Experimental animals

Two cohorts of sex and age-matched WT non-transgenic littermates (C57Bl/6) and TDP-43A315T mice [46] (n = 60/genotype) were used in this study to conduct histology (n = 30/genotype), proteomic and molecular biology techniques (n = 30/genotype). In both cases, the ALS-like disease was divided into three stages (n = 5/stage): asymptomatic, onset (defined as the last day of individual peak body weight before a gradual loss occurs) and the end-stage of disease (defined as when weight is 20% below the initial weight on three consecutive days), which is typically reached 2–4 weeks after symptom onset. To avoid the ambiguity associated with reported sex-related differences in mean survival time of this mouse model of ALS [20, 46], only male mice were used. Animals were euthanized independently on each stage of disease. Animals expressing the human TDP-43 (hTDP-43) transgene were confirmed via PCR according to the distributor’s protocol.

To monitor disease onset and progression, all mice were weighed and assessed three times per week until the disease onset-stage, after which they were checked daily in the morning until the disease end-stage. The maintenance and use of mice and all experimental procedures were approved by the Animal Ethics Committee of the Hospital Nacional de Parapléjicos, Toledo (Spain) (Approval No 26/OH 2018) in accordance with the Spanish Guidelines for the Care and Use of Animals for Scientific Purposes. All analyses were conducted by personnel blinded to the animal genotype.

Perfusion and tissue collection

Animals were terminally anesthetized with sodium pentobarbitone (140 mg/kg, intraperitoneally) and transcardially perfused with 0.01 M PBS (pH 7.4). For immunohistochemistry, subcutaneous WAT (scWAT) and perigonadal WAT (pgWAT) were immediately dissected, rinsed in cold phosphate buffered saline (PBS), postfixed 70% ethanol, and stored at 4ªC until paraffin embedding using an Automatic Tissue Processor (ATP 1000, Histo-line, Italy), for further use. For molecular biology experiments, scWAT and pgWAT tissues of each animal were split into two fractions and processed independently, for real time qPCR and Western blot analysis, or proteomics methods. Samples were immediately frozen on dry ice and stored at − 80 °C for later analysis.

Immunohistochemistry (IHC)

Paraffin-embedded Sects. (4 μm thick) of scWAT and pgWAT were deparaffinized in xylene and rehydrated through descending grades of ethanol (100%, 95%, 90%, 80% and 70%) to water. Sections were stained with hematoxylin and eosin (H&E), dehydrated through ascending grades of ethanol (75%, 95%, and 100%), cleared in xylene and finally mounted in dibutyl phthalate xylene (DPX). For analysis, digital photomicrographs of the entire scWAT and pgWAT tissue Sects. (20x; Leica CTR 6000, Leica, Mannheim, Germany) were used to quantify the histochemical staining in 10 random fields for each sample different regions to assess the regional heterogeneity in tissue samples. The regions were outlined using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.net/ij/, 1997–2018). All slides were analysed using the same morphologic criteria for the quantification of crown-like structures (CLSs), defined by the clustering of macrophages (identified by each morphology) to surround a dying adipocyte, as a sign of adipose-tissue inflammation. This criterion was: presence of a ring of mononuclear cells surrounding an adipocyte vacuole. In addition, the number of blood vessels (BV) was also determined to assess vascularity as an index of angiogenesis.

Finally, by semiquantitative methods we determined inflammatory markers such as mononuclear infiltrate and fibrosis, and necrosis markers such as adipocyte normal membrane shape and tissue integrity. Semiquantitative scores were assigned by an observer based on predefined morphologic criteria: 1 (< 25%), 2 (25–50%), 3 (51–75%), and 4 (> 75%). Measure 10 fields at 10 × in simple scWAT and pgWAT tissues (n = 60). All analyses were conducted by personnel blinded to animal genotype.

