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Professor Bryce Vissel


Bryce Vissel is a Professor of Neuroscience at UTS and Director of Neuroscience and Regenerative Medicine in the Faculty of Science. His work spans neurodegenerative diseases including Alzheimer’s, Parkinson’s and spinal disorders as well as studies of the neural basis of learning and memory and movement mechanisms. 
 After being awarded his PhD in medical genetics from the University of Melbourne in 1991, Professor Vissel joined Garvan’s Neuroscience Division, where he was subsequently awarded a National Health and Medical Research Council CJ Martin Fellowship to pursue neuroscience research with Professor Stephen Heinemann at the world-leading Salk Institute in San Diego, USA.
He spent 10 years at Salk, where he authored a number of seminal studies describing molecular mechanisms that regulate synaptic function and their role in behaviour and neurological diseases.
He also received several prestigious awards – a Human Frontiers Award, a Fulbright Award and a Lieberman Award.
In late 2002, Professor Vissel returned to Garvan taking up a position as Head of the Neurodegenerative Diseases group in the Neuroscience Division. His team’s work at the Garvan since then has, among other things, provided new insights into synapse function and shown that the brain has far greater potential for regeneration and repair than previously thought. Professor Vissel is also an important contributor to a number of organisations. He is Chair of the Advisory Board of Cellmid Ltd, a member of the Board of Parkinson’s NSW, and scientific advisor to Alzheimer’s Australia and SpinalCure Australia.
Image of Bryce Vissel
Professor of Neuroscience, School of Life Sciences

Research Interests

  • Neuroscience
  • Neurodegenerative diseases – Alzheimers Disease, Parkinson’s Disease
  • Regenerative medicine - spinal cord injury
  • Synaptic plasticity
Can supervise: Yes

Journal articles

Stayte, S., Rentsch, P., Tröscher, A.R., Bamberger, M., Li, K.M. & Vissel, B. 2017, 'Activin A Inhibits MPTP and LPS-Induced Increases in Inflammatory Cell Populations and Loss of Dopamine Neurons in the Mouse Midbrain In Vivo.', PLoS One, vol. 12, no. 1, p. e0167211.
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Parkinson's disease is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta region and a subsequent loss of dopamine within the striatum. A promising avenue of research has been the administration of growth factors to promote the survival of remaining midbrain neurons, although the mechanism by which they provide neuroprotection is not understood. Activin A, a member of the transforming growth factor superfamily, has been shown to be a potent anti-inflammatory following acute brain injury and has been demonstrated to play a role in the neuroprotection of midbrain neurons against MPP+-induced degeneration in vitro. We hypothesized that activin A may offer similar anti-inflammatory and neuroprotective effects in in vivo mouse models of Parkinson's disease. We found that activin A significantly attenuated the inflammatory response induced by both MPTP and intranigral administration of lipopolysaccharide in C57BL/6 mice. We found that administration of activin A promoted survival of dopaminergic and total neuron populations in the pars compacta region both 8 days and 8 weeks after MPTP-induced degeneration. Surprisingly, no corresponding protection of striatal dopamine levels was found. Furthermore, activin A failed to protect against loss of striatal dopamine transporter expression in the striatum, suggesting the neuroprotective action of activin A may be localized to the substantia nigra. Together, these results provide the first evidence that activin A exerts potent neuroprotection and anti-inflammatory effects in the MPTP and lipopolysaccharide mouse models of Parkinson's disease.
Leake, J., Zinn, R., Corbit, L. & Vissel, B. 2017, 'Dissociation between complete hippocampal context memory formation and context fear acquisition.', Learn Mem, vol. 24, no. 4, pp. 153-157.
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Rodents require a minimal time period to explore a context prior to footshock to display plateau-level context fear at test. To investigate whether this rapid fear plateau reflects complete memory formation within that short time-frame, we used the immediate-early gene product Arc as an indicator of hippocampal context memory formation-related activity. We found that hippocampal Arc expression continued to increase well past the minimal time required for plateau-level fear. This raises the possibility that context fear conditioning occurs more rapidly than complete memory formation. Thus, animals may be able to condition robustly to both complete and incomplete contextual representations.
Wright, A.L. & Vissel, B. 2016, 'CAST your vote: is calpain inhibition the answer to ALS?', Journal of neurochemistry, vol. 137, no. 2, pp. 140-141.
A publication in the Journal of Neurochemistry by Rao et al. (2016) suggests that the overexpression of the calpain inhibitor, calpastatin (CAST) rescues neuron loss and increases survival of the amyotrophic lateral sclerosis (ALS) mouse model, hSOD1G93A. The findings of Rao et al. (2016) provide an insight into the mechanisms that lead to neuronal loss in ALS and suggest a cell loss pathway common to several neurodegenerative disorders that may be therapeutically targeted. Here, we highlight the findings of Rao et al. (2016) and discuss some key considerations required prior to assessing the potential use of calpain inhibitors in the clinic. Read the highlighted article 'Calpastatin inhibits motor neuron death and increases survival in hSOD1(G93A) mice' on page 253.
Morris, G.P., Wright, A.L., Tan, R.P., Gladbach, A., Ittner, L.M. & Vissel, B. 2016, 'A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice.', PloS one, vol. 11, no. 2, pp. e0148503-e0148503.
The intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice and rats has been plagued by inconsistency, owing in part to the multitude of variables requiring control. In this study we investigated the impact of several major variables on survival rate, lesion volume, neurological scores, cerebral blood flow (CBF) and body weight including filament width, time after reperfusion, occlusion time and the choice of surgical method. Using the Koizumi method, we found ischemic injury can be detected as early as 30 min after reperfusion, to a degree that is not statistically different from 24 h post-perfusion, using 2,3,5-Triphenyltetrazolium chloride (TTC) staining. We also found a distinct increase in total lesion volume with increasing occlusion time, with 30-45 min a critical time for the development of large, reproducible lesions. Furthermore, although we found no significant difference in total lesion volume generated by the Koizumi and Longa methods of MCAO, nor were survival rates appreciably different between the two at 4 h after reperfusion, the Longa method produces significantly greater reperfusion. Finally, we found no statistical evidence to support the exclusion of data from animals experiencing a CBF reduction of <70% in the MCA territory following MCAO, using laser-Doppler flowmetry. Instead we suggest the main usefulness of laser-Doppler flowmetry is for guiding filament placement and the identification of subarachnoid haemorrhages and premature reperfusion. In summary, this study provides detailed evaluation of the Koizumi method of intraluminal filament MCAO in mice and a direct comparison to the Longa method.
Clark, I.A. & Vissel, B. 2016, 'Excess cerebral TNF causing glutamate excitotoxicity rationalizes treatment of neurodegenerative diseases and neurogenic pain by anti-TNF agents.', Journal of neuroinflammation, vol. 13, no. 1, p. 236.
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The basic mechanism of the major neurodegenerative diseases, including neurogenic pain, needs to be agreed upon before rational treatments can be determined, but this knowledge is still in a state of flux. Most have agreed for decades that these disease states, both infectious and non-infectious, share arguments incriminating excitotoxicity induced by excessive extracellular cerebral glutamate. Excess cerebral levels of tumor necrosis factor (TNF) are also documented in the same group of disease states. However, no agreement exists on overarching mechanism for the harmful effects of excess TNF, nor, indeed how extracellular cerebral glutamate reaches toxic levels in these conditions. Here, we link the two, collecting and arguing the evidence that, across the range of neurodegenerative diseases, excessive TNF harms the central nervous system largely through causing extracellular glutamate to accumulate to levels high enough to inhibit synaptic activity or kill neurons and therefore their associated synapses as well. TNF can be predicted from the broader literature to cause this glutamate accumulation not only by increasing glutamate production by enhancing glutaminase, but in addition simultaneously reducing glutamate clearance by inhibiting re-uptake proteins. We also discuss the effects of a TNF receptor biological fusion protein (etanercept) and the indirect anti-TNF agents dithio-thalidomides, nilotinab, and cannabinoids on these neurological conditions. The therapeutic effects of 6-diazo-5-oxo-norleucine, ceptriaxone, and riluzole, agents unrelated to TNF but which either inhibit glutaminase or enhance re-uptake proteins, but do not do both, as would anti-TNF agents, are also discussed in this context. By pointing to excess extracellular glutamate as the target, these arguments greatly strengthen the case, put now for many years, to test appropriately delivered ant-TNF agents to treat neurodegenerative diseases in randomly controlled trials.
Richards, L.R., Michie, P.T., Badcock, D.R., Bartlett, P.F., Bekkers, J.M., Bourne, J.A., Castles, A., Egan, G.F., Fornito, A., Hannan, A.J., Hickie, I.B., Mattingley, J.B., Schofield, P.R., Shum, D.H.K., Stuart, G.J., Vickers, J.C. & Vissel, B. 2016, 'Australian Brain Alliance', Neuron, vol. 92, no. 3, pp. 597-600.
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&copy; 2016 Elsevier Inc.A proposal for an Australian Brain Initiative (ABI) is under development by members of the Australian Brain Alliance. Here we discuss the goals of the ABI, its areas of research focus, its context in the Australian research setting, and its necessity for ensuring continued success for Australian brain research.
Clark, I.A. & Vissel, B. 2015, 'A Neurologist's Guide to TNF Biology and to the Principles behind the Therapeutic Removal of Excess TNF in Disease.', Neural plasticity, vol. 2015, p. 358263.
