Dysmyelinated lower motor neurons retract and regenerate dysfunctional synaptic terminals.
ABSTRACT Axonal degeneration is the major cause of permanent neurological disability in individuals with inherited diseases of myelin. Axonal and neuronal changes that precede axonal degeneration, however, are not well characterized. We show here that dysmyelinated lower motor neurons retract and regenerate dysfunctional presynaptic terminals, leading to severe neurological disability before axonal degeneration. In addition, dysmyelination led to a decreased synaptic quantal content, an indicator of synaptic dysfunction. The amplitude and rise time of miniature endplate potentials were also increased, but these changes were primarily consistent with an increase in the passive membrane properties of the transgenic muscle fibers. Maintenance of synaptic connections should be considered as a therapeutic target for slowing progression of neurological disability in primary diseases of myelin.
- [Show abstract] [Hide abstract]
ABSTRACT: The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when proteolipid protein (PLP) replaced P0 protein as the most abundant protein of CNS myelin. However, PLP did not replace P0 in peripheral nervous system (PNS) myelin. To investigate the possible consequences of a PLP to P0 shift in PNS myelin, we engineered mice to express PLP instead of P0 in PNS myelin (PLP-PNS mice). PLP-PNS mice had severe neurological disabilities and died between 3 and 6 months of age. Schwann cells in sciatic nerves from PLP-PNS mice sorted axons into one-to-one relationships but failed to form myelin internodes. Mice with equal amounts of P0 and PLP had normal PNS myelination and lifespans similar to wild-type (WT) mice. When PLP was overexpressed with one copy of the P0 gene, sciatic nerves were hypomyelinated; mice displayed motor deficits, but had normal lifespans. These data support the hypothesis that while PLP can co-exist with P0 in PNS myelin, PLP cannot replace P0 as the major structural protein of PNS myelin. GLIA 2014.Glia 07/2014; · 5.47 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Twenty-nine patients underwent single (n=15) or double (n=14) nerve transfer for post-traumatic elbow flexion palsy. Patients averaged 30.2 years, with a mean preoperative delay of six months and postoperative follow-up of 34.2 months. Sixty per cent of the single transfer patients recovered to BMRC grade M4 after an average of follow-up of 13.2 months. Eighty-five percent of double nerve transfer patients reached grade M4 after an average follow-up of 11 months. There were no significant differences between groups. Clinical assessment revealed motor or sensory deficit in seven cases, which did not cause any impairment. Patients with a C5-C6 injury had shorter recovery times and better strength in comparison with those with C5-C6-C7 injury. By restoring shoulder function, elbow flexion will be indirectly improved. This improvement can be partially attributed to the base of the arm being more stable.Chirurgie de la Main 03/2014; · 0.25 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: This paper analyses the insufficiencies of the conventional initial reaction rate method based on the measurements in a batch reactor. Using simulations, the plausibility of pseudo-steady state approximation is primarily assessed for different values of the ratio of the total volume of bulk solution and particles. A concept of critical substrate concentration is suggested to characterise achieving the pseudo-steady state. A new approach is proposed for the correction of the measured temporal derivative of the substrate bulk concentration in order to obtain a true value of the reaction rate. For the Michaelis-Menten kinetics, the correction factor is correlated with the effectiveness factor using a simple empirical equation.Progress in Biotechnology - PROGR BIOTECHNOL. 01/1996; 11:328-335.
neurons retract and regenerate dysfunctional presynaptic terminals, leading to severe neurological disability before axonal degenera-
tion. In addition, dysmyelination led to a decreased synaptic quantal content, an indicator of synaptic dysfunction. The amplitude and
Axons are myelinated by Schwann cells in the PNS and by oligo-
dendrocytes in the CNS. Point mutations, duplications, and de-
cells cause dysmyelination and neurological disability (Scherer,
1999). Although dysmyelination can significantly slow nerve
conduction velocities, irreversible and progressive neurological
disabilities in most inherited diseases of myelin are caused by
axonal degeneration. Axonal pathology and axonal degeneration
have been documented morphologically in many inherited and
acquired diseases of myelin (Trapp et al., 1998, 1999; Scherer,
1999; Garbern et al., 2002). The timing of clinical disease onset
and rate of disease progression depend on the mutation and the
severity of dysmyelination. Individuals with congenital hypomy-
elination or De ´je ´rine-Sottas syndrome have an early onset and
often fatal clinical course. Dysmyelination, neurological disabil-
ity, and axonal degeneration in many forms of Charcot-Marie
Yin et al., 1998; Lappe-Siefke et al., 2003), indicating that long-
term survival of axons depends on glial-derived support after
myelination is complete. Degeneration of chronically dysmyeli-
nated axons, therefore, is a major cause of permanent neurolog-
ical disability associated with inherited diseases of myelin
Little is known about the molecular mechanisms responsible
apies have been established. Axonal pathologies that precede ax-
onal degeneration in animal models of dysmyelination include
atrophy, swelling, altered axonal cytoskeleton, reduced axonal
transport (de Waegh et al., 1992; Griffiths et al., 1998; Yin et al.,
misregulation of gene expression by the dysmyelinated neuron
it seems likely that synaptic connections of dysmyelinated neu-
eral lines of transgenic mice that overexpress P0protein (P0
Overexpressed P0protein prevented spiral and longitudinal my-
elin growth (Yin et al., 2000) and caused neurological disability,
myelination. These mice resemble and serve as an animal model
ofthehumanperipheralneuropathiesDe ´je ´rine-Sottassyndrome
al., 2000). The transgene was not expressed by oligodendrocytes,
the pathogenesis of neurological disability during chronic dys-
myelination, we analyzed the synaptic connections of lower mo-
tor neurons (LMNs) in P0
myelination leads to structural and functional changes at
tgmice. We report here that PNS dys-
This work was supported by National Institutes of Health Grants NS38186 (B.D.T.), NS38517 (R.J.B.-G.), and
NS41319 and NS45630 (M.L.F., L.W.) and Telethon Italy Grant GGP030074 (L.W.). We thank Victoria Pickett for
3890 • TheJournalofNeuroscience,April14,2004 • 24(15):3890–3898
P0-overexpressing mice. The transgene consisted of all introns and exons
of the mouse P0gene, 6 kb of the promoter, and the natural polyadenyl-
ation signal. Generation and characterization of mice expressing this
highest transgene copy number and most severe peripheral neuropathy.
