum samples24,34
2.4 Protein extraction
Radioinmuno precipitation Assay Buffer (RIPA) solution was pre-
pared with a protease inhibitor (cOmplete Protease Inhibitor Cocktail,
RESEARCH IN CONTEXT
1. Systematic review: Inflammation is a critical component
of frontotemporal dementia (FTD). We explore the liter-
ature using sources such as PubMed. We found only five
publications under the term’s “microglia”, “cerebrospinal
fluid”, and “frontotemporal dementia”. Therefore, the clin-
ical practice suffers from a notable absence of reliable
microglial proinflammatory markers associated with FTD.
2. Interpretation: We have uncovered evidence of a sub-
stantial upregulation of the microglial marker galectin-3
(Gal-3) in patients with FTD, thereby emphasizing the piv-
otal role of neuroinflammation in FTD pathogenesis and
the utility of microglial markers as biomarkers. Notably,
disparities in FTD subtypes, particularly the elevated lev-
els of Gal-3 observed in Microtubule-associated protein
Tau (MAPT) mutation carriers, suggest the possibility of
FTD subtype-specific neuroinflammatory patterns.
3. Future directions: Future research must prioritize cere-
brospinal fluid (CSF) longitudinal and independent cohort
studies to determine the Gal-3-dependent neuroinflam-
matory response in the course of FTD. In addition, neu-
ropathological investigations are needed to identify brain
regions wherein microglial activation manifests most
prominently.
Roche) and a phosphatase inhibitor (PhosphoStop, Roche). Frozen
human tissue samples of the hippocampus and cerebral cortex were
homogenized in RIPA buffer (1 mL/100 mg of tissue, Sigma-Aldrich,
Germany) and sonicated briefly in ice. The pellet was ultracentrifuged
subsequently at 25000 relative centrifugal force (rcf) for 25 min. The
supernatant was isolated and used for analysis. Protein concentration
was determined using a BCA Kit (Bio-Rad) according to the manu-
facturer’s protocols. All the samples were normalized to the same
concentration prior to the analysis.
2.5 CSF biomarker analysis
The samples were processed within 2 h from needle-to-freezer (mean
time 45 min). Both CSF and blood samples were centrifuged at 2000 ×
gfor 10 min at 4◦C. Then they were stored in polypropilene tubes and
kept at −80◦C until use: Storage tubes for CSF (eppendorf 0.5 mL Ref.
72.730.007 (Sarstedt)) and for serum (cryotubes 2 mL Ref. 363401PK
(Nunc)).
Core AD biomarker concentrations were measured with INNOTEST
ELISAs following the manufacturer’s instructions (Fujirebio, Ghent,
Belgium). CSF neurofilament light chain (NfL) concentration was mea-
sured using the ELISA kit of Uman Diagnostics distributed by IBL
International (Hamburg, Germany). For γ−14-3-3 protein, we used the

1518 BORREGO–ÉCIJA ET AL.
TAB L E 1 Group summaries are given as each measure’s mean and standard deviation in brackets.
