Surge of neurophysiological coupling and connectivity of gamma oscillations in the dying human brain.

The brain is assumed to be hypoactive during cardiac arrest. However, animal models of cardiac and respiratory arrest demonstrate a surge of gamma oscillations and functional connectivity. To investigate whether these preclinical findings translate to humans, we analyzed electroencephalogram and electrocardiogram signals in four comatose dying patients before and after the withdrawal of ventilatory support. Two of the four patients exhibited a rapid and marked surge of gamma power, surge of cross-frequency coupling of gamma waves with slower oscillations, and increased interhemispheric functional and directed connectivity in gamma bands. High-frequency oscillations paralleled the activation of beta/gamma cross-frequency coupling within the somatosensory cortices. Importantly, both patients displayed surges of functional and directed connectivity at multiple frequency bands within the posterior cortical "hot zone," a region postulated to be critical for conscious processing. This gamma activity was stimulated by global hypoxia and surged further as cardiac conditions deteriorated in the dying patients. These data demonstrate that the surge of gamma power and connectivity observed in animal models of cardiac arrest can be observed in select patients during the process of dying.

In vitro deacetylation of N-acetylserotonin by arylacetamide deacetylase.

Arylacetamide deacetylase (AADAC) is a deacetylation enzyme present in the mammalian liver, gastrointestinal tract, and brain. During our search for mammalian enzymes capable of metabolizing N-acetylserotonin (NAS), AADAC was identified as having the ability to convert NAS to serotonin. Both human and rodent recombinant AADAC proteins can deacetylate NAS in vitro, although the human AADAC shows markedly higher activity compared with rodent enzyme. The AADAC-mediated deacetylation reaction can be potently inhibited by eserine in vitro. In addition to NAS, recombinant hAADAC can deacetylate melatonin (to form 5-methoxytryptamine) and N-acetyltryptamine (NAT) (to form tryptamine). In addition to the in vitro deacetylation of NAS by the recombinant AADAC proteins, liver (mouse and human) and brain (human) extracts were able to deacetylate NAS; these activities were sensitive to eserine. Taken together, these results demonstrate a new role for AADAC and suggest a novel pathway for the AADAC-mediated metabolism of pineal indoles in mammals.

Concentration of non-myocyte proteins in arterial media of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

The most common inherited cause of vascular dementia and stroke, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is caused by mutations in NOTCH3. Post-translationally altered NOTCH3 accumulates in the vascular media of CADASIL arteries in areas of the vessels that exhibit profound cellular degeneration. The identification of molecules that concentrate in the same location as pathological NOTCH3 may shed light on processes that drive cytopathology in CADASIL. We performed a two phase immunohistochemical screen of markers identified in the Human Protein Atlas to identify new proteins that accumulate in the vascular media in a pattern similar to pathological NOTCH3. In phase one, none of 16 smooth muscle cell (SMC) localized antigens exhibited NOTCH3-like patterns of expression; however, several exhibited disease-dependent patterns of expression, with antibodies directed against FAM124A, GZMM, MTFR1, and ST6GAL demonstrating higher expression in controls than CADASIL. In contrast, in phase two of the study that included 56 non-SMC markers, two proteins, CD63 and CTSH, localized to the same regions as pathological NOTCH3, which was verified by VesSeg, a customized algorithm that assigns relative location of antigens within the layers of the vessel. Proximity ligation assays support complex formation between NOTCH3 fragments and CD63 in degenerating CADASIL media. Interestingly, in normal mouse brain, the two novel CADASIL markers, CD63 and CTSH, are expressed in non-SMC vascular cells. The identification of new proteins that concentrate in CADASIL vascular media demonstrates the utility of querying publicly available protein databases in specific neurological diseases and uncovers unexpected, non-SMC origins of pathological antigens in small vessel disease.

Indolethylamine N-methyltransferase (INMT) is not essential for endogenous tryptamine-dependent methylation activity in rats.