In addition, a lower expressing line (Tg 80.4) was examined and crossed
with P0-null mice (Wrabetz et al., 2000) for rescue experiments.
Behavior testing. Body weight, running speed, and grip strength were
(Chatillion DFIS-2; AMETEK, Paoli, PA). The maximal grip strength of
best of three trials) to run 75 cm and is calculated in centimeters per
established previously (Mitsumoto et al., 1994).
(n ? 2). The muscles examined include the following: gluteus maximus
(proximal hindlimb extensor), gastrocnemius (distal hindlimb flexor),
triceps (proximal forelimb extensor), and forepaw flexors (distal fore-
acquired with a TECA Sapphire EMG using a concentric 25 gauge elec-
ined. Fibrillation potentials (50 ?V or greater in amplitude) were quan-
tified per second.
Confocal analysis of neuromuscular junctions. A minimum of six glu-
at P15, P45, and P90. The muscles were fixed and permeabilized as de-
scribed previously (Son and Thompson, 1995). Specimens were labeled
jugated ?-bungarotoxin (Molecular Probes, Eugene, OR) and immuno-
stained for axons with a combination of neurofilament (SMI31; Stern-
berger Monoclonals, Baltimore, MD) and synaptophysin (Sigma, St.
Louis, MO) antibodies. Additional sections were tripled labeled for ax-
ons, acetylcholine receptors, and P0protein. Primary antibodies were
detected using FITC and Texas Red-conjugated secondary antibodies
?-bungarotoxin was detected using Cy5-avidin (Jackson ImmunoRe-
search). Sections were mounted in Vectashield (Vector Laboratories,
Burlingame, CA) and examined on a Leica (Heidelberg, Germany) con-
focal microscope (TCS-NT system). Either Leica TCS-NT acquisition
software or Scion (Frederick, MD) Image software was used to recon-
struct z-series images into maximum intensity projections. Images were
prepared for publication using Photoshop 7 (Adobe Systems, San Jose,
The relationship between terminal axons and bungarotoxin staining
was investigated in gluteus muscles using whole-mount preparations.
The numbers of NMJs analyzed from a minimum of six WT and P0
200 WT and 222 P0
were divided into three categories: those normally innervated; those in
which axons were separated from bungarotoxin staining (partially de-
nervated); and those junctions without axons (totally denervated). Ax-
onal sprouting was quantified by determining the percentage of NMJs
analyzed for sprouts were as follows: at P15, 83 WT and 142 P0
110 WT and 223 P0
analyzed by Student’s t test. Similar analysis was performed on an addi-
mice bred to P0-null mice (n ? 80 NMJs from three mice).
Nerve transection. Four WT and four P0
tized, and their left sciatic nerves were transected at the sciatic notch.
Both soleus muscles were removed 48 hr later, immersion fixed, immu-
tgand WT littermates at P37 (n ? 6), P72 (n ? 4), and P111
Grove, PA). Biotinylated
tg; and at P90, 292 WT and 263 P0
tg. The junctions
tg; at P45,
tg. Data were
tg; and at P90, 106 WT and 275 P0
tgmice (P30) were anesthe-
nostained for axons and postsynaptic specializations, and examined by
confocal microscopy as described above.
Electrophysiological analyses of neuromuscular synaptic transmission.
Two WT and six P0
injection of 0.05 cc of a mixture of 17.4 mg/ml ketamine and 2.6 mg/ml
cle and its innervation were dissected under oxygenated (95% O2, 5%
CO2) Rees’ Ringer’s solution (Rees, 1978) (110 mM NaCl, 5 mM KCl, 1
mM MgCl2, 25 mM NaHCO3, 2 mM CaCl2, 11 mM glucose, 0.3 mM gluta-
mate, 0.4 mM glutamine, 5 mM BES [N,N -bis(2-hydroxyethyl)-2-
aminoethanesulfonic acid] buffer, 0.434 ?M cocarboxylase, and 36 ?M
choline chloride). Muscles were pinned in a Sylgard-lined Petri dish and
superfused with oxygenated Ringer’s solution, and the muscle nerve was
placed into a suction electrode.
Physiological analyses were performed as described by Kopp et al.
(2000). Briefly, to determine whether nerve-evoked muscle contractions
were present, the nerve was stimulated with 0.1–0.2 msec duration rect-
10 V, and muscle contractions were visually monitored through a dis-
excitable, muscle fibers were placed into a suction electrode and were
stimulated with 0.5 msec duration rectangular pulses delivered at 0.5–1
muscle contractions were visually monitored.