Healthy Controls All FTD bvFTD svPPA nfvPPA p-value*
N18 115 62 28 25 –
NCSF 18 99 36 26 16 –
NSerum 13 107 50 24 20 –
Sex (male/female) 6/12 57/58 32/30 15/13 10/15 0.20
Age, years, mean (SD) 54.3 (4.8) 63.5 (9.1) 63.0 (9.2) 64.0 (8.4) 64.2 (9.9) <0.001
Duration, years, mean (SD) NA 3.4 (2.3) 3.8 (2.7) 2.9 (1.3) 3.0 (2.1) –
Mean MMSE score (SD) 29.1 (1.1) 23.6 (5.8) 23.4 (6.3) 23.5 (5.9) 24.3 (4.7) <0.001
Genetic (%) 0 (0%) 35 (30%) 25 (40%) 2 (7%) 8 (32%) <0.001
Serum Gal-3, median [Q1, Q3] 1428.7 [1296.6, 1644.4] 1463.3 [1231.0, 1758.4] 1491.6 [1292.1, 1834.4] 1450.6 [1211.0, 1565.9] 1502.8 [1163.2, 1727.3] 0.80
CSF Gal-3, median [Q1, Q3] 459.0 [433.9, 501.6] 545.6 [437.2, 667.2] 567.4 [434.0, 701.1] 532.8 [505.0, 635.4] 484.0 [428.0, 652.6] <0.05
CSF Aβ42, median [Q1, Q3] 901.8 [664.7, 1088.5] 761.7 [589.9, 944.5] 788.5 [550.4, 979.5] 730.5 [627.2, 896.8] 682.0 [589.9, 876.0] 0.13
CSF p-tau, median [Q1, Q3] 44.7 [37.4, 57.1] 40.5 [32.1, 55.2] 39.0 [32.0, 54.0] 37.3 [32.8, 49.0] 46.0 [35.3, 58.3] 0.30
CSF t-tau, median [Q1, Q3] 192.4 [165.5, 266.9] 276.4 [210.1, 409.5] 290.0 [214.0, 427.2] 260.5 [214.9, 327.5] 269.2 [209.4, 473.5] <0.01
CSF YKL-40, median [Q1, Q3] 214.5 [170.5, 245.3] 289.0 [207.6, 371.5] 321.6 [193.1, 391.3] 275.8 [252.0, 317.4] 296.0 [214.5, 381.0] <0.05
CSF NfL, median [Q1, Q3] 401.8 [358.2, 501.5] 2232.6 [1286.2, 3962.0] 2390.3 [1244.7, 4023.6] 1912.9 [1391.0, 2579.6] 2537.1 [1191.0, 4102.7] <0.001
CSF 14-3-3, median [Q1, Q3] 2571.9 [2231.2, 3001.4] 4075.0 [3042.0, 5301.3] 4133.4 [3042.0, 5509.7] 3539.9 [3155, 4997.2] 3835.9[2968.1, 4782.7] <0.001
The p-value is from the comparison between controls and all the FTD patients.
Abbreviations: bvFTD, behavioralvariant frontotemporal dementia; nfvPPA, non-fluent variant primary progressive aphasia; svPPA, semantic variant primary progressive aphasia. *Between all FTD and controls.

BORREGO–ÉCIJA ET AL.1519
TAB L E 2 Group summaries of cortical samples, both genetic and sporadic, used in the study..
Healthy
Controls MAPT GRN C9orf72 sFTD-tau sFTD-TPD-43 All FTD p-value*
N10 4 5 8 10 9 37
Sex (male/female) 5/5 3/1 2/3 4/5 4/6 6/3 20/18
Age, years, mean (SD) 81.3 (12.3) 59.5 (3.1) 66.4(4.8) 70.0 (11.9) 72.1 (5.5) 72.0 (7.9) 67.5 (2.1)
Disease duration, mean (SD) –6.3 (2.1) 6.6 (2.4) 5.3(4.08) 8 (3.5) 11 (4.24) 8.05 (3.9)
Brain Gal-3, median [Q1 – Q3] 472 [449 – 524] 1509 [1156 – 1837] 1900 [1860 – 1947] 714 [454 – 767] 1137 [1046 –1263] 589 [430 – 735] 974 [669 – 1286] <0.001
Abbreviations: MAPT, microtubule-associated Protein Tau; GRN, Granulin; C9orf72, chromosome 9 open reading frame 72; sFTD, sporadic Frontotemporal dementia; TDP-43, Tar DNA binding protein 43.
* Between all FTD and controls.
ELISA kit CircuLex 14-3-3 gamma (MBL International Corporation,
Woburn, MA, USA) with a CSF sample dilution of 1:5. CSF YKL-40 con-
centration was measured with an ELISA from QUIDEL (San Diego, CA,
USA) using a CSF sample dilution of 1:2.5. The antibodies for the detec-
tion of these four biomarkers have been used by us and other authors
on previous studies with CSF samples in neurodegenerative demen-
tias. These biomarkers have been also studied individually using other
technologies or antibodies.35–38
All analyses were performed by duplicate and experienced labora-
tory personnel blinded to clinical diagnosis. We are participants of the
Alzheimer’s Association QC program,5and Aβ42, total tau (t-tau), and
phosphorylated tau 181 (p-tau181) levels obtained in our laboratory
have been consistently within mean ±2SD.
