Could lipocalin 2 be involved in the effects of iron overload and a high-fat diet on neuronal death? An exploratory study

doi:10.21203/rs.3.rs-2753493/v1

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Could lipocalin 2 be involved in the effects of iron overload and a high-fat diet on neuronal death? An exploratory study

https://doi.org/10.21203/rs.3.rs-2753493/v1

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Lipocalin 2 (LCN2) controls iron levels, inflammation, cell death and is associated with neurodegenerative conditions. Moreover, obesity and insulin resistance modulate LCN2 expression. In this study we explored the effects of neonatal iron overload and a high-fat diet (HFD) after weaning on gene expression of LCN2, its receptor 24p3R, and the pro-apoptotic BCL-2-interacting mediator of cell death (BIM), besides evaluating the levels of LCN2 and of the anti-apoptotic protein B-cell lymphoma 2 (BCL2). Male Wistar rats received vehicle or carbonyl iron (30mg/kg) from the 12th to the 14th postnatal day. After weaning animals were treated with a HFD or a standard diet. At 9 months animal were euthanized and the hippocampus collected for RT-qPCR analysis of gene expression and Western Blot analysis of protein levels. The results indicate that iron overload during the neonatal period induced an increase in the gene expression for LCN2, its receptor 24p3R, and BIM, besides an increase of LCN2 protein levels. The exposure to a HFD throughout life, increased animals’ body weight and led to the decrease on BIM mRNA and BCl2 protein levels. Moreover, the combination of iron overload and HFD exacerbated the increase in LCN2 levels. In conclusion, the results of this study give support to the hypothesis that early life iron overload and a high fat diet are potential risk factors (each one alone and together) for neuronal death mediated by LCN2.

neutrophil gelatinase-associated lipocalin (NGAL)

apoptosis

Iron

neurodegeneration

brain-type organic cation transporter (BOCT)

transcriptional repression–induced atypical death (TRIAD)

Iron, a micronutrient essential for several biological processes, plays an important role in the synthesis of neurotransmitters, generation of ATP and myelination processes during the development of the Central Nervous System (CNS) (Zecca et al. 2004). However, in neurodegenerative disorders, including Alzheimer's Disease (AD), high levels of iron have been reported in specific brain areas, such as the hippocampal region (Ward et al. 2014). The role played by iron accumulation in the pathogenesis of neurodegenerative disorders is not completely elucidated. However, it is known that iron catalyzes the formation of Reactive Oxygen Species (ROS), due to its ability of donate electrons in the Fenton reaction, leading to oxidative damage (Jakaria et al. 2021). More recently, consistent evidence indicates that ferroptosis, a type of iron-dependent cell death, is implicated in neurodegenerative disorders, such as AD (Ashraf et al. 2020). Previous studies by our research group, using an animal model, showed that iron administration during the neonatal period induces the accumulation of this metal in specific brain regions, and induces memory impairments, in addition to oxidative damage (De Lima et al. 2005), decreased BDNF levels (Alcalde et al. 2018), increased expression of apoptotic markers (Miwa et al. 2011; Da Silva et al. 2014; da Silva et al. 2018b), and accumulation of ubiquitinated proteins in the hippocampus of adult rats (Figueiredo et al. 2016). In agreement, elevated iron intake during development has been considered a risk factor for neurodegenerative diseases (Hare et al. 2015). Multiple risk factors have been reported for AD, including hypertension, diabetes, and obesity (Ashraf et al. 2020). Studies call attention to important links between AD and diabetes-related insulin signaling abnormalities, considering AD as a neuroendocrine disease known as “type 3 diabetes” or “brain diabetes (H. Ferreira-Vieira et al. 2016). Elements related to the metabolic syndrome cause excessive and repeated inflammatory responses, which can result in the recruitment of peripheral innate immune cells, which significantly contribute to local inflammatory processes (Kierdorf and Prinz 2013; Peferoen et al. 2014).

