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Department of Neurology, Hadassah University Hospital and Hebrew University Hadassah Medical School, Jerusalem, Israel 91120; and
* Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel 91904
2Correspondence: The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail: soreq{at}shum.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neuromuscular function • MG • acetylcholine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and motoneuron diseases like amyotrophic lateral sclerosis (ALS, ref 2
), congenital myasthenias (3)
, and myasthenia gravis (MG), in which autoantibodies to the nicotinic ACh receptor (nAChR) induce neuromuscular junction (NMJ) malfunction (4)
.
Recent information indicates that nAChR and AChE functions in NMJs are intimately related. For example, compound mutagenesis of the ACHE gene and the gene for the 
subunit of nAChR limits the severity of the impaired neuromuscular phenotype that is caused in zebrafish by ACHE disruption alone (5)
. Based on this and related information, we hypothesized that distortions in the AChE-nAChR balance may be relevant to several neuromuscular diseases. In the mammalian nerve and muscle, for example, imbalanced cholinergic neurotransmission induces enhanced transcription and shifted splicing of AChE pre-mRNA, leading to overproduction and accumulation of the normally rare readthrough AChE-R variant (6)
. In the immediate response to either acute stress or exposure to anticholinesterases, elevated AChE-R attenuates the initial hyperexcitation (7)
. However, continued accumulation of AChE-R may be detrimental, as it prolongs the state of cholinergic impairment (8
, 9)
, increases adhesive and morphogenic noncatalytic activities of AChE (10
, 11)
, and is associated with muscle pathologies (12)
, predicting additional long-term effects under AChE-R overproduction. The synaptic AChE-S variant forms multimers that are attached to the membrane through the proline-rich PRiMA anchor (13)
. In contrast, AChE-R appears as soluble, secretory monomers that lack the carboxyl-terminal cysteine that is essential for linkage to PRiMA (14)
. Thus, each AChE variant is specialized: AChE-S for hydrolysis at the synapse and controlling its properties and AChE-R for nonsynaptic hydrolysis and morphogenesis. Consequently, the signaling action of ACh at the receptor, its cessation by hydrolysis, and the morphogenic activity of AChE may all be involved in the maintenance of neuromuscular functioning in a manner dependent on the composition of AChE variants.
MG of humans (4
, 15)
and experimental autoimmune MG (EAMG) of rats (16)
present valuable systems for testing the involvement of AChE variants in the pathophysiology of NMJ diseases. Loss of receptors induces severe abnormalities in cholinergic neurotransmission, which cause neuromuscular weakness and accelerated, readily measurable muscle fatigue. Symptoms can be corrected by anticholinesterase therapy; however, therapy is effective for only short periods and chronic abnormalities in cholinergic neurotransmission persist. Our previous studies (8)
(12)
demonstrated AChE-R accumulation in muscle with impaired cholinergic neurotransmission. Therefore, we tested the accumulation of AChE-R in MG and EAMG and used variant-selective antisense agents to suppress AChE-R accumulation. Our findings demonstrate a role for AChE-R in EAMG pathophysiology and emphasize the advantage of targeting AChE-R mRNA in neuromuscular diseases that involve a shift in pre-mRNA splicing.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials
Unless otherwise specified, materials were purchased from Sigma Chemical Co. (St. Louis, MO).
Animals
EAMG was induced in female Lewis rats (120–150 g, Jackson Laboratory, Bar Harbor, ME) in accordance with NIH guidelines. FVB/N mice (Harlan Biotech Rehovot, Israel) and transgenic FVB/N mice that overexpress human (h)AChE-R were as described (17)
.
Oligonucleotides
Lyophilized, HPLC-purified, GLP grade oligodeoxynucleotides (purity >90% as verified by capillary electrophoresis; Hybridon, Inc., Worcester, MA) were resuspended in sterile double distilled water (24 mg/mL) and stored at -20°C. Their sequences were:
EN101 5'-CTGCGATATTTTCTTGTA*C*C*-3';
EN102 5'-GGGAGAGGAGGAGGAAGA*G*G*-3' ; and
invEN102 5'-GGAGAAGGAGGAGGAGAG*G*G*-3'.
The three 3'-terminal residues (*) were substituted with oxymethyl groups at the 2' positions. The EN101 and EN102 antisense oligonucleotides (AS-ONs) are complementary to the coding sequence of the rat AChE mRNA sequence (GeneBank accession no. S50879) common to all variants (18)
. The third is inverse (inv) to EN102 (19)
, and served as a negative control.