To determine whether spontaneous and evoked neurotransmitter re-
lease was present, skeletal muscle fibers were cut at each end to prevent
muscle contractions attributable to muscle fiber depolarization (Glavi-
novic, 1979; Ribchester et al., 1994). Intracellular recordings were per-
formed using glass microelectrodes filled with 3 M KCl (30–570 M?
resistance). All experiments were performed at room temperature. A
total of 43 junctions from six P0
type littermates were studied.
Electrical potentials were amplified using an Axoprobe 1A amplifier
(Axon Instruments, Union City, CA), low-pass filtered at 1 kHz, and
Instruments) and interactive software (Axoscope; Axon Instruments).
Muscle fiber resting membrane potential was continuously monitored,
and only fibers with resting potentials more hyperpolarized than ?40
mV and in which the resting potential did not change by ?5 mV during
the course of the experiment were studied further. This was the mini-
mum resting potential that allowed a sufficient signal-to-noise ratio to
detect miniature endplate potentials (mepps). Muscle fiber input resis-
tance was calculated as voltage–current after injection of a 100 msec
hyperpolarizing current pulse. mepps were recorded for 10–20 min and
GA). To characterize nerve-evoked endplate potentials, the stimulation
tal content was determined under conditions of reduced Ca2?(1 mM
CaCl2) and elevated Mg2?(10 mM MgCl2) using the method of failures
(Del Castillo and Katz, 1954; Kopp et al., 2000) or expressed as the ratio
of mean endplate potential amplitude/mean mepp amplitude. The epp
amplitudes were corrected for nonlinear summation (McLachlan and
Martin, 1981) using an f value of 0.8. The quantal content, i.e., the num-
each NMJ by dividing the mean corrected epp amplitude by the mean
mepp amplitude. (McLachlan and Martin, 1981). Statistical analyses
were performed using Microsoft (Seattle, WA) Excel software. Data are
presented as mean ? SEM.
6) and P0
nerves were dissected under cold oxygenated (95% O2, 5% CO2) mam-
Muscles were placed in 5 ?M FM1-43 (Molecular Probes) in oxygenated
Ringer’s solution at room temperature. The nerve was placed into a
suction electrode and was stimulated with 5 sec trains of 0.1 msec dura-
tion pulses delivered at 30 Hz, repeated every 20 sec for 5 min. The
stimulus intensity used was the minimum that reliably caused muscle
contraction, typically 0.1–0.5 V. After stimulation, muscles were rinsed
tgmice at P60 were anesthetized by intraperitoneal
tgmice and 25 junctions from five wild-
tg(n ? 10) mice were anesthetized, and the soleus muscle and
Yinetal.•DysmyelinationCausesSynapticDysfunctionJ.Neurosci.,April14,2004 • 24(15):3890–3898 • 3891
with 10 min washes in oxygenated Ringer’s so-
lution and 2 min washes in Ringer’s solution
with 1 mM ADVASEP-7 (Biotium, Hayward,
CA) over a 1 hr period. After FM1-43 labeling,
AChRs were labeled by incubation in 10 ?g/ml
Ringer’s solution for 4 min. Muscles were
mounted onto glass slides and coverslipped.
Muscles were mounted onto glass slides and
junctions) were imaged using a laser scanning
confocal microscope (Leica TCS 4D system).
The percentage of AChR staining apposed by
FM1-43 was determined using interactive soft-
ware (MetaMorph; Universal Imaging Corpo-
ration, West Chester, PA).
Quantification of AChR subunit mRNA.
was isolated using the Total RNA isolation kit
(Promega, Madison, WI), quantified using
standard spectrophotometric measurements
and run on an Agilent (Palo Alto, CA) 2100
Bioanalyzer to ensure RNA quality. RNA (de-
void of DNA) was reverse transcribed and am-
plified using gene-specific primers for AChR ?
subunit (GenBank accession number M17640;
reverse, AATCGACCCATTGCTGTTTC), ?
subunit (GenBank accession number M14537;
forward, AGCCTGAACGAGAAGGATGA; re-
verse, AGCAGTGATGCGGAGAGAAT), and
GCTCTC; reverse, TCTGGGATTGGAAGAT-
GAGG). PCR fragments were subsequently li-
gated to the T7 polymerase promoter sequence
(Lig-n-scribe kit; Ambion, Austin, TX) and PCR amplified. Five micro-
grams of total RNA were used for Northern analysis using the Northern
Max kit (Ambion) protocols. Radiolabeled ([32P]UTP) antisense RNA,
transcribed using T7 polymerase (Maxiscript kit; Ambion) was used as
probe. Hybridized membranes were exposed to Eastman Kodak (Roch-
Image software and normalized to glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) levels. After detection, membranes were stripped
? subunit, followed by ? subunit).
Electron microscopy. Three WT and mutant mice were perfused at P15
and P90 with 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.08 M
Sorensen’s buffer. The gluteus maximus muscle and L4 and L5 ventral
tions, and Schwann cells was investigated in electron micrographs from
18 WT NMJs and 16 P0
in serial sections. Montages of the entire left and right L4 and L5 ventral
roots from three P90 WT and P0
micrographs. Total axons were quantified in the montages. The data
were analyzed using Student’s t test.
tgNMJs. Three of the P0
tgNMJs were examined
tgmice were generated from electron
Several lines of mice that overexpress P0protein were generated
(Wrabetz et al., 2000). The present study focuses on the most
severely affected line (Tg 80.2). A progressive decline in motor
performance accompanied reduced body weight and muscle at-
rophy (Fig. 1A). Running speed in P0
P30 and 80% at P60, and the mice were non-ambulatory at P90.
