II-3
BIOLOGY OF SOME NEUROMUSCULAR
DISORDERS
BIOLOGIJA NEKATERIH ŽIVČNOMIŠIČNIH BOLEZNI
Gerta Vrbová1, Irena Hausmanowa-Petrusewicz2
1 Department of Anatomy and Developmental Biology, University College London, Great Britain
2 Neuromuscular Unit, Medical Research Center, Polish Academy of Science, Warsaw, Poland
Arrived 2004-02-16, accepted 2004-05-10; ZDRAV VESTN 2004; 73: Suppl. II: 3–10
Key words: 5 not in title Duchenne dystrophy; adaptation;
SMA; delayed maturation
Abstract – In order to understand and possibly interfere/
treat neuromuscular disorders it is important to analyze the
biological events that may be causing the disability. We illus-
trate such attempts on two examples of genetically determined
neuromuscular diseases: 1) Duchenne muscular dystrophy
(DMD), and 2) Spinal muscular atrophy (SMA).
DMD is an x-linked hereditary muscle disease that leads to
progressive muscle weakness. The altered gene in DMD
affects dystrophin, a muscle membrane associated proteine.
Attempts were made to replace the deficient or missing gene/
protein into muscles of Duchenne children. Two main strate-
gies were explored: 1) Myoblast and stem cell transfer and 2)
Gene delivery. The possible use of methods other than the
introduction of the missing gene for dystrophin into muscle
fibres are based on the knowledge about the adaptive poten-
tial of muscle to different functional demands and the ability
of the muscle to express a new set of genes in response to such
stimuli. Stretch or overload is now known to lead to changes
of gene expression in normal muscle, and the success of
muscle stretch in the management of Duchenne boys is most
likely to be due to such adaptive changes. Electrical stimula-
tion of muscles is also a powerful stimulus for inducing the
expression of new genes and this method too has produced
beneficial effects on the progress of the disease in mice and
men.
SMA is a heterogeneous group of hereditary neuromuscular
disorders where the loss of lower motoneurones leads to pro-
gressive weakness and muscle atrophy. The disease subdivides
into 3 forms according to the severity of the symptoms and
age of onset. All three forms of SMA have been mapped to
chromosome 5q11.2-13.2. Clinical features of all these forms
of SMA include hypotonia shortly after birth, symmetrical
muscle weakness and atrophy, finger tremor, areflexia or
hyporeflexia and later contractures. In patients with SMA 1
and 2 the development of all parts of the motor unit is slower.
The rate of maturation is critical for the survival of both
motoneurone and muscle and that events that interfere with
the time course of maturation cause both motoneurone and
muscle fibre death. The proposal that the SMN gene/protein is
involved in the process to developmental changes in cells and
therefore crucial for their survival is put forward. The under-
standing of the developmental changes and their influence
on motoneurone and muscle survival may help to devise thera-
peutic interventions. These may include a) protection of the
motoneurone cell body during a critical period of its develop-
ment by reducing its excitability or enhancing its defences by
Ključne besede: Duchenova distrofija; adaptacija; SMA; za-
poznelo dozorevanje
Izvleček – Živčnomišične bolezni bomo bolje razumeli in
morebiti zdravili le na podlagi analize bioloških procesov, ki
povzročajo prizadetost. V prispevku predstavljamo poskuse
takšne analize na dveh primerih iz skupine dednih živčno-
mišičnih bolezni: 1) Duchennove mišične distrofije (DMD)
in spinalne mišične atrofije (SMA).
DMD je na x-kromosom vezana dedna bolezen mišic, ki pov-
zroča napredujočo mišično šibkost. Patološko spremenjeni
gen, ki povzroča DMD, določa na mišično celično membra-
no vezani protein distrofin. Poskusi nadomestiti patološko
spremenjeni gen/protein v mišicah so bili že napravljeni z
uporabo prenosa mioblastov in zarodnih celic ter neposred-
nega vnosa dednine/gena v mišice dečkov z DMD. Drugi
smiselni terapevtski pristopi bi lahko temeljili na razumeva-
nju adaptacijskih zmožnosti skeletne mišice na različne funk-
cijske zahteve in dokazane zmožnosti ekspresije novih nizov
genov v mišičnih celicah v odgovor na takšne zahteve. Nateg
ali prekomerna obremenitev normalnih mišičnih celic vodita
v spremenjeno ekspresijo genov v mišicah, verjetno pa lahko
terapevtske uspehe razteznih vaj oziroma uporabe ortoz v te
namene pri dečkih z DMD pripišemo istemu mehanizmu
mišične adaptacije. Tudi električna stimulacija je močan
stimulus indukcije ekspresije novih genov. Njeni ugodni učin-
ki na napredovanje mišične distrofije so že dokazani tako na
živalskem modelu (miš) kot na človeku.
Spinalna mišična atrofija je klinično heterogena skupina ded-
nih živčnomišičnih bolezni, za katero je značilna izguba spod-
njih motoričnih nevronov z napredujočo mišično šibkostjo
in mišičnimi atrofijami. SMA se deli v tri oblike glede na stop-
njo izraženosti bolezni in na starost ob pojavu bolezni. Vse
tri oblike SMA so vezane na peti kromosom (5q11.2-13.2), to
je na regijo, ki določa gen SMN (»survival motoneuron gene«).
Klinične značilnosti vseh oblik bolezni so zmanjšan mišični
napon (hipotonija; lahko že ob ali zgodaj po rojstvu), sime-
trična šibkost in atrofije mišic, tremor prstov in jezika, hipore-
fleksija ali arefleksija tetivnih refleksov ter kasneje v poteku
bolezni izrazite kontrakture. Pri pacientih s SMA1 in SMA2 je
razvoj vseh delov motorične enote upočasnjen. Pravilen raz-
voj oziroma časovno usklajeno dozorevanje motoričnih
nevronov in mišic je kritičnega pomena za njihovo preživetje,
dogodki, ki posežejo v časovni potek njihovega dozorevanja,
pa povzročijo smrt tako motoričnih nevronov kot mišičnih
vlaken. V prispevku utemeljujemo, da je gen/protein SMN
kritično udeležen v procesu razvojnih sprememb celic in tako
presoden za njihovo preživetje. Razumevanje razvojnih spre-
memb in njihovega vpliva na preživetje motoričnih nevronov
ZDRAV VESTN 2004; 73: II-3–10
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upregulating heat shock proteins, b) stabilizing neuromuscu-
lar junctions to enhance and prolong the retrograde influen-
ces from the muscle that affect motoneurone survival, c) pro-
tecting muscle fibres from apoptosis, as well as stimulating
their maturation by activity appropriate to their younger age
that results from their delayed development.
These approaches should be considered in addition to or in
conjunction with possible interference with the gene and its
product.