Lipocalin 2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a member of the lipocalin family of soluble proteins, that participate in the modulation of the immune system and in inflammatory responses (Jha et al. 2015). In the CNS, LCN2 can bind and transport iron. It has been proposed that, under certain pathological conditions, LCN2 is expressed by components of the CNS, including glia and neurons, and that chronic immune response triggers the secretion of LCN2, which in turn, regulates neuroinflammation. In addition, studies indicate that LCN2 plays an important role in CNS abnormalities, including neuroinflammatory conditions, neurodegeneration, gliomas, autoimmune disorders, brain injury, encephalitis, intracerebral hemorrhage, schizophrenia, and spinal cord injury (Jha et al. 2015).

Yang and colleagues (2002) describe LCN2 as an alternative pathway for iron release and absorption and suggest that LCN2 could deliver iron through a transferrin receptor-independent mechanism (Yang et al. 2002). According to Devireddy et al., (2005) and Richardson et al. (2005) the modulation of cellular iron levels by LCN2 occurs through the interaction between the LCN2 receptor, known as 24P3R, with mediators of pro-apoptotic cell death, such as BIM (BIM) (Devireddy et al. 2005; Richardson 2005). The accumulation of iron at toxic levels in neurons causes the exacerbation of neuroinflammatory conditions, favoring cell death by apoptosis (Jin et al. 2020).

Studies have shown that cognitive impairment is associated with neuroinflammation in obesity, which promotes a state of low-grade systemic inflammation that contributes to the development of various comorbidities, such as type 2 diabetes, dyslipidemia, cardiovascular disease, and neurodegenerative disorders (Gregor and Hotamisligil 2011). High-fat diets (HFD) are positively correlated with the development of obesity and significantly impact cognitive function (Solas et al. 2017). Experimental models of obesity and diabetes demonstrate that circulating levels of LCN2 are associated with hyperglycemia, insulin resistance (IR), metabolic syndrome, and cardiovascular diseases (Song and Kim 2018). HFD may be associated with brain insulin resistance, suggesting that LCN2 could mediate neuroinflammation and oxidative stress, leading to neurodegeneration (Kothari et al. 2017; Shin et al. 2022).

Although there is preclinical and clinical evidence indicating the involvement of excess iron and LCN2 in neurodegenerative processes, little is known about the interaction between these two factors in vivo.

Considering that LCN2 controls iron levels, inflammation and cell death in the CNS, and that risk factors such as obesity and insulin resistance may influence LCN2 expression, in the present study, we evaluated the effect of neonatal iron overload, as an additional risk factor, followed by a HFD after weaning, on LCN2 levels, and the expression of its receptor, 24p3R, and pro- (BIM) and anti-apoptotic (BCL2) markers.

2.1 Animals

Pregnant Wistar rats (CrlCembe:WI) were obtained from the Centro de Modelos Biológicos Experimentais (CeMBE), Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brasil. After birth, each litter was adjusted within 48 h to contain eight rat pups including offspring of both genders. Each pup was kept together with its mother in a plastic cage with sawdust bedding in a room temperature of 21 ± 1°C and a 12/12 h light/dark cycle. At the age of 3 weeks, pups were weaned, and the males were selected and maintained in groups of three to five in individually ventilated cages with sawdust bedding up to 9 months of age. As this study was a subproject of a greater investigation (n = 62 adult animals), aimed to understand the interactions between iron overload and high-fat diet on insulin resistance and neurodegenerative mechanisms, only a subset of the adult animals (n = 17) was used for the biochemical and molecular analysis presented in this article.

All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition 2011) and the Brazilian Guidelines for the Care and Use of Animals in Research and Teaching (DBCA, published by CONCEA, MCTI, Brazil). This study was approved by the Institutional Ethics Committee of the Pontifical Catholic University 9331 and all efforts were made to minimize the number of animals and their suffering.

2.2 Experimental Design and Treatments

The experimental design of this study is depicted in Fig. 1. Twelve-day-old rat pups received orally a single daily dose (10 mL/kg solution volume) of vehicle (5% sorbitol in water; control group) or 30 mg/kg of body weight of Fe2+ (iron carbonyl, Sigma-Aldrich, São Paulo, Brazil) via a metallic gastric tube, over 3 days (postnatal days 12th–14th). The period of treatment was chosen based on studies that show that iron uptake by the brain increases rapidly in the first 15 days of life, declining thereafter.