P90. Body weight was reduced at P30, when pelvic girdle muscle
leling more generalized muscle atrophy. EMG of girdle, hind-
limb, and forelimb muscles was performed to test for muscle
denervation. Fibrillation potentials (Fig. 1B, inset), an indicator
of muscle denervation, were graded quantitatively in WT and
were not detected in WT muscles. At P37, fibrillations were
prominent in the pelvic girdle (gluteus) muscles, minimal in dis-
tal hindlimb flexor (gastrocnemius) muscles, and absent in fore-
limb distal flexors. By P72, fibrillations were present in all mus-
cles examined but were most prominent in distal hindlimb
muscles. At P111, fibrillation potentials persisted in girdle and
cles. These data support a progressive denervation of P0
culature that parallels the proximal-to-distal gradient of muscle
To determine whether degeneration of LMNs or their proxi-
mal axons contribute to the muscle denervation, axons in the L4
and L5 ventral roots from P90 WT (Fig. 1C) and P0
mice were quantified by electron microscopy (Fig. 1E). Despite
proximal axons, therefore, do not contribute to the neurological
deficits in P0
tgwas reduced by 50% at
tgmice at P37, P72, and P111 (Fig. 1B). Fibrillation potentials
muscles. C–E, Comparison of toluidine blue-stained Epon sections of ventral roots from P90 wild-type (C) and P0
P0overexpression causes severe neurological phenotypes and muscle fibrillation but not proximal motor axon
3892 • J.Neurosci.,April14,2004 • 24(15):3890–3898 Yinetal.•DysmyelinationCausesSynapticDysfunction
During normal development, two or more motor axons initially
and Colman, 2000). By the end of the second postnatal week,
however, most NMJs are innervated by single axons. We com-
pared innervation of the gluteus maximus and soleus muscles at
P15, P45, and P90 in P0
tissue immunostained for terminal axons with neurofilament
P15, NMJs in both mouse strains (Fig. 2A,B) were innervated by
mice were appropriately apposed by axonal terminals (Fig. 2C).
tgand WT mice by confocal analysis of
the postsynaptic specialization were un-
opposed by axons (Fig. 2F, asterisks),
Twenty-five percent of the NMJs in P15
nervated (Table 1). The preterminal ax-
ons, however, did not degenerate or form
axonal retraction bulbs, the stereotypic
appearance of preterminal axonal with-
drawal at NMJs (Balice-Gordon et al.,
1993). On the basis of these observations,
NMJs in P0
development. However, synaptic connec-
tions between terminal axons and muscle
fibers begin to break down by P15.
Terminal axonal withdrawal from
NMJs increased as the mutant mice aged
ined were partially denervated, and 7%
were totally denervated (Table 1). At P90,
only 3% of the mutant NMJs appeared
normal, 89% were partially denervated,
and 7% were entirely devoid of presynap-
tic nerve terminals and were thus dener-
vated. In addition, the normal pretzel-like
shape of AChR clusters (Fig. 2C) became
punctate and discontinuous (Fig. 2F–I),
and the terminal axons extended multiple
intrajunctional branches to the many
both the gluteus maximus (Fig. 2H) and
soleus (Fig. 2I) muscles. Axonal sprouts
also extended to neighboring NMJs (Fig.
2F–I). In muscles from P15–P90 WT
mice, ?3% of NMJs extended and/or re-
ceived interjunctional axonal sprouts. In
were significantly increased at both P45
(45%) and P90 (89%). Although dysmy-
elinated axons cannot maintain normal
NMJs, they retain the ability to sprout to
The relationship between synaptic ab-
normalities and axonal degeneration was
investigated by examining NMJ innerva-
tion after transection of P30 WT and P0
tggluteus maximus were partially de-
tgmice form normally during
tgmice, however, these percentages
sciatic nerves. Whereas bungarotoxin staining was unaffected,
axonal terminals were eliminated by 48 hr after transection in
both strains of mice (data not shown). Synaptic withdrawal and
sprouting at the NMJ, therefore, precedes axonal degeneration
and is not a response of axons disconnected from their somas.
capable of releasing neurotransmitter, the ability of junctions to
internalize the fluorescent dye FM1-43 was evaluated in the so-
leus muscle from 2-month-old P0
tion without muscle or nerve damage. Nerves were stimulated in
short trains of pulses delivered at high frequency in Ringer’s so-
tgand WT mice. The soleus
devoid of axons (F, asterisks). AChR staining appeared normal in D and E and was omitted to demonstrate axonal differences.
Yinetal.•DysmyelinationCausesSynapticDysfunctionJ.Neurosci.,April14,2004 • 24(15):3890–3898 • 3893
cular junctions were analyzed with confocal microscopy.
In 10 of 10 NMJs examined in P60 WT muscles, an average of
43-stained presynaptic nerve terminals (Fig. 3A). However, in
NMJs in the soleus muscle of P60 P0
of the postsynaptic AChR cluster area was occupied by FM1-43-
stained nerve terminals (Fig. 3B,C), and 5 of 22 junctions exam-
ined were entirely devoid of FM1-43-stained nerve terminals.