Tumor necrosis factor (TNF) is an ancient and widespread cytokine required in small amounts for much physiological function. Higher concentrations are central to innate immunity, but if unchecked this cytokine orchestrates much chronic and acute disease, both infectious and noninfectious. While being a major proinflammatory cytokine, it also controls homeostasis and plasticity in physiological circumstances. For the last decade or so these principles have been shown to apply to the central nervous system as well as the rest of the body. Nevertheless, whereas this approach has been a major success in treating noncerebral disease, its investigation and potential widespread adoption in chronic neurological conditions has inexplicably stalled since the first open trial almost a decade ago. While neuroscience is closely involved with this approach, clinical neurology appears to be reticent in engaging with what it offers patients. Unfortunately, the basic biology of TNF and its relevance to disease is largely outside the traditions of neurology. The purpose of this review is to facilitate lowering communication barriers between the traditional anatomically based medical specialties through recognition of shared disease mechanisms and thus advance the prospects of a large group of patients with neurodegenerative conditions for whom at present little can be done.
Clark, I.A. & Vissel, B. 2015, 'Amyloid : one of three danger-associated molecules that are secondary inducers of the proinflammatory cytokines that mediate Alzheimer's disease.', British journal of pharmacology, vol. 172, no. 15, pp. 3714-3727.
This review concerns how the primary inflammation preceding the generation of certain key damage-associated molecular patterns (DAMPs) arises in Alzheimer's disease (AD). In doing so, it places soluble amyloid (A), a protein hitherto considered as a primary initiator of AD, in a novel perspective. We note here that increased soluble A is one of the proinflammatory cytokine-induced DAMPs recognized by at least one of the toll-like receptors on and in various cell types. Moreover, A is best regarded as belonging to a class of DAMPs, as do the S100 proteins and HMBG1, that further exacerbate production of these same proinflammatory cytokines, which are already enhanced, and induces them further. Moreover, variation in levels of other DAMPs of this same class in AD may explain why normal elderly patients can exhibit high A plaque levels, and why removing A or its plaque does not retard disease progression. It may also explain why mouse transgenic models, having been designed to generate high A, can be treated successfully by this approach.
Stayte, S., Rentsch, P., Li, K.M. & Vissel, B. 2015, 'Activin A protects midbrain neurons in the 6-hydroxydopamine mouse model of Parkinson's disease.', PloS one, vol. 10, no. 4, p. e0124325.
Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) and a subsequent loss of dopamine (DA) within the striatum. Despite advances in the development of pharmacological therapies that are effective at alleviating the symptoms of PD, the search for therapeutic treatments that halt or slow the underlying nigral degeneration remains a particular challenge. Activin A, a member of the transforming growth factor superfamily, has been shown to play a role in the neuroprotection of midbrain neurons against 6-hydroxydopamine (6-OHDA) in vitro, suggesting that activin A may offer similar neuroprotective effects in in vivo models of PD. Using robust stereological methods, we found that intrastriatal injections of 6-OHDA results in a significant loss of both TH positive and NeuN positive populations in the SNpc at 1, 2, and 3 weeks post-lesioning in drug nave mice. Exogenous application of activin A for 7 days, beginning the day prior to 6-OHDA administration, resulted in a significant survival of both dopaminergic and total neuron numbers in the SNpc against 6-OHDA-induced toxicity. However, we found no corresponding protection of striatal DA or dopamine transporter (DAT) expression levels in animals receiving activin A compared to vehicle controls. These results provide the first evidence that activin A exerts potent neuroprotection in a mouse model of PD, however this neuroprotection may be localized to the midbrain.
Morris, G.P., Clark, I.A. & Vissel, B. 2014, 'Inconsistencies and controversies surrounding the amyloid hypothesis of Alzheimer's disease.', Acta neuropathologica communications, vol. 2, p. 135.
The amyloid hypothesis has driven drug development strategies for Alzheimer's disease for over 20 years. We review why accumulation of amyloid-beta (A) oligomers is generally considered causal for synaptic loss and neurodegeneration in AD. We elaborate on and update arguments for and against the amyloid hypothesis with new data and interpretations, and consider why the amyloid hypothesis may be failing therapeutically. We note several unresolved issues in the field including the presence of A deposition in cognitively normal individuals, the weak correlation between plaque load and cognition, questions regarding the biochemical nature, presence and role of A oligomeric assemblies in vivo, the bias of pre-clinical AD models toward the amyloid hypothesis and the poorly explained pathological heterogeneity and comorbidities associated with AD. We also illustrate how extensive data cited in support of the amyloid hypothesis, including genetic links to disease, can be interpreted independently of a role for A in AD. We conclude it is essential to expand our view of pathogenesis beyond A and tau pathology and suggest several future directions for AD research, which we argue will be critical to understanding AD pathogenesis.
Stayte, S. & Vissel, B. 2014, 'Advances in non-dopaminergic treatments for Parkinson's disease.', Frontiers in neuroscience, vol. 8, p. 113.
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Since the 1960's treatments for Parkinson's disease (PD) have traditionally been directed to restore or replace dopamine, with L-Dopa being the gold standard. However, chronic L-Dopa use is associated with debilitating dyskinesias, limiting its effectiveness. This has resulted in extensive efforts to develop new therapies that work in ways other than restoring or replacing dopamine. Here we describe newly emerging non-dopaminergic therapeutic strategies for PD, including drugs targeting adenosine, glutamate, adrenergic, and serotonin receptors, as well as GLP-1 agonists, calcium channel blockers, iron chelators, anti-inflammatories, neurotrophic factors, and gene therapies. We provide a detailed account of their success in animal models and their translation to human clinical trials. We then consider how advances in understanding the mechanisms of PD, genetics, the possibility that PD may consist of multiple disease states, understanding of the etiology of PD in non-dopaminergic regions as well as advances in clinical trial design will be essential for ongoing advances. We conclude that despite the challenges ahead, patients have much cause for optimism that novel therapeutics that offer better disease management and/or which slow disease progression are inevitable.
Stayte, S. & Vissel, B. 2014, 'Corrigendum: Advances in non-dopaminergic pharmacological treatments of Parkinson's disease', Frontiers in Neuroscience, no. 8 AUG.
Clark, I.A. & Vissel, B. 2014, 'Inflammation-sleep interface in brain disease: TNF, insulin, orexin.', Journal of neuroinflammation, vol. 11, p. 51.
The depth, pattern, timing and duration of unconsciousness, including sleep, vary greatly in inflammatory disease, and are regarded as reliable indicators of disease severity. Similarly, these indicators are applicable to the encephalopathies of sepsis, malaria, and trypanosomiasis, and to viral diseases such as influenza and AIDS. They are also applicable to sterile neuroinflammatory states, including Alzheimer's disease, Parkinson's disease, traumatic brain injury, stroke and type-2 diabetes, as well as in iatrogenic brain states following brain irradiation and chemotherapy. Here we make the case that the cycles of unconsciousness that constitute normal sleep, as well as its aberrations, which range from sickness behavior through daytime sleepiness to the coma of inflammatory disease states, have common origins that involve increased inflammatory cytokines and consequent insulin resistance and loss of appetite due to reduction in orexigenic activity. Orexin reduction has broad implications, which are as yet little appreciated in the chronic inflammatory conditions listed, whether they be infectious or sterile in origin. Not only is reduction in orexin levels characterized by loss of appetite, it is associated with inappropriate and excessive sleep and, when dramatic and chronic, leads to coma. Moreover, such reduction is associated with impaired cognition and a reduction in motor control. We propose that advanced understanding and appreciation of the importance of orexin as a key regulator of pathways involved in the maintenance of normal appetite, sleep patterns, cognition, and motor control may afford novel treatment opportunities.
Morris, G.P., Clark, I.A., Zinn, R. & Vissel, B. 2013, 'Microglia: a new frontier for synaptic plasticity, learning and memory, and neurodegenerative disease research.', Neurobiology of learning and memory, vol. 105, pp. 40-53.
We focus on emerging roles for microglia in synaptic plasticity, cognition and disease. We outline evidence that ramified microglia, traditionally thought to be functionally "resting" (i.e. quiescent) in the normal brain, in fact are highly dynamic and plastic. Ramified microglia continually and rapidly extend processes, contact synapses in an activity and experience dependent manner, and play a functionally dynamic role in synaptic plasticity, possibly through release of cytokines and growth factors. Ramified microglial also contribute to structural plasticity through the elimination of synapses via phagocytic mechanisms, which is necessary for normal cognition. Microglia have numerous mechanisms to monitor neuronal activity and numerous mechanisms also exist to prevent them transitioning to an activated state, which involves retraction of their surveying processes. Based on the evidence, we suggest that maintaining the ramified state of microglia is essential for normal synaptic and structural plasticity that supports cognition. Further, we propose that change of their ramified morphology and function, as occurs in inflammation associated with numerous neurological disorders such as Alzheimer's and Parkinson's disease, disrupts their intricate and essential synaptic functions. In turn altered microglia function could cause synaptic dysfunction and excess synapse loss early in disease, initiating a range of pathologies that follow. We conclude that the future of learning and memory research depends on an understanding of the role of non-neuronal cells and that this should include using sophisticated molecular, cellular, physiological and behavioural approaches combined with imaging to causally link the role of microglia to brain function and disease including Alzheimer's and Parkinson's disease and other neuropsychiatric disorders.
Wright, A.L., Zinn, R., Hohensinn, B., Konen, L.M., Beynon, S.B., Tan, R.P., Clark, I.A., Abdipranoto, A. & Vissel, B. 2013, 'Neuroinflammation and neuronal loss precede A plaque deposition in the hAPP-J20 mouse model of Alzheimer's disease.', PloS one, vol. 8, no. 4, p. e59586.
