Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination.

Abstract

Mutations in dynamin 2 (DNM2) lead to dominant intermediate Charcot-Marie-Tooth neuropathy type B, while a different set of DNM2 mutations cause autosomal dominant centronuclear myopathy. In this study, we aimed to elucidate the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B and to find explanations for the tissue-specific defects that are associated with different DNM2 mutations in dominant intermediate Charcot-Marie-Tooth neuropathy type B versus autosomal dominant centronuclear myopathy. We used tissue derived from Dnm2-deficient mice to establish an appropriate peripheral nerve model and found that dominant intermediate Charcot-Marie-Tooth neuropathy type B-associated dynamin 2 mutants, but not autosomal dominant centronuclear myopathy mutants, impaired myelination. In contrast to autosomal dominant centronuclear myopathy mutants, Schwann cells and neurons from the peripheral nervous system expressing dominant intermediate Charcot-Marie-Tooth neuropathy mutants showed defects in clathrin-mediated endocytosis. We demonstrate that, as a consequence, protein surface levels are altered in Schwann cells. Furthermore, we discovered that myelination is strictly dependent on Dnm2 and clathrin-mediated endocytosis function. Thus, we propose that altered endocytosis is a major contributing factor to the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B.

Full text

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BRAIN
A JOURNAL OF NEUROLOGY
Dynamin 2 mutations in Charcot–Marie–Tooth
neuropathy highlight the importance of
clathrin-mediated endocytosis in myelination
Pa ´ris N. M. Sidiropoulos,1Michaela Miehe,1Thomas Bock,2,3Elisa Tinelli,1Carole I. Oertli,1
Rohini Kuner,4Dies Meijer,5Bernd Wollscheid,2,3Axel Niemann1,* and Ueli Suter1,*
1 Department of Biology, Institute of Molecular Health Sciences, Chair in Cell Biology, ETH Zurich, CH-8093 Zurich, Switzerland
2 Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, CH-8093 Zurich, Switzerland
3 National Centre of Competence in Research (NCCR)–Neuro Centre for Proteomics, UZH/ETH Zurich, CH-8093 Zurich, Switzerland
4 Institute for Pharmacology, Heidelberg University, D-69120 Heidelberg, Germany
5 Department of Cell Biology and Genetics, Erasmus University Medical Centre, 3000 CA Rotterdam, The Netherlands
*These authors contributed equally to this work
Correspondence to: Prof. Dr. Ueli Suter,
Institute of Molecular Health Sciences, Chair in Cell Biology,
ETH Zurich, ETH-Ho ¨nggerberg, HPM E39,
Schafmattstrasse 18,
CH-8093 Zurich, Switzerland
E-mail: usuter@cell.biol.ethz.ch
Mutations in dynamin 2 (DNM2) lead to dominant intermediate Charcot–Marie–Tooth neuropathy type B, while a different set
of DNM2 mutations cause autosomal dominant centronuclear myopathy. In this study, we aimed to elucidate the disease
mechanisms in dominant intermediate Charcot–Marie–Tooth neuropathy type B and to find explanations for the tissue-specific
defects that are associated with different DNM2 mutations in dominant intermediate Charcot–Marie–Tooth neuropathy type
B versus autosomal dominant centronuclear myopathy. We used tissue derived from Dnm2-deficient mice to establish an
appropriateperipheralnervemodeland found thatdominant
B-associated dynamin 2 mutants, but not autosomal dominant centronuclear myopathy mutants, impaired myelination. In
contrast to autosomal dominant centronuclear myopathy mutants, Schwann cells and neurons from the peripheral nervous
system expressing dominant intermediate Charcot–Marie–Tooth neuropathy mutants showed defects in clathrin-mediated endo-
cytosis. We demonstrate that, as a consequence, protein surface levels are altered in Schwann cells. Furthermore, we discovered
that myelination is strictly dependent on Dnm2 and clathrin-mediated endocytosis function. Thus, we propose that altered
endocytosis is a major contributing factor to the disease mechanisms in dominant intermediate Charcot–Marie–Tooth neur-
opathy type B.
intermediate Charcot–Marie–Toothneuropathytype
Keywords: Charcot–Marie–Tooth disease; hereditary motor and sensory neuropathy; myelination; endocytosis; dynamin 2
Abbreviations: CMT = Charcot–Marie–Tooth; CNM = centronuclear myopathy; EGFP = enhanced green fluorescent protein
doi:10.1093/brain/aws061Brain 2012: Page 1 of 17 |
1
Received June 20, 2011. Revised December 13, 2011. Accepted January 5, 2012.
? The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

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Introduction
Charcot-Marie-Tooth disease (CMT), also called hereditary motor
and sensory neuropathy, is one of the most common monogenetic
diseases of the nervous system (Skre, 1974). CMT is clinically and
genetically diverse (for further information see http://neuromus-
cular.wustl.edu/time/hmsn.html) and has been classified by elec-
trophysiological and histopathological means into demyelinating or
dysmyelinating (CMT1, CMT3, CMT4) and axonal forms (CMT2)
(Dyck et al., 1993). Axonal CMT shows slightly reduced to normal
nerve conduction velocity, but reduced compound muscle action
potential due to major loss of myelinated axons. Cellular effects of
disease mutations leading to axonal forms are thought to originate
on the neuronal side, whereas demyelinating or dysmyelinating
CMT is generally assumed to start with an insult affecting myeli-
nating Schwann cells. Characteristic for the myelin-affecting forms
is decreased nerve conduction velocity, followed by axonal degen-
eration and reduced compound muscle action potential as the
main correlate to clinical disability (Suter and Scherer, 2003;
Scherer et al., 2008).
Intermediate forms of CMT have also been described (Nicholson
et al., 2006) with uncertainty of whether these are primarily
demyelinating or axonal disorders, or both. Within this group,
dynamin 2 (DNM2) mutations have been linked to dominant-
intermediate CMT subtype B (Zuchner et al., 2005). Patients
with dominant-intermediate CMT subtype B are characterized by
mildly reduced nerve conduction velocity, loss of myelinated
axons, rare segmental demyelination and remyelination with
onion bulb formation, and focal hypermyelination (Zuchner
et al., 2005). The contributions of mutated forms of Dnm2 to
the disease in neurons, in Schwann cells, and in the crucial
dynamics of Schwanncell–axon
(Nicholson et al., 2006; Claeys et al., 2009).
Dynamins are highly conserved, large GTPases that belong to
the dynamin superfamily (Heymann et al., 2009). In general, they
hydrolyze GTP enabling them to act as mechanochemical proteins
to mediate, or contribute to, membrane fission. The core group of
classical dynamins consists of the three isoforms: Dnm1, Dnm2
and Dnm3. In addition, the family of large GTPases includes the
subfamily of dynamin-related proteins with mitofusin 2 (CMT2A
causing gene) and OPA1 (mutated in autosomal dominant optic
atrophy) as prominent members. Classical dynamins share a highly
conserved catalytic N-terminal GTPase domain, a middle domain
important for oligomerization, a pleckstrin homology domain crit-
ical for the interaction with membrane phosphoinositides and the
regulation of GTPase activity (Kenniston et al., 2010), and a
GTPase effector domain, which is thought to act as GTPase
activating-domain upon assembly of dynamin oligomers into
higher order structures (Ramachandran, 2007). The less well-con-
served C-terminus contains an arginine-/proline-rich domain,
which is a major partner for interacting proteins (Durieux et al.,
2010) (Fig. 1). The vast majority of CMT-associated DNM2
mutations are clustered in the N-terminal part of the pleckstrin
homology domain (Fig. 1).
