MDA Research Update #80

Muscular Dystrophy Association - USA
Research and Program Services Department
03/29/99

CREATINE AND NEUROMUSCULAR DISEASE

Creatine can be synthesized in the body by an interaction between two amino acids (arginine and glycine), with an additional methyl group added. Creatine is also available in the diet from meat, fish and milk. When a phosphate group is added to the creatine molecule, a very important energy molecule called phosphocreatine is produced. This helps maintain the supply of another high-energy molecule, ATP, important in driving many reactions maintaining various cellular functions and overall health of the cell. Creatine supplementation in athletes has shown increased lean body mass, high-intensity power output and strength. The mechanism of strength and performance enhancement is not yet well understood, but may include more energy availability due to increasing levels of phosphocreatine, or there may be stimulation of muscle protein synthesis important in building muscle mass. Upon metabolic breakdown of creatine and phosphocreatine molecules, creatinine is produced as a waste product, which is cleared from the blood by the kidney to appear in urine. The level of this metabolic product in urine may be an indicator of muscle mass. A decline in creatinine level in urine over time can indicate a loss in muscle mass, as seen in some dystrophies. (Champe, PC & Harvey, RA. Biochemistry 2nd Edition. J.B. Lippincott Co., Philadelphia. 1987.)

SPECIAL NOTE TO READERS: Creatine, even though readily available over the counter, hasn't yet been shown to be without risk when used in high doses or over a long period of time. Caution is strongly advised before individuals attempt to treat themselves for the disorders listed in the following creatine supplementation research reports. Most important, a proper dosage for maximal benefit in any disorder hasn't been established in humans. While the long-term effects of creatine supplementation are unknown, there are suspicions that kidney damage may occur. MDA is working with researchers to begin a controlled clinical trial in AMYOTROPHIC LATERAL SCLEROSIS (ALS) and other NEUROMUSCULAR DISORDERS as soon as possible. Please visit the MDA Web site for future announcements.

CREATINE SUPPLEMENTATION IN AN ALS MOUSE MODEL

Effects of creatine were studied in a mouse model of FAMILIAL AMYTROPHIC LATERAL SCLEROSIS (FALS). Mitochondria, the energy-producing organelles within all cells, are particularly vulnerable to oxidative stress. Mitochondrial swelling and vacuolization are among the earliest pathological features seen in the mouse model of FALS carrying the human SOD1 gene mutations. One of these mouse models (G93A) shows altered mitochondrial electron transport enzymes, whose normal activity results in the production of high-energy molecules to power all kinds of cellular activity. The mutant SOD1 in cultured cells show a loss of mitochondrial membrane potential and elevated calcium concentration, which can cause disruption of mitochondria function.* Mitochondrial dysfunction may lead to ATP depletion, contributing to cell death. Evidence now suggests that creatine kinase enzyme acting upon creatine and phosphocreatine connects mitochondria (the sites of energy production) with sites of energy consumption. Additional evidence indicates creatine administration stabilizes the mitochondrial creatine kinase, allowing it to function and to also inhibit the opening of the mitochondrial transition pore, through which calcium may enter.

Oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival time in the FALS mouse model, protecting these mice from loss of both motor neurons and substantia nigra neurons typical of the disease in these mice at 120 days of age. (Life span of normal healthy mice is about 2 years.) The data suggest that creatine administration may be a new therapuetic strategy for ALS.

The diet in these mice was supplemented with 1 percent or 2 percent creatine. Mean survival without supplement was 143.7 days in the mutant mice while 1 percent supplement allowed a mean of 157.2 days, and 169.3 days of survival with 2 percent creatine, increasing life expectancy in this mouse model of 13 days and 26 days, respective to dose. Studies with Riluzole in these mice show only a 13-day increase in life expectancy. Performance tests also showed an increase in abilities to perform physical activities.

