Iron is an important micronutrient that participates in several vital cellular functions such as oxygen transport and cellular respiration. Moreover, iron is a cofactor for numerous enzymes. Despite its importance, free iron is known to cause oxidative damages in cellular environment due to its ability to generate free radicals through Fenton reaction (Zecca et al., 2004; Belaidi and Bush, 2015). Indeed, abnormal accumulation of iron in brain has been pointed out as one of the factors that accelerate the aggregation of proteins associated with neurodegenerative diseases, such as cellular prion protein (PrP^C) and α-synuclein (Golts et al., 2002; Das et al., 2010). Moreover, iron can bind to protein aggregates and enhance their toxicity by attributing redox activity to these aggregates (Liu et al., 2011).

On the other hand, iron deficiency can turn out the aerobic respiration inefficient leading to reduced energy production and accumulation of reactive oxygen species, which can culminate into mitochondrial dysfunction (Masini et al., 1994; Walter et al., 2002). Iron deficiency can also cause deregulation of monoaminergic system. Inhibition of iron uptake into dopaminergic neurons not only caused mitochondrial damage, but also reduced dopamine levels and evoked abnormal activity of dopamine receptors (Matak et al., 2016). Several studies have also reported that nutritional iron deficiency alters dopaminergic neurotransmission, increasing the concentration of extracellular dopamine and reducing the activity of dopamine transporter (DAT) and D2 receptor in striatum (Youdim et al., 1989; Beard et al., 1994; Bianco et al., 2008; Unger et al., 2014). Reduced DAT expression has been also reported in the ventral midbrain (Bianco et al., 2008). These alterations appear to contribute to the memory impairment, attention deficiency and learning problems frequently observed in iron-deficient population (Youdim et al., 1989; Asanuma et al., 2003; Radlowski and Johnson, 2013; Gupta, 2016; Scott and Murray-Kolb, 2016). However, how nutritional iron deficiency affects the brain iron metabolism, particularly in dopaminergic regions, is poorly understood. Brain appears to have a greater tendency to retain iron and resist to nutritional iron deficiency due to the slow exchange of iron out of brain (Youdim et al., 1989; Bradbury, 1997). However, a few studies have demonstrated that dietary iron restriction reduces the iron levels of ventral midbrain and striatum in young rats (Bianco et al., 2008; Unger et al., 2014), while others claimed that iron levels of striatum were not altered after similar treatment (Erikson et al., 1997). Thus, the effects of dietary iron on the iron metabolism in dopaminergic neurons need to be better investigated.

Dopamine is a neurotransmitter that participates in the regulation of motor coordination, responses to rewarding experiences, attention and mood (Russo and Nestler, 2013; Bissonette and Roesch, 2016). At the same time, dopamine can be neurotoxic, being readily oxidized by non-enzymatic reactions, which generate reactive molecules, such as dopamine quinone (Lotharius and Brundin, 2002; Asanuma et al., 2003). Oxidative dopamine metabolites are known to promote the aggregation of α-synuclein into non-ordered toxic oligomers, and these molecular alterations are closely associated with pathogenesis of Parkinson's disease (Lotharius and Brundin, 2002; Yamakawa et al., 2010). Dopamine can also induce the aggregation of PrP^C, another aggregation-prone protein associated with transmissible spongiform encephalopathies, a fatal neurodegenerative disease (da Luz et al., 2015).

Altogether, these previous findings suggest that the aggregation of α-synuclein and PrP^C can be promoted either by iron or by dopamine, and both excess and deficiency of iron can be a risk factor for conformational diseases through distinct molecular mechanisms. It is possible that the distinct subcellular localization of the proteins and the site of iron and/or dopamine accumulation contribute to the specificity of pathogenic pathways. Thus, in this study, we evaluated how dietary iron restriction influences brain ferritin (a main iron storage protein) and dopamine metabolism in striatum, hippocampus and prefrontal cortex, three regions that receive afferents from midbrain dopaminergic neurons (Yetnikoff et al., 2014) and investigated how altered iron and dopamine metabolism can affect the expression level and solubility of PrP^C, a protein that is anchored to the outer leaflet of the plasma membrane. As mentioned above, dietary iron restriction is known to increase the concentration of extracellular dopamine. Since the accumulation of α-synuclein occur due to the intracellular dopamine, in order to check the specificity of the iron restriction effects on proteins of distinct subcellular comportments, we also analyzed the level of α-synuclein, which is a cytosolic protein.

