Nicotinic Achr In The Muscle example essay topic

Proteins involved in formation of the neuromuscular junction The neuromuscular junction is a specialized junction, where a motor nerve forms its synaptic terminal with a muscle fibre, one of many fibres that make up a whole muscle. The mature neuromuscular junction is composed of three types of cells - a motor nerve terminal, a muscle fibre and a Schwann cell covering the junction. All three of these cells are highly differentiated and specialized for their functions (Kandel 2000, p. 1089). The events that occur during the formation of the neuromuscular junction have been extensively studied and are the most comprehensively understood of any nerve-to-target cell contact.

Prior to formation of the synapse all three components of the neuromuscular junction develop and acquire identities independently. The muscle cells are derived from the mesoderm and migrate from the dermomyotomal portion of the somite. Motoneuron's migrate from the ventricular zone of the neural tube to a ventral-lateral location before axons grow out of the spinal cord. Schwann cells are glial cells, which insulate the axons outside of the spinal cord, they are derived from neural crest cells and associate with axons from the somite onwards to the peripheral target.

At the time of the first axons reaching the developing muscle, the muscle fibres are myoblasts that have just fused to form multinucleated myotubes, there is no evidence to suggest that motor neurons prefer certain site on the developing myotube or that there is a predetermined site for the formation of the synapse, on the contrary, synapse formation can occur on most, if not all of the myotube surface (Licht man et al in Zigmond et al 2000, pp. 547-8). Acetylcholine receptors (AChR) are found uniformly dispersed over the surface of the myotube until the nerve approaches the myotube. As the nerve approaches the myotube a protein known as agrin, which is synthesized in the motor neuron, is transported down the axon to the synaptic cleft where it is released from the nerve terminal, here it is deposited in the synaptic basal lamina. The major components of the basal lamina are laminin's, which are made up of α , β and γ chains; it forms a continuous non-myelin layer over the nerve terminal and is a potent promoter of axon outgrowth.

The basal lamina is present (at least components of the basal lamina are present) prior to the arrival of the nerve and is rich in the enzyme acetylcholinesterase (AChE), an enzyme which catalyses the breakdown of acetylcholine (ACh) the major neurotransmitter at the neuromuscular junction. Agrin acts through receptors in the plasma membrane of the muscle fibre, it brings about the localization of the AChR to the region below the nerve terminal, the aggregation of AChR is essential for the development of the neuromuscular junction. Agrin also regulates the distribution of other synaptic proteins, such as AChE, rapsyn, utrophin and neuregulin (NRG) receptors, this indicates agrin has a central role in synaptic differentiation. The agrin gene is expressed in a variety of cell types, for example agrin is also synthesized by muscle cells, however, the neuronal isoforms of agrin are a thousand times more active in the aggregation of AChR. It has been shown that agrin is an essential component for AChR clustering, by using antibodies against agrin; it was shown AChR clustering is blocked and the neuromuscular junction cannot mature (Reist et al 1992, p. 867).

The exact mechanism of agrin-mediated AChR clustering is not yet known, but a receptor tyrosine kinase known as muscle-specific kinase or MuSK has been shown to be a vital component of the agrin receptor complex. MuSK is normally found aggregated at the synaptic sites. However, agrin does not bind directly to MuSK, so the agrin receptor may contain an additional subunit. Exactly how agrin interacts with MuSK is not yet known. It has been shown however that MuSK is an essential component for AChR clustering, as mice with the MuSK gene which has been genetically disrupted, lack normal neuromuscular junctions (Gautam et al 1996, p. 531) The steps that follow MuSK activation and that lead to postsynaptic differentiation are not yet known. However, it seems likely that another molecule is involved in agrin-mediated signaling, a protein termed rapsyn.

Agrin stimulates clustering of rapsyn in myotubes in cell culture, and clustering of rapsyn and AChR appears to occur coincidentally at synapse formation (Burden, S.J. 1985, p. 8272) Rapsyn has been shown to be critical for synapse formation, as mice lacking rapsyn expression, die within a few hours after birth (Gautam et al 1995, p. 234) The clustering of AChR at the post-synaptic site is maintained further via: 1) A mechanism of increased AChR production by nuclei at the synaptic site due to the release of a nerve derived component, termed neuregulin (NRG) that acts via ErbB receptors on the muscle fibre, this is shown in figure 1 2) Reduction of AChR production at nuclei in extra-synaptic locations, i.e. nuclei outside the synaptic site produce less AChR. NRG is capable of stimulating expression of the genes for the acetylcholine receptor subunit, by stimulating transcription of the protein, via the ErbB receptors found in the postsynaptic membrane at neuromuscular synapses. NRG is synthesized in the motor neurons as well as the muscle fibre, similar to agrin. Summary of the formation of the neuromuscular junction -The three cells types that comprise the mature neuromuscular junction all develop independently prior to the formation of the synapse. As the growth cone of the motor neuron approaches the developing muscle, the multinucleated myotube has just been formed by the fusion of the myoblasts and elements of the basal lamina are already present. -The terminal accumulates synaptic vesicles containing ACh the main neurotransmitter at the synapse.