Total protein preparation and mass spectrometry analysisSample preparation for library generation

Proteins were separated on precast gel, 4–20% MiniPROTEAN TGX (BioRad) and visualized by Coomassie staining. The entire gel lane was manually cut into 8 sections and subjected to in-gel tryptic digestion. The digestion was performed according to Schevchenko et al. [40] with minor modifications: gel slices were incubated with 10 mM dithiothreitol (DTT; Sigma Aldrich) in 50 mM ammonium bicarbonate (99% purity; Scharlau) for 60 min at 37ºC and after reduction, alkylation with 55 mM iodoacetamide (IAA; Sigma Aldrich) in 50 mM ammonium bicarbonate was carried out for 20 min at RT. Gel plugs were washed with 50 mM ammonium bicarbonate in 50% methanol (gradient, HPLC grade, Scharlau), rinsed in acetonitrile (ACN, gradient, HPLC grade, Scharlau) and dried in a Speedvac. Dry gel pieces were then embedded in sequencing grade modified porcine trypsin (Promega, Madison, WI, USA) at a final concentration of 12.5 ng/µL in 20 mM ammonium bicarbonate. After digestion at 37 ºC overnight, peptides were extracted with 60% acetonitrile in 0.5% formic acid (FA, 99.5% purity; Sigma Aldrich) and the samples were resuspended in 10µL [98% water with 2% formic acid and 2% ACN].

Sample preparation for SWATH analysis

Samples lysates were digested using Single-pot solid-phase-enhanced sample preparation (SP3) according to the protocol of Hughes et al. The lysates were reduced and alkylated using DTT and IAA, respectively. After reduction and alkylation, 6 µL of the prepared bead mix was added to the lysate and made up to 30 µL using H2O. Afterward, EtOH was added to a final concentration of 70% (v / v) and the samples were left stirring at 1000 rpm and room temperature for 20 min. Subsequently, the beads were immobilized by incubation on a magnetic rack for 2 min. The supernatant was recovered in a new vial, and the entire procedure was repeated. The pellet was rinsed with 80% (v / v) EtOH in water several times. The beads were resuspended in 300 µl of 100 mM NH4HCO3 supplemented with trypsin in an enzyme to protein ratio of 1:25 (w / w). After digestion overnight at 37 °C and 1000 rpm, the samples are centrifuged at 20,000 g, the supernatant is collected and acidified using 2% FA.

LCMSMS Analysis for library, DDA and SWATH

In order to build the spectral library, the peptides extract were analysed by a shotgun approach by nanoLC-MS/MS. Samples were pooled and 3 µg was separated into a Ekspert™ nanoLC425 (Eksigent, Dublin, CA, USA) using a C18 column (ChromXPC18, 3 µm, 120 Å 0.075 × 150 mm, Eksigent) at a flow rate of 300nL/min in combination with a precolumn (NanoLC Trap ChromXP C18, 3 µm 120 Å, Eksigent) at a flow rate of 5µL/min. The buffers being used were: A = 0.1%FA 2%ACN and B = 98% ACN in water with 0.1% FA. Peptide were desalted for 3 min with 0.1%FA/2% ACN on the precolumn, followed by a separation for 85 min using gradient from 5 to 30% solvent B, 30–95% for 0.1 min, and finally 95%B for 5 min. Column was then regenerated with 5%B for 10 additional minutes. Peptides eluted were directly injected into a hybrid quadrupole-TOF mass spectrometer TripleTOF® 6600 + (Sciex, Redwood City, CA, USA). Sample was ionized in a source type Optiflow < 1µL Nano applying 3.0 kV to the spray emitter at 200ºC. Analysis was carried out in a data-dependent positive ion mode (DDA). Survey MS1 scans were acquired 350–1400 m/z for 250 ms. The TripleTOF was operated in SWATH mode, in which a 50 ms TOF MS scan from 350 to 1400 m/z was performed, followed by 50 ms product ion scans from 100 to 1500 m/z on the 70 variable windows from 350 to 1400 Da (2.20 s/cycle). The individual SWATH injections were randomized.

Protein data analysis

Peptide and protein identifications were performed using ProteinPilot™ Software V 5.0 (Sciex) and the Paragon algorithm [41]. Each MS/MS spectrum was searched against the uniprot-proteome_MusMusculus_2021_04 database, with the fixed modification of carbamidomethyl—labelled cysteine parameter enabled. Other parameters such as the tryptic cleavage specificity, the precursor ion mass accuracy and the fragment ion mass accuracy, are TripleTOF® 6600plus built in functions of the ProteinPilot software. SWATH Acquisition MicroApp v.2.0 was used for building a peptide spectral library containing the peptide identified in the database search with confidence score above 95%. SWATH Acquisition MicroApp was used for extracting the ion chromatogram traces from the SWATH raw files and using the previously generated spectral library, and the following parameters: 20 peptides/protein; 6 fragment ions/peptide; extraction windows of 5 min and 25 ppm; peptide FDR of 1% and confidence score threshold of 95%. Normalisation of the protein abundance signal as measured by SWATH was carried out using MarkerView (v1.2.1, Sciex).