Indolethylamine N-methyltransferase (INMT) is a transmethylation enzyme that utilizes the methyl donor S-adenosyl-L-methionine to transfer methyl groups to amino groups of small molecule acceptor compounds. INMT is best known for its role in the biosynthesis of N,N-Dimethyltryptamine (DMT), a psychedelic compound found in mammalian brain and other tissues. In mammals, biosynthesis of DMT is thought to occur via the double methylation of tryptamine, where INMT first catalyzes the biosynthesis of N-methyltryptamine (NMT) and then DMT. However, it is unknown whether INMT is necessary for the biosynthesis of endogenous DMT. To test this, we generated a novel INMT-knockout rat model and studied tryptamine methylation using radiometric enzyme assays, thin-layer chromatography, and ultra-high-performance liquid chromatography tandem mass spectrometry. We also studied tryptamine methylation in recombinant rat, rabbit, and human INMT. We report that brain and lung tissues from both wild type and INMT-knockout rats show equal levels of tryptamine-dependent activity, but that the enzymatic products are neither NMT nor DMT. In addition, rat INMT was not sufficient for NMT or DMT biosynthesis. These results suggest an alternative enzymatic pathway for DMT biosynthesis in rats. This work motivates the investigation of novel pathways for endogenous DMT biosynthesis in mammals.

A midposition NOTCH3 truncation in inherited cerebral small vessel disease may affect the protein interactome.

Mutations in NOTCH3 underlie cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited cerebral small vessel disease. Two cleavages of NOTCH3 protein, at Asp80 and Asp121, were previously described in CADASIL pathological samples. Using monoclonal antibodies developed against a NOTCH3 neoepitope, we identified a third cleavage at Asp964 between an Asp-Pro sequence. We characterized the structural requirements for proteolysis at Asp964 and the vascular distribution of the cleavage event. A proteome-wide analysis was performed to find proteins that interact with the cleavage product. Finally, we investigated the biochemical determinants of this third cleavage event. Cleavage at Asp964 was critically dependent on the proline adjacent to the aspartate residue. In addition, the cleavage product was highly enriched in CADASIL brain tissue and localized to the media of degenerating arteries, where it deposited with the two additional NOTCH3 cleavage products. Recombinant NOTCH3 terminating at Asp964 was used to probe protein microarrays. We identified multiple molecules that bound to the cleaved NOTCH3 more than to uncleaved protein, suggesting that cleavage may alter the local protein interactome within disease-affected blood vessels. The cleavage of purified NOTCH3 protein at Asp964 in vitro was activated by reducing agents and NOTCH3 protein; cleavage was inhibited by specific dicarboxylic acids, as seen with cleavage at Asp80 and Asp121. Overall, we propose homologous redox-driven Asp-Pro cleavages and alterations in protein interactions as potential mechanisms in inherited small vessel disease; similarities in protein cleavage characteristics may indicate common biochemical modulators of pathological NOTCH3 processing.

Overlapping Protein Accumulation Profiles of CADASIL and CAA: Is There a Common Mechanism Driving Cerebral Small-Vessel Disease?

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and cerebral amyloid angiopathy (CAA) are two distinct vascular angiopathies that share several similarities in clinical presentation and vascular pathology. Given the clinical and pathologic overlap, the molecular overlap between CADASIL and CAA was explored. CADASIL and CAA protein profiles from recently published proteomics-based and immuno-based studies were compared to investigate the potential for shared disease mechanisms. A comparison of affected proteins in each disease highlighted 19 proteins that are regulated in both CADASIL and CAA. Functional analysis of the shared proteins predicts significant interaction between them and suggests that most enriched proteins play roles in extracellular matrix structure and remodeling. Proposed models to explain the observed enrichment of extracellular matrix proteins include both increased protein secretion and decreased protein turnover by sequestration of chaperones and proteases or formation of stable protein complexes. Single-cell RNA sequencing of vascular cells in mice suggested that the vast majority of the genes accounting for the overlapped proteins between CADASIL and CAA are expressed by fibroblasts. Thus, our current understanding of the molecular profiles of CADASIL and CAA appears to support potential for common mechanisms underlying the two disorders.

Electrocardiomatrix Facilitates Accurate Detection of Atrial Fibrillation in Stroke Patients.