A second distinct set of DNM2 mutations, mainly found in the
middle domain and at the pleckstrin homology–GTPase effector
interactions,areunclear
domain boundary, is linked to autosomal-dominantcentronuclear
myopathy (Kenniston et al., 2010) (Fig. 1). Autosomal-dominant
centronuclear myopathy (CNM) is characterized by slowly pro-
gressive muscular weakness and wasting with centrally located
nuclei in a large number of skeletal muscle fibres, radial arrange-
ment of sarcoplasmic strands and predominance of hypotrophy of
type 1 fibres (Fischer et al., 2006; http://neuromuscular.wustl
.edu/musdist/distal.html#distal).
Dnm2 is ubiquitously expressed and is involved in several
cellular processes including various endocytotic pathways, vesicle
trafficking from and to the trans-Golgi network, centrosome
cohesion and actin- and microtubular dynamics (Praefcke et al.,
2004; Liu et al., 2008; Ferguson et al., 2009). The three classical
dynamin isoforms share some functions and individual members
can, at least partially, compensate for each other, while other
functions are unique (Ferguson et al., 2007, 2009; Liu et al.,
2008; Durieux et al., 2010). However, Dnm1 expression is largely
restricted to neuronal cells, and Dnm3 is found in brain, heart,
lung and testis. At the organism level, Dnm1-deficient mice die
within 2 weeks after birth and show poor motor coordination
(Ferguson et al., 2007). Furthermore, ablation of Dnm1 correlates
with severe impairment of synaptic vesicle reformation during
intense activity. In contrast, ubiquitous deletion of Dnm2 results
in early embryonic lethality indicating a pivotal role of Dnm2 in
early mouse development (Ferguson et al., 2009; unpublished
data).
The consequences of some mutations associated with CMT or
CNM have been studied in vitro and in different model systems.
In vitro, at least some CNM-associated DNM2 mutants (CNM
mutants) alter dynamin’s GTPase activity, while retaining binding
to phospholipids (Kenniston et al., 2010). In contrast, Zuchner
et al. (2005) proposed diminished binding to membranes of
selected CMT-associated
DNM2
Furthermore, several CNM mutants form abnormally stable poly-
mers (Wang et al., 2010). At the cellular level, interference with
centrosome cohesion was suggested as potential disease mechan-
ism for CNM mutants R369W and R465W, whereas expression of
the CMT mutant D555_E557? resulted in altered microtubule
dynamics and defects in mature Golgi complex formation
(Zuchner et al., 2005; Tanabe et al., 2009).
A comparison of different mutants in Cos7 cells revealed inhib-
ition of transferrin uptake upon expression of CNM mutants
R465W, V625?, E650K and of CMT mutant K562E, whereas
the CMT mutant D555_E557? had no impact (Bitoun et al.,
2009; Tanabe et al., 2009). Impairment of clathrin-mediated
endocytosis was also found in fibroblasts from a patient with
CNM carrying the R465W mutant (Bitoun et al., 2009). Despite
these data, mouse embryonic fibroblasts with a heterozygous
Dnm2 R465W knock-in mutation did not reveal a clathrin-
mediated endocytosis defect, whereas homozygous cells did
(Durieux et al., 2010).
Notwithstanding recent progress on biochemical malfunctions
caused by DNM2 mutations (Kenniston et al., 2010; Wang
et al., 2010), a thorough understanding of the disease mechan-
isms and the development of pathologies requires detailed analysis
in appropriate cellular systems. We were particularly interested in
addressing why and how CMT-causing DNM2 mutations have
mutants(CMTmutants).
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a selective deleterious effect on myelinated peripheral nerves.
By comparing CMT- with CNM-causing mutants, we aimed to
reveal disease-specific mechanistic differences and/or cell type-
specific effects. Using lentiviral gene transfer in combination with
dissociated dorsal root ganglia explant cultures from ubiquitous
and cell type-specific Dnm2-deficient mice, we have established
a peripheral nerve disease model. We demonstrate that CMT
mutants, in contrast to CNM mutants, have a destructive effect
by impairing myelination. In line with these results, we found that
CMT mutants inhibit clathrin-mediated endocytosis in Schwann
cells and differentiated motor neurons, whereas CNM mutants
do not. Indeed, clathrin-mediated endocytosis was found to
beessentialfor peripheralnerve
clathrin-mediated endocytosis appears to be a major contributor
to the disease mechanism underlying dominant-intermediate CMT
subtype B.
myelinationand altered
Materials and methods
Mice
Analysis of transgenic mice with exon 2 of Dnm2 flanked by LoxP sites
will be described elsewhere. Dnm2wt/0mice were generated using a
Cre deleter mouse line. Mice heterozygous (Dnm2wt/flox) or homozy-
gous (Dnm2flox/flox) for the Dnm2 floxed allele were crossed with
established mouse lines expressing Cre recombinase under the control
of desert hedgehog (Dhh) (Jaegle et al., 2003) or NaV1.8 sodium
channel (SNS) (Agarwal et al., 2004) gene regulatory elements.
Genotypes of mice were determined by polymerase chain reaction
on genomic DNA derived from ear biopsies using Dnm2- and Cre-
specific primer pairs (Supplementary material). Experiments with ani-
mals followed approved protocols (Veterinary Office, Canton Zurich,
Switzerland).
Plasmids
Rat Dnm2 (in the splice form denoted aa) was kindly provided by Dr
Ari Helenius (ETH Zurich, Switzerland). Dnm2 CMT and CNM
disease-causing mutations (Fig. 1) were generated by site-directed mu-
tagenesis (QuikChange?mutagenesis kit, Stratagene; for primers, see
Supplementary material). Constructs were cloned into the enhanced
green fluorescent protein (EGFP) vector pEGFP-N1(Clontech) or the
lentiviral vector pSicoR-GFP (Addgene) for eukaryotic expression. Rat
Dnm2 K44A-EGFP (splice form aa) was a kind gift from Dr Sandra
Schmid (Scripps Research Institute, La Jolla, USA) and rat EGFP-tagged
Dnm1 (splice form aa) and Dnm3 (in the splice form denoted aaa)
were kindly supplied by Dr Marc McNiven (Mayo Clinic, Rochester,
USA). All dynamin constructs used in this study were N-terminally
tagged with EGFP. Short hairpin RNAs targeting clathrin heavy chain
and AP-2 subunit m2 (AP50) were from Sigma and subcloned into the
Ubc-TurboGFPTMvector (Sigma).
Antibodies and labelled transferrin
Primary
Glycerinaldehyd-3-phosphat-Dehydrogenase (GAPDH; Hytest) mouse
monoclonal anti-?-tubulin (Sigma), rat monoclonal anti-myelin basic
protein(MBP;Serotec), mouse
medium chain (NF-160;Sigma),
(BectonDickinson),mouse monoclonal
(Serotec), rabbit polyclonal anti-ErbB2 (Abcam), rabbit polyclonal
anti-integrin
b1
(Abcam),mouse
Systems), mouse monoclonal anti-Tuj-1 (Lucerna Chem). Secondary
antibodies and detection reagents: horseradish peroxidase-conjugated
goatanti-mouse, goat anti-rabbit
conjugated goat anti-mouse IgG (all Promega) for immunoblotting.
Alexa Fluor 488 (Molecular Probes), Cy3-coupled streptavidin, Cy3
and Cy5-conjugated goat anti-rabbit, anti-rat and anti-mouse IgG
antibodies(Jackson ImmunoResearch)
Alexa Fluor 555- and Alexa Fluor 647-coupled transferrin were from
Invitrogen.
antibodies usedwere:mouse monoclonal anti-
monoclonal
mouse
anti-neurofilament
monoclonal
anti-transferrin
anti-AP50
receptor
monoclonalanti-Sox10 (R&D
andalakalinephosphatase-
forimmunocytochemistry.