Antibodies
Rabbit polyclonal Abs against the carboxyl-terminal sequence of human AChE-R have been described (17)
. Goat polyclonal anti-nAChR (S.C.-1448) was from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated donkey anti-rabbit Ab (Chemicon International, Temecula, CA) and biotinylated donkey anti-goat Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary Abs.
Induction of EAMG
Torpedo ACh receptor (T-AChR) purified from T. californica electroplax on neurotoxin-Sepharose resin (20)
was subcutaneously injected in the hind footpads of rats (40 µg T-AChR and 1 mg of M. tuberculosis H37Ra, Difco, Detroit MI, emulsified in complete Freund’s adjuvant). Booster injection of the same amount was administered after 30 days. Animals that did not develop EAMG after the second injection received a third one. Animals were weighed and inspected weekly during the first month, and daily after the booster immunization to evaluate muscle weakness. Their clinical status was graded as follows: (0) no weakness or fatigue, treadmill running time, 23 ± 3 min; (1) mildly decreased activity, weak grip with fatigue, weight loss >3% of body weight during a week, > 10 min running time on treadmill; (2) moderate weakness accompanied by weak grip, weight loss of 5–10%, 3–5 min running on treadmill; (3) moderate-severe weakness, hunched back posture at rest, head down and forelimb digit flexed, tremulous ambulation, 10% body weight loss, 1–2 min run on treadmill; and (4) severe general weakness, no audible complaint or grip, treadmill running time <1 min, weight loss >10%.
Serum analyses
Nondenaturing gel and catalytic activity measurements of AChE were as described (7)
. Iso-OMPA (tetraisopropylpyrophosphoramide) was used to block butyrylcholinesterase activity in serum samples (5·10-5 M) and polyacrylamide gels (5·10-6 M).
Anti-AChR Ab determination
Sera from EAMG animals and MG patients were assayed by direct radioimmunoassay for anti-Torpedo (T)-AChR, rat (R)-AChR, or human (H)-AChR (16
, 20)
. All the EAMG rats displayed high anti-T-AChR and/or anti-R-AChR levels, with serum mean ± SE values of 82.1 ± 16.0 nM for anti-T-AChR Abs and 19.9 ± 1.8 nM for anti-R-AChR. MG patient sera were assayed by the same radioimmunoassay using H-AChR as antigen and displayed 1–60 nM Ab titers.
Quantification of nAChR
AChR concentration in the gastrocnemius and tibialis muscles was determined using 125I--bungarotoxin binding, followed by precipitation in saturated ammonium sulfate as described (21)
.
In situ hybridization
In situ hybridization was performed with fully 2'-O-methylated AChE-R- or AChE-S-specific 50-mer cRNA probes complementary to ACHE pseudointron 4 or exon 6, respectively (9)
. Detection was with alkaline phosphatase and Fast RedTM substrate (Molecular Probes, Eugene, OR). DAPI (Hoechst) counterstaining served to visualize nuclei.
Immunohistochemistry
Immunohistochemistry was performed on 7 µm paraffin-embedded muscle sections as described elsewhere (22)
. Primary Abs were diluted 1:100 and 1:30 for detecting rabbit anti-AChE-R and goat anti-AChR, respectively.
Drug administration
Intravenous (i.v.) injection and blood sampling for anti-nAChR Ab determination were via the jugular vein under anesthesia. For oral administration, a curved intubation feeding needle with a ball end (Stoelting, Wood Dale, IL) was used. Pyridostigmine (Mestinon BromideTM, Hoffmann-La Roche, Basel, Switzerland) was administered in a dose of 1 mg/kg per treatment, the highest dose that does not cause cholinergic crisis.
Electromyography
Rats were anesthetized by i.p. injection of 2.5 mg/kg pentobarbital, immobilized, and subjected to repetitive sciatic nerve stimulation at 3 Hz, using a pair of concentric needle electrodes. Baseline compound muscle action potentials (CMAPs) were recorded by electrodes placed in the gastrocnemius muscle following a train of repetitive nerve stimulations at supramaximal intensity. Normal muscle shows no decrease in action potentials. Decreases (percent) in the amplitude of the fifth vs. the first muscle action potential were determined in two sets of repetitive stimulations for each animal. A reduction of 10% or more indicated neuromuscular dysfunction (16)
. To assess the efficacy of the treatment, the reduced CMAP in each animal was taken as a baseline and changes were presented as comparison to this value. Thus, an improvement of baseline decrement of 0.87 to 1.0 would yield a value of the CMAP ratio of 115%.