In order to understand and possibly interfere/treat neuro-
muscular disorders it is important to analyze the biological
events that may be causing the disability. In this presentation
I would illustrate such attempts on two examples of geneti-
cally determined neuromuscular diseases: 1) Duchenne mu-
scular dystrophy, and 2) Spinal muscular atrophy.
in mišic bi utegnilo pomagati pri razvoju terapevtskih inter-
vencij. Te bi lahko bile: a) zaščita motoričnih nevronov v
kritičnem času njihovega dozorevanja z zmanjševanjem nji-
hove vzdražnosti ali krepitvijo obrambnih zmožnosti s pove-
čano sintezo (»up-regulation«) posameznih proteinov (»heat-
shock«), b) stabilizacija živčnomišičnega stika, da bi tako
okrepili in podaljšali retrogradne vplive mišice na preživetje
motoričnih nevronov, c) zaščititi mišična vlakna pred apo-
ptozo, kot tudi spodbuditi njihovo dozorevanje s primerno
aktivnostjo glede na stopnjo upočasnjenega razvoja.
Navedeni pristopi bi lahko pomenili dodatne ali dopolnilne
terapevtske metode ob manipulaciji genov oziroma njihovih
produktov.
Duchenne muscular dystrophy
Duchenne muscular dystrophy is an x-linked hereditary
muscle disease that leads to progressive muscle weakness.
The gene that is altered in this condition is a very large gene,
dystrophin, that codes for a protein deficient in muscles of
patients suffering from this disease. The mode of inheritance
is illustrated in Figure 1. Since the discovery of the genetic
nature of the disease attempts were made to design therapies
that would replace the missing gene/protein.
Gene therapy
Duchenne muscular dystrophy was the first neuromuscular
disease in which the genetic and biochemical abnormality was
identified (1). Soon after the genetic abnormality was estab-
lished the product of the abnormal gene was identified and
named dystrophin (2). Having established these basic facts,
efforts were made to replace the deficient or missing gene/
protein into muscles of Duchenne children, with the hope
that such treatment may halt the relentless progression of the
disease. Two main strategies were explored: 1) Myoblast and
stem cell transfer and 2) Gene delivery. A brief summary of
these attempts follows.
Myoblast and stem cell transfer
This approach was based on the hope that normal myoblasts
or myotubes when transferred into the diseased muscle may
produce sufficient amounts of dystrophin to allow the diseased
muscle to function (3). However, attempts to cure dystrophic
muscles by myoblast transfer on children suffering from Duch-
enne dystrophy failed (4, 5).
More recently, attempts were made to isolate muscle derived
stem cells that could be used for transfer into diseased muscle,
with the hope of inducing the formation of normal muscle
fibres (6). So far it does not appear that enough healthy
muscle fibres can be introduced by this method to counter the
progress of muscle weakness.
Delivery of genes
Efforts were made to deliver to the muscle fibres either a full-
length DNA construct of the very large dystrophin gene or its
truncated forms using various vectors to carry the genetic
material into the cell. The lack of success of this approach may
have been due to a combination of factors, such as technical
problems, immunological rejection and responses of the
muscle fibre itself when confronted with new, unusual proteins
and vectors that were used to introduce them (7). Nevertheless,
recent results based on the use of adeno-associated virus for treat-
ment of an animal model homologous to limb girdle muscular
dystrophy (LGMD) warrant some optimism (8, 9).
A slightly different approach has been used in an attempt to
improve the condition of mdx mice that lack dystrophin. The
expression of a protein with a similar function to dystrophin,
i. e. utrophin, was upregulated and this resulted in some bene-
ficial effects in the mdx mouse. Upregulation of utrophin in
mdx mice seemed to have improved their condition while a
total absence of utrophin in mdx KO mice increased the se-
verity of the symptoms, thus indicating that utrophin may be
able to compensate for the absence of dystrophin (10).
Encouraging results were recently reported by Moll et al. (11)
where the introduction of an agrin minigene alleviated the
symptoms in a mouse model of congenital muscular dystro-
phy. The rationale for this approach was that agrin binds both
to the basement membrane and to alpha dystroglycans and
may amend the membrane deficit responsible for the disease.
These results indicate that the function of dystrophin can be
replaced by other proteins.
In all these experiments introduction of new molecules or the
upregulation of existing molecules used genetic manipulations,
a method with many problems that are not yet fully under-
stood.
In view of the relatively little success obtained using the me-
thods of direct gene manipulations, it may be timely to ex-
plore alternatives where the gene expression of the muscle
fibre itself is modified without introducing an alien substance
into the system. Most cells, and muscle is no exception, have a
tremendous adaptive potential that enables them to produce,
given the right stimulus, their own alternative isoforms of pro-
teins and molecules (12). Inducing the expression of new pro-
teins and upregulation of some organelles such as mitochon-
dria in the diseased muscle fibres may possibly protect them
from degeneration and thus halt the progress of the disease. It
is the exploitation of this avenue that is being considered. In
order to achieve this aim it is important to examine the func-
tion of dystrophin.
Function of dystrophin
The cell membrane is a lipid bilayer which ensures a more or
less stable internal environment of the cell by regulating the
communication between the extracellular and intracellular
compartment. Muscle fibres are unique, for due to their me-
chanical activity during contraction and relaxation unusually
severe mechanical stresses are imposed upon their membrane.
The fragile membrane which consists of a lipid bilayer is pro-
tected by a) the thick basal lamina on the external surface of
the muscle fibre and b) a complex subsarcolemmal network
on the inside of the membrane of which actin filaments are the
major component. A device that keeps these two layers to-
gether is provided by dystrophin and dystrophin associated
proteins (DAP) (13). In the absence of dystrophin and DAP
d13.p65 5.6.2004, 3:054
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the lipid membrane when subjected to excessive stresses
ceases to be a reliable barrier between the extra- and intra-
cellular compartment. Thus excess calcium may enter the
muscle fibre and cause damage, while other molecules that
are usually not allowed to leave the muscle fibre, such as the
enzyme creatinkinase, leak out in excessive amounts. Such a
disturbance of the internal environment of the muscle fibre
may then have disastrous consequences (14).
It could be that the undesirable consequences of the lack of
dystrophin and DAP may be alleviated by inducing muscle fi-
bres to upregulate other molecules such as agrin or interme-
diate filaments that can be produced by the diseased muscle
fibre, for the muscles do have the genes coding for these mole-
cules. Inducing the muscle fibres to produce these molecules
by using the muscles’ own resources may strengthen the
muscle membrane and cytoskeleton, so as to protect it from
the consequences of repeated membrane damage. In addi-
tion, the muscle fibre can be induced to increase the number
of mitochondria therefore giving it a better chance to buffer
Ca++ as well as to maintain a relatively better supply of energy,
which may be reduced due to the loss of creatinkinase.
