Bernard J. Jasmin

Chronic Alterations in Dopaminergic Neurotransmission Produce a Persistent Elevation of ΔFosB-like Protein(s) in both the Rodent and Primate Striatum

Using an antibody that recognizes the products of all known members of the fos family of immediate early genes, it was demonstrated that destruction of the nigrostriatal pathway by 6‐hydroxydopamine (6‐OHDA) lesions of the medial forebrain bundle produces a prolonged (>3 months) elevation of Fos‐like immunoreactivity in the striatum. Using retrograde tract tracing techniques, we have previously shown that this increase in Foslike immunoreactivity is located predominantly in striatal neurons that project to the globus pallidus. In the present study, Western blots were performed on nuclear extracts from the intact and denervated striatum of 6‐OHDA‐lesioned rats to determine the nature of Fos‐immunoreactive protein(s) responsible for this increase. Approximately 6 weeks after the 6‐OHDA lesion, expression of two Fos‐related antigens with apparent molecular masses of 43 and 45 kDa was enhanced in the denervated striatum. Chronic haloperidol administration also selectively elevated expression of these Fos‐related antigens, suggesting that their induction after dopaminergic denervation is mediated by reduced activation of D2‐like dopamine receptors. Western blot immunostaining using an antibody which recognizes the N‐terminus of FosB indicated that the 43 and 45 kDa Fos‐related antigens induced by dopaminergic denervation and chronic haloperidol administration may be related to a truncated from of FosB known as ΔFosB. Consistent with this proposal, retrograde tracing experiments confirmed that ΔFosB‐like immunoreactivity in the deafferented striatum was located predominantly in striatopallidal neurons. Gel shift experiments demonstrated that elevated AP‐1 binding activity in denervated striata contained FosB‐like protein(s), suggesting that enhanced ΔFosB levels may mediate some of the effects of prolonged dopamine depletion on AP‐1‐regulated genes in striatopallidal neurons. In contrast, chronic administration of the D1‐like receptor agonist CY 208–243 to 6‐OHDA‐lesioned rats dramatically enhanced ΔFosB‐like immunoreactivity in striatal neurons projecting to the substantia nigra. Western blot immunostaining revealed that ΔFosB and, to a lesser extent, FosB are elevated by chronic D1‐like agonist administration. Both the quantitative reverse transcriptase‐polymerase chain reaction and the ribonuclease protection assay demonstrated that Δfos B mRNA levels were substantially enhanced in the denervated striatum by chronic D1‐like agonist administration. Lastly, we examined the effects of chronic administration of D1‐like and D2‐like dopamine receptor agonists on striatal ΔFosB expression in the 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) primate model of Parkinsons disease. In monkeys rendered Parkinsonian by MPTP, there was a modest increase in ΔFosB‐like protein(s), while the development of dyskinesia produced by chronic D1‐like agonist administration was accompanied by large increases in ΔFosB‐like protein(s). In contrast, administration of the long‐acting D2‐like agonist cabergoline, which alleviated Parkinsonian symptoms without producing dyskinesia reduced ΔFosB levels to near normal. Taken together, these results demonstrate that chronic alterations in dopaminergic neurotransmission produce a persistent elevation of ΔFosB‐like protein(s) in both the rodent and primate striatum.

Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies

Although the molecular defect causing Duchenne/Becker muscular dystrophy (DMD/BMD) was identified nearly 20 years ago, the development of effective therapeutic strategies has nonetheless remained a daunting challenge. Over the years, a variety of different approaches have been explored in an effort to compensate for the lack of the DMD gene product called dystrophin. This review not only presents some of the most promising molecular, cellular, and pharmacological strategies but also highlights some issues that need to be addressed before considering their implementation. Specifically, we describe current strategies being developed to exogenously deliver healthy copies of the dystrophin gene to dystrophic muscles. We present the findings of several studies that have focused on repairing the mutant dystrophin gene using various approaches. We include a discussion of cell‐based therapies that capitalize on the use of myoblast or stem cell transfer. Finally, we summarize the results of several studies that may eventually lead to the development of appropriate drug‐based therapies. In this context, we review our current knowledge of the mechanisms regulating expression of utrophin, the autosomal homologue of dystrophin. Given the complexity associated with the dystrophic phenotype, it appears likely that a combinatorial approach involving different therapeutic strategies will be necessary for the appropriate management and eventual treatment of this devastating neuromuscular disease. Chakkalakal J. V., Thompson J., Parks R. J., Jasmin B. J. Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies. FASEB J. 19, 880–891 (2005)

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling.

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated β-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.