Exercise training on treadmill
Animals were placed on an electrically powered treadmill (23)
running at a rate of 25 m/min, an effort of moderate physical intensity, until visibly fatigued. The time rats were able to run was recorded before and after AS-ON or pyridostigmine treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Serum from FVB/N mice subjected to forced swim stress (9)
displayed higher levels than control mice of a rapidly migrating mAChE-R band, which was inhibitable by 5·10-6 M 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51), an AChE-specific inhibitor, but not by the butyrylcholinesterase-specific inhibitor iso-OMPA. Sera from transgenic mice, which overexpress human (h)AChE-R (17)
, displayed an even more rapidly migrating band, possibly due to the differences in the carboxyl-terminal sequences between human and mouse AChE-R or because of changes in the glycosylation of these two enzymes. Figure 1
A presents these iso-OMPA-resistant activity patterns. Similarly, fast-migrating AChE was observed in the sera of 10 of 19 MG patients whereas sera from 3 patients with an unrelated disease (e.g., hepatitis) or from 2 healthy nonstressed humans showed primarily a slower migrating AChE form. Figure 1A
further demonstrates this fast-migrating AChE variant in sera from 2 of 4 MG patients. There was no apparent correlation between the intensity of staining in the gels and anti-AChR Ab titers of the analyzed patients (an average ±SE of 22±16 nM vs. 1.0 nM in healthy subjects). There was no correlation with total serum AChE activity, which, on average, was unchanged from levels in normal subjects. These observations suggested that the faster migrating bands represent murine and human AChE-R (mR and hR, respectively) and that AChE-R is over-represented in the blood of some MG patients. A different method to detect AChE-R in serum is by immunoblot analysis. In the serum of EAMG vs. healthy rats, there was a massive increase in immunopositive rat AChE-R (rR) that comigrated with hAChE-R (Fig. 1B
).
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AChE-R and AChE-R mRNA accumulate in muscles of EAMG rats
Expression of alternative AChE variants (Fig. 2
A) was tested in control and EAMG rats. In a quantitative binding of 125I--bungarotoxin, muscle nAChR was reduced by 48 ± 7% from normal values in 10 mildly affected rats (disease grade 1–2) and by 75 ± 5% in 10 severely affected rats (grade 4) compared with controls. Immunohistochemistry with antibodies to nAChR visualized this depletion in muscle sections from EAMG rats compared with control rats (Fig. 2B
1, 2). Immunohistochemical staining with a polyclonal antiserum that selectively detects AChE-R (17)
revealed positive signals in some but not all muscle fibers of control rats. Similar patterns appeared under treatment with the inert, inversely oriented oligonucleotide invEN102 (9
; see Fig. 2B
3 for invEN102-treated control muscle). In EAMG rats (Fig. 2B
4), the same staining procedure showed dispersed cytoplasmic localization that is characteristic of this isoform, which is also secreted (6)
and contrasts with the membrane-associated clusters of the synaptic variant (24)
. The level of expression and cellular distribution of muscle AChE-S were similar in EAMG and control, untreated, and invEN102-treated rats (data not shown).
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Using in situ hybridization with variant-selective probes, we observed similarly distributed AChE-S mRNA in muscles from untreated and invEN102-treated healthy and EAMG rats (see Fig. 2B
5, 6 for invEN102 treated muscle). In contrast, normal rats displayed only weak and diffuse labeling of the AChE-R mRNA transcript whereas pronounced punctuate labeling of AChE-R mRNA accumulations appeared in triceps muscles of EAMG rats, unaffected by invEN102 treatment (Fig. 2B
7, 8). This accumulation indicated a selective overexpression of AChE-R in muscles of EAMG rats.
