News Article | May 4, 2017
Working with mouse, fly and human cells and tissue, Johns Hopkins researchers report new evidence that disruptions in the movement of cellular materials in and out of a cell's control center -- the nucleus -- appear to be a direct cause of brain cell death in Huntington's disease, an inherited adult neurodegenerative disorder. Moreover, they suggest, laboratory experiments with drugs designed to clear up these cellular "traffic jams" restored normal transport in and out of the nucleus and saved the cells. In the featured article published online on April 5 in Neuron, the researchers also conclude that potential treatments targeting the transport disruptions they identified in Huntington's disease neurons may also work for other neurodegenerative diseases, such as ALS and forms of dementia. Huntington's disease is a relatively rare fatal inherited condition that gradually kills off healthy nerve cells in the brain, leading to loss of language, thinking and reasoning abilities, memory, coordination and movement. Its course and effects are often described as Alzheimer's disease, Parkinson's disease and ALS rolled into one, making Huntington's disease a rich focus of scientific investigation. "We're trying to get at the heart of the mechanism behind neurodegenerative diseases and with this research believe we've found one that seems to be commonly disrupted in many of them, suggesting that similar drugs may work for some or all of these disorders," says Jeffrey Rothstein, M.D., Ph.D., a professor of neurology and neuroscience, and director of the Brain Science Institute and the Robert Packard Center for ALS Research at the Johns Hopkins University School of Medicine. In 2015, Rothstein's team found out how a mutation in a gene -- implicated in 40 percent of inherited ALS cases and 25 percent of inherited frontotemporal dementia cases -- gums up transport in and out of the nucleus in neurons, ultimately shutting the cell down and leading to its death. The mutant gene makes RNA molecules that stick to a transport protein, RanGAP1. RanGAP1 in turn helps move molecules through nuclear pores that serve as passageways in the nucleus, letting proteins and genetic material flow in and out of it. Jonathan Grima, currently a fourth-year neuroscience graduate student in Rothstein's laboratory, learned that this same mutation is also the most common cause of another disorder in which patients have Huntington's -like symptoms without having the causative Huntington's disease mutation. Additionally, he realized that other researchers previously showed that mutations in the nuclear pore protein NUP62 caused Huntington's disease-like pathology. Because of such clues from others' research, Grima took on the task of investigating whether problems with nuclear transport and the nuclear pores also happened in neurons with Huntington's disease. Huntington's disease is caused by a mutation in the Huntingtin protein, resulting in too many repeats of the amino acid glutamine in the protein's sequence, making the protein sticky and clumpy. Grima used two mouse models of Huntington's disease: one with a human version of the mutant Huntingtin protein and another with an aggressive form of the disease that contains only the first portion of the mouse Huntingtin protein. By using antibodies with glowing markers that bind to specific proteins and viewing the neurons under the microscope, Grima saw that the mutant Huntingtin protein clumped up in the same location of the cell as abnormal clumps of RanGAP1, the nuclear transport protein. It also clumped up in the same location as abnormal clumps of nuclear pore proteins NUP88 and NUP62. "This finding was quite tantalizing given the fact that mutations in the NUP62 protein were shown by other researchers to cause an infantile form of Huntington's disease called infantile bilateral striatal necrosis," says Grima. Grima also observed this same clumping of Huntingtin protein with RanGAP1 and nuclear pore proteins to the wrong place in the cell in brain tissue and cultured brain cells derived from deceased patients with Huntington's disease. To further explore nuclear transport's role in Huntington's disease, Grima took lab-grown mouse neurons and