2.6 Statistical analysis
All FTD-related variables from the cortical tissue were analyzed with
the Mann–Whitney test to compare independent groups. For multi-
ple comparisons, the Kruskal–Wallis test followed by Dunn test was
used as a post hoc correction to identify the pair-wise group differ-
ences. Receiver-operating characteristic (ROC) curves analysis was
performed to assess the diagnostic accuracy of the Gal-3. For the sta-
tistical analysis of the CSF and serum levels of Gal-3, permutation tests
with age and sex added as covariables were used. The p-values of these
results were correct for multiple comparisons with the Benjamini &
Hochberg correction. We compared the HCs, genetic FTD, and spo-
radic FTD patients with the same procedure. Finally, we studied in
detail group differences for the different FTD groups (clinical pheno-
type and genetic form) and HCs with the same methodology. Multiple
linear regression corrected by age and sex were applied to evaluate
the association between the CSF Gal-3 and the other CSF FTD-related
markers levels (Aβ42, t-tau, p-tau181, 14-3-3, YKL-40, and NfL levels)
for abovementioned groups. For all the analyses, statistical significance
was set at p-value <0.05. Statistical analyses were carried out using
GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA),
SPSS v. 26 (IBM Corp., Armonk, NY, USA) software, and the language R
in R-studio version 4.2.1 (https://www.r-project.org).
3RESULTS
3.1 Demographic and clinical characteristics of
participants
Demographic information of the study population and group statis-
tics are shown in Table 1. Controls were younger than FTD patients
(p-value <0.001). Because Gal-3 levels in CSF showed a moderate cor-
relation with age in the whole cohort (r =0.42, p-value <0.001), further
statistical analyses were corrected for age. There were statistically sig-
nificant differences in Gal-3 levels grouping by sex in the whole cohort;
thus the analyses were also corrected for sex. We did not find a signifi-
cant link between Gal-3 and disease duration. However, it is important

1520 BORREGO–ÉCIJA ET AL.
FIGURE 1 Galectin-3 (Gal-3)protein levels in brain cortex. (A) Control vs FTD cases. Gal-3 levels were significantly increased in FTD compared
with controls. (B) FTD-tau vs FTD-TDP-43. FTD cases with tau pathology had higher brain levels of Gal-3 than those with TDP-43 pathology. (C)
Brain Gal-3 levels in genetic carriers. MAPT and GRN carriers showed increased Gal-3 levels in brain. Cortical brain tissue was analyzed with the
Mann–Whitney Uand the Kruskal–Wallis test (multiple comparison) followed by Dunn’s test used as a post hoc correction to identify the pair-wise
group differences. (See Section 2.6 for further statistical analysis description.
to mention that most samples in our study were collected at the time
of diagnosis, with only a few collected during the later stages of the
disease.
3.2 Galectin-3 levels are upregulated in cortical
tissue from FTD
First, we evaluated Gal-3 levels in FTD brain samples. Gal-3 level was
upregulated in patients with FTD as a whole compared to control sam-
ples (Figure 1A). When compared according to their neuropathological
substrate, FTD-tau showed higher Gal-3 levels compared to FTD-TDP-
43 cases (here we included genetic and sporadic cases with tau or
TDP-43 deposition in the comparison) (Figure 1B). Finally, we evalu-
ated Gal-3 levels in genetic cases and found increased Gal-3 levels in
MAPT and GRN carriers compared to controls (Figure 1C). Both MAPT
and GRN genetic cases also displayed higher Gal-3 values compared
to C9orf72 (Figure 1C). In contrast, Gal-3 levels in C9orf72 expansion
carriers did not differ from controls.
3.3 Elevated Gal-3 levels in CSF in FTD samples
Following the brain sample analyses, we measured Gal-3 levels in
CSF samples from genetic and sporadic FTD patients (Figure 2).
CSF Gal-3 levels were elevated in FTD comparison to HC samples
(Figure 2A). For the next analysis, we separate sporadic by clinical
phenotype (bvFTD, svPPA, and nfvFTD) and genetic samples by type
of mutation (MAPT,GRN,andC9orf72)(Figure2B, C). The analysis of
the sporadic variants of FTD resulted in higher Gal-3 levels in bvFTD
compared to svPPA (Figure 2B), nfvFTD (Figure 2B), and HC samples
(Figure 2B).
