Browsing by Subject "Neuron"
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Item APOE4 Drives Impairment in Astrocyte-Neuron Coupling in Alzheimer's Disease and Works Through Mechanisms in Early Disease to Influence Pathology(2023-05) Brink, Danika Marie Tumbleson; Lamb, Bruce; Bissel, Stephanie; Herbert, Brittney-Shea; Landreth, Gary; Puntambekar, Shweta; Saykin, Andrew; Zhang, ChiAlzheimer’s disease (AD) is a neurodegenerative disorder resulting in progressive memory loss, brain atrophy, and eventual death. AD pathology is characterized by the accumulation of neurotoxic amyloid-beta (Aβ) plaques, synapse loss, neurofibrillary tangles (NFTs), and neurodegeneration. The APOE4 allele is associated with a 3-fold increased risk for AD and results in increased Aβ plaque deposition, reduced Aβ clearance, and reduced synaptic plasticity. Although APOE expression is upregulated in microglia in AD, APOE is expressed primarily by astrocytes in the CNS. It is not well understood how astrocytic APOE drives the mechanisms that result in worsened AD outcomes. Here, digital spatial profiling and bioinformatics data suggest that APOE4 causes transcriptional dysregulation in early AD and may disrupt neuronal processes via astrocytes. Whole transcriptome data from plaque and non-plaque regions in the cortices and hippocampus of 4- and 8-month-old AD model mice expressing humanized APOE4/4 or APOE3/3 (control) were analyzed. Transcriptional dysregulation was increased in APOE4/4 AD mice compared to that in APOE3/3 at 4 but not 8 months of age, suggesting that early dysregulation of APOE4-driven disease mechanisms may shape degenerative outcomes in late-stage AD. Additionally, APOE4/4 potentially functions via plaque-independent mechanisms to influence neuronal function in early AD before the onset of pathology. Single-nuclei RNA sequencing data were obtained from human post-mortem astrocytes and the bioinformatic analyses revealed a novel astrocyte subtype that highly expresses several top genes involved in functional alterations associated with APOE4, including neuronal generation, development, and differentiation, and synaptic transmission and organization. Overall, our findings indicate that APOE4 may drive degenerative outcomes through the presented astrocyte candidate pathways. These pathways represent potential targets for investigations into early intervention strategies for APOE4/4 patients.Item 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 MedicineWe 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.Item Circadian rhythms in bipolar disorder patient-derived neurons predict lithium response: Preliminary studies(Springer Nature, 2021) Mishra, Himanshu K.; Ying, Noelle M.; Luis, Angelica; Wei, Heather; Nguyen, Metta; Nakhla, Timothy; Vandenburgh, Sara; Alda, Martin; Berrettini, Wade H.; Brennand, Kristen J.; Calabrese, Joseph R.; Coryell, William H.; Frye, Mark A.; Gage, Fred H.; Gershon, Elliot S.; McInnis, Melvin G.; Nievergelt, Caroline M.; Nurnberger, John I.; Shilling, Paul D.; Oedegaard, Ketil J.; Zandi, Peter P.; The Pharmacogenomics of Bipolar Disorder Study; Kelsoe, John R.; Welsh, David K.; McCarthy, Michael J.; Psychiatry, School of MedicineBipolar disorder (BD) is a neuropsychiatric illness defined by recurrent episodes of mania/hypomania, depression and circadian rhythm abnormalities. Lithium is an effective drug for BD, but 30–40% of patients fail to respond adequately to treatment. Previous work has demonstrated that lithium affects the expression of “clock genes” and that lithium responders (Li-R) can be distinguished from non-responders (Li-NR) by differences in circadian rhythms. However, circadian rhythms have not been evaluated in BD patient neurons from Li-R and Li-NR. We used induced pluripotent stem cells (iPSCs) to culture neuronal precursor cells (NPC) and glutamatergic neurons from BD patients characterized for lithium responsiveness and matched controls. We identified strong circadian rhythms in Per2-luc expression in NPCs and neurons from controls and Li-R, but NPC rhythms in Li-R had a shorter circadian period. Li-NR rhythms were low-amplitude and profoundly weakened. In NPCs and neurons, expression of PER2 was higher in both BD groups compared to controls. In neurons, PER2 protein levels were higher in BD than controls, especially in Li-NR samples. In single cells, NPC and neuron rhythms in both BD groups were desynchronized compared to controls. Lithium lengthened period in Li-R and control neurons but failed to alter rhythms in Li-NR. In contrast, temperature entrainment increased amplitude across all groups, and partly restored rhythms in Li-NR neurons. We conclude that neuronal circadian rhythm abnormalities are present in BD and most pronounced in Li-NR. Rhythm deficits in BD may be partly reversible through stimulation of entrainment pathways.Item Critical and Ictal Phases in Simulated EEG Signals on a Small-World Network(Frontiers Media, 2021-01-08) Nemzer, Louis R.; Cravens, Gary D.; Worth, Robert M.; Motta, Francis; Placzek, Andon; Castro, Victor; Lou, Jennie Q.; Mathematical Sciences, School of ScienceHealthy brain function is marked by neuronal network dynamics at or near the critical phase, which separates regimes of instability and stasis. A failure to remain at this critical point can lead to neurological disorders such as epilepsy, which is associated with pathological synchronization of neuronal oscillations. Using full Hodgkin-Huxley (HH) simulations on a Small-World Network, we are able to generate synthetic electroencephalogram (EEG) signals with intervals corresponding to seizure (ictal) or non-seizure (interictal) states that can occur based on the hyperexcitability of the artificial neurons and the strength and topology of the synaptic connections between them. These interictal simulations can be further classified into scale-free critical phases and disjoint subcritical exponential phases. By changing the HH parameters, we can model seizures due to a variety of causes, including