Erica L. Unger

Locomotor hyperactivity and alterations in dopamine neurotransmission are associated with overexpression of A53T mutant human α-synuclein in mice

Genetic and biochemical abnormalities associated with alpha-synuclein are implicated in the etiology of Parkinsons disease (PD). In this study, altered locomotor behavior linked to the expression of mutant or wildtype human alpha-synuclein was investigated. A53T alpha-synuclein transgenic (A53T-tg) mice exhibited normal activity at 5 months of age; however, by 7 months, they developed marked hyperactivity that remained evident until 19 months. By contrast, mice expressing human wildtype or A30P mutant alpha-synuclein showed no locomotor alterations. Hyperactivity in A53T-tg mice was reversed by the D1 receptor antagonist SCH 23390. Furthermore, A53T-tg mice were supersensitive to the D1 receptor agonist SKF 81297 but not to the serotonin1B receptor agonist RU 24969. Hyperactivity in A53T-tg mice was also associated with increased D1 receptor expression in the substantia nigra and decreased dopamine transporter expression in the nucleus accumbens and striatum. Finally, striatal dopamine uptake measured by high-speed chronoamperometry was reduced by 40% in A53T-tg mice. Thus, expression of A53T mutant human alpha-synuclein in mice results in adult-onset hyperactivity associated with D1 receptor and dopamine transporter-mediated alterations in dopamine neurotransmission.

Motor restlessness, sleep disturbances, thermal sensory alterations and elevated serum iron levels in Btbd9 mutant mice

Restless legs syndrome (RLS), also known as Willis-Ekbom disease, is a sensory-motor neurological disorder with a circadian component. RLS is characterized by uncomfortable sensations in the extremities, generally at night or during sleep, which often leads to an uncontrollable urge to move them for relief. Recently, genomic studies identified single-nucleotide polymorphisms in BTBD9, along with three other genes, as being associated with a higher risk of RLS. Little is known about the function of BTBD9 or its potential role in the pathophysiology of RLS. We therefore examined a line of Btbd9 mutant mice we recently generated for phenotypes similar to symptoms found in RLS patients. We observed that the Btbd9 mutant mice had motor restlessness, sensory alterations likely limited to the rest phase, and decreased sleep and increased wake times during the rest phase. Additionally, the Btbd9 mutant mice had altered serum iron levels and monoamine neurotransmitter systems. Furthermore, the sensory alterations in the Btbd9 mutant mice were relieved using ropinirole, a dopaminergic agonist widely used for RLS treatment. These results, taken together, suggest that the Btbd9 mutant mice model several characteristics similar to RLS and would therefore be the first genotypic mouse model of RLS. Furthermore, our data provide further evidence that BTBD9 is involved in RLS, and future studies of the Btbd9 mutant mice will help shine light on its role in the pathophysiology of RLS. Finally, our data argue for the utility of Btbd9 mutant mice to discover and screen novel therapeutics for RLS.

Behavior and Monoamine Deficits in Prenatal and Perinatal Iron Deficiency Are Not Corrected by Early Postnatal Moderate-Iron or High-Iron Diets in Rats

Developmental iron deficiency anemia (IDA) causes brain and behavioral deficits in rodent models, which cannot be reversed when treated at periods equivalent to later infancy in humans. This study sought to determine whether earlier iron treatment can normalize deficits of IDA in rats and what iron dose is optimal. The offspring of dams with IDA during gestation were cross-fostered at postnatal d (P) 8 to dams receiving diets with 1 of 3 iron concentrations until weaning (P21): 0.003-0.01 g/kg [totally iron deficient (TID)]; 0.04 g/kg [formerly iron deficient (FID-40)]; or 0.4 g/kg (FID-400). Always iron-sufficient control dams (CN-40) received a 0.04-g/kg iron diet. At P21, TID pups received a 0.01 g iron/kg diet; all others received a 0.04 g iron/kg diet. Hematocrit and brain iron and monoamine concentrations were assessed at P21 and P100. Pup growth, development, activity, object recognition, hesitancy, and watermaze performance were evaluated. Regional brain iron was restored by iron treatment. Regional monoamine and metabolite concentrations were elevated in FID-40 rats and reduced in FID-400 and TID rats compared with CN-40 rats. FID-40 offspring had motor delays similar to TID during lactation and FID-400 rats had elevated thigmotaxis similar to TID rats at P25 and P100 in the spatial watermaze. In conclusion, iron treatment at P8 in rats did not normalize all monoamine or behavioral measures after early IDA. Moderate iron treatment improved adult behavior, but higher iron treatment caused brain and behavioral patterns similar to TID in the short and long term.