After weaning, animals of the control and iron treatment groups were divided into two subgroups, which received a standard commercial chow (NUVILAB CR-1, Quintia, Paraná, Brazil) or a high-fat diet, which was handcrafted and composed of 40% standard commercial chow, 25% whole powdered milk, 20% soy oil and 15% corn syrup (Yoshimura et al. 2016). Thus, four groups composed the experimental design of this study: vehicle + standard diet group; vehicle + high-fat diet; iron + standard diet group; iron + high-fat diet. At nine months of age animals were euthanized by decapitation and the hippocampi were rapidly removed for further biochemical and molecular analysis.

Animals’ body weight was assessed at weaning and in the day the animals were euthanized.

3.1 Preparation of samples for analysis

After euthanasia the hippocampi were quickly dissected and placed in a cooled RNA-later solution (Sigma-Aldrich, São Paulo, Brazil) for RT-qPCR assays, and in a cooled protease inhibitor solution (Complete Mini, Roche Applied Science, Mannheim, Germany) for the Western blot assays. Samples were stored at − 80°C for subsequent molecular analysis.

3.2 Western Blot Analysis

Hippocampi obtained from rats euthanized by decapitation were homogenized in ice-cold lysis buffer containing 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 100 mM NaCl, 1 mM Na3VO4, protease inhibitor cocktail, Triton X-100 0.5%, and SDS 0.1%. After 30 min in ice, samples were centrifuged at 13,500 rpm for 10 min (Da Silva et al. 2014; Figueiredo et al. 2016). The supernatant was collected, and the protein content was determined using Bradford assay (Bradford, 1976). Aliquots were stored at – 20°C. Thirty µg of protein was separated on a 12% SDS polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. Membranes were blocked with 5% albumin in TBS containing 0.05% Tween 20 and were incubated overnight with one of the following antibodies: anti-LCN2 (1:1,000; ab63929, Abcam), anti-Bcl2 (1: 500; ab196495, Abcam), anti-GAPDH (1: 2000; ab37168, Abcam), or anti-β actin (1: 2000; ab8227, Abcam) which were dissolved in TBS-T with 5% bovine serum albumin. Membranes were then washed 3 times with TBS-T and incubated for 120 min at room temperature in TBS-T with 1% skimmed milk containing anti- rabbit IgG H&L (HPR) (Abcam, Cambridge, UK) secondary antibody, detected using ECL Western Blotting Substrate Kit (Abcam, Cambridge, UK). Pre-stained molecular weight protein marker (SuperSignal Molecular Weight Protein Ladder, THER-84785, Thermo Scientific, Rockford, USA) was used to determine the detected bands’ molecular weight. The image acquisition for densitometric analysis was performed using L-Pix Chemi Express (Loccus Biotechnology, São Paulo, Brazil), followed by quantification using ImageJ software (http://rsb.info.nih.gov/ij/), by an experimenter blind to samples experimental condition (Da Silva et al. 2014; Figueiredo et al. 2016; da Silva et al. 2018b). Protein levels were quantified by normalizing the optical density of its band to a loading control band (GAPDH or β actin).

3.3 Quantitative reverse transcription PCR (RT-qPCR) analysis

RT-qPCR was used to analyze the mRNA levels of LCN2, 24p3R, and BIM, as previously described (Da Silva et al. 2014). PCR primer sequences are available in Table 1. Molecular analysis was performed following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines for RT-qPCR experiments (Bustin et al. 2009) Minimum information for publication. Extraction of total RNA with the TRIzol reagent (Invitrogen, Carlsbad, USA) from hippocampus of rats was performed according to the manufacturer's instructions. The purity (Abs 260/280nm ~ 2.0) and concentration of the RNA were determined by NanoDrop Lite (Thermo Fisher Scientific) and after that, samples were treated with Deoxyribonuclease I – Amplification Grade (Sigma-Aldrich, São Paulo, Brazil) to eliminate genomic DNA contamination. All samples were tested by 1% agarose gel electrophoresis with Gel Red nucleic acid dye (Biotium, Hayward, USA). The cDNA species were synthesized ImProm-II™ Reverse Transcription System (Promega Corporation, Promega Biotecnologia do Brasil, Ltda, São Paulo, Brazil) from 1 µg of the total RNA, following the manufacturer’s instruction.

