Mouse model of CMT2L
AbstractWe previously found that the K141N mutation in heat shock protein B8 (HSPB8) was responsible
for Charcot-Marie-Tooth disease type 2L in a large Chinese family. The objective of the
present study was to generate a transgenic mouse model bearing the K141N mutation in the
human HSPB8 gene, and to determine whether this K141NHSPB8 transgenic mouse model would
manifest the clinical phenotype of Charcot-Marie-Tooth disease type 2L, and consequently be
suitable for use in studies of disease pathogenesis. Transgenic mice overexpressing K141NHSPB8
were generated using K141N mutant HSPB8 cDNA cloned into a pCAGGS plasmid driven by a
human cytomegalovirus expression system. PCR and western blot analysis confirmed integration
of the K141NHSPB8 gene and widespread expression in tissues of the transgenic mice. The
K141NHSPB8 transgenic mice exhibited decreased muscle strength in the hind limbs and impaired
motor coordination, but no obvious sensory disturbance at 6 months of age by behavioral assessment.
Electrophysiological analysis showed that the compound motor action potential amplitude
in the sciatic nerve was significantly decreased, but motor nerve conduction velocity remained
normal at 6 months of age. Pathological analysis of the sciatic nerve showed reduced myelinated
fiber density, notable axonal edema and vacuolar degeneration in K141NHSPB8 transgenic mice,
suggesting axonal involvement in the peripheral nerve damage in these animals. These findings
indicate that the K141NHSPB8 transgenic mouse successfully models Charcot-Marie-Tooth disease
type 2L and can be used to study the pathogenesis of the disease.
Key Words: nerve regeneration; peripheral nerve injury; axonal injury; animal models; Charcot-Marie-
Tooth disease type 2L; gene mutation; pronuclear injection; transgenic model; small heat shock
protein B8; NSFC grant; neural regeneration
Charcot-Marie-Tooth disease is the most common inherited
peripheral neuropathy, with an estimated prevalence of 17–
40/10,000[1-2]. Charcot-Marie-Tooth is characterized by distal
muscle weakness and atrophy, distal sensory loss, depressed
tendon reflexes, and pes cavus. There are two main forms:
the demyelinating type 1 (Charcot-Marie-Tooth disease type
1) and the axonal type 2 (Charcot-Marie-Tooth disease type
2). Autosomal-dominant inheritance is most common, but
X-linked and autosomal-recessive forms are also seen[1-3].
There is considerable genetic heterogeneity, and more than
40 genes have been identified (https://neuromuscular.wustl.
edu/time/hmsn.html); however, the underlying pathogenetic
mechanisms remain unclear[4-5].
Tang et al. identified an autosomal dominant familial
form of Charcot-Marie-Tooth disease type 2 in a Chinese
family, linked to 12q24 by genome-wide screening, and
designated Charcot-Marie-Tooth disease type 2L (OMIM
Number 608673). A positional candidate cloning study
showed that the G423T (K141N) mutation in the small
heat shock protein B8 (HSPB8) gene co-segregated with the
Charcot-Marie-Tooth disease type 2L phenotype, and that it
was the causative mutation. The expression of K141NHSPB8
in SHSY5Y cells results in intracellular aggregates that are
mainly distributed in the cytoplasm and that are colocalized
with HSPB1 and neurofilament light polypeptide
To provide insight into the molecular pathogenesis of
the disease, in the present study, the transgenic plasmid
pCAGGS-HA-K141NHSPB8 was constructed, and K141NHSPB8
transgenic mice were generated by pronuclear injection.
Behavioral, electrophysiological, and pathological analyses
were performed on K141NHSPB8 transgenic mice to deter-mine whether they manifest the clinical phenotype of Charcot-
Marie-Tooth disease type 2L and would be suitable for
studies on disease pathogenesis.
Generation of K141NHSPB8 transgenic mice
We used PCR combined with sequencing for genotyping.
The amplification yielded a 976-bp genomic fragment with
primers CAG1 and CAG2, and a 516-bp genomic fragment
with primers CAG1 and HSP (Figure 1A–C). Sequencing of
the 976-bp amplification product showed the presence of
the G423T mutation when aligned with the HSPB8 sequence
(Figure 1C). Western blot assay revealed extensive expression
of the HA-tagged 22-kDa human HSPB8 in heart and
gastrocnemius muscle of founder mice (Figure 1D). Three
founder males were generated and bred normally to establish
lines of K141NHSPB8 transgenic mice.