Nonsupplemented mutant mice showed a significant loss of neurons (49.3 percent) in the ventral horn region of the spinal cord, which contains the spinal motor neurons commanding muscle function, all due to disease progression, at 120 days. Those mutants fed 1 percent creatine supplementation showed complete protection of these important motor neurons and appeared as if they were normal animals at the same 120 days of age. In addition, muscle samples examined histologically from the supplemented group appeared essentially normal.

Another finding may have application in Parkinson's disease as well. The same FALS mutant mice were found to have lost neurons in a part of the brain called the substantia nigra, that affects motor control. The loss of neurons in this part of the brain is also seen in Parkinson's disease. In the creatine-supplemented FALS mutant mice, these cells were also protected.

Researchers suggest that creatine administration may help both to buffer intracellular energy stores and to inhibit mitochondrial transition pore opening. The latter is linked to both excitotoxic and apoptotic cell death. Creatine-stabilized mitochondrial creatine kinase appears to inhibit the opening of the mitochondrial transition pore, allowing calcium to enter. Other researchers have noted excess calcium can disrupt the mitochondrial membrane causing the release of factors known to be involved in cell suicide cascade, also known as apoptosis. (See the two reports below by Susin and Earnshaw.) Creatine can also stimulate mitochondrial respiration and phosphocreatine synthesis. Phosphocreatine is another high-energy molecule which can help to maintain needed ATP levels driving chemical reactions throughout the cell, including glutamate uptake into vesicles which may aid protection from excitotoxicity due to excess glutamate implicated in SPORACID AMYOTROPHIC LATERAL SCLEROSIS (SALS).

This study adds additional evidence to the importance of mitochondrial function to the health of the cell. In indicates that when this function is disrupted it may trigger the death of the cell. (Klivenyi, P., et al. Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. Nature Medicine. 5:(3):347-350:1999.)

CREATINE SUPPLEMENTATION IN NEUROMUSCULAR DISORDERS

It's been reported that some patients show reduced phosphocreatine in muscles in such neuromuscular disorders as mitochondrial and inflammatory myopathies, and some muscular dystrophies. In a Canadian study, the effects of creatine supplementation were tested in people with various neuromuscular disorders for improvement in muscle strength. In this study, researchers supplemented diets in those with such disorders as MYOTONIC (DM), LIMB-GIRDLE (LGMD) and BECKER (BMD) MUSCULAR DYSTROPHIES, SPINAL MUSCULAR ATROPHY (SMA), as well as some MITOCHONDRIAL (MITO), CONGENITAL (CMD and MC) AND INFLAMMATORY MYOPATHIES (PM/DM).

Researchers provided patients with creatine monohydrate supplementation at 20g daily for five days followed by 10g daily for another five to seven days in two different studies to determine effects on muscle strength. On day one of the study, patients were asked to perform certain strength-measuring tests. During days of supplementation after day one, no changes in normal daily routines, including diet and any exercise regimens, were allowed. The same tests administered on day one were then administered on the last day of supplementation. One part of the study was comparison with a placebo.

It was reported that there was a significant increase in isometric (dorsiflexion) ankle strength, and that fatigue was lessened in the same test in those given supplementation. Handgrip strength was also significantly higher with supplementation and, even though a tendency to increase strength was seen with placebo, supplementation produced significantly greater hand strength than placebo. Knee strength was higher with creatine, while no effect was seen with placebo. Changes in these neuromuscular disorders are significant in that there may be an increase in protein synthesis with supplementation, where these disorders normally have muscle wasting.

This study couldn't show any differences between neuromuscular disorders. The authors make the point, which readers should also keep in mind, that this study is only preliminary for the disorders studied and that it covered only a short period of time. Authors strongly suggest that clinical studies into the specific disorders over longer periods are needed to determine benefits and risk factors. (Tarnopolsky, M. & Martin, J. Creatine monohydrate increases strength in patients with neuromuscular disease. Neurology. 52:854-857:1999.)