Conformational changes in proteins and deposition of their aggregates are common hallmarks of several neurodegenerative diseases. Thus, identification of conditions that lead to protein aggregation is important not only for elucidation of mechanism of pathogenesis, but also to establish diverse strategies of prevention and treatment. Many studies have demonstrated a close relationship between iron and dopamine metabolism and neurodegenerative diseases (Belaidi and Bush, 2015; Hare and Double, 2016). In this study, we aimed to disturb dopamine metabolism by restricting dietary iron and investigate how altered iron and dopamine metabolism affect proteostasis of PrP^C and α-synuclein.

Previous studies have demonstrated that 30 or less days of dietary iron restriction were sufficient to mimic iron deficiency in animal models with reduced iron and ferritin levels in liver and serum (Youdim et al., 1989; Kamei et al., 2013). Indeed, we observed reduced iron and ferritin levels in liver of IR group as expected. However, we did not observe alterations in serum iron levels although serum ferritin level was reduced. This lack of correlation between total iron pool and ferritin levels in serum can be explained by abundant presence of other iron binding proteins such as transferrin, albumin and free hemoglobin. Indeed, red blood cells are considered one of largest iron pools. Considering that half-life time of red blood cells or hemoglobin of mice is more than 30 days and most of iron derived from red blood cells are recycled and not excreted (Vanputten, 1958; De Domenico et al., 2008; Muñoz et al., 2009), it is reasonable to expect that 30 days of nutritional iron restriction do not significantly deplete the iron pool in blood. In addition, lack of a significant increase of transferrin receptor in all tissues evaluated in this study also indicates that our experimental condition was not sufficient to fully exhaust iron stores (Skikne et al., 1990; Cook, 2005). In younger animals, shorter period of iron restriction can robustly increase the expression of transferrin receptor (Moos et al., 1998), suggesting that age is an important factor that influence the responses to dietary iron restriction.

In addition to partial depletion of peripheral iron pools, we also observed reduced ferritin levels in hippocampus, indicating that brain iron metabolism was also affected by our experimental protocol. Concomitant with ferritin reduction, higher degree of lipid peroxidation was observed in the hippocampus of iron restricted animals. Iron deficiency is known to reduce cytochrome C activity and energy metabolism, leading to increased production of reactive oxygen species (de Deungria et al., 2000; Carlson et al., 2009; Srinivasan and Avadhani, 2012). Moreover, iron deficiency can reduce mTOR activity in hippocampus via REDD1, a signaling molecule that can be activated by reactive oxygen species (Ohyashiki et al., 2009; Waibel et al., 2009). Thus, our observations appear to corroborate these previous findings that show increased oxidative stress in hippocampus under iron deficient condition.

Intriguingly, ferritin level was increased in the striatum of iron-deprived animals. This result was somewhat similar to the results reported in previous study, which showed that the treatment of younger rats with iron deficient diet reduced the iron level in hippocampus, but not in striatum (Erikson et al., 1997). Iron distribution in the normal brain is known to be heterogeneous (Hill and Switzer, 1984; Rouault, 2013), and striatum is one of the regions that have higher iron levels in the adult brain and is one of the sites where iron accumulates during normal aging (Martin et al., 1998). Altogether, these findings suggest that each brain region has distinct iron requirements, and under iron deficient condition, each region might develop distinct capacity of iron storage by altering the ferritin expression level. Nonetheless, abnormal ferritin accumulation in the striatum has been referred to as an early event in neurological motor dysfunction such as Parkinsonism and Huntington disease (Vidal et al., 2004; Oakley et al., 2007; Simmons et al., 2007). Although underlying mechanisms needs to be further investigated, our data show that chronic nutritional iron deficiency can trigger molecular alterations observed in early stage of neurological disorders that involve degeneration of dopaminergic neurons.