Quantitative PCR was performed using SYBR® Green I (Thermo Fisher Scientific, Waltham, USA) to detect double-strand cDNA synthesis on the 7500 Real-time PCR System (Applied Biosystems). The PCR cycling conditions were the same as described previously (da Silva et al. 2018a). All real time assays were carried out in quadruplicate and, in all cases, a reverse transcriptase negative control was included by substituting the templates for DNase/RNase-free distilled water in each PCR reaction. HPRT1 (Hypoxanthine Phosphoribosyl Transferase 1) was used as reference gene for normalization. The efficiency per sample was calculated using LinRegPCR 2018.0 Software (http://LinRegPCR.nl). Relative mRNA expression levels were determined using the 2-ΔΔCq method (Bustin et al. 2009; Da Silva et al. 2014).

Table 1

The list of primers sequences for RT-qPCR experiments included in the study
PRIMER	FORWARD PRIMER	REVERSE PRIMER	REFERENCE
LCN2	5’-TCACCCTGTACGGAAGAACC-3’	5’-TCGGTGGGAACAGAGAAAAC-3’	Mesquita et al.,2014
24p3R	5’-AATGACTCTCACGGGGATTG-3’	5’-AGTGGTGGGGATGACTTCAG-3’	Devireddy et al.,2005
BIM	5’-GTTCCTCCTGAGACTGCCTT-3’	5’-TGCTACCAGATCCCCACTTT-3’	Mesquita et al.,2014
HPRT1	5′-GCAGACTTTGCTTTCCTTGG-3′	5′- CGAGAGGTCCTTTTCACCAG-3′	Bonefeld et al., 2008

All variables were analyzed by two-way ANOVA, with treatment (vehicle or iron) and diet (standard or high-fat) as fixed factors. Data are expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

To examine the effects of iron overload during the neonatal period, the exposure to a high-fat diet throughout life, or the combination of these two risk factors, iron- or vehicle-treated animals were further divided into groups fed with a normal standard commercial rat chow or a high fat diet, from weaning to 9-months of age. Table 2 shows the body weights of the animal groups from which we randomly selected the dissected hippocampi of 17 animals for the biochemical and molecular analysis. No significant differences in body weight were found between the iron and vehicle treatment groups at the weaning day, as indicated by the independent samples t-test [t (60) = -1.44; p = 0.154]. As expected, animals fed with the high fat diet showed a significant increase in body weight when adults. Two-way ANOVA comparison of the body weights immediately before euthanasia, indicated a significant main effect of diet (F(1,58) = 12.08, p = 0.001, Table 2), while no significant main effect of iron treatment (F(1,58) = 0.068, p = 0.796) or interaction were observed (F(1,54) = 2.02, p = 0.161). These results suggest that iron treatment does not affect body weight, whilst the high fat diet induces weight gain.

Table 2

Body weights at weaning and before euthanasia of animals from different experimental groups
NEONATAL TREATMENT GROUP	BODY WEIGHT AT WEANING DAY (MEAN ± S.D.)	N	DIET GROUP	BODY WEIGHT BEFORE EUTHANASIA (MEAN ± S.D.)	N
Vehicle	103.21 ± 12.26	33	Standard diet	689.87 ± 73.72	16
Vehicle	103.21 ± 12.26	33	High fat diet	731.52 ± 70.30 **	17
Iron	108.66 ± 17.29	29	Standard diet	655.78 ± 68.51	14
Iron	108.66 ± 17.29	29	High fat diet	755.07 ± 102.07**	15

Comparisons of body weights at weaning day revealed no significant differences between the groups (iron x vehicle, independent samples’ t-test). Animals’ weights before euthanasia were affected by diet. ** indicates a significant main effect of diet p < 0.01 (two-way ANOVA).

