1985 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Anticonvulsant Mechanisms of Action @ University of Michigan At Ann Arbor
Murine spinal cord and cortical neurons in primary dissociated cell culture and rat hippocampal pyramidal neurons in brain slice will be used to investigate the mechanisms of action of the anticonvulsant drugs phenytoin (Dilantin) and carbamazepine (Tegretol). Intracellular recording techniques will be used. We will examine the hypotheses that: 1) phenytoin and carbamazepine have anticonvulsant action by limiting sustained, high frequency repetitive firing and 2) that anticonvulsant limitatio fo repetitive firing is due to use (frequency) dependent sodium channel block. We will investigate carbamazepine actions on spinal cord neurons in cell culture. We will determine if carbamazepine alters: 1) spontaneous activity and convulsant-induced paroxysmal bursting, 2) resting membrane potential or conductance, 3) calcium-dependent action potentials and 4) postsynaptic amino acid responses. We will determine if phenytoin and carbamazepine limit sustained repetitive firing of cortical neurons in cell culture and hippocampal pyramidal neurons in brain slice. We will determine the mechanism of phenytoin- and carbamazepine-induced limitation of sustained repetitive firing in spinal cord neurons in cell culture.
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0.948 |
1986 — 1988 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Opioid Peptides--Receptor-Ion Channel Coupling @ University of Michigan At Ann Arbor
Murine dorsal root ganglion (DRG) neurons grown in primary dissociated cell culture have mu-, delta- and kappa- opioid receptors on their somatic surfaces. Mu- and delta- opioid receptors are coupled to a voltage and/or calcium-dependent potassium conductance while kappa- opioid receptors are coupled to a voltage-dependent calcium channel. In addition, opioid receptors are coupled to adenylate cyclase. Binding of opioids to opioid receptors results in the activation of the inhibitory GTP-binding regulatory protein, GI, resulting in an inhibition of adenylate cyclase and thus to a reduction of intracellular cyclic AMP concentration. Furthermore, binding of opioids to their receptors is regulated by guanine nucleotides. In the present application, we propose to investigate the coupling of opioid receptors to their ion channels. First, we propose to characterize the specific potassium channel which is enhanced by mu- and delta- opioid receptor activation. Second, we intend to characterize the calcium channel which is inhibited by kappa-opioid receptor activation. Third, we intend to determine whether or not the coupling between mu-, delta- and kappa- opioid receptors is via a second messenger system, specifically the adenylate cyclase/cyclic AMP system. We will use intracellular recording, voltage clamp and patch clamp techniques to characterize whole cell currents and single channel currents in this project.
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0.948 |
1989 — 1992 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Opioid Peptides-Receptor Ion Channel Coupling @ University of Michigan At Ann Arbor
Murine primary afferent neurons have mu- and delta- and kappa-opioid receptors on their somatic surfaces. mu- and delta-opioid receptors are coupled to a voltage- and/or calcium-dependent potassium channels while kappa-opioid receptors are coupled to a voltage-dependent calcium channel. The identity of the potassium channels coupled to opioid receptors on prima y afferent neurons is uncertain,m but the kappa-opioid receptor is coupled to the N-type calcium channel. Opioid receptors have been shown to be coupled to the inhibitory GTP-binding regulatory protein, Gi, resulting in inhibiti n of adenylate cyclase, and thus, to a reduction of intracellular cAMP and protein kinase A activity. The N calcium current has been demonstrated to be reduced by application of forskolin and phorbol esters, compounds which lead to increased activity of protein kinase A and protein kinase C. These results suggest that kappa-opioid receptors are coupled in calcium current and that N calcium channels are subject to regulation by several forms of protein kinases. In the present application, we will investigate the coupling of opioid receptors to their ion channels. First, we propose to determine whether or not coupling of mu-, delta- and kappa-opioid receptors to their ion channel is via a pertussis toxin sensitive G protein of the Go or G1 type. Second, we will determine whether or not nonhydrolyzable GTP analogs result in reduction of N calcium current, presumably by irreversibly releasing a G protein alpha-subunit. Third, we will determine whether activated alpha o, alpha i1 or alpha i2 G protein alpha-subunits can directly reduce N calcium current. Four, we will determine whether or not the catalytic subunit of protein kinase A will reduce the N calcium current, and if so, which of the two types of catalytic subunit is effective. Five, we will determine wheth r protein kinase C regulates the N calcium current, and if so, which of the various channels. Seven, we will determine whether or not the gating properties of N calcium channels are influenced by dynorphin A, GTP analogs G protein alpha-subunits, protein kinase A and protein kinase C. These experiments will lead, in general, to a better understanding of the coupling of G protein coupled receptors to ion channels and will elucidate the regulatory roles of receptor phosphorylation produced by protein kinase A and protein kinase C. It will lead also to a better understanding of the regulatory functions of G proteins and of protein kinase A and C.
