HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease modifier for Parkinson's disease. Moreover, the finding that the HFE mutant mice are resistant to paraquat may provide a model in which to study resistant mechanisms to neurotoxicants.

Statins accelerate disease progression and shorten survival in SOD1(G93A) mice.

INTRODUCTION: HMG-CoA reductase inhibitors (statins) and H63D HFE polymorphism may modify amyotrophic lateral sclerosis (ALS). We hypothesized that statins worsen phenotype in ALS mice, dependent on HFE genotype. METHODS: Mice harboring SOD1(G93A) heterozygous for H67D Hfe (homologous to human H63D HFE) were administered simvastatin and/or coenzyme Q10, and were allowed to reach end stage. Disease progression was measured by grip strength. A separate group of animals was administered simvastatin and euthanized at the symptomatic 120-day time-point. Mitochondria from gastrocnemius muscle and lumbar spine were analyzed. RESULTS: Simvastatin and H67D Hfe accelerated disease progression. Simvastatin decreased survival. Coenzyme Q10 did not rescue statin-induced effects. Statins did not alter mitochondrial protein levels. CONCLUSIONS: Statins and Hfe genotype alter disease course in the ALS mouse model. Because the H63D HFE polymorphism is present in 30% of patients with ALS, studying disease progression in patients who receive statins, stratified for HFE genotype, may guide therapy. Muscle Nerve, 2016 Muscle Nerve 54: 284-291, 2016.

H63D HFE genotype accelerates disease progression in animal models of amyotrophic lateral sclerosis.

H63D HFE is associated with iron dyshomeostasis and oxidative stress; each of which plays an important role in amyotrophic lateral sclerosis (ALS) pathogenesis. To examine the role of H63D HFE in ALS, we generated a double transgenic mouse line (SOD1/H67D) carrying the H67D HFE (homologue of human H63D) and SOD1(G93A) mutations. We found double transgenic mice have shorter survival and accelerated disease progression. We examined parameters in the lumbar spinal cord of double transgenic mice at 90days (presymptomatic), 110days (symptomatic) and end-stage. Transferrin receptor and L-ferritin expression, both indicators of iron status, were altered in double transgenic and SOD1 mice starting at 90days, indicating loss of iron homeostasis in these mice. However, double transgenic mice had higher L-ferritin expression than SOD1 mice. Double transgenic mice exhibited increased Iba-1 immunoreactivity and caspase-3 levels, indicating increased microglial activation which would be consistent with the higher L-ferritin levels. Although both SOD1 and double transgenic mice had increased GFAP expression, the magnitude of the increase was higher in double transgenic mice at 110days, suggesting increased gliosis in these mice. Increased hemeoxygenase-1 and decreased nuclear factor E2-related factor 2 levels in double transgenic mice strongly suggest the accelerated disease process could be associated with increased oxidative stress. There was no evidence of TAR-DNA-binding protein 43 mislocalization to the cytoplasm in double transgenic mice; however, there was evidence suggesting neurofilament disruption, which has been reported in ALS. Our findings indicate H63D HFE modifies ALS pathophysiology via pathways involving oxidative stress, gliosis and disruption of cellular functions.

A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice.

Because of the increasing evidence that H63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences of H63D HFE in vivo using mice that carry H67D HFE (homologous to human H63D). Although total brain iron concentration did not change significantly in the H67D mice, brain iron management proteins expressions were altered significantly. The 6-month-old H67D mice had increased HFE and H-ferritin expression. At 12 months, H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia in H67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-old H67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12 months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in the H67D mice. These findings demonstrate that the H63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. The H67D knock-in mouse can be used as a model to evaluate how the H63D HFE mutation contributes to neurodegenerative diseases.

Neuropeptide Y is associated with changes in appetite-associated hypothalamic nuclei but not food intake in a hypophagic avian model.

