Browsing by Author "Bernabe, Cristian S."

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    Characterization of the A1527G variant of ABCA7 in an animal model for late‐onset Alzheimer’s disease
    (Wiley, 2025-01-03) Bernabe, Cristian S.; Kotredes, Kevin P.; Pandey, Ravi S.; Carter, Gregory W.; Sasner, Michael; Oblak, Adrian L.; Howell, Gareth R.; Lamb, Bruce T.; Territo, Paul R.; MODEL-AD consortium; Medicine, School of Medicine
    Background: Genome‐wide association studies (GWAS) identified the ATP binding cassette subfamily A member 7 (ABCA7) gene as increasing risk for Alzheimer’s disease (AD). ABC proteins transport various molecules across extra and intra‐cellular membranes. ABCA7 is part of the ABC1 subfamily and is expressed in brain cells including neurons, astrocytes, microglia, endothelial cells and pericytes. However, the mechanisms by which variations in ABCA7 increase risk for AD are not known. Method: The IU/JAX/PITT MODEL‐AD Center identified the A1527G variant in ABCA7 (ABCA7*A1527G) as a putative LOAD risk factor. CRISPR/CAS9 was first used to introduce Abca7*A1527G variant to B6.APOE4.Trem2*R47H (LOAD1) mice to assess the transcriptional profiling on brain hemispheres from different ages. The Abca7*A1527G was then incorporated into B6.APOE4.Trem2*R47H.hAb (LOAD2) mice to further evaluate its contribution to LOAD. Female and male LOAD2.Abca7*A1527G and LOAD2 mice were characterized at 4, 12, and 24 months using the following phenotyping pipeline: behavior, PET/CT, multi‐omics, fluid biomarkers, electrophysiology, cognition, and neuropathology. Result: Brain transcriptional profiling showed that Abca7*A1527G induced changes in gene expression that are similar to some of those observed in human AD (e.g., granulocyte/neutrophil migration, and insulin receptor signaling). LOAD2.Abca7*A1527G showed no aging cognitive deficit but did show significant sex‐ and region‐dependent increases in brain glycolysis paralleled by reduced tissue perfusion yielding progressive age‐related uncoupled phenotypes between 4‐12 and 4‐24 months. While multi‐resolution consensus clustering of regional covariance matrices revealed an increase in cluster number and organization in LOAD2.Abca7*A1527G over LOAD2 for both sexes at 4 months, the cluster number and complexity were reduced by 24 months. Importantly, LOAD2.Abca7*A1527G, but not LOAD2, displayed a similar age‐dependent reduction in cluster number for both sexes. Consistent with the uncoupled phenotype, IL6, IL10, and TNFα were elevated in plasma with genotype, but were not age dependent. Conversely, brain levels of IL4, IL12, TNFα, and CXCL1 were decreased, whereas IL2 and IL10 were elevated in LOAD2.Abca7*A1527G relative to LOAD2. Lastly, assessment of plasma levels of Ab40‐Ab42 revealed an age‐dependent increase in both genotypes. Conclusion: Data collected to date support a model whereby variations in ABCA7 exert risk for AD through interactions between cerebrovasculature, microglia, and peripheral immune cells.
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    Evaluation of Low versus High Volume per Minute Displacement CO₂ Methods of Euthanasia in the Induction and Duration of Panic-Associated Behavior and Physiology
    (MDPI, 2016-08) Hickman, Debra L.; Fitz, Stephanie D.; Bernabe, Cristian S.; Caliman, Izabela F.; Haulcomb, Melissa M.; Federici, Lauren M.; Shekhar, Anatatha; Johnson, Philip L.; Department of Cellular & Integrative Physiology, IU School of Medicine
    Current recommendations for the use of CO ₂ as a euthanasia agent for rats require the use of gradual fill protocols (such as 10% to 30% volume displacement per minute) in order to render the animal insensible prior to exposure to levels of CO ₂ that are associated with pain. However, exposing rats to CO ₂ , concentrations as low as 7% CO ₂ are reported to cause distress and 10%-20% CO ₂ induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO ₂ concentrations are at least 40%. This suggests that the use of the currently recommended low flow volume per minute displacement rates create a situation where rats are exposed to concentrations of CO ₂ that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO ₂ for a prolonged period of time as compared to room air controls. It demonstrated that rats exposed to this experimental condition displayed clinical signs consistent with significantly increased panic-associated behavior and physiology during CO ₂ exposure. When atmospheric air was then again delivered, there was a robust increase in respiration rate that coincided with rats moving to the air intake. The rats exposed to CO ₂ also displayed behaviors consistent with increased anxiety in the behavioral testing that followed the exposure. Next, this study assessed the behavioral and physiologic responses of rats that were euthanized with 100% CO ₂ infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO ₂ and oxygen in the euthanasia chamber and the behavioral responses of the rats suggest that the use of the very low flow volume per minute displacement rate (10%) may prolong the duration of panicogenic ranges of ambient CO ₂ , while the use of the higher flow volume per minute displacement rate (100%) increases agitation. Therefore, of the volume displacement per minute rates evaluated, this study suggests that 30% minimizes the potential pain and distress experienced by the animal.