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0.948 |
1993 |
Macdonald, Robert Louden |
R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Opioid,Benzodiazepine,Barbiturate Regulation Ca++,Gaba-A @ University of Michigan At Ann Arbor
This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.
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0.948 |
1994 — 1997 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Opioid Regulation of Calcium Channels @ University of Michigan At Ann Arbor
Opioids have been used to relieve pain for centuries. However, the abuse of opioids is a major health care problem in the world. The bases for the behavioral effects of these drugs of abuse will be explained by studies of their effects on brain systems, and then ultimately, on specific ion or neurotransmitter receptor channels. It is the long term goal of the proposed project to focus on the latter aspect, to determine the action of opioids on ion and neurotransmitter receptor channels. On primary afferent neurons, kappa and mu opioid receptors are coupled to specific voltage-dependent calcium channels which likely regulate release of neurotransmitter from primary afferent terminals. We are interested in the coupling of kappa and mu opioid receptors to specific calcium channels and the regulation of those calcium channels by phosphorylation. Our specific aims are to determine: 1) which pertussis toxin sensitive G protein couples kappa receptors to calcium channels using specific antibodies directed at the G protein alpha subunits, alphao, alphai1, alphai2 and alphai3, to block the coupling; 2) the specific high threshold calcium channel coupled to mu and kappa receptors using a combination of specific calcium channel blockers and voltage clamp protocols; 3) which high threshold calcium currents are regulated by phosphorylation by protein kinase A (PKA) or protein kinase C (PKC); and 4) whether phosphorylation by PKA and PKC regulates the coupling between opioid receptors and calcium channels. The hypotheses to be tested are that mu and kappa receptors couple to a specific high threshold transient calcium channel by a specific G protein and that phosphorylation of the calcium channel increases the number of channels available for regulation and enhances the opioid receptor reduction of calcium current.
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0.948 |
1995 — 1998 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Allosteric Regulation--Recombinant/Native Gabaa Receptor @ University of Michigan At Ann Arbor
DESCRIPTION: (adapted from Applicant's Abstract) The specific aims are to determine: 1) the biophysical properties of native GABA receptors (GABARs) from identified CNS neurons. Single channel properties will be characterized and a kinetic gating scheme developed for GABARs from each neuronal type; 2) the pharmacological regulation of native GABARs from identified CNS neurons; 3) the functional expression of recombinant GABAR subtypes into GABAR isoforms; 4) the biophysical properties of recombinant GABAR isoforms assembled from GABAR subtypes which have been shown to be coassembled in hippocampal dentate granule cells, pyramidal neurons, cerebellar granule cells, and Purkinje cells; 5) the pharmacological regulation of these.
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0.948 |
1996 — 1999 |
Macdonald, Robert Loughlin |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Endothelin and Oxidant Stress in Cerebral Vasospasm
Cerebral vasospasm is delayed onset of vasoconstriction occurring days after aneurysmal subarachnoid hemorrhage. It is a major cause of stroke and death after subarachnoid hemorrhage. Hemoglobin and oxidant stress induced by it may be important in the pathogenesis of vasospasm. We found that heme oxygenase-1 messenger ribonucleic acid (mRNA) and protein are increased in smooth muscle cells exposed to hemoglobin. Heme oxygenase-1 protein is also increased in vasospastic arteries. Heme oxygenase-1 expression is known to be induced by oxidative stress and by heme in other types of cells, where it may protect against these cytotoxic stimuli. The long-term objective of this work is to determine the role of heme oxygenase-1 in vasospasm. The specific aims are: (1) To define the time course and dose-dependence of induction of heme oxygenase-1 and ferritin mRNA and protein in response to hemolysate and purified hemoglobin in cultured cerebrovascular smooth muscle cells. Hypothesis: Heme oxygenase-1 and ferritin are induced by solutions that contain heme, such as hemolysate and purified hemoglobin. (2) To define the time course of induction of heme oxygenase- 1 and ferritin mRNA and protein in the rat basilar artery after subarachnoid hemorrhage. Hypothesis: Vasospasm is associated with hemolysis, exposure of smooth muscle cells to heme, and induction of heme oxygenase-1 and ferritin. The induction of heme oxygenase-1 and ferritin is associated with the resolution of vasospasm. (3) To determine the effects of induction of heme oxygenase-1 and ferritin on vasospasm. Hypothesis: Induction of heme oxygenase-1 and ferritin decrease vasospasm.
