The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradati... more The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, β-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-...
In Parkinson's disease, dopamine‐containing nigrostriatal neurons undergo profound degenerati... more In Parkinson's disease, dopamine‐containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build‐up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH‐over‐expressing mice (TH‐HI) using a BAC‐transgenic approach that results in over‐expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH‐HI mice had a 3‐fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH‐HI mice showed increased striatal production of H2O2 and reduced glutathione levels. In addition, TH‐HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4‐dihydroxyphenylacetaldehyde and 5‐S‐cysteinyl‐dopamine and were more susceptible than wild‐type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.
Background Abnormalities of cellular signaling are well characterized in neuropsychiatric illness... more Background Abnormalities of cellular signaling are well characterized in neuropsychiatric illnesses, including schizophrenia. Changes in signaling pathways reflect the underlying genetic, environmental, and epigenetic perturbations driving disease phenotypes. A shortcoming of most signaling studies is a focus on one or a few protein kinases at a time, a limitation since protein kinases work in networks with other kinases, phosphatases, and regulatory molecules to effect signaling events. We addressed this challenge by employing a kinome array platform that simultaneously measures protein kinase activity at hundreds of reporter peptide substrates. We then developed a novel bioinformatics pipeline to identify protein kinase nodes, signaling networks, upstream biological pathways, and drug candidates that “reverse” kinomic disease signatures. Methods Postmortem DLPFC brain samples from subjects with schizophrenia (n = 20 per group, 10 males and 10 females per group), were compared to a...
Social isolation raises the risk for mood disorders associated with serotonergic disruption. Yet,... more Social isolation raises the risk for mood disorders associated with serotonergic disruption. Yet, the underlying mechanisms by which the stress of social isolation increases risk are not well understood. Men and women are differently vulnerable; however, this modulating role of sex is challenging to study in humans under carefully controlled conditions. Therefore, we investigated this question in mice of both sexes, asking how the long-term stress of social isolation (from weaning into adulthood) affects the excitability of serotonin neurons in the dorsal raphe nucleus as well as mouse behaviour. The electrophysiological experiments and the first set of behavioural tests were conducted in young adult mice, with additional behavioural assays completed as the mice matured to assess the stability of their behavioural phenotype. We found that social isolation exerted seemingly-opposite effects in male and female mice, relative to their respective group-housed littermate controls. This distinctive pattern was observed for the effect of social isolation on the control of serotonergic neuron excitability via the SK family of calcium-activated potassium channels. Furthermore, we observed a similar and consistent pattern on tests relevant to assessing the efficacy of anti-depressant medicines, including the forced swim test, the novelty-suppressed feeding test, and the sucrose preference test. These findings underscore the concept that stress-elicited illness manifests distinctly in males and females and that treatments aimed at restoring serotonergic function may require a sex-specific approach.
NMDA receptors are important for cognition and are implicated in neuropsychiatric disorders. GluN... more NMDA receptors are important for cognition and are implicated in neuropsychiatric disorders. GluN1 knockdown (GluN1KD) mice have reduced NMDA receptor levels, striatal spine density deficits, and cognitive impairments. However, how NMDA depletion leads to these effects is unclear. Since Rho GTPases are known to regulate spine density and cognition, we examined the levels of RhoA, Rac1, and Cdc42 signaling proteins. Striatal Rac1-pathway components are reduced in GluN1KD mice, with Rac1 and WAVE-1 deficits at 6 and 12 weeks of age. Concurrently, medium spiny neuron (MSN) spine density deficits are present in mice at these ages. To determine whether WAVE-1 deficits were causal or compensatory in relation to these phenotypes, we intercrossed GluN1KD mice with WAVE-1 overexpressing (WAVE-Tg) mice to restore WAVE-1 levels. GluN1KD-WAVE-Tg hybrids showed rescue of striatal WAVE-1 protein levels and MSN spine density, as well as selective behavioral rescue in the Y-maze and 8-arm radial ma...