Figure 1 Dominant inherited mutations in DNM2 that cause CMT or CNM. A schematic representation of the Dnm2 protein illustrates that
DNM2 mutations leading to dominant-intermediate CMT subtype B are mainly clustered in the N-terminal part of the pleckstrin homology
(PH) domain. DNM2 autosomal-dominant CNM mutations are localized within the middle domain (MD), pleckstrin homology domain and
GTPase effector domain (GED). Mutations highlighted in red were cloned by site-directed mutagenesis and used in this article. The
dominant-negative point mutation K44A (blue) within the GTPase domain leads to a catalytic-deficient protein and was used as control.
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Cell culture and transfections
HEK293T, HeLa cells, proliferating NSC-34 and RT4-D6P2T (RT4)
cellswereculturedinDulbecco’s
with GlutaMAXTM(Invitrogen) supplemented with 10% foetal calf
serum (Brunswick). Culture medium for primary rat Schwann cells add-
itionally contained 4mg/ml glial growth factor (Harlan) and 8.2mg/ml
forskolin (Sigma). Differentiation of NSC-34 cells was induced
by Dulbecco’s modified Eagle medium/Ham’s F12 supplemented
with 1% foetal calf serum and 1% minimum essential medium-
non-essential amino acids. Lipofectamine?2000 (Invitrogen) and
FugGENE?
6 (Roche)wereused
protocols.
modifiedEagle medium
according to manufacturers’
Lentiviral stocks
High-titre lentivirus was produced as described (Stendel et al., 2010).
Additionally, lentivirus was titrated in HEK293T cells to obtain optimal
transduction efficiencies. Primary Schwann cells, purified dorsal
root ganglia neurons and dorsal root ganglia explant cultures were
infected using 1.0 ? 108TU/ml of high-titre lentivirus, if not indicated
differently.
Immunochemistry and microscopy
Cells were fixed with 4% paraformaldehyde in phosphate-buffered
saline for 20min at room temperature in the dark, washed with
phosphate-buffered saline andpermeabilized with 0.2%
X-100 (Sigma), blocked with phosphate-buffered saline containing
10% goat serum for 1h at room temperature, followed by incubation
with primary antibodies in blocking buffer for an additional hour at
room temperature. After washing with phosphate-buffered saline, cells
were incubated with fluorescent antibodies in blocking buffer for 1h at
room temperature. Cells were washed with phosphate-buffered saline,
if indicated incubated for 5min with 40,60-diamidino-2-phenylindole
dihydrochloride (DAPI) (Sigma), washed again and mounted on cover-
slips (Immu-Mount; Thermo Scientific). Images were generated with
an epifluorescence Zeiss Axioplan microscope equipped with a Zeiss
MRM camera or a confocal inverted microscope (Leica SP2-AOBS)
using argon and helium/neon lasers. Images were imported to
Photoshop CS4 (Adobe) for appropriate pseudo-colouring, linear
contrast adjustment, cropping and merging.
Triton
Dorsal root ganglia explant cultures
Dissociated dorsal root ganglia explant cultures were generated,
allowed to myelinate, processed for immunochemistry, and quantified
as described previously (Stendel et al., 2010). Transduction efficiency
was monitored by EGFP fluorescence. Quantification of myelination: at
least nine fields per coverslip were randomly acquired (?10 magnifi-
cation) and meanCy3fluorescence
basic protein-positive segments was determined within the field
of view using ImageJ software (National Institute of Health).
Cy3-fluorescence of pSicoR-GFP control-infected dorsal root ganglia
explant cultures was set to 100%. Results of wild-type-infected cul-
tures were compared with cultures expressing Dnm2 mutant proteins.
In rescue experiments, wild-type conditions were set to 100%.
Experiments were done in independent triplicates and at least two
coverslips per condition were analysed in each experiment.
representing themyelin
Surface biotinylation and
immunoblotting analysis
Cells were cooled on ice for 5min and washed with phosphate-
buffered saline. Biotin-3-sulpho-N-hydroxy succinimide (Sigma) was
dissolved in water (1mg/100ml) and 2ml cold phosphate-buffered
saline including 100ml biotin was added per plastic 35mm cell culture
dish (Nunc). Cells were incubated for 30min on ice while gently shak-
ing, washed with phosphate-buffered saline and unbound biotin was
quenched with cold 0.1M glycine in phosphate-buffered saline for
5min. Subsequently, cells were lysed with RIPA buffer (50mM Tris–
HCl pH 7.4, 150mM NaCl, 0.1% sodium dodecyl sulphate, 1%
NP-40, 0.5% deoxycholic acid, 5mM EDTA) containing protease
inhibitor cocktail (Sigma), scraped off and sonicated. Cell lysates
wererecoveredafter centrifugation
streptavidin-coupled sepharose beads (GE Healthcare) overnight at
4?C. After intensive washing, the beads were boiled in sodium dodecyl
sulphate sample buffer (80mM Tris–HCl pH 6.8, 10% glycerol, 2%
sodium dodecyl sulphate, 0.005% bromophenol blue), resolved by
sodium dodecyl sulphate–polyacrylamide gel electrophoresis and
immunoblotted onpolyvinylidene
(Millipore). Immunoblots were incubated with primary antibodies of
interest followed by horseradish peroxidase or alkaline phosphatase
chemiluminescence detection. Protein levels on western blots were
quantified by densitometry using Quantity One?software (BioRad).
and incubatedwith
fluoridetransfer membranes
Transferrin assay for
fluorescence-activated flow cytometry
RT4 cells were transfected with expression constructs and incubated in
growth medium for 24h. NSC-34 cells were additionally differentiated
for 72h. Subsequently, cells were serum-starved for 30min at 37?C,
followed by incubation with 8.4mg/ml Alexa Fluor 647-labelled trans-
ferrin in serum-free Dulbecco’s modified Eagle medium for 15min at
37?C. Cells were washed, acid-stripped (0.2M Na2HPO4, 0.1M citric
acid), detached with 0.25% trypsin (Gibco) and fixed with 4% paraf-
ormaldehyde. After washing and pelleting, the cells were resuspended
in phosphate-buffered saline containing 20mM EDTA, 2% foetal calf
serum and 0.002% NaN3and subjected to flow cytometry. If indi-
cated, dynasore (kindly provided by Dr Ari Helenius) or its solvent
ethanol was added during serum starvation and transferrin incubation.
Data acquisition: FACSCalibur flow cytometer using CellQuest soft-
ware (BD Biosciences). Fluorescence intensity of internalized transferrin
was measured for 2000 GFP (green fluorescent protein)-positive cells.
The results using cells with high GFP expression are shown, if not
indicated otherwise.
Biosynthetic assay
Primary rat Schwann cells were transduced with lentiviral vectors for
96h. Cells were starved in Dulbecco’s modified Eagle medium lacking
L-methionine/L-cysteine (Gibco) for 30min before pulsing with
0.2mCi/ml
additional 20min. If indicated, 120mM dynasore, 10mg/ml brefeldin
A or 0.25% dimethyl sulphoxide were added. Cells were surface
biotinylated followed by cell lysis in RIPA-buffer and incubation with
streptavidin-coupled sepharose overnight at 4?C. Beads were washed,
boiled insamplebufferand
polyacrylamide gel electrophoresis. Gels were dried and analysed by
autoradiography (STORM-800 PhosphorImager; GE Healthcare).
35S-L-methionine/L-cysteine in starvation medium for
resolved by sodiumdodecyl-
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Primary rat Schwann cell surface
proteome analysis
Primary rat Schwann cells were grown to 90% density and 20 million
cells were used. Cell-surface capturing technology was performed as
described previously (Wollscheid et al., 2009; Hofmann et al., 2010).