used chemical switches to a) turn on both an additional healthy copy of the RanGAP1 gene and a mutant version of Huntingtin; b) just turn on the mutant Huntingtin; or c) just turn on a healthy version of Huntingtin. He then measured cell death and found that neurons with the healthy version of Huntingtin had about 17 percent of the neurons die off. Neurons with only the mutant version of Huntingtin were more likely to die, with about 33 percent dying off, but in neurons with both the mutant Huntingtin and the RanGAP1, only 24 percent of the neurons died off. The researchers think that some of the extra healthy RanGAP1 they introduced into diseased cells wasn't bound up to the mutant Huntingtin and resumed normal nuclear transport. Next, Grima looked at cell death in cultured neurons with a healthy or a mutant form of Huntingtin, or with a mutant form of Huntingtin that was treated with small amounts of an experimental drug called KPT-350, one that prevents a nuclear export protein, Exportin-1, from shuttling proteins and RNA out of the nucleus. Neurons with the healthy version of Huntingtin had about 18 percent die off, and neurons with the mutant version of Huntingtin had about 38 percent die off. Those treated with the nuclear export blocking drug had improved survival, with only about 22 percent of the cells die off. Blocking nuclear export seemed to prevent cells from dying and counteracted the defects in neurons with mutant Huntingtin, the researchers say. "Our studies show that broken-down components of the nuclear transport machinery lead to traffic jams within brain neurons of essential information and eventually brain cell death," says Grima. "We believe that the reestablishment of proper cell transport could provide a promising therapeutic target for Huntington's disease, and potentially other neurodegenerative disorders." "Although the disrupted nuclear transport seems to be killing neurons in multiple neurodegenerative diseases, these diseases have very different properties and symptoms," cautions Rothstein. "We need to do more work to find out why one disease causes a certain set of symptoms and another disease causes others with respect to what is happening with nuclear transport." According to the researchers, there is an average of 2000 nuclear pores per cell and each individual nuclear pore consists of multiple copies of more than 30 different proteins that each serve different functions. It may be that nuclear pores on neurons and other types of brain cells like glia are constructed of different combinations of these proteins, some of which may be more or less critical in various neurodegenerative diseases. Grima is currently working on answering this question using a new mouse model developed at Johns Hopkins that will allow him to isolate these nuclear pore proteins from different cell types in the mouse brain to identify whether these nuclear pore components are in fact different based on brain cell types and brain locations. "We sincerely hope our new findings may help bring us a step closer to treating this and potentially other horrific neurodegenerative disorders," says Grima. According to the Huntington's Society of America, about 30,000 people in the United States have Huntington's symptoms and 200,000 people are at risk of inheriting the disease from a parent. Additional authors include J. Gavin Daigle, Nicolas Arbez, Kathleen Cunningham, Ke Zhang, Jenna Glatzer, Jacqueline Pham, Ishrat Ahmed, Qi Peng, Harsh Wadhwa, Olga Pletnikova, Juan Troncoso, Wenzhen Duan, Solomon Snyder, Thomas Lloyd, and Christopher Ross of Johns Hopkins Medicine; Joseph Ochaba, Charlene Geater, Eva Morozko, Jennifer Stocksdale and Leslie Thompson of University of California, Irvine; and Laura Ranum of University of Florida, Gainesville. This work was funded by grants from the National Institute of Neurological Disorders and Stroke (R01NS094239 and R01NS085207); CIRM Training Grant; National Science Foundation Graduate Research Fellowship Award; Thomas Shortman Training Fund Graduate Award; Axol Science Award; NIH Training in Neurotherapeutics Discovery and Development for Academic Scientists; MDI Laboratory QFM Chroma Fellowship Award; and the Johns Hopkins Brain Science Institute.