We observed a significant elevation of Gal-3 levels in MAPT carrier
samples compared to GRN carriers (Figure 2C), C9orf72 (Figure 2C),
and HC samples (Figure 2C). No statistically significant differences
were found between GRN and C9orf72 groups. CSF Gal-3 levels were
significantly higher for symptomatic carriers than presymptomatic
MAPT mutation carriers (Figure 2C). In our cohort, Gal-3 CSF could be
used to differentiate FTD from controls: ROC curve (area under the
curve [AUC] 0.67).
3.4 Levels in serum
Serum Gal-3 levels were also increased in FTD patients compared to
controls (Figure 2D). The ROC curve, however, showed a poor perfor-
mance of serum Gal-3 differentiating FTD from controls (AUC: 0.55).
No significant differences were found between any of the sporadic syn-
dromes of FTD and HCs (Figure 2E). No difference in serum Gal-3 levels
were found between the different causal mutations of genetic FTD and
HCs (Figure 2F).
3.5 Association of CSF Gal-3 with other
biomarkers
When studying the multiple linear regressions between different CSF
biomarkers (Aβ42, t-tau, p-tau181, 14-3-3, YKL-40, and NfL) and Gal-
3, we observed a significant association for FTD patients but not
for the HCs (Table 3). The t-tau levels for FTD presented a moder-
ate association with CSF Gal-3 (R =0.43, p-value adjusted <0.001)
and with 14-3-3 levels (R =0.45, p-value adjusted <0.001, respec-
tively). Gal-3 also presented a weak relationship with Aβ42 for
FTD patients (R =0.39, p-value adjusted <0.001). No statistically

BORREGO–ÉCIJA ET AL.1521
FIGURE 2 Galectin-3 levels in CSF (A, B, C) and serum (D, E, F). The p-values of the plots were adjusted for multiple comparisons and corrected
by age and sex. (A) CSF Gal-3 levels in FTD cases are increased compared with controls. (B) Comparing FTD clinical syndromes, the bvFTD group
showed higher levels of CSF Gal-3 than controls, svPPA, and nfvPPA. (C) CSFGal-3 levels in mutation carriers revealed higher levels in MAPT
carriers. (D) Serum levels of Gal-3 were elevated in FTD patients. (E) Serum Gal-3 levels between clinical syndromes showed higher levels in
nfvPPA than in svPPA, with no differences in other comparisons. (F) No differences in serum Gal-3 levels were found between mutation carriers.
(See Section 2.6 for further statistical analysis description.)
significant relationship was found between Gal-3 and p-tau181, NfL, or
YKL-40. Table 3shows the coefficients details of all the multiple linear
regressions.
4DISCUSSION
In this study, we examined Gal-3, a microglial marker, across the
FTD spectrum in neuropathological and clinical cohorts (CSF and
serum levels) of both sporadic and genetic FTD patients. Our findings
revealed elevated Gal-3 levels in FTD subjects’ brains, CSF, and serum,
thereby highlighting neuroinflammation’s significance and the role of
Gal-3-expressing microglia in FTD’s neurodegenerative mechanism.
Galectins play a crucial role in the brain’s neuroinflammatory response
by identifying glycan structures and sensing their modifications both
intracellularly and extracellularly. Despite the importance if galectins,
the regulation of their expression remains elusive.39 Recent investiga-
tions found substantial upregulation of Gal-3 in GRN knockout Induced
Pluripotent stem cells (iPSC)-derived microglia.40 Gal-3 was detected
in human studies and FTD mouse models with GRN gene knockout,
emerging as the primary upregulated protein alongside GPNMB.29
Other galectins such as galectin-1 (Gal-1) and galectin-9 (Gal-9) have
been involved in the regulation of neuroinflammatory processes.41,42
However, Gal-1 has been shown to deactivate microglial activation,41
thereby reducing the associated inflammatory response. On the other
hand, Gal-9 is produced mainly by astrocytes but not microglial cells
and it has been shown to indirectly promote microglial activity.42 Gal-9
CSF levels have been shown to be increased in secondary progressive
multiple sclerosis43 and have been also linked with central nervous
system (CNS) immune activation and poor cognitive performance in
human immunodeficiency virus (HIV) infected individuals.44
Due to FTD’s substantial heterogeneity, significant differences
emerged among clinical, genetic, and neuropathological subtypes. A
relevant maker of microglial activation in disease conditions is TREM2,
which is implicated in the neuroinflammatory response in AD, and
has shown a strong association with FTD-tau.45–48 We demonstrated

1522 BORREGO–ÉCIJA ET AL.
TAB L E 3 Multiple linear regression coefficients for assessing the different CSF biomarkers trajectories by CSF Gal-3 level according to age and sex.
