2012 — 2016 |
Dulla, Chris G |
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. |
Impact of Astrocytic Glutamate Transport On Epilepsy Associated With Developmenta @ Tufts University Boston
DESCRIPTION (provided by applicant): Diseases of cortical malformation cause approximately 25% of all cases of epilepsy. They are also the most common cause for surgical resection of epileptic brain tissue. Almost 80% of people with a cortical malformation suffer from epilepsy and greater than 70% of those people have seizures which are not managed by anti-epileptic drugs. Novel treatment strategies are urgently needed to treat this problematic group of epilepsies. In this proposal we will address the hypothesis that loss of astrocytic glutamate reuptake during the development of a cortical malformation acutely disrupts glutamate homeostasis and has long term effects on synaptic connectivity and cortical network function. Normally, astrocytes remove the neurotransmitter glutamate via glutamate transporters. In diseases of cortical malformation, however, astrocytes become reactive which we believe decreases their ability to remove extracellular glutamate. In the developing cortex glutamate directly drives synapse formation. Therefore, we hypothesize that loss of astrocytic glutamate reuptake during the development of a cortical malformation increases extracellular glutamate levels which promotes excitatory synapse formation and leads to long term cortical hyperexcitability. We will test our hypothesis utilizing cutting-edge imaging techniques, electrophysiological recording from astrocytes, in vivo assays of cortical excitability and molecular disruption and augmentation of astrocyte glutamate transport. Our experiments are extremely innovative. We have developed a novel rodent model of cortical malformation which closely replicates focal cortical dysplasia type 1, a disease with no current animal model. We will utilize exciting, novel glutamate biosensor imaging techniques to assay network function and astrocytic glutamate reuptake. We will record cortical glutamate transporter currents, which have not previously been investigated, and we will do so in the malformed cortex. We will utilize laser-scanning photostimulation to spatially map how astrocytic glutamate reuptake is altered in the malformed cortex. Utilizing molecular modulation of astrocytic glutamate transport we will test whether developmental loss of astrocytic glutamate transport is sufficient to induce cortical hyperexcitability and whether increasing glutamate reuptake in the malformed cortex interrupts epileptogenic processes which we believe lead to later network dysfunction. Importantly, we will utilize drugs which are already clinically available to increase glutamate reuptake. Should this approach successfully attenuate cortical hyperexcitability it could be rapidly translated into a potential anti-epileptogenic clinical tool.
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0.931 |
2016 — 2021 |
Dulla, Chris G |
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. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
The Role of Beta-Catenin in the Pathophysiology of Infantile Spasms @ Tufts University Boston
Project summary Infantile spasms (IS, also known as West Syndrome) is a catastrophic childhood epilepsy syndrome characterized by spasms which progress into seizures later in life. Spasms are typified by spontaneous flexion/extension of the head, neck, and limbs and occur first between 4 and 8 months of age. The current treatment options for IS are often ineffective and are associated with significant side effects. Therefore, novel treatment strategies are essential. One limiting factor in identifying new treatment approaches is a paucity of pre-clinical animal models. We have identified and characterized a novel rodent model with many phenotypic characteristics of human IS. The model was generated by breeding male mice containing a floxed version of the Adenomatous polyposis coli (APC) gene with female mice expressing the Cre-recombinase gene under the control of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter. The offspring of this cross, which lack APC in CaMKIIa-positive neurons, are known as APC conditional knockouts (APC cKOs). APC cKO animals have been shown to have increased excitatory synaptic communication and an increased density of excitatory spines on hippocampal CA1 pyramidal neurons. APC is the main inhibitory regulator of a large signaling pathway known as the ?-catenin/Wnt pathway. APC is part of the ?-catenin destruction complex, targeting ?-catenin for degradation. When APC is lost, ?-catenin levels rise and 1) increase transcription of a large family of genes, and 2) increase the stability of excitatory synapses. We began by examining APC cKO animals for phenotypes consistent with human IS. We found that they exhibit spontaneous behavioral spasms at postnatal days 8-11, they have an ictal EEG correlate of spasm behavior similar to human ictal activity in IS, and as adults, they have spontaneous electrographic and behavioral seizures. Interestingly, APC heterozygous mutations in humans are linked to both developmental and seizure disorders. Furthermore, many of the genes linked to IS are either part of the ?-catenin/Wnt pathway or are reciprocally regulated by it. In this proposal, we will specifically examine the role of ?-catenin in the pathophysiology of infantile spasms. We will examine the effects of increasing ?-catenin (by deleting APC and independently of APC) on spasm behavior, seizures, and electrographic brain activity. Next, we will perform careful pharmacokinetic, pharmacodynamic, and adverse effect analysis of manipulating ?-catenin during development with a drug called G007-LK. Lastly, we will determine if restoring ?-catenin levels to normal attenuates spasms and seizures later in life. This proposal will address the role of ?-catenin in the pathophysiology of spasms, provide a new mouse model for pre-clinical analysis, and introduce a large set of new potential therapeutic targets for the treatment of IS.