BDNF Is Expressed in Skeletal Muscle Satellite Cells and Inhibits Myogenic Differentiation

In skeletal muscle, brain-derived neurotrophic factor (BDNF) has long been thought to serve as a retrograde trophic factor for innervating motor neurons throughout their lifespan. However, its localization in mature muscle fibers has remained elusive. Given the postulated roles of BDNF in skeletal muscle, we performed a series of complementary experiments aimed at defining the localization of BDNF and its transcripts in adult muscle. By reverse transcription-PCR, in situ hybridization, and immunofluorescence, we show that BDNF, along with the receptor p75NTR, is not expressed at significant levels within mature myofibers and that it does not accumulate preferentially within subsynaptic regions of neuromuscular junctions. Interestingly, expression of BDNF correlated with that of Pax3, a marker of muscle progenitor cells, in several different adult skeletal muscles. Additionally, BDNF was expressed in Pax7+ satellite cells where it colocalized with p75NTR. In complementary cell culture experiments, we detected high levels of BDNF and p75NTR in myoblasts. During myogenic differentiation, expression of BDNF became drastically reduced. Using small interfering RNA (siRNA) technology to knock down BDNF expression, we demonstrate enhanced myogenic differentiation of myoblasts. This accelerated rate of myogenic differentiation seen in myoblasts expressing BDNF siRNA was normalized by administration of recombinant BDNF. Collectively, these findings show that BDNF plays an important regulatory function during myogenic differentiation. In addition, the expression of BDNF in satellite cells is coherent with the notion that BDNF serves a key role in maintaining the population of muscle progenitors in adult muscle.

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997)J. Biol. Chem. 272, 8117–8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24–48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of β-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5′ from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.

Glucocorticoid treatment alleviates dystrophic myofiber pathology by activation of the calcineurin/NF-AT pathway

Duchenne muscular dystrophy (DMD) is a progressive and ultimately fatal skeletal muscle disease. Currently, the most effective therapy is the administration of a subclass of glucocorticoids, most notably deflazacort. Although deflazacort treatment can attenuate DMD progression, extend ambulation, and maintain muscle strength, the mechanism of its action remains unknown. Prior observations have shown that activation of a JNK1‐mediated signal transduction cascade contributes to the progression of the DMD phenotype, in part by phosphorylation and inhibition of a calcineurin sensitive NF‐ATc1 transcription factor. Here, we observed that deflazacort treatment restored myocyte viability in muscle cells with constitutive activation of JNK1 and in dystrophic mdx mice. However, deflazacort treatment did not alter JNK1 activity itself, but rather led to an increase in the activity of the calcineurin phosphatase and an up‐regulation of NF‐ATc1‐dependent gene expression. The prophylactic effect of deflazacort treatment was associated with increased expression of NF‐ATc1 target genes such as the dystrophin homologue utrophin. Moreover, the muscle sparing effects of deflazacort were completely abolished when used in conjunction with the calcineurin inhibitor cyclosporine. Collectively, these results show that deflazacort attenuates loss of dystrophic myofiber integrity by up‐regulating the activity of the phosphatase calcineurin, which in turn negates JNK1 inhibition of NF‐ATc1‐mediated phosphorylation and nuclear exclusion of NF‐ATc1.

Local transcriptional control of utrophin expression at the neuromuscular synapse.

Recently, the use of a transgenic mouse model system for Duchenne muscular dystrophy has demonstrated the ability of utrophin to functionally replace dystrophin and alleviate the muscle pathology (see Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). However, there is currently a clear lack of information concerning the regulatory mechanisms presiding over utrophin expression during normal myogenesis and synaptogenesis. Using in situ hybridization, we show that utrophin mRNAs selectively accumulate within the postsynaptic sarcoplasm of adult muscle fibers. In addition, we demonstrate that a 1.3-kilobase fragment of the human utrophin promoter is sufficient to confer synapse-specific expression to a reporter gene. Deletion of 800 base pairs from this promoter fragment reduces the overall expression of the reporter gene and abolishes its synapse-specific expression. Finally, we also show that utrophin is present at the postsynaptic membrane of ectopic synapses induced to form at sites distant from the original neuromuscular junctions. Taken together, these results indicate that nerve-derived factors regulate locally the transcriptional activation of the utrophin gene in skeletal muscle fibers and that myonuclei located in extrasynaptic regions are capable of expressing utrophin upon receiving appropriate neuronal cues.

Brain-derived Neurotrophic Factor Regulates Satellite Cell Differentiation and Skeltal Muscle Regeneration

In this study, muscle-specific BDNF knockout animals were generated and compared with BDNF−/− knockouts. Our findings show that muscle-derived BDNF plays an important role in 1) regulating satellite cell proliferation and differentiation and 2) early regeneration after muscle injury.

Distinct regions in the 3′ untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells

In this study, we have sought to determine whether utrophin transcripts are targeted to a distinct subcellular compartment in skeletal muscle cells, and have examined the role of the 3′ untranslated region (UTR) in regulating the stability and localization of utrophin transcripts. Our results show that utrophin transcripts associate preferentially with cytoskeleton-bound polysomes via actin microfilaments. Because this association is not evident in myoblasts, our findings also indicate that the localization of utrophin transcripts with cytoskeleton-bound polysomes is under developmental influences. Transfection of LacZ reporter constructs containing the utrophin 3′UTR showed that this region is critical for targeting chimeric mRNAs to cytoskeleton-bound polysomes and controlling transcript stability. Deletion studies resulted in the identification of distinct regions within the 3′UTR responsible for targeting and stabilizing utrophin mRNAs. Together, these results illustrate the contribution of posttranscriptional events in the regulation of utrophin in skeletal muscle. Accordingly, these findings provide novel targets, in addition to transcriptional events, for which pharmacological interventions may be envisaged to ultimately increase the endogenous levels of utrophin in skeletal muscle fibers from Duchenne muscular dystrophy (DMD) patients.