Recent human trials of treatments for Alzheimer's disease (AD) have been largely unsuccessful, raising the idea that treatment may need to be started earlier in the disease, well before cognitive symptoms appear. An early marker of AD pathology is therefore needed and it is debated as to whether amyloid-A? plaque load may serve this purpose. We investigated this in the hAPP-J20 AD mouse model by studying disease pathology at 6, 12, 24 and 36 weeks. Using robust stereological methods, we found there is no neuron loss in the hippocampal CA3 region at any age. However loss of neurons from the hippocampal CA1 region begins as early as 12 weeks of age. The extent of neuron loss increases with age, correlating with the number of activated microglia. Gliosis was also present, but plateaued during aging. Increased hyperactivity and spatial memory deficits occurred at 16 and 24 weeks. Meanwhile, the appearance of plaques and oligomeric A were essentially the last pathological changes, with significant changes only observed at 36 weeks of age. This is surprising given that the hAPP-J20 AD mouse model is engineered to over-expresses A. Our data raises the possibility that plaque load may not be the best marker for early AD and suggests that activated microglia could be a valuable marker to track disease progression.
Clark, I.A. & Vissel, B. 2013, 'Treatment implications of the altered cytokine-insulin axis in neurodegenerative disease.', Biochemical pharmacology, vol. 86, no. 7, pp. 862-871.
The disappointments of a series of large anti-amyloid trials have brought home the point that until the driving force behind Alzheimer's disease, and the way it causes harm, are firmly established and accepted, researchers will remain ill-equipped to find a way to treat patients successfully. The origin of inflammation in neurodegenerative diseases is still an open question. We champion and expand the argument that a shift in intracellular location of -synuclein, thereby moving a key methylation enzyme from the nucleus, provides global hypomethylation of patients' cerebral DNA that, through being sensed by TLR9, initiates production of the cytokines that drive these cerebral inflammatory states. After providing a background on the relevant inflammatory cytokines, this commentary then discusses many of the known alternatives to the primary amyloid argument of the pathogenesis of Alzheimer's disease, and the treatment approaches they provide. A key point to appreciate is the weight of evidence that inflammatory cytokines, largely through increasing insulin resistance and thereby reducing the strength of the ubiquitously important signaling mediated by insulin, bring together most of these treatments under development for neurodegenerative disease under the one roof. Moreover, the principles involved apply to a wide range of inflammatory diseases on both sides of the blood brain barrier.
Zelikowsky, M., Bissiere, S., Hast, T.A., Bennett, R.Z., Abdipranoto, A., Vissel, B. & Fanselow, M.S. 2013, 'Prefrontal microcircuit underlies contextual learning after hippocampal loss.', Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 24, pp. 9938-9943.
Specific brain circuits have been classically linked to dedicated functions. However, compensation following brain damage suggests that these circuits are capable of dynamic adaptation. Such compensation is exemplified by Pavlovian fear conditioning following damage to the dorsal hippocampus (DH). Although the DH normally underlies contextual fear and fear renewal after extinction, both can be learned in the absence of the DH, although the mechanisms and nature of this compensation are currently unknown. Here, we report that recruitment of alternate structures, specifically the infralimbic and prelimbic prefrontal cortices, is required for compensation following damage to the hippocampus. Disconnection of these cortices in DH-compromised animals and immediate early gene induction profiles for amygdala-projecting prefrontal cells revealed that communication and dynamic rebalancing within this prefrontal microcircuit is critical. Additionally, the infralimbic cortex normally plays a role in limiting generalization of contextual fear. These discoveries reveal that plasticity through recruitment of alternate circuits allows the brain to compensate following damage, offering promise for targeted treatment of memory disorders.
Wright, A. & Vissel, B. 2012, 'The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain.', Frontiers in molecular neuroscience, vol. 5, p. 34.
-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are comprised of different combinations of GluA1-GluA4 (also known asGluR1-GluR4 and GluR-A to GluR-D) subunits. The GluA2 subunit is subject to RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Gln; Q), present in the GluA2 gene, to a codon for arginine (Arg; R) found in the mRNA. AMPA receptors are calcium (Ca(2+))-permeable if they contain the unedited GluA2(Q) subunit or if they lack the GluA2 subunit. While most AMPA receptors in the brain contain the edited GluA2(R) subunit and are therefore Ca(2+)-impermeable, recent evidence suggests that Ca(2+)-permeable AMPA receptors are important in synaptic plasticity, learning, and disease. Strong evidence supports the notion that Ca(2+)-permeable AMPA receptors are usually GluA2-lacking AMPA receptors, with little evidence to date for a significant role of unedited GluA2 in normal brain function. However, recent detailed studies suggest that Ca(2+)-permeable AMPA receptors containing unedited GluA2 do in fact occur in neurons and can contribute to excitotoxic cell loss, even where it was previously thought that there was no unedited GluA2.This review provides an update on the role of GluA2 RNA editing in the healthy and diseased brain and summarizes recent insights into the mechanisms that control this process. We suggest that further studies of the role of unedited GluA2 in normal brain function and disease are warranted, and that GluA2 editing should be considered as a possible contributing factor when Ca(2+)-permeable AMPA receptors are observed.
Clark, I., Atwood, C., Bowen, R., Paz-Filho, G. & Vissel, B. 2012, 'Tumor necrosis factor-induced cerebral insulin resistance in Alzheimer's disease links numerous treatment rationales.', Pharmacological reviews, vol. 64, no. 4, pp. 1004-1026.
The evident limitations of the amyloid theory of the pathogenesis of Alzheimer's disease are increasingly putting alternatives in the spotlight. We argue here that a number of independently developing approaches to therapy-including specific and nonspecific anti-tumor necrosis factor (TNF) agents, apolipoprotein E mimetics, leptin, intranasal insulin, the glucagon-like peptide-1 mimetics and glycogen synthase kinase-3 (GSK-3) antagonists-are all part of an interlocking chain of events. All these approaches inform us that inflammation and thence cerebral insulin resistance constitute the pathway on which to focus for a successful clinical outcome in treating this disease. The key link in this chain presently absent is a recognition by Alzheimer's research community of the long-neglected history of TNF induction of insulin resistance. When this is incorporated into the bigger picture, it becomes evident that the interventions we discuss are not competing alternatives but equally valid approaches to correcting different parts of the same pathway to Alzheimer's disease. These treatments can be expected to be at least additive, and conceivably synergistic, in effect. Thus the inflammation, insulin resistance, GSK-3, and mitochondrial dysfunction hypotheses are not opposing ideas but stages of the same fundamental, overarching, pathway of Alzheimer's disease pathogenesis. The insight this provides into progenitor cells, including those involved in adult neurogenesis, is a key part of this approach. This pathway also has therapeutic implications for other circumstances in which brain TNF is pathologically increased, such as stroke, traumatic brain injury, and the infectious disease encephalopathies.
Götz, J., Schonrock, N., Vissel, B. & Ittner, L.M. 2011, 'Alzheimer's disease selective vulnerability and modelling in transgenic mice', Advances in Alzheimer's Disease, vol. 1, pp. 49-58.
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Neurodegenerative diseases are characterized by 'hot spots' of degeneration. The regions of primary vulnerability vary between different neurodegenerative diseases. Within these regions, some neurons are lost whereas others that are morphologically indiscriminable survive. The enigma of this selective vulnerability is tightly linked to two fundamental problems in the neurosciences. Firstly, it is not understood how many neuronal cell types make up the mammalian brain; estimates are in the order of more than thousand. Secondly, the mechanisms by which some nerve cells undergo functional impairment followed by degeneration while others do not, remain elusive. Understanding the basis for this selective vulnerability has significant implications for understanding the pathogenesis of disease and for developing treatments. Here, we review what is known about selective vulnerability in Alzheimer's disease, frontotemporal dementia and Parkinson's disease. We suggest, since transgenic animal models of disease reproduce aspects of selective vulnerability, that these models offer a valuable system for future investigations into the physiological basis of selective vulnerability. &copy; 2011 The authors and IOS Press. All rights reserved.
McRoberts, J.A., Ennes, H.S., Marvizón, J.C., Fanselow, M.S., Mayer, E.A. & Vissel, B. 2011, 'Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test.', Neuroscience, vol. 172, pp. 474-482.
The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a 75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.
Clark, I.A., Alleva, L.M. & Vissel, B. 2011, 'TNF and leptin tell essentially the same story in Alzheimer's disease.', Journal of Alzheimer's disease : JAD, vol. 26, no. 2, pp. 201-205.
Both tumor necrosis factor (TNF) and leptin are, independently, under investigation as the central mechanism for Alzheimer's disease. The wider literature provides every indication that both mediators are part of the same pathways thought to cause functional loss in this condition. This association, which has not been specifically addressed in the Alzheimer's disease literature, may be a useful link to expedite future study into the pathogenesis of this condition.
Clark, I.A., Alleva, L.M. & Vissel, B. 2010, 'The roles of TNF in brain dysfunction and disease.', Pharmacology & therapeutics, vol. 128, no. 3, pp. 519-548.
Certain cytokines, the prototype being the highly pleiotropic TNF, have many homeostatic physiological roles, are involved in innate immunity, and cause inflammation when in excess. These cytokines have long been accepted to have central roles in the pathogenesis of systemic or local non-cerebral disease states, whether acute or chronic, and whether or not caused by infectious agents. Over the last decade they have also been appreciated to be broadly important in brain physiology. As in other organs, excessive levels in brain are harmful, and its physiological complexity leads to correspondingly complex dysfunction. This review summarizes the burgeoning literature on this topic, and how the functions of these molecules, particularly TNF, are influencing the outlook of researchers on the pathophysiology of these diseases. Basic brain physiology is thus informing knowledge of the brain dysfunction that characterizes such apparently diverse states as Alzheimer's disease, trauma (mostly, but not only, to the brain), Parkinson's disease, and severe systemic infectious states, including malaria, sepsis, viral diseases and major depression. The implication is that the anti-cytokine therapies now in use, typically directed at TNF, warrant testing in these diseases in circumstances in which the therapeutic agent enters the cerebrospinal fluid. Routinely administering such drugs to patients exhibiting the neurological changes discussed in this review would simply add another organ system to what is already a very successful strategy in the treatment of inflammatory disease at other sites, such as joints, skin and gut. Clearly, the most relevant research is focussed on Alzheimer's disease, but the principles may also apply to other encephalopathies.
Wiltgen, B.J., Royle, G.A., Gray, E.E., Abdipranoto, A., Thangthaeng, N., Jacobs, N., Saab, F., Tonegawa, S., Heinemann, S.F., O'Dell, T.J., Fanselow, M.S. & Vissel, B. 2010, 'A role for calcium-permeable AMPA receptors in synaptic plasticity and learning.', PloS one, vol. 5, no. 9, p. e744.
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A central concept in the field of learning and memory is that NMDARs are essential for synaptic plasticity and memory formation. Surprisingly then, multiple studies have found that behavioral experience can reduce or eliminate the contribution of these receptors to learning. The cellular mechanisms that mediate learning in the absence of NMDAR activation are currently unknown. To address this issue, we examined the contribution of Ca(2+)-permeable AMPARs to learning and plasticity in the hippocampus. Mutant mice were engineered with a conditional genetic deletion of GluR2 in the CA1 region of the hippocampus (GluR2-cKO mice). Electrophysiology experiments in these animals revealed a novel form of long-term potentiation (LTP) that was independent of NMDARs and mediated by GluR2-lacking Ca(2+)-permeable AMPARs. Behavioral analyses found that GluR2-cKO mice were impaired on multiple hippocampus-dependent learning tasks that required NMDAR activation. This suggests that AMPAR-mediated LTP interferes with NMDAR-dependent plasticity. In contrast, NMDAR-independent learning was normal in knockout mice and required the activation of Ca(2+)-permeable AMPARs. These results suggest that GluR2-lacking AMPARs play a functional and previously unidentified role in learning; they appear to mediate changes in synaptic strength that occur after plasticity has been established by NMDARs.
Galbraith, S., Daniel, J.A. & Vissel, B. 2010, 'A study of clustered data and approaches to its analysis.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 30, no. 32, pp. 10601-10608.
Statistical analysis is critical in the interpretation of experimental data across the life sciences, including neuroscience. The nature of the data collected has a critical role in determining the best statistical approach to take. One particularly prevalent type of data is referred to as "clustered data." Clustered data are characterized as data that can be classified into a number of distinct groups or "clusters" within a particular study. Clustered data arise most commonly in neuroscience when data are compiled across multiple experiments, for example in electrophysiological or optical recordings taken from synaptic terminals, with each experiment providing a distinct cluster of data. However, there are many other types of experimental design that can yield clustered data. Here, we provide a statistical model for intracluster correlation and systematically investigate a range of methods for analyzing clustered data. Our analysis reveals that it is critical to take data clustering into account and suggests appropriate statistical approaches that can be used to account for data clustering.
Daniel, J.A., Galbraith, S., Iacovitti, L., Abdipranoto, A. & Vissel, B. 2009, 'Functional heterogeneity at dopamine release sites.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 29, no. 46, pp. 14670-14680.
Although drugs used to treat several neurological diseases are presumed to target synapses that secrete dopamine (DA), relatively little is known about synaptic vesicle (SV) release mechanisms at single DA synapses. We found that the relative probability of release (Pr) varied between individual DA synapses. Furthermore, DA terminals generally exhibited lower Pr than glutamatergic hippocampal (Hpc) terminals, suggesting that DA release is less reliable than the release of glutamate. Our mathematical model of fluorescence loss shows that Pr is regulated by two independent and heterogeneous elements. First, the size of the recycling SV pool regulates Pr. Second, Pr is also independently regulated by additional factors, which are reflected in the time constant of FM 1-43 destaining, tau. We found that the observed difference in Pr between Hpc and DA neurons results because the recycling SV pool is smaller in DA neurons than in Hpc neurons. However, tau does not vary between these two neuron populations. We also identified a population of functional nonsynaptic boutons in DA axons, which are not associated with a postsynaptic element and which are not functionally different from boutons that formed conventional synapses. Our work provides a new approach to the study of SV exocytosis in DA neurons and shows that synaptic terminals of DA neurons are functionally heterogeneous and differ from excitatory terminals in terms of Pr.
Abdipranoto-Cowley, A., Park, J.S., Croucher, D., Daniel, J., Henshall, S., Galbraith, S., Mervin, K. & Vissel, B. 2009, 'Activin A is essential for neurogenesis following neurodegeneration.', Stem cells (Dayton, Ohio), vol. 27, no. 6, pp. 1330-1346.
It has long been proposed that excitotoxicity contributes to nerve cell death in neurodegenerative diseases. Activin A, a member of the transforming growth factor-beta superfamily, is expressed by neurons following excitotoxicity. We show for the first time that this activin A expression is essential for neurogenesis to proceed following neurodegeneration. We found that intraventricular infusion of activin A increased the number of newborn neurons in the dentate gyrus, CA3, and CA1 layers of the normal adult hippocampus and also, following lipopolysaccharide administration, had a potent inhibitory effect on gliosis in vivo and on microglial proliferation in vivo and in vitro. Consistent with the role of activin A in regulating central nervous system inflammation and neurogenesis, intraventricular infusion of follistatin, an activin A antagonist, profoundly impaired neurogenesis and increased the number of microglia and reactive astrocytes following onset of kainic acid-induced neurodegeneration. These results show that inhibiting endogenous activin A is permissive for a potent underlying inflammatory response to neurodegeneration. We demonstrate that the anti-inflammatory actions of activin A account for its neurogenic effects following neurodegeneration because co-administration of nonsteroidal anti-inflammatory drugs reversed follistatin's inhibitory effects on neurogenesis in vivo. Our work indicates that activin A, perhaps working in conjunction with other transforming growth factor-beta superfamily molecules, is essential for neurogenesis in the adult central nervous system following excitotoxic neurodegeneration and suggests that neurons can regulate regeneration by suppressing the inflammatory response, a finding with implications for understanding and treating acute and chronic neurodegenerative diseases.
Götz, J., Schonrock, N., Vissel, B. & Ittner, L.M. 2009, 'Alzheimer's disease selective vulnerability and modeling in transgenic mice.', Journal of Alzheimer's disease : JAD, vol. 18, no. 2, pp. 243-251.
Neurodegenerative diseases are characterized by 'hot spots' of degeneration. The regions of primary vulnerability vary between different neurodegenerative diseases. Within these regions, some neurons are lost whereas others that are morphologically indiscriminate survive. The enigma of this selective vulnerability is tightly linked to two fundamental problems in the neurosciences. First, it is not understood how many neuronal cell types make up the mammalian brain; estimates are in the order of more than a thousand. Second, the mechanisms by which some nerve cells undergo functional impairment followed by degeneration while others do not, remain elusive. Understanding the basis for this selective vulnerability has significant implications for understanding the pathogenesis of disease and for developing treatments. Here, we review what is known about selective vulnerability in Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. We suggest, since transgenic animal models of disease reproduce aspects of selective vulnerability, that these models offer a valuable system for future investigations into the physiological basis of selective vulnerability.
Abdipranoto, A., Wu, S., Stayte, S. & Vissel, B. 2008, 'The role of neurogenesis in neurodegenerative diseases and its implications for therapeutic development.', CNS & neurological disorders drug targets, vol. 7, no. 2, pp. 187-210.
Neurodegenerative diseases are characterised by a net loss of neurons from specific regions of the central nervous system (CNS). Until recently, research has focused on identifying mechanisms that lead to neurodegeneration, while therapeutic approaches have been primarily targeted to prevent neuronal loss. This has had limited success and marketed pharmaceuticals do not have dramatic benefits. Here we suggest that the future success of therapeutic strategies will depend on consideration and understanding of the role of neurogenesis in the adult CNS. We summarize evidence suggesting that neurogenesis is impaired in neurodegenerative diseases such as Parkinson's, Alzheimer's and Amyotrophic Lateral Sclerosis, while it is enhanced in stroke. We review studies where stimulation of neurogenesis is associated with restored function in animal models of these diseases, suggesting that neurogenesis is functionally important. We show that many current therapeutics, developed to block degeneration or to provide symptomatic relief, serendipitously stimulate neurogenesis or, at least, do not interfere with it. Importantly, many receptors, ion channels and ligand-gated channels implicated in neurodegeneration, such as NMDA, AMPA, GABA and nicotinic acetylcholine receptors, also play an important role in neurogenesis and regeneration. Therefore, new therapeutics targeted to block degeneration by antagonizing these channels may have limited benefit as they may also block regeneration. Our conclusion is that future drug development must consider neurogenesis. It appears unlikely that drugs being developed to treat neurodegenerative diseases will be beneficial if they impair neurogenesis. And, most tantalizing, therapeutic approaches that stimulate neurogenesis might stimulate repair and even recovery from these devastating diseases.
Youn, D.H., Royle, G., Kolaj, M., Vissel, B. & Randi, M. 2008, 'Enhanced LTP of primary afferent neurotransmission in AMPA receptor GluR2-deficient mice.', Pain, vol. 136, no. 1-2, pp. 158-167.
Ca(2+)-permeable-AMPA receptors (AMPARs) are expressed in the superficial dorsal horn (SDH, laminae I/II) of the spinal cord, the area involved in transmission and modulation of sensory information, including nociception. A possible role of Ca(2+)-permeable-AMPARs in synaptic strengthening has been suggested in postnatal DH cultures, but their role in the long-lasting activity-dependent synaptic plasticity of primary afferent neurotransmission in the adult mouse SDH has not been investigated. In the present study the role of Ca(2+)-permeable-AMPARs in the regulation of long-lasting synaptic plasticity, specifically long-term potentiation (LTP) and long-term depression (LTD) in the SDH, was investigated using mice deficient in AMPAR GluR2 subunit. We show here that the GluR2 mutants exhibited no changes in passive membrane properties, but a significant increase in rectification of excitatory postsynaptic currents, the finding suggesting increased expression of Ca(2+)-permeable-AMPARs. In the absence of GluR2, high-frequency stimulation (HFS) of small-diameter primary afferent fibers induced LTP that is enhanced and non-saturating in the SDH at both primary afferent Adelta- and/or C-fibers monosynaptic and polysynaptic pathways, whereas neuronal excitability and paired-pulse depression were normal. The LTP could be induced in the presence of the NMDA receptor antagonist d-AP5, and L-type Ca(2+) channel blockers, suggesting that Ca(2+)-permeable-AMPARs are sufficient to induce LTP in the SDH neurons of adult mouse spinal cord. In contrast, the induction of HFS-LTD is reduced in the SDH of GluR2 mutants. These results suggest an important role for AMPAR GluR2 subunit in regulating synaptic plasticity with potential relevance for long-lasting hypersensitivity in pathological states.
Hinshelwood, R.A., Huschtscha, L.I., Melki, J., Stirzaker, C., Abdipranoto, A., Vissel, B., Ravasi, T., Wells, C.A., Hume, D.A., Reddel, R.R. & Clark, S.J. 2007, 'Concordant epigenetic silencing of transforming growth factor-beta signaling pathway genes occurs early in breast carcinogenesis.', Cancer research, vol. 67, no. 24, pp. 11517-11527.
Human mammary epithelial cells (HMEC) grown under standard cell culture conditions enter a growth phase referred to as selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection or variant HMECs, may be derived from progenitor cells found in normal mammary epithelium that subsequently acquire premalignant lesions, including p16(INK4A) promoter hypermethylation. Epigenetic silencing of tumor suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. A major challenge is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs but reactivated after treatment with the demethylation agent 5-aza-2'-deoxycytidine. We found that several members of the transforming growth factor beta (TGF-beta) signaling pathway were consistently down-regulated in the post-selection HMEC populations, and this was associated with a marked decrease in Smad4 nuclear staining. Gene suppression was not associated with DNA methylation but with chromatin remodeling, involving a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation. These results show for the first time that TGF-beta2, its receptors TGF-beta R1 and TGF-beta R2, and activator thrombospondin-1 are concordantly suppressed early in breast carcinogenesis by histone modifications and indicate that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy.
Gray, E.E., Fink, A.E., Sariñana, J., Vissel, B. & O'Dell, T.J. 2007, 'Long-term potentiation in the hippocampal CA1 region does not require insertion and activation of GluR2-lacking AMPA receptors.', Journal of neurophysiology, vol. 98, no. 4, pp. 2488-2492.
Activity-dependent insertion of AMPA-type glutamate receptors is thought to underlie long-term potentiation (LTP) at Schaffer collateral fiber synapses on pyramidal cells in the hippocampal CA1 region. Although it is widely accepted that the AMPA receptors at these synapses contain glutamate receptor type 2 (GluR2) subunits, recent findings suggest that LTP in hippocampal slices obtained from 2- to 3-wk-old rodents is dependent on the transient postsynaptic insertion and activation of Ca(2+)-permeable, GluR2-lacking AMPA receptors. Here we examined whether LTP in slices prepared from adult animals exhibits similar properties. In contrast to previously reported findings, pausing synaptic stimulation for as long as 30 min post LTP induction had no effect on LTP maintenance in slices from 2- to 3-mo-old mice. LTP was also not disrupted by postinduction application of a selective blocker of GluR2-lacking AMPA receptors or the broad-spectrum glutamate receptor antagonist kynurenate. Although these results suggest that the role of GluR2-lacking AMPA receptors in LTP might be regulated during postnatal development, LTP in slices obtained from 15- to 21-day-old mice also did not require postinduction synaptic stimulation or activation of GluR2-lacking AMPA receptors. Thus the insertion and activation of GluR2-lacking AMPA receptors do not appear to be fundamental processes involved in LTP at excitatory synapses in the hippocampal CA1 region.
Skvorak, K., Vissel, B. & Homanics, G.E. 2006, 'Production of conditional point mutant knockin mice.', Genesis (New York, N.Y. : 2000), vol. 44, no. 7, pp. 345-353.
Genetically engineered mice with point mutations in endogenous genes (i.e., knockin mice) are extremely useful tools for dissecting gene function. Currently available methodologies for creating knockin mice are limited in that the introduced mutation is globally present in all cells of the animal from conception through adulthood. In this report, we describe a strategy for creating mice in which a point mutant allele replaces the wild type allele in a conditional manner, e.g., in a tissue-specific and/or temporally restricted pattern. As proof of concept, we created mice that conditionally harbor a point mutated gamma-aminobutyric acid receptor subunit. In the absence of Cre recombinase, the engineered allele produces only wild type product with no evidence of expression of the mutant. In contrast, following Cre-mediated recombination, only the point mutant product is produced. By restricting Cre expression to subpopulations of neurons of postnatal animals, we demonstrate tissue-specific regulation of the point mutant knockin. This strategy will be useful for a wide variety of studies that require precise conditional replacement of an endogenous wild type gene with a point mutant.
Thomas, C.G., Krupp, J.J., Bagley, E.E., Bauzon, R., Heinemann, S.F., Vissel, B. & Westbrook, G.L. 2006, 'Probing N-methyl-D-aspartate receptor desensitization with the substituted-cysteine accessibility method.', Molecular pharmacology, vol. 69, no. 4, pp. 1296-1303.
Several forms of macroscopic N-methyl-D-aspartate (NMDA) receptor desensitization affect the amplitude and duration of postsynaptic responses. In addition to its functional significance, desensitization provides one means to examine the conformational coupling of ligand binding to channel gating. Segments flanking the ligand binding domain in the extracellular N terminus of the NMDA receptor NR2 subunit influence the glycine-independent form of desensitization. The NR2A pre-M1 region, the linker between the glutamate binding domain and the channel pore, plays a critical role in desensitization. Thus, we used the substituted-cysteine accessibility method to scan the accessibility of residues in the pre-M1 region and the first transmembrane domain (M1) of NR2A. Cysteine mutants were expressed with NR1 in human embryonic kidney 293 cells and were assayed by whole-cell recording. With activation of the receptor by glutamate and glycine, only a single mutant, V557C, which is located at the beginning of M1, led to irreversible inhibition by the methanethiosulfonate derivative methanethiosulfonate ethyltrimethylammonium (MTSET). The NR2 ligand glutamate was insufficient on its own to induce modification of V557C by MTSET, suggesting that the change in accessibility required channel gating. The rate of MTSET modification of the homologous residue on NR1 (NR1-1a(L562C)/NR2A) was much slower than V557C. We also substituted cysteine in the V557 site of mutant subunits that exhibit either enhanced or reduced desensitization. Modification by MTSET correlated with the degree of desensitization for these subunits, suggesting that V557C is a sensitive detector of desensitization gating.
Hauser, K.F., Aldrich, J.V., Anderson, K.J., Bakalkin, G., Christie, M.J., Hall, E.D., Knapp, P.E., Scheff, S.W., Singh, I.N., Vissel, B., Woods, A.S., Yakovleva, T. & Shippenberg, T.S. 2005, 'Pathobiology of dynorphins in trauma and disease.', Frontiers in bioscience : a journal and virtual library, vol. 10, pp. 216-235.
Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.
Sonner, J.M., Vissel, B., Royle, G., Maurer, A., Gong, D., Baron, N.V., Harrison, N., Fanselow, M. & Eger, E.I. 2005, 'The effect of three inhaled anesthetics in mice harboring mutations in the GluR6 (kainate) receptor gene.', Anesthesia and analgesia, vol. 101, no. 1, pp. 143-148.
Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.
Petralia, R.S., Sans, N., Wang, Y.X., Vissel, B., Chang, K., Noben-Trauth, K., Heinemann, S.F. & Wenthold, R.J. 2004, 'Loss of GLUR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor subunit differentially affects remaining synaptic glutamate receptors in cerebellum and cochlear nuclei.', The European journal of neuroscience, vol. 19, no. 8, pp. 2017-2029.
The alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) type of ionotropic glutamate receptor is the major mediator of fast neurotransmission in the brain and spinal cord. Most AMPA receptors are impermeable to calcium because they contain the GluR2 subunit. However, some AMPA receptors lack GluR2 and pass calcium which can mediate synaptic plasticity and, in excess, neurotoxicity. Previously, we showed a decrease in the density of synaptic AMPA receptors in the hippocampus of mice lacking GluR2. In this study, using these GluR2-lacking mice, we examined other areas of the brain that differ in the amount of GluR2 normally present. Like hippocampal spines, cerebellar Purkinje spines normally express AMPA receptors with high GluR2 and showed a decrease in synaptic AMPA receptors in mutant mice. In contrast, neurons that normally express AMPA receptors with little or no GluR2, such as in the anteroventral cochlear nucleus, showed no decrease in AMPA receptors and even showed an increase in one AMPA receptor subunit. These two different patterns may relate to preadaptations to prevent calcium neurotoxicity; such mechanisms might be absent in Purkinje and hippocampal spines so that these neurons must decrease their total expression of synaptic AMPA receptors (calcium permeable in mutant mice) to prevent calcium neurotoxicity. In addition, we found that another glutamate receptor, GluRdelta2, which is abundant only in parallel fibre synapses on Purkinje cells and in the dorsal cochlear nucleus, is up-regulated at these synapses in mutant mice; this probably reflects some change in GluRdelta2 targeting to these synapses.
Sonner, J.M., Antognini, J.F., Dutton, R.C., Flood, P., Gray, A.T., Harris, R.A., Homanics, G.E., Kendig, J., Orser, B., Raines, D.E., Rampil, I.J., Trudell, J., Vissel, B. & Eger, E.I. 2004, 'Erratum: Inhaled Anesthetics and Immobility: Mechanisms, Mysteries, and Minimum Alveolar Anesthetic Concentration (Anesthesia and Analgesia (September 2003) 97 (718-740))', Anesthesia and Analgesia, vol. 98, no. 1, p. 29.
Sonner, J.M., Antognini, J.F., Dutton, R.C., Flood, P., Gray, A.T., Harris, R.A., Homanics, G.E., Kendig, J., Orser, B., Raines, D.E., Rampil, I.J., Trudell, J., Vissel, B. & Eger, E.I. 2003, 'Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration.', Anesthesia and analgesia, vol. 97, no. 3, pp. 718-740.
Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific amino acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., gamma-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and alpha(2)-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-D-aspartate, and sodium) remain credible candidates.
Sans, N., Vissel, B., Petralia, R.S., Wang, Y.X., Chang, K., Royle, G.A., Wang, C.Y., O'Gorman, S., Heinemann, S.F. & Wenthold, R.J. 2003, 'Aberrant formation of glutamate receptor complexes in hippocampal neurons of mice lacking the GluR2 AMPA receptor subunit.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 28, pp. 9367-9373.
The number and type of receptors present at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Because most neurons receive multiple and distinct synaptic inputs and contain several different subtypes of receptors stimulated by the same neurotransmitter, the assembly and trafficking of receptors in neurons is a complex process involving many levels of regulation. To investigate the mechanism that neurons use to regulate the assembly of receptor subunits, we studied a GluR2 knock-out mouse. GluR2 is a critical subunit that controls calcium permeability of AMPA receptors and is present in most native AMPA receptors. Our data indicate that in the absence of GluR2, aberrant receptor complexes composed of GluR1 and GluR3 are formed in the hippocampus, and that there is an increased number of homomeric GluR1 and GluR3 receptors. We also show that these homomeric and heteromeric receptors are less efficiently expressed at the synapse. Our results show that GluR2 plays a critical role in controlling the assembly of AMPA receptors, and that the assembly of subunits may reflect the affinity of one subunit for another or the stability of intermediates in the assembly process. Therefore, GluR1 may have a greater preference for GluR2 than it does for GluR3.
South, S.M., Kohno, T., Kaspar, B.K., Hegarty, D., Vissel, B., Drake, C.T., Ohata, M., Jenab, S., Sailer, A.W., Malkmus, S., Masuyama, T., Horner, P., Bogulavsky, J., Gage, F.H., Yaksh, T.L., Woolf, C.J., Heinemann, S.F. & Inturrisi, C.E. 2003, 'A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 12, pp. 5031-5040.
To determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, the NMDAR1 (NR1) subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein are reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, are reduced 86-88% in lamina II neurons. The spatial NR1 knock-out does not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. Our results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury.
Sekirnjak, C., Vissel, B., Bollinger, J., Faulstich, M. & du Lac, S. 2003, 'Purkinje cell synapses target physiologically unique brainstem neurons.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 15, pp. 6392-6398.
The cerebellum controls motor learning via Purkinje cell synapses onto discrete populations of neurons in the deep cerebellar nuclei and brainstem vestibular nuclei. In the circuitry that subserves the vestibulo-ocular reflex, the postsynaptic targets of Purkinje cells, termed flocculus target neurons (FTNs), are thought to be a critical site of learning. Little is known, however, about the intrinsic cellular properties of FTNs, which are sparsely distributed in the medial vestibular nucleus. To identify these neurons, we used the L7 promoter to express a tau-green fluorescent protein fusion protein selectively in Purkinje cells. Fluorescent Purkinje cell axons and terminal boutons surrounded the somata and proximal dendrites of a small subset of neurons, presumed FTNs, in the medial vestibular nucleus. Targeted intracellular recordings revealed that FTNs fired spontaneously at high rates in brain slices (mean, 47 spikes/sec) and exhibited dramatic postinhibitory rebound firing after the offset of membrane hyperpolarization. These intrinsic firing properties were exceptional among brainstem vestibular nucleus neurons but strikingly similar to neurons in the deep cerebellar nuclei, indicating a common role for intrinsic firing mechanisms in cerebellar control of diverse behaviors.
Kaspar, B.K., Vissel, B., Bengoechea, T., Crone, S., Randolph-Moore, L., Muller, R., Brandon, E.P., Schaffer, D., Verma, I.M., Lee, K.F., Heinemann, S.F. & Gage, F.H. 2002, 'Adeno-associated virus effectively mediates conditional gene modification in the brain.', Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 4, pp. 2320-2325.
The Cre/loxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates beta-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.
Vissel, B., Krupp, J.J., Heinemann, S.F. & Westbrook, G.L. 2002, 'Intracellular domains of NR2 alter calcium-dependent inactivation of N-methyl-D-aspartate receptors.', Molecular pharmacology, vol. 61, no. 3, pp. 595-605.
At central excitatory synapses, the transient elevation of intracellular calcium reduces N-methyl-D-aspartate (NMDA) receptor activity. Such 'calcium-dependent inactivation' is mediated by interactions of calcium/calmodulin and alpha-actinin with the C terminus of NMDA receptor 1 (NR1) subunit. However, inactivation is also NR2-subunit specific, because it occurs in NR2A- but not NR2C-containing receptors. We examined the molecular basis for NR2-subunit specificity using chimeric and mutated NMDA receptor subunits expressed in HEK293 cells. We report that the intracellular loop immediately distal to the pore-forming P-loop M2 (M2-3 loop), as well as a short region in the C terminus, are involved in NR2-subunit specificity. Within the M2-3 loop, substitution of residue 619 in NR2A (valine) for the corresponding NR2C residue (isoleucine) reduced inactivation without affecting calcium permeability of the channel. In contrast, a Q620E mutation in NR2A reduced the relative calcium permeability without altering inactivation. Mutation of three serine/threonine residues in the M2-3 loop also reduced inactivation, as did substitution of the intracellular C terminus of NR2A for NR2C. We speculate that the M2-3 loop of NR2 modulates calcium-dependent inactivation by interacting with the NR1 C terminus, a region known to be essential for inactivation.
Luo, J.H., Fu, Z.Y., Losi, G., Losi, G., Kim, B.G., Prybylowski, K., Vissel, B. & Vicini, S. 2002, 'Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture.', Neuropharmacology, vol. 42, no. 3, pp. 306-318.
We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.
Krupp, J.J., Vissel, B., Thomas, C.G., Heinemann, S.F. & Westbrook, G.L. 2002, 'Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors.', Neuropharmacology, vol. 42, no. 5, pp. 593-602.
Phosphatase IIb (calcineurin, CaN) can reduce N-methyl-D-aspartate (NMDA) synaptic responses by enhancing glycine-independent desensitization. We examined the action of CaN on desensitization in recombinant NMDA receptors comprised of NMDA receptor 1 (NR1) and NR2A subunits. The C-terminus of NR2A, but not NR1, was critical for modulation of desensitization by CaN. Alanine-scanning mutagenesis indicated that serines 900 and 929 in NR2A altered desensitization, as did inhibition of tyrosine phosphatases. Our data suggest that dephosphorylation-dependent regulation of the C-terminus of NR2A increases desensitization of NMDA receptors, providing an additional mechanism for modulation of synaptic signals.
Vissel, B., Krupp, J.J., Heinemann, S.F. & Westbrook, G.L. 2001, 'A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux.', Nature neuroscience, vol. 4, no. 6, pp. 587-596.
Tyrosine phosphorylation can upregulate NMDA receptor activity during pathological and physiological alterations of synaptic strength. Here we describe downregulation of recombinant NR1/2A receptors by tyrosine dephosphorylation that requires agonist binding, but is independent of ion flux. The tyrosine residues involved in this new form of NMDA receptor modulation likely form a 'ring' adjacent to the last transmembrane domain. The downregulation was due to a reduction in the number of functional channels, and was blocked by co-expressing a dominant-negative mu2-subunit of the clathrin-adaptor protein AP-2. Our results provide a mechanism by which synaptic NMDA receptors can be modulated in a use-dependent manner even when the postsynaptic membrane is not sufficiently depolarized to relieve channel block by magnesium ions.
