Developmental regulation of amyloid precursor protein at the neuromuscular junction in mouse skeletal muscle.

Amyloid precursor protein (APP), associated with Alzheimer's disease plaques, is known to be present in synapses of the brain and in the adult neuromuscular junction (NMJ). In the present study we examined protein and gene expression of APP during the development of mouse skeletal muscle. Using immunocytochemical approaches, we found that APP is first detected in myotube cytoplasm at embryonic day 16 and becomes progressively concentrated at the NMJ beginning at birth until adulthood. The colocalization between APP and acetylcholine receptors at the NMJ is only partial at birth, but becomes complete upon reaching adulthood. We observed that all APP isoforms, including the Kunitz-containing (protease inhibitor or KPI) forms, are up-regulated from birth to postnatal day 5 and then decreased to reach the low levels observed in the adult. This suggests the involvement of APP during the events which lead to a mature mono-innervated synapse. A 92-kDa band, characteristic of a cleaved APP695 isoform and not due to a new muscle-specific alternative spliced form, was observed from postnatal day 15 following completion of polyneuronal synapse elimination. Taken together, these data suggest that skeletal muscle APP may well play a role in the differentiation of skeletal muscle and in the formation and maturation of NMJs.

Rapid and reversible effects of activity on acetylcholine receptor density at the neuromuscular junction in vivo.

Quantitative fluorescence imaging was used to study the regulation of acetylcholine receptor (AChR) number and density at neuromuscular junctions in living adult mice. At fully functional synapses, AChRs have a half-life of about 14 days. However, 2 hours after neurotransmission was blocked, the half-life of the AChRs was now less than a day; the rate was 25 times faster than before. Most of the lost receptors were not quickly replaced. Direct muscle stimulation or restoration of synaptic transmission inhibited this process. AChRs that were removed from nonfunctional synapses resided for hours in the perijunctional membrane before being locally internalized. Dispersed AChRs could also reaggregate at the junction once neurotransmission was restored. The rapid and reversible alterations in AChR density at the neuromuscular junction in vivo parallel changes thought to occur in the central nervous system at synapses undergoing potentiation and depression.

Ultrastructural localization of cellular prion protein (PrPc) at the neuromuscular junction.

We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti-PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc-specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse.

Developmental regulation of the serpin, protease nexin I, localization during activity-dependent polyneuronal synapse elimination in mouse skeletal muscle.

During vertebrate neuromuscular development, all muscle fibers are transiently innervated by more than one neuron. Among the numerous factors shown to potentially influence the passage from poly- to mononeuronal innervation, serine proteases and their inhibitors appear to play important roles. In this regard, protease nexin I (PNI), a potent inhibitor of the serine protease, thrombin, is highly localized to the neuromuscular junction (NMJ). In turn, thrombin is responsible for activity-dependent synapse elimination both in an in vitro model, and in vivo. In the present study, we used a monospecific anti-PNI polyclonal antibody to study the developmental kinetics of PNI expression in mouse leg skeletal muscle. By using immunoblotting, we detected PNI at embryonic day 16 (E16), as a 48-kDa band. This 48-kDa PNI band became prominent in leg muscle extracts at postnatal day 5 (P5) and remained so in extracts from adult muscle. In contrast, a higher molecular weight immunoreactive PNI band, which was sodium dodecyl sulfate- and beta-mercaptoethanol-resistant, was first detected at E16, increased at birth (P0), and then decreased at P15, i.e., after the wave of polyneuronal synapse elimination had occurred in these muscles. The results of an enzyme-linked immunosorbent assay, measuring active, complexed, and truncated PNI, correlated with Western blot data. We used immunocytochemistry to probe the localization of PNI at the NMJ and found that PNI was present in the cytoplasm of myotubes at E16, but neither then nor at birth did it colocalize with acetylcholine receptors. PNI became localized at NMJs by P5 and increased by P15, after which it remained stably concentrated there in the adult. Finally, we studied the gene expression of PNI mRNA, by using Northern blotting, and showed that PNI mRNA was present in skeletal muscle and remained stable throughout the time-course studies, suggesting that developmental regulation of muscle PNI occurs principally at the translational and/or post-translational levels. These results suggest that the localization of PNI, through a binding site or "receptor" may play an important role in differentiation and maintenance of synapse.

Neural thrombin and protease nexin I kinetics after murine peripheral nerve injury.

We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2-4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase--polymerase chain reaction (RT-PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I-labeled target protease and found peak activity occurring later, 6-9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti-PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.

Myoblast fusion promotes the appearance of active protease nexin I on human muscle cell surfaces.

Protease nexin I (PNI) is a 43- to 50-kDa glycoprotein capable of inhibiting a number of serine proteases and belongs to the serpin superfamily. PNI is identical to glia-derived nexin, a neurite outgrowth promoter by virtue of its thrombin-inhibiting activity. Of particular relevance to neuromuscular biology and pathology, PNI was the first serpin shown to be highly localized to the neuromuscular junction and it maps to precisely the same locus as autosomal recessive amyotrophic lateral sclerosis (ALSJ) at chromosome 2q33-35. In the present report, we now show that in cultures of human skeletal muscle, PNI protein is expressed only after myoblast fusion into multinuclear myotubes and is localized in patches on their surfaces. We performed complex formation experiments with labeled thrombin, another target protease for PNI, with intact human muscle cells in culture. We detected specific SDS-stable PNI/thrombin complexes in myotube extracts only, indicating that active PNI was bound to their surfaces. We studied the gene expression of PNI mRNA using a 300-bp cDNA synthesized from the published sequence of human PNI. Confirming the protein data, upregulation of PNI appears in myotubes using Northern blot analysis. The current results reinforce the hypothesis that the regulation of the balance of serine proteases and serpins, such as PNI, is involved in muscle differentiation. They also prompt us to explore PNI abnormalities in several neuromuscular diseases, including ALSJ.

Serine proteinase inhibitors in human skeletal muscle: expression of beta-amyloid protein precursor and alpha 1-antichymotrypsin in vivo and during myogenesis in vitro.

The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-alpha 1-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-beta APP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both alpha 1-ACT and beta APP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, alpha 1-ACT and beta APP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both alpha 1-ACT and beta APP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for alpha 1-ACT and beta APP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation.

Beneficial effects of insulin-like growth factor-I on wobbler mouse motoneuron disease.

Recombinant human insulin-like growth factor-I (IGF-I) is being considered as a possible therapeutic agent for the treatment of motoneuron diseases like amyotrophic lateral sclerosis. The neurological mutant mouse wobbler, carries an autosomal recessive gene (wr) and has been characterized as a model of lower motoneuron disorders with associated muscle atrophy, denervation and reinnervation. The purpose of the present study was to determine the possible beneficial effect of IGF-I administration in this mouse model. Upon diagnosis at 4 weeks of age, affected mice and their control normal littermates received human recombinant IGF-I (1 mg/kg) or vehicle solution, once a day, for 6 weeks. Body weight and grip strength were evaluated periodically during the treatment period. Mean muscle fiber diameter on biceps brachii sections, choline acetyltransferase activity in muscle extracts, and motoneuron numbers in spinal cord sections were determined. IGF-I treated wobbler mice showed a marked weight increase from 3 to 6 weeks of treatment in comparison with placebo treated mutant mice. At the end of the treatment, grip strength, estimated by dynamometer resistance, was 40% higher in IGF-I treated versus placebo treated animals. Mean muscle fiber diameter which is smaller in wobbler mice than in normal mice was increased in IGF-I treated mutants. However, in this study the muscle choline acetyltransferase activity and the number of spinal cord motoneurons were unchanged. Thus, IGF-I administration mainly results in a significant effect on the behavioral and skeletal muscle histochemical parameters of the wobbler mouse mutant.

Apolipoprotein E expression at neuromuscular junctions in mouse, rat and human skeletal muscle.