Quantitative analysis of the experimental mice
Nine 6-month-old K141NHSPB8 transgenic mice, and nine agematched
C57BL mice, as control, were used for behavioral
assessments, and data from all mice were included in the final analyses. Three of the nine 6-month-old K141NHSPB8 transgenic
mice and three age-matched C57BL mice were used for sciatic
nerve electrophysiological and histopathological studies.
Impaired motor performance in K141NHSPB8 transgenic
All K141NHSPB8 transgenic mice were normal at birth and
showed normal weaning and grooming behavior. The frequency
at birth of the various genotypes exhibited a normal
Mendelian inheritance pattern. At 6 months of age, the
mice displayed an unsteadiness of gait, reminiscent of the
“steppage” gait in Charcot-Marie-Tooth disease patients.
Tremor and seizures were absent. When walking over a flat
surface in a straight line, the transgenic mice moved clumsily
and displayed outward positioning of their hind paw
with a significantly more open hind paw angle than wild
type mice (P < 0.05; Figure 2A–C). In the fixed-bar test,
all the wild type mice balanced and supported their body
weight easily, and moved agilely on the bar. In contrast, the
transgenic mice either remained motionless or crawled with
difficulty using only their forelimbs, dragging their hind
limbs behind. Two of the transgenic mice dropped off the
bar in all eight consecutive trials within 120–240 seconds. Inthe rotarod test, the K141NHSPB8 transgenic mice remained on
the rotating roller for a significantly shorter period of time
than wild type mice (P < 0.05; Figure 3). However, in the
pain threshold experiment involving plantar electric shocks,
there were no significant differences between K141NHSPB8
mice and age-matched wild type mice.
Electrophysiological alterations in the proximal and distal
sciatic nerve in K141NHSPB8 transgenic mice
K141NHSPB8 transgenic mice showed a significant decrease
in the peak-to-peak amplitude of compound motor action
potentials in both proximal and distal sciatic nerve at the age
of 6 months, confirming the axonal basis of the motor neuropathy
(P < 0.01; Figure 4, Table 1). Motor nerve conduction
velocity was normal, indicating that the myelin sheath
remained basically intact (Table 1).
Histopathological alterations in the distal portion of the
sciatic nerve in K141NHSPB8 transgenic mice
Semithin sections of the distal portion of the sciatic nerve
from 6-month-old K141NHSPB8 transgenic mice revealed a
decrease in the number of axons compared with wild type
control mice (P < 0.05; Figure 5A–C). No signs of demyelination
or remyelination were observed. Under the electron
microscope, ultrathin sections of the sciatic nerve showed
the presence of notable axonal edema and degeneration, although
the myelin sheath remained relatively intact (Figure
In the present study, we created a transgenic mouse model
of Charcot-Marie-Tooth disease type 2L by overexpressing
human HSPB8 containing the K141N mutation. The ubiquitous-
expression pCAGGS plasmid, driven by the human
cytomegalovirus expression system, has proven to be suitable
for the creation of transgenic mouse models. Therefore,
we chose this plasmid for the generation of the K141NHSPB8
transgenic mice[10-12]. PCR and western blot assays on the
K141NHSPB8 transgenic founder mice confirmed that the human
G423T (K141N) mutant HSPB8 gene had successfully
integrated into the mouse chromosome and was widely expressed.
Footprint analysis, a behavioral assessment, mainly reflects
the motor function status of the hind limbs. Because
the outward positioning of the hind paw helps to control
the forward momentum generated during walking, hindpaw angle was used as a specific index to evaluate the state
of muscle relaxation of the hind limbs[13-14]. The increased
angle of paw placement in K141NHSPB8 mice (6 months of
age, equivalent to human 16.1 years) suggested motor nerve
impairment in the hind limbs. In both the fixed-bar and
rotarod tests, the K141NHSPB8 mice showed notable impairment
in motor coordination. However, the pain threshold
experiment involving plantar electric shocks did not show
a significant difference between K141NHSPB8 mice and agematched
wild type mice. This suggests that sensory function
in K141NHSPB8 mice was not significantly affected.
Electrophysiological study is mainly used to examine the
physiological status of large-diameter nerve fibers. Demyelination
of peripheral nerve results in a substantial reduction in
conduction velocity, and other typical changes in motor nerve
conduction, including a notable increase in motor nerve
latency and conduction block. However, axonal deficit is
characterized by notable decreases in the amplitude of compound
motor action potentials[15-16]. The electrophysiological
study of the peripheral nerve in K141NHSPB8 mice revealed a
significantly decreased amplitude of compound motor action
potentials, and a relatively normal nerve conduction velocity.
This suggests that the phenotype of K141NHSPB8 transgenic
mice is caused by axonal deficits, rather than myelin abnormalities.
