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Gustina A.S.,Program in Neuroscience | Gustina A.S.,University of Maryland, Baltimore | Trudeau M.C.,University of Maryland, Baltimore
Journal of General Physiology | Year: 2011

Human ether-á-go-go-related gene (hERG) potassium channels have voltage-dependent closing (deactivation) kinetics that are unusually slow. A Per-Arnt-Sim (PAS) domain in the cytoplasmic N-terminal region of hERG regulates slow deactivation by making a direct interaction with another part of the hERG channel. The mechanism for slow deactivation is unclear, however, because the other regions of the channel that participate in regulation of deactivation are not known. To identify other functional determinants of slow deactivation, we generated hERG channels with deletions of the cytoplasmic C-terminal regions. We report that hERG channels with deletions of the cyclic nucleotide-binding domain (CNBD) had accelerated deactivation kinetics that were similar to those seen in hERG channels lacking the PAS domain. Channels with dual deletions of the PAS domain and the CNBD did not show further acceleration in deactivation, indicating that the PAS domain and the CNBD regulate deactivation by a convergent mechanism. A recombinant PAS domain that we previously showed could directly regulate PAS domain-deleted channels did not regulate channels with dual deletions of the PAS domain and CNBD, suggesting that the PAS domain did not interact with CNBD-deleted channels. Biochemical protein interaction assays showed that glutathione S-transferase (GST)-PAS (but not GST) bound to a CNBD-containing fusion protein. Coexpression of PAS domain-deleted subunits (with intact C-terminal regions) and CNBD-deleted subunits (with intact N-terminal regions) resulted in channels with partially restored slow deactivation kinetics, suggesting regulatory intersubunit interactions between PAS domains and CNBDs. Together, these data suggest that the mechanism for regulation of slow deactivation in hERG channels is an interaction between the N-terminal PAS domain and the C-terminal CNBD. © 2011 Gustina and Trudeau. Source


News Article
Site: http://phys.org/biology-news/

The paper, published today in Current Biology, is the first to confirm that a key aspect of human memory impaired in memory disorders exists in the type of pre-clinical animal models that influence major decisions about drug development. The study's results, which required over a year's worth of intensive data collection and analysis, could hold important insights for drug companies. That's because selecting less relevant data early in the research process can create costly errors later in the "translational pipeline" that connects basic science to new treatments and therapies. "There is a huge history of translation failure in memory disorders caused by companies trying to develop compounds based on therapies that produce relief in pre-clinical animal models but later fail during early clinical trials," said Jonathon Crystal, professor in the IU Bloomington College of Arts and Sciences' Department of Psychological and Brain Sciences and director of the Program in Neuroscience, who led the study. "We're working to create stronger pre-clinical models of the types of memory systems that are impaired in human diseases." The conclusions are notable in light of the federal government's recent 60 percent increase in Alzheimer's disease research funding, or $350 million in new spending. No treatment currently exists to halt or reverse the long-term effects of Alzheimer's disease, estimated to affect 5.3 million people in the United States alone. The IU study, conducted in rats, shows for the first time that the animals possess two independent "working memory" resources, or the ability to remember more information across two categories versus a single category. In humans, working memory consists of two memory resources: visual and auditory information. The average person, for example, cannot recall a phone number longer than seven characters despite easily remembering both the audio and video on a television show. To test these forms of memory in animals, Crystal's team challenged rats to memorize odors and spatial information. To test rats' ability to remember spatial data, IU scientists had them find food pellets inside an eight-arm maze. To test their ability to remember new odors, they used pellets inside containers scented by up to 100 common household spices, with only new odors yielding food. Across numerous trials, IU scientists consistently showed that the rats could recall significantly more details in combination—scents and spaces—compared to trying to remember a single type of information. "We saw high-level performance because the animals were encoding information in two dedicated memory resources," Crystal said. "This is the defining quality of working memory in people; and for the first time, we've shown animals have this property of independent memory systems as well." The results also suggest that this form of memory arose evolutionarily much earlier than previously thought. Historically, Crystal said almost all investigations on the genetics of Alzheimer's disease depend upon spatial memory research because these studies are easier to carry out. Yet treatments based solely upon spatial memory data aren't likely to strike at the heart of what's so cruel about memory disorders. It's critical to also investigate more complex forms of memory, including working memory. "What researchers are doing now is akin to coming up with a plan for developing a drug which, if successful after spending billions of dollars, helps your grandmother find her reading glasses or car keys," Crystal said. "Those symptoms aren't the most debilitating aspect of Alzheimer's disease. We need solutions that address the inability to remember significant things, like memories of the past or personal exchanges with friends and family, whose loss is so distressing to sufferers of the disease and their loved ones." The IU study was made possible in part by student researchers from the College of Arts and Sciences whose work helped overcome the significant time investment required to perform complex memory trials in animals. IU students who contributed to the project were first author Alexander Bratch, now a graduate student at the University of Minnesota, who wrote his undergraduate thesis on the work; Diana Arman, Austin Dunn, Shiloh Cooper, Hannah Corbin, Stefan Dalecki and Spencer Kann, all undergraduates, and Alexandra Smith, a graduate student. Explore further: New discovery on animal memory opens doors to research on memory impairment diseases