Which molecules could help to substitute the missing dystro-
phin? Possible candidates for such a role are peptides usually
referred to as intermediate filaments (spectrin, vimentin,
desmin and others). These are known to be important for the
integration of various cellular structures within the sarcomere
and cytoplasm as well as for connecting them to the sub-
sarcolemmal network of actin (15). Interestingly, these mole-
cules are abundant in skeletal muscle fibres during develop-
ment and are downregulated when the muscle matures (15).
It could be that this downregulation of the intermediate fila-
ment expression with age coincides in normal muscle with
the upregulation of dystrophin and DAP and that the integrity
of the muscle fibre then depends entirely upon this system.
Such a sequence of events may explain observations on Duch-
enne children where muscle deterioration occurs after the
first few years of life, and observations in mdx mice where a
dramatic muscle degeneration can be observed during the 2nd
and 3rd week of their life (16), a time when in mice neonatal
isoforms of proteins are exchanged for their more mature
counterparts (17). Unfortunately there are no precise data to
correlate these clinical observations with the presence of in-
termediate filaments, or in normal muscles with the develop-
mental changes of dystrophin.
Nevertheless it may be beneficial for muscles that lack dystro-
phin to prevent the downregulation of intermediate filaments
that normally occurs with development as well as to find ways
to upregulate the expression of these polypeptides in
muscles of Duchenne patients. This may be possible to achieve
by altering the activity of the muscle. Appropriate alterations
of muscle activity are known to induce/upregulate specific
isoforms of muscle proteins and genes coding for them.
Induction of the expression of new
genes by altered muscle function
Modification of gene expression by stretch/increased
load
It has long been known that increased load imposed upon
skeletal muscle or muscle stretch leads to muscle hypertro-
phy as well as to changes in oxidative enzymes and capillary
density (18–20). In addition to these changes stretch/over-
load also modifies gene expression, and some of these chan-
ges include alterations in structural proteins of the sarcomere
such as myosin heavy chains, and possibly those involved in
maintaining the structural integrity of the sarcomere (21–23).
Thus, stretch has an important morphogenetic role in regulat-
ing muscle phenotype.
Modification of gene expression by contractile
activity
Changing the pattern of activity of skeletal muscles has pro-
found effects on the phenotype of skeletal muscle fibres. Re-
ducing or abolishing continued activity typical for slow con-
tracting postural muscles will transform them into fast con-
tracting muscles that are activated intermittently. Moreover,
applying a tonic pattern of activity on such a modified slow
muscle by electrical stimulation will maintain its characteristic
slow properties. These findings inspired experiments in which
fast muscles that are naturally activated intermittently were
subjected to electrical stimulation typical of a slow postural
muscle. Results showed that this particular type of continuous
activity turned fast muscles into slow muscles (24, 25).
The ability of skeletal muscle fibres to respond to altered acti-
vity has been particularly well explored in fast twitch muscles
that were converted into slow twitch fibres by chronic low
frequency electrical stimulation (CLFS) (for review see 12,
23). The model of CLFS of fast twitch muscles allowed the
study of the adaptive potential of skeletal muscle and defines
a) the various physiological, biochemical and molecular chan-
ges that occur in skeletal muscles in response to this distinct,
patterned activity regime and the molecular changes that
could account for the transformation, b) the changes of gene
expression induced by CLFS and finally c) the mechanism that
induces changes of gene expression.
The main functional changes that are induced by electrical
stimulation of fast muscles are as follows: a) a change in the
time course of contraction and relaxation, b) a change in the
force/frequency relationship of the muscle, in that more force
is developed at relatively lower rates of stimulation, c) a dra-
matic increase of fatigue resistance and the ability of the
muscle to work for long periods of time. Various molecular
changes in the different compartments of the muscle fibre are
responsible for these functional alterations (Figure 2 molecu-
lar changes related to function).
The changes in the time course of contraction and relaxation
induced by CLFS are likely to be linked to alterations of pro-
teins and receptors of the sarcoplasmic reticulum (SERCA) as
well as to alterations of isoforms of myofibrillar proteins, i. e.
myosin heavy and light chains, and the troponin complex. The
isoforms of myofibrillar proteins in typical fast muscles are
gradually exchanged for their slow counterparts. This ex-
change involves the rebuilding of the sarcomere and a dra-
matic reorganization of its structure to make it more suitable
for sustained and long-term activity.
An important part of the adaptation to prolonged activity is
the strengthening of the cytoskeletal infrastructure of the
muscle fibre. Thus a several fold increase in vimentin, desmin
(26) and a change in the alpha actinin reflected also in the
thickening of the Z line have been reported (27).
CLFS leads to a dramatic increase in fatigue resistance, and this
may be to some extent correlated with the transition from
anaerobic to aerobic metabolism as well as with the increase
in capillary density and blood supply. Nevertheless other fac-
tors such as the steep increase in hexokinase II and the rise in
GLUT 4, which lead to an increased capacity of glucose uptake
and phosphorylation, probably also contribute to the estab-
lishment of this physiological change.
Remodelling of the muscle fibres involves altered
gene expression
In the previous section some of the functional and biochemical
changes that are induced by electrical stimulation were listed
and discussed in relation to function. The transition from fast to
slow phenotype in response to electrical stimulation involves
processes of coordinated expression and repression of proteins
within the same or different cellular structures or compartments.
VRBOVÁ G, HAUSMANOWA-PETRUSEWICZ I. BIOLOGY OF SOME NEUROMUSCULAR DISORDERS
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Examination of changes at the mRNA level revealed that alte-
rations of mRNA levels for the various proteins that are induced
to change in response to electrical stimulation precede the
changes in proteins. Thus there is now clear evidence that the
gene expression for various isoforms of proteins of the muscle
fibres can be modified by patterned electrical activity. Although
the change of gene expression controlling different molecular
elements of the muscle is not synchronous it can nevertheless
be concluded that the gene expression for most proteins of the
muscle fibre changes with activity.
Thus these experimental results taken together clearly show
that it is possible to alter the gene expression of post-mitotic
excitable cells by activity.
The mechanisms involved in this change in gene expression
are under discussion, but it seems to be related to the duration
of the Ca++ transients (28) possibly mediated by activation of a
calcineurin and MEF2 signal NFAT (29).
These experimental results obtained on healthy muscles
either by stretch or activity illustrate the ability of the muscle to
activate a new programme of gene expression. They provide
the background and rationale for attempting to study whether
such manipulations of muscle function are beneficial for
diseased muscles of mice and men.