These data are consistent with the partial denervation observed
after immunostaining. This observation supports widespread
To determine how the loss of functional nerve terminals in
intracellular recording using standard methods. Spontaneous
tgmice, an average of ?40%
tgmice suggested by FM1-43 staining affected neuromuscular
mepps were recorded for 5–10 min. In the soleus muscle from
2-month-old mice, the frequency of spontaneous mepps was
similar between P0
spontaneous release of neurotransmitter quanta is relatively un-
affected. However, mepp amplitude was increased 2.3-fold ( p ?
0.005) compared with WT mice (Fig. 4C–E).
The increase in mepp size might be attributable to muscle
fiber atrophy and thus reflect an increase in muscle fiber input
tgand WT mice (Fig. 4A,B), suggesting that
?95% of the postsynaptic AChR cluster area (left) was occupied in its entirety by FM1-43-
FM1-43 labeling demonstrates defects in synaptic vesicle recycling and loss of
bution of the amplitude of all events from muscle fibers shown in A. Distributions are not
tg, but quantal content was reduced (I), and epp rise time (J) and latency (K) were
3894 • J.Neurosci.,April14,2004 • 24(15):3890–3898 Yinetal.•DysmyelinationCausesSynapticDysfunction
resistance as has been observed after denervation, inactivity, and
as a consequence of neuromuscular disease (Tonge, 1974; Wein-
resistance showed that input resistance was greater in P0
significant muscle fiber atrophy measured by muscle fiber cross-
in part for the differences in mepp amplitude.
To characterize nerve-evoked neurotransmitter release, the
The quantal content, i.e., the number of ACh quanta released
after a single nerve impulse, was calculated as the mean epp am-
plitude, corrected for nonlinear summation (McLachlan and
Martin, 1981), divided by the mean mepp amplitude. In the so-
leus muscle from 2-month-old mice, epp amplitude was similar
type soleus muscle (Fig. 4I). Moreover, in P0
WT littermates, a small percentage of junctions in P0
(?5%) had significantly reduced epp amplitude. In addition, no
evoked epps or spontaneous mepps were observed in ?10% of
muscle fibers were completely denervated. These observations
or completely denervated after FM1-43 labeling (see above) and
suggest that synaptic transmission is reduced in P0
The increased mepp amplitude and rise time observed in the
in the expression of postsynaptic AChR subunits, as has been
observed after denervation and reinnervation (Gu and Hall,
1988; Sanes and Lichtman, 1999). To evaluate this possibility,
the ? AChR subunit mRNA were significantly increased in P0
muscle at P30 (sixfold) and P90 (fourfold). The ? subunit was
increased twofold at P30 and was unchanged at P90. The embry-
onic ? subunit was abundant in WT newborn muscle (Fig. 5),
at other time points in either WT or P0
tgmuscles but not
tgmuscle total RNA ex-
tracts. These data suggest that the changes observed in mepp
in addition to a change in muscle fiber input resistance.
In the soleus muscle, epp rise time was significantly greater
than that observed in WT mice (Fig. 4G,J), as was observed for
mepps. One possible explanation is that the muscle fiber mem-
brane time constant is altered in P0
muscles from P0
evoked epps has been reported under circumstances in which
AChR density is low (Katz and Miledi, 1973; Albuquerque et al.,
1976; Betz and Osborne, 1977; Kidokoro, 1980; Colman et al.,
1997). A third possibility is that there is an increased separation
between nerve terminals and postsynaptic AChRs at junctions in
retracting. A slowing of mepp and epp rise times has been sug-
gested to be attributable to the increased time that would be
required for acetylcholine molecules to bind to unoccupied
AChRs (Katz and Miledi, 1973; Betz and Osborne, 1977;
time, the latency from the stimulus artifact to epp onset was
delayed by more than fourfold in the soleus muscle of P0
compared with WT (Fig. 4K). This is consistent with the dysmy-
elination and slowing of axonal conduction velocity observed
after P0overexpression (Wrabetz et al., 2000). Together, these
the soleus muscle of P0
tion and reinnervation by terminal sprouts (Fig. 2).
tgmice as a consequence of
tgmice, because a slowing of spontaneous and
tgmice, as would be expected to occur if nerve terminals were
tgmice, consistent with partial denerva-
When NMJs were examined in P90 gluteus maximus from an-
other P0-overexpressing line (Tg 80.4) with less severe dysmyeli-
Tg 80.4 line was rescued by breeding to P0-null mice (Wrabetz et
al., 2000). We investigated whether the NMJ abnormalities in Tg
mus from P90 Tg 80.4 ? P0-null mice (P0R), muscle fibrillations
that dysmyelination is the primary cause of NMJ denervation in
Confocal studies were performed to determine whether the
Schwann cells that cap P0
ity terminated 10–30 ?m before the entry of the axon into the
NMJ (Fig. 6A,B). In WT mice, P0-expressing cells near NMJs
formed myelin sheaths that closely apposed axons (Fig. 6A). In
the mutant mice, P0-expressing Schwann cell perikarya (Fig. 6B)
were larger and covered shorter axonal segments than myelinat-
ing Schwann cells in WT mice. P0protein was not detected by
confocal microscopy in terminal Schwann cells of the NMJ in
vations, we compared the ultrastructure of NMJs from P15 WT
(Fig. 6C) and P0
percent of NMJs from P15 P0
tinguishable from WT NMJs (Fig. 6C). The axon terminal was
appropriately apposed to postsynaptic specializations, and
Schwann cells capped the surface of the axon terminal. Six of the
tgNMJs play a primary role in NMJ
tg(Fig. 6D) gluteus maximus muscles. Sixty-five
tgmice (10 of 16 NMJs) were indis-
Yinetal.•DysmyelinationCausesSynapticDysfunctionJ.Neurosci.,April14,2004 • 24(15):3890–3898 • 3895
erating presynaptic terminals (Fig. 6D),
identified by an abundance of synaptic
vesicles (Fig. 6D, inset). Regions of the
postsynaptic membrane were unapposed
by axons or Schwann cells (Fig. 6D). Ter-
minal Schwann cells or their processes
were not detected between P0
confocal imaging that terminal Schwann
cells are not playing a primary role in sep-
arating presynaptic and postsynaptic
components of the NMJ. The ultrastruc-
ture of NMJs in P90 P0
of postsynaptic membranes were unap-
posed by terminal axons. Terminal axons
of varying diameters (Fig. 6E) were
present and ensheathed by Schwann cells.