Similar to ferritin levels that varied in a region-specific manner, dopamine metabolism of each region also generated distinct outcomes in animals subjected to dietary iron restriction. In hippocampus, which receives relatively small portion of dopaminergic innervation from VTA, iron restriction did not cause any significant alterations. In prefrontal cortex, which receives innervation from same region, dopamine or DOPAC levels were also not altered in IR group. However, HVA levels were increased probably due to the increased MAO-A activity. Previous studies have demonstrated that iron chelators can reduce the MAO activity, suggesting that iron bioavailability can affect the MAO activity (Zheng et al., 2005; Gal et al., 2006). The underlying mechanism of induction of MAO-A activity in IR group is still unclear, but these results can help to explain the symptoms of depression reported in iron-deficient population (Corwin et al., 2003), since increased MAO-A activity has been linked to mood disorders (Meyer et al., 2009; Sacher et al., 2015; Kolla et al., 2016).

Reduced levels of dopamine and/or its metabolites in striatum of IR group were rather expected based on previous observations (Unger et al., 2014; Matak et al., 2016). It is tempting to speculate that this reduction occurred due to the compromised dopamine synthesis, since iron is a cofactor for tyrosine hydroxylase, an enzyme that catalyzes limiting step of dopamine synthesis. On the other hand, iron deficiency is known to evoke abnormal signaling via D2 receptor (Youdim et al., 1989; Erikson et al., 2001; Matak et al., 2016). Activation of D2 auto receptors regulates dopamine release and TH activity (Ford, 2014). Thus, it is possible that abnormal D2 receptor signaling contributed to reduced levels of dopamine metabolites in striatum of iron deprived animals.

Since extensive studies have demonstrated the involvement of PrP^C and α-synuclein in dopamine and iron metabolism, we evaluated how these divergent outcomes of dietary iron restriction in each region affected the proteostasis of both proteins. Regarding the expression levels, only PrP^C of the striatum was significantly increased after iron restriction. Previous studies have demonstrated that high concentration of dopamine can promote oxidation and oligomerization of PrP^C (Shiraishi et al., 2005; da Luz et al., 2015). PrP^C can also regulate dopamine metabolism by suppressing the TH expression (Beckman et al., 2015; da Luz et al., 2016). However, in this study, we observed that iron restriction reduced the levels of dopamine metabolites in the striatum, without affecting TH levels or PrP^C solubility. Thus, increased PrP^C levels observed in this region might not be related with dopamine metabolism. On the other hand, PrP^C is known to play roles in iron uptake by acting as ferrireductase, and its expression appears to correlate with ferritin expression (Singh et al., 2013; Haldar et al., 2015; Tripathi et al., 2015). Thus, the increase of PrP^C in striatum of IR group is more likely related to iron recruitment into the striatum leading to the increased ferritin level. Moreover, the regulation of ferritin and PrP^C expression might play important roles for intracellular iron buffering capacity since dietary iron restriction did not alter expression levels of α-synuclein in any brain regions, despite previous report showing reduced translation of α-synuclein upon iron chelation (Febbraro et al., 2012). These results also reinforce specificity of the iron restriction effects on proteins of distinct subcellular comportments.

Altogether, our results demonstrate that insufficient dietary iron supply can establish molecular features that resemble several neurological disorders, such as abnormal ferritin accumulation in striatum observed in parkinsonian syndromes and Huntington disease (Vidal et al., 2004; Simmons et al., 2007), increased MAO-A activity in prefrontal cortex which is associated with depression (Meyer et al., 2009) and increased degree of lipid peroxidation in hippocampus that is related with most of conformational diseases (Sultana et al., 2013). By showing these molecular alterations, this study emphasizes the importance of adequate iron supplementation for brain health and prevention of neurological diseases. Moreover, these brain region-specific responses can be useful information to better understand the diversity of the disorders that can be generated by altered iron metabolism.

JP conducted all experiments. Md assisted in animal handling and western blotting. HA and SG assisted in animal handling and sample collection. VM helped with the study design and interpretation of data, and revised the manuscript. KL conceived and supervised the study, and wrote the manuscript. All authors contributed to final version of the manuscript.

This study was supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo: 2016/04297-6), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico: 467566/2014-3), CAPES (Coordenação de Aperfeiçoamento de pessoal de Nível Superior) and EMU (programa de equipamentos multiusuários).