Malfunctioning mitochondria are notorious culprits in a variety of neurological diseases. A growing body of literature indicates that their reach extends to painful peripheral neuropathies, including those caused by genetic defects, diabetes, and drugs for cancer and HIV (e.g., see Baloh et al., 2008; Fernyhough et al., 2010; Bennett, 2010; Joseph and Levine, 2009; Osio et al., 2006). Here we present a roundup of three recent studies in rodent models that take on the question of how mitochondrial functions, or dysfunctions, impact the health and activities of sensory neurons. All three studies support the idea that mitochondria hold an untapped cache of therapeutic possibilities for pain.
The first study, from the lab of Jon Levine at the University of California at San Francisco, indicates that mitochondrial division, also called fission, plays a critical role in the painful neuropathies brought on by cancer chemotherapy and anti-HIV medication. In the second, Jeffrey Milbrandt and colleagues at Washington University School of Medicine in St. Louis, Missouri, find that a loss of mitochondrial function in Schwann cells leads to a progressive peripheral neuropathy that starts with degeneration of pain-sensing small fibers. Finally, Kyungsoon Chung at the University of Texas Medical Branch, Galveston, and colleagues present evidence that mitochondrial calcium uptake and production of reactive oxygen species are necessary for pain-related synaptic changes to occur in dorsal horn neurons in response to elevated intracellular calcium. The papers all appeared in the Journal of Neuroscience.
Known primarily as the powerhouses of cells, mitochondria are crucial to the nervous system, probably because neurons have high energy demands. Cancer chemotherapy and anti-HIV medicines are thought to trigger painful neuropathies by damaging peripheral nerve mitochondria and thus starving the cells for energy. That idea is supported by recent work showing that respiration and ATP production in mitochondria from peripheral nerves is reduced in rat models of neuropathy induced by the chemotherapeutic agents paclitaxel and oxaliplatin (Zheng et al., 2011).
But mitochondria are “more than energy boxes,” Levine told PRF. He pointed out that the organelles are involved in a wide array of cellular processes, including calcium regulation, cell death, and generation of reactive oxygen species (ROS), and evidence is growing that all of these play parts in various forms of neuropathic pain (Reichling and Levine, 2011; also see PRF related discussion).
In their new study, Levine and his group focused on the process of mitochondrial fission as a possible mediator of painful neuropathies induced by cancer and HIV drugs and oxidative stress. To inhibit mitochondrial fission in rats, first author Luiz Ferrari and coworkers targeted dynamin-related protein 1 (Drp1), a GTPase that is required for fission. They shut down Drp1 in sensory neurons either by intrathecal administration of an antisense oligonucleotide or by local injection of a small molecule Drp1 inhibitor, mdivi-1, into the skin. Drp1 inhibition decreased mechanical hyperalgesia brought on by the anti-HIV drug 2,3-dideoxycytidine (ddC) or the cancer drug oxaliplatin. Drp1 inhibition also mitigated hypersensitivity caused by ROS (produced by injection of hydrogen peroxide into the skin). In addition, the researchers showed that Drp1 inhibition reversed mechanical hyperalgesia caused by mediators of neuropathic and inflammatory pain, namely tumor necrosis factor alpha (TNFα), glial-derived neurotrophic factor (GDNF), and the nitric oxide donor NOR-3.
The effects of blocking Drp1 suggest that mitochondrial fission is a crucial contributor to at least some forms of peripheral neuropathic and inflammatory pain, but the exact relationship between nociception and fission remains to be seen. “The signaling pathways by which pronociceptive agonists activate Drp1 and how Drp1 in particular and the fission process in general interact with classic mitochondrial functions (e.g., ATP synthesis, reactive oxygen species generation, calcium buffering, and apoptotic signaling) in terms of nociceptor function are still open questions,” Levine said. “Fission is likely integrating various aspects of the role of the mitochondrion in the cell.”
Interestingly, Drp1 inhibition did not affect mechanical hyperalgesia induced by nerve growth factor (NGF) or epinephrine. However, a separate study from the Levine lab implicates other mitochondrial functions in NGF-induced hypersensitivity (Chu et al., 2011), suggesting that the organelles can alter pain pathways in various ways.
Levine sees mitochondrial fission and fusion as promising targets for therapeutic control, and as alternatives to antioxidant therapy, which, although showing therapeutic potential for peripheral neuropathy (for a recent example, see Pace et al., 2010), has not been as effective as hoped or expected, he said. Given that fission inhibitors like mdivi-1 are now being developed (Lackner and Nunnari, 2010), the possibility of testing this strategy in patients “may not be remote,” Levine said.