tggluteus was sig-
Here we describe a mouse model in which
chronically dysmyelinated lower motor
neurons retract and regenerate dysfunc-
tional synaptic terminals. These synaptic
changes precede the axonal degeneration
that becomes prominent by 6 months of
age and that reduces the lifespan of the
mice by ?50% (Wrabetz et al., 2000).
ing early postnatal development and were
crossed to P0-null mice. Terminal axonal
withdrawal and NMJ dysfunction in P0
mice, therefore, are pathological conse-
tenance of synaptic connectivity should
thus be considered as a therapeutic target
in primary diseases of myelin.
Schwann cells have essential roles in
Schwann cell function, therefore, could
cause neurological disability by several
mechanisms. Although peripheral myelin
does not form, axons in P0
nerves are ensheathed by Schwann cells
(Yin et al., 2000). This Schwann cell en-
sheathment promotes maturation of axons, including appropri-
surface of muscle. Molecular requirements for formation of the
NMJ include axonally derived agrin, which orchestrates the
postsynaptic distribution and organization of acetylcholine re-
ceptors by interacting with MuSK (muscle-specific receptor ty-
rosine kinase) (McMahan, 1990; DeChiara et al., 1996). Our
studies indicate that these molecular events are intact in P0
in which an early downstream effect of dysmyelination was par-
tial withdrawal of axon terminals from the NMJs. The pretermi-
nal axons initially retained one-to-one relationships with NMJs
ters at NMJs. These structural changes have functional conse-
quences, as evidenced by a decrease in quantal content in P0
observed after P0overexpression (Wrabetz et al., 2000). After
rapidly degenerated in both WT and P0
mice aged, intrajunctional and interjunctional sprouts increased
to the point at which 96% of NMJs examined at P90 were abnor-
mal. Many junctions in P90 P0
of CNS synaptic terminal arbors, with multiple sprouts contact-
also increased the expression of mRNA encoding the ? and ?
tgmice. Synaptic changes
tgNMJs, therefore, precede axonal degeneration. As the
tgmuscle attained the appearance
3896 • J.Neurosci.,April14,2004 • 24(15):3890–3898Yinetal.•DysmyelinationCausesSynapticDysfunction
an increase in extrajunctional AChR expression, similar to that
observed after denervation or inactivity (Fambrough, 1974; Berg
and Hall, 1975; Blondet et al., 1989).
A number of studies support the existence of unidentified
molecules or signals that are required to maintain axon–muscle
contact once NMJs are formed (Lichtman and Colman, 2000).
The separation of maintenance of axon–muscle contact from
ports this hypothesis and raises the possibility that chronic dys-
myelination preferentially alters the unidentified signals that
maintain NMJs. Altered axonal cytoskeleton and impaired ax-
of mutations in myelin protein genes (de Waegh et al., 1992;
Griffiths et al., 1998; Yin et al., 1998; Lappe-Siefke et al., 2003). It
is possible, therefore, that dysmyelination in P0
tenance of the NMJ. This may initiate a response by the muscle
that eventually implements terminal axon sprouting. As the P0
mice age, progressive muscle atrophy, reduced muscle fibrilla-
of the sprouting phenotype. Synaptic stripping by terminal
Schwann cells has been described in laminin ?2-deficient mice
focal and EM analysis, did not appear to contribute to the dener-
vation of P0
back distal axonopathy (Fig. 1B), because the longest fibers were
distal axonopathy that affects proximal muscles before distal
muscles. The progression of proximal-to-distal muscle pheno-
type and of predominately slow muscle implicates muscle usage
as a contributor to the NMJ phenotype in P0
anism has also been suggested to contribute to NMJ dysfunction
in hereditary canine spinal muscular atrophy (HCSMA), a neu-
al., 1998). Physiological and morphological NMJ phenotypes,
dogs, which exhibit motor unit tetanic failure before structural
and functional alterations in NMJs are apparent (Balice-Gordon
et al., 2000; Rich et al., 2002).
Mechanisms of neuronal degeneration are highly compart-
mentalized (Gillingwater and Ribchester, 2001). Injury or depri-
vation of growth factors can lead to apoptotic death of the neu-
ronal cell body, followed by rapid degeneration of dendrites,
Axonal transection results in rapid degeneration of the isolated
distal axon by a caspase-independent mechanism (Raff et al.,
2002). Rapid axonal degeneration after transection is delayed in
mice with the Wldsmutation (Brown et al., 1992). Whether this
mutation, which involves a proteosomal protein, would protect
against the NMJ changes in P0
We described here significant alteration in the synaptic connec-
tions of chronically dysmyelinated neurons that precedes axonal
degeneration. Our data have important implications regarding
the progression of neurological disability in primary myelin dis-
eases because it provides proof of principle for gradual muscle
deafferentiation by chronically dysmyelinated or demyelinated
neurons. Gradual and progressive neuronal diaschsis may con-
tgmice. This mech-
tgmice remains to be determined.