Altered activity can protect dystrophic skeletal
muscles
Since a change in gene expression of normal muscles can be
induced by altering muscle function such as stretch or a diffe-
rent pattern of contractile activity, it may be pertinent to
examine the possibility that switching on novel genes by alter-
ing muscle function may reduce the rate of deterioration of
diseased skeletal muscle fibres.
Mechanical interference
Empirical observations of the effects of stretch and muscle
load on the progress of the deterioration of muscles in Duch-
enne boys indicate that these methods of treatment are per-
haps the most successful yet.
Regular stretching of muscles of Duchenne boys resulted in a
slowing of the deterioration of the diseased muscles (30). The
use of orthotic devices has also proved beneficial, and in-
creased the life expectancy of the patients treated in this way
(31). The use of such orthotic devices is likely to act through
stretching the hamstrings and other muscle groups. In con-
trast to these positive effects of load and stretch, unloading the
calf muscles by lengthening the achilles tendon has deleterious
effects on the long-term prospect of the patients (32). It is also
well recognized that assisted respiration improves the con-
dition of the patients. While the most likely effect of this treat-
ment is the improved respiratory function and the effect of
this on the cardiovascular and other systems affected by chronic
hypoventilation and ensuing lack of oxygen, a possible by-pro-
duct of assisted ventilation is a stretch of respiratory muscles.
This in addition to the other beneficial effects of assisted venti-
lation may improve the condition of the patient. These obser-
vations, though carefully documented in Duchenne children,
had not hitherto had a rational explanation. However, when
considering the effects of stretch on gene expression, content
of oxidative enzymes and capillary density on normal skeletal
muscles (19, 20, 23), these results on improved muscle func-
tion of Duchenne children induced by stretch now have a ra-
tional explanation.
Electrical stimulation
Experiments on mutant mice that suffer from severe muscle
weakness (C57BL dy/2J/dy2J) that is much more severe than
that of dystrophin deficient mdx mouse have shown that a
particular pattern of activity imposed upon their muscles leads
to reduction of the progress of deterioration of their muscles.
These mutant mice have a defect of laminin M and some of the
DAP molecules and there are some similarities between these
mutants and congenital muscular dystrophy (CMD) (33). Elec-
trical stimulation of leg muscles in these animals at low fre-
quencies for several weeks led to a reduction of the rate of
loss of force and muscle bulk characteristic of this disease in
mice (17, 34). Moreover, electrical stimulation reverses the
changes in enzyme activities caused by the disease process in
muscles of these dystrophic animals (35, 36).
The encouraging results on dystrophic mice led to attempts to
investigate the effects of low frequency electrical stimulation on
muscles of patients with Duchenne or Becker dystrophy. The
results demonstrated that electrical stimulation reduces the rate
of deterioration of ankle dorsiflexors and quadriceps muscles in
boys with Duchenne dystrophy, provided the stimulation was
started before the muscles became excessively weak (30, 37–
39). Moreover, the pattern of stimulation was critical, for only
stimulation at slow frequencies (i. e. 10 Hz or less) caused im-
provement while stimulation at frequencies in excess of 30 Hz,
which is well within the range of normal firing frequency of
motoneurones to these muscles, not only did not improve the
muscle function, but enhanced the deterioration of the muscle
(30). It could be argued that the low frequency electrical stimu-
lation may lead to increases of Ca++ levels of the stimulated
muscle fibres, and it is therefore counterintuitive that this pro-
cedure would have a beneficial effect on the muscle. However,
the results show that even if such increases did occur they did
not prevent the improvement induced by electrical stimulation.
Perhaps this is not surprising, for as our knowledge about the
role of Ca++ in biological processes increases, it is becoming clear
that the particular characteristics of the Ca++ dynamics, i. e. the
frequency, amplitude and duration of the Ca++ transients, could
be more important for the function of the cell than the resting
concentration of this ion (see 40).
Manipulating ion channels
Recently, evidence has been presented that manipulating Cl–
channels could be beneficial to mdx mice where dystrophin is
lacking (41, 42).
The firing pattern of skeletal muscle fibres is controlled by the
activity that reaches them via their axons and in normal condi-
tions each action potential in an axon elicits a single muscle
action potential and a single twitch contraction. This response
of the muscle fibre depends on the excitability of its mem-
brane and is controlled by the passive membrane properties
of the muscle fibre which depends on the conductance of
some of the ion channels. If the conductance of the Cl– channel
is low, then the muscle fibre will be repeatedly activated by a
single nerve impulse. Nature has provided us with such a mo-
del in the form of the myotonic adr mouse mutant. Recently
two groups independently produced a mutant where the mdx
mouse was combined with an adr mouse and surprisingly the
mdx mouse benefited from this crossbreeding and showed
longer life expectancy and other improvements of its perfor-
mance (41, 42). The most likely explanation of this result is
that the muscles of the mdx mouse were undergoing chronic
stimulation, since each impulse in an axon produced several
discharges in the muscle membrane. In terms of hypothetical
treatment this is a very exciting result, since it shows a new
way how to influence most muscles of Duchenne patients with-
out having to stimulate each individual muscle electrically.
Spinal muscular atrophy (SMA)
Current status of SMA
SMA is a heterogeneous group of hereditary neuromuscular
disorders where the loss of lower motoneurones leads to pro-
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gressive weakness and muscle atrophy. The most common of
these disorders is the infantile and juvenile proximal SMA, with
an incidence of 1/10–15000 newborns and carrier frequency
of 1/50 individuals (43). SMA represents the second most com-
mon fatal autosomal recessive disorder of children, cystic fi-
brosis being first. The disease usually subdivides into 3 forms
according to the severity of the symptoms and age of onset
(44, 45).
SMA1 is the most severe form, SMA2 intermediate and SMA3
the relatively mild form. There are some common features of
all these which include hypotonia shortly after birth, symme-
trical muscle weakness and atrophy, finger tremor, areflexia
or hyporeflexia and later contractures. Not all muscle groups
are affected to the same extent; the diaphragm, extraoccular
muscles and the myocardium are usually spared, while the
proximal muscles of the limbs are affected earlier than the
others and the atrophy is more severe.
Molecular genetics
All three forms of SMA have been mapped by linkage analysis to
chromosome 5q11.2-13.2 (46–48). Although several other ge-
nes have been localized to this region the SMN gene
appears to be the most important, since around 95% of SMA
patients carry homozygous deletions on this gene. The SMN gene
in humans exists in 2 copies: SMN1 localized at the telomeric part
of the chromosome and SMN2 at the centromeric part of the
chromosome. Both copies are identical apart from 2 base pairs
on exon 7 and 8 which are present only in the SMN1 copy and
absent in the SMN2 copy of the SMN gene. In SMA patients the
SMN1 form lacks 2 base pairs on exon 7 and 8.
