IU Indianapolis ScholarWorks :: Browsing by Author "Brustovetsky, Nickolay"

Browsing by Author "Brustovetsky, Nickolay"

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Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol
(Oxford University Press, 2016-08) Patel, Reesha R.; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Cummins, Theodore R.; Biology, School of Science
Resurgent sodium currents arise from channel reopening during repolarisation, and are predicted to increase neuronal excitability. Patel et al. show that epilepsy-associated mutations in the voltage-gated sodium channel Nav1.6, but not Nav1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy., Resurgent sodium currents arise from channel reopening during repolarisation, and are predicted to increase neuronal excitability. Patel et al. show that epilepsy-associated mutations in the voltage-gated sodium channel Nav1.6, but not Nav1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy. , Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 μM Navβ4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.
Ca(2+) handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease
(Wiley, 2015-08) Pellman, Jessica J.; Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Department of Pharmacology and Toxicology, IU School of Medicine
We investigated Ca(2+) handling in isolated brain synaptic and non-synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non-synaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca(2+) uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca(2+) capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca(2+) uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen because of mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca(2+) followed by recovery to near resting levels. Following recovery of cytosolic Ca(2+), mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca(2+), suggesting similar Ca(2+) release and, consequently, Ca(2+) loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca(2+) handling in brain mitochondria of YAC128 mice. We demonstrate that mutant huntingtin (mHtt) binds to brain synaptic and nonsynaptic mitochondria and the amount of mitochondria-bound mHtt correlates with increased mitochondrial Ca(2+) uptake capacity. We propose that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage.
Collapsin response mediator protein 2 (CRMP2) interacts with N-methyl-D-aspartate (NMDA) receptor and Na+/Ca2+ exchanger and regulates their functional activity
(ASBMB, 2014-03-14) Brustovetsky, Tatiana; Pellman, Jessica J.; Yang, Xiao-Fang; Khanna, Rajesh; Brustovetsky, Nickolay; Department of Pharmacology and Toxicology, IU School of Medicine
Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.
CRMP2 Is Involved in Regulation of Mitochondrial Morphology and Motility in Neurons
(MDPI, 2021-10-17) Brustovetsky, Tatiana; Khanna, Rajesh; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of Medicine
Regulation of mitochondrial morphology and motility is critical for neurons, but the exact mechanisms are unclear. Here, we demonstrate that these mechanisms may involve collapsin response mediator protein 2 (CRMP2). CRMP2 is attached to neuronal mitochondria and binds to dynamin-related protein 1 (Drp1), Miro 2, and Kinesin 1 light chain (KLC1). Treating neurons with okadaic acid (OA), an inhibitor of phosphatases PP1 and PP2A, resulted in increased CRMP2 phosphorylation at Thr509/514, Ser522, and Thr555, and augmented Drp1 phosphorylation at Ser616. The CRMP2-binding small molecule (S)-lacosamide ((S)-LCM) prevented an OA-induced increase in CRMP2 phosphorylation at Thr509/514 and Ser522 but not at Thr555, and also failed to alleviate Drp1 phosphorylation. The increased CRMP2 phosphorylation correlated with decreased CRMP2 binding to Drp1, Miro 2, and KLC1. (S)-LCM rescued CRMP2 binding to Drp1 and Miro 2 but not to KLC1. In parallel with CRMP2 hyperphosphorylation, OA increased mitochondrial fission and suppressed mitochondrial traffic. (S)-LCM prevented OA-induced alterations in mitochondrial morphology and motility. Deletion of CRMP2 with a small interfering RNA (siRNA) resulted in increased mitochondrial fission and diminished mitochondrial traffic. Overall, our data suggest that the CRMP2 expression level and phosphorylation state are involved in regulating mitochondrial morphology and motility in neurons.
CRMP2 Participates in Regulating Mitochondrial Morphology and Motility in Alzheimer’s Disease
(MDPI, 2023-04-29) Brustovetsky, Tatiana; Khanna, Rajesh; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of Medicine
Mitochondrial bioenergetics and dynamics (alterations in morphology and motility of mitochondria) play critical roles in neuronal reactions to varying energy requirements in health and disease. In Alzheimer’s disease (AD), mitochondria undergo excessive fission and become less motile. The mechanisms leading to these alterations are not completely clear. Here, we show that collapsin response mediator protein 2 (CRMP2) is hyperphosphorylated in AD and that is accompanied by a decreased interaction of CRMP2 with Drp1, Miro 2, and Mitofusin 2, which are proteins involved in regulating mitochondrial morphology and motility. CRMP2 was hyperphosphorylated in postmortem brain tissues of AD patients, in brain lysates, and in cultured cortical neurons from the double transgenic APP/PS1 mice, an AD mouse model. CRMP2 hyperphosphorylation and dissociation from its binding partners correlated with increased Drp1 recruitment to mitochondria, augmented mitochondrial fragmentation, and reduced mitochondrial motility. (S)-lacosamide ((S)-LCM), a small molecule that binds to CRMP2, decreased its phosphorylation at Ser 522 and Thr 509/514, and restored CRMP2′s interaction with Miro 2, Drp1, and Mitofusin 2. This was paralleled by decreased Drp1 recruitment to mitochondria, diminished mitochondrial fragmentation, and improved motility of the organelles. Additionally, (S)-LCM-protected cultured cortical AD neurons from cell death. Thus, our data suggest that CRMP2, in a phosphorylation-dependent manner, participates in the regulation of mitochondrial morphology and motility, and modulates neuronal survival in AD.
Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria
(American Society for Biochemistry and Molecular Biology, 2018-10-05) Hamilton, James; Brustovetsky, Tatiana; Rysted, Jacob E.; Lin, Zhihong; Usachev, Yuriy M.; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of Medicine