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.

Ceruloplasmin deficiency results in an anxiety phenotype involving deficits in hippocampal iron, serotonin, and BDNF

J. Neurochem. (2012) 120, 125–134.

Dopamine D2 Receptor Expression Is Altered by Changes in Cellular Iron Levels in PC12 Cells and Rat Brain Tissue

Iron deficiency anemia in early life alters the development and functioning of the dopamine neurotransmitter system, but data regarding the specific effects of brain iron loss on dopamine D(2) receptor regulation are lacking. Cell culture and animal models were employed in this study to determine whether D(2) receptor expression is altered when cellular iron levels are depleted. Endogenous D(2) receptor-expressing PC12 cells exposed to increasing concentrations of the iron chelator desferrioxamine (25-100 micromol/L) exhibited dose-dependent decreases in total D(2) receptor protein concentrations (20-65%), but there were minimal effects on D(2) receptor mRNA levels. When iron-deficient cells were repleted with ferric ammonium citrate for 24 h, D(2) receptor protein densities were similar to control. Dietary iron deficiency for 6 wk in weanling rats also reduced regional iron concentrations by nearly 50% in the ventral midbrain and caudate but did not affect D(2) receptor mRNA levels in the ventral midbrain. Iron deficiency significantly reduced membrane D(2) receptor protein levels by >70% in caudate, whereas cytosolic concentrations showed only 25% losses. D(2) receptor protein densities and regional iron concentrations were restored within 2 wk of dietary iron repletion. These results support the concept that D(2) receptor gene expression is not significantly changed by iron deficiency, whereas dopamine receptor trafficking is affected and is likely related to known dopamine system alterations in iron deficiency.

Diurnal cycle influences peripheral and brain iron levels in mice

Iron movement between organ pools involves a dynamic equilibrium of iron efflux and uptake, and homeostatic mechanisms are likely involved in providing iron to cells and organs when required. Daily iron levels in the plasma pool fluctuate with the diurnal cycle, but clear explanations regarding the objectives and regulation of the flux are lacking. The association between diurnal cycle and iron flux is relevant in the disease of restless legs syndrome (RLS), where individuals display diurnal deficits in motor control, have impaired brain iron metabolism, and perhaps altered iron uptake from the plasma pool. The goal of the present study was to examine diurnal variations in peripheral and regional brain iron to evaluate iron flux between organs in iron-sufficient and iron-deficient mice. In mice fed control diet, liver iron was elevated 30-40%, and plasma iron was reduced 20-30% in the active dark period compared with the inactive light phase. Dietary iron deficiency eliminated this variation in liver iron in male and female mice and in plasma iron in male mice. Reductions in ventral midbrain and nucleus accumbens iron and ferritin were apparent in iron-deficient mice during both diurnal phases, but only during the light phase was an approximately 25% reduction in whole brain iron observed, suggesting different brain iron requirements between phases. These data demonstrate that iron flux between organs is sensitive to diurnal regulatory biology. Importantly, variations in brain iron may have temporal implications regarding neural functioning and may contribute to the diurnal cycle-dependent symptoms of RLS.