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0.948 |
1999 — 2002 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Recombinant/Native Gabaa Receptors @ University of Michigan At Ann Arbor
DESCRIPTION (from applicant's abstract) GABA is the major inhibitory neurotransmitter in the brain. Fast inhibitory post-synaptic potentials are mediated by GABAA receptors (GABARs), which contain binding sites for many clinically relevant drugs such as benzodiazepines, barbiturates, and general anesthetics. GABAR currents are also modulated by neurosteroids and lanthanum and antagonized by penicillin, picrotoxin, bicuculline, furosemide, and zinc. The GABAR is a hetero-oligomeric protein complex composed of five subunits which together form a transmembrane chloride ion channel. Four different subunit families (alpha, beta, gamma, delta) have been studied extensively and two new subunit families pi and epsilon have been identified recently. Each subunit family is composed of one or more subtypes. Six alpha (alpha1-alpha6), three beta (beta1-beta3), three gamma(gamma1-gamma3), and delta(delta1), one e (e1) and one pi(pi1) subunit subtypes have been identified. Pharmacological studies of recombinant receptors have shown that individual subtypes confer different sensitivities to GABAR modulators such as benzodiazepines, barbiturates, propofol, loreclezole, alcohol, furosemide, zinc, other divalent cations and lanthanum. The hypotheses to be tested are the following: 1) GABAR subunit subtypes contain binding and modulatory sites that are subtype specific. 2) Allosteric modulators bind to N-terminal, extracellular portions of M2 or M2-M2 extracellular domains. 3) Binding of allosteric modulators bind to a restricted number of amino acid residues on these extracellular domains. 4) The kinetic properties of GABARs, including gating and desensitization, are subunit subtype specific. 5) Specific functional domains are present in the transmembrane portion of GABAR subunit subtypes that determine their kinetic properties. The specific aims are to determine: 1) Binding site(s) on GABAR beta and/or alpha subtypes for zinc and other divalent cations. 2) Modulatory sites on alpha, gamma, delta, and epsilon subtypes that regulate sensitivity to zinc and other divalent cations. 3) Binding sites on GABAR subtypes for lanthanum enhancement and inhibitions of GABAR current. 4) Binding sites on GABAR subtypes for furosemide inhibition of GABAR current. 5) Binding sites on GABAR subtypes for barbiturate enhancement of GABAR current and direct activation of current. 6) Biophysical properties of recombinant GABAR isoforms assembled from GABAR subtypes that are expressed in hippocampal dentate granule cells. 7) Structural bases for the biophysical properties of recombinant GABAR isoforms that are assembled from GABAR subtypes expressed in hippocampal dentate granule cells.
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1 |
2000 — 2005 |
Macdonald, Robert Loughlin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathophysiology of Chronic Cerebral Vasospasm
[unreadable] DESCRIPTION (provided by applicant): We are investigating cerebral vasospasm which is an important cause of cerebral ischemia after subarachnoid hemorrhage (SAH). The long-term objective of this grant is to determine the mechanism of vasospasm after SAH and to thereby develop treatments that will prevent and/or reverse it. We have shown that hemoglobin causes vasospasm and that vasospasm is associated with impaired arterial relaxation. One mechanism of hemoglobin-induced vasospasm may be the binding and removal of nitric oxide (NO). We have used electron paramagnetic resonance (EPR) spectroscopy to detect nitrosyl hemoglobin in the subarachnoid space after SAH, proving that this mechanism occurs. We will therefore test the hypothesis that there is an NO-reversible component of vasospasm by: 1) defining the extent to which vasospasm is reversible with NO donors in a monkey model of SAH; 2) measuring heme-NO adducts (nitrosyl hemoglobin) by EPR spectroscopy in clots removed from the subarachnoid space of monkeys at different times after SAH; 3) quantifying NO in the perivascular space at different times after SAH in monkeys; and 4) defining the time course of changes in and the immunohistochemical locations of the 3 isoforms of NOS in cerebral arteries and perivascular blood clot after SAH in monkeys. Second, because vasospasm does not seem to be completely preventable by NO donors, we will investigate mechanisms of NO-independent vasospasm by: 1) measuring protein kinase G messenger ribonucleic acid, protein and activity during the time course of vasospasm in monkeys; and 2) assessing calcium sensitivity of monkey cerebral arteries during the time course of vasospasm. In a rat model, we will assess the contribution of other downstream effectors of NO-induced relaxation by: 1) assessing potassium channel function during vasospasm (calcium-activated potassium channel density, single channel conductance, and open probability will be assessed using whole cell and single channel patch clamp recordings of isolated vasospastic rat cerebrovascular smooth muscle cells); and 2) measuring whole cell calcium currents during vasospasm in rats because assessment of potassium channel function requires knowledge of intracellular calcium and because smooth muscle calcium homeostasis may be altered during vasospasm after SAH.