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    Identification of a novel perifornical-hypothalamic-area-projecting serotonergic system that inhibits innate panic and conditioned fear responses
    (Springer Nature, 2024-01-25) Bernabe, Cristian S.; Caliman, Izabela F.; de Abreu, Aline R. R.; Molosh, Andrei I.; Truitt, William A.; Shekhar, Anantha; Johnson, Philip L.; Anatomy, Cell Biology and Physiology, School of Medicine
    The serotonin (5-HT) system is heavily implicated in the regulation of anxiety and trauma-related disorders such as panic disorder and post-traumatic stress disorder, respectively. However, the neural mechanisms of how serotonergic neurotransmission regulates innate panic and fear brain networks are poorly understood. Our earlier studies have identified that orexin (OX)/glutamate neurons within the perifornical hypothalamic area (PFA) play a critical role in adaptive and pathological panic and fear. While site-specific and electrophysiological studies have shown that intracranial injection and bath application of 5-HT inhibits PFA neurons via 5-HT1a receptors, they largely ignore circuit-specific neurotransmission and its physiological properties that occur in vivo. Here, we investigate the role of raphe nuclei 5-HT inputs into the PFA in panic and fear behaviors. We initially confirmed that photostimulation of glutamatergic neurons in the PFA of rats produces robust cardioexcitation and flight/aversive behaviors resembling panic-like responses. Using the retrograde tracer cholera toxin B, we determined that the PFA receives discrete innervation of serotonergic neurons clustered in the lateral wings of the dorsal (lwDRN) and in the median (MRN) raphe nuclei. Selective lesions of these serotonergic projections with saporin toxin resulted in similar panic-like responses during the suffocation-related CO2 challenge and increased freezing to fear-conditioning paradigm. Conversely, selective stimulation of serotonergic fibers in the PFA attenuated both flight/escape behaviors and cardioexcitation responses elicited by the CO2 challenge and induced conditioned place preference. The data here support the hypothesis that PFA projecting 5-HT neurons in the lwDRN/MRN represents a panic/fear-off circuit and may also play a role in reward behavior.
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    LOAD1 and LOAD2: Longitudinal characterization of mouse models carrying human‐relevant risk factors for late‐onset Alzheimer’s disease
    (Wiley, 2025-01-03) Bernabe, Cristian S.; Kotredes, Kevin P.; Pandey, Ravi S.; Carter, Gregory W.; Sasner, Michael; Oblak, Adrian L.; Howell, Gareth R.; Lamb, Bruce T.; Territo, Paul R.; MODEL-AD consortium; Medicine, School of Medicine
    Background: Alzheimer’s disease (AD) is the most common form of dementia, yet the effectiveness of disease‐modifying interventions is inconclusive. Although exceptional progress in our understanding of AD neuropathology has been made via transgenic mouse models bearing familial mutations, they often fail to recapitulate the disease progression of late‐onset AD (LOAD). To address this, MODEL‐AD has developed LOAD1 and LOAD2 mouse models which carry the most common human‐relevant risk factors for AD. In‐depth, longitudinal characterization through aging will reveal useful insights to develop novel treatments for LOAD. Method: APOEε4 and Trem2*R47H, two risk factors for LOAD, were incorporated into C57BL/6J mice to produce the double homozygous LOAD1 model, whereas LOAD2 also contains humanized amyloid‐beta (Aβ) yielding a triple homozygous model. Cohorts of LOAD1 and LOAD2 mice were aged on multiple sites to 4‐, 12‐, 18‐, and 24‐month timepoints and both sexes were characterized using behavior, PET/CT, cytokines/Aβ40‐42 immunoassays, and astrocyte and microglia immunohistochemistry. Result: Although aging LOAD1 and LOAD2 mice did not display significant cognitive deficits, there was a genotype‐dependent increase of plasma levels of Ab40‐Ab42. Both sexes across genotypes showed significant region‐dependent increases in brain glycolysis and tissue perfusion between 4 and 24 months. Consistent with the increased brain metabolism and perfusion neurovascular coupling phenotypes, immunopathology analyses revealed an age‐dependent increased number of astrocytes across genotypes that was restricted to cortical regions. Similarly, the total number of activated microglia was slightly elevated in cortical regions of aging mice, even though the results were only marginally significant. Lastly, aged LOAD1 mice displayed increased brain levels of the pro‐inflammatory cytokine IL‐12p70 when compared to LOAD2. Longitudinal analyses of brain and plasma cytokines are still in progress. Conclusion: LOAD1 and LOAD2 PET/CT analyses revealed phenotypes which are in line with imaging profiles of patients at prodromal stages of AD. Combined with the astrogliosis, these strains are promising venues that can be used to test early disease‐modifying therapeutic targets and can also serve as platform to incorporate additional human‐relevant AD risk factors.
