Dopaminergic neurons, restless legs, iron and manganese (2020/2021 research)


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BTBD9 is a novel component of IGF signaling and regulates manganese-induced dopaminergic dysfunction

View ORCID ProfilePan Chen, Fuli Zheng, Shaojun Li, Hong Cheng, Julia Bornhorst, Yunhui Li, Bobo Yang, Kun He Lee, Tao Ke, Tanja Schwerdtle, Xiaobo Yang, Aaron B. Bowman, Michael Aschner



Restless legs syndrome (RLS) is a common neurological disorder associated with iron deficiency and dopaminergic (DAergic) neuronal dysfunction.

BTBD9 is a genetic risk factor for RLS.

However, its molecular function remains largely unknown.
Here, we report the interaction between BTBD9, manganese (Mn) and insulin/insulin-like growth factor (IGF) signaling in Caenorhabditis elegans, mouse Neuro2a cells and humans.

We found that elevated Mn downregulated BTBD9 mRNA levels; in turn, BTBD9 expression attenuated Mn-induced cellular stress and dopaminergic neurodegeneration.

As Mn is a known co-factor for insulin receptor and IGF-1 receptor, which activates IGF signaling, we posited that BTBD9 negatively regulates IGF signaling.

Our results showed that the protective effects of BTBD9 against Mn toxicity were dependent on the forkhead box O (FOXO) protein.

Furthermore, BTBD9 overexpression significantly elevated FOXO level and decreased PKB level, while phosphoinositide-dependent kinase-1 (PDK1) level remained unchanged. We conclude that BTBD9 acts as a key component in the IGF signaling pathway.

Meanwhile, the roles of Mn in DAergic neurotoxicity and regulating BTBD9 shed new light on the etiology of RLS.


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This is not a research article but it enlights the interplay between iron manganese and zinc in dopaminergic neurons viability

Connections Between Manganese Neurotoxicity and Neurological Disease
Abstract Manganese (Mn) is an essential nutrient for the human body but is associated with neurotoxicity in excess.

Here we discuss evidence support hypotheses that deviations in brain Mn homeostasis are associated with the etiology of neurodegenerative and neurodevelopmental diseases like manganism, Huntington's disease, restless legs syndrome (RLS), attention deficit hyperactive disorder (ADHD) and Tourette syndrome (TS).

Most of these neurological disorders have overarching motor symptoms—indicating a role of the basal ganglia and by extension the dopaminergic system.

Brain Mn levels are normally enriched in the basal ganglia, and excessive levels are associated with dopaminergic dysfunction.

Although the etiology and cellular pathophysiology of RLS, ADHD and TS are not defined, all three have a strong genetic component, a role for dopaminergic dysfunction, and show comorbidities with each other. Genetic studies have indicated overlapping single nucleotide polymorphisms and genes between RLS and ADHD and Tourette's.

Recent evidence has linked changes in brain Mn to the associated genotypes or phenotypes of these disorders and support hypotheses that Mn dyshomeostasis may contribute to their pathophysiology.

It is important to consider how changes in other divalent metals within the brain affect Mn distribution, since Mn shares transporters with iron, zinc and other metals.

This review will collate the current knowledge about the potential function of Mn and dopamine in RLS, ADHD and TS, and the interplay between these three disorders.


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Hyperactivity, dopaminergic abnormalities, iron deficiency and anemia in an in vivo opioid receptors knockout mouse: Implications for the restless legs syndrome
Shangru Lyu 1, Mark P DeAndrade 1, Stefan Mueller 2, Alexander Oksche 3, Arthur S Walters 4, Yuqing Li 5
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Free PMC article 2019


Previous studies have uncovered a potential role of the opioid system in iron hemostasis and dopamine metabolism.

Abnormalities in both of these systems have been noted in human RLS.

Autopsy studies of human RLS have shown an endogenous opioid deficiency in the thalamus. Opioids, particularly prolonged-release oxycodone/naloxone, have been approved in Europe to be a second-line therapy for severe restless legs syndrome (RLS).

To study the role of opioid receptors in the pathogenesis of RLS, we used a triple knockout (KO) mouse strain that lack mu, delta, and kappa opioid receptors and explored the behavioral and biochemical parameters relevant to RLS.

The triple KO mice showed hyperactivity and a trend of increased probability of waking during the rest period (day) akin to that in human RLS (night).