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0.948 |
2000 — 2003 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gabaa Receptor Channel Alteration by Prolonged Seizures
DESCRIPTION: (Verbatim from the Applicant's Abstract) While most partial and generalized seizures are relatively brief in duration, during some seizures, early termination fails and a prolonged epileptic state occurs that has been termed status epilepticus. Status epilepticus is relatively common, has a high morbidity and mortality and is a medical emergency requiring immediate treatment. Spontaneous seizure termination may involve activation of gamma-aminobutyric acid (GABA) receptor (GABAR)-mediated inhibition. If the GABAergic inhibition fails to terminate the seizure, a progressive reduction of GABAR-mediated inhibition develops that, when severe enough, results in a prolonged seizure. Status epilepticus in humans is treated acutely with benzodiazepines as well as barbiturates, which enhance GABAR-mediated inhibition. However, benzodiazepines are often efficacious early but not late in status epilepticus. We have shown that properties of dentate granule cell GABAR are altered during prolonged seizures in rats, with a reduction in benzodiazepine and zinc sensitivity without a change in GABA or pentobarbital sensitivity. These observations suggest that GABAR function changes during prolonged seizures, extending seizure duration and producing refractoriness to benzodiazepine treatment. Development of an understanding of this seizure-induced receptor plasticity would enhance understanding of spontaneous seizure termination and permit development of new treatment strategies for status epilepticus. The hypothesis to be tested is that during prolonged seizures, hippocampal dentate granule cell GABAR pharmacological and biophysical properties change due either to a change in receptor subtype composition or to receptor phosphorylation. The specific aims are to determine the : 1) dependence on seizure duration of development of insensitivity to benzodiazepines, 2) time course of development of decreased sensitivity of granule cell GABAR currents to diazepam and zinc, 3) sensitivity of granule cell GABAR currents GABAR modulators following prolonged seizures, 4) transient and steady state kinetic properties of granule cell GABAR single channel currents following prolonged seizures, 5) rate of recovery of regulation by diazepam and zinc of granule cell GABAR current following prolonged seizures, 6) rate of recovery of regulation by allosteric regulators of granule cell GABAR current following prolonged seizures, 7) dependence on PKA of recovery of granule cell GABAR current following prolonged seizures, 8) dependence on other kinases of recovery of granule cell GABAR current following prolonged seizures and 9) regulation of benzodiazepine, zinc and other allosteric regulator sensitivity of GABARs by phosphorylation.