AChE-R and AChE-R mRNA levels in muscle respond to EN101
The soluble, secretory nature of AChE-R predicted it would degrade ACh before it reached the postsynaptic membrane, limiting receptor activation. To test this hypothesis, we used the EN101 AS-ON, which is capable of selective suppression of AChE-R production (9
, 25)
. Rat EN101 efficacy was tested in healthy and EAMG rats. EAMG muscle presented reduced nAChR staining (Fig. 2C
1, 2). One day after a single i.v. injection of 250 µg/kg EN101, immunohistochemical staining demonstrated that AChE-R was significantly reduced in muscles from control and EAMG rats (Fig. 2C
3, 4). In contrast, AChE-S remained essentially unchanged (data not shown). Under rEN101 treatment, receptor labeling intensity remained high in healthy rats and low in EAMG animals, similar to untreated animals and animals treated with invEN102 (compare Fig. 2B
1, 2 with Fig. 2C
1, 2). In situ hybridization indicated that AChE-S mRNA labeling was only nominally affected by EN101, suggesting that neuromuscular transmission would be maintained under this treatment (Fig. 2C
5, 6). In contrast, EN101 reduced AChE-R mRNA labeling almost to the limit of detection in control and myasthenic rats (Fig. 2C
7, 8).
Suppression of AChE-R restores normal CMAP ratios in EAMG rats
Immunoblot analysis followed by densitometric quantification confirmed the increase of serum AChE-R in EAMG. A single i.v. injection of 250 µg/kg EN101, but not invEN102, reduced the serum level of AChE-R 24 h later in control and EAMG animals (Fig. 3
A). To evaluate the physiological outcome of this suppression, we recorded CMAPs from the gastrocnemius muscle. EAMG rats, but never control animals, displayed a decrement in CMAP during repeated stimulation at 3 Hz. The baseline decrement, the percent of difference in the heights of the fifth and the first evoked potentials, ranged from 10% to 36% (mean±SE=13.0±2.5%, Fig. 3B
) compared with 4.0 ± 0.9% in healthy controls. Standard therapy for MG is administration of an anti-cholinesterase that elevates ACh levels to a threshold enabling receptor activation. Accordingly, we administered i.p. neostigmine bromide (ProstigmineTM, Hoffmann-La Roche, 75 µg/kg). This rapidly corrected the CMAP decrement in EAMG rats from 87.6% of the first basal stimulation in untreated EAMG animals, which was considered as 100% baseline, to > 105% of the first evoked potential (i.e., 120% of the baseline). The effects of the cholinesterase blockade were evident starting 15 min after injection yet lasted only 2 h, after which time CMAP reverted to the decrement baseline (Fig. 3C
).
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Unlike anticholinesterases, which block all AChE variants, EN101 was shown to selectively suppress muscle AChE-R production (12)
. Therefore, retrieval of stable CMAP peaks in EN101-teated EAMG rats could attest to the causal role of AChE-R in myasthenic neuromuscular malfunctioning. Injection (i.v.) of EN101 at doses ranging from 50 to 500 µg/kg (2–20 nmol/rat) did not affect CMAP in control animals, but in EAMG rats retrieved stable, normalized CMAP ratios within 1 h (Fig. 3B
, Fig. 4
A, and Table 1
). CMAP normalization was accompanied by increased mobility, upright posture, stronger grip, and reduced tremulousness of ambulation. The extent and the duration of CMAP correction were dose dependent; for example, 500 µg/kg conferred 
72 h rectification of CMAP up to 125% of baseline whereas 50 µg/kg was effective for only 24 h. EN102, a similarly 3' protected AS-ON targeting another sequence in AChE mRNA (AS1 in ref 19
), produced similar rectification of CMAP decrements in EAMG rats, supporting the relevance of AChE-R as a contributing element to these decrements. Comparable amounts of invEN102, an inverted sequence with no target in the database, did not improve muscle function, attesting to the sequence specificity of the EN101 effect (Table 1)
. Dose response curves revealed that up to 5 h after an injection, EN101 produced a saturable response with an IC50 of <10 µg/kg. This effect appeared to be superimposed on a much longer lasting and less concentration-dependent effect that showed no saturation in the range we studied (Fig. 4B
), possibly reflecting altered muscle and/or NMJ properties under the stable CMAP retrieval afforded by EN101.