Surprisingly, triple KO mice also exhibit decreased serum iron concentration, evidence of anemia, a significant dysfunction in dopamine metabolism akin to that noted in human RLS, as well as an increased latency in response to thermal stimuli.

To our knowledge, this is the first study to demonstrate that the endogenous opioid system may play a role in iron metabolism and subsequently in the pathogenesis of anemia.

It is also the first study showing that opioid receptors are involved in the production of motor restlessness with a circadian predominance.

Our findings support the role of endogenous opioids in the pathogenesis of RLS, and the triple KO mice can be used to understand the relationship between iron deficiency, anemia, dopaminergic dysfunction, and RLS.


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Restless legs syndrome and iron deficiency in adults with attention-deficit/hyperactivity disorder


Study Objective
The association between restless legs syndrome (RLS), periodic leg movements during sleep (PLMS) and iron deficiency has been reported in children with attention-deficit/hyperactivity disorder (ADHD); however little is known in adults. The aim of this study was to assess frequencies of RLS, PLMS and other leg movements (LM) and iron deficiency and their relationships with ADHD phenotype in adults with ADHD.


Two hundred adults with ADHD (112 males, median age 31 years) were evaluated on lifetime ADHD symptoms and sleep characteristics.

RLS was diagnosed according to standard criteria. Serum ferritin levels were measured, with iron deficiency defined as <50 ng/mL.

A subgroup of 48 ADHD patients with RLS, 48 ADHD without RLS and 48 controls underwent a polysomnography to record sleep, LM, and PLMS.

RLS was diagnosed in 33.0%, associated with earlier onset of ADHD, hyperactive presentation and more severe lifetime ADHD symptoms.

Iron deficiency was found in 35.5% with higher frequency in patients with RLS.

LM were more frequent in ADHD patients, with higher LM periodicity levels in those with comorbid RLS in comparison to controls. However, PLMS index did not differ between groups.

Patients with ADHD and RLS had higher frequency of iron deficiency than other groups.


In a large sample of adults with ADHD, we individualized a subgroup characterized by earlier and severe ADHD symptoms, RLS, higher LM during sleep and iron deficiency.

This endophenotype may reflect a different neurobiological mechanism that remains to be further studied.


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Correlates of Nonanemic Iron Deficiency in Restless Legs Syndrome
Neurol., 30 April 2020 |

Xiao-Ying Zhu1†, Ting-Ting Wu1†, Hong-Ming Wang2, Xuan Li1, Ling-Yan Ni1, Tian-Jiao Chen1, Meng-Yao Qiu1, Jun Shen2, Te Liu3, William G. Ondo4 and Yun-Cheng Wu1*
  • 1Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Objective: Iron deficiency anemia (IDA) is a well-known cause of secondary restless legs syndrome (RLS). Iron deficiency without anemia (IDNA) is insidious, and its association with RLS is less evaluated. We investigate prevalence and features of IDNA in a consecutive cohort of patients with RLS.

Methods: We included sequential primary RLS patients and RLS patients with IDA. We also recruited age- and gender-matched healthy controls. RLS mimics and other comorbidities were carefully excluded.


One-hundred and ninety-six RLS patients without anemia, 26 RLS patients with IDA, and 63 controls were included.

42.3% of RLS patients without anemia had iron deficiency.

Women were much more susceptible for IDNA with a relative risk of 5.51 (p < 0.0001).

Women with IDNA and RLS had younger age both at interview and at RLS onset compared to women with RLS without iron deficiency (NID) (P < 0.01).

IDNA RLS patients showed a tendency to higher risk of severe/very severe tiredness or sleepiness during the day as compared to NID RLS patients.

Furthermore, IDNA RLS patients had longer duration of RLS (P < 0.01 in men, P < 0.05 in women) and younger age at onset (only in men, P < 0.05) compared to IDA RLS patients.


IDNA is frequent in RLS and iron deficiency may be severe despite a normal hemoglobin level. Women are at much higher risk for IDNA, and IDNA in women presents some specific clinical features.

Features of IDNA RLS are different from IDA RLS. Regular screening of peripheral iron parameters even in patients with normal blood counts is recommended for timely optimal management.


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Brain Iron Deficiency lowers Adenosine A1R/A2R ratio: Implications for Restless Legs Syndrome​

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS).

BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype.

Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients.

It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs).

It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs.

Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats.

It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.