Histopathological study of the peripheral nerve in
K141NHSPB8 transgenic mice showed reduced numbers of
myelinated fibers, axonal vacuoles and degeneration. There
was no thickening of the myelin sheath or onion-bulb like
structures, which further suggests that peripheral neuropathy
is an axonal neuropathy rather than a demyelinating one.
The behavioral, electrophysiological and histopathological
characteristics of the transgenic mice are similar to the features
in patients with familial Charcot-Marie-Tooth disease
type 2L, in whom the onset of disease occurs at young or
middle age. In these patients, impairment mainly involves
the lower limbs, and electrophysiological and histopathological
studies show an axonal neuropathy.
The G423T (K141N) mutation in HSPB8 causes Charcot-
Marie-Tooth disease type 2L. Interestingly, the K141N
and K141E mutations in HSPB8 were also identified in families
with distal hereditary motor neuropathy (dHMN)[17-18].
The dHMNs comprise a heterogeneous group of diseases
that share the common feature of a length-dependent predominant
motor neuropathy. Many forms of dHMN have
minor sensory abnormalities and/or a significant upper
motor neuron component, and there is often an overlap
with the axonal forms of Charcot-Marie-Tooth disease type
2 and with juvenile forms of amyotrophic lateral sclerosis
and hereditary spastic paraplegia. Patients in the Charcot-
Marie-Tooth disease type 2L family had mild sensory
impairments, including deficits in pain and touch, but no
evidence of painless injury or ulceration. While both start
in adulthood, distal HMN type II progresses more rapidly,
and complete paralysis of all distal muscles of the lower extremities
occurs within 5 years, while Charcot-Marie-Tooth
disease type 2L progresses slowly and all patients remain
ambulant. It remains unclear how the K141N mutation in
HSPB8 produces different phenotypes in the two different
diseases. In the present study, the K141NHSPB8 transgenic
mice developed slowly progressing hind limb weakness, and
had no obvious sensory impairment at the age of 6 months,
indicating relatively mild disease progression, with little or
no sensory disturbance.We consider the K141NHSPB8 transgenic mice that we
generated to be a suitable model of human Charcot-Marie-
Tooth disease type 2L. However, the degree of sensory
involvement in the peripheral nerves needs to be further
investigated. Further study on dorsal root ganglion neurons
is needed to clarify whether the sensory system is affected to
varying degrees in individual transgenic mice[20-21].
In a previous study, combined expression of HSPB8
K141N and HSPB8 K141E mutant proteins in motor neurons
resulted in neurite degeneration, manifested by a
reduction in number of neurites per cell and a reduction
in average length of neurites. In early passage, primary fibroblast
cultures derived from dHMN patient skin biopsies,
HSPB8 protein aggregates were present and mitochondrial
membrane potential was reduced. Cytoplasmic aggregateswere observed when K141NHSPB8 was transiently expressed in
cultured cells, and cell viability was impaired after heat shock
treatment. Overexpression of mutant HSPB8 was found to
result in autophagosomes that colocalized with protein aggregates,
but failed to colocalize with lysosomes. HSPB8 and
Bag3 form a chaperone complex that stimulates degradation
of protein substrates by macroautophagy. Thus, defects in
HSPB8-mediated autophagy are likely to be pathogenic[26-30].
We anticipate that the novel K141NHSPB8 transgenic mouse
model, coupled with cell and tissue culture systems, will be
a valuable research tool for elucidating the cellular and molecular
pathogenesis of Charcot-Marie-Tooth disease type 2,
and should advance the development of novel therapeutic
strategies for this neurological disorder.
Materials and Methods
Establishment of a transgenic mouse model.
Time and setting
Major components of experiments were performed at
the State Key Laboratory of Medical Genetics and Third
Xiangya Hospital, Central South University, China; parts of
experiments were performed at Institute of Neuroscience,
Shanghai Institute for Biological Sciences and Kunming Institute
of Zoology, Chinese Academy of Sciences, China from
March 2008 to August 2012.
A total of nine clean K41NHSPB8 transgenic mice (four females,
five males), aged 6 months, weighing 23–28 g, and nine
clean wild type C57BL mice (four females, five males), aged 6
months, weighing 23–28 g, as controls, were provided by the
Institute of Laboratory Animal Sciences, Chinese Academy
of Medical Sciences in China (license No. SCXK (Jing) 2005-
0013). Animals were housed at the Laboratory in the Animal
Facility of Third Xiangya Hospital, Central South University
in China. Animals were reared in 17 cm × 26 cm cages, each
containing two or three mice, under a 12-hour light/dark
cycle (lights off between 18:00–06:00), at an average temperature
of 22°C, with free access to food and water. This study
was approved by the Animal Ethics Committee, Third Xiangya
Hospital, Central South University, China.