News Article | December 22, 2016
A clump of just a few thousand brain cells, no bigger than a mustard seed, controls the daily ebb and flow of most bodily processes in mammals -- sleep/wake cycles, most notably. Now, Johns Hopkins scientists report direct evidence in mice for how those cell clusters control sleep and relay light cues about night and day throughout the body. A summary of their study of the brain region known as the suprachiasmatic nucleus, or SCN, will be published online in the journal Current Biology on Dec. 22. "Light has a strong, negative and direct effect on sleep in humans. We experience this every evening when we turn out the lights before we go to bed and every morning when we open the curtains to let light in. However, very little was known about how this happens. Learning that the SCN is indeed required for light to directly regulate sleep is an important piece of the circadian rhythm puzzle," says Seth Blackshaw, Ph.D., professor of neuroscience at the Johns Hopkins University School of Medicine. "Our chances of finding treatments for people with sleep disorders, or just jet lag, improve the more we understand the details about how sleep is controlled." Blackshaw says scientists have known for a while that the SCN functions as a master clock to synchronize sleep and other so-called circadian rhythms in humans and other mammals. But its importance in the more immediate regulation of sleep, like when a bright light wakes someone up, remained debatable because the experiments needed to show its role in a living animal were essentially impossible. "If you surgically removed the SCN in mice, their sleeping and waking were no longer immediately influenced by light, but you can't remove the SCN without also severing the optic nerve that brings light information to it from the retina. So no one knew if this resistance to light was due to the missing SCN or the missing optic nerve," says Blackshaw. In experiments first reported several years ago, Blackshaw's team found a way to disrupt the normal function of the SCN without physically removing it and damaging the optic nerve. The researchers were trying to identify genes involved in the development of the mouse hypothalamus, the area of the brain that includes the SCN. They identified one such gene, dubbed LHX1, that seemed to be the earliest to "turn on" in the development of the fetal SCN. For the new round of experiments, the scientists used a customized genetic tool to delete LHX1 just from cells that make up the SCN. They found that the mice experienced severely disrupted circadian rhythms, although they could still be weakly synchronized to light cycles. And the cells of the SCN no longer produced six small signaling proteins known to coordinate and reinforce their efforts, a biochemical process known as coupling. Whether the mice were kept in constant light, constant darkness or normal cycles of both, their sleep times and duration became random. Cumulatively, they slept for the same amount of time, about 12 hours each 24-hour period, like normal mice, but there was no pattern to the cycle. "This experiment showed that the SCN is critical to light's immediate effect on sleep," says Blackshaw. The scientists also noticed that in the SCN-impaired mice, core body temperatures didn't cycle normally. The average body temperature for humans is 37 degrees Celsius, but it fluctuates throughout the day by about 1 degree Celsius, being highest in the afternoon and lowest just before dawn. A similar pattern occurs in mice. These small temperature fluctuations can have a big influence on processes that occur outside the brain that are also under circadian control, such as glucose usage and fat storage, and it has been speculated that they may be the main way by which the SCN controls these bodily rhythms. In contrast, one of the hallmarks of the body's circadian processes, including cycles in core body temperature, is that they aren't generally disturbed by large temperature changes. "Otherwise, you would feel jet-lagged every time you got a fever," says Blackshaw. But it wasn't clear from mouse experiments if the SCN was responsible for this resistance to strong temperature changes in living animals. Normal SCN cells in the lab keep cycling in synchrony without regard to temperature pulses, but research from another group showed that they could be "reset" by temperature changes if they could no longer signal to each other. Knowing that the SCN cells in their LHX1-deficient mice were similarly impaired, a graduate student in Blackshaw's lab, Joseph Bedont, reasoned that their mice might now be able to return to normal temperature cycles if given pulses of heat. To try that, they injected the mice -- kept in the dark -- with a molecule found in bacterial cell walls, which makes them run a fever in response to the perceived threat. Fever is a first-line infection fighter in humans as well. As suspected, their regular core temperature cycling came back. "These results suggest that the SCN is indeed responsible for the temperature resistance of circadian rhythms in live animals, and it shows us how important SCN coupling is," says Blackshaw. "It also bolsters the idea that the body's other physiologic cycles, such as hunger and hormone secretion, are synchronized by the SCN through its regulation of core body temperature." Additional experiments identified several molecules that may be directing these vital signals. The Blackshaw team plans to follow up by studying each one to determine their roles. With that information, drug developers will have a better idea which component to target and how. To treat jet lag, for example, Blackshaw says that one hypothetical option would be to briefly block LHX1 so that the SCN cells uncouple and become easier to reset, either by light or temperature. But no one knows yet if that plan would produce undesirable side effects or the desired outcomes. Other authors of the report include Abhijith Bathini, Jonathan Ling, Benjamin Bell, Mark Wu, Philip Wong and Samer Hattar of the Johns Hopkins University School of Medicine; Tara LeGates of The Johns Hopkins University; Ethan Buhr and Russell Van Gelder of the University of Washington, Seattle; and Valerie Mongrain of the University of Montreal. This work was supported by grants from the Johns Hopkins Brain Science Institute, the Wilmer Eye Institute Visual Neuroscience Training Program, the National Science Foundation, the Fonds de Recherche du Quebec-Sante, the Canadian Institutes of Health Research, the W.M. Keck Foundation, Research to Prevent Blindness and the National Eye Institute (EY001730).