Vissel, B., Royle, G.A., Christie, B.R., Schiffer, H.H., Ghetti, A., Tritto, T., Perez-Otano, I., Radcliffe, R.A., Seamans, J., Sejnowski, T., Wehner, J.M., Collins, A.C., O'Gorman, S. & Heinemann, S.F. 2001, 'The role of RNA editing of kainate receptors in synaptic plasticity and seizures.', Neuron, vol. 29, no. 1, pp. 217-227.
The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.
Krupp, J.J., Vissel, B., Thomas, C.G., Heinemann, S.F. & Westbrook, G.L. 1999, 'Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 19, no. 4, pp. 1165-1178.
Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and alpha-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of alpha-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of alpha-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or alpha-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from alpha-actinin by two distinct but converging calcium-dependent processes: competitive displacement of alpha-actinin by calmodulin and reduction in the affinity of alpha-actinin for C0 after binding of calcium to alpha-actinin.
Krupp, J.J., Vissel, B., Heinemann, S.F. & Westbrook, G.L. 1998, 'N-terminal domains in the NR2 subunit control desensitization of NMDA receptors.', Neuron, vol. 20, no. 2, pp. 317-327.
Recent molecular studies of glutamate channels have provided increasingly detailed models of the agonist-binding site and of the channel pore. However, little information is available on the domains involved in channel gating. We examined the molecular determinants for the NR2-subunit specificity of glycine-independent desensitization of NMDA channels using NR2C/NR2A chimeric subunits expressed in HEK 293 cells. We show that glycine-independent desensitization is controlled by N-terminal domains of the NR2 subunit that flank the putative agonist-binding domain: a four amino acid (aa) segment immediately preceding the first transmembrane domain (M1) and a region containing the leucine/isoleucine/valine-binding protein-like (LIVBP-like) domain. Our results provide evidence for a functional role of the region containing the LIVBP-like domain in glutamate receptor channels. We suggest that the pre-M1 segment, presumably situated near the entrance to the pore, serves as a dynamic link between ligand binding and channel gating.
Westbrook, G.L., Krupp, J.J. & Vissel, B. 1997, 'Cytoskeletal interactions with glutamate receptors at central synapses', Journal of General Physiology, no. 50TH ANN. SYMP., pp. 163-175.
The data presented here are clearly just the beginning of any comprehensive understanding of the set of regulatory and cytoskeletal proteins that interact with membrane receptors in the postsynaptic density. They do, however, indicate that both glutamate channels at central excitatory synapses are involved in complex protein-protein interactions. For example, while NR2A is important for Ca-dependent in activation of NMDA receptors, studies in several systems suggest that the other major NR2 subunit in hippocampal neurons, NR2B, predominates at critical times during synapse formation. In addition, the COOH terminus of NR2B binds to several novel cytoskeletal proteins. These results provide circumstantial evidence that NR2B may play specific roles in function and localization of receptors at excitatory synapses. The possible role of NR2B in early synaptic function gains additional support from functional data suggesting that NMDA receptors have specific roles during development (Komuro and Rakic, 1993; Rabacchi et al., 1992: Yen et al., 1993). The essential role of NR1 and NR2B in development is graphically demonstrated by the neonatal death of transgenic mice lacking either of these two subunits (Forrest et al., 1994: Kutsuwada et al., 1996) whereas NR2A and NR2C-deficient mice are less severely affected (Sakimura et al., 1995: Ebralidze et al., 1996).
Westbrook, G.L., Krupp, J.J. & Vissel, B. 1997, 'Cytoskeletal interactions with glutamate receptors at central synapses.', Society of General Physiologists series, vol. 52, pp. 163-175.
The data presented here are clearly just the beginning of any comprehensive understanding of the set of regulatory and cytoskeletal proteins that interact with membrane receptors in the postsynaptic density. They do, however, indicate that both glutamate channels at central excitatory synapses are involved in complex protein-protein interactions. For example, while NR2A is important for Ca-dependent inactivation of NMDA receptors, studies in several systems suggest that the other major NR2 subunit in hippocampal neurons, NR2B, predominates at critical times during synapse formation. In addition, the COOH terminus of NR2B binds to several novel cytoskeletal proteins. These results provide circumstantial evidence that NR2B may play specific roles in function and localization of receptors at excitatory synapses. The possible role of NR2B in early synaptic function gains additional support from functional data suggesting that NMDA receptors have specific roles during development (Komuro and Rakic, 1993; Rabacchi et al., 1992; Yen et al., 1993). The essential role of NR1 and NR2B in development is graphically demonstrated by the neonatal death of transgenic mice lacking either of these two subunits (Forrest et al., 1994; Kutsuwada et al., 1996) whereas NR2A and NR2C-deficient mice are less severely affected (Sakimura et al., 1995; Ebralidze et al., 1996).
Krupp, J.J., Vissel, B., Heinemann, S.F. & Westbrook, G.L. 1996, 'Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific.', Molecular pharmacology, vol. 50, no. 6, pp. 1680-1688.
Intracellular Ca2+ can reversibly reduce the activity of native N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons, a phenomenon termed Ca2+-dependent inactivation. We examined inactivation in heteromeric NMDA receptors expressed in human embryonic kidney (HEK) 293 cells using whole-cell recording. NR1-1a/2A heteromers showed reversible inactivation that was very similar to native NMDA receptors in cultured hippocampal neurons. Inactivation was dependent on the extracellular Ca2+ concentration and the degree of intracellular Ca2+ buffering. In 2 mM extracellular Ca2+, inactivation resulted in a 46.1 +/- 12.6% reduction in the whole-cell current during a 5-sec agonist application. Inactivation of NR1-1a/2A heteromers was unaffected by calcineurin inhibitors, staurosporine, or phalloidin. NR1-1a/2D heteromers also showed a similar degree of inactivation. In contrast, NR1-1a/2B and NR1-1a/2C heteromers showed no significant inactivation. At saturating concentrations of NMDA (1 mM), NR1-1a/2A heteromers also showed Ca- and glycine-independent desensitization, as seen in native hippocampal neurons. Ca(2+)- and glycine-independent desensitization was less pronounced in NR1-1a/2B heteromers and absent in NR1-1a/2C heteromers. Activation of NR1-1a/2C heteromers triggered intracellular Ca2+ transients similar to NR1-1a/2A heteromers as verified by combined Ca2+ imaging and whole-cell recording. Thus differences in Ca2+ permeability were not responsible for the lack of inactivation in NR1-1a/2C heteromers. Our results show that inactivation of recombinant NMDA receptors requires either the NR2A or NR2D subunit, whereas both inactivation and desensitization were absent in NR2C-containing receptors. The gating of inactivating NMDA receptors is more likely to be influenced by ongoing NMDA receptor activity and Ca2+ transients, perhaps consistent with the prominent expression of NR2A in hippocampus and cerebral cortex.
Kalitsis, P., Earle, E., Vissel, B., Shaffer, L.G. & Choo, K.H. 1993, 'A chromosome 13-specific human satellite I DNA subfamily with minor presence on chromosome 21: further studies on Robertsonian translocations.', Genomics, vol. 16, no. 1, pp. 104-112.
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We describe a new human satellite I DNA subfamily (pTRI-6) which is composed of 72 copies of monomeric repeating units of 42 basepairs (bp). These repeating units are tandemly organized into a higher order structure of 2.97 kilobases (kb). Sequencing of this DNA revealed base substitutions, deletions and insertions, and an overall conservation of 85% among the monomers. The sequence has a high AT content of 77%. Under low-stringency in situ hybridization conditions, satellite I is found on the pericentric regions of chromosomes 3 and 4 and on all the acrocentric chromosomes. On the acrocentric chromosomes, satellite I is further detected on the distal p13 region. Analysis of somatic cell hybrids under high stringency indicates the presence of the pTRI-6 subfamily predominantly on chromosome 13. Chromosome 21 shows a 50- to 100-fold reduced amount of this subfamily and the presence of other sequences closely related to pTRI-6. Investigation of a group of 11 human t(14q21q) Robertsonian translocations revealed the retention of satellite I DNA around the breakpoints in all cases. These results extend our understanding of these translocations and of the general structural organization of the cen-pter regions of the acrocentric chromosomes.
Trowell, H.E., Nagy, A., Vissel, B. & Choo, K.H. 1993, 'Long-range analyses of the centromeric regions of human chromosomes 13, 14 and 21: identification of a narrow domain containing two key centromeric DNA elements.', Human molecular genetics, vol. 2, no. 10, pp. 1639-1649.
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Alpha-satellite, satellite 3 and satellite 1 DNA have been proposed as candidate components of a functional human centromere. Multiple subfamilies of these three DNA have recently been identified at the pericentric regions of the human acrocentric chromosomes. Using pulsed field gel electrophoresis, we have constructed long-range maps of the various centromeric markers for chromosomes 13, 14 and 21. These maps cover approximately 2.3 megabases of sequence for each chromosome, and the results demonstrate that within this centromeric region, chromosomes 13 and 21 have a similar organization that is partially shared by chromosome 14. A discrete satellite 3 domain was identified on each chromosome within the boundaries of the alpha-satellite DNA. No satellite 1 was detected within the defined centromeric regions suggesting that satellite 1 is not essential for centromere function.
Vissel, B. & Choo, K.H. 1992, 'Evolutionary relationships of multiple alpha satellite subfamilies in the centromeres of human chromosomes 13, 14, and 21.', Journal of molecular evolution, vol. 35, no. 2, pp. 137-146.
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Using Southern and in situ hybridization analyses, we have earlier defined four different subfamilies of alpha satellite DNA (designated pTRA-1, -2, -4, and -7), each of which has a unique higher order structure represented almost identically on human chromosomes 13, 14, and 21. Here we present the complete sequence of single isolates of these four subfamilies, representing approximately 12 kb of sequence information. Sequences of the individual 171-bp monomers that constitute these four subfamilies (and a fifth subfamily, Alpha-R1, that is known to be present on chromosomes 13 and 21) were compared both within and between the different clones. The results indicate that, at the level of their primary sequence, the five alpha subfamilies are characterized by structures that are as unrelated to each other as the different alpha subfamilies from other chromosomes. However, sequence comparisons between monomers of these clones indicate the possibility that pTRA-2, -4, and -1 may have arisen, at least in part, from a common ancestral alphoid sequence. We also provide evidence that exchange of pTRA-1 between nonhomologous centromeres and its homogenization throughout the population, perhaps by unequal exchange mechanisms, could have occurred after the divergence of humans and chimpanzees. The evolution of multiple alphoid subfamilies within a single centromere suggests that unequal exchange mechanisms may be restricted to specific domains. This may in turn contribute to some requirement for subregional pairing of sequences along the length of the centromeres of these chromosomes.
Vissel, B., Nagy, A. & Choo, K.H. 1992, 'A satellite III sequence shared by human chromosomes 13, 14, and 21 that is contiguous with alpha satellite DNA.', Cytogenetics and cell genetics, vol. 61, no. 2, pp. 81-86.
We report the isolation of a clone (pTR9) from a human chromosome 21 lambda phage library, which was found to contain two distinct components: (1) a previously unreported subfamily of human satellite III (pTR9-s3; 1,485 bp) and (2) an alpha satellite sequence (pTR9-alpha; 250 bp) containing 1.5 copies of a 171-bp alphoid unit that shows 88.4% homology to a previously reported alpha satellite consensus sequence. The two components are separated by two direct repeats of 9 bp. Use of the polymerase chain reaction (PCR) to amplify across the junction between pTR9-s3 and pTR9-alpha established that these two sequences are contiguous in total human genomic DNA and in DNA derived from somatic cell hybrids carrying human chromosomes 13, 14, or 21. A related, but considerably more diverged, sequence was also detected on chromosome 15. Southern analysis of somatic cell hybrids at high stringency revealed a common structure of the pTR9-s3 sequence on chromosomes 13, 14, and 21 but not on 15 or 22. This sequence should be useful for the study of the structural organisation of the centromere of these chromosomes and the mechanism of their involvement in Robertsonian translocations.
Choo, K.H., Earle, E., Vissel, B. & Kalitsis, P. 1992, 'A chromosome 14-specific human satellite III DNA subfamily that shows variable presence on different chromosomes 14.', American journal of human genetics, vol. 50, no. 4, pp. 706-716.
We describe a new subfamily of satellite III DNA (pTRS-63), which, by a combination of in situ hybridization to human metaphase chromosomes and analysis of a panel of somatic cell hybrids, is shown to be specific for human chromosome 14. This DNA has a basic 5-bp repeating unit of diverged GGAAT which is tandemly repeated and organized into either one of two distinct higher-order structures of 5 kb (designated the "L" form) or 4.8 kb (designated the "S" form). In addition, a third (Z) form, representing no detectable levels of this satellite III subfamily, is found. Results from five somatic cell hybrid lines and from a number of informative human individuals suggest that, on any one chromosome 14, only one of the three forms may exist. Subchromosomally, this sequence has been mapped to the p11 region and is distal to the domain occupied by another previously described satellite III subfamily (pTRS-47) found on chromosome 14. The pTRS-63 sequence described adds to the understanding of the structural organization of the short arm of human chromosome 14 and should be useful for the investigation of the molecular etiology of the frequently occurring t(13q14q) and t(14q21q) Robertsonian translocations.
Vissel, B. & Choo, K.H. 1991, 'Four distinct alpha satellite subfamilies shared by human chromosomes 13, 14 and 21.', Nucleic acids research, vol. 19, no. 2, pp. 271-277.
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We describe the characterisation of four alpha satellite sequences which are found on a subset of the human acrocentric chromosomes. Direct sequence study, and analysis of somatic cell hybrids carrying specific human chromosomes indicate a unique 'higher-order structure' for each of the four sequences, suggesting that they belong to different subfamilies of alpha DNA. Under very high stringency of Southern hybridisation conditions, all four subfamilies were detected on chromosomes 13, 14 and 21, with 13 and 21 showing a slightly greater sequence homology in comparison to chromosome 14. None of these subfamilies were detected on chromosomes 15 and 22. In addition, we report preliminary evidence for a new alphoid subfamily that is specific for human chromosome 14. These results, together with those of earlier published work, indicate that the centromeres of the five acrocentric chromosomes are characterised by a number of clearly defined alphoid subfamilies or microdomains (with at least 5, 7, 3, 5 and 2 different ones on chromosomes 13, 14, 15, 21 and 22, respectively). These microdomains must impose a relatively stringent subregional pairing of the centromeres of two homologous chromosomes. The different alphoid subfamilies reported should serve as useful markers to allow further 'dissection' of the structure of the human centromere as well as the investigation of how the different nonhomologous chromosomes may interact in the aetiology of aberrations involving these chromosomes.
Choo, K.H., Vissel, B., Nagy, A., Earle, E. & Kalitsis, P. 1991, 'A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence.', Nucleic acids research, vol. 19, no. 6, pp. 1179-1182.
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Choo, K.H., Vissel, B., Nagy, A., Earle, E. & Kalitsis, P. 1991, 'A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence', Nucleic Acids Research, vol. 19, no. 8, p. 1979.
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It should be noted that the accession number accompanying the above article has been deleted by EMBL Data Library. However, the sequence data reported is available on the EMBL netserver under the reference DS6O53. &copy; 1991 Oxford University Press.
Choo, K.H., Earle, E., Vissel, B. & Filby, R.G. 1990, 'Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15.', Genomics, vol. 7, no. 2, pp. 143-151.
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We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.
Choo, K.H., Vissel, B. & Earle, E. 1989, 'Evolution of alpha-satellite DNA on human acrocentric chromosomes.', Genomics, vol. 5, no. 2, pp. 332-344.
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In situ hybridization of five new and one previously described alpha-satellite sequences isolated from chromosome 21 libraries gave the following chromosomal distribution patterns: (a) two sequences (pTRA-1 and -4) hybridizing to chromosomes 13, 14, 15, 21, and 22 (also 19 and 20); (b) one sequence (pTRA-7) hybridizing to chromosome 14; and (c) three sequences (pTRA-2, -11 and -15) hybridizing to chromosomes 13, 14, and 21, with significant but weaker signals on 15 and 22. These results suggested the sharing of alphoid domains between different acrocentric chromosomes and the coexistence of multiple domains on each chromosome. Analysis of somatic cell hybrids carrying a single human acrocentric chromosome using pTRA-2 demonstrated a higher-order repeating structure common to chromosomes 13, 14, and 21, but not to 15 and 22, providing direct evidence for sequence homogenization in this domain among the former three chromosomes. We present a model of evolution and genetic exchange of alpha sequences on the acrocentric chromosomes which can satisfactorily explain these and previous observations of (a) two different alphoid subfamilies, one common to chromosomes 13 and 21 and the other common to chromosomes 14 and 22, (b) a different alphoid subfamily on chromosome 22, and (c) nonrandom participation of chromosomes 13 and 14, and 14 and 21 in Robertsonian translocations.
Vissel, B. & Choo, K.H. 1989, 'Mouse major (gamma) satellite DNA is highly conserved and organized into extremely long tandem arrays: implications for recombination between nonhomologous chromosomes.', Genomics, vol. 5, no. 3, pp. 407-414.
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We have isolated and sequenced 30 independent clones derived from MnlI digestion of purified mouse major (gamma) satellite DNA. These clones contained between 0.9 and 1.1 gamma monomeric units derived presumably from random chromosomal sources. Individual clones showed a mean deviation from the mouse consensus satellite sequence of 3.9%, with a range of 0.9-9.1%. Cleavage of total mouse LTK cell genomic DNA with three different restriction enzymes (HindIII, BglII, BamHI) that do not cut within satellite monomers, followed by Southern and pulsed-field gel electrophoretic analyses, showed that the majority of monomers were organized into largely uninterrupted arrays that varied from a minimum of 240 kb to greater than 2000 kb in length. We suggest that the high degree of conservation of the mouse gamma-satellite sequences throughout the mouse genome results from frequent recombinational exchange between nonhomologous chromosomes. Further, this same process, facilitated by the all-acrocentric constitution of the typical mouse karyotype, and the extremely long and homologous gamma-satellite arrays, may be related to the common occurrence of Robertsonian translocation in mouse.
Choo, K.H., Vissel, B., Brown, R., Filby, R.G. & Earle, E. 1988, 'Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.', Nucleic acids research, vol. 16, no. 4, pp. 1273-1284.
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We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.
Vissel, B. & Choo, K.H. 1988, 'Altered activity of restriction endonuclease Mn1-I cleavage of mouse satellite DNA.', Nucleic acids research, vol. 16, no. 10, p. 4731.
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Vissel, B. & Choo, K.H. 1987, 'Human alpha satellite DNA--consensus sequence and conserved regions.', Nucleic acids research, vol. 15, no. 16, pp. 6751-6752.
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