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0.931 |
2017 — 2018 |
Dulla, Chris G |
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.) |
Preserving Inhibitory Cortical Networks Following Tbi: Attenuating Excitation Using Inhibitors of Glycolysis @ Tufts University Boston
Project summary Traumatic brain injuries (TBI) are the leading cause of death and disability in children and the aged. Cognitive and motor dysfunction, as well as post-traumatic epilepsy (PTE), often occurs following TBI. There are limited therapeutic options for TBI, none of which have proven to be efficacious in improving neurological outcomes across diverse groups of TBI patients. Therefore, developing new therapeutic tools based on mechanistic rationale are critical to finding treatments to improve patient outcome following TBI. Recently, we reported that the controlled cortical impact (CCI) model of TBI resulted in a significant loss of parvalbumin-positive inhibitory interneurons in the cortex. Parvalbumin-positive interneurons provide a bulk of cortical inhibition which constrains neuronal activity. When parvalbumin-positive interneurons were lost following TBI, uncontrolled glutamatergic activity was seen along with increased excitatory and decreased inhibitory synaptic inputs. Based on these findings, we set out to develop approaches to preserve interneurons following TBI. Based on published data showing areas of increased glycolytic activity in the brain following TBI, and known linkages between glycolysis and neuronal activity, we set out to determine if inhibiting glycolysis following TBI would attenuate loss of parvalbumin interneurons. We hypothesized that TBI leads to glycolysis-dependent increases in excitatory neuron activity. This would lead to hyper-activation of inhibitory interneurons and their subsequent excitotoxic cell death. We propose to interrupt glycolysis to attenuate excitatory neuronal activity following TBI. Using 2-deoxyglucose (2DG), an inhibitor of hexokinase (the rate-limiting enzyme of glycolysis), we have begun to test this hypothesis. Our preliminary data suggests that 2DG can acutely attenuate cortical hyperexcitability in brain slices 2-4 weeks following TBI and that in vivo treatment with 2DG following TBI attenuates both network hyperexcitability and parvalbumin-positive cell loss. Our preliminary data also suggests that 2DG attenuates excitatory, but not inhibitory, neuron excitability. Here we propose to further these studies by demonstrating that 2DG reduces parvalbumin-positive interneuron cell death and reduces changes in synaptic communication in the cortex following injury. We also propose to test the hypothesis that inhibition of glycolysis attenuates excitatory, but not inhibitory, cell excitability. Furthermore, we aim to determine whether there is differential expression of glycolytic and related proteins in excitatory neurons vs. inhibitory interneurons via single-cell qPCR. This aspect of the proposal is both high-risk and high-reward. Our studies will determine if 2DG is able to preserve interneurons following TBI, will begin to establish 2DG's mechanism of action, and will potentially demonstrate a novel form of cell type-specific coupling of metabolic and electrical activity. Based on these studies, we will be better able to manipulate neuronal excitability with cell type-specific metabolic disruption and to design therapeutic strategies to reduce TBI-associated pathology.
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0.931 |
2017 |
Dulla, Chris G |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
The Role of Glutamate Signaling in Developmental Cortical Malformations @ Tufts University Boston
Project summary Epilepsy is the most common neurological disorder and has associated costs >$15 billion in the US annually (CDC). We focus on developmental cortical malformations (DCMs), a group of pediatric epilepsy syndromes (80% of patients with DCMs have epilepsy) characterized by reorganization of cortical structure. DCMs include polymicrogyria, focal cortical dysplasia, and schizencephaly and are caused by early life insults and genetic mutation. DCMs are the most common cause of pediatric refractory epilepsy (76% of DCM patients do not respond to treatment) and often require surgical intervention. Due to diffuse regions of hyperexcitability, even surgical resection is of limited usefulness. DCMs demand new treatment options. We believe that DCMs arise from disrupted glutamate signaling during early development. The precise timing of glutamate signaling during development is essential to network maturation, therefore, disruptions may have significant long-term consequences. Our studies using the neonatal freeze-lesion (FL) model of DCM demonstrate that early life cortical insult disrupts glutamate levels in the developing cortex. We hypothesize that this alters interneuron activation, disrupting their maturation and later functional properties. Intriguingly, pharmacologically blocking a subtype of NMDA receptors mimics multiple FL-associated pathologies and alters interneuron maturation. Taken together, we hypothesize that disruptions in ambient glutamate during early postnatal development alter interneuron activity at a critical time, lead to disruption of inhibitory networks, and cause lasting cortical network hyperexcitability. To address our hypothesis we will utilize a combination of whole-cell electrophysiological recording, glutamate biosensor imaging, extracellular field recording of network activity, EEG analysis of cortical hyperexcitability, and analysis of inhibitory interneurons. We will ask whether 1) ambient glutamate affects interneuron maturation, 2) FL alters ambient glutamate and leads to changes in IN maturation, and 3) whether altering interneuron activity during early neonatal development (using pharmacological and chemogenetic approaches) disrupts interneuron maturation and induces network hyperexcitability. At the completion of these studies, we will know if disruption interneuron activity during development leads to long term inhibitory hypofunction. We will be poised to engage 2C/D-NMDARs as a preclinical target to improve clinical outcomes for individuals with epilepsy due to DCMs. Additionally, our studies will provide new insight into the relationship between glutamatergic activity, astrocytes, and interneurons in the developing cerebral cortex.