There is some correlation between the number of copies of
the SMN2 gene and the severity of the disease so that a higher
number of copies of this gene is associated with the milder
form of the disease (49). Thus the SMN2 gene, in spite of the
absence of the crucial base pairs of exon 7 and 8, is able to
influence to some extent the phenotype of the disease.
A further important step in elucidating the possible cause of
the symptoms in SMA patients was the isolation of the protein
coded for by the SMN gene. It is expressed in most tissues and
is believed to be involved in mRNA metabolism, in particular
pre-mRNA splicing (50–52). This cellular function indicates
that the SMN protein may be involved in situations where the
phenotype of cells changes, such as during development, and
the finding that the expression of the SMN protein is highest
during development is consistent with such a role. Thus it could
be that this protein by controlling mRNA splicing and meta-
bolism may influence and bring about developmental changes
of the tissues. In systems where the timing of these changes is
critical such as motoneurone and muscle, which have to achieve
a certain degree of maturity to be integrated into the develop-
ing neuronal networks, this function of the SMN protein may
be of particular importance.
Provided that the SMN protein is involved in bringing about
developmental changes of motoneurones and muscles it could
be expected that the progress of maturation of the neuromus-
cular system may be delayed in SMA patients.
Signs of immaturity of the neuromuscular system in
SMA children
In a thorough study on a large number of SMA patients Prof.
Hausmanowa’s group examined all 3 components of the motor
unit and showed clear signs of immaturity of all 3 components.
A characteristic feature of the spinal cord taken from autop-
sies of patients with SMA is the absence of motoneurones in
the ventral horn. Nevertheless, the remaining motoneurones
show some signs of immaturity and are smaller with less devel-
oped Nissl substance than in normal fetuses of comparable
age (53–55).
In addition, continuous EMG activity with a firing rate of 9–18
Hz can be recorded from muscles of SMA patients, even dur-
ing rest and sleep (56–58). This altered activity of motoneuro-
nes is similar to that found in animals where maturation of
motoneurones was retarded by temporarily disrupting the
interaction between the motor nerve and muscle (55). These
findings indicate a profound change in the excitability of the
spinal motoneurones.
Consistent with the findings on motoneurones signs of delayed
development have also been described for motor axons (59).
In the ventral roots from SMA patients there were many multi-
axonal bundles enwrapped by a single Schwann cell, a low
density of axons/mm and a relatively high proportion of poor-
ly myelinated thin axons. Small atrophic ventral roots were
previously described in material from SMA patients, without
signs of sparse myelination (60). In addition, axons have a pro-
longed latency of the response and reduced conduction velo-
city (61).
Finally signs of immaturity can also be seen in muscles from
SMA children. In SMA1 many undifferentiated, extremely small
muscle fibres were present which displayed characteristics of
fetal muscles (60). There were also some myogenic cells that
resembled myotubes and several of these were often enclosed
in the same basal lamina. The fetal character of these very
small muscle fibres was indicated by the finding that when
stained with acridine orange they displayed a vivid orange co-
lour possibly associated with the high content of RNA. Perhaps
the most compelling evidence of their immaturity is the pre-
sence of desmin and vimentin, known to be downregulated
during normal muscle development and absent in denervated
muscles (63–65).
An interesting feature of muscles from SMA children is their
different response to injury. Whereas more mature muscles
respond to injury by necrosis, muscles from SMA children re-
spond by apoptotic changes to the same insult.
In conclusion, there is strong evidence that the neuromuscu-
lar system in SMA children is immature. However, why de-
layed maturation should result in the specific pathology seen
in SMA children needs to be explained. Such an explanation
may be provided by understanding the role of accurate timing
of motoneurone and muscle maturation during normal de-
velopment of the neuromuscular system.
Survival of motoneurones depends on maturation
Motoneurones are among the first cells to be generated in the
spinal cord. Like in other neuronal populations the initial gene-
ration of these neurones is followed by death of a proportion
of these motoneurones. This event is referred to as naturally
occurring cell death, and the regulation of this event is not
clear.
Early studies indicated a relationship between the number of
surviving motoneurones and the amount of muscle tissue avail-
able for innervation (66).
After the ‘naturally occurring cell death’ has been completed,
motoneurones remain critically dependent on contact and
active interaction with their target. If the connection between
the motoneurone and target is disrupted during early post-
natal development either surgically by cutting the axon and
removing the target, or pharmacologically by preventing neu-
romuscular interactions a considerable number of motoneu-
rones die. The extent of motoneurone death depends on the
timing of the insult, i. e. when after birth it is inflicted and the
duration of the interruption of neuromuscular contacts (67).
What are the changes induced by the target that allow moto-
neurone survival? During the developmental period when the
motoneurone needs contact with its target muscle many chang-
es occur within the CNS and spinal cord. The motoneurone is
initially activated by relatively few and weak afferent inputs
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which is amplified by the high excitability of the small moto-
neurones as well as by the electrotonic coupling of the moto-
neurones to each other (68). With further development the
strength and efficiency of the afferent inputs to motoneurones
increase. In order for the motoneurone to be able to deal with
this increase of excitatory stimuli mediated by glutamate it has
to acquire new properties that allow it to deal with these new
functions.
Glutamate, the mediator of most excitatory afferent inputs to
the motoneurone, excites the motoneurone through various
receptors. The most studied of these is the NMDA receptor.
The role of this receptor in excitotoxic cell death is well estab-
lished. Immature motoneurones die when exposed to gluta-
mate, and interaction with the target is necessary to induce
changes that allow them to survive exposure to glutamate
(69).
When neuromuscular interactions of immature motoneurones
are prevented they revert to their less mature behaviour and
are killed by exogenously applied NMDA (70). In view of these
findings, we proposed that target deprived postnatal moto-
neurones fail to achieve the degree of maturity that enables
them to withstand exposure to glutamate. Since glutamate is
released by the ever more abundant afferent inputs that im-
pinge upon the motoneurone, such motoneurones are then
likely to die as a result of this exposure (69). Figure 3 illu-
strates schematically this hypothesis.
Further support for this hypothesis was provided by results
that permanent survival of motoneurones destined to die
after axotomy can be achieved if during the period of sepa-
ration from the target muscle the cell body is protected from
the excitotoxic effects of glutamate by blocking NMDA recep-
tors (71).
These findings clearly show the importance of the precise
timing of the maturation of the motoneurone in relation to the
development of the rest of the spinal cord circuitry for the
normal development of the system and any interference with
this will have devastating effects on the immature motoneu-
rone. The possibility that the SMN protein and its partners are
important for the correct timing of these events may explain
their importance for the survival of developing motoneurones.
Although these results bring us a step further and pinpoint the
importance of the target for motoneurone maturation and
survival, the mechanisms by which the target muscle achieves
this are not clear.