While neuropeptide Y (NPY) has been studied extensively per its pronounced role in food intake stimulation as well as its role in central pathways governing eating disorders, it has to our knowledge not been studied in polygenic models of hypo- and hyperphagia. Thus, the present study was designed to measure central NPY-associated food intake in lines of chickens that have undergone long-term genetic selection for low (LWS) or high (HWS) body weight and exhibit hypo- and hyperphagia, respectively. LWS chicks did not respond with any magnitude of altered food intake to any dose of NPY tested, while HWS chicks responded to all doses of NPY at similar magnitudes throughout the duration of observation. Both lines responded with similar increases in c-Fos immunoreactivity in the lateral hypothalamus and both divisions of the paraventricular nucleus; there were no significant line or line by treatment interactions. These data support the hypothesis that differences exist in the central NPY system of chicks from LWS and HWS lines and may provide novel insight for understanding NPY control of appetite.

HFE gene variants affect iron in the brain.

Iron accumulation in the brain and increased oxidative stress are consistent observations in many neurodegenerative diseases. Thus, we have begun examination into gene mutations or allelic variants that could be associated with loss of iron homeostasis. One of the mechanisms leading to iron overload is a mutation in the HFE gene, which is involved in iron metabolism. The 2 most common HFE gene variants are C282Y (1.9%) and H63D (8.9%). The C282Y HFE variant is more commonly associated with hereditary hemochromatosis, which is an autosomal recessive disorder, characterized by iron overload in a number of systemic organs. The H63D HFE variant appears less frequently associated with hemochromatosis, but its role in the neurodegenerative diseases has received more attention. At the cellular level, the HFE mutant protein resulting from the H63D HFE gene variant is associated with iron dyshomeostasis, increased oxidative stress, glutamate release, tau phosphorylation, and alteration in inflammatory response, each of which is under investigation as a contributing factor to neurodegenerative diseases. Therefore, the HFE gene variants are proposed to be genetic modifiers or a risk factor for neurodegenerative diseases by establishing an enabling milieu for pathogenic agents. This review will discuss the current knowledge of the association of the HFE gene variants with neurodegenerative diseases: amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and ischemic stroke. Importantly, the data herein also begin to dispel the long-held view that the brain is protected from iron accumulation associated with the HFE mutations.

Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability.

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Prolyl-peptidyl isomerase, Pin1, phosphorylation is compromised in association with the expression of the HFE polymorphic allele, H63D.

There is substantial interest in HFE gene variants as putative risk factors in neurodegenerative diseases such as Alzheimer disease (AD). Previous studies in cell models have shown the H63D HFE variant to result in increased cellular iron, oxidative stress, glutamate dyshomeostasis, and an increase in tau phosphorylation; all processes thought to contribute to AD pathology. Pin1 is a prolyl-peptidyl cis/trans isomerase that can regulate the dephosphorylation of the amyloid and tau proteins. Hyperphosphorylation of these later proteins is implicated in the pathogenesis of AD and Pin1 levels are reportedly decreased in AD brains. Because of the relationship between Pin1 loss of function by oxidative stress and the increase in oxidative stress in cells with the H63D polymorphism it was logical to interrogate a relationship between Pin1 and HFE status. To test our hypothesis that H63D HFE would be associated with less Pin1 activity, we utilized stably transfected human neuroblastoma SH-SY5Y cell lines expressing the different HFE polymorphisms. Under resting conditions, total Pin1 levels were unchanged between the wild type and H63D HFE cells, yet there was a significant increase in phosphorylation of Pin1 at its serine 16 residue suggesting a loss of Pin1 activity in H63D variant cells. To evaluate whether cellular iron status could influence Pin1, we treated the WT HFE cells with exogenous iron and found that Pin1 phosphorylation increased with increasing levels of iron. Iron exposure to H63D variant cells did not impact Pin1 phosphorylation beyond that already seen suggesting a ceiling effect. Because HFE H63D cells have been shown to have more oxidative stress, the cells were treated with the antioxidant Trolox which resulted in a decrease in Pin1 phosphorylation in H63D cells with no change in WT HFE cells. In a mouse model carrying the mouse equivalent of the H63D allele, there was an increase in the phosphorylation status of Pin1 providing in vivo evidence for our findings in the cell culture model. Thus, we have shown another cellular mechanism that HFE polymorphisms influence; further supporting their role as neurodegenerative disease modifiers.

The threshold of amylin-induced anorexia is lower in chicks selected for low compared to high juvenile body weight.