Briefly, cells were oxidized with sodium metaperiodate on the cell cul-
ture plate at room temperature in the dark for 10min, scraped after
removal of the oxidative reagent and labelled with biocytin hydrazide
at room temperature for 1h. Cells were mechanically lysed in cold
hypotonic buffer, cell membranes enriched by ultracentrifugation,
and solubilized by detergent lysis (0.1% RapiGest) and indirect sonic-
ation. Carbamidomethylation of cysteine-containing proteins was per-
formed [reduction:5mM tris(2-carboxyethyl)phosphine
alkylation: 10mM iodoacetamide] prior to tryptic digestion into pep-
tides. Biotin-labelled N-glycopeptides were affinity enriched using
streptavidin and further released by enzymatic cleavage of the
carbohydrate chain (PnGaseF). Desalted peptide mixtures (Ultra
MicroTIP Columns, The Nest Group) were dried in a vacuum concen-
trator. Priorto analysis, peptides
Chromatography-Mass Spectrometry (LC-MS) grade water supple-
mented with 2% acetonitrile and 0.1% formic acid.
(TCEP),
weresolubilizedin Liquid
Statistical analysis
Data shown represent the average and standard deviation of three
independent experiments. Statistical significance was assessed using a
two-tailed unpaired Student’s
t-test
***P50.0001).
(*P50.05, **P50.01or
Results
CMT-associated Dnm2 mutants impair
myelination whereas CNM-associated
mutants do not
We used dorsal root ganglia cultures to assess functional effects of
Dnm2 disease-causing mutations in a relevant peripheral nerve
model. To this end, mouse embryonic Day 13.5 dorsal root ganglia
were explanted, dissociated and infected with lentiviral vectors
encoding either EGFP (pSicoR), EGFP-tagged wild-type Dnm2,
the artificially generated GTPase-deficient Dnm2 K44A mutant,
selected Dnm2 CMT mutants, or CNM mutants (Fig. 1). After
induction of myelination, immunocytochemistry for neurofilaments
(NF-160; neurons) and myelin basic protein (for myelin) was used
for analyses (Stendel et al., 2010).
We chose to employ dorsal root ganglia explants derived from
embryos carrying a heterozygous Dnm2 null allele (Dnm2wt/0) to
mimic closely the genetic situation in affected patients who carry a
wild-type and a mutant DNM2 allele. In this setting, we found
that expression of the CMT mutant K562E strongly reduced mye-
lination (Fig. 2A). In contrast, expression of the CNM mutant
E560K had no discernable effect, equivalent to uninfected
(data not shown), control-infected (pSicoR) and wild-type-
overexpressing cultures. To determine whether the observed
impairment is common to CMT mutants, we expanded the ana-
lysis to the mutants G358R, G537C, K559?, K562? and L570H.
Indeed, quantitative analysis revealed that all but the K562?
mutation affected myelination (Fig. 2B). Also Dnm2 K44A strongly
impaired myelination, but both CNM mutants tested (R465W and
E560K) had no significant effect.
To test the demyelinating potential of different mutants
(Woodhoo et al., 2009), we infected robustly myelinated dorsal
root ganglia explant cultures of Dnm2wt/0embryos with appropri-
ate recombinant lentiviral vectors. Cultures were kept for an add-
itional 8 days after the start of infection, and myelin was
subsequently assessed by MBP immunostaining. Expression of
the CMT mutant K562E led to myelin damage and induced
myelin loss, in contrast to the CNM mutant E560K, control-
infection (pSicoR) and wild-type-overexpression (Fig. 3A). Quan-
titative analysis revealed reduced myelin in cultures expressing
Dnm2 K44A or both tested CMT mutants (G358R, K562E),
whereas expression of the CNM mutant E560K had no detectable
effect (Fig. 3B).
In our experiments, we found an effect on myelin formation
and maintenance but no apparent alterations in Schwann cell
numbers, neuronal survival or axonal outgrowth (data not
shown). To check for potential effects of Dnm2 mutants on neu-
rons closer, Dnm2wt/0dorsal root ganglia neurons were purified
and cultured. Expression of CMT mutants G358R, G537C, K562E,
L570H or the CNM mutant E560K in these cultures did not
result in neuritic beading or altered microtubule acetylation
(Supplementary Fig. 1). We conclude that CMT mutants have
no overt effects on neurons in our experimental setup.
In the course of carrying out the initial set of myelination
experiments, we found unexpectedly that expression of the
Dnm2 K44A mutant and the CMT mutant K562E had no effect
in our model if dorsal root ganglia from Dnm2wt/wtembryos were
used (Supplementary Fig. 2). These findings indicated that en-
dogenous Dnm2 gene dosage, and presumably different Dnm2
protein expression levels, have a critical influence on the functional
effects of CMT mutants. Thus, we performed a series of studies
that were aimed at examining potential cell type-specific effects of
partial, or full loss of Dnm2 in neurons or Schwann cells in this
experimental context.
To address the impact of Dnm2 loss in dorsal root ganglia neu-
rons, we used transgenic mice expressing Cre recombinase under
the control of sensory neuron-specific NaV1.8 (SNS) gene regula-
tory elements (Agarwal et al., 2004). Loss of protein was deter-
mined in purified neurons obtained from dorsal root ganglia of
SNSCre+; Dnm2flox/floxembryos by immunoblotting. Recombina-
tion was further confirmed in mixed dorsal root ganglia cultures by
polymerase chain reaction (data not shown). First, we used het-
erozygous SNSCre+; Dnm2wt/floxdorsal root ganglia cultures and
lentiviral expression of Dnm2 mutants. No significantly altered
myelination was observed for the Dnm2 K44A mutant, CMT mu-
tants G358R, G537C, K559?, K562E, K562?, L570H and CNM
mutants R465W and E560K (Fig. 4A and B), compared with con-
trol or wild-type overexpression. Note that these observations
differ from our findings with Dnm2wt/0dorsal root ganglia,
where most CMT mutants and the Dnm2 K44A mutant displayed
deleterious effects (Fig. 2). Next, we ablated Dnm2 completely in
sensory neurons. SNSCre+; Dnm2flox/flox
cultures displayed normal levels of myelination compared with
dorsal root ganglia
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control or wild-type-overexpressing cultures, also without detect-
able neuronal defects (Fig. 4C). Expression of Dnm2 K44A and the
CMT mutants G358R, G537C, K559?, K562E and L570H
impaired myelination (Fig. 4D), but the effects were less promin-
ent if compared with the corresponding experiments using
heterozygous Dnm2wt/0dorsal root ganglia cultures (Fig. 2B).
Again, the CNM mutants R465W and E560K showed no effects.
Incomplementaryexperiments,
specifically in Schwann cells using transgenic mice in which Cre
expressionis controlled by
Dhh gene regulatory
we ablated
Dnm2
elements
Figure 2 CMT-causing Dnm2 mutants impair myelination of heterozygous Dnm2wt/0dorsal root ganglia explant cultures. (A) Dorsal root
ganglia explants dissected from Dnm2wt/0embryos were infected with lentivirus encoding GFP alone (pSicoR), or EGFP-tagged Dnm2
(wild-type), CMT mutant K562E, or CNM mutant E560K. Confocal projection images of Dnm2 K562E (CMT)-expressing cultures show
strongly reduced myelination as assessed by myelin basic protein (MBP) immunostaining 12 days after induction of myelination. Control,
Dnm2 wild-type and E560K (CNM)-infected cultures display normal myelination. A comparable, extensive neuritic network was present in
all conditions (immunostaining for neurofilaments; NF-160). (B) Quantification of MBP staining revealed significantly reduced myelination
for cultures expressing Dnm2 CMT mutants (except for K562?) and for Dnm2 K44A. No significant alterations were found for CNM
mutants R465W and E560K in comparison with controls (wild-type and pSicoR). Values represent means ? SD of three independent
experiments: *P50.05, **P50.01, Student’s t-test. Scale bars: 75mm.