It is possible that in SMA patients the motoneurone is receiv-
ing the signal from the muscle and all the messages required
for its maturation but it is unable to do so for the molecules that
are necessary to transform the motoneurone from an imma-
ture to a mature cell are missing. An adequate number of co-
pies of the SMN gene and its gene product are therefore likely
to be needed to carry out the changes involved in the transi-
tion of an immature to a mature motoneurone.
Critical dependence of muscle development and
differentiation on continued contact with the motor
nerve
Although many aspects of muscle development can proceed
without innervation, and even immature muscle fibres can
survive denervation for considerable periods of time, they are
nevertheless critically dependent on functional innervation
during early postnatal development. If the muscle is denerva-
ted during the early periods of postnatal development and
then reinnervated by its own nerve about 60% of its muscle
fibres die after reinnervation. The most likely explanation of
this devastating effect reinnervation has on survival of imma-
ture muscles is the arrest of differentiation and maturation of
these muscle fibres during the period of denervation. Such
immature muscle fibres are unable to withstand the vigorous
activity that is imposed upon them by the now more mature
motoneurone and are destroyed by it. Thus if the timing be-
tween the maturation of the muscle and that of the motoneu-
rone and its activity is disrupted muscle fibres are unable to
survive.
Thus, like the motoneurone the immature muscle too has to
become competent to cope with functions that are changing
with the development of the locomotor system.
Why is the motor unit so uniquely susceptible to the
lack of SMN protein?
Unfortunately, in the absence of hard evidence the answers to
this problem will be largely speculative, but these specula-
tions can nevertheless be useful since they can now be tested
in the various transgenic animal models of the disease. When
considering the possibility that the SMN gene and protein may
play a pivotal role during changes of a cell’s or neurone’s phe-
notype the obvious question is: why is the motoneurone
affected more than any other cell? To account for this unique
need of motoneurones for the SMN gene it may be useful to
list the particular features of the motoneurone:
a) The motoneurone is a cholinergic cell, i. e. the transmitter
it synthesizes and releases is cholinergic. However other
cholinergic cells in the CNS, even those with large projec-
tions and relatively long axons such as neurones in the Mey-
nert nucleus, are not affected by the lack of the smn gene.
Thus neither the cholinergic nature of the motoneurone
nor its exceptionally large projections can account for its
special need for the SMN gene.
b) The axon of the motoneurone is outside the CNS in the
periphery. This distinguishes it from many other neurones.
Nevertheless, sensory neurones too have their long pro-
cesses outside the CNS and are not dramatically affected
by the absence of the SMN gene. Moreover, even cholin-
ergic cells with axons in the periphery such as many neu-
rones of the autonomic nervous system are not critically
dependent on the presence of the SMN gene and seem to
survive in patients with SMA.
c) The special relationship of motoneurone and muscle. The
last and probably most unique feature of the motoneurone
is its special relationship with skeletal muscle fibres.
Indeed, it appears that this feature distinguishes the moto-
neurone from all other neurones of the central and
peripheral nervous system and therefore points to the
cause of its unique involvement in SMA.
Why does a neurone that interacts with the muscle have a
special requirement for the SMN gene and protein? Perhaps
the answer to this question can be obtained when considering
results discussing the dependence of the developing normal
motoneurone on contact and interaction with the muscle. Two
main points are relevant to this problem: 1) The motoneurone
in order to become successfully integrated and survive the
increasing excitatory inputs of the maturing spinal cord has to
undergo a change of phenotype where many of the immature
molecules are replaced by their more mature counterparts.
2) This transition is induced by interaction with the muscle and
if it fails to occur in time the motoneurones’ survival is at risk.
Conclusions
Duchenne muscular dystrophy
The possible use of methods other than the introduction of the
missing gene for dystrophin into muscle fibres is discussed.
These methods are based on the knowledge about the adap-
tive potential of muscle to different functional demands and
the ability of the muscle to express a new set of genes in
response to such stimuli. Stretch or overload is now known to
d13.p65 5.6.2004, 3:058
II-9
lead to changes of gene expression in normal muscle, and the
success of muscle stretch in the management of Duchenne
boys is most likely to be due to such adaptive changes. Electri-
cal stimulation of muscles is also a powerful stimulus for induc-
ing the expression of new genes and this method too has pro-
duced beneficial effects on the progress of the disease in mice
and men. The article also illustrates that the understanding of
the cellular mechanisms involved in the consequences of lack
of dystrophin is essential for proposing rational treatment.
Spinal muscular atrophy
The present review provides evidence that the development
of all parts of the motor unit is slower in patients with SMA1
and 2. It also argues that the rate of maturation is critical for the
survival of both motoneurone and muscle and that events that
interfere with the time course of maturation cause both moto-
neurone and muscle fibre death. The proposal that the SMN
gene/protein is involved in the process to developmental
changes in cells and therefore crucial for their survival is put
forward.
The understanding of the developmental changes and their
influence on motoneurone and muscle survival may help to
devise therapeutic interventions. These may include a) pro-
tection of the motoneurone cell body during a critical period
of its development by reducing its excitability or enhancing its
defences by upregulating heat shock proteins, b) stabilizing
neuromuscular junctions to enhance and prolong the retro-
grade influences from the muscle that affect motoneurone
survival, c) protecting muscle fibres from apoptosis, as well as
stimulating their maturation by activity appropriate to their
younger age that results from their delayed development.
These approaches should be considered in addition to or in
conjunction with possible interference with the gene and its
product.
Acknowledgements
I am grateful to Debbie Bartram for her help in preparing this
manuscript.
References
1. Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM.
Isolation of candidate cDNAs for portions of the Duchenne muscular dystro-
phy gene. Nature 1986; 323: 646–50.
2. Hoffman EP, Brown RH, Kunkel LM. Dystrophin: the protein product of the
Duchenne muscular dystrophy locus. Cell 1987; 51: 919–28.
3. Partridge TA. Invited review: Myoblast transfer: A possible therapy for
inherited myopathies? Muscle Nerve 1991; 14: 197–212.
4. Law PK, Goodwin TG, Li HJ, Ajamoughli G, Chen M. Myoblast transfer im-
proves muscle genetics/structure/function and normalizes the behavior
and life-span of dystrophic mice. Adv Exp Med Biol 1990; 280: 75–87.
5. Karpati G, Ajdukovic D, Arnold D, Gledhill RB, Guttmann R, Holland P, Koch
PA, Shoubridge E, Spence D, Vanasse M, Watters GV, Abrahamowicz M, Duff
C, Worton RG. Myoblast transfer in Duchenne muscular dystrophy. Ann
Neurol 1993; 34: 8–17.