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0.931 |
2017 — 2018 |
Dulla, Chris G |
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.) |
Voltage-Dependent Modulation of Astrocyte Glutamate Transport Induced by Neuronal Activity @ Tufts University Boston
Astrocytes remove the excitatory neurotransmitter glutamate from the extracellular space following neuronal activity via sodium-driven, voltage-dependent excitatory amino acid transporters (EAATs). Robust glutamate uptake by EAATs ensures the temporal and spatial fidelity of glutamate signaling. Interestingly, we recently found that neuronal activity rapidly (within milliseconds) and reversibly slows glutamate uptake in the adult cerebral cortex. This slowing prolongs neuronal NMDA responses, consistent with prolonged extracellular glutamate dynamics, and is highly dependent on the frequency and duration of stimulation. Additionally, glutamate clearance can be modulated by neuronal activity with synapse specificity, even within a single astrocyte. We believe this may have important consequences on neurotransmission, extrasynaptic receptor activation, and synaptic plasticity. Based on that exciting finding, we hypothesized that neuronal activity induces microdomain-level changes in astrocyte membrane potential (Vm) that locally modulate EAAT function. GLT1 is the predominant astrocytic EAAT in the adult forebrain, is abundantly expressed, and ensures that glutamate in the extracellular space is rapidly sequestered by EAAT binding. Once bound to EAATs, the transport of glutamate into the astrocyte is both sodium-driven and voltage-dependent. Under normal conditions, astrocytes are hyperpolarized (-80 mV) due to their high permeability to potassium. However, neuronal activity increases extracellular potassium, [K ]e, and astrocyte Vm is especially sensitive to [K ]e changes. Therefore, it stands to reason that neuronal activity can alter EAAT function by depolarizing astrocytes. Changes in astrocytic Vm may be especially relevant in fine astrocytic processes, where EAATs are concentrated, and where small intracellular volumes may amplify changes in Vm, as compared to soma. A major challenge to testing our hypothesis, however, is an inability to monitor astrocyte Vm at distal processes due to low membrane resistance and process morphology. Overcoming this challenge is important because astrocyte distal processes are the site of synaptic interaction and EAATs localization. In order to detect distal changes in astrocyte Vm, we have begun to develop an approach to image Vm in astrocyte processes using QuasAr, a genetically-encoded voltage sensor. Dr. Adam Cohen, who developed QuasArs, is a collaborator on this proposal. Utilizing astrocyte and neuron electrophysiological recording, optogenetic manipulation of astrocyte Vm, and QuasAr imaging of astrocyte membrane potential we have generated preliminary data that supports our hypothesis that EAAT function can be modulated by activity-induced changes in astrocyte Vm.