NMDA receptor dysfunction is central to the encephalopathies caused by missense mutations in the ... more NMDA receptor dysfunction is central to the encephalopathies caused by missense mutations in the NMDA receptor subunit genes. Missense variants of GRIN1, GRIN2A, and GRIN2B cause similar syndromes with varying severity of intellectual impairment, autism, epilepsy, and motor dysfunction. To gain insight into possible biomarkers of NMDAR hypofunction, we asked whether a loss-of-function variant in the Grin1 gene would cause structural changes in the brain that could be detected by MRI. We also studied the developmental trajectory of these changes to determine whether structural changes coincided with reported cognitive impairments in the mice. We performed magnetic resonance imaging in male Grin1-/- knockdown mice (GluN1KD) that were three, six, or twelve weeks old. Deformation-based morphometry was used to assess neuroanatomical differences. Volumetric reductions were detected in substantia nigra and striatum of GluN1KD mice at all ages. Changes in limbic structures were only evident at six weeks of age. Reductions in white matter volumes were first evident at three weeks, and additional deficits were detected at six and twelve weeks. FluoroJade immunofluorescence revealed degenerating neurons in twelve-week old GluN1KD mice. We conclude that Grin1 loss-of-function mutations cause volume reductions in dopaminergic structures early in development, while changes to limbic and white matter structures are delayed and are more pronounced in post-adolescent ages. The evidence of degenerating neurons in the mature brain indicates an ongoing process of cell loss as a consequence of NMDAR hypofunction.
Schizophrenia is a devastating illness that affects over 2 million people in the United States an... more Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays. Laser-capture microdissection-quantitative polymerase chain reaction was used to examine these pathways at the cellular level. We found decreases in hexokinase (HXK) and phosphofructokinase (PFK) activity in the DLPFC, as well as decreased PFK1 mRNA expression. In pyramidal neurons, we found an increase in monocarboxylate transporter 1 mRNA expression, and decreases in HXK1, PFK1, glucose transporter 1 (GLUT1), and GLUT3 mRNA expression. These results suggest abnormal bioenergetic function, as well as a neuron-specific defect in glucose utilization, in the DLPFC in schizophrenia.
Postoperative delirium is associated with poor long-term outcomes and increased mortality. Genera... more Postoperative delirium is associated with poor long-term outcomes and increased mortality. General anesthetic drugs may contribute to delirium because they increase cell-surface expression and function of α5 subunit-containing γ-aminobutyric acid type A receptors, an effect that persists long after the drugs have been eliminated. Dexmedetomidine, an α2 adrenergic receptor agonist, prevents delirium in patients and reduces cognitive deficits in animals. Thus, it was postulated that dexmedetomidine prevents excessive function of α5 γ-aminobutyric acid type A receptors. Injectable (etomidate) and inhaled (sevoflurane) anesthetic drugs were studied using cultured murine hippocampal neurons, cultured murine and human cortical astrocytes, and ex vivo murine hippocampal slices. γ-Aminobutyric acid type A receptor function and cell-signaling pathways were studied using electrophysiologic and biochemical methods. Memory and problem-solving behaviors were also studied. The etomidate-induced s...
Synaptic neurotransmission relies on maintenance of the synapse and meeting the energy demands of... more Synaptic neurotransmission relies on maintenance of the synapse and meeting the energy demands of neurons. Defects in excitatory and inhibitory synapses have been implicated in schizophrenia, likely contributing to positive and negative symptoms as well as impaired cognition. Recently, accumulating evidence has suggested that bioenergetic systems, important in both synaptic function and cognition, are abnormal in psychiatric illnesses such as schizophrenia. Animal models of synaptic dysfunction demonstrated endophenotypes of schizophrenia as well as bioenergetic abnormalities. We report findings on the bioenergetic interplay of astrocytes and neurons and discuss how dysregulation of these pathways may contribute to the pathogenesis of schizophrenia, highlighting metabolic systems as important therapeutic targets.