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1 |
2000 — 2003 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pka &Pkc Regulation of Nt2-N &Recombinant Gabars
GABA is the most important CNS inhibitory neurotransmitter. GABAA receptors (GABARs) are composed of subtypes from six families (alphal-6, beta1-3, gamma1-3, delta, epsilon, pi). Cytoplasmic loops between the 3rd and 4th transmembrane domains of several subtypes contain phosphorylation consensus sequences. Protein kinase A (PKA) and C (PKC) variably modified GABAR current in different neuronal and recombinant preparations. Effects of GABAR phosphorylation in neurons are uncertain since multiple undetermined GABAR isoforms are present in neurons, and biochemical studies of specific cell type GABARs has not been possible since pure populations of specific neurons cannot be obtained. To overcome these problems, we propose to use human stem (NT2) and neuronal (NT2-N) cell lines and recombinant GABARs expressed in mammalian cells. The hypotheses to be tested are: 1) Activation of PKA or PKC enhances NT2-N neuronal and NT2 precursor cell GABAR currents. 2) Phosphorylation of a single beta subunit subtype serine (S409 in the beta1, S410 in the beta2 and S408 in the beta3 subtype) by PKA or PKC enhances recombinant alphax-betay-gamma3 GABAR currents (x = 2, 3 or 5; y = 1, 2 or 3). 3) Phosphorylation of GABARs on a single beta subtype serine (S408 in the beta3 subtype) by PKA or PKC produces enhancement of NT2-N neuronal and NT2 precursor cell GABAR current. 4) Phosphorylation of GABAR channels by PKA or PKC enhances GABAR current by modifying rates of entry into and/or out of desensitized states, thus altering the time course of GABAR currents. The specific aims are to determine: 1) The GABAR subtype proteins in NT2 and NT2-N cells. 2) The pharmacological, steady state single channel kinetic and transient kinetic properties of "NT2 and NT2-N" GABARs expressed in mammalian fibroblasts. 3) The functional consequences of activation of PKA and PKC on recombinant alpha-betax-gamma3 GABAR currents recorded from mammalian cells. 4) The effects of phosphorylation of beta2 and beta3 subtype serines on alpha-betax-gamma3 GABAR currents. S) The biophysical mechanisms of PKA and PKC modification of recombinant GABAR currents. 6) The functional consequences of activation of PKA and PKC on GABAR currents in NT2 and NT2-N cells. 7) The NT2 and NT2-N GABAR subtype protein phosphorylated by PKA and PKC. 8) The biophysical mechanisms PKA and PKC modification of NT2 and NT2-N GABAR currents.
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1 |
2004 — 2007 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Abyabo Gabaar Function/Structure and Epilepsy Mutations
DESCRIPTION (provided by applicant): Characterization of intrinsic factors that determine GABAA receptor (GABAR) inhibitory postsynaptic current (IPSC) shape and the magnitude of extrasynaptic tonic inhibitory current are fundamental issues in inhibitory synaptic physiology. Alterations in phasic, synaptic (? subunit-containing) and tonic, extrasynaptic (d subunit-containing) inhibition due to mutations of ? and d subunits have been shown to underlie several types of genetic generalized epilepsies. To understand the bases for genetic epilepsies involving GABAR gene mutations, it is necessary to understand the functional properties of GABARs that mediate phasic and tonic inhibition. The goals of this proposal are to determine the GABAR functional properties and structural domains that regulate phasic and tonic GABAR currents and to characterize the functional deficits in phasic and tonic GABAerqic inhibition produced by ?2S and d subunit epilepsy mutations. The hypotheses to be tested are that: a) The structural determinants of aa? and aad GABAR gating modes involve non-channel (non-M2) structures including M2/M3, M1 and the N-terminus loop 2. b) Fast/intermediate and slow/ultraslow desensitization of GABAR currents are determined by distinct, structurally separate domains, c) The ?2L(R42Q) mutation reduces "synaptic" a1a2?2L(R42Q) current by reducing cell surface expression, d) The ?2L(K289M) mutation reduces "synaptic" a1a2?2L (K289M) current by destabilizing channel open states, e) The ?2L(Q351X) mutation reduces "synaptic" a1a2?2L (Q351X) current by impairing receptor assembly and decreasing surface expression, f) The d(mutant) reduces "tonic" a4a2d(mutant) current by destabilizing channel open states. The specific aims are to determine: a) The structural determinants of aa? and aad GABAR gating modes, b) The structural determinants of desensitization of aa? and aad GABARs. c) The basis for the reduced "synaptic" current in heterozygous a1a2?2L(R42Q) GABARs. d) The basis for the reduced "synaptic" current in heterozygous a1a2?2L(K289M) GABARs. e) The basis for the reduced "synaptic" current in heterozygous a1a2?2L(Q351X) GABARs. f) The basis for the reduced "synaptic" current in heterozygous a4a2d(mutant) GABARs.