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(Jaegle et al., 2003). We first examined dorsal root ganglia cultures
from DhhCre+; Dnm2wt/floxembryos toevaluate if loss ofone Dnm2
allele in Schwann cells is sufficient to perturb myelination (Fig. 5A).
Dorsal root ganglia cultures from this genotype myelinated normally,
as did wild-type-overexpressing cultures. Myelination was impaired,
however, by CMT mutants G358R, G537C, K559?, K562E, L570H
and Dnm2 K44A (Fig. 5B). In contrast, the CNM mutants R465W
and E560K, as well as the CMT mutant K562? had no significant
effect. These results are strikingly similar to the data obtained with
Dnm2wt/0dorsal root ganglia cultures (Fig. 2B). Thus, the reduced
myelination observed in the latter experiments is most likely due to
the reduced Dnm2 levels specifically in Schwann cells.
Figure 3 CMT-causing Dnm2 mutants cause demyelination in heterozygous Dnm2wt/0dorsal root ganglia explant cultures. (A) Dorsal
root ganglia explants dissected from Dnm2wt/0embryos were induced to myelinate for 12 days (uninfected culture at day of infection;
left). Sister plates of myelinated cultures were infected on the 12th day of culture with lentivirus encoding GFP alone (pSicoR),
EGFP-tagged Dnm2 (wild-type), CMT mutant K562E or CNM mutant E560K. Confocal projection images of Dnm2 K562E (CMT)-
expressing cultures show aberrant myelin structures including signs of myelin swellings (arrows) and degradation (arrowheads), assessed
by myelin basic protein (MBP) immunostaining 8 days after infection. The neuritic network (NF-160 immunostaining) was not detectably
altered in all conditions. (B) MBP staining revealed significantly reduced MBP-fluorescence intensities for cultures expressing the Dnm2
CMT mutants G358R, K562E and for Dnm2 K44A. No significant alteration was found for CNM mutant E560K in comparison with
controls (wild-type and pSicoR). Values represent means ? SD of three independent experiments: *P50.05, Student’s t-test. Scale bars:
75mm.
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Next, we examined dorsal root ganglia cultures with complete
Dnm2 ablation in Schwann cells (DhhCre+; Dnm2flox/flox). Unin-
fected (data not shown) and control-infected dorsal root ganglia
cultures of this genotype failed totally to myelinate (Fig. 5C and
D). The defect could be rescued, however, by overexpression of
Dnm2 wild-type. This finding is important since it shows that the
loss of Dnm2 in Schwann cells had not damaged the cultures
irreversibly. In contrast to Dnm2 wild-type, CMT mutants
G358R, G537C, K559?, K562E, L570H and Dnm2 K44A were
unable to rescue myelination (Fig. 5C and D). The CNM mutants
R465W and E560K, however, rescued myelination analogously to
Dnm2 wild-type protein. The CMT mutant K562? showed partial
rescue activity. Interestingly, we found also that overexpression of
Dnm1 or Dnm3 was able to restore myelination in DhhCre+;
Dnm2flox/floxcultures (Supplementary Fig. 3). Thus, the missing
molecular function(s) of Dnm2 in this experimental setting is
shared by all three dynamin isoforms.
Taken together, we have shown that Dnm2 CMT—but not
CNM-mutants have a destructive effect in a peripheral nerve dis-
ease model. Furthermore, Schwann cell-expressed Dnm2 is
required for myelination, whereas Dnm2 expression by sensory
neurons is dispensable. Schwann cell-specific ablation of Dnm2
rendered the peripheral nerve model more sensitive to CMT mu-
tants than analogous ablation, specifically in sensory neurons. In
both cell types, the effect of CMT mutants is dependent on the
endogenous Dnm2 gene dosage.
Figure 4 Dnm2 CMT mutants have a minor detrimental effect on myelination of dorsal root ganglia explants with specifically altered
Dnm2 gene dosage in sensory neurons. (A and B) Heterozygous ablation of Dnm2 specifically in sensory neurons (SNSCre+; Dnm2wt/flox)
revealed no significantly altered myelination [myelin basic protein (MBP) staining] in dorsal root ganglia explant cultures upon expression
of Dnm2 CMT and CNM disease mutants compared with control-infected (pSicoR) and wild-type-infected settings. (C) In dorsal root
ganglia explant cultures homozygously deleted for Dnm2 in sensory neurons (SNSCre+; Dnm2flox/flox), myelination was indistinguishable
between uninfected (not shown), control-infected and wild-type-transduced cultures. Expression of CMT mutant K562E impairs mye-
lination, whereas CNM mutant E560K has no influence. Immunostaining for the neuritic network (NF-160) revealed no differences in all
conditions. (D) MBP staining was decreased significantly in cultures expressing Dnm2 K44A and CMT mutants G358R, G537C, K559?,
K562E and L570H, but not for CNM mutants R465W and E560K; and K562? (CMT). Quantified values represent means ? SD of three
independent experiments: *P50.05, **P50.01, Student’s t-test. Scale bars: 75mm. WT = wild-type.
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Inhibition of clathrin-mediated
endocytosis by Dnm2 CMT-causing
mutants
Dnm2 has a well-known critical function in endocytotic processes,
and CMT and CNM mutants impaired clathrin-mediated endo-
cytosis in various heterologous cell lines (Altschuler et al., 1998;
Zuchner et al., 2005; Bitoun et al., 2009; Durieux et al., 2010).
We hypothesized, partially based on our results with dorsal root
ganglia cultures, that the effects of the different mutations may be
cell type-specific with regard to endocytosis. Thus, we investigated
the consequences of mutant Dnm2 protein expression on endo-
cytotic pathways in disease-relevant Schwann cells and differen-
tiated mouse motor neuronal NSC-34 cells. To obtain definitive
quantitative data, we established an uptake assay with labelled
transferrin tomeasureclathrin-mediated
fluorescence-activated flow cytometry. Alexa Fluor 647-labelled
transferrin was measured in correlation to protein (EGFP) levels.
This experimental set up allowed us to functionally characterize
endocytosisusing
Figure 5 Schwann cells require Dnm2 for myelination and reduced endogenous Dnm2 gene dosage in Schwann cells renders dorsal root
ganglia cultures highly susceptible to harmful effects of CMT mutants. (A) Myelination [myelin basic protein (MBP) staining] of dorsal root
ganglia explants with a DhhCre+; Dnm2wt/floxbackground was reduced by expression of the Dnm2 CMT mutant K562E, but not by the
CNM mutant E560K, compared with control (pSicoR) or WT-infected cultures. (B) MBP staining was significantly decreased in cultures
expressing Dnm2 K44A and CMT mutants G358R, G537C, K559?, K562E and L570H. Dnm2 K562? (CMT) and CNM mutants R465W
and E560K did not have a significant influence. (C) Complete ablation of Dnm2 in Schwann cells (DhhCre+; Dnm2flox/floxgenotype)
resulted in loss of myelination as revealed by myelin basic protein staining in pSicoR control-infected cultures. This effect was rescued by
expression of Dnm2 WT and CNM mutant E560K, whereas the expression of the Dnm2 CMT mutant K562E was not able to rescue the
defect. The neuritic network (NF-160 immunostaining) was not altered in all conditions. (D) Dnm2 K44A and CMT mutants G358R,
G537C, K559?, K562E and L570H showed virtually no rescue capabilities, whereas expression of the CMT mutant K562? led to partial
rescue. Expression of CNM mutants R465W or E560K was able to fully rescue the defect, comparable with WT expression. Data represent
means ? SDofthreeindependentexperiments:*P50.05,**P50.01,***P50.001,Student’st-test.Scalebars:75mm.WT = wild-type.