We aimed to investigate whether iron overload, the exposure to a high-fat diet, or the combination of these two factors, would alter Lcn2 in the hippocampus of rats. We first examined protein content using western blot, and the results are presented in Fig. 2A. Two-way ANOVA indicated a significant main effect of iron treatment [F(1, 15) = 7.81, p = 0.016] and a significant interaction of iron and high-fat diet [F(1, 15) = 4.89, p = 0.047]. We also analyzed Lcn2 mRNA expression and found similar results (Fig. 2B). Two-way ANOVA revealed a significant main effect of iron overload [F(1, 16) = 5.08, p = 0.042], although the interaction between iron and diet felt short of significance [F(1, 16) = 3.91, p = 0.070]. Taken together these results suggest that iron overload in the neonatal period increases Lipocalin 2 gene expression and protein levels, while the association of iron with a high fat diet strengthen this effect.

We next decided to evaluate the effects of iron, high-fat diet or the combination of iron with the diet on Lipocalin 2 receptor, 24p3R, mRNA expression. Interestingly, we also found a significant main effect of iron overload (Fig. 3), increasing 24p3R expression [F(1, 16) = 17.26, p = 0.001]. No main effect of diet [F(1, 16) = 0.24, p = 0.636] nor significant interactions were found [F(1, 16) = 1.56, p = 0.234].

Lipocalin 2 is involved in a wide range of cellular physiological and pathophysiological processes, including apoptosis. Considering that Lipocalin and 24p3R were affected by iron overload and a high-fat diet, we decided to verify whether these conditions would be accompanied by alterations in the expression of proteins involved in the apoptotic cascade. We first evaluated the mRNA expression of the pro-apoptotic protein BIM (Fig. 4). Two-way ANOVA revealed a significant main effect of iron neonatal treatment [F(1, 16) = 9.76, p = 0.008] enhancing the expression of the pro-apototic protein BIM. Strikingly, a significant main effect of the high-fat diet decreasing BIM expression [F(1, 16) = 21.21, p < 0.0001] was found. No significant interaction was observed [F(1, 16) = 2.16, p = 0.166].

We next evaluated protein levels of the anti-apoptotic BCl2. Two way-ANOVA showed a significant main effect of the high-fat diet, which decreased BCl2 levels [F(1, 19) = 30.09, p < 0.0001], Fig. 5. No significant main effect of iron neonatal treatment [F(1, 19) = 0.004, p = 0.953] nor a significant interaction [F(1, 19) = 0.12, p = 0.739] was observed.

In this study we explored the effects of iron overload during the neonatal period, the exposure to a high-fat diet (HFD) throughout life, or the combination of these two risk factors, on the hippocampal gene expression and levels of LCN2, 24p3R and anti and pro-apoptotic proteins of the BCL2 family, namely BIM and BCL2, respectively. The results indicate that iron overload during the neonatal period induced an increase in the gene expression for LCN2 and its receptor 24p3R, and an increase of LCN2 protein and BIM mRNA levels, suggesting a role for LCN2 on the intrinsic apoptotic pathway. The exposure to a high-fat diet throughout life increased animals’ weight and led to the decrease on BIM mRNA and BCl2 protein levels, which could be associated to the transcriptional repression–induced atypical death (TRIAD) and apoptosis, respectively. Moreover, the combination of iron overload and HFD exacerbated the increase in LCN2 levels.

Studies on the role of LCN2 on iron homeostasis indicate that this protein can have dual effects, being capable to increase or decrease cellular iron content (Bhusal et al. 2019). Although the roles of LCN2 in different tissues under physiological and pathological conditions are under debate, it was already shown that the level of this protein increases in neurodegenerative diseases known to be associated with iron accumulation in specific brain structures, including the hippocampus (for a review see Denkens et al., 2021) (Dekens et al. 2021). In this study, animals were submitted to iron overload in a period known to have increased iron uptake by the brain, due to characteristics of the blood-brain barrier and the need of iron for neurodevelopment (Dwork et al. 1990; Rao et al. 2003). In accordance with former studies, it is expected that this neonatal iron overload would induce iron accumulation and disrupt iron homeostasis in the brain (Fredriksson et al. 1999; Hoshino et al. 2006; Dornelles et al. 2010). Therefore, the increase of LCN2 mRNA, LCN2 protein and 24p3R mRNA levels in the hippocampus of adult animals, which were submitted to iron overload during the neonatal period, is expected to be occurring in a condition of iron excess in the brain. Such condition would favor the increase of holo-LCN2 (LCN2 plus iron-siderophore complex), which interacts with the 24p3R receptor to be internalized in neurons (Chia et al. 2015).Giving support for a role of LCN2 on iron accumulation in the hippocampus, Denkens and colleagues showed that the iron content in this structure was significantly decreased in a transgenic amyloid precursor protein (APP) overexpressing AD mouse model (J20) with knock-out of the LCN2 gene (J20xLCN2KO), in comparison to J20 mice (Dekens et al. 2018).