Aβ42 p-Tau181 Total tau NfL YKL-40 14-3-3
βCI 95% p-value βCI 95% p-value βCI 95% p-value βCI 95% p-value βCI 95% p-value βCI 95% p-value
R20.1365 0.2143 0.05659 0.01183 0.1927 0.2590
Gal3 0.6 0.3– 0.9 <0.001 0.0 0.0– 0.10.022 0.4 0.2– 0.6 <0.001 1.0 -1.9– 3.8 0.50 0.1 -0.1– 0.3 0.40 4.7 2.5– 7.0 <0.001
Age -6.5 -12.0– −1.4 0.013 -0.1 -0.7– 0.50.80 1.6 -1.5– 4.8 0.30 12.0 -38.0–62.0 0.60 5.8 2.4– 9.3 0.0010 41.0 4.1– 78.0 0.030
Sex
(Female
vs. male)
-11.0 -107.0 –85.0 0.80 -6.9 -17.0–3.50.20 -59.0 -117.0–0.1 0.050 16.1 -779.0–1102.0 0.70 16.0 -41.0–73.0 0.60 -527.0 -1186.0– 132.0 0.12
previously that Gal-3 expressed by microglial cells can act as a TREM2
ligand.21 In addition, we have shown a clear association of Gal-3 with
tau and p-tau181 in CSF in AD and have demonstrated a clear co-
localization of microglial cells expressing Gal-3 with tau protein in asso-
ciation of Aβplaques.24 Therefore, Gal-3′s unique expression, pivotal
role in microglial activation, and relevance in FTD progression mark it
as a key molecule for future exploration, differentiating it from other
galectins in understanding neuroinflammation in neurodegenerative
disorders.
Microglial activation correlates strongly with FTD progression and
cognitive decline.11,12 Therefore, the reactive microgliosis observed
in FTD might contribute to an upregulation of Gal-3 levels. In our
study, FTD-tau exhibited higher Gal-3 levels in the brain than FTD-
TDP-43. Moreover,the patients we analyzed carrying MAPT mutations,
which cause tau pathology, showed increased Gal-3 levels in the brain.
Previous neuropathological works have shown that FTD-MAPT cases
present strong microglial cell activation, even more than other FTD
cases.49,50 Indeed, reactive gliosis is also prevalent in tauopathies and
FTD mouse models.27–29,51,52 Likewise, the elevation of Gal-3 in MAPT
carriers was evident in CSF but not serum samples, indicating Gal-
3 CSF’s superior performance in these cases. Gal-3 serum levels did
not distinguish FTD clinical forms or genetic samples, likely due to
its peripheral origin (e.g., monocytes), rather than CNS microglia,53,54
due to posttranslational modification, like phosphorylation, hampering
bloodstream release55–57 and its upregulation in other comorbidities
outside the brain, such as heart disease.58
Regarding Gal3 levels in patients with genetic FTD, symptomatic
MAPT carriers had higher Gal-3 levels than presymptomatic individu-
als, implying that Gal-3 could be a biomarker for MAPT carrier clinical
onset or progression, pending larger longitudinal validation. In vivo
evidence for presymptomatic neuroinflammation in a MAPT mutation
carrier59 has been found in recently.The study indicated that microglial
activation is a better marker for discriminating MAPT mutation carriers
from controls than tau protein aggregation at this pre-symptomatic
disease stage of FTD.59 This result might indicate that microglial acti-
vation in MAPT mutation carriers might be an early event rather than
a consequence of protein dysregulation,59 which might open up new
possibilities for early anti neuroinflammatory treatments. In mouse
models, Van Olst and colleagues investigated neuroinflammation in
P301S MAPT mice and found that microglia changes started after neu-
ronal p-tau deposition in the early stages of tau processing.52 In this
model, microglia adopted a p-tau-associated phenotype, morpholog-
ical and functionally distinct from wild-type microglia, after neuronal
p-tau accumulation was initiated. Other studies have revealed the
pivotal role of microglial cells and apolipoprotein E gene (APOE)in
driving neurodegeneration in a mouse model of tauopathy,60 under-
scoring the potential critical significance of microglia in the context
of FTD.