traumatic brain injury (TBI), congenital channelopathies, and idiopathic etiologies, as well as the effects of anticonvulsant drugs. The results of this work may be used to help identify parameters from actual patient EEG or electrocorticographic (ECoG) data associated with ictogenesis, as well as generating simulated data for training machine-learning seizure prediction algorithms.Item 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 MedicineRegulation 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.Item 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.Item Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects(BMC, 2018-07-11) Mysore, Keshava; Li, Ping; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineBACKGROUND: Sophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms. However, we lack comparable genetic tools for analysis of non-model organisms, including Aedes aegypti, a vector mosquito which displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) recently facilitated genome-wide discovery of putative A. aegypti cis-regulatory elements (CREs), many of which could be used to manipulate gene expression in mosquito neurons and other tissues. The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system, a tissue of vector importance. RESULTS: Eight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. Six A. aegypti strains, including strains displaying transgene expression in all ORNs, subsets of these neurons, or in a sex-specific fashion, were isolated. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. CRE activity in A. aegypti was found to be comparable to that observed in D. melanogaster reporter assays. CONCLUSIONS: These results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.Item In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging(2014-02-25) Freeman, Kim Renee; Rubart-von der Lohe, Michael; Atkinson, Simon; Hurley, Thomas D., 1961-; Gattone II, Vincent H.The sympathetic nervous system strongly modulates the contractile and electrical function of the heart. The anatomical underpinnings that enable a spatially and temporally coordinated dissemination of sympathetic signals within the cardiac tissue are only incompletely characterized. In this work we took the first step of unraveling the in situ 3D microarchitecture of the cardiac sympathetic nervous system. Using a combination of two-photon excitation fluorescence microscopy and computer-assisted image analyses, we reconstructed the sympathetic network in a portion of the left ventricular epicardium from adult transgenic mice expressing a fluorescent reporter protein in all peripheral sympathetic neurons. The reconstruction revealed several organizational principles of the local sympathetic tree that synergize to enable a coordinated and efficient signal transfer to the target tissue. First, synaptic boutons are aligned with high density along much of axon-cell contacts. Second, axon segments are oriented parallel to the main, i.e., longitudinal, axes of their apposed cardiomyocytes, optimizing the frequency of transmitter release sites per axon/per cardiomyocyte. Third, the local network was partitioned into branched and/or looped sub-trees which extended both radially and tangentially through the image volume. Fourth, sub-trees arrange to not much overlap, giving rise to multiple annexed innervation domains of variable complexity and configuration. The sympathetic network in the epicardial border zone of a chronic myocardial infarction was observed to undergo substantive remodeling, which included almost complete loss of fibers at depths >10 µm from the surface, spatially heterogeneous gain of axons, irregularly shaped synaptic boutons, and formation of axonal plexuses composed of nested loops of variable length. In conclusion, we provide, to the best of our knowledge, the first in situ 3D reconstruction of the local cardiac sympathetic network in normal and injured mammalian myocardium. Mapping the sympathetic network connectivity will aid in elucidating its role in sympathetic signal transmisson and processing.Item Involvement of CRMP2 in Regulation of Mitochondrial Morphology and Motility in Huntington’s Disease(MDPI, 2021-11-15) Brustovetsky, Tatiana; Khanna, Rajesh; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of MedicineMitochondrial morphology and motility (mitochondrial dynamics) play a major role in the proper functioning of distant synapses. In Huntington's disease (HD), mitochondria become fragmented and less motile, but the mechanisms leading to these changes are not clear. Here, we found that collapsin response mediator protein 2 (CRMP2) interacted with Drp1 and Miro 2, proteins involved in regulating mitochondrial dynamics. CRMP2 interaction with these proteins inversely correlated with CRMP2 phosphorylation. CRMP2 was hyperphosphorylated in postmortem brain tissues of HD patients, in human neurons derived from induced pluripotent stem cells from HD patients, and in cultured striatal neurons from HD mouse model YAC128. At the same time, CRMP2 interaction with Drp1 and Miro 2 was diminished in HD neurons. The CRMP2 hyperphosphorylation and dissociation from Drp1 and Miro 2 correlated with increased fission and suppressed motility. (S)-lacosamide ((S)-LCM), a small molecule that binds to CRMP2, decreased its phosphorylation at Thr 509/514 and Ser 522 and rescued CRMP2's interaction with Drp1 and Miro 2. This was accompanied by reduced mitochondrial fission and enhanced mitochondrial motility. Additionally, (S)-LCM exerted a neuroprotective effect in YAC128 cultured neurons. Thus, our data suggest that CRMP2 may regulate mitochondrial dynamics in a phosphorylation-dependent manner and modulate neuronal survival in HD.Item A New Mechanism of Serotonin Transporter Regulation by Simvastatin and the Isoprenylation Pathway(2021-07) Deveau, Carmen Marie; Yamamoto, Bryan K.; Sheets, Patrick L.; Sullivan, William J., Jr.; Atwood, Brady K.; Brustovetsky, NickolayThe serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.