SAN DIEGO—A series of recent studies suggests that oxidative damage to neuronal mitochondria—leading to mitochondrial fission and, ultimately, to apoptosis of the neuron—is a key element in the pathogenesis of diabetic neuropathy, reported Eva Feldman, MD, PhD, at the 130th Annual Meeting of the American Neurological Association. The findings not only shed light on why individuals with diabetes are so vulnerable to neuropathy but also point to possible therapeutic targets for preventing or treating the condition.

“I think that mitochondrial oxidative stress is one of the fundamental mechanisms that underlie glucose-mediated injury to the nervous system,” said Dr. Feldman, who is Professor of Neurology at the University of Michigan, Ann Arbor. She and her colleagues began studying the role of mitochondria in diabetic neuropathy a decade ago, when electron microscopy of dorsal root ganglion neurons from diabetic rats revealed some intracellular structures that appeared to be vacuoles. It turned out, Dr. Feldman said, that the structures were enlarged mitochondria, which “swell in the presence of some sort of toxic insult.” In this case, further research revealed that the toxic insult comes from superoxides (free radicals) created when hyperglycemia overwhelms the mitochondrial machinery responsible for energy production—notably, the electron transport system that synthesizes adenosine triphosphate (ATP). “Think of it as a tube clogged with too many electrons,” Dr. Feldman said. The result of this mitochondrial congestion is the formation and accumulation of superoxides rather than of molecular oxygen.

Because free radicals are highly reactive, they can cause intracellular damage. In one series of experiments, the researchers examined neuronal DNA from rats with a genetic form of diabetes. After three months of diabetes, 20% of the neurons had evidence of DNA damage. “This clearly indicates that the neurons are injured,” Dr. Feldman asserted in an interview with Neurology Reviews. “And an injured neuron isn’t going to function as well as a healthy one. It can’t maintain sufficient protein production for axonal transport, and it has multiple metabolic impairments” that “we believe lead to diabetic neuropathy.”

When Dr. Feldman and colleagues first presented such findings at a diabetes conference, the “endocrinologists in the audience were skeptical that high glucose could induce this type of damage. So we elected to try to better understand this process by going to an in vitro model.” The investigators added glucose to rat dorsal root ganglion neurons to simulate the diabetic milieu; the neurons responded “with a burst of ATP” but then “simply petered out with exhaustion of the electron transport chain,” leading to increases in multiple parameters of oxidative stress and ATP deficiency. This was followed by caspase activation and, finally, apoptosis.

The latter process appears to involve the Bcl family of apoptotic proteins, Dr. Feldman reported. When dorsal root ganglion neurons were exposed in vitro to glucose for three hours at levels comparable to those in the serum of patients with diabetes, the expression of one pro-apoptotic Bcl protein rose by about 50%, the researchers found.

FISSION CONTROL

However, another mitochondrial mechanism also appears to come into play. Work by Dr. Feldman and others has shown that mitochondria are not in a static state; instead, they undergo fission and fusion, depending on circumstances. Fission serves to create new mitochondria when cells are metabolically active and to provide energetics along the axon.

Dr. Feldman hypothesized that fusion and fission are always occurring in neurons and that abnormalities in these processes—induced, for example, by elevated glucose levels—might lead to cellular dysfunction and, eventually, neuropathy. In vitro experiments revealed that after three to six hours of high glucose exposure, neuronal expression of a key fission protein, Drp1, increases by 20% and that this protein is transported to the mitochondria, where it helps spur fission. If glucose levels remain high and the need for further fission continues, Bcl proteins such as Bax are called into the mitochondrial membrane, and apoptosis may occur.

This loss of mitochondria was quite apparent, Dr. Feldman said, when the Michigan researchers recently conducted some simple counting experiments in neurons exposed to high glucose levels. “We had never done this,” Dr. Feldman said, but preliminary results indicate a decrease in the number of intact mitochondria and a “marked increase in disrupted mitochondria.”

Overall, these findings provide a striking picture of the cellular basis for diabetic neuropathy. First, high glucose levels increase the need for mitochondria; expression of pro-fission proteins such as Drp1 increases, causing existing mitochondria to divide. Although this temporarily satisfies the cell’s needs, eventually the stress from high glucose overwhelms the cells and results in the production of superoxides, which begin to exert toxic effects, including promoting mitochondrial apoptotic fission. Caspases are activated, mitochondrial fragmentation occurs, and the neuron undergoes apoptosis.

Most likely, this scenario is not specific to diabetic neuropathy, according to Dr. Feldman. “This idea of mitochondrial fission and fusion is likely going to be very important for other neurodegenerative diseases. It likely underlies Charcot-Marie-Tooth disease type 2a,” she said, “and we have very preliminary data that a similar process occurs in amyotrophic lateral sclerosis.”

AN ANTIOXIDANT COCKTAIL

The elucidation of these mitochondrial mechanisms raises some intriguing therapeutic possibilities—notably, the potential to prevent or halt free radical damage by treating patients with antioxidants. Already, Dr. Feldman reported, “we can rescue neurons [from oxidative damage] in vitro and in diabetic animal models.” Buoyed by such findings, the Michigan researchers have launched a clinical trial supported by the Juvenile Diabetes Research Foundation designed to halt oxidative damage in patients with type 1 diabetes mellitus who have autonomic and peripheral neuropathy. During the two-year, placebo-controlled trial, patients in the active treatment group will receive a cocktail of three antioxidants: nicotinamide, allopurinol, and a-lipoic acid.

The study’s primary end point is amelioration of autonomic neuropathy, though the researchers are also looking at whether there are improvements in peripheral neuropathy and whether treatment prevents retinopathy and nephropathy. Enrollment should be completed in 2006.

“Our hope,” Dr. Feldman said, “is that if we can show that this triple-antioxidant therapy is effective in halting or even ameliorating neuropathy, then patients may be placed on antioxidants prophylactically when they are diagnosed with diabetes.”

Another approach may be to use viral vectors to provide neurons with genes for anti-apoptotic proteins. Experiments have shown that this strategy can rescue neurons in vitro and in animal models, at least initially, Dr. Feldman said, but it hasn’t been tried in patients with diabetic neuropathy.

NR

—Peter Doskoch

Suggested Reading
Vincent AM, McLean LL, Backus C, Feldman EL. Short-term hyperglycemia produces oxidative damage and apoptosis in neurons. FASEB J. 2005;19:638-640.

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