The identification of circulating autoantibodies against neuronal receptors in neuropsychiatric d... more The identification of circulating autoantibodies against neuronal receptors in neuropsychiatric disorders has fostered new conceptual and clinical frameworks. However, detection reliability, putative presence in different diseases and in health have raised questions about potential pathogenic mechanism mediated by autoantibodies. Using a combination of single molecule-based imaging approaches, we here ascertain the presence of circulating autoantibodies against glutamate NMDA receptor (NMDAR-Ab) in about 20% of psychotic patients diagnosed with schizophrenia and very few healthy subjects. NMDAR-Ab from patients and healthy subjects do not compete for binding on native receptor. Strikingly, NMDAR-Ab from patients, but not from healthy subjects, specifically alter the surface dynamics and nanoscale organization of synaptic NMDAR and its anchoring partner the EphrinB2 receptor in heterologous cells, cultured neurons and in mouse brain. Functionally, only patients' NMDAR-Ab prevent ...
NMDA receptors (NMDAR) are important in the formation of activity-dependent connections in the br... more NMDA receptors (NMDAR) are important in the formation of activity-dependent connections in the brain. In sensory pathways, NMDAR disruption during discrete developmental periods has enduring effects on wiring and function. Yet, it is not clear whether NMDAR-limited critical periods exist for higher-order circuits governing mood and cognition. This question is urgent for neurodevelopmental disorders, like schizophrenia, that have NMDAR hypofunction and treatment-resistant cognitive symptoms. As proof of concept, we developed a novel mouse model where developmental NMDAR deficits can be ameliorated by inducible Cre recombinase. Rescue of NMDARs in either adolescence or adulthood yields surprisingly strong improvements in higher-order behavior. Similar levels of behavioral plasticity are observed regardless of intervention age, with degree of plasticity dependent on the specific behavioral circuit. These results reveal higher-order brain function as amenable to treatment in adulthood a...
The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradati... more The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, β-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-...
In Parkinson's disease, dopamine‐containing nigrostriatal neurons undergo profound degenerati... more In Parkinson's disease, dopamine‐containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build‐up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH‐over‐expressing mice (TH‐HI) using a BAC‐transgenic approach that results in over‐expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH‐HI mice had a 3‐fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH‐HI mice showed increased striatal production of H2O2 and reduced glutathione levels. In addition, TH‐HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4‐dihydroxyphenylacetaldehyde and 5‐S‐cysteinyl‐dopamine and were more susceptible than wild‐type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.
Background Abnormalities of cellular signaling are well characterized in neuropsychiatric illness... more Background Abnormalities of cellular signaling are well characterized in neuropsychiatric illnesses, including schizophrenia. Changes in signaling pathways reflect the underlying genetic, environmental, and epigenetic perturbations driving disease phenotypes. A shortcoming of most signaling studies is a focus on one or a few protein kinases at a time, a limitation since protein kinases work in networks with other kinases, phosphatases, and regulatory molecules to effect signaling events. We addressed this challenge by employing a kinome array platform that simultaneously measures protein kinase activity at hundreds of reporter peptide substrates. We then developed a novel bioinformatics pipeline to identify protein kinase nodes, signaling networks, upstream biological pathways, and drug candidates that “reverse” kinomic disease signatures. Methods Postmortem DLPFC brain samples from subjects with schizophrenia (n = 20 per group, 10 males and 10 females per group), were compared to a...