(Trapp et al., 1999; Herndon, 2002). Maintenance of synaptic
connectivity should be considered as a therapeutic target in in-
herited and acquired diseases of myelin.
Albuquerque EX, Rash JE, Mayer RF, Satterfield JR (1976) An electrophys-
iological and morphological study of the neuromuscular junction in pa-
tients with myasthenia gravis. Exp Neurol 51:536–563.
Balice-Gordon RJ, Chua CK, Nelson CC, Lichtman JW (1993) Gradual loss
of synaptic cartels precedes axon withdrawal at developing neuromuscu-
lar junctions. Neuron 11:801–815.
Balice-Gordon RJ, Smith DB, Goldman J, Cork LC, Shirley A, Cope TC,
Pinter MJ (2000) Functional motor unit failure precedes neuromuscu-
lar degeneration in canine motor neuron disease. Ann Neurol
Berg DK, Hall ZW (1975) Loss of alpha-bungarotoxin from junctional and
extrajunctional acetylcholine receptors in rat diaphragm muscle in vivo
and in organ culture. J Physiol (Lond) 252:771–789.
Betz W, Osborne M (1977) Effects of innervation on acetylcholine sensitiv-
ity of developing muscle in vitro. J Physiol (Lond) 270:75–88.
Blazej RG, Mellersh CS, Cork LC, Ostrander EA (1998) Hereditary canine
from human spinal muscular atrophy. J Hered 89:531–537.
Blondet B, Rieger F, Verdiere-Sahugue M (1989) Activity-independent
modulation of acetylcholine receptor levels in rat skeletal muscle follow-
ing neonatal denervation. Neurosci Lett 102:273–278.
Brady ST, Witt AS, Kirkpatrick LL, de Waegh SM, Readhead C, Tu PH, Lee
VM (1999) Formation of compact myelin is required for maturation of
the axonal cytoskeleton. J Neurosci 19:7278–7288.
Brown MC, Lunn ER, Perry VH (1992) Consequences of slow Wallerian
degeneration for regenerating motor and sensory axons. J Neurobiol
Cochilla AJ, Angleson JK, Betz WJ (1999) Monitoring secretory membrane
with FM1-43 fluorescence. Annu Rev Neurosci 22:1–10.
Colman H, Nabekura J, Lichtman JW (1997) Alterations in synaptic
strength preceding axon withdrawal. Science 275:356–361.
de Waegh SM, Lee VM, Brady ST (1992) Local modulation of neurofila-
ment phosphorylation, axonal caliber, and slow axonal transport by my-
elinating Schwann cells. Cell 68:451–463.
Thomas S, Kinetz E, Compton DL, Rojas E, Park JS, Smith C, DiStefano
PS, Glass DJ, Burden SJ, Yancopoulos GD (1996) The receptor tyrosine
kinase MuSK is required for neuromuscular junction formation in vivo.
DelCastilloJ,KatzB (1954) Quantalcomponentsoftheend-platepotential.
J Physiol (Lond) 124:560–573.
Dyck PJ, Karnes JL, Lambert EH (1989) Longitudinal study of neuropathic
sory neuropathy type 1. Neurology 39:1302–1308.
FambroughDM (1974) Acetylcholinereceptors.Revisedestimatesofextra-
junctional receptor density in denervated rat diaphragm. J Gen Physiol
Frei R, Motzing S, Kinkelin I, Schachner M, Koltzenburg M, Martini R
(1999) Loss of distal axons and sensory Merkel cells and features indica-
tive of muscle denervation in hindlimbs of P0-deficient mice. J Neurosci
Garbern JY, Yool DA, Moore GJ, Wilds IB, Faulk MW, Klugmann M, Nave
KA, Sistermans EA, van der Knaap MS, Bird TD, Shy ME, Kamholz JA,
GriffithsIR (2002) PatientslackingthemajorCNSmyelinprotein,pro-
teolipid protein 1, develop length-dependent axonal degeneration in the
absence of demyelination and inflammation. Brain 125:551–561.
GillingwaterTH,RibchesterRR (2001) Compartmentalneurodegeneration
and synaptic plasticity in the Wld(s) mutant mouse. J Physiol (Lond)
Glavinovic MI (1979) Voltage clamping of unparalysed cut rat diaphragm
for study of transmitter release. J Physiol (Lond) 290:467–480.
Griffiths I, Klugmann M, Anderson T, Yool D, Thomson C, Schwab MH,
Axonal swellings and degeneration in mice lacking the major proteolipid
of myelin. Science 280:1610–1613.
Gu Y, Hall ZW (1988) Immunological evidence for a change in subunits of
Yinetal.•DysmyelinationCausesSynapticDysfunctionJ.Neurosci.,April14,2004 • 24(15):3890–3898 • 3897
Herndon RM (2002) Medical hypothesis: why secondary progressive mul-
Katz B, Miledi R (1973) The binding of acetylcholine to receptors and its
removal from the synaptic cleft. J Physiol (Lond) 231:549–574.