Background and Purpose- Cardiac telemetry is a routine part of inpatient ischemic stroke/transient ischemic attack evaluation to assess for atrial fibrillation (AF). Yet, tools to assist stroke clinicians in the evaluation of the large quantities of telemetry data are limited. The investigators developed a new method to evaluate electrocardiographic signals, electrocardiomatrix, that was applied to stroke unit telemetry data to determine its feasibility, validity, and usefulness. Electrocardiomatrix displays telemetry data in a 3-dimensional matrix that allows for more accurate and less time consuming P-wave analysis. Methods- In this single-center, prospective, observational study conducted in a stroke unit, all telemetry data from ischemic stroke and transient ischemic attack patients were collected (April 2017-January 2018) for examination facilitated by electrocardiomatrix. AF>30 seconds was identified through review of electrocardiomatrix-generated matrices by a nonphysician researcher. Electrocardiomatrix results were compared with the clinical team's medical record documentation of AF identified through telemetry. A study cardiologist reviewed the standard telemetry associated with all AF episodes identified by electrocardiomatrix and each case of disagreement. Results- Telemetry data (median 46 hours [interquartile range: 22-90]) were analyzed among 265 unique subjects (88% ischemic stroke). Electrocardiomatrix was successfully applied in 260 (98%) of cases. The positive predictive value of electrocardiomatrix compared with the clinical documentation was 86% overall and 100% among the subset with no prior history of AF. For the 5 false-positive and 5 false-negative cases, expert overview disagreed with the clinical documentation and confirmed the electrocardiomatrix-based diagnosis. Conclusions- The application of electrocardiomatrix to stroke unit-acquired telemetry data is feasible and appears to have superior accuracy compared with traditional monitor analysis by noncardiologists.

Biosynthesis and Extracellular Concentrations of N,N-dimethyltryptamine (DMT) in Mammalian Brain.

N,N-dimethyltryptamine (DMT), a psychedelic compound identified endogenously in mammals, is biosynthesized by aromatic-L-amino acid decarboxylase (AADC) and indolethylamine-N-methyltransferase (INMT). Whether DMT is biosynthesized in the mammalian brain is unknown. We investigated brain expression of INMT transcript in rats and humans, co-expression of INMT and AADC mRNA in rat brain and periphery, and brain concentrations of DMT in rats. INMT transcripts were identified in the cerebral cortex, pineal gland, and choroid plexus of both rats and humans via in situ hybridization. Notably, INMT mRNA was colocalized with AADC transcript in rat brain tissues, in contrast to rat peripheral tissues where there existed little overlapping expression of INMT with AADC transcripts. Additionally, extracellular concentrations of DMT in the cerebral cortex of normal behaving rats, with or without the pineal gland, were similar to those of canonical monoamine neurotransmitters including serotonin. A significant increase of DMT levels in the rat visual cortex was observed following induction of experimental cardiac arrest, a finding independent of an intact pineal gland. These results show for the first time that the rat brain is capable of synthesizing and releasing DMT at concentrations comparable to known monoamine neurotransmitters and raise the possibility that this phenomenon may occur similarly in human brains.

Surge of corticocardiac coupling in SHRSP rats exposed to forebrain cerebral ischemia.

Sudden death is an important but underrecognized consequence of stroke. Acute stroke can disturb central control of autonomic function and result in cardiac dysfunction and sudden death. Previous study showed that bilateral common carotid artery ligation (BCCAL) in the spontaneously hypertensive stroke-prone rat strain (SHRSP) is a well-established model for forebrain ischemic sudden death. This study aims to investigate the temporal dynamic changes in electrical activities of the brain and heart and functional interactions between the two vital organs following forebrain ischemia. EEG and ECG signals were simultaneously collected from nine SHRSP and eight Wistar-Kyoto (WKY) rats. RR interval was analyzed to investigate the cardiac response to brain ischemia. EEG power and coherence (CCoh) analysis were conducted to study the cortical response. Corticocardiac coherence (CCCoh) and directional connectivity (CCCon) were analyzed to determine brain-heart connection. Heart rate variability (HRV) was analyzed to evaluate autonomic functionality. BCCAL resulted in 100% mortality in SHRSP within 14 h, whereas no mortality was observed in WKY rats. The functionality of both the brain and the heart were significantly altered in SHRSP compared with WKY rats after BCCAL. SHRSP, but not WKY rats, exhibited intermittent surge of CCCoh, which paralleled the elevated CCCon and reduced HRV, following the onset of ischemia until sudden death. Elevated brain-heart coupling invariably associated with the disruption of the autonomic nervous system and the risk of sudden death. This study may improve our understanding of the mechanism of forebrain ischemia-induced sudden death. NEW & NOTEWORTHY This study demonstrates a marked surge of corticocardiac coupling in rats dying from focal cerebral ischemia, consistent with our earlier data in rats exposed to fatal asphyxia. Since the bidirectional electrical signal coupling (corticocardiac coherence) and communication (corticocardiac connectivity) between the brain and the heart are only identified in dying animals, they could be used as potential biomarkers to predict the risk of sudden death.

