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Hope for paralyzed people as mice with spinal cord injury made to walk again

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*Scientists reverse ageing-associated skin wrinkles, hair loss in mouse model
Most people with spinal cord injury are paralyzed from the injury site down, even when the cord isn’t completely severed. Why don’t the spared portions of the spinal cord keep working? Researchers at Boston Children’s Hospital now provide insight into why these nerve pathways remain quiet. They also show that a small-molecule compound, given systemically, can revive these circuits in paralyzed mice, restoring their ability to walk.

The study, led by Zhigang He, PhD, in Boston Children’s F.M. Kirby Neurobiology Center, United States (U.S.), was published online July 19 by the journal Cell.

“For this fairly severe type of spinal cord injury, this is most significant functional recovery we know of,” says He. “We saw 80 percent of mice treated with this compound recover their stepping ability.”

Many animal studies looking to repair spinal cord damage have focused on getting nerve fibers, or axons, to regenerate, or to getting new axons to sprout from healthy ones. While impressive axon regeneration and sprouting have been achieved, by He’s lab and others, their impacts on the animals’ motor function after a severe injury are less clear. Some studies have tried using neuromodulators such as serotonergic drugs to simulate the spinal circuits, but have gotten only transient, uncontrolled limb movement.

He and colleagues took another approach, inspired by the success of epidural electrical stimulation-based strategies, the only treatment known to be effective in patients with spinal cord injury. This treatment applies a current to the lower portion of the spinal cord; combined with rehabilitation training, it has enabled some patients to regain movement.

“Epidural stimulation seems to affect the excitability of neurons,” says He. “However, in these studies, when you turn off the stimulation, the effect is gone. We tried to come up with a pharmacologic approach to mimic the stimulation and better understand how it works.”

Also, researchers have reversed wrinkled skin and hair loss, hallmarks of aging, in a mouse model. When a mutation leading to mitochondrial dysfunction is induced, the mouse develops wrinkled skin and extensive, visible hair loss in a matter of weeks. When the mitochondrial function is restored by turning off the gene responsible for mitochondrial dysfunction, the mouse returns to smooth skin and thick fur, indistinguishable from a healthy mouse of the same age.

Keshav Singh, Ph.D., and colleagues have done just that, in a mouse model developed at the University of Alabama at Birmingham (UAB), United States (U.S.). When a mutation leading to mitochondrial dysfunction is induced, the mouse develops wrinkled skin and extensive, visible hair loss in a matter of weeks. When the mitochondrial function is restored by turning off the gene responsible for mitochondrial dysfunction, the mouse returns to smooth skin and thick fur, indistinguishable from a healthy mouse of the same age.

“To our knowledge, this observation is unprecedented,” said Singh, a professor of genetics in the UAB School of Medicine.

The study was published in the journal Cell Death & Disease.

Importantly, the mutation that does this is in a nuclear gene affecting mitochondrial function, the tiny organelles known as the powerhouses of the cells. Numerous mitochondria in cells produce 90 percent of the chemical energy cells need to survive.

In humans, a decline in mitochondrial function is seen during aging, and mitochondrial dysfunction can drive age-related diseases. A depletion of the DNA in mitochondria is also implicated in human mitochondrial diseases, cardiovascular disease, diabetes, age-associated neurological disorders and cancer.

Meanwhile, He, first author Bo Chen and colleagues selected a handful of compounds that are already known to alter the excitability of neurons, and are able to cross the blood-brain barrier. They gave each compound to paralyzed mice in groups of 10 via intraperitoneal injection. All mice had severe spinal cord injury, but with some nerves intact. Each group (plus a control group given placebo) was treated for eight to ten weeks.

One compound, called CLP290, had the most potent effect, enabling paralyzed mice to regain stepping ability after four to five weeks of treatment. Electromyography recordings showed that the two relevant groups of hindlimb muscles were active. The animals’ walking scores remained higher than the controls’ up to two weeks after stopping treatment. Side effects were minimal.