Wozniak K.M.,Johns Hopkins Brain Science Institute |
Nomoto K.,Eisai Inc |
Lapidus R.G.,University of Maryland, Baltimore |
Wu Y.,Johns Hopkins Brain Science Institute |
And 7 more authors.
Cancer Research | Year: 2011
Chemotherapy-induced neurotoxicity is a significant problem associated with successful treatment of many cancers. Tubulin is a well-established target of antineoplastic therapy; however, tubulin-targeting agents, such as paclitaxel and the newer epothilones, induce significant neurotoxicity. Eribulin mesylate, a novel microtubule-targeting analogue of the marine natural product halichondrin B, has recently shown antineoplastic activity, with relatively low incidence and severity of neuropathy, in metastatic breast cancer patients. The mechanism of chemotherapy-induced neuropathy is not well understood. One of the main underlying reasons is incomplete characterization of pathology of peripheral nerves from treated subjects, either from patients or preclinically from animals. The current study was conducted to directly compare, in mice, the neuropathy-inducing propensity of three drugs: paclitaxel, ixabepilone, and eribulin mesylate. Because these drugs have different potencies and pharmacokinetics, we compared them on the basis of a maximum tolerated dose (MTD). Effects of each drug on caudal and digital nerve conduction velocity, nerve amplitude, and sciatic nerve and dorsal root ganglion morphology at 0.25 x MTD, 0.5 x MTD, 0.75 x MTD, and MTD were compared. Paclitaxel and ixabepilone, at their respective MTDs, produced significant deficits in caudal nerve conduction velocity, caudal amplitude and digital nerve amplitudes, as well as moderate to severe degenerative pathologic changes in dorsal root ganglia and sciatic nerve. In contrast, eribulin mesylate produced no significant deleterious effects on any nerve conduction parameter measured and caused milder, less frequent effects on morphology. Overall, our findings indicate that eribulin mesylate induces less neuropathy in mice than paclitaxel or ixabepilone at equivalent MTD-based doses. ©2011 AACR.
Barinka C.,Academy of Sciences of the Czech Republic |
Rojas C.,Johns Hopkins Brain Science Institute |
Slusher B.,Johns Hopkins Brain Science Institute |
Pomper M.,Johns Hopkins Medical Institutions
Current Medicinal Chemistry | Year: 2012
Glutamate carboxypeptidase II (GCPII) is a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. Throughout the nervous system, glia-bound GCPII is intimately involved in the neuronneuron and neuron-glia signaling via the hydrolysis of N-acetylaspartylglutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. The inhibition of the GCPII-controlled NAAG catabolism has been shown to attenuate neurotoxicity associated with enhanced glutamate transmission and GCPII-specific inhibitors demonstrate efficacy in multiple preclinical models including traumatic brain injury, stroke, neuropathic and inflammatory pain, amyotrophic lateral sclerosis, and schizophrenia. The second major area of pharmacological interventions targeting GCPII focuses on prostate carcinoma; GCPII expression levels are highly increased in androgenindependent and metastatic disease. Consequently, the enzyme serves as a potential target for imaging and therapy. This review offers a summary of GCPII structure, physiological functions in healthy tissues, and its association with various pathologies. The review also outlines the development of GCPII-specific small-molecule compounds and their use in preclinical and clinical settings. © 2012 Bentham Science Publishers.
Dixon S.J.,Columbia University |
Dixon S.J.,Stanford University |
Patel D.,Columbia University |
Welsch M.,Columbia University |
And 9 more authors.
eLife | Year: 2014
Exchange of extracellular cystine for intracellular glutamate by the antiporter system Xc - is implicated in numerous pathologies. Pharmacological agents that inhibit system Xc - activity have long been sought, but have remained elusive. Here, we report that the small molecule erastin is a potent, selective inhibitor of system Xc -. RNA sequencing revealed that inhibition of cystine-glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system Xc - inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system Xc - function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis.