Transcript Document

General Comments
Golden Age of neuroscience has also been
translated to RLS research
Fe is central to brain function and RLS
etiology
Vast complexity = vast opportunity
More answers = more questions
Expect surprises
Take home message:
RLS is most likely associated with
decreased brain Fe levels.
There is an alteration in Fe
transport, metabolism and storage
proteins.
But should you just focus on Fe?
Yes and no
Not exhaustive, but what’s known

Decreased transferrin receptor expression in RLS
(Connor et al., Neurology 62:1563-7, 2004).

Ferritin subunits in CSF are decreased in RLS
(Clardy et al., J Lab Clin Invest 147:167-173).

“At this time, it cannot be determined if the lower
levels of pro-hepcidin in the CSF represent a
compensatory response to the decreased levels of
iron in the brain or a defective signaling
mechanism in RLS” (Clardy et al., J Neurol Sci 247:173-179,
2006).

CSF ferritin poorly correlates with serum ferritin in
RLS (Earley et al., Sleep 28:1069-1075, 2005).
Iron uptake occurs via the transferrin receptor and DMT1.
Steap3 is a ferrireductase.
Ferroportin is the only known cellular iron exporter, and ceruloplasmin is a ferroxidase
mediating efficient cellular iron release.
Iron homeostasis is regulated by hepcidin, a circulating peptide that binds to
ferroportin, mediating uptake and degradation of this exporter.
Iron enters mitochondria via mitoferrin, and frataxin is a mitochondrial protein
mediating Fe-S cluster formation and heme biosynthesis.
The human DMT1 gene, maps to chromosome 12q
near the RLS1 locus (Xiong et al., Neurology 144, 911-917, 2007).
Samples from RLS families with compatible linkage to
the RLS1 locus on 12q were sequenced in both the
coding regions and the long stretches of UTR
sequences.
No detectable difference in DMT1 protein levels
between RLS patient lymphoblastoid cell lines and
controls.
Linkage analyses failed to identify any significant
linkage signals within the DMT1 gene region.
Sequencing did not detect any sequence variant(s)
compatible with DMT1 harboring RLS causative
mutation(s).
No association between ten SNPs, spanning the
whole DMT1 gene region, and RLS status.
Sequence analysis and association between SNPs in
Fe associated genes and RLS
Iron uptake occurs via the transferrin receptor and DMT1.
Steap3 is a ferrireductase.
Ferroportin is the only known cellular iron exporter, and ceruloplasmin is a ferroxidase
mediating efficient cellular iron release.
Iron homeostasis is regulated by hepcidin, a circulating peptide that binds to
ferroportin, mediating uptake and degradation of this exporter.
Iron enters mitochondria via mitoferrin, and frataxin is a mitochondrial protein
mediating Fe-S cluster formation and heme biosynthesis.
Mn MRI and AAS correlations
R1 vs. [Mn] for CN and MnT
1.1
Cerebellum
Brainstem
1
Midbrain
Hippocampus
R1 (1/s)
0.9
Striatum
Cortex
0.8
0.7
0.6
0.5
0
0.05
0.1
0.15
0.2
0.25
[Mn] (mmol/kg tissue)
0.3
0.35
MRI findings - limitations
R1 was strongly correlated with tissue Mn levels.
However, the slopes of the linear regression fits
varied significantly among different brain regions
A simple linear model failed to explain the changes in
relaxation rate when both Mn and Fe brain contents
changed.
When Mn and Fe are both changing, their combined
influence on MRI signals is complicated.
In such case, the simple linear model is not suitable
to explain the change of MRI relaxation rates
Other metals may contribute to the R1s, R2s.
ID is associated with increased brain
copper levels
Garcia et al., Toxicol Sci 95:205-14, 2007
Complimentary Experimental Models
Surdej P, Richman L, Kühn LC. Differential translational
regulation of IRE-containing mRNAs in Drosophila
melanogaster by endogenous IRP and a constitutive human
IRP1 mutant. Insect Biochem Mol Biol 38:891-4, 2008.
Missirlis F, Kosmidis S, Brody T, Mavrakis M, Holmberg S,
Odenwald WF, Skoulakis EM, Rouault TA. Homeostatic
mechanisms for iron storage revealed by genetic manipulations
and live imaging of Drosophila ferritin. Genetics 177:89-100,
2007.
Bagheri N, Stelling J, Doyle FJ 3rd. Circadian phase resetting
via single and multiple control targets. PLoS Comput Biol. 4;4
2008.
Power of genetics (screen for suppressor,
complementation, forward and reverse genetics)
combined with availability of multiple knockout
or loss-of-function mutation GFP expressing
strains, as well as behavioral tests
Role of DMT-1 in Mn-induced death
in C. elegans
Mn Concentrations in Smf
mutants
smf-3 expression pattern by
transcriptional and
translational GFP fusions
E) smf-3::GFP expression in the intestine.
F) smf-3::GFP expression in the epidermis hyp7.
G) smf-3::GFP expression in the epidermis hyp1 to hyp6.
Working model of Mn transport in the worm
SMF-1 and SMF-3 are the DMT1 isoforms responsible
for Mn uptake.
SMF-2 is involved in Mn sensing and uptake regulation
via inhibition of pharyngeal pumping.
Schematic model of a C. elegans DA neuron
containing known and predicted genes involved
in DA biosynthesis and metabolism
TH, tyrosine hydroxylase;
GTPCH, GTP cyclohydrolase;
AAAD, aromatic L-amino
acid decarboxylase; MAO,
monoamine oxidase; VMAT,
vesicular monoamine
transporter; DAT-1,
dopamine transporter;
CeDOP1, D1-like DA
receptor; CeDOP2, D2-like
DA receptor; COMT, cacholO-methyltransferase.
CeDOP2
CeDOP2
Genetic mutants are
indicated in italics.
Selective Sensitivity of C. elegans to Mn
Future Objectives
Identify the neuropathological mechanisms of RLS?
Focus on Fe, but not all the keys may be under the
streetlight.
Studies in animals exposed to precipitants of RLS, e.g., Fe
depletion.
How can complimentary models be utilized for better
understanding of the pathophysiology?
Are their promising models that can be used, and what is
the best strategy for utilizing these models to assess the
genetic predisposition and environmental influences
(gene x environment interactions?)
Interactions with other metals.
studies to determine how dopamine pathways are altered
in RLS and relationships to circadian rhythms.
Future Objectives
Brain bank of deceased RLS patients?
Sequence analysis and association between SNPs in Fe
(metals?) associated genes and RLS.
Family studies to distinguish genetic and non-genetic
forms and to map, identify, and characterize genes
involved in the etiology of RLS.
Clinical trials are needed to study the efficacy of iron
supplementation in RLS.
Develop surrogate measures of Fe status
Transferrin synthesis is not specific to Fe availability as it is decreased
during inflammation and other conditions.
The serum soluble transferrin receptor, a truncated form of the
membrane receptor produced by enzymatic cleavage is a direct
reflection of cellular transferrin receptors, showing little variability.
The hereditary disorders of brain
iron metabolism
Aceruloplasminemia
Neuroferritinopathy
Genetics
Autosomal recessive loss-offunction mutations
ceruloplasmin gene
Autosomal dominant (dominantnegative) mutations ferritin light chain
gene
Presentation
Third decade—diabetes
Third through sixth decade
Fifth decade—neurologic
Defect
Brain iron recycling
Brain iron storage
Pathogenesis
Brain iron accumulation
Brain iron accumulation
Systemic iron accumulation
Clinical
Diabetes, anemia, dementia
Dementia, dystonia, dysarthria
Dystonia, dysarthria
Pathology
Iron accumulation in astrocytes
Iron accumulation in astrocytes
Neuronal loss
Neuronal loss