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the effects of disease-causing mutations on clathrin-mediated
endocytosis compared with transgene expression levels (Supple-
mentary Fig. 4A). We validated dynamin-dependent internaliza-
tion of transferrin in our experimental system using dynasore, a
potentinhibitoroflarge GTPases
Dynasore-treated Schwann cells (rat Schwannoma cell line RT4)
showed reduced transferrin uptake from 80mM (25 ? 2.9%) to
120mM (80 ? 4.9%) in a concentration-dependent manner com-
pared with solvent-treated (ethanol) cells (Supplementary Fig. 4B).
For the analysis of Dnm2 mutants, we first expressed disease-
associated Dnm2 mutants in differentiated motor neurons (cell
line NSC-34). Qualitative analyses by confocal microscopy re-
vealed strongly reduced transferrin uptake by cells expressing
the CMT mutant K562E, whereas the CNM mutant E560K had
no detectable defect compared with the control and wild-type
situation (Fig. 6A). We extended the analyses to groups of CMT
and CNM mutants using quantitative flow cytometry (Fig. 6B).
The CMT mutants G358R, G537C, K559?, K562E, K562?,
L570H and Dnm2 K44A showed impaired clathrin-mediated endo-
cytosis, whereas all 11 CNM mutants analysed in parallel did not
alter transferrin uptake. Next, we carried out the same experi-
ments in Schwann cells (RT4 cell line). Again, CMT mutants im-
paired transferrin uptake, with the exception of K562? (Fig. 6C;
note that the same mutant had already shown a peculiar behav-
iour with effects ranging from ‘weak to not significant’ in dorsal
root ganglia cultures). As in the motor neuron cell line, CNM
mutants did not evoke detectable alterations of transferrin
uptake in the Schwann cell line (Fig. 6C). Selected mutants were
also tested qualitatively in rat primary Schwann cells using labelled
transferrin and confocal microscopy. Dnm2 K44A and the CMT
mutants G358R, G537C, K562E and L570H inhibited transferrin
internalization. In contrast, the CNM mutant E560K showed no
detectable effect (Supplementary Fig. 5). These results confirm the
data obtained with RT4 cells.
Decreased cell surface levels of transferrin receptor may also
contribute to reduced transferrin uptake. To investigate this
issue, we transfected RT4 cells transiently with EGFP-tagged
Dnm2 disease-associated mutants and examined the cells 24h
later by live cell immunostaining and qualitative confocal micros-
copy for transferrin receptor cell surface levels. Control, Dnm2
wild-type and CNM mutant E560K-transfected cells revealed a
comparable punctuated cell surface transferrin receptor staining,
whereas expression of the CMT mutant K562E increased this
staining (Fig. 7A). We extended the analysis to additional CMT
and clathrin-mediated endocytosis mutants and quantified trans-
ferrin receptor cell surface levels by flow cytometry. Overall, none
of the tested mutants reduced the transferrin receptor surface
levels. The CMT mutants G358R, G537C, K562E and Dnm2
K44A even induced a significant increase of relative transferrin
receptor cell surface levels compared with Dnm2 wild-type
transfected cells (Fig. 7B). We interpreted these results in that
elevated transferrin receptor cell surface levels are due to receptor
trapping on the surface by impaired endocytosis. Alternatively,
transport of membrane proteins to the cell surface could be
affected in general. To examine this possibility, we infected rat
primary Schwann cells with lentiviral vectors to achieve expression
of mutants and controls. After 4 days, the cells were pulse-labelled
(Macia
etal., 2006).
with
surface biotinylation and purification of biotinylated proteins.
35S-labelledproteins (input)
35S-labelled proteins (beads) were analysed by sodium dodecyl sul-
phate–polyacrylamide gel electrophoresis (Supplementary Fig. 6A),
detected by autoradiography and quantified. The amounts of
labelled cell surface proteins in relation to totally labelled proteins
were determined (Supplementary Fig. 6B). We found no signifi-
cant differences between cells that expressed Dnm2 K44A, CMT
mutants (G358R, K562E) or the CNM mutant E560K compared
with control or wild-type overexpressing cells indicating no overt
alteration of transport of the newly synthesized proteins to the
plasma membrane. In contrast and as expected, brefeldin A and
dynasore-treated cells reduced trafficking of newly synthesized
proteins to the plasma membrane compared with controls
(Supplementary Fig. 6C and D).
We conclude that in Schwann cells, CMT but not CNM mutants
inhibitclathrin-mediated endocytosis
transport of cell surface proteins to the plasma membrane.
Furthermore, impaired transferrin uptake appears not to be the
result of decreased transferrin receptor surface levels, but the con-
sequence of direct inhibition of clathrin-mediated endocytosis as
shown before for Dnm2 K44A (Altschuler et al., 1998).
35S-L-methionine/L-cysteine for 20min followed by cell
andcell surface-biotinylated
without affectingthe
CMT-associated Dnm2 mutations alter
membrane receptor surface level of
proteins essential for Schwann cell
biology
Schwann cells require precise amounts of cell surface receptors for
correct migration, myelination, survival and interaction with the
accompanying axon (Pereira et al., 2012). Such processes are
likely disturbed by Dnm2 mutations in dominant-intermediate
CMT subtype B. Thus, we hypothesized that besides the transfer-
rin receptor, other surface proteins might be affected by impaired
clathrin-mediated endocytosis due to Dnm2 mutations. We
therefore first determined the rat primary Schwann cell surface
glycoproteome with an antibody-independent, mass spectrome-
try-based cell-surface capturing approach (Wollscheid et al.,
2009; Hofmann et al., 2010). In total, 211 glycoproteins of the
rat primary Schwann cell surface proteome were identified
(Supplementary Fig. 7A and Supplementary Table 3). Out of
these, integrin b1 (ITB1_RAT, CD29) and the receptor tyrosine-
protein kinase ErbB-2 (ERBB2_RAT, CD340) were further ana-
lysed. We selected integrin b1 as the first potentially Dnm2
mutant-affected protein, since ?6b1-integrin is internalized via a
dynamin-dependent pathway in colonic epithelial cells (Vassilieva
et al., 2008), and Schwann cell-expressed integrin b1is critical for
peripheral nerve function (Berti et al., 2006). Rat primary Schwann
cells were infected with lentiviral vectors encoding EGFP (pSicoR),
Dnm2 wild-type or the CMT mutant K562E, followed 4 days later
by cell surface biotinylation and purification of biotinylated plasma
membrane proteins. We found that surface integrin b1levels were
significantly increased on K562E expressing rat primary Schwann
cells compared with wild-type over-expressing cells or controls
(Fig. 7C). Parallel staining with Cy3-coupled streptavidin of
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Figure 6 Dnm2 CMT mutants impair clathrin-mediated endocytosis in motor neurons and Schwann cells, whereas CNM mutants have no
effect. (A) NSC-34 motor neurons were transfected in proliferative conditions to express EGFP, EGFP-tagged wild-type, or mutant Dnm2
proteins. After 24h, cells were differentiated for an additional 72h. After serum starvation, transferrin uptake (20mg/ml Alexa Fluor
555-labelled transferrin) was allowed (15min), followed by immunostaining for the neuronal marker Tuj-1. Confocal images illustrate an
inhibition of transferrin uptake for cells expressing Dnm2 K562E (CMT) (arrowheads) compared with EGFP control-transfected and
EGFP-Dnm2 wild-type expressing cells (arrows). Expression of the CNM mutant E560K does not inhibit transferrin uptake (arrow). Scale
bars: 15mm. (B) Quantification by fluorescence-activated flow cytometry revealed impaired transferrin uptake by Dnm2 mutant K44A and
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untreated and detergent-treated cells expressing EGFP-tagged
Dnm2 wild-type validated the experimental setting (Supplemen-
tary Fig. 7B). Next, we examined ErbB2, an essential regulator of
Schwann cell–axon interactions (Newbern and Birchmeier, 2010),
which is internalized in a clathrin-dependent manner (Michailov
et al., 2004; Sorkin et al., 2008). Using the same basic experi-
mental set up as for the integrin b1studies, the timing of ErbB2
internalization after stimulation with NRG-1 type III was deter-
mined (Supplementary Fig. 7C). A total of 1h of stimulation
was established to be optimal for our purpose. Under these con-
ditions, the levels of ErbB2 on the cell surface of Dnm2 K44A or
CMT mutant K562E expressing cells were significantly higher than
on control- or wild-type over-expressing cells (Fig. 7D). These
results demonstrate an important role for Dnm2-dependent recep-
tor internalization in Schwann cells that is altered by the CMT
mutant K562E.