Whether a cell survives or dies by apoptosis depends on the balance between anti-apoptotic and pro-apoptotic signals (O’Brien and Kirby 2008). Two important proteins of the intrinsic apoptotic pathway are BIM and BCL2. In this study, the increased expression and protein levels of LCN2 seen in animals treated with iron were accompanied by an increase in the expression of the pro-apoptotic protein BIM. Since the levels of the anti-apoptotic protein BCL2 were not affected by the iron treatment, our results suggest an alteration in the balance of anti and pro-apoptotic signaling, favoring apoptosis. A putative mechanism for the alteration in this balance is the increased uptake and intracellular release of iron by the holo-LCN2-24p3R complex, as discussed earlier. The excess of intracellular iron could generate reactive oxygen species and upregulate BIM expression (Chia et al. 2015). Former studies with this animal model of neonatal iron overload have already shown an increase in oxidative stress and apoptosis (De Lima et al. 2005; Miwa et al. 2011; da Silva et al. 2018b). Moreover, Chia and colleagues (2015) showed that neurons treated with holo-LCN2 show an increase in BIM mRNA levels and decreased survival.

Animals submitted to the HFD from weaning to nine months of age showed an increased weight and decreased BIM mRNA and BCL2 levels. The high-fat and hypercaloric diet used in this study was already shown to induce weight gain and hyperglycemia in rodents (Yoshimura et al. 2016). Obesity and hyperglycemia are closely related morbidities and increase the risk for type 2 diabetes. They lead to an inflammatory phenotype in the brain characterized by gliosis, immune cell infiltration, and a surge in inflammatory cytokine levels(Bhusal et al. 2019). In these conditions LCN2 may show an upregulation in the hippocampus and promote neuroinflammation and cognitive dysfunctions in animal models (Jeon et al. 2016; Elizabeth et al. 2017). However, whether this change directly contributes to inflammatory responses or promotes neuroprotection remains an open question (for a review see Bhusal et al., 2019).

Although no significant alterations of LCN2 mRNA and protein levels were seen in our study after eight months of the HFD, our results indicate that this diet exacerbated the increase of LCN2 levels seen in animals submitted to iron overload during the neonatal period. Different studies have already indicated an association between iron overload and peripheral insulin resistance (Bozzini et al. 2005) and between metabolic syndrome/diabetes and a peripheral increase of LCN2 and pro-inflammatory cytokines (Auguet et al. 2011; Guo et al. 2013). Thus, the possibility that the additional effect of HFD on LCN2 levels in the hippocampus of iron treated animals is the result of an influx of LCN2 to the brain, through a more permeable blood-brain barrier due to inflammation, cannot be ruled out.