MDA RESEARCH COMMENTS ON CREATINE:

The two papers summarized above have stimulated considerable interest in getting larger, longer-term clinical trials begun on the effect of creatine in human neuromuscular diseases. MDA/ALS researchers are presently planning a large multi-center trial of creatine in ALS patients based upon the very strong implications in the Klivenyi study. Various researchers have been invited to submit studies for funding by MDA in order to assess the effects on specific disorders over longer time periods. It's of some concern that, in some dystrophies, increased strength may be of greater detriment to an already severely weakened muscular membrane than may have been indicated in the 11 days of the preliminary study reported here.

Little is known about the safety of long-term creatine supplementation in people with neuromuscular disorders, and effective doses haven't been determined. It's important to approach all of these drugs and supplements from a very controlled research method in order to rule out any placebo type of effect and to indicate if there actually is any beneficial effect or, on the other side, any risk in long-term usage. Your participation in MDA clinical trials can help provide this valuable information. If you're interested in taking creatine, please continue to monitor the MDA Web site for clinical trial announcements, and participate in a controlled study if you can. Also, please don't begin taking any supplement without first consulting your physician.

For more information, please go to MDA Answers Frequently Asked Questions About Creatine.

MITOCHONDRIA AND APOPTOSIS

Several key PROGRAMMED CELL DEATH (PCD, APOPTOSIS) factors have been found in the space between the inner and outer membranes of mitochondria. Apoptosis is driven by classes of proteases (enzymes destroying a variety of proteins) known as caspases (cysteine aspartases). Under certain circumstances these caspases can be activated, initiating a cascade of activation of other proapoptotic (cell suicide) factors. Various caspases act as executioners by destroying key life-sustaining proteins, turning off cell-survival mechanisms and activating additional cell suicide mechanisms in a cascade of increasing destruction. One of these factors was surprisingly discovered to be cytochrome-c, which has long been known to be a factor in energy production supporting the life of the cell. In apoptosis, cytochrome-c is found to double as a switch to turn on the cell suicide machinery.

Factors such as the newly discovered APOPTOSISIN-INDUCING FACTOR (AIF) (discussed below) or cytochrome c that promote cell death are released by mitochondria. In some cells, it appears that mitochondria are the reservoirs for some procaspases (caspases in an inactive, dormant state) that, when activated, begin the cascade of cell death. It would appear that the intermembrane space of mitochondria is a storehouse, where cell death factors are stored without causing any harm to cellular components. Once factors allow the release of these factors by some alteration of permeability of the outer membrane or by opening of pores allowing such destructive factors as calcium ions* to enter, the apoptotic mechanism is activated and the cascade of cell destruction begins. (Earnshaw, WC. A cellular poison cupboard. Nature. News and Views. 397:387-388:1999.)

Recent evidence is indicating a more prominent role of mitochondria in the regulation of APOPTOSIS (Programmed Cell Death, PCD). Mitochondria have both an inner and outer membrane, and the space between these membranes has been found to contain several proteins that, when liberated through the outer membrane, participate in a cascade of molecular events that essentially destroy the entire cell from the inside out. This process is referred to as apoptosis. Researchers have identified an additional protein called apoptosis-inducing factor, AIF5, which is sufficient to induce apoptosis by itself. AIF is normally confined to the intramembrane space of mitochondria, which also contains other apoptogenic factors, including cytochrome-c and procaspases 2, 3 and 9.

When the mitochondrial membrane is disrupted, it releases AIF, which finds its way to the nucleus, where it causes condensation of chromosome material and results in fragmenting of DNA into smaller denser pieces characteristic of apoptosis. AIF was also found to cause the mitochondria to release other proapoptotic proteins, cytochrome c and caspase-9. Caspase inhibitors don't block this effect, but overexpression of Bcl-2, a known inhibitor of apoptosis, prevents the release of AIF from the mitochondria.

Opening of the mitochondrial permeability transition pore, which is under the control of members of the Bcl-2 family, is one of the decisive events of the apoptotic process. This causes an increase in the permeability of the outer mitochondrial membrane and the release of soluble proteins, such as proapoptotic proteins from the intermembrane space.

The AIF gene is linked to the X-chromosome region Xq25-26. Researchers conclude that AIF provides a new molecular link between mitochondrial membrane permeabilization and nuclear apoptosis. (Please see accompanying discussion below.) (Susin, SA., et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 397:441-446:1999.)