DiLeonardi A.M.,Program in Neuroscience | Huh J.W.,Childrens Hospital of Philadelphia | Raghupathi R.,Program in Neuroscience | Raghupathi R.,Drexel University
Journal of Neuropathology and Experimental Neurology | Year: 2012

Diffuse axonal injury is a major component of traumatic brain injury in children and correlates with long-term cognitive impairment. Traumatic brain injury in adult rodents has been linked to a decrease in compound action potential (CAP) in the corpus callosum, but information on trauma-associated diffuse axonal injury in immature rodents is limited.We investigated the effects of closed head injury on CAP in the corpus callosum of 17-day-old rats. The injury resulted in CAP deficits of both myelinated and unmyelinated fibers in the corpus callosum between 1 and 14 days postinjury (dpi). These deficits were accompanied by intra-axonal dephosphorylation of the 200-kDa neurofilament subunit (NF200) at 1 and 3 dpi, a decrease in total NF200 at 3 dpi and axonal degeneration at 3 and 7 dpi. Although total phosphatase activity decreased at 1 dpi, calcineurin activity was unchanged. The calcineurin inhibitor, FK506, significantly attenuated the injury-induced NF200 dephosphorylation of NF200 at 3 dpi and axonal degeneration at 3 and 7 dpi but did not affect the decrease in NF200 protein levels or impaired axonal transport. FK506 had no effect on CAP deficits at 3 dpi but exacerbated the deficit in only the myelinated fibers at 7 dpi. Thus, in contrast to adult animals, FK506 treatment did not improve axonal function in brain-injured immature animals, suggesting that calcineurin may not contribute to impaired axonal function. Copyright © 2012 by the American Association of Neuropathologists, Inc. Source


Kulakowski S.A.,Program in Neuroscience | Parker S.D.,Health Science University | Personius K.E.,Program in Neuroscience | Personius K.E.,Health Science University
Journal of Applied Physiology | Year: 2011

Acute blockade of signaling through the tyrosine kinase receptor B (TrkB) attenuates neuromuscular transmission and fragments postsynaptic acetylcholine receptors (AChRs) in adult mice, suggesting that TrkB signaling is a key regulator of neuromuscular function. Using immunohistochemical, histological, and in vitro muscle contractile techniques, we tested the hypothesis that constitutively reduced TrkB expression would disrupt neuromuscular pre- and postsynaptic structure, neurotransmission, muscle fiber size, and muscle function in the soleus muscle of 6- to 8-mo-old TrkB +/ - mice compared with age-matched littermates. Age-like expansion of postsynaptic AChR area, AChR fragmentation, and denervation was observed in TrkB +/ - mice similar to that found in 24-mo-old wild-type mice. Neurotransmission failure was increased in TrkB +/ - mice, suggesting that these morphologic changes were sufficient to alter synaptic function. Reduced TrkB expression resulted in decreased muscle strength and fiber cross-sectional area. Immunohistochemical and muscle retrograde labeling experiments show that motor neuron number and size are unaffected in TrkB +/ - mice. These results suggest that TrkB- signaling at the neuromuscular junction plays a role in synaptic stabilization, neurotransmission, and muscle function and may impact the aging process of sarcopenia. © 2011 the American Physiological Society. Source


Conner K.R.,Program in Neuroscience | Forbes M.E.,Medical Center Boulevard | Lee W.H.,Virginia Polytechnic Institute and State University | Lee Y.W.,Virginia Polytechnic Institute and State University | And 2 more authors.
Radiation Research | Year: 2011

Blockers of the renin-angiotensin-aldosterone system (RAAS) ameliorate cognitive deficits and some aspects of brain injury after whole-brain irradiation. We investigated whether treatment with the angiotensin II type 1 receptor antagonist L-158,809 at a dose that protects cognitive function after fractionated whole-brain irradiation reduced radiation-induced neuroinflammation and changes in hippocampal neurogenesis, well-characterized effects that are associated with radiation-induced brain injury. Male F344 rats received L-158,809 before, during and after a single 10-Gy dose of radiation. Expression of cytokines, angiotensin II receptors and angiotensin-converting enzyme 2 was evaluated by real-time PCR 24 h, 1 week and 12 weeks after irradiation. At the latter times, microglial density and proliferating and activated microglia were analyzed in the dentate gyrus of the hippocampus. Cell proliferation and neurogenesis were also quantified in the dentate subgranular zone. L-158,809 treatment modestly increased mRNA expression for Ang II receptors and TNF-α but had no effect on radiation-induced effects on hippocampal microglia or neurogenesis. Thus, although L-158,809 ameliorates cognitive deficits after whole-brain irradiation, the drug did not mitigate the neuroinflammatory microglial response or rescue neurogenesis. Additional studies are required to elucidate other mechanisms of normal tissue injury that may be modulated by RAAS blockers. © 2011 by Radiation Research Society. Source

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