ACh receptors are uniformly spread over the myotube prior to the arrival of the motor neuron, but as the motor neuron approaches it synthesizes and secretes the protein agrin into the synaptic cleft. -The agrin binds to the receptors on the muscle surface and initiates clustering of the AChR to the synapse, an intracellular protein called rapsyn appears to play a pivotal role in this process. A muscle specific tyrosine kinase (MuSK) is also essential in the clustering of AChR, it is thought to be a part of the agrin receptor complex, although agrin does not bind directly to it -Both agrin and MuSK are essential for the normal development of the neuromuscular junction. -As the muscle matures, multiple axons form synapses with the one muscle fibre, however strong synaptic inputs influence the muscle to decrease intracellular components that are supporting synapses from axons that weakly influence the post-synaptic site, this is illustrated in figure 2.

One hypothesis is that active synapses punish inactive neighbours by secreting molecules that have been termed synaptomedians. -As the neuromuscular junction matures the extra axons retreat and eventually only one motor neuron synapses with a fibre. The motor neuron terminal is rich in vesicles containing ACh; many of these vesicles are clustered in dense patches on the presynaptic membrane called active zones. -The postsynaptic membrane on the muscle fibre has indents which lie directly opposite to these active sites, the indents are called junctional folds and are rich in AChR. -An illustration of the mature neuromuscular junction is shown in figure 3. Disorders of the neuromuscular junction Myasthenia Gravis There are several disorders of the neuromuscular junction; one of these is myasthenia gravis (which means severe weakness of muscle).

There are two major forms of myasthenia gravis; the first and most prevalent is the autoimmune form. The autoimmune form is one of few diseases that fulfill the strict criteria for an immune-mediated disease. Which are, (1) an antibody is present in almost all cases. (2) The antibody acts with an antigen that is important in the pathophysiology of the disease.

(3) By transferring antibodies to experimental animals, features of the disease can be reproduced. (4) An experimental form of the illness can be induced, by immunizing animals with the antigen. (4) Therapeutic reduction of antibody levels relieves symptoms (Kandel et al 2000, p. 298). The second and less common form of myasthenia gravis is congenital and heritable; it is not autoimmune and is heterogeneous. Firstly we will be concentrating on the more common autoimmune form of myasthenia gravis. In the autoimmune form, antibodies are produced against the nicotinic AChR in the muscle.

The AChR antibodies appear to produce the neuromuscular transmission defect in MG by 1) binding to the AChR and affecting its function, 2) accelerating the degradation rate of AChR and thereby lowering the concentration of AChR, and 3) causing complement-mediated lysis of the muscle endplate (Boonyapisit et al 1999, p. 102) The first mechanism appears to be relatively insignificant, but dramatic exceptions do exist that may be clinically important. Their effect appears to be a direct blockade of ACh-induced opening of the ion channel. In patients such antibodies appear to be only a small percentage of total AChR antibodies, and their effect on the endplate potential is small. The antibodies can cross-link AChR and increase their degradation rate; the reduction of AChR concentration at the endplate is brought about in part by an increase in AChR degradation By far the most significant effect of the AChR antibody is complement-mediated destruction of the neuromuscular junction. The geometry of the end-plate is also disturbed by this; the normal infolding at the junctional folds is reduced and the synaptic cleft is enlarged which leads to loss of AChR-rich membrane (Yo han et al 2001, pp. 6-10).

Alteration in the architecture of the junctional folds decreases the amount of membrane surface available for AChR insertion, this is shown in figure 4. All these effects would lead to a decrease in the safety factor for neuromuscular transmission. The safety factor is the difference between the threshold needed to elicit an action potential, which is usually around -45 mV and the actual end-plate potential amplitude, which is usually 70-80 mV, this is shown in figure 5. As a result of the reduced number of functional AChR, the skeletal muscle becomes weakened. This weakness has four special characteristics: 1. It generally affects the cranial muscles; e.g. eyelids and the eye muscles, an example of this is shown in figure 6.2.

The severity of the symptoms varies in the course of a single day and over longer periods 3. There are no conventional clinical signs of d enervation 4. Drugs that inhibit acetylcholinesterase, the enzyme that breaks down ACh, reverse the weakness. It was shown experimentally in animals that antibodies, which cause myasthenia are usually active against either of the two peptide sequences on the protein; the bungarotoxin-binding site or an area on the α -subunit termed the main immunogenic region.

Circulating antibodies in humans are often directed against the main immunogenic region. It was found that the causative factor of myasthenia is found in the plasma, because draining of the lymph from the thoracic lymph ducts improves the symptoms in the patient, but if the patients own lymphatic fluid is re-injected into the patient the symptoms recur. Through experimentation it has been shown that antibodies to the α -subunit of ACh receptors have a pivotal role in the pathogenesis of myasthenia. However, questions still remain unanswered, like what stimulates production of the antibodies to the AChR? One theory is that viral or bacterial antigens may share epitopes with the AChR. Therefore, when a person is infected with the bacteria or virus the antibodies produced also work on the AChR.

The molecular similarity of the antigens is called molecular mimicry. The way that myasthenia affects the neuromuscular junction is that it decreases the number of functional ACh receptors in the postsynaptic membrane as well as decreasing the geometry of the junctional folds and therefore reducing the area to insert new ACh receptors. This means that ACh only has a limited number of receptors to bind to and since the acetylcholinesterase is hydrolyzing it the neurotransmitter at the same time repeated stimulation means that the excitatory potential amplitude may not reach threshold to elicit an action potential. There are two main ways to treat this disease; this first is by injecting acetylcholinesterase inhibitors, such as neostigmine into the muscle and therefore the ACh won't be degraded as quickly and there will be a higher concentration of the neurotransmitter in the synaptic cleft to bind to the receptors. The effectiveness of this treatment is shown in figure 4.

Another way of treating the symptoms of myasthenia is by injecting antigens to the antibodies into the muscle. Soon after the identification of the disease it was found that in 15% of the adult cases the patient also had a benign tumour of the thymus. In 1939 Alfred Blalock reported that when the thymoma was removed the patients symptoms improved. Later in the 1950's Blalock and Harvey found that patients with myasthenia gravis experience improved symptoms with the removal of the thymus even if the patient didn't have a thymoma.

This procedure was known as a thymectomy and has become standard practice in the treatment of myasthenia gravis. The second and less common form of myasthenia is congenital myasthenia (CMS), which can arise from presynaptic, synaptic or postsynaptic defects. The effect of this form of myasthenia is similar to the autoimmune form in that the specific defect compromises the safety factor of neuromuscular transmission. As already stated congenital myasthenic syndromes are heterogenous disorders, meaning there is a lot of variety with in the disease. It was found that mutations in the AChR that increase or decrease the synaptic response to ACh are a common cause of the postsynaptic CMS. The increased response to ACh is called a slow channel mutation, which is characterized by prominent limb weakness with little weakness of the cranial muscles.

There are eleven slow channel mutations to date. The consequences of slow channel CMS mutations, stem from prolonged opening periods of the AChR ion channel. This causes (1) cationic overloading pf the junctional sarco plasm and an end plate myopathy with loss of AChR due to the degradation of the junctional folds and (2) a depolarization block due to staircase summation of prolonged end plate potentials (Engel et al 1999, pp. 165). The end plate potentials of the slow channel mutation are similar to that observed in AChE deficiency. A decreased response to ACh arises from a low affinity, fast channel mutation. In patients with this syndrome the response to ACh is markedly decreased, even though the number of AChR per end plate is normal.

Studies have shown that with this syndrome comes infrequent AChR channel events, abnormally brief activation episodes due to decreased channel re openings during ACh occupancy, and an increased resistance to desensitization by ACh. CMS can also arise from AChE deficiency at the end plate. In this deficiency there is a detrimental response of the compound action potential evoked in muscle by repetitive stimulation of the nerve, the same as in the autoimmune form. However, the muscle responds repetitively to a single stimulus, a feature that is not observed in other conditions. There are also other abnormalities, including abnormalities of the presynaptic nerve terminal, this results in impaired release of ACh from the terminals, and the result of this is similar to that of fast channel mutations. Conclusion As can be seen from this report the formation of the neuromuscular junction is very well understood the most comprehensively understood of any nerve-to-target cell contact, but yet there is still much more to learn.

We know that the protein agrin, which is synthesized in the motor axon, plays a major role in the formation of the synapse. The muscle-specific kinase, MuSK, found on the muscle cells and concentrated at the synaptic site also plays a key role. Agrin triggers the aggregation of AChR on the muscle cell at the synapse, an intracellular protein known as rapsyn plays a pivotal role in this process. The motor axon also secreted neuregulin, which stimulates expression of the AChR and its insertion into the postsynaptic membrane. There are also several diseases that affect the neuromuscular junction; the two that were concentrated on in this paper were the two forms of myasthenia; the autoimmune form and the congenital form. The autoimmune form is the more common of the two, and fits the criteria for an autoimmune disease perfectly, antibodies are produced that act against the AChR and break them down.

The symptoms of this disease vary greatly and misdiagnosis is common, it generally affects the cranial muscle and abnormal fatigability is a common symptom. The result of this disorder is that the number of AChR's on the postsynaptic membrane is decreased by increased breakdown of the receptors and degradation of the junctional folds, which decreases the surface area to insert the receptors. The symptoms of this disorder can be treated by injection of AChE inhibitors into the muscle. There is still a lot to learn about this disease, like why the antibodies are produced in the first place, there are theories to explain this, but there is no strong evidence to back them up. The second type of myasthenia is the less common congenital form, which can arise from presynaptic, synaptic or postsynaptic defects.

Mutations in the AChR can lead to an increase or a decrease in response of the receptor to ACh. A decreased response is known as a fast channel mutation, while an increased response is known as a slow channel mutation. The congenital form of myasthenia can also arise from AChE deficiency and defects on the presynaptic membrane which inhibit the ACh being released. There are still many things that need to be known about the two diseases and the only way this can be done is with more research.

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