Electrocardiomatrix facilitates qualitative identification of diminished heart rate variability in critically ill patients shortly before cardiac arrest.

BACKGROUND: Although heart rate variability (HRV) has diagnostic and prognostic value for the assessment of cardiac risk, HRV analysis is not routinely performed in a hospital setting. Current HRV analysis methods are primarily quantitative; such methods are sensitive to signal contamination and require extensive post hoc processing. METHODS AND RESULTS: Raw electrocardiogram (ECG) data from the Sleep Heart Health Study was transformed into electrocardiomatrix (ECM), in which sequential cardiac cycles are aligned, in parallel, along a shared axis. Such juxtaposition facilitates the visual evaluation of beat-to-beat changes in the R-R interval without sacrificing the morphology of the native ECG signal. Diminished HRV, verified by traditional methods, was readily identifiable. We also examined data from a cohort of hospitalized patients who suffered cardiac arrest within 24 h of data acquisition, all of whom exhibited severely diminished HRV that were visually apparent on ECM display. CONCLUSIONS: ECM streamlines the identification of depressed HRV, which may signal deteriorating patient condition.

Accurate detection of atrial fibrillation and atrial flutter using the electrocardiomatrix technique.

BACKGROUND: Atrial fibrillation (AFIB) and atrial flutter (AFL) are two common cardiac arrhythmias that predispose patients to serious medical conditions. There is a need to accurately detect these arrhythmias to prevent diseases and reduce mortality. Apart from accurately detecting these arrhythmias, it is also important to distinguish between AFIB and AFL due to differing clinical treatments. METHODS: In this study, we applied a new technology, the electrocardiomatrix (ECM) invented in our lab, in detecting AFIB and AFL in human patients. ECM converts 2D ECG signals into a 3D color matrix, which renders arrhythmia detection intuitive, fast, and accurate. Using ECM, we analyzed the ECG signals from the online MIT-BIH Atrial Fibrillation Database (PhysioNet), and compared our ECM-based results to manual annotations based on ECG by physicians. RESULTS: Results demonstrate that ECM and PhysioNet annotations of AFIB and AFL agree more than 99% of the time. The sensitivities of the ECM for AFIB and AFL detection were 99.2% and 98.0%, respectively, and the specificities of the ECM for AFIB and AFL were both at 99.8% and 99.8%. CONCLUSIONS: This study demonstrates that ECM is a reliable method for accurate identification of AFIB and AFL.

Adrenergic Blockade Bi-directionally and Asymmetrically Alters Functional Brain-Heart Communication and Prolongs Electrical Activities of the Brain and Heart during Asphyxic Cardiac Arrest.

Sudden cardiac arrest is a leading cause of death in the United States. The neurophysiological mechanism underlying sudden death is not well understood. Previously we have shown that the brain is highly stimulated in dying animals and that asphyxia-induced death could be delayed by blocking the intact brain-heart neuronal connection. These studies suggest that the autonomic nervous system plays an important role in mediating sudden cardiac arrest. In this study, we tested the effectiveness of phentolamine and atenolol, individually or combined, in prolonging functionality of the vital organs in CO2-mediated asphyxic cardiac arrest model. Rats received either saline, phentolamine, atenolol, or phentolamine plus atenolol, 30 min before the onset of asphyxia. Electrocardiogram (ECG) and electroencephalogram (EEG) signals were simultaneously collected from each rat during the entire process and investigated for cardiac and brain functions using a battery of analytic tools. We found that adrenergic blockade significantly suppressed the initial decline of cardiac output, prolonged electrical activities of both brain and heart, asymmetrically altered functional connectivity within the brain, and altered, bi-directionally and asymmetrically, functional, and effective connectivity between the brain and heart. The protective effects of adrenergic blockers paralleled the suppression of brain and heart connectivity, especially in the right hemisphere associated with central regulation of sympathetic function. Collectively, our results demonstrate that blockade of brain-heart connection via alpha- and beta-adrenergic blockers significantly prolonged the detectable activities of both the heart and the brain in asphyxic rat. The beneficial effects of combined alpha and beta blockers may help extend the survival of cardiac arrest patients.