Clathrin-mediated endocytosis is
essential for myelination of dorsal root
ganglia cultures
Our data indicated an inhibition of clathrin-mediated endocytosis
by CMT mutants. Thus, we asked whether altered endocytosis is
sufficient to explain the impairment of myelination in our periph-
eral nerve model (dorsal root ganglia cultures). We decided to
approach this question by knocking down the expression of the
specific clathrin adaptor at the plasma membrane, AP-2 subunit m2
(AP-2), to inhibit clathrin-mediated endocytosis (Motley et al.,
2003). For this purpose, we developed two short hairpin RNAs
targeting AP-2. Both of them, after delivery into rat primary
Schwann cells with lentiviral vectors, resulted in significant
decrease of AP-2 protein levels 4 days post-infection (Fig. 8A).
As expected, AP-2 knock-down resulted in severe inhibition of
transferrin uptake as qualitatively analysed by confocal microscopy
(data not shown). These results were confirmed and quantified by
flow cytometry in RT4 cells (Fig. 8B). Cell surface biotinylation of
pulse
cells revealed that AP-2 knock-down does not influence transport
of cell surface proteins to the plasma membrane (Fig. 8C and D).
We used the established AP-2 short hairpin RNAs in dorsal root
ganglia cultures to test for effects of AP-2 knock-down on mye-
lination. Dorsal root ganglia cultures infected with lentiviral vectors
encoding the AP-2 targeting short hairpin RNA 747 survived well,
but failed to myelinate, whereas control DsRed2 short hairpin
RNA-infected cultures myelinated normally (Fig. 8E). Cultures in
which the more efficient AP-2 targeting short hairpin RNA 748
(Fig. 8A) was used did not survive, indicating that a too strong
reduction of clathrin-mediated endocytosis is not tolerated in this
35S-L-methionine/L-cysteine-labelled rat primary Schwann
experimental setting (data not shown). This interpretation is sup-
ported by comparable experiments employing short hairpin RNAs
against clathrin, in which the cultures also did not survive (data
not shown). We conclude that proper functional clathrin-mediated
endocytosis is required for myelination in our model. These find-
ings are consistent with the observation that CMT-associated
Dnm2 mutants that affect clathrin-mediated endocytosis in cul-
tured neurons and Schwann cells are also deleterious in dorsal
root ganglia myelination cultures.
Discussion
Dnm2 is a ubiquitously expressed protein with various cellular
functions. Despite these pleiotropic roles, DNM2 mutations are
associated with distinct diseases and affect a limited number of
cell types such as Schwann cells and/or neurons of the PNS in
dominant-intermediate CMT subtype B, or muscle cells in auto-
somal dominant CNM. In this study, we compared the effects of
CMT and CNM mutants in a PNS model and in cell cultures of
PNS-derived cells to elucidate disease mechanisms in dominant-
intermediate CMT subtype B and to address the intriguing ques-
tion of why CNM mutants do not affect the PNS.
We established a disease-related peripheral nerve model using
tissue from Dnm2wt/0transgenic mice and showed that expression
of CMT mutants had strikingly harmful effects, whereas expres-
sion of CNM mutants was without consequences. These results
validated the model for additional investigations. Importantly, fur-
ther analyses discovered that changes in endogenous Dnm2 gene
dosage have a profound influence. In particular, expression of
CMT mutants on a Dnm2wt/wtbackground did not cause defects
demonstrating that the endogenous gene dosage modulates the
consequences of CMT mutants. Further analyses using cell type-
specific modulation of endogenous gene dosage revealed that
such alterations in Schwann cells had a more pronounced effect
compared with sensory neurons. However, the system was sensi-
tive to such changes in both cell types with respect to the effects
of CMT mutants. Complete ablation of Dnm2 in Schwann cells
caused a total loss of myelin formation demonstrating that
Schwann cell Dnm2 function is indispensable for myelination.
Re-expression of Dnm2 rescued the phenotype, while CMT mu-
tants could not. Expression of CNM mutants, however, was able
to rescue myelination efficiently indicating that CMT mutant pro-
teins have lost an essential PNS function that CNM mutants can
still provide. Interestingly, expression of Dnm1 or Dnm3 was also
able to rescue myelination in this experimental setting indicating
that the critical Dnm2 function is shared by the dynamin isoforms.
The cellular and molecular mechanisms underlying the observed,
at least partially cell type-specific, gene dosage sensitivities remain
Figure 6 Continued
by all tested CMT mutants compared with the wild-type overexpression setting. CNM mutants did not alter transferrin uptake. (C) RT4
Schwann cells were transfected to express the proteins indicated and 24h later, transferrin uptake was analysed as described above using
flow cytometry. Transferrin uptake was reduced by the expression of Dnm2 K44A and CMT mutants G358R, G537C, 551?3, K559?,
K562E and L570H. Dnm2 K562? (CMT) and all CNM mutants tested had no significant effect. Values represent means ? SD of three
independent experiments: *P50.05, **P50.01, ***P50.001, Student’s t-test. WT = wild-type.
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Figure 7 Transferrin receptor-, ErbB2- and integrin b1cell surface levels are elevated on Schwann cells expressing Dnm2 CMT mutants.
(A) RT4 Schwann cells were transfected with EGFP-tagged Dnm2, Dnm2 mutants, or control constructs followed by live cell transferrin
receptor immunostaining 24h later. Confocal images of control (pEGFP), Dnm2 wild-type (WT) and CNM mutant E560K-expressing cells
show a punctuate transferrin receptor cell surface pattern (arrows). Expression of Dnm2 CMT mutant K562E results in an increased cell
surface level of transferrin receptor (arrowhead). Scale bars: 15mm. (B) Transferrin receptor cell surface amounts were quantified by flow
cytometry on cells with mid-range expression of exogenous proteins (Supplementary Fig. 4). Transferrin receptor cell surface levels were
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to be elucidated. We speculate, however, that endogenous ex-
pression levels of the classical dynamin family members in the
presence of Dnm2 mutants are likely to be a key issue.