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    Orexin Depolarizes Central Amygdala Neurons via Orexin Receptor 1, Phospholipase C and Sodium-Calcium Exchanger and Modulates Conditioned Fear
    (Frontiers, 2018-12-18) Dustrude, Erik T.; Caliman, Izabela F.; Bernabe, Cristian S.; Fitz, Stephanie D.; Grafe, Laura A.; Bhatnagar, Seema; Bonaventure, Pascal; Johnson, Philip L.; Molosh, Andrei I.; Shekhar, Anantha; Psychiatry, School of Medicine
    Orexins (OX), also known as hypocretins, are excitatory neuropeptides with well-described roles in regulation of wakefulness, arousal, energy homeostasis, and anxiety. An additional and recently recognized role of OX is modulation of fear responses. We studied the OX neurons of the perifornical hypothalamus (PeF) which send projections to the amygdala, a region critical in fear learning and fear expression. Within the amygdala, the highest density of OX-positive fibers was detected in the central nucleus (CeA). The specific mechanisms underlying OX neurotransmission within the CeA were explored utilizing rat brain slice electrophysiology, pharmacology, and chemogenetic stimulation. We show that OX induces postsynaptic depolarization of medial CeA neurons that is mediated by OX receptor 1 (OXR1) but not OX receptor 2 (OXR2). We further characterized the mechanism of CeA depolarization by OX as phospholipase C (PLC)- and sodium-calcium exchanger (NCX)- dependent. Selective chemogenetic stimulation of OX PeF fibers recapitulated OXR1 dependent depolarization of CeA neurons. We also observed that OXR1 activity modified presynaptic release of glutamate within the CeA. Finally, either systemic or intra-CeA perfusion of OXR1 antagonist reduced the expression of conditioned fear. Together, these data suggest the PeF-CeA orexinergic pathway can modulate conditioned fear through a signal transduction mechanism involving PLC and NCX activity and that selective OXR1 antagonism may be a putative treatment for fear-related disorders.
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    Using loss- and gain-of-function approaches to target amygdala-projecting serotonergic neurons in the dorsal raphe nucleus that enhance anxiety-related and conditioned fear behaviors
    (Sage, 2020) Bernabe, Cristian S.; Caliman, Izabela F.; Truitt, William A.; Molosh, Andrei I.; Lowry, Christopher A.; Hay-Schmidt, Anders; Shekhar, Anantha; Johnson, Philip L.; Anatomy, Cell Biology and Physiology, School of Medicine
    Background: The central serotonergic system originating from the dorsal raphe nucleus (DR) plays a critical role in anxiety and trauma-related disorders such as posttraumatic stress disorder. Although many studies have investigated the role of serotonin (5-HT) within pro-fear brain regions such as the amygdala, the majority of these studies have utilized non-selective pharmacological approaches or poorly understood lesioning techniques which limit their interpretation. Aim: Here we investigated the role of amygdala-projecting 5-HT neurons in the DR in innate anxiety and conditioned fear behaviors. Methods: To achieve this goal, we utilized (1) selective lesion of 5-HT neurons projecting to the amygdala with saporin toxin conjugated to anti-serotonin transporter (SERT) injected into the amygdala, and (2) optogenetic excitation of amygdala-projecting DR cell bodies with a combination of a retrogradely transported canine adenovirus-expressing Cre-recombinase injected into the amygdala and a Cre-dependent-channelrhodopsin injected into the DR. Results: While saporin treatment lesioned both local amygdalar 5-HT fibers and neurons in the DR as well as reduced conditioned fear behavior, optical activation of amygdala-projecting DR neurons enhanced anxious behavior and conditioned fear response. Conclusion: Collectively, these studies support the hypothesis that amygdala-projecting 5-HT neurons in the DR represent an anxiety and fear-on network.