Social isolation raises the risk for mood disorders associated with serotonergic disruption. Yet,... more Social isolation raises the risk for mood disorders associated with serotonergic disruption. Yet, the underlying mechanisms by which the stress of social isolation increases risk are not well understood. Men and women are differently vulnerable; however, this modulating role of sex is challenging to study in humans under carefully controlled conditions. Therefore, we investigated this question in mice of both sexes, asking how the long-term stress of social isolation (from weaning into adulthood) affects the excitability of serotonin neurons in the dorsal raphe nucleus as well as mouse behaviour. The electrophysiological experiments and the first set of behavioural tests were conducted in young adult mice, with additional behavioural assays completed as the mice matured to assess the stability of their behavioural phenotype. We found that social isolation exerted seemingly-opposite effects in male and female mice, relative to their respective group-housed littermate controls. This distinctive pattern was observed for the effect of social isolation on the control of serotonergic neuron excitability via the SK family of calcium-activated potassium channels. Furthermore, we observed a similar and consistent pattern on tests relevant to assessing the efficacy of anti-depressant medicines, including the forced swim test, the novelty-suppressed feeding test, and the sucrose preference test. These findings underscore the concept that stress-elicited illness manifests distinctly in males and females and that treatments aimed at restoring serotonergic function may require a sex-specific approach.
NMDA receptors are important for cognition and are implicated in neuropsychiatric disorders. GluN... more NMDA receptors are important for cognition and are implicated in neuropsychiatric disorders. GluN1 knockdown (GluN1KD) mice have reduced NMDA receptor levels, striatal spine density deficits, and cognitive impairments. However, how NMDA depletion leads to these effects is unclear. Since Rho GTPases are known to regulate spine density and cognition, we examined the levels of RhoA, Rac1, and Cdc42 signaling proteins. Striatal Rac1-pathway components are reduced in GluN1KD mice, with Rac1 and WAVE-1 deficits at 6 and 12 weeks of age. Concurrently, medium spiny neuron (MSN) spine density deficits are present in mice at these ages. To determine whether WAVE-1 deficits were causal or compensatory in relation to these phenotypes, we intercrossed GluN1KD mice with WAVE-1 overexpressing (WAVE-Tg) mice to restore WAVE-1 levels. GluN1KD-WAVE-Tg hybrids showed rescue of striatal WAVE-1 protein levels and MSN spine density, as well as selective behavioral rescue in the Y-maze and 8-arm radial ma...
NMDA receptor dysfunction is central to the encephalopathies caused by missense mutations in the ... more NMDA receptor dysfunction is central to the encephalopathies caused by missense mutations in the NMDA receptor subunit genes. Missense variants of GRIN1, GRIN2A, and GRIN2B cause similar syndromes with varying severity of intellectual impairment, autism, epilepsy, and motor dysfunction. To gain insight into possible biomarkers of NMDAR hypofunction, we asked whether a loss-of-function variant in the Grin1 gene would cause structural changes in the brain that could be detected by MRI. We also studied the developmental trajectory of these changes to determine whether structural changes coincided with reported cognitive impairments in the mice. We performed magnetic resonance imaging in male Grin1-/- knockdown mice (GluN1KD) that were three, six, or twelve weeks old. Deformation-based morphometry was used to assess neuroanatomical differences. Volumetric reductions were detected in substantia nigra and striatum of GluN1KD mice at all ages. Changes in limbic structures were only evident at six weeks of age. Reductions in white matter volumes were first evident at three weeks, and additional deficits were detected at six and twelve weeks. FluoroJade immunofluorescence revealed degenerating neurons in twelve-week old GluN1KD mice. We conclude that Grin1 loss-of-function mutations cause volume reductions in dopaminergic structures early in development, while changes to limbic and white matter structures are delayed and are more pronounced in post-adolescent ages. The evidence of degenerating neurons in the mature brain indicates an ongoing process of cell loss as a consequence of NMDAR hypofunction.
Schizophrenia is a devastating illness that affects over 2 million people in the United States an... more Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays. Laser-capture microdissection-quantitative polymerase chain reaction was used to examine these pathways at the cellular level. We found decreases in hexokinase (HXK) and phosphofructokinase (PFK) activity in the DLPFC, as well as decreased PFK1 mRNA expression. In pyramidal neurons, we found an increase in monocarboxylate transporter 1 mRNA expression, and decreases in HXK1, PFK1, glucose transporter 1 (GLUT1), and GLUT3 mRNA expression. These results suggest abnormal bioenergetic function, as well as a neuron-specific defect in glucose utilization, in the DLPFC in schizophrenia.