Our data demonstrated a positive association between CSF Gal-3
levels and two markers of neuronal dysfunction, 14-3-3, and t-tau.61–63
Indeed, neuroinflammatory response has been linked to synaptic
dysfunction.64 We also demonstrated a positive relationship between
CSF Gal-3 levels and t-tau in AD patients along with GAP-43 and

BORREGO–ÉCIJA ET AL.1523
neurogranin, markers of synaptic dysfunction.24 However, in this study
no association was found between Gal-3 and p-tau 181, contrary to
our previous study in AD patients where we observed a significant
association.24 This suggests that the mechanism of neuroinflammatory
response is similar but not identical in AD compared to FTD. The timing
of neuroinflammation in FTD, whether preceding neuronal dysfunction
or ensuing it, is presently unclear.
Regarding GRN mutation, brain Gal-3 levels were also elevated in
FTD subjects carrying GRN mutations but not in C9orf72 expansion
carriers. Other markers of inflammation, such as Glial Fibrillary Acid
Protein (GFAP), have been shown to be differentially elevated in GRN
carriers.65 The GRN gene encodes the progranulin protein, which is
involved in many biological processes, including inflammation, particu-
larly in deactivating glial cells.30 Mutations in GRN result in progranulin
haploinsufficiency, suggesting that deficiency of progranulin in GRN
mutation carriers may lead to pro-inflammatory glial activation and
increased levels of Gal-3.30 Indeed, recent research has highlighted
the crucial role of activated microglia in GRN knockout mice, which
drive disease progression by inducing neurodegeneration and TDP-43
protein aggregation during aging.66 Of interest, proteomic analysis of
GRN KO identified GPNMB and Gal-3 as two of the most enriched pro-
teins in the GRN KO brain proteome, particularly in aged animals29 and
substantial upregulation of Gal-3 was found in GRN KO iPSC-derived
microglia.40 Notwithstanding, the finding of increased brain Gal-3 lev-
els in the cortex of GRN cases was not reflected in CSF or serum in our
study. This may be related to different magnitudes or dynamics of Gal-
3 levels in these tissues, or due to Gal-3 upregulation occurring only in
the latest stages of the disease in GRN carriers. More research would
be needed to elucidate the role of Gal-3 in GRN mutation carriers.
Recent work from Woollacott and colleagues evaluated three glia-
derived biomarkers in CSF: TREM2, YKL-40, and chitotriosidase in
183 participants from the Genetic FTD Initiative (GENFI), includ-
ing C9orf72,GRN,andMAPT mutation carriers and controls. Only
chitotriosidase showed increased levels in symptomatic GRN muta-
tion carriers; the other group comparisons failed to show statistically
significant differences.65,67
The differences mentioned above between neuropathological and
genetic subgroups of FTD points to Gal-3 as a promising biomarker
to distinguish between molecular subtypes of FTD. Although our clin-
ical cohort did not include cases with confirmed neuropathology, our
results indicated that clinical phenotypes usually associated with FTD-
tau (i.e., nfvPPA) showed increased levels of CSF Gal-3 compared with
clinical phenotypes usually associated with FTD-TDP-43 (i.e., svPPA).
When determining the diagnostic significance of Gal-3 in distinguish-
ing between FTD patients and controls, our analysis of ROC curves
revealed that both serum and CSF Gal-3 levels displayed less accu-
racy compared to more established biomarkers such as NfL for this
comparison.68–71
We note several limitations in our work. First, despite the consider-
able sample size of our cohort of patients with FTD, the heterogeneity
of this disease makes smaller clinical or genetic subgroups leading
to a lack of statistical power needed to explore subtle differences
between subgroups. Second, even though we included a neuropatho-
logical cohort where we found differences in brain Gal-3 levels,
the participants included in the clinical cohort lack neuropatholog-
ical confirmation. Acknowledging the need for validation through
a replication cohort, we are aware of limitations in obtaining CSF
genetic samples from FTD individuals. In addition, we recognize
the constrained sensitivity of the applied Gal-3 ELISA, as more
advanced/validated platforms like single molecule array (Simoa,
Quanterix) or Mesoscale Discovery platform (Mesoscale Diagnos-
tics) lack specific Gal-3 assays. In addition, there was a significant
age difference between FTD patients and controls, which may have
been a confounding factor, although the analysis was adjusted for
age difference. Although we did not find any significant correlation
between Gal-3 and disease duration, it is important to mention that
most samples in our study were collected at the time of the clinical
diagnosis, with few samples collected in the later stages of the disease.