Iron Deficiency Alters the Day‐Night Variation in Monoamine Levels in Mice

Monoamine metabolism in the central nervous system is altered by dietary iron deficiency, with a stronger effect seen during the active than rest span of the circadian cycle. In this report, we examined changes in intracellular and extracellular monoamine levels, synthetic enzymes, transporter and receptor densities, and responses to amphetamine‐induced dopamine (DA) efflux in iron‐deficient and iron‐sufficient mice. Extracellular striatal DA levels were 15–20% higher in all groups during the active dark phase compared to the inactive light phase, with correspondingly lower dopamine transporter (DAT) and higher tyrosine hydroxylase levels. Iron deficiency decreased DAT density by 20% and 28% in the light and dark phases, respectively, and elevated the DOPAC/DA ratio only in the dark, indicating that iron deficiency does interact with the normal diurnal cues for cyclicity. Enhanced DA efflux after amphetamine stimulation indicates no limitation on monoamine synthesis and release and is consistent with altered synaptic efficacy and perhaps recycling of DA in iron deficiency. These experimental findings provide new evidence that brain iron insufficiency does have a differential effect on the DA system at different biological times of the day and night and may be causally related to the phasic motor symptoms observed in Restless Legs Syndrome.

Iron deficiency alters expression of dopamine-related genes in the ventral midbrain in mice

A clear link exists between iron deficiency (ID) and nigrostriatal dopamine malfunction. This link appears to play an important role in at least restless legs syndrome (RLS) if not several other neurological diseases. Yet, the underlying mechanisms remain unclear. The effects of ID on gene expression in the brain have not been studied extensively. Here, to better understand how exactly ID alters dopamine functioning, we investigated the effects of ID on gene expression in the brain, seeking to identify any potential transcription-based mechanisms. We used six strains of recombinant inbred mice (BXD type) known to differ in susceptibility to ID in the brain. Upon weaning, we subjected mice from each strain to either an iron-deficient or iron-adequate diet. After 100 days of dietary treatment, we measured the effects of ID on gene expression in the ventral midbrain, a region containing the substantia nigra. The substantia nigra is the base of the nigrostriatal dopamine pathway and a region particularly affected by iron loss in RLS. We screened for ID-induced changes in expression, including changes in that of both iron-regulating and dopamine-related genes. Results revealed a number of expression changes occurring in ID, with large strain-dependent differences in the genes involved and number of expression changes occurring. In terms of dopamine-related genes, results revealed ID-induced expression changes in three genes with direct ties to nigrostriatal dopamine functioning, two of which have never before been implicated in an iron-dopamine pathway. These were stromal cell-derived factor 1 (Cxcl12, or SDF-1), a ferritin regulator and potent dopamine neuromodulator, and hemoglobin, beta adult chain 1 (Hbb-b1), a gene recently shown to play a functional role in dopaminergic neurons. The extent of up-regulation of these genes varied by strain. This work not only demonstrates a wide genetic variation in the transcriptional response to ID in the brain, but also reveals two novel biochemical pathways by which iron may potentially alter dopamine function.

Genetic-based, differential susceptibility to paraquat neurotoxicity in mice

Paraquat (PQ) is an herbicide used extensively in agriculture. This agent is also suspected to be a risk factor for Parkinsons disease (PD) by harming nigro-striatal dopamine neurons. There is likely, genetic-based, individual variability in susceptibility to PQ neurotoxicity related PD. In this study, we measured the delivery of PQ to the brain after three weekly injections of PQ at 5 mg kg(-1), PQ-related neural toxicity after three weekly injections of PQ at 1 mg kg(-1)or 5 mg kg(-1), PQ-related iron accumulation and PQ-related gene expression in midbrain of DBA/2J (D2) and C57BL/6J (B6) inbred mouse strains after a single injection of PQ at 15 mg kg(-1) and 10 mg kg(-1), respectively. Results showed that compared to controls, PQ-treated B6 mice lost greater numbers of dopaminergic neurons in the substantia nigra pars compacta than D2 mice; however, distribution of PQ to the midbrain was equal between the strains. PQ also significantly increased iron concentration in the midbrain of B6 but not D2 mice. Microarray analysis of the ventral midbrain showed greater PQ-induced changes in gene expression in B6 compared to D2 mice. This is the first study to report genetically-based differences in susceptibility to PQ neurotoxicity and to understanding individual differences in vulnerability to PQ neurotoxicity and its relation to PD in humans.