Creation and genotyping of K141NHSPB8 transgenic mice
The pEGFPN1-K141NHSPB8 vector constructed in our previous
study was used as a template to clone HA-tagged
K141NHSPB8 cDNA using the EcoRI restriction endonuclease
into the pCAGGS plasmid. An expression vector driven by
a human cytomegalovirus immediate-early enhancer linked
to the chicken β-actin promoter was used. We excised a
2,896-bp fragment from the pCAGGS-HA-K141NHSPB8 vector
with the restriction endonucleases SalI and HindIII, and
then with BsaXI. We purified the fragment from an agarose
gel with the QIAquick Gel Extraction Kit (Qiagen, Hilden,
Germany), dialyzed it against injection buffer, and diluted it
to a concentration of 2 ng/μL. After preparing C57BL female
mice for ovulation and egg fertilization, a 0.5-μL aliquot of
DNA was microinjected into the fertilized eggs. Injected zygotes
were maintained overnight and transferred into pseudo-
pregnant C57BL females. The mice were placed in individually
ventilated cages. The tail tips of 1-week-old pups
were collected (1–2 cm/pup), and the phenol-chloroform
method was used to extract DNA. Genotyping of transgenic
animals was performed with two primer sets: forward
primer (CAG1), 5′-GCC ACC ATG TAC CCA TAC G-3′ and
reverse primer (CAG2), 5′-GCA GGA GGC TGT TTC ATA-
3′ to amplify the transgenic construct including the whole
HSPB8 gene; forward primer CAG1 and reverse primer
(HSP), 5′-TGG GGA AAG TGA GGC AAA TA-3′ to amplify
the transgenic construct including part of the HSPB8 gene
to confirm that HSPB8 had inserted into the mouse genome.
PCR products were separated by 8% polyacrylamide gel
electrophoresis, and DNA sequencing was performed using
an ABI PRISM 3100 sequencer (Perkin Elmer, Waltham, MA,
USA). Sequencing results were analyzed with DNASTAR Lasergene.
v7.1 software (DNASTAR. Inc, Madison, WI, USA).
The transgenic founders were identified and subsequently
transferred to the animal facility of Third Xiangya Hospital,
Central South University in China.Western blot assay for HA-tagged HSPB8 in K141NHSPB8
Three 10-month-old K141NHSPB8 founder mice and one
wild type C57BL control were killed by cervical dislocation,
and flash-frozen tissue samples were maintained at –80 °C.
Frozen tissues from heart and gastrocnemius muscles were
homogenized in radio immunoprecipitation assay buffer
(containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl
sulfate and phosphatase inhibitors). Protein concentrations
were determined using the microBCA kit (Pierce Chemical
Co., Rockford, IL, USA) according to the manufacturer’s
instructions. Western blot assay was performed as described
before. Goat anti-HSPB8 antibody (1:500; Sigma-Aldrich,
Milwaukee, WI, USA), mouse anti-HA-Tag antibody
(1:2,000; Sigma-Aldrich) and mouse anti-β-actin antibody
(1:500; Sigma-Aldrich) were used as primary antibodies
and incubated with the blots at 4°C for 2 hours. Rabbit anti-
goat IgG (1:2,000; Sigma-Aldrich) and goat anti-mouse
lgG (1:6,000 or 1:10,000; Sigma-Aldrich) were the secondary
antibodies, and were incubated with the blots at 4°C for 2
hours. The target bands were visualized using an enhanced
chemiluminescence detection kit (BioRad, Hercules, CA,
USA) and then exposed to X-ray film in a dark room. The
X-ray film signal was scanned on an imaging analysis system
(Bio-Rad), and absorbance values were analyzed.Behavioral assessments of K141NHSPB8 transgenic mice
For each behavioral experiment, each mouse was tested
twice a day for 4 days in a row to ensure the repeatability
and reliability of the data. Footprint analysis was performed
as described by Sereda et al.. Waterproof black ink wasapplied to the hind paws and footprints were taken on watercolor
paper. The animal was allowed to walk forward in a
12-cm-wide lane. The footprints were then scanned and the
angle of deviation of the median foot axis with regard to the
movement axis (i.e., the hind paw angle) was measured for
10 clearly visible footprints per mouse.
Fixed-bar test was performed as described by Norreel et
al.[31-32]. For the fixed-bar test, a round wooden bar (diameter:
1.5 cm; length: 50 cm) was positioned 40 cm above
the cage floor. Mice were placed on the middle of the bar.