A longitudinal approach would be needed to determine the association
between Gal-3 level and the progression of the pathology. Finally, the
presence of other co-pathologies might also induce Gal-3 elevation.
However, the individuals in our cohort underwent measurements of
AD-related biomarkers, and their values indicated the absence of AD
pathology.
To sum up, our study robustly establishes heightened Gal-3 levels
in patients with FTD, underscoring its pivotal role in neuroinflamma-
tion and potentially driving the disease pathogenesis. This deepens our
comprehension of FTD’s mechanisms, highlighting microglial markers
as valuable biomarkers. Notably, FTD subtype variations, particularly
a unique Gal-3 increase in MAPT mutation carriers, signifying subtype-
specific neuroinflammation. Our findings align with preclinical models,
accentuating neuroinflammation’s acceleration of FTD progression.
This accentuates potential immunomodulatory therapies and suggests
evaluating microglial activation for refined clinical trial participant
selection.
ACKNOWLEDGMENTS
The authors thank patients, their relatives, and healthy controls for
participating in the research. This work was supported by Instituto
de Salud Carlos III, Spain (grant no. PI20/0448 to Dr R. Sanchez-
Valle, Instituto de Salud Carlos III, Spain, co-funded by the EU (FEDER)
“Una manera de hacer Europa” and PI19/00449 to Dr Lladó) and
Generalitat de Catalunya (SGR 2021-01126). Dr S. Borrego-Écija is a
recipient of the Joan Rodés Josep Baselga grant from FBBVA. Anto-
nio Boza-Serrano, PhD is recipient of the Vetenskapsrådet grant,
2019-0633, Kungliga Fysiografiska Sällskapet i Lund, 20191114ABS
and 20211129ABS, Greta och Johan Kocks stiftelser, 20201201ABS,
and Juan de la Cierva Incorporación—IJC2019-040731-I. Professor
Jose Luis Venero is recipient of Spanish Ministerio de Ciencia e
Innovación /FEDER/UE (PID2021-124096OB-I00). Professor Javier
Vitorica is recipient of Instituto de Salud Carlos III, Union PI18/01556,
PI21/00914. Professor Tomas Deierborg is recipient of: Swedish
Demensfonden, The Strategic Research Area MultiPark (Multidisci-
plinary Research in neurodegenerative diseases) at Lund University,
the Swedish Brain Foundation, Crafoord Foundation, Swedish Demen-
tia Association, G&J Kock Foundation, Olle Engkvist Foundation,

1524 BORREGO–ÉCIJA ET AL.
Gamla Tjänarinnor Foundation, the Swedish Medical Research Council,
the Swedish Parkinson Foundation, the Swedish Parkinson Research
Foundation, the A.E. Berger Foundation.
CONFLICT OF INTEREST STATEMENT
R.S.V. has served in advisory boards meetings for Wave Life Sciences,
Ionis, UCB, Prevail, Pfizer, and Novo Nordisk and has received personal
fees for participating in educational activities from Roche Diagnostics
and Neuroxpharma. The other authors declare no conflicts of interest;
they declare that the research was conducted without any commercial
or financial relationships that could be construed as a potential conflict
of interest. Conflicts of Interest Author disclosures are available in the
Supporting Information.
CONSENT STATEMENT
All the subjects provided informed consent and the study was
approved by the Hospital Clínic de Barcelona Ethics Committee (HCB
2019/0105).
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SUPPORTING INFORMATION
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ing Information section at the end of this article.
How to cite this article: Borrego–Écija S, Pérez-Millan A,
Antonell A, et al. Galectin-3 is upregulated in frontotemporal
dementia patients with subtype specificity. Alzheimer’s Dement.
2024;20:1515–1526. https://doi.org/10.1002/alz.13536