6. Asakura A. Stem cells in adult skeletal muscle. Trends Cardiovasc Med 2003;
13: 123–8.
7. Ferrer A, Wells KE, Wells DJ. Immune responses to dystropin: implications
for gene therapy of Duchenne muscular dystrophy. Gene Ther 2000; 7: 1439–
46.
8. Xiao X, Li J, Tsao YP, Dressman D, Hoffman EP, Watchko JF. Full functional
rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associ-
ated virus vector-directed gene therapy. J Virol 2000; 74: 1436–42.
9. Akkaraju GR, Huard J, Hoffman EP, Goins WF, Pruchnic R, Watkins SC, Cohen
JB, Glorioso JC. Herpes simplex virus vector-mediated dystrophin gene
transfer and expression in MDX mouse skeletal muscle. J Gene Med 1999; 1:
280–89.
10. Tinsley JM, Potter AC, Phelps SR, Fisher R, Trickett JI, Davies KE. Amelioration
of the dystrophic phenotype of mdx mice using a truncated utrophin trans-
gene. Nature 1996; 384: 349–53.
11. Moll J, Barzaghi P, Lin S, Bezakova G, Lochmüller H, Engvall E, Müller U, Ruegg
MA. An agrin minigene rescues dystrophic symptoms in a mouse model for
congenital muscular dystrophy. Nature 2001; 413: 302–7.
12. Pette D, Vrbová G. What does chronic electrical stimulation teach us about
muscle plasticity? Muscle Nerve 1999; 22: 666–77.
13. Crawford GE, Faulkner JA, Crosbie RH, Campbell KP, Froehner SC, Chamber-
lain JS. Assembly of the dystrophin-associated protein complex does not
require the dystrophin COOH-terminal domain. J Cell Biol 2000; 150: 1399–
410.
14. Ozawa E, Noguchi S, Hizumo Y, Hagiwara Y, Yoshida M. From dystrophino-
pathy: Evolution of a concept of muscular dystrophy. Muscle Nerve 1998; 21:
421–38.
15. Small JV, Furst DO, Thornell LE. The cytoskeletal lattice of muscle cells. Eur J
Biochem 1992; 208: 559–72.
16. Dangain J, Vrbová G. Muscle development in MDX mutant mice. Muscle
Nerve 1984; 7: 700–4.
17. Dangain J, Pette D, Vrbová G. Developmental changes in succinate dehydro-
genase (SDH) activity in muscle fibres from normal and dystrophic mice. J
Physiol 1984; 351: 38P.
18. Frischknecht R, Vrbová G. Adaptation of rat extensor digitorum longus to
overload and increased activity. Pflügers Arch 1991;419: 319–26.
19. Frischknecht R, Belverstone D, Vrbová G. The response of adult and develop-
ing rat plantaris muscle to overload. Pflügers Arch 1995; 431: 204–11.
20. Zhou AL, Egginton S, Brown MD, Hudlická O. Capillary growth in overload-
ed, hypertrophic adult rat skeletal muscle: an ultrastructural study. Anat Rec
1998; 252: 49–63.
21. Goldspink G, Scutt A, Loughna PT, Wells DJ, Jaenicke T, Gerlach GF. Gene
expression in skeletal muscle in response to stretch and force generation.
Am J Physiol 1992; 262: R356–63.
22. Goldspink G. Selective gene expression during adaptation of muscle in
response to different physiological demands. Comp Biochem Physiol B
Biochem Mol Biol 1998; 120: 5–15.
23. Goldspink G. Gene expression in skeletal muscle. Biochem Soc Trans 2002;30:
285–90.
24. Vrbová G. The effect of tenotomy on the speed of contraction of fast and
slow mammalian muscle. J Physiol 1963; 166: 241–50.
25. Salmons S, Vrbová G. The influence of activity on some contractile charac-
teristics of mammalian fast and slow muscles. J Physiol 1969; 201: 535–49.
26. Baldi JC, Reiser PJ. Intermediate filament proteins increase during chronic
stimulation of skeletal muscle. J Muscle Res Cell Motil 1995; 16: 587–94.
27. Eisenberg BR, Salmons S. The reorganization of subcellular structure in
muscle undergoing fast-to-slow type transformation. A stereological study.
Cell Tissue Res 1981; 220: 449–71.
28. Chen G, Carroll S, Racay P, Dick J, Pette D, Traub I, Vrbova G, Eggli P, Celio M,
Schwaller B. Deficiency in parvalbumin increases fatigue resistance in fast-
twitch muscle and upregulates mitochondria. Am J Physiol Cell Physiol 2001;
281: C114–22.
29. Wu H, Rothermel B, Kanatous S, Rosenberg P, Naya FJ, Shelton JM, Hutcheson
KA, DiMaio JM, Olson EN, Bassel-Duby R, Williams RS. Activation of MEF2 by
muscle activity is mediated through a calcineurin-dependent pathway. EMBO
J 2001; 20: 6414–23.
30. Scott OM, Hyde SA, Goddard C, Dubowitz V. Prevention of deformity in
Duchenne muscular dystrophy. A prospective study of passive stretching
and splintage. Physiotherapy 1981; 67: 177–80.
31. Hyde SA, Scott OM, Goddard CM, Dubowitz V. Prolongation of ambulation
in Duchenne muscular dystrophy by appropriate orthoses. Physiotherapy
1982; 68: 105–8.
32. Manzur AY, Hyde SA, Rodillo E, Heckmatt JZ, Bentley G, Dubowitz V. A
randomized controlled trial of early surgery in Duchenne muscular dystro-
phy. Neuromuscul Disord 1992; 2: 379–87.
33. Arahata K, Engvall E, Hayashi YK et al. Defect of laminin M in skeletal and
cardiac muscles and peripheral nerve of the homozygous dystrophic dy/dy
mice. Proc Japan Acad 1993; 69: 259–64.
34. Luthert P, Vrbová G, Ward KM. Effects of slow frequency electrical stimu-
lation on muscles of dystrophic mice. J Neurol Neurosurg Psychiatry 1980; 43:
803–9.
35. Reichmann H, Pette D, Vrbová G. Effects of low frequency electrical stimu-
lation on enzyme and isozyme patterns of dystrophic mouse muscle. FEBS
Lett 1981; 128: 55–8.
36. Reichmann H, Pette D, Vrbová G. Electrostimulation-induced changes in the
enzyme activity pattern of dystrophic mouse muscles. Acta Histochem Suppl
1983; 28: 125–7.
37. Scott OM, Hyde SA, Vrbová G, Dubowitz V. Therapeutic possibilities of
chronic low frequency electrical stimulation in children with Duchenne
muscular dystrophy. J Neurol Sci 1990; 95: 171–82.