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0.931 |
2019 — 2021 |
Dulla, Chris G |
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. |
Voltage Imaging of Astrocyte-Neuron Interactions @ Tufts University Boston
Project summary Astrocytes remove the excitatory neurotransmitter glutamate from the extracellular space following neuronal activity via sodium-driven, voltage-dependent excitatory amino acid transporters (EAATs). Robust glutamate uptake by EAATs ensures the temporal and spatial fidelity of glutamate signaling. Interestingly, we recently found that neuronal activity rapidly (within milliseconds) and reversibly slows glutamate uptake in the adult cerebral cortex. This slowing prolongs neuronal NMDA responses, consistent with prolonged extracellular glutamate dynamics, and is highly dependent on the frequency and duration of stimulation. Additionally, glutamate clearance can be modulated by neuronal activity with synapse specificity, even within a single astrocyte. We believe this may have important consequences on neurotransmission, extrasynaptic receptor activation, and synaptic plasticity. Based on this finding, we hypothesized that neuronal activity induces microdomain-level changes in astrocyte membrane potential (Vm) that locally modulate EAAT function. GLT1 is the predominant astrocytic EAAT in the adult forebrain, is abundantly expressed, and ensures that glutamate in the extracellular space is rapidly sequestered by EAAT binding. Once bound to EAATs, the transport of glutamate into the astrocyte is both sodium-driven and voltage-dependent. Under normal conditions, astrocytes are hyperpolarized (-80 mV) due to their high permeability to potassium. However, neuronal activity increases extracellular potassium, [K+]e, and astrocyte Vm is especially sensitive to [K+]e changes. Therefore, it stands to reason that neuronal activity can alter EAAT function by depolarizing astrocytes. Changes in astrocytic Vm may be especially relevant in fine astrocytic processes, where EAATs are concentrated, and where small intracellular volumes may amplify changes in Vm, as compared to soma. We will also explore alternative mechanisms including voltage-independent modulation of EAATs by increases in [K+]e. A major challenge to testing our hypothesis, however, is an inability to monitor astrocyte Vm at distal processes due to low membrane resistance and process morphology. Overcoming this challenge is important because astrocyte distal processes are the site of synaptic interaction and EAATs localization. In order to detect distal changes in astrocyte Vm, we developed an approach to image Vm in astrocyte processes using genetically-encoded voltage indicator (GEVI) imaging. Utilizing astrocyte and neuron electrophysiological recording, optogenetic manipulation of astrocyte Vm, and GEVI imaging of astrocyte membrane potential we have generated preliminary data that supports our hypothesis that EAAT function can be modulated by activity-induced changes in astrocyte Vm.
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0.931 |
2020 |
Dulla, Chris G Jacobs, Muazzam Raimondo, Joseph Valentino (co-PI) [⬀] |
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.) |
Utilizing Single Cell Biological Approaches to Understand Cns Tb @ Tufts University Boston
PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative pathogen in tuberculosis (TB). TB is the leading cause of death from infectious disease globally and is especially prevalent in individuals infected with HIV. While normally thought of as a respiratory disease, TB also infects other organ systems. Infection of the central nervous system (CNS-TB) is the most severe form of the disease, and has a mortality rate of nearly 50%, despite aggressive clinical intervention. CNS-TB is associated with severe neurological dysfunction including cranial nerve palsies, cognitive impairment, stroke, and seizures. The brain?s molecular, cellular, and network level response to CNS-TB is almost completely unknown. We hypothesize CNS-TB leads to significant activation of neuroinflammatory signaling, as well as glial and neuronal dysfunction. The risk of developing CNS-TB is markedly increased under conditions of immune suppression, evident in HIV infected individuals, who have a higher occurrence of TB meningitis (TBM). A regulated tumor necrosis factor (TNF) response is a critical feature of immune competence necessary for protection against TB, and is lost in progressive HIV infection. The importance of a proper TNF response is clinically validated by the reactivation of TB in patients on anti-TNF treatment for autoimmune diseases. We recently reported that TNF deficiency (a model of immune suppression) in mice (TNF-/-), promotes CNS-TB infection, a hyper-inflammatory response, gross brain pathology, and mortality. We will utilize a realistic mouse model of CNS-TB in which active Mycobacterium tuberculosis (Mtb) will be injected into the brains of control and immune compromised (TNF-/-) mice to study how resident CNS cell respond. Although Mtb primarily infects microglia and astrocytes, human and mouse neurons also act as host cells for Mtb. Therefore, multiple CNS cells react both directly and indirectly to brain infection, creating a complex cellular- and tissue-level response. To deal with this complexity, we will utilize single cell RNA-seq, a cutting edge genomic approach, which allows transcriptional analysis of the response to CNS-TB on a cell-by-cell basis. This approach enables the identification of individual types of cells, for example astrocytes, and analysis of their unique transcriptional response. In addition, single cell RNA-seq allows investigation of the heterogeneity of the cellular response to CNS-TB by analyzing individual cells. We will utilize single cell RNA-seq to determine how the lack of a proper immune response regulates neuroinflammatory signaling and leads to broad-scale disruption of CNS resident cells in a mouse model of CNS-TB. In doing so, we will establish a partnership between The University of Cape Town and Tufts University. Emphasis will be placed on training South African scientists in neuro- immune interactions, single cell RNA-seq, and advanced genomic analysis approaches, thereby helping develop robust research infrastructure in South Africa.
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0.931 |