Chicks that have undergone long-term selection for low body weight responded to intracerebroventricular amylin injection with reduced food intake at a dose considerably lower and with a greater magnitude suppression than those selected for high body weight. Behaviors unrelated to ingestion were not affected. These data support the thesis of correlated amylin system responses to selection for low or high body weight, with possible implications to other species.

Differential feed intake responses to central corticotrophin releasing factor in lines of chickens divergently selected for low or high body weight.

Effects of intracerebroventricular (ICV) injection of corticotrophin releasing factor (CRF) on feed intake were evaluated in two lines of White Plymouth Rock chickens that have been selected from a common base population for high (HWS) or low (LWS) juvenile body weight. Both lines responded with reduced feed intake after ICV CRF; however, the threshold of response was lower in line LWS than HWS. Additionally, the effects of two receptor antagonists, astressin and alpha-helical CRF (9-41; alpha-CRF), and the effect of CRF fragment 6-33, (which displaces CRF from its binding protein), were evaluated in these lines. Although all three antagonists increased feed intake in line LWS but not line HWS, they attenuated the appetite-reducing effects of CRF only in line HWS. Peripheral plasma corticosterone concentrations after an acute stressor were higher in line LWS than in line HWS. These data support the thesis of correlated responses in the CRF system to selection for high or low juvenile body weight. These differences may contribute to differential feed intake, and hence altered body weights.

Effect of calcitonin gene-related peptide (CGRP) on avian appetite-related processes.

Calcitonin gene-related peptide (CGRP) is released from the gastrointestinal tract following ingestion and causes satiety in mammals. Its effects on appetite in non-mammalian vertebrates are unreported. In Experiment 1, fasted chicks reduced food and water intake after central injection of CGRP. These effects were not associated with increased plasma corticosterone concentration. In Experiment 2, we showed that the effect on water intake was independent of food intake. In Experiment 3, central CGRP caused increased c-Fos immunoreactivity in the arcuate (ARC) nucleus, paraventricular nucleus (PVN), periventricular (PHN) and ventromedial (VMH) hypothalamic nuclei. The results of Experiment 4 demonstrate that intraperitoneal injection of CGRP also causes reduced food and water intake. c-Fos immunoreactivity was increased in the ARC, PHN, PVN and VMH in Experiment 5 after intraperitoneal injection of CGRP. Lastly in Experiment 6, we showed that central CGRP changes the type of pecks from feeding to exploratory, and reduces the number of escape attempts. The effect of CGRP appears to be primary on appetite in chicks. In conclusion, the mechanisms of CGRP induced satiety have some similarities and differences between avian and rodent models. The results presented here provide new insight into the evolution of vertebrate satiety regulatory mechanisms.

Differential feeding responses to central alpha-melanocyte stimulating hormone in genetically low and high body weight selected lines of chickens.

This study was conducted to compare the effects of central alpha-MSH, a potent anorexigenic signal, in lines of chickens that have undergone long-term divergent selection for low (LWS) or high (HWS) body weight. Chicks from both lines were centrally injected with 0, 24, 120 or 600 pmol alpha-MSH and feed and water intake were concurrently measured thereafter for a total of 180 min. The LWS line responded to all doses of alpha-MSH with a similar potent decrease in feed intake at all observation times. The HWS line only responded to 600 pmol alpha-MSH with decreased feed intake. alpha-MSH did not influence water intake in either line. To determine if differential hypothalamic signaling was associated with the anorexigenic effect, c-Fos immunoreactivity was measured in appetite-related hypothalamic nuclei after 600 pmol central alpha-MSH injections. c-Fos immunoreactivity was increased in the dorsomedial hypothalamus, paraventricular nucleus (PVN) and ventromedial hypothalamus in both lines after alpha-MSH; however, the magnitude of increase was greater in LWS than in HWS chicks at the PVN (136% vs. 47% increase over controls, respectively). Based on behavior observations, the number of feeding and exploratory pecks is decreased with greater magnitude after alpha-MSH in the LWS line. Additionally, alpha-MSH was associated with increased deep rest in both lines, and may be a secondary effect to reduced ingestion. These data support that the LWS line has a lower threshold for the anorexigenic effect of central alpha-MSH while in the HWS line this threshold is higher, and that this difference may be associated with differential hypothalamic signaling. Genetic variation exists in the threshold of anorexigenic response for central alpha-MSH in LWS and HWS lines of chickens with possible implications to other species including humans.