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Antisense prevention of AChE-R accumulation promotes stamina in EAMG rats
Placed on a treadmill at 25 m/min, control rats ran for 23.0 ± 3.0 min, then displayed visible signs of fatigue. Starting at 5 h and for at least 24 h after i.v. administration of 250 µg/kg EN101, EAMG rats demonstrated improved performance on the treadmill. Running time increased from 247 ± 35, 179 ± 21, and 32 ± 6 s to 488 ± 58, 500 ± 193, and 212 ± 59 s for animals at disease grades 2, 3, and 4, respectively (average values for 6–9 animals per group). Healthy animals, in contrast, were not significantly affected by EN101 injection.
2'-Oxymethyl protected AS-ON agents are efficient under oral administration (26)
. The dose of 50 µg/kg of EN101 was administered to EAMG rats once a day via an intubation feeding needle and CMAP measured 1, 5, and 24 h later. Orally administered 50 µg/kg EN101 and EN102 were as effective as 25 µg/kg EN101 administered i.v. (Table 1
and Fig. 4
and Fig. 5
), but EN102 effects appeared somewhat delayed compared with EN101. Oral pyridostigmine (1000 µg/kg) restored CMAP for up to several hours whereas invEN102 had no significant effect (Table 1)
.
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Long-term antisense AChE-R suppression modifies the course of EAMG pathophysiology
The long-term maintenance of stable CMAP under EN101 enabled us to test whether the cholinergic impairment contributes to the progressive physiological deterioration in EAMG. Rats were first treated with EN101 once a day for 5 days, CMAPs being determined before each treatment. The efficacy of EN101 and its capacity to reduce the inter-animal variability in CMAP values were comparable to those of pyridostigmine (Fig. 5A, B
). However, the onset of response to pyridostigmine was more rapid (Table 1)
whereas that observed with EN101 was longer lasting. Daily oral or i.v. administration of EN101 stabilized CMAPs over the entire course of treatment (Fig. 5B
). In contrast, the effect of pyridostigmine wore off within several hours, causing pronounced fluctuations in muscle status (Table 1
, Fig. 5
).
Five of six animals with grade 3 severity of EAMG symptoms treated once daily with pyridostigmine died within 5 days. In contrast, six of eight animals with similar disease severity treated orally once daily with EN101 survived the full 5 day period. To exclude the possibility that this difference reflects the susceptibility of EAMG rats to the repeated anesthesia and CMAP measurements, we subjected groups of moderately sick animals to 1 month of daily oral treatment with minimal interference. EAMG rats receiving daily oral doses of EN101, presented significant improvement in survival, clinical status, and treadmill performance compared with pyridostigmine- and saline-treated animals (Fig. 6
; P<0.041 for 4 wk survival incidence, Fisher exact test, AS-ON vs. other treatments). One-way repeated measures ANOVA yielded P < 0.05 for all other measures (AS vs. other treatments at 4 wk). Rats in the saline- and pyridostigmine-treated groups lost weight (13.5 and 11 g/animal, respectively) whereas animals in the EN101-treated group gained weight, on average 13 g/animal, during the treatment period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cholinergic balance considerations
Neuromuscular homeostasis involves, among other elements, a balance between NMJ-bound AChE and AChR that regulates neuromuscular maintenance and functioning (3
, 5)
. When this balance is impaired, notably in the cases of inherited or acquired MG and EAMG or under exposure to organophosphates (27)
, deterioration of muscle performance ensues. Figure 7
presents a scheme of the key elements controlling the cholinergic balance in NMJs and the putative mechanism through which it may be retrieved in MG and EAMG.
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Pharmacological inhibition of AChE, a primary treatment of MG for many decades, assumed that AChE plays no role in the etiology or pathophysiology of MG; and that long-term use of AChE inhibitors does not contribute to MG or its symptoms. By damping the destruction of ACh by AChE, anti-cholinesterase therapy can transiently compensate for the reduced numbers of AChRs at NMJs (28)
. However, AChE-S blockade by the nondiscriminative anticholinesterases induces AChE-R overproduction (7)
. This would limit the duration of anticholinesterases’ capacity to retrieve stable CMAP and intensify the disease-associated cholinergic imbalance.