The movements and the time (in seconds) that the animals
remained on the bar were monitored using videos. A maximum
of 5 minutes per trial was allowed. Motor coordination
was further assessed with the rotarod test, as described
by Zhao et al.. The apparatus (DXP-2, Institute of Materia
Medica, Chinese Academy of Medical Sciences, Beijing,
China), consisting of a base platform and a rotating rod
with a diameter of 2.5 cm, was subdivided into four equal
sections. The mice were trained to reach their performance
baseline with 5 sessions of 2-minute periods of walking at
4 r/min per day for two days before the test. After training,
each mouse was placed on the rod and allowed to rest for
30 seconds, and then the rod was rotated at 2 r/min. The
rotation speed was increased every 30 seconds to 4, 8, 12,
16, 24, 28, 32 and 36 r/min. In a series of six trials per animal,
the time (in seconds) that the mouse remained on the
rod was measured. Pain threshold test was performed as
previously described[34-35]. Mice were placed in a Threshold
Activity Monitoring system (Med Associates Inc., Pittsburgh,
PA, USA). Electric shocks began at 0.2 mA, 0.5 seconds, and
were increased by 0.1 mA in 1-min intervals until the mouse
had the first pain response (either limb flick or jump). The
amplitude of the current at which the mouse had the first
pain response was recorded.
Nerve electrophysiology in K141NHSPB8 transgenic mices
Following anesthesia with pentobarbital (240 mg/kg), mice
were fixed on a thermostat-controlled heating plate at 37°C.
The sciatic nerve on one side was carefully isolated. The sciatic
and posterior tibial nerves were stimulated with needle
electrodes inserted alongside the nerves at the sciatic notch
and at the hock. The distance between the two points was
1 cm. The square pulses (1 Hz, 3 mV) were delivered five
times per site using the BL-420E+ biological and functional
experimental system (Pclab, Chengdu, China). Compound
motor action potentials were recorded from an intrinsic foot
muscle using a concentric needle electrode. The latencies of
compound motor action potentials, elicited by stimulation at
both proximal (hip) and distal (hock) sites, were measured
from the stimulus artifact to the first negative deflection.
Amplitudes were determined as the maximum peak-to-peak
voltage. Motor nerve conduction velocity was calculated
using the distance between the two points of stimulation
(1 cm) and the difference in the latencies between the two
points (conduction time).
Sciatic nerve histopathology in K141NHSPB8 transgenic mice
After nerve electrophysiology, anesthetized mice were perfused
through the left ventricle with saline followed by 4%
paraformaldehyde in cacodylate buffer (pH 7.2). The sciatic
nerves on the other side were carefully isolated and dissected
from the region of the sciatic notch to the hock. Nerve specimens
were placed in either 4% paraformaldehyde (0.05 mol/L
PBS, pH 7.2) or 2.5% glutaraldehyde. Specimens fixed in 4%
paraformaldehyde were embedded in paraffin, subjected to
semi-thin (1-μm) sectioning, stained with 1% toluidine blue
at 80°C for 30–45 seconds, then observed with an Olympus
CX31 light microscope (Olympus Corporation, Tokyo, Japan).
The number of axons was counted on four slides from
each mouse. Specimens in 2.5% glutaraldehyde were embedded
in porous rubber bodies, subjected to ultrathin (50-nm)
sectioning, double stained with uranyl acetate and lead nitrate,
and then observed with an H-7500 transmission electron
microscope (Hitachi, Tokyo, Japan) and photographed.Statistical analysis
Data were expressed as mean ± SEM, and processed with
SPSS 19.0 software (SPSS, Chicago, IL, USA). Differences
between repeated measures of different genotypes were
compared using two-way analysis of variance followed by
Bonferroni’s post hoc test. Comparisons of means were made
using a two-sample t-test. A value of P < 0.05 was considered
Author contributions: Zhang RX and Zhang FF participated in
the study conception and design. Zhang FF, Li XB, Huang SX, Zi
XH, Liu T, Liu SM and Li XN provided the data and ensured its
integrity. Zhang RX and Zhang FF analyzed the data. Zhang RX
wrote the manuscript and was in charge of manuscript revision.
Zhang RX and Zhang FF obtained funding. Xia K, Pan Q and
Tang BS provided technical support. All authors approved the final
version of this paper.
Conflicts of interest: None declared.
Peer review: Although progress in molecular genetics research is
significant, the pathological mechanism of Charcot-Marie-Tooth
still waits to be unveiled. In this study, the K141NHSPB8 transgenic
mouse model is successfully generated, and can be used as a model
to study the pathogenesis of Charcot-Marie-Tooth disease type 2L.References
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