The HFD also caused a decrease in BCL2 levels, suggesting a pro-apoptotic effect, since the reduction of BCL2 means a decrease in its buffering capacity of pro-apoptotic proteins, favoring apoptotic priming (Singh et al. 2019) The gene expression of BIM also decreased in animals treated with the HFD. This finding is in line with the decrease of BIM expression seen in adipocytes of obese people and mice submitted to a HFD protocol. According to Wang and Cull (2022), this decrease in BIM expression could be caused by the activation of the transcriptional cofactors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ‐ binding motif) and the increase in the translocation of the YAP/TAZ complex to the nucleus, where it would downregulate the expression of the BIM gene and protect the adipocytes against apoptosis (Wang and Cull 2022). Although there are no studies about the association of HFD and increased activity on the YAP/TAZ complex in the nervous system, Hoshino and colleagues (2006) found a neuron specific isoform of YAP, the YAPdeltaC, that protects against apoptosis and shows increased levels in animal models of Huntington Disease, suggesting a pro-survival effect for YAPdeltaC (Hoshino et al. 2006). This hypothesis is reinforced by the finding that the levels of YAPdeltaC decrease as Amyotrophic Lateral Sclerosis progresses, whereas the normal length YAP (which has a pro-apoptotic effect) doesn’t change during the evolution of the disease (Greggio et al. 2009). Although apoptosis is one of the most studied forms of neuronal death, the slow and progressive profile of neuronal death observed in neurodegenerative diseases suggests that apoptosis is not the only mechanism to determine neuronal fate in these conditions (Liberski et al. 2004). YAPdeltaC is associated with transcriptional repression–induced atypical death (TRIAD), which results in transcriptional dysfunctions and extremely slow neuronal death progression (Hoshino et al. 2006). Future studies should evaluate the possibility that TRIAD and YAPdeltaC are involved in the decrease in BIM expression observed in the hippocampus of animal treated with HFD.

The main limitation of this investigation is intrinsically associated with its exploratory nature, i.e., the study was designed to investigate a possible association between iron overload and diet on a LCN2 mediated apoptosis, rather than analyze in detail the signaling pathways of neuronal death. As such, only a few variables were investigated. Moreover, the interpretation of our results would be more accommodating in the presence of evidence of the functional impact of iron overload and HFD treatments, such as neuronal and behavioral plasticity evaluation. Even so, the objective of the study was accomplished, and the results warrant further investigations.

In conclusion, the results of this study give support to the hypothesis that early life iron overload and a high fat diet are potential risk factors (each one alone and together) for neuronal death mediated by LCN2. This is especially important if we consider that preventive iron supplementation with iron-fortified formulae is common in childhood, showing adverse effects on different cognitive domains (Lozoff et al. 2012) and that high fat and hypercaloric diets are common and associated to diabetes, especially type 2, which also has important functional impacts on mental health (Kirvalidze et al. 2022). Future studies should evaluate in more detail the interaction of neonatal iron overload and a high fat diet on neuronal death and inflammation pathways potentially associated to LCN2, as well as the functional outcomes resulting from the association of these two treatments.

Competing Interests

The authors declare that they have no financial or non-financial competing interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted (Institutional Ethics Committee of the Pontifical Catholic University, permit number SIPESQ #9331).

Acknowledgements

This research was supported by the National Council for Scientific and Technological Development (CNPq; grant numbers 308290/2015-1 and 421643/2016-1 to N.S., and grant number 307442/2018-7 to E.B.); the National Institute for Translational Medicine (INCT-TM – grant number 465458/2014-9); the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance code 001, and by the Pontifical Catholic University of Rio Grande do Sul. N.S. and E.B. are CNPq research fellows. M.P.P. had a CAPES fellowship, F.V.N. had a CNPq fellowship, L.K. had a BPA-PUCRS fellowship, and C.S.S had a Research Foundation of the State of Rio Grande do Sul (FAPERGS) fellowship. The funding source(s) were not involved in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Author contributions

Conceptualization and methodology: M.P.P., N.S., and E.B.; Data acquisition: M.P.P., B.S.F, F.V.N, L.K., and C.S.S.; Data analysis: M.P.P., B.S.F, N.S., and E.B; Writing - original draft preparation: M.P.P., N.S., and E.B.; Writing - review and editing final version: E.B.; Resource acquisition: N.S. and E.B. Supervision: E.B.; all authors participated in the results’ discussion, and read and approved the final manuscript.

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Could lipocalin 2 be involved in the effects of iron overload and a high-fat diet on neuronal death? An exploratory study

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Abstract

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1 Introduction

2 Materials And Methods

2.1 Animals

2.2 Experimental Design and Treatments

3. Biochemical And Molecular Analysis

3.1 Preparation of samples for analysis

3.2 Western Blot Analysis

3.3 Quantitative reverse transcription PCR (RT-qPCR) analysis

4. Statistical Analysis

5. Results

6. Discussion

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