Previous studies have suggested, based on analyses in fibro-
blasts, that both CMT (K562E) and CNM (R465W, V625?,
E650K) mutants impair clathrin-mediated endocytosis (Bitoun
et al., 2009; Durieux et al., 2010). We extended these studies
and found that in HeLa cells, none of the tested CNM mutants
(E368K, E368Q, E369Q, E369W, R465W, E560K, A618T, S619L,
S619W, V625? and E650K), including the mutants tested in fibro-
blasts previously, inhibited clathrin-mediated endocytosis even if
highly expressed (data not shown). In contrast, some CMT mu-
tants (G358R, G537C, K559? and K562E) significantly inhibited
clathrin-mediated endocytosis while others (D555_E557?, K562?
and L570H) did not (data not shown). The variability of these
data, obtained using heterologous, non-disease-relevant cell
types, indicates the importance of examining the effects of
Dnm2 mutants in appropriate cellular settings, since these mutants
appear toaffectclathrin-mediated
depending on the cell type used for analysis. Thus, we focused
our experiments on cell lines and primary cells that originate from
CMT-related tissue. As in HeLa cells, we found that CMT mutants
G358R, G537C, K559?, K562E and additionally L570H impair
clathrin-mediated endocytosis in Schwann cells and in motor
neurons, whereas CNM mutants do not show a detectable
effect. Interestingly, the CMT mutant K562? had no significant
impact on clathrin-mediated endocytosis in Schwann cells and only
a minor effect in motor neurons suggesting that this mutant has
different characteristics compared with the bulk of the CMT mu-
tants. These data are consistent with the divergent effects of the
K562? mutant compared with the other CMT mutants in our
peripheral nerve model, and warrant further investigations into a
mutant-specific disease mechanism. However, we found that CMT
and CNM mutants have distinct consequences on clathrin-
mediated endocytosis in cells originating from the PNS; CMT
mutants affect clathrin-mediated endocytosis in CMT-relevant
cell types, whereas CNM mutants have no effect. The CMT
mutant-specific effect on clathrin-mediated endocytosis in cells
derived from the PNS are coherent with the fact that CMT
mutants lead to dominant-intermediate CMT subtype B and indi-
cate that altered clathrin-mediated endocytosis contributes to the
endocytosisdifferently,
dominant-intermediate CMT subtype B disease mechanism. In fur-
ther support of this hypothesis, we demonstrated that short hair-
pin RNA-mediated knock-down of the plasma membrane-specific
clathrin adaptor AP-2 is sufficient to inhibit myelination in dorsal
root ganglia cultures comparable with the effects caused by CMT
mutants. We conclude that clathrin-mediated endocytosis is critical
for proper peripheral nerve myelination and defects in this cellular
function, caused by CMT mutants, are likely to contribute to the
observed PNS pathophysiology in dominant-intermediate CMT
subtype B.
Wehave focusedourstudies
CMT subtype B and the PNS. With regard to CNM mutants,
our data indicate that they do not cause CMT, since they do
not affect clathrin-mediated endocytosis in cells of the PNS.
The findings that CNM mutants, but not CMT mutants, are
able to rescue the phenotype of Dnm2 loss in Schwann cells
in our peripheral nerve model lend further support to this
notion. In contrast, CNM mutants have been shown to alter
clathrin-mediated endocytosis in fibroblast, whereas we have
not found such effects in HeLa cells. These divergent results
may be explained by the specific cell biology of epithelial-like
HeLa cells that are more closely related to polarized cells of the
PNS including myelinating Schwann cells (Ozcelik et al., 2010)
than fibroblasts and myoblasts. Taken together, it is conceivable
butspeculativethat CNM
clathrin-mediated endocytosis in skeletal muscle cells by a com-
bined mutant- and cell type-specific mechanism that contributes
to the disease. However, other mechanisms are also possible
(Kenniston et al., 2010; Wang et al., 2010) and thus, the ques-
tion of how CNM mutants cause a specific disease in skeletal
muscles remains open.
The elucidation of disease mechanisms is of considerable value
to learn more about the basic biology of cells and tissues, but such
studies also ultimately aim at providing suitable targets for thera-
peutic strategies. In this respect, clathrin-mediated endocytosis is a
rather pleiotropic pathway and difficult to target without the
expectation of major side effects. Though, specific plasma mem-
brane proteins with critical PNS functions are likely to be affected
and to contribute to the disease development. As a prerequisite to
identify such candidates, we employed the mass spectrometry-
based cell surface capturing strategy to generate a snapshot
ondominant-intermediate
mutantsmightalso affect
Figure 7 Continued
found to be significantly increased by the expression of Dnm2 K44A and Dnm2 CMT mutants G358R, G537C and K562E compared with
cells expressing Dnm2 wild-type. Values represent means ? SD of three independent experiments: *P50.05, Student’s t-test. (C) To
examine cell surface levels of b1-integrin, rat primary Schwann cells were control-infected (pSicoR), or infected with lentivirus encoding
Dnm2 wild-type or K562E (CMT) for 96h, followed by surface biotinylation and purification of biotinylated proteins (beads). Western blot
analysis revealed elevated integrin b1cell surface levels for cells expressing Dnm2 CMT mutant K562E compared with control and wild-
type infected conditions. ?-Tubulin served as negative control for the purification of surface proteins. The graph represents values of cell
surface integrin b1levels (beads) in relation to total (input) amounts as means ? SD of three independent experiments: *P50.05,
Student’s t-test. (D) To examine clathrin-mediated endocytosis-dependent internalization of ErbB2, rat primary Schwann cells were either
control-infected (pSicoR), or infected with lentivirus encoding Dnm2 WT, K44A, or K562E (CMT) and incubated for 96h. Cells were
serum-starved for 4h and stimulated with 20ng/ml NRG-1 type III for 1h, followed by surface biotinylation. Dnm2 K44A and K562E
(CMT) decreased ErbB2 internalization significantly compared with Dnm2 WT as indicated by increased cell surface levels of ErbB2.
GAPDH served as negative control for surface biotinylation. Quantitation represents means ? SD of three independent experiments:
**P50.01, Student’s t-test.
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Figure 8 Expression knock-down of the plasma membrane-specific clathrin adaptor protein AP-2 impairs clathrin-mediated endocytosis
in Schwann cells and prevents myelination of dorsal root ganglia cultures. (A) Rat primary Schwann cells were infected with lentiviral
vectors (Ubc-TurboGFP) expressing AP-2 (m2)-targeting short hairpin RNA 747 and short hairpin RNA 748 that result in AP-2 (m2) protein
knock-down with different efficiencies in comparison with control-infected (short hairpin RNA targeting DsRed2) cells [4 days
post-infection (dpi)] as shown by immunoblotting. GAPDH served as loading control. Graph shows relative AP-2 protein expression levels
(means ? SD of three independent infections: ***P50.001, Student’s t-test). (B) Transferrin uptake was inhibited in RT4 Schwann cells
expressing short hairpin RNAs against AP-2. On completing 4 days after transfection with short hairpin RNA expression constructs, also
expressing EGFP under a separate promoter (Ubc-TurboGFP), cells were serum-starved and incubated with Alexa Fluor 647-labelled
transferrin. Quantification by flow cytometry showed that transferrin uptake was significantly reduced for both short hairpin RNAs
targeting AP-2 compared with control short hairpin RNA. Values represent means ? SD of three independent experiments: **P50.01,
Student’s t-test. (C and D) Rat primary Schwann cells infected with lentiviral vectors (Ubc-TurboGFP) expressing AP-2 (m2)-targeting short
hairpin RNA 747 and short hairpin RNA 748 were pulse labelled with35S-L-methionine/L-cysteine for 20min, 4days post-infection
followed by surface biotinylation. Purified biotinylated surface proteins (beads) and total labelled proteins (input) were analysed by sodium
DNM2 mutations in CMTBrain 2012: Page 15 of 17 |
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