*Stout, AK., et al. Glutamate-induced neuron death requires mitochondrial calcium uptake. Nature Neuroscience. 1:(5):366-373:1998.

EMERY-DREIFUSS MUSCULAR DYSTROPHY & THE NUCLEAR MEMBRANE

All cells of the body are enclosed by a membrane that is selective. It allows some elements to enter the cell and keeps others out. It's basically a boundary that maintains an environment within the membrane where cellular functions can be performed while keeping harmful, disruptive elements outside the cell. But there is another membrane inside each cell that surrounds the nucleus where the genetic material DNA is separated from the rest of the cell, providing an environment allowing gene interactions to occur. Recent research is indicating that this nuclear membrane serves other purposes besides keeping internal cellular activities separated. In both the outer cellular membrane and the nuclear membrane, a basic structure is found, providing a framework upon which functionally important proteins may be found organized in such a way as to allow important reactions to proceed. This basic structure is referred to as a nuclear lamina, or fibrous layer, found on the inner side of the nuclear membrane facing the nuclear contents (genetic material). This membrane is thought to provide a framework for the nuclear envelope and its associated proteins, such as emerin.

Emerin is a nuclear membrane protein missing in EMERY-DREIFUSS MUSCULAR DYSTROPHY (EDMD). Emerin is one member of a family of lamina-associated proteins which includes LAP1, LAP2 and lamin B receptor (LBR). Lamins, as the name implies, may serve as a layering, or framework, for other structural proteins, or acts as a lattice for interaction of various proteins. Monoclonal antibodies (mAbs) serve to "find," or hunt down, bind to and thus "tag" a very specific protein or part of a protein in such a way that it can be "seen" and localized within a cell under special kinds of microscopes. Determining the location of a protein within a cell can provide indications of its functions in the cell.

Researchers have made several kinds of mAbs to bind to specific parts of the emerin protein in order to locate its position within a cell. When mAbs are made for specific parts of a protein and that part of the emerin protein is missing, the mAb will not be able to bind and tag to indicate the presence of that part of the protein. The "strength" of a tag indicates the amount of the protein present and/or that all parts of the protein are present. In general, the more individual protein present in a particular location within the cell, the more mAb tags will be seen at that location in the cell when viewed under special microscopes.

Although emerin in normal individuals is expressed in all tissues and cell lines tested, its absence in EDMD affects two tissues in particular--heart and skeletal muscle. Myopathic changes are evident in some skeletal muscles, and early contractures are seen in neck, elbows and Achilles tendon. In the heart, conduction defects occur and the patient may need a pacemaker. As reported in earlier studies, one hypothesis has been that emerin located at the intercalated discs (where heart cells meet in a dense line or "disc") could provide the basis the suggestion of cardiac conduction defects in EDMD was due to emerin absence at these intercalated discs. However, researchers using better antibodies for emerin now report that, in normal heart tissue, emerin wasn't found in intercalated discs, but was found instead on cardiomyocyte (heart muscle cell) nuclei.

The data reported cast some doubt on the hypothesis that cardiac defects in EDMD are due to the absence of emerin from intercalated discs, where this data indicate emerin may not normally be present in intercalated discs. Lamin B1 was also found absent from cardiomyocyte nuclei in EDMD and is also almost completely absent from skeletal muscle nuclei. In EDMD, where emerin is absent, the additional absence of lamin B1 from heart and skeletal muscle nuclei may offer an alternative explanation of why these tissues are particularly affected. Data suggest that the absence of lamin B1 from cardiac and skeletal muscle nuclei may indicate the greater importance of emerin in nuclear function in those cells lacking lamin B1 and, thus, normal interactions with LAP2 and lamin B receptor.