CMAP increases occurred under anticholinesterase treatment (for a few hours) and in EN101-treated animals (>24 h after treatment). The rapid onset of CMAP improvement under EN101 is most likely due to its easy accessibility to muscle tissue and a rapid turnover of its target mRNA. The long-lasting functioning of the AS-ON may indicate that there is no physiological change until AChE-R reaccumulates to a level at which it can cause NMJ malfunction. Under daily EN101 treatment, the steady reversal of the decremental change likely enabled sustained recovery processes, unlike the fluctuating values under conventional therapy. Recently, ACh was shown to suppress interleukin (IL) 1ß production in macrophages (29
, 30)
. The causal involvement of IL-1ß in the myasthenic phenotype (31)
suggested that the EN101 effect could be partially mediated by its persistent capacity to reduce AChE and therefore increase ACh and suppress IL-1ß levels. Interrelationships between NMJ cholinergic imbalance and stress-induced change in the cytokine balance, recently reported to affect EAMG pathophysiology (31
32
33)
, may explain at least part of the continuously improving clinical scores under prolonged EN101 treatment.
Drug dose and time dependence
By directing nanomolar doses of an orally delivered AS-ON drug to the long-known MG target AChE, we achieved rapid yet long-lasting clinical improvement, associated with stable reversal of the CMAP decrement response at 3 Hz nerve stimulation and increased muscle stamina in the treadmill test. The beneficial effect of AS-ON injection on the CMAP response began within 1 h post-treatment and lasted many more hours than the effect of anticholinesterases, likely reflecting mechanistic differences between these two groups of drugs.
Chemical anticholinesterases stoichiometrically inhibit the large number of AChE-S molecules present in the NMJ (3000–5000 molecules/µm2; ref 34
). By contrast, AChE mRNA chains exist in far lower quantities than their protein products (35)
. AChE-R mRNA normally is the least abundant of the alternative splice variants of AChE pre-mRNA (6)
. AS-ON agents that target the AChE-R mRNA transcripts can operate repeatedly, i.e., one AS-ON is responsible for hydrolysis of many mRNAs (36)
. This explains the > 100-fold difference in the molar dose of AS-ON vs. pyridostigmine that is effective in relieving EAMG weakness.
Because of the intrinsic instability of the AChE-R mRNA transcript (37)
, AS-ON agents targeted at the AChE mRNA sequence that is shared by all transcripts, such as EN101 or EN102, will destroy primarily the longer, less G,C-rich AChE-R mRNA (9)
. This provides selectivity toward this normally rare transcript, while protecting most of the synaptic AChE-S variant from the antisense effect. Consequently, neuromuscular function is maintained while the disease-associated variant is efficiently prevented from being synthesized. The 3' terminal 2'-oxymethyl protection of AS-ON chains increases the hybridization affinity while minimizing toxicity (25)
, explaining its efficacy after i.v. as well as oral administration. In comparison, conventional anticholinesterases would nonselectively inhibit the catalytic activity of the two variants and induce a robust, multi-tissue feedback response of AChE-R overproduction (7)
. This increases the noncatalytic activities of AChE (5)
that are not necessarily blocked by anticholinesterases (5
, 6)
. The long-term survival and improved clinical status and stamina of animals receiving AS-ON by daily oral treatment compared with the poor survival of severely diseased animals under the physiologically fluctuating effects of a single daily dose of placebo or pyridostigmine, highlight these differences.
Potential relevance to other diseases
The pathogenesis of MG and EAMG is primarily related to the destructive effect of anti-AChR Abs on the NMJ (4)
. Neuromuscular weakness associated with cholinergic imbalance is also known in patients with congenital myasthenic syndromes (3)
, where absence of the membrane anchor results in synaptic AChE-S being the only form missing, as well as after exposure to anticholinesterases, e.g., in Gulf War veterans (38)
. Other diseases with peripherally imbalanced cholinergic neurotransmission include muscle dystrophy (1)
, amyotrophic lateral sclerosis and post-traumatic stress disorder (2)
. As AS-ONs targeted to AChE mRNA provide relief of cholinergic muscle malfunction, they may be found useful for alleviating the weakness in these and other muscle malfunctions syndromes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 18, 2002. Accepted for publication October 13, 2002.
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REFERENCES |
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J. X. S. Jiang, R. C. Y. Choi, N. L. Siow, H. H. C. Lee, D. C. C. Wan, and K. W. K. Tsim Muscle Induces Neuronal Expression of Acetylcholinesterase in Neuron-Muscle Co-culture: TRANSCRIPTIONAL REGULATION MEDIATED BY cAMP-DEPENDENT SIGNALING J. Biol. Chem., November 14, 2003; 278(46): 45435 - 45444. [Abstract] [Full Text] [PDF]
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