Ca2+ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca2+ uptake machinery. The MCU is a Ca2+-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca2+ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca2+ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca2+ ionophore ETH129 significantly accelerated Ca2+ uptake. The residual Ca2+ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na+/Ca2+ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca2+ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca2+ uptake pathway in brain mitochondria that mediates residual Ca2+ influx and induction of PTP in a fraction of the mitochondrial population.
The effect of mitochondrial calcium uniporter and cyclophilin D knockout on resistance of brain mitochondria to Ca2+-induced damage
(Elsevier, 2021) Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of Medicine
The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major signs of Ca2+-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca2+ uptake and diminished extent of PTP induction. The Ca2+ uptake by mitochondria from CyD-KO mice was increased compared with mitochondria from WT mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca2+, but it did not prevent Ca2+-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca2+, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca2+-activated mitochondrial phospholipase A2, attenuated and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca2+.
The effects of CaMKII signaling on neuronal viability
(2013-12-10) Ashpole, Nicole M.; Hudmon, Andrew; Brustovetsky, Nickolay; Hurley, Thomas D., 1961-; Russell, Weihua Lee, 1956-; Oxford, G. S.
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical modulator of synaptic function, plasticity, and learning and memory. In neurons and astrocytes, CaMKII regulates cellular excitability, cytoskeletal structure, and cell metabolism. A rapid increase in CaMKII activity is observed within the first few minutes of ischemic stroke in vivo; this calcium-dependent process is also observed following glutamate stimulation in vitro. Activation of CaMKII during pathological conditions is immediately followed by inactivation and aggregation of the kinase. The extent of CaMKII inactivation is directly correlated with the extent of neuronal damage. The studies presented here show that these fluctuations in CaMKII activity are not correlated with neuronal death; rather, they play a causal role in neuronal death. Pharmacological inhibition of CaMKII in the time immediately surrounding glutamate insult protects cultured cortical neurons from excitotoxicity. Interestingly, pharmacological inhibition of CaMKII during excitotoxic insult also prevents the aggregation and prolonged inactivation of the kinase, suggesting that CaMKII activity during excitotoxic glutamate signaling is detrimental to neuronal viability because it leads to a prolonged loss of CaMKII activity, culminating in neuronal death. In support of this, CaMKII inhibition in the absence of excitotoxic insult induces cortical neuron apoptosis by dysregulating intracellular calcium homeostasis and increasing excitatory glutamate signaling. Blockade of the NMDA-receptors and enzymatic degradation of the extracellular glutamate signal affords neuroprotection from CaMKII inhibition-induced toxicity. Co-cultures of neurons and glutamate-buffering astrocytes also exhibit this slow-induced excitotoxicity, as CaMKII inhibitors reduce glutamate uptake within the astrocytes. CaMKII inhibition also dysregulates calcium homeostasis in astrocytes and leads to increased ATP release, which was neurotoxic when applied to naïve cortical neurons. Together, these findings indicate that during aberrant calcium signaling, the activation of CaMKII is toxic because it supports aggregation and prolonged inactivation of the kinase. Without CaMKII activity, neurons and astrocytes release stores of transmitters that further exacerbate neuronal toxicity.
Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock
(Elsevier, 2015-06-18) Mantel, Charlie R.; O'Leary, Heather A.; Chitteti, Brahmananda R.; Huang, XinXin; Cooper, Scott; Hangoc, Giao; Brustovetsky, Nickolay; Srour, Edward F.; Lee, Man-Ryul; Messina-Graham, Steve; Haas, David M.; Falah, Nadia; Kapur, Reuben; Pelus, Louis M.; Bardeesy, Nabeel; Fitamant, Julien; Ivan, Mircea; Kim, Kye-Seong; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of Medicine
Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.
Epilepsy Mutations in Different Regions of the Nav1.2 Channel Cause Distinct Biophysical Effects
(2020-06) Mason, Emily R.; Cummins, Theodore; Sullivan, William J., Jr.; Brustovetsky, Nickolay; Sheets, Patrick; Hashino, Eri
While most cases of epilepsy respond well to common antiepileptic drugs, many genetically-driven epilepsies are refractory to conventional antiepileptic drugs. Over 250 mutations in the Nav1.2 gene (SCN2A) have been implicated in otherwise idiopathic cases of epilepsy, many of which are refractory to traditional antiepileptic drugs. Few of these mutations have been studied in vitro to determine their biophysical effects on the channels, which could reveal why the effects of some are refractory to traditional antiepileptic drugs. The goal of this dissertation was to characterize multiple epilepsy mutations in the SCN2A gene, which I hypothesized would have distinct biophysical effects on the channel’s function. I used patch-clamp electrophysiology to determine the biophysical effects of three SCN2A epilepsy mutations (R1882Q, R853Q, and L835F). Wild-type (WT) or mutant human SCN2A cDNAs were expressed in human embryonic kidney (HEK) cells and subjected to a panel of electrophysiological assays. I predicted that the net effect of each of these mutations was enhancement of channel function; my results regarding the L835F and R1882Q mutations supported this hypothesis. Both mutations enhance persistent current, and R1882Q also impairs fast inactivation. However, examination of the same parameters for the R853Q mutation suggested a decrease of channel function. I hypothesized that the R853Q mutation creates a gating pore in the channel structure through which sodium leaks into the cell when the channel is in its resting conformation. This hypothesis was supported by electrophysiological data from Xenopus oocytes, which showed a significant voltage-dependent leak current at negative potentials when they expressed the R853Q mutant channels. This was absent in oocytes expressing WT channels. Overall, these results suggest that individual mutations in the SCN2A gene generate epilepsy via distinct biophysical effects that may require novel and/or tailored pharmacotherapies for effective management.

Browsing by Author "Brustovetsky, Nickolay"

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