Postoperative delirium is associated with poor long-term outcomes and increased mortality. Genera... more Postoperative delirium is associated with poor long-term outcomes and increased mortality. General anesthetic drugs may contribute to delirium because they increase cell-surface expression and function of α5 subunit-containing γ-aminobutyric acid type A receptors, an effect that persists long after the drugs have been eliminated. Dexmedetomidine, an α2 adrenergic receptor agonist, prevents delirium in patients and reduces cognitive deficits in animals. Thus, it was postulated that dexmedetomidine prevents excessive function of α5 γ-aminobutyric acid type A receptors. Injectable (etomidate) and inhaled (sevoflurane) anesthetic drugs were studied using cultured murine hippocampal neurons, cultured murine and human cortical astrocytes, and ex vivo murine hippocampal slices. γ-Aminobutyric acid type A receptor function and cell-signaling pathways were studied using electrophysiologic and biochemical methods. Memory and problem-solving behaviors were also studied. The etomidate-induced s...
Synaptic neurotransmission relies on maintenance of the synapse and meeting the energy demands of... more Synaptic neurotransmission relies on maintenance of the synapse and meeting the energy demands of neurons. Defects in excitatory and inhibitory synapses have been implicated in schizophrenia, likely contributing to positive and negative symptoms as well as impaired cognition. Recently, accumulating evidence has suggested that bioenergetic systems, important in both synaptic function and cognition, are abnormal in psychiatric illnesses such as schizophrenia. Animal models of synaptic dysfunction demonstrated endophenotypes of schizophrenia as well as bioenergetic abnormalities. We report findings on the bioenergetic interplay of astrocytes and neurons and discuss how dysregulation of these pathways may contribute to the pathogenesis of schizophrenia, highlighting metabolic systems as important therapeutic targets.
The identification of circulating autoantibodies against neuronal receptors in neuropsychiatric d... more The identification of circulating autoantibodies against neuronal receptors in neuropsychiatric disorders has fostered new conceptual and clinical frameworks. However, detection reliability, putative presence in different diseases and in health have raised questions about potential pathogenic mechanism mediated by autoantibodies. Using a combination of single molecule-based imaging approaches, we here ascertain the presence of circulating autoantibodies against glutamate NMDA receptor (NMDAR-Ab) in about 20% of psychotic patients diagnosed with schizophrenia and very few healthy subjects. NMDAR-Ab from patients and healthy subjects do not compete for binding on native receptor. Strikingly, NMDAR-Ab from patients, but not from healthy subjects, specifically alter the surface dynamics and nanoscale organization of synaptic NMDAR and its anchoring partner the EphrinB2 receptor in heterologous cells, cultured neurons and in mouse brain. Functionally, only patients' NMDAR-Ab prevent ...
NMDA receptors (NMDAR) are important in the formation of activity-dependent connections in the br... more NMDA receptors (NMDAR) are important in the formation of activity-dependent connections in the brain. In sensory pathways, NMDAR disruption during discrete developmental periods has enduring effects on wiring and function. Yet, it is not clear whether NMDAR-limited critical periods exist for higher-order circuits governing mood and cognition. This question is urgent for neurodevelopmental disorders, like schizophrenia, that have NMDAR hypofunction and treatment-resistant cognitive symptoms. As proof of concept, we developed a novel mouse model where developmental NMDAR deficits can be ameliorated by inducible Cre recombinase. Rescue of NMDARs in either adolescence or adulthood yields surprisingly strong improvements in higher-order behavior. Similar levels of behavioral plasticity are observed regardless of intervention age, with degree of plasticity dependent on the specific behavioral circuit. These results reveal higher-order brain function as amenable to treatment in adulthood a...
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Papers by Amy Ramsey