It's also found that the distribution of lamin A is most similar to emerin, and has been considered a candidate for the autosomal dominant form of EDMD, which, clinically, is indistinguishable from the X-linked EDMD, in which it's emerin that is absent. Here is a case where emerin is normal but the absence of lamin A produces the same symptoms, which strongly indicates a very important interaction between lamin A and emerin where neither one alone can compensate for the other. Lamin A maps to chromosome 1q21.3 and the locus of autosomal EDMD lies within the chromosome segment 1q11-23. (Manilal, S., et al. Distribution of emerin and lamins in the heart and implications for Emery-Dreifuss muscular dystrophy. Human Molecular Genetics. 8:(2):353-359:1999.)

EDMD, MEMBRANE LAMINS AND LIMB-GIRDLE MUSCULAR DYSTROPHY

EDMD symptoms are early contractures of elbows and Achilles tendons, slowly progressive muscle wasting and weakness, and a cardiomyopathy with conduction block, which can be life threatening. Two modes of inheritance are found, X-linked (X-EDMD) affecting the emerin gene, and an autosomal dominant (EDMD-AD) form. EDMD-AD is clinically identical to the X-linked form. The locus for EDMD-AD is 1q11-23. This region contains the lamin A/C gene (LMNA), a candidate gene encoding two proteins important in formation of nuclear lamina, lamins A & C. These different forms of lamin are produced by alternative splicing of the same gene. These results are the first identification of mutations in a component of the nuclear lamina as a cause of inherited muscle disorder. Together with mutations in EDMD, they underscore the potential importance of the nuclear envelope components in the pathogenesis of neuromuscular disorders.

The interval of chromosome 1q is in the locus described in families with autosomal dominant LIMB-GIRDLE MUSCULAR DYSTROPHY (LGMD) with cardiac involvement (LGMD1B), which may represent the same entity as EDMD-AD. However, the clinical description of LGMD1B differs by not showing significant contractures, predominance of proximal limb weakness and the occasional calf hypertrophy. Whether these disorders represent different alleles (different mutations in the same gene) is yet to be determined.

Lamins A & C are members of the intermediate filament family and are present in fully differentiated (mature) cells. Lamins form dimers through their rod domains and interact with chromatin and integral proteins of the inner nuclear membrane (lamin B receptor, LAPs and emerin). Mutations found in lamin A/C suggest the lamins are deficient in their interactions with chromatin and/or integral membrane proteins, or in filament assembly.

Data represent the first report of a disease caused by mutations in the lamin family. The gene encoding lamin B1 (LMNB1) also maps to chromosome 5q23.3-31.3 in corresponding to the locus of another dominantly inherited myopathy, LGMD1A. These findings show that interactions between nuclear membrane components, nuclear lamina and chromatin of chromosomes are important to skeletal and cardiac muscle function, and that loss of integrity of the nuclear envelope is an underlying cause of muscular dystrophy. (Bonne, G., et al. Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nature Genetics. 21:285-288:1999.)

A NEW FORM OF LIMB-GIRDLE AND STILL COUNTING!!

LGMD (all forms) has an incidence of about 1/100,000. The classification includes a heterogeneous group of disorders characterized by proximal muscle weakness first affecting the hip and shoulder girdle, elevated creatine kinase values, and absent or reduced deep-tendon reflexes. Both recessive and dominant forms are documented, and new sporadic cases also appear.

Patients linked to this new locus on chromosome 7q (terminal region) show progressive proximal leg weakness with or without proximal arm weakness, absent ankle deep-tendon reflexes and elevated creatine kinase values. Microscopic muscle changes were seen in some patients. Neither electromyography nor muscle biopsy demonstrated any pathognomonic features seen in other disorders. Mean age of onset was 27.1 ± 8.5 years. Those family members with normal appearance and with creatine kinase levels within normal range were considered gene-carrier risks based on age at time of examination.

Researchers report that, during this study, other families with no linkage to any known forms of LGMD were found. Thus, more genetic forms of LGMD type 1 (autosomal dominant) are expected to be determined. (Speer, MC, et al. Identification of a new autosomal dominant Limb-Girdle Muscular Dystrophy locus on chromosome 7. American Journal Human Genetics. 64:556-562:1999.)

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