RNA Isolation and qPCR analysis

Total RNA was isolated from WAT using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Complementary DNA (cDNA) (0.5 µg of total RNA) synthesis and the relative quantification of TDP-43, UCP-1, C/EBPβ and PPARγ were performed as described previously [14]. The 18S rRNA was used as a control to normalize gene expression [13]. The reactions were run on an CFX96 Real-Time System instrument and software (CFX Manager 3.0) (BioRad) according to the manufacturer’s protocol. Primers were designed using NCBI/Primer-BLAST software (Table 1). Relative quantification for each gene was performed by the ∆∆Ct method [28].

Table 1 List of RT-qPCR primersProtein extraction and western-blot analysis

Proteins from WAT were extracted using RIPA buffer (Sigma Aldrich) containing a cocktail of protease inhibitors (Roche) as described previously [16]. Denatured protein samples (20 µg) from each group were electrophoresed into Bolt® Bis–Tris Plus gels (Invitrogen), transferred to PVDF membranes (BioRad and incubated with rabbit anti-TDP-43 (1:1000; Proteintech) overnight. Subsequently, anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Vector Laboratories) was used as described previously (https://doi.org/10.3390/ijms221910305). Mouse anti-actin (1:1000; Cell Signalling) was used as a loading control and band intensity was measured as the integrated intensity using ImageJ software (v1.4; NIH). All data were normalized to control values on each membrane.

Human plasma samples

All procedures performed in studies involving human samples (plasma) were in accordance with the.

Ethics Committee (783/23/98) of the University CEU-San Pablo, Madrid, Spain. Human plasma samples were provided by the Biobank HUB-ICO-IDIBELL, integrated in the ISCIII Biobanks and Biomodels Platform and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees.

Patients were eligible for inclusion if they had diagnosis of ALS based on Gold Coast criteria [39]. Controls were also provided by the Biobank HUB-ICO-IDIBELL. Samples were collected at the hospital using Lithium heparin collecting tubes at the time of diagnosis. Samples were centrifuged at 10,000 RPM for 10 min. The supernatant was collected and frozen at − 80 degrees Celsius.

ALS patients were classified by sex, BMI and survival. Patients were classified as nonobese (BMI, < 25) and obese (BMI, ≥ 25). BMI was calculated at the time of diagnosis. Patients were classified as slow progressors when survival was higher than 5 years, as normal progressors when survival was between 3 and 5 years, and as fast progressors when survival was less than 3 years. All the human samples included were of patients who were already deceased at the time of the study.

Measurement of plasma leptin by ELISA

ELISAs were performed as suggested by the manufacturer’s protocol. Leptin plasma levels were measured using a Human Leptin ELISA Kit PicoKine® (Boster Biological Technology) with samples diluted 1:20 for males and 1:10 for females. Each patient’s samples were processed in duplicate. The limit of detection was 62.5 pg/mL, and the within-assay and between-assay coefficient of variability (CVs) were 7.8% and 6.5%, respectively.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software v10.2.0. Normality of datasets was assessed by Kolmogorov–Smirnov Test. Outliers were removed with ROUT method with Q = 1%. For IHC Mann–Whitney test was used. For molecular biology analysis, two-way ANOVA was used followed by Dunett’s post hoc test to compare all groups with control WT asymptomatic mice, while Tukey’s post hoc test was used for multiple comparisons between all groups. To compare within the same group, t-test test was used. For ELISA, two-way ANOVA was used followed by Tukey’s post hoc test for multiple comparisons between all groups. Mann–Whitney test was used to compare within the same group. A Spearman correlation coefficient (rho) was employed to assess the correlation between quantitative variables, with significance set at a p-value of ≤ 0.05 (n = 78). This analysis was performed with SPSS Statistics. Values were reported as means ± standard error of the mean (SEM). For all comparisons, significant results were taken when p value < 0.05.