KidokoroY (1980) Developmentalchangesofspontaneoussynapticpoten-
tial properties in the rat neuromuscular contact formed in culture. Dev
Kopp DM, Perkel DJ, Balice-Gordon RJ (2000a) Disparity in neurotrans-
mitter release probability among competing inputs during neuromuscu-
lar synapse elimination. J Neurosci 20:8771–8779.
Lappe-Siefke C, Goebbels S, Gravel M, Nicksch E, Lee J, Braun PE, Griffiths
IR, Nave KA (2003) Disruption of Cnp1 uncouples oligodendroglial
functions in axonal support and myelination. Nat Genet 33:366–374.
Lichtman JW, Colman H (2000) Synapse elimination and indelible mem-
ory. Neuron 25:269–278.
McLachlanEM,MartinAR (1981) Non-linearsummationofend-platepo-
tentials in the frog and mouse. J Physiol (Lond) 311:307–324.
McMahan UJ (1990) The agrin hypothesis. Cold Spring Harb Symp Quant
Mitsumoto H, Ikeda K, Holmlund T, Greene T, Cedarbaum JM, Wong V,
Lindsay RM (1994) The effects of ciliary neurotrophic factor on motor
dysfunction in wobbler mouse motor neuron disease. Ann Neurol
Noakes PG, Gautam M, Mudd J, Sanes JR, Merlie JP (1995) Aberrant dif-
ferentiation of neuromuscular junctions in mice lacking s-laminin/lami-
nin beta 2. Nature 374:258–262.
PettmannB,HendersonCE (1998) Neuronal
Raff MC, Whitmore AV, Finn JT (2002) Axonal self-destruction and neu-
rodegeneration. Science 296:868–871.
Rees D (1978) A non-phosphate-buffered physiological saline for in vitro
electrophysiological studies on the mammalian neuromuscular junction.
J Physiol (Lond) 278:8P–9P.
Ribchester RR, Mao F, Betz WJ (1994) Optical measurements of activity-
dependent membrane recycling in motor nerve terminals of mammalian
skeletal muscle. Proc R Soc Lond B Biol Sci 255:61–66.
Rich MM, Wang X, Cope TC, Pinter MJ (2002) Reduced neuromuscular
quantal content with normal synaptic release time course and depression
in canine motor neuron disease. J Neurophysiol 88:3305–3314.
Roy RR, Zhong H, Monti RJ, Vallance KA, Kim JA, Edgerton VR (2000)
Mechanical properties and fiber type composition of chronically inactive
muscles. J Gravit Physiol 7:103–104.
Sack Jr GH, Cork LC, Morris JM, Griffin JW, Price DL (1984) Autosomal
dominant inheritance of hereditary canine spinal muscular atrophy. Ann
Sahenk Z, Chen L, Mendell JR (1999) Effects of PMP22 duplication and
deletions on the axonal cytoskeleton. Ann Neurol 45:16–24.
SanchoS,MagyarJP,AguzziA,SuterU (1999) Distalaxonopathyinperiph-
eral nerves of PMP22-mutant mice. Brain 122:1563–1577.
SanesJR,LichtmanJW (1999) Developmentofthevertebrateneuromuscu-
lar junction. Annu Rev Neurosci 22:389–442.
Scherer S (1999) Axonal pathology in demyelinating diseases. Ann Neurol
SonYJ,ThompsonWJ (1995) Schwanncellprocessesguideregenerationof
peripheral axons. Neuron 14:125–132.
Tonge DA (1974) Chronic effects of botulinum toxin on neuromuscular
cle of the mouse. J Physiol (Lond) 241:127–139.
Trachtenberg JT, Thompson WJ (1997) Nerve terminal withdrawal from
rat neuromuscular junctions induced by neuregulin and Schwann cells.
J Neurosci 17:6243–6255.
Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L (1998) Ax-
onal transection in the lesions of multiple sclerosis. N Engl J Med
Trapp BD, Ransohoff RM, Fisher E, Rudick RA (1999) Neurodegeneration
in multiple sclerosis: relationship to neurological disability. The Neuro-
Warner LE, Hilz MJ, Appel SH, Killian JM, Kolodny EH, Karpati G, Carpen-
C, Lupski JR (1996) Clinical phenotypes of different MPZ (P0) muta-
tions may include Charcot-Marie-Tooth type 1B, Dejerine-Sottas, and
congenital hypomyelination. Neuron 17:451–460.
Weinstein SP (1980) A comparative electrophysiological study of motor
end-plate diseased skeletal muscle in the mouse. J Physiol (Lond)
Wrabetz L, Feltri ML, Quattrini A, Imperiale D, Previtali S, D’antonio M,
Martini R, Yin X, Trapp BD, Zhou L, Chiu SY, Messing A (2000) P0
glycoprotein overexpression causes congenital hypomyelination of pe-
ripheral nerves. J Cell Biol 148:1021–1034.
Yin X, Crawford TO, Griffin JW, Tu P-H, Lee VMY, Li C, Roder J, Trapp BD
(1998) Myelin-associated glycoprotein is a myelin signal that modulates
the caliber of myelinated axons. J Neurosci 18:1953–1962.
Yin X, Kidd GJ, Wrabetz L, Feltri ML, Messing A, Trapp BD (2000)
tein. J Cell Biol 148:1009–1020.
3898 • J.Neurosci.,April14,2004 • 24(15):3890–3898 Yinetal.•DysmyelinationCausesSynapticDysfunction