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1 |
2007 — 2010 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Function and Trafficking of Gaba Receptor With Gamma2 Subunit Truncation
DESCRIPTION (provided by applicant): Epilepsy affects more than 0.5 % of the population in the world. Genetic factors play an important role in many of the idiopathic generalized epilepsies (IGEs) and in some partial epilepsies. GABAA receptors (GABARs) are the major inhibitory receptors in the CNS, and mutations in ?2, d and al GABAR genes are associated with IGEs. To understand the bases for IGEs associated with GABAR mutations, it is necessary to determine the functional, assembly and trafficking errors produced by the mutations. Many of these are single nucleotide missense mutations, but recently mutations that introduce a premature translation- termination codon (PTC) and mutations in splice donor sites have been reported. PTCs might produce nonsense mediated decay (NMD) of mRNA if the mutation is not in the last exon or produce a truncated subunit if it is in the last exon. Splice donor site mutations produce mutant protein by: (1) exon skipping, (2) use of cryptic splice site within the down stream intron, or (3) intron inclusion if the intron is small. It is possible all three mechanisms would generate a PTC, thus triggering NMD. The goals of this proposal are to characterize the altered expression, function and trafficking produced by ?2 epilepsy PTC and splice donor site mutations. Hypotheses are that the: a) ?2S(Q351X) PTC mutation reduces het al[unreadable]2?2S(Q351X) current by producing a C-terminal truncated subunit that assembles to form mutant receptors that reduce trafficking of wt receptors, are co trafficked with wt receptors to the cell surface and have altered function, b) ?2S(Q1X) PTC mutation reduces het al[unreadable]2?2L(Q1X) receptor current by triggering NMD of mutant mRNA, thus producing haploinsufficiency. c) ?2S (IVS6+2T-G) splice donor site mutation reduces het al[unreadable]2?2L (IVS6+2T-G) receptor current via haploinsufficiency by triggering NMD through either exon 6 skipping, resulting in a PTC at the joining site of exon 5 and exon 7, or by using intron 6 downstream cryptic splice sites, also resulting in a PTC, or by generating a truncated protein due to incomplete NMD (NMD inefficiency). Specific aims are to determine the pathophysiological alterations in translation, trafficking, surface expression and pharmacological and biophysical properties of het and horn a) al[unreadable]2?2 (Q351X), b) al[unreadable]2?2 (Q1X), and c) al[unreadable]2?2 (IVS6+2T-G) receptors expressed in fibroblasts and ?2S siRNA treated cultured hippocampal neurons.
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1 |
2009 — 2012 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Abg and Abd Gabaar Assembly/Trafficking/Function and Epilepsy Missense Mutations
DESCRIPTION (provided by applicant): GABAA receptors (GABARs) are the primary mediators of fast inhibitory synaptic transmission and tonic extrasynaptic inhibition. Synaptic GABARs are composed of 1, 2, and 32 subunits while extrasynaptic GABARs are generally composed of 1, 2, and 4 subunits. Mutations and variants in GABAR 32 and 4 subunit genes have recently been associated with idiopathic generalized epilepsies (IGEs). Our long-term goals are to understand how these mutations and variants disrupt the normal assembly, surface trafficking, synaptic and extrasynaptic targeting, and surface stability of GABARs;to characterize the effects of these mutations on GABAR functional properties;and ultimately, to provide a mechanistic foundation for development of novel therapeutic strategies. Hypotheses to be tested are: 1) Assembly, trafficking, and functional properties of synaptic 1(1,2,3,4)2232 GABARs have strict subunit and cellular requirements;2) Assembly, trafficking, and functional properties of extrasynaptic 1(1,4)224 and 152232 GABARs have strict subunit and cellular requirements;and 3) 32 subunit mutations and 4 subunit variants promote neuronal hyperexcitability by altering assembly, surface trafficking, and/or function of synaptic 1(1,2,3,4)2232 and extrasynaptic 1(1,4)224 and 152232 GABARs. Specific aims are: 1) To determine how synaptic 1(1,2,3,4)2232 GABARs are assembled and trafficked to the cell surface and to characterize their functional properties;2) Specific Aim 2: To determine how extrasynaptic 1(1,4)224 and 152232 GABARs are assembled and trafficked to the cell surface and to characterize their functional properties;and 3) : To determine how 32 subunit mutations and 4 subunit variants associated with IGEs disrupt subunit assembly, trafficking, and/or functional properties of synaptic and extrasynaptic GABARs. PUBLIC HEALTH RELEVANCE Epilepsy affects more than 0.5 % of the world's population and genetic factors play an important role in many generalized and in some partial epilepsies. At the present time there is treatment for genetic epilepsies with antiepileptic drugs but there is no cure. This proposal seeks to determine the basis for the genetic epilepsies associated with mutations in the inhibitory neurotransmitter GABAA receptor subunit genes that will provide a mechanistic foundation for development of novel therapeutic strategies.