Mechanisms of xenin-induced anorectic response in chicks (Gallus gallus).

We recently reported that the 25 amino acid peptide xenin caused reduced feed intake when centrally injected in chicks. The present study was designed to explore possible mechanisms of the xenin-induced anorexigenic response in chicks. In Experiments 1 and 2, chicks were implanted with cannulas and xenin injections were made directly into the ventromedialis hypothalami (VMH). Chicks responded with reduced feed intake and increased c-Fos immunoreactivity at the VMH. In Experiment 3 chicks that received co-intracerebroventricular (ICV) injection of naloxone and a dose of xenin (100 pmol), that alone does not affect feed intake, had reduced feed intake. In Experiment 4, chicks responded to ICV xenin with reduced feed- but increased exploratory-pecking. Thus, we conclude that xenin may mediate its effect directly at the VMH and that the endogenous opioid system may counter anorexigenic effects of low xenin doses in chicks. Xenin also caused increased exploration of a novel environment, an effect that may be competitive with feeding. Taken together, these results suggest that xenin regulation of chick appetite is the result of several central and behavioral mechanisms acting in synergism.

Central oxyntomodulin causes anorexigenic effects associated with the hypothalamus and alimentary canal in chicks (Gallus gallus).

Several peptides that are derived from proglucagon including glucagon, glucagon-like peptide-1 (GLP-1), and oxyntomodulin (OXM) cause satiety in mammals. Glucagon and GLP-1 also cause satiety in the avian, but the effects of OXM on avian appetite-related processes are not reported. Thus, this study was conducted to elucidate whether OXM induces satiety in chicks and to determine its mechanism of induction. Intracerebroventricular (ICV) OXM, in a linear-dose dependent manner, potently decreased feed and water intake. However, we found that the effect on water intake was secondary to a reduction in feed intake. Chicks treated with ICV OXM had decreased c-Fos immunoreactivity in the regio lateralis hypothalami, but the nucleus infundibuli hypothalami (homologue to the mammalian arcuate nucleus) had increased c-Fos immunoreactivity. ICV OXM also caused total alimentary canal transit time to be decreased. We conclude that changes in the hypothalamus and gut may contribute to anorexigenic effects after ICV OXM in chicks. Through divergent evolution of birds and mammals, the central anorexigenic effects of OXM may have been conserved.

Amylin causes anorexigenic effects via the hypothalamus and brain stem in chicks.

This study was conducted to determine the effects of amylin on appetite-related processes in chicks. Broiler chicks were centrally and peripherally injected with amylin, and feed and water intake were quantified. Feed intake was reduced after both central and peripheral amylin, but water intake was not affected. To determine if the hypothalamus and brainstem were involved in the anorexigenic effect, chicks were centrally and peripherally injected with amylin, and c-Fos immunoreactivity was quantified in the lateral hypothalamus (LH), ventromedial hypothalamus (VMH), area postrema (AP) and the nucleus of the solitary tract (NTS). Amylin decreased c-Fos immunoreactivity in the LH, did not affect the VMH, and increased c-Fos immunoreactivity in the AP and NTS. To determine if alimentary transit time was affected, chicks received central amylin and were gavaged with chicken feed slurry containing a visible marker. Amylin-treated chicks had increased alimentary canal transit time. Chicks also responded to central amylin with increased anxiety-related behaviors and increased plasma corticosterone concentration. These results demonstrate that amylin affects feeding, alimentary canal transit, and behavior through hypothalamic and brainstem mechanisms in chicks.

Central visfatin causes orexigenic effects in chicks.