CLP290 is known to activate a protein called KCC2, found in cell membranes, that transports chloride out of neurons. The new research shows that inhibitory neurons in the injured spinal cord are crucial to recovery of motor function. After spinal cord injury, these neurons produce dramatically less KCC2. As a result, He and colleagues found, they can’t properly respond to signals from the brain. Unable to process inhibitory signals, they respond only to excitatory signals that tell them to keep firing. And since these neurons’ signals are inhibitory, the result is too much inhibitory signaling in the overall spinal circuit. In effect, the brain’s commands telling the limbs to move aren’t relayed.

By restoring KCC2, with either CLP290 or genetic techniques, the inhibitory neurons can again receive inhibitory signals from the brain, so they fire less. This shifts the overall circuit back toward excitation, the researchers found, making it more responsive to input from the brain. This had the effect of reanimating spinal circuits disabled by the injury.

“Restoring inhibition will allow the whole system to be excited more easily,” He explains.

“Too much excitation not good, and too much inhibition is not good either. You really need to get a balance. This hasn’t been demonstrated in a rigorous way in spinal cord injury before.”

He and colleagues are now investigating other compounds that act as KCC2 agonists. They believe such drugs, or perhaps gene therapy to restore KCC2, could be combined with epidural stimulation to maximize a patient’s function after spinal cord injury.

“We are very excited by this direction,” says He. “We want to test this kind of treatment in a more clinically relevant model of spinal cord injury and better understand how KCC2 agonists work.”

Meanwhile, Singh said: “This mouse model should provide an unprecedented opportunity for the development of preventive and therapeutic drug development strategies to augment the mitochondrial functions for the treatment of aging-associated skin and hair pathology and other human diseases in which mitochondrial dysfunction plays a significant role.”

The mutation in the mouse model is induced when the antibiotic doxycycline is added to the food or drinking water. This causes depletion of mitochondrial DNA because the enzyme to replicate the DNA becomes inactive.

In four weeks, the mice showed gray hair, reduced hair density, hair loss, slowed movements and lethargy, changes that are reminiscent of natural aging. Wrinkled skin was seen four to eight weeks after induction of the mutation, and females had more severe skin wrinkles than males.

Dramatically, this hair loss and wrinkled skin could be reversed by turning off the mutation. The photos below show the hair loss and wrinkled skin after two months of doxycycline induction, and the same mouse a month later after doxycycline was stopped, allowing restoration of the depleted mitochondrial DNA.

Little change was seen in other organs when the mutation was induced, suggesting an important role for mitochondria in skin compared to other tissues.

The wrinkled skin showed changes similar to those seen in both intrinsic and extrinsic aging — intrinsic aging is the natural process of aging, and extrinsic aging is the effect of external factors that influence aging, such as skin wrinkles that develop from excess sun or long-term smoking.

Among the details, the skin of induced-mutation mice showed increased numbers of skin cells, abnormal thickening of the outer layer, dysfunctional hair follicles and increased inflammation that appeared to contribute to skin pathology. These are similar to extrinsic aging of the skin in humans. The mice with depleted mitochondrial DNA also showed changed expression of four aging-associated markers in cells, similar to intrinsic aging.

The skin also showed disruption in the balance between matrix metalloproteinase enzymes and their tissue-specific inhibitor — a balance of these two is necessary to maintain the collagen fibers in the skin that prevent wrinkling.

The mitochondria of induced-mutation mice had reduced mitochondrial DNA content, altered mitochondrial gene expression, and instability of the large complexes in mitochondria that are involved in oxidative phosphorylation.

Reversal of the mutation restored mitochondrial function, as well as the skin and hair pathology. This showed that mitochondria are reversible regulators of skin aging and loss of hair, an observation that Singh calls “surprising.”

“It suggests that epigenetic mechanisms underlying mitochondria-to-nucleus cross-talk must play an important role in the restoration of normal skin and hair phenotype,” Singh said, who has a secondary UAB appointment as professor of pathology. “Further experiments are required to determine whether phenotypic changes in other organs can also be reversed to wildtype level by restoration of mitrochondrial DNA.”


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