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1 |
2011 — 2014 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathophysiology and Treatment of Gabrg2 Nonsense Epilepsy Mutations
DESCRIPTION (provided by applicant): Epilepsy affects more than 0.5% of the population worldwide. Genetic factors play an important role in many of the epilepsy syndromes. GABAA receptors (GABAARs) are the major inhibitory receptors in the brain and mutations in GABAAR subunit genes (GABRs) coding for the 32, 11 and 4 GABAAR subunits are associated with idiopathic generalized epilepsy syndromes (IGEs). To understand the bases for IGEs associated with GABR mutations, it is necessary to determine the errors in receptor biogenesis and function that are produced by the mutations. Many of them are single nucleotide missense mutations, but recently nonsense mutations that introduce a premature translation-termination codon (PTC) and mutations in splice donor sites have been reported. PTCs might produce nonsense mediated mRNA decay (NMD) if the mutation is not in the last exon or produce a truncated subunit if it is in the last exon that could have dominant negative effects on wt subunits. Splice donor site mutations produce mutant subunits by: (1) exon skipping, (2) use of a cryptic splice site within the downstream intron, or (3) intron inclusion if the intron is small. It is possible that all three mechanisms would generate a PTC, thus triggering NMD. There are several approaches that can be taken to treat patients with nonsense epilepsy mutations. If the mutation produces no functional protein and therefore is haplo-insufficient, the subunit could be over expressed in neurons to overcome the loss of subunit or if the PTC is in frame, a read through strategy could be applied to read through the PTC and express a full length functional subunit. If the mutation produces a truncated, nonfunctional dominant negative protein, the subunit could be over expressed in neurons to overcome the loss of subunit or a read through strategy could be applied to read through the PTC and express a full length functional subunit. The goals of this proposal are to characterize the altered receptor biogenesis and function produced by GABRG2 epilepsy PTC and splice donor site mutations and to develop strategies to overcome the resultant deficits as potential therapies for the associated epilepsies. The hypotheses of the proposal are: 1) The GABRG2(IVS6+2T`G) mutation leads to aberrant mRNA splicing that creates a PTC and activates NMD and produces a stable, nonfunctional, truncated dominant negative protein whose effects can be reversed by siRNA knock down and/or GABRG2 over expression; 2) The GABRG2(Q1X) mutation activates NMD to degrade the mutant mRNA, producing loss of function that can be reversed by PTC read through and/or GABRG2 over expression; and 3) The GABRG2(Q351X) mutation produces a stable, nonfunctional, truncated dominant negative protein that can be reversed by PTC read through and/or GABRG2 over expression. The specific aims of the proposal are: 1) To determine the aberrant mRNA splicing pattern produced by the GABRG2(IVS6+2T`G) mutation and how the mutant translation products affect g2 subunit expression and GABAAR function and to evaluate potential treatment strategies; 2) To determine the impairment of g2 subunit biogenesis produced by the GABRG2(Q1X) mutation and to evaluate potential treatment strategies, and 3) To characterize GABRG2(Q351X) knock in mice and evaluate PTC read through and/or GABRG2 over expression as potential treatment strategies.
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1 |
2013 — 2016 |
Macdonald, Robert Louden |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba(a) Receptor Assembly/Trafficking/Function and Epilepsy Missense Mutations
DESCRIPTION (provided by applicant): Epilepsy affects more than 0.5% of the population worldwide, and genetic factors play an important role in the idiopathic generalized epilepsy syndromes (IGEs). Many monogenic mutations associated with IGEs are in ion channel genes. GABAA receptors (GABAARs) are the major inhibitory receptors in the brain and mutations in GABAAR subunit genes (GABRs) coding for the y2, ¿1 and ¿3 subunits are associated with IGEs. We have classified the known monogenic GABR mutations into 6 classes: those that reduce subunit expression due to: 1) impaired transcription; 2) impaired translation, 3) misfolding and degradation, 4) truncation and ER retention with or without a dominant negative effect on other subunits or 5) ER retention of functional receptors. A final class of mutations 6) reduces surface receptor function. This classification is useful for developing treatment strategies for severe IGEs. Mutations in GABR¿3 (P11S, S15F) and GABR?2 (N79S, R82Q, P83S, R177G) have been associated with IGEs, and P11S has also been associated with autism. In Specific Aim 1 we continue our strategy of characterizing