38. Zupan A. Long-term electrical stimulation of muscles in children with Duch-
enne and Becker muscular dystrophy. Muscle Nerve 1992; 15: 362–7.
39. Zupan A, Gregoric M, Valencic, Vandot S. Effects of electrical stimulation on
muscles of children with Duchenne and Becker muscular dystrophy. Neuro-
pediatrics 1993; 24: 189–92.
40. Berridge MJ, Dupont C. Spatial and temporal signalling by calcium. Curr
Opin Cell Biol 1994; 6: 267–74.
41. Heimann P, Augustin M, Wieneke S, Heising S, Jockusch H. Mutual interfer-
ence of myotonia and muscular dystrophy in the mouse: A study on ADR-
MDX double mutants. Neuromuscular Disord 1998; 8: 551–60.
42. Kramer R, Lochmuller H, Abicht A, Rudel R, Brinkmeier H. Myotonic ADR-
MDX mutant mice show less severe muscular dystrophy than MDX mice.
Neuromuscular Disord 1998; 8: 542–50.
VRBOVÁ G, HAUSMANOWA-PETRUSEWICZ I. BIOLOGY OF SOME NEUROMUSCULAR DISORDERS
d13.p65 5.6.2004, 3:059
II-10 ZDRAV VESTN 2004; 73: SUPPL II
43. Pearn J. Incidence, prevalence, and gene frequency studies of chronic
childhood spinal muscular atrophy. J Med Genet 1978; 15: 409–13.
44. Dubowitz V. Muscle disorders in childhood. Major Probl Clin Pediatr 1978;
16: 1–282.
45. Hausmanowa-Petrusewicz I, Fidziańska A. The clinical features of spinal
infantile and juvenile muscular atrophy. In: Gamstorp I, Sarnat HB eds.
Progressive spinal muscular atrophies. New York: Raven Press, 1984: 31–42.
46. Brzustowicz LM, Lehner T, Castilla LH et al. Genetic mapping of chronic
childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3. Na-
ture 1990; 344: 540–1.
47. Melki J, Abdelhak S, Sheth P et al. Gene for chronic proximal spinal muscular
atrophies maps to chromosome 5q. Nature 1990; 344: 767–8.
48. Lefebvre S, Burglen L, Reboullet S et al. Identification and characterization of
a spinal muscular atrophy-determining gene. Cell 1995; 80: 155–65.
49. Lefebvre S, Burlet P, Liu Q et al. Correlation between severity and SMN
protein level in spinal muscular atrophy. Nat Genet 1997; 16: 265–9.
50. Liu Q, Dreyfuss G. A novel nuclear structure containing the survival of motor
neurons protein. EMBO J 1996; 15: 3555–65.
51. Fischer U, Liu Q, Dreyfuss G. The SMN-SIP1 complex has an essential role in
spliceosomal snRNP biogenesis. Cell 1997; 90: 1023–9.
52. Young PJ, Man NT, Lorson CL, Le TT, Androphy EJ, Burghes AH, Morris GE.
The exon 2b region of the spinal muscular atrophy protein, SMN, is involved
in self-association and SIP1 binding. Hum Mol Genet 2000; 9: 2869–77.
53. Malamud W. Infantile progressive muscular atrophy. In: Minckler J ed. Patho-
logy of the nervous system. McGrow Book Company, 1968: 45–54.
54. Malinsky J. Ultrastructural changes of cell elements during the differentiation
of neuroblasts and glioblasts in human spinal cord. Acta Univ Olumunic
Faculty Med 1972; 64: 183–93.
55. Buchthal F, Olsen PZ. Electromyography and muscle biopsy in infantile
spinal muscular atrophy. Brain 1970; 93: 15–30.
56. Hausmanowa-Petrusewicz I, Karwańska A. Electromyographic findings in
different forms of infantile and juvenile proximal spinal muscular atrophy.
Muscle Nerve 1986; 9: 37–46.
57. Hausmanowa-Petrusewicz I, Friedman A, Kowalski J, Skalski M. Spontaneous
motor unit firing in spinal muscular atrophy of childhood. Electromyogr
Clin Neurophysiol 1987; 27: 259–64.
58. Navarrete R, Vrbová G. Differential effect of nerve injury at birth on the
activity pattern of reinnervated slow and fast muscles of the rat. J Physiol
1984; 351: 675–85.
59. Drac H. Histological changes in peripheral nerves in spinal Werdnig-Hoff-
mann muscular atrophy. Neuropatol Pol 1977; 15: 1–16 (Polish).
60. Greenfield JC, Stern RO. The anatomical identity of the Werdnig-Hoffmann
and Oppenheim forms of infantile muscular atrophy. Brain 1927; 50: 652–86.
61. Krajewska G, Hausmanowa-Petrusewicz I. Abnormal nerve conduction ve-
locity as a marker of immaturity in childhood muscle spinal atrophy. Folia
Neuropathol 2002; 40: 67–74.
62. Sarnat HB. Pathology of spinal muscular atrophy. In: Gamstorp I, Sarnat HB,
eds. Progressive Spinal Muscular Atrophy. New York: Raven Press, 1974: 91–
110.
63. Fidziańska A, Goebel HH, Warlo I. Acute infantile spinal muscular atrophy.
Muscle apoptosis as a proposed pathogenetic mechanism. Brain 1990; 113:
433–45.
64. Sarnat HB. Vimentin and desmin in maturing skeletal muscle and develop-
mental myopathies. Neurology 1992; 42: 1616–24.
65. Goebel HH, Bornemann A. Desmin pathology in neuromuscular diseases.
Virchows Arch B Cell Pathol Incl Mol Pathol 1993; 64: 127–35.
66. Hamburger V. The developmental history of the motor neuron. Neurosci Res
Program Bull 1977; 15: 1–37.
67. Lowrie MB, Vrbová G. Dependence of postnatal motoneurones on their
targets: review and hypothesis. Trends Neurosci 1992; 15: 80–4.
68. Navarrete R, Vrbová G. Activity-dependent interactions between motoneu-
rones and muscles: their role in the development of the motor unit. Prog
Neurobiol 1993; 41: 93–124.
69. Greensmith L, Vrbová G. Motoneurone survival: a functional approach.
Trends Neurosci 1996; 19: 450–5.
70. Greensmith L, Hasan HI, Vrbová G. Nerve injury increases the susceptibility
of motoneurons to N-methyl-D-aspartate-induced neurotoxicity in the de-
veloping rat. Neuroscience 1994a; 58: 727–33.
71. Greensmith L, Mentis GZ, Vrbová G Blockade of N-methyl-D-aspartate recep-
tors by MK-801 (dizocilpine maleate) rescues motoneurones in developing
rats. Dev Brain Res 1994b; 81: 162–70.
d13.p65 5.6.2004, 3:0510