Apolipoprotein E (ApoE)-epsilon 4 allele has been associated with late onset familial Alzheimer's disease (AD). In both familial and sporadic AD brain, ApoE is localized to the vessel walls, senile amyloid plaques, and neurofibrillary tangles. ApoE is also an 'injury-response' macromolecule in peripheral nerves and was reported to increase in response to injury. We have demonstrated that Alzheimer beta-amyloid precursor protein and a serpin alpha 1-antichymotrypsin also found accumulated in senile plaques in AD brain, were also localized at neuromuscular junctions (NMJs). Using immunocytochemistry, our present results indicate that ApoE is found in normal mouse, rat and human skeletal muscle and concentrated at the NMJs as it is in the senile plaques in AD brain. Such experiments may shed light on the mechanism of synapse loss, as well as plaque formation in this neurodegenerative disease.

Neurotrophic regulation of mouse muscle beta-amyloid protein precursor and alpha 1-antichymotrypsin as revealed by axotomy.

Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP), known also as protease nexin II (PNII), and alpha 1-antichymotrypsin (alpha 1-ACT), a serpin, are important components of the serine protease and inhibitor balance in many tissues. In the nervous system, this balance may have trophic or growth factor activity at different stages of development, after injury and in disease states. In the current study, using immunocytochemistry and Western blotting with antibodies against the human homologues, we analyzed whether denervation affected the localization of beta APP and alpha 1-ACT in adult mouse muscle following axotomy. In mouse muscle, anti-human alpha 1-ACT antibody detected a 60 kD immunoreactive band and anti-human beta APP antibody a band at 92 kD in both normal and denervated extracts. beta APP was present in normal mouse muscle at both neuromuscular junctions and within intramuscular nerves. alpha 1-ACT was also detected at neuromuscular junctions, on the perineurium and endothelial cell surfaces. Following axotomy, both beta APP and alpha 1-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction, alpha 1-ACT decreased more rapidly with beta APP lingering before disappearing. Since both alpha 1-ACT as well as beta APP are present within senile plaques in Alzheimer's disease brains such experiments with the nicotinic, cholinergic neuromuscular synapse in denervated muscle may help to focus experiments on the mechanism of synapse loss as well as plaque deposition in this disease.

Serine proteases and their serpin inhibitors in Alzheimer's disease.

Our article documents recent studies in the proteolytic processing of the Alzheimer's beta-amyloid protein precursor (beta-APP), as well as the role of thrombin and its potent inhibitor, protease nexin I in Alzheimer's disease (AD). Since synapse loss correlates best with cognitive decline in AD, we also present in detail, our model of synapse formation and elimination, reviewing recent findings related to the subject as well as our own original data. Recent exciting findings concerning the involvement of thrombin-like activity in synapse elimination, which we feel to be important in neural plasticity are also discussed.

[Serine proteases and their inhibitors: their role in the differentiation in neuromuscular system]. / Sérine protéases et leurs inhibiteurs: leur rôle dans la différenciation du système neuromusculaire.

It is now well established that some serine proteases, such as plasminogen activators and thrombin, as well as their inhibitors, have roles in the development of both central and peripheral nervous systems. We have shown that muscle plasminogen activators, activated after denervation, were able to digest some components of the muscle basement membrane. We have also shown that several inhibitors of serine proteases were concentrated at the neuromuscular junction. These are protease nexin, I, also called glia-derived nexin, protease nexin II, a secreted form of the beta-amyloid precursor protein (APP), and alpha 1-antichymotrypsine (ACT). These results leads us to propose a model in which serine proteases would favor plasticity of motor nerve endings during neuromuscular development. On the contrary, the inhibitors of serine proteases would act to provide and secure maintenance of the synaptic contact. A dysequilibrium between serine proteases and their inhibitors might underlie one or more motor neuron diseases.

The influence of denervation on beta-amyloid protein precursor and alpha 1-antichymotrypsin in mouse skeletal muscle.

A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of the balance between serine proteases and inhibitors in the nervous system. In the current report we studied whether axotomy affected the localization of beta APP and alpha 1-ACT in adult mouse muscle. Immunocytochemical experiments indicated that beta APP was present in normal muscle both at neuromuscular junctions and within intramuscular nerves. alpha 1-ACT was also present at neuromuscular junctions, on the perineurium of nerves and endothelial cell surfaces. Following axotomy, both beta APP and alpha 1-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction alpha 1-ACT decreased more rapidly with beta APP lingering before disappearing.