Intracerebroventricular injection of visfatin caused increased feed intake and pecking efficiency, but did not affect water intake in chicks. Visfatin-treated chicks had increased c-Fos immunoreactivity in the lateral hypothalamus, decreased reactivity in the ventromedial hypothalamus and the dorsomedial hypothalamus, infundibular nucleus, periventricular nucleus, paraventricular nucleus were not affected. A low dose of visfatin increased locomotion. We conclude that intracerebroventricular injection of visfatin causes orexigenic effects in chicks.

Central neuropeptide FF reduces feed consumption and affects hypothalamic chemistry in chicks.

Information on the physiological functions of neuropeptide FF; NPFF, a morphine modulating octapeptide in avians is lacking. Thus, we designed a study to investigate the effects of central NPFF with particular emphasis on appetite-related processes. Cobb-500 chicks were intracerebroventricularly (ICV) injected with 0, 4.16, 8.32 or 16.6nmol NPFF, and feed and water intake were quantified. Feed intake was linearly decreased as NPFF dose increased, and this effect decayed over time and was not significant by 120min post-injection. Water intake was not affected by ICV NPFF. In a second exp, we observed that naloxone completely reversed the NPFF-induced decrease in feed intake. The amount of time a visible marker took to travel through the total length of the alimentary canal linearly increased as NPFF dose increased. We measured neuronal activation in the lateral hypothalamus (LH), paraventricular nucleus (PVN) dorsomedial nucleus (DMN) and ventromedial hypothalamus (VMN) of the hypothalamus, and nucleus dorsomedialis posterior thalami (DMP) of the thalamus. The DMN, DMP, PVN and VMH were all activated by ICV NPFF while the LH was not affected. Finally, we determined that the anorexigenic effect of ICV NPFF is primarily behavior specific, since behaviors unrelated to ingestion were not increased the same duration of time as was consumatory pecking. We conclude that NPFF causes anorexigenic effects in chicks that are primarily behavior specific.

Anorexigenic effects of central neuropeptide S involve the hypothalamus in chicks (Gallus gallus).

Neuropeptide S (NPS) affects appetite-related processes in mammals. However, its role in avian biology is unreported. We hypothesized that intracerebroventricular (ICV) NPS would cause anorexigenic effects in chicks (Gallus gallus). To evaluate this, Cobb-500 chicks were centrally injected with multiple doses (0, 0.313, 0.625 and 1.250 mug) of NPS. NPS-treated chicks responded with decreased feed and water intake. The effect on water intake was secondary to feed intake, because fasted NPS-treated chicks did not reduce water intake. ICV NPS injection also reduced plasma corticosterone concentration. We monitored behavior and found decreased ingestive and exploratory pecking, jumping, locomotion, and increased time spent in deep rest. We hypothesized that the anorexigenic effects were hypothalamic in origin and quantified c-Fos reactivity in the lateral hypothalamus (LH), paraventricular nucleus (PVN) and ventromedial hypothalamus (VMH) after NPS treatment. NPS was associated with decreased c-Fos reactivity in the LH, increased reactivity in the PVN and had no effect in the VMH. When NPS was injected directly into the LH and PVN, chicks responded with decreased feed and water intake, suggesting that effects were directly mediated by these nuclei. We conclude that ICV NPS causes anorexigenic effects in chicks, without directly affecting water intake, and the hypothalamus is involved.

Xenin reduces feed intake by activating the ventromedial hypothalamus and influences gastrointestinal transit rate in chicks.

This study was conducted to determine the effects of xenin on appetite related processes in chicks. Chicks were centrally and peripherally administered xenin, and feed and water intake were quantified. Chicks responded with a linear dose-dependent decrease in feed intake to central xenin and had a quadratic type response to peripheral administration. Water intake was not affected by treatment. To determine if the lateral hypothalamus (LH) or ventromedial hypothalamus (VMH) was involved in this effect, chicks were both centrally and peripherally injected with xenin and an immunocytochemistry assay for c-Fos was conducted. Central and peripheral xenin caused increased activation of the VMH but had no effect on the LH. Finally, to determine if gastrointestinal transit rate was affected, chicks received central xenin and were gavaged with chicken feed slurry containing a visible marker. Chicks exhibited a quadratic dose-dependent response to transit rate after central xenin. These results suggest that xenin affects feeding and gastrointestinal motility through hypothalamic interactions in chicks.