Neurons, of course, do not work alone. In the periphery, nerve axons associate intimately with Schwann cells, glial cells that are best known for producing the myelin sheaths that wrap large-diameter axons. Milbrandt and his colleagues wondered whether mitochondria in Schwann cells might provide crucial help to maintain the stability of long peripheral axons. “We know about…the mysterious mothering that glia do, and we thought some of that could have to do with metabolic support,” Milbrandt told PRF. “This is a beginning attempt to ask, If you have a mitochondrial deficiency in glia, what are the consequences for the axon?”
First author Andreu Viader and coworkers generated mice with the nuclear-encoded mitochondrial protein transcription factor A (TFAM) deleted specifically in Schwann cells.The mice showed depletion of mitochondrial DNA and mitochondrial-encoded transcripts in sciatic nerves. Moreover, Schwann cell mitochondria displayed evidence of impaired respiration and abnormal morphology. Starting at three months of age, mutant mice developed a progressive peripheral neuropathy marked by motor problems, reduced nerve conduction velocity, and loss of axons in muscle tissue. Large myelinated nerve fibers showed extensive demyelination and degeneration, consistent with defects in Schwann cell function.
Before the effects on large myelinated fibers became apparent, however, something intriguing happened: At one to two months, unmyelinated small fibers (C-fibers) and the non-myelinating Schwann cells that normally wrap around them developed structural abnormalities, and the axons appeared to be degenerating. In agreement with C-fibers’ function as high-threshold sensory receptors, the researchers found that by two months of age, mutant mice had an impaired response to painful heat. Thus, before motor symptoms appeared, the defects in Schwann cell mitochondria meddled with the animals’ pain responses.
The authors note that the early loss of pain-sensing fibers they saw is reminiscent of the early loss of small fibers that occurs in patients with painful polyneuropathies, including those associated with diabetes and HIV/AIDS. In addition, in a variety of neuropathies, abnormal mitochondria appear preferentially associated with Schwann cells rather than neurons (Kalichman et al., 1998; Schröder et al., 1993); the Milbrandt group’s study now offers evidence that dysfunction in the Schwann cell mitochondria may actually drive the diseases. Milbrandt said that his group is now working to pinpoint particular features of Schwann cell mitochondrial metabolism that promote axon stability.
Broadening the participation of mitochondria past the periphery, researchers have recently identified new roles for the organelles in spinal pain processing. The central sensitization that contributes to chronic pain involves increased synaptic strength in the dorsal horn of the spinal cord, where incoming sensory afferents meet central neurons (Ji et al., 2003). In the strengthening process, known as spinal long-term potentiation or LTP, activation of NMDA receptors on dorsal horn neurons causes an increase in intracellular Ca2+ concentration, which in turn activates downstream signaling pathways, leading ultimately to central sensitization.
In their new study, Chung and colleagues insert mitochondria into this crucial signaling pathway, by providing evidence that incoming Ca2+ ions must first enter mitochondria to produce subsequent signaling events. First author Hee Young Kim and colleagues inhibited spinal mitochondrial Ca2+ uptake in mice using several different pharmacological strategies and found that the inhibition blocked NMDA-induced activation of downstream protein kinases involved in spinal synaptic plasticity, and reduced induction of LTP. In behavioral studies, they showed that inhibition of mitochondrial Ca2+ uptake also prevented animals from developing mechanical hyperalgesia in response to intrathecal NMDA or intradermal capsaicin injection.
Mitochondrial Ca2+ uptake boosts ATP production, which generates ROS, namely superoxide (O2), as a byproduct. In earlier studies, the group had found that intrathecally injected scavengers of superoxide dramatically alleviated mechanical hypersensitivity in a rat model of neuropathic pain (Kim et al., 2004). They went on to show that superoxide from mitochondria, in particular, leads to hypersensitivity after capsaicin injection (Schwartz et al., 2009). In the current study, the researchers found that decreasing the levels of superoxide using either a superoxide scavenger or an inhibitor of mitochondrial electron transport blocked NMDA- or capsaicin-induced spinal LTP and hyperalgesia. Those findings suggest that ROS production could be the means by which mitochondrial Ca2+ uptake contributes to spinal LTP.
Whereas there is growing evidence that in the periphery, pain can be produced by mitochondrial pathology (from chemotherapy, diabetes, and other factors), the study from Chung and colleagues indicates that in the spinal cord, physiological functioning of mitochondria may contribute to the induction of chronic pain. ROS is known as a toxic material for cells, Chung told PRF. However, she said, “We’re thinking that in our system, ROS is not causing cell death, but it’s causing signal transduction pathway changes.” Further, she said they suspect that mitochondrial ROS might play a similar part in other types of LTP, such as the synaptic plasticity that underlies learning and memory in the hippocampus.