effects of monogenic GABR mutations associated with IGEs on functional properties and/or biogenesis of GABAARs, focusing on the mutations in ¿3 and ?2 subunits. It is important, also, to determine the effects of these mutations in vivo on thalamocortical network function and mouse behavior. In addition and to, characterize the adaptive neuronal plasticity that occurs in response to the loss of inhibition for each mutation. Since the GABR¿3 (P11S) mutation has been associated with both epilepsy and autism, it is a particularly important mutation, and in Specific Aim 2 we will study a class 3 ¿3+/P11S KI mouse and a ¿3+/- mouse for comparison. We will determine if: 1) the KI mice develop a generalized epilepsy and altered autism-like behavior due to haploinsufficiency or due also to a dominant negative effect of the mutant subunit, 2) the mutation alters cortical and thalamic inhibition, 3) mut ¿3 (P11S) subunits are reduced in mouse brain, and 4) the mutation causes altered transcriptional signatures of cellular plasticity in compensation for the loss of b3 subunits. The basis for most IGEs has not been found since about 98% are polygenic, and thus, likely due to the presence of multiple nsSNPs (nonsynonymous single nucleotide polymorphisms that change aa coding). Thus, new strategies are needed for identification of nsSNPs that contribute to IGEs with complex inheritance. Among GABR genes, monogenic mutations associated with IGEs have been found in the hEP genes, GABR¿1, GABR¿3 and GABR?2. Exome sequencing of candidate ion channel genes, including GABAR genes, from well characterized IGE cases and controls identified rare nsSNPs in non-hEP genes in cases but not in controls. Also, with the Exome Variant Server, we found rare nsSNPs in the hEP genes. In Specific Aim 3 we will characterize the effects of the rare nsSNPs found in non hEP genes only in cases or in hEP genes on functional properties and/or biogenesis of GABAARs.
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2016 — 2017 |
Gallagher, Martin J Macdonald, Robert Louden Zhou, Chengwen [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Impaired Homeostatic Potentiation of Gabaergic Currents Initiates Seizures @ Vanderbilt University Medical Center
Seizures affect almost 3 million peoples in US and in two thirds of patients, the causes are not known (possible genetic origins). Idiopathic generalized epilepsy(IGE) has been recognized as its genetic origins which include many single-nucleotide polymorphisms (SNPs) or mutation of ionotropic receptors, such as GABAergic receptor (GABAR) subunit mutations (Gabrg2Q390X and Gabra1A322D) linked to severe Dravet epilepsy syndrome and child absence epilepsy, and cognitive comorbidity in patients. In contrast to acquired seizures study, IGE seizure and epileptogenesis mechanism remains largely elusive. Moreover, seizures and sleep influence/interact with each other in their physiology mechanism, which imposes a challenge to IGE study and patient treatment. In one recent human patient study, sleep-like slow-wave oscillation has been shown to facilitate epileptic seizure activity. Therefore, we hypothesize that sleep-related slow-wave hyperpolarization-depolarization oscillation(SWO) can drive homeostatic potentiation(HSP) of excitatory synaptic currents, not inhibitory synaptic currents in cortical neurons of thalamocortical circuitry in IGE animal models with Gabrg2Q390X or Gabra1A322D mutation, and therefore create an escaped excitatory synaptic currents (without balancing from inhibitory synaptic currents) in cortical neurons during sleep and sleep-wake transition. This critical step of gabaergic current HSP impairment induced by SWOs can lead to seizure occurrence/initiation and also contribute to epileptogenesis in IGE models. This work will fill a critical void in our understanding of seizure mechanism, plus epileptogenesis, and potentially generate a new seizure therapy. First, we will study whether SWO-induced HSP of inhibitory GABAR-mediated currents is impaired, but not excitatory AMPAR-mediated currents in layer V-VI cortical neurons in vitro from heterozygous Gabrg2+/Q390X or Gabra1+/A322D knock-in mice and whether this impairment results in neuronal elevated firing. Second, we will determine whether light-induced SWOs in vivo causally initiate epileptic activity in cortex from mice expressing halorhodopsin (NpHR) and Gabrg2Q390X or Gabra1A322D mutation. Last, we will use a retinoid acid synthesis blocker DEAB to maintain the dynamic HSP balance between synaptic excitatory and inhibitory currents during SWOs in IGE models, which will provide a proof of principle for a potential seizure therapy. The information generated in these studies will substantially alter our view of seizure/epileptogenesis regarding its interaction with sleep waves and eventually lead to a new seizure therapy in IGE patients.
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