Chronic circadian advance shifts abolish melatonin secretion for days in rats.

Melatonin deficiency has been proposed to underlie higher risks for cardiovascular and several other diseases in humans experiencing prolonged shiftwork. However, melatonin secretion has not been monitored longitudinally during consecutive shifts of the light:dark (LD) cycles in the same individuals (animals or humans) and the extent of melatonin deficiency is unknown in individuals experiencing consecutive LD shifts. We investigated the effect of consecutive LD shifts on melatonin secretion in adult F344 rats using continuous online pineal-microdialysis. The rats were entrained to the 12 h:12 h LD cycle before the shifts. The LD cycle was then advanced (n=5) or delayed (n=4) for six hours every four days for four consecutive times. The rats exhibited marked asymmetry in response to delay or advance LD shifts. While rats exposed to the repeated LD delay shifts always exhibited melatonin secretion throughout the entire periods, repeated LD advance shifts suppressed nocturnal melatonin secretion for several consecutive days in the middle of the 3-week period. Moreover, melatonin offset after LD delay and melatonin onset after LD advance determined the rate of circadian pacemaker reentrainment. Additionally, melatonin offset was phase locked at the new dark/light junctions for days following LD advance. These data demonstrate that chronic LD shifts are deleterious to melatonin rhythms, and that this effect is much more pronounced during advance shifts. These data may enhance our understanding of impact of LD shifts on our circadian timing system and benefit better design of shiftwork schedules to avoid melatonin disruption.

Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway.

Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.

Asphyxia-activated corticocardiac signaling accelerates onset of cardiac arrest.

The mechanism by which the healthy heart and brain die rapidly in the absence of oxygen is not well understood. We performed continuous electrocardiography and electroencephalography in rats undergoing experimental asphyxia and analyzed cortical release of core neurotransmitters, changes in brain and heart electrical activity, and brain-heart connectivity. Asphyxia stimulates a robust and sustained increase of functional and effective cortical connectivity, an immediate increase in cortical release of a large set of neurotransmitters, and a delayed activation of corticocardiac functional and effective connectivity that persists until the onset of ventricular fibrillation. Blocking the brain's autonomic outflow significantly delayed terminal ventricular fibrillation and lengthened the duration of detectable cortical activities despite the continued absence of oxygen. These results demonstrate that asphyxia activates a brainstorm, which accelerates premature death of the heart and the brain.

Surge of neurophysiological coherence and connectivity in the dying brain.

The brain is assumed to be hypoactive during cardiac arrest. However, the neurophysiological state of the brain immediately following cardiac arrest has not been systematically investigated. In this study, we performed continuous electroencephalography in rats undergoing experimental cardiac arrest and analyzed changes in power density, coherence, directed connectivity, and cross-frequency coupling. We identified a transient surge of synchronous gamma oscillations that occurred within the first 30 s after cardiac arrest and preceded isoelectric electroencephalogram. Gamma oscillations during cardiac arrest were global and highly coherent; moreover, this frequency band exhibited a striking increase in anterior-posterior-directed connectivity and tight phase-coupling to both theta and alpha waves. High-frequency neurophysiological activity in the near-death state exceeded levels found during the conscious waking state. These data demonstrate that the mammalian brain can, albeit paradoxically, generate neural correlates of heightened conscious processing at near-death.

LC/MS/MS analysis of the endogenous dimethyltryptamine hallucinogens, their precursors, and major metabolites in rat pineal gland microdialysate.

We report a qualitative liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous analysis of the three known N,N-dimethyltryptamine endogenous hallucinogens, their precursors and metabolites, as well as melatonin and its metabolic precursors. The method was characterized using artificial cerebrospinal fluid (aCSF) as the matrix and was subsequently applied to the analysis of rat brain pineal gland-aCSF microdialysate. The method describes the simultaneous analysis of 23 chemically diverse compounds plus a deuterated internal standard by direct injection, requiring no dilution or extraction of the samples. The results demonstrate that this is a simple, sensitive, specific and direct approach to the qualitative analysis of these compounds in this matrix. The protocol also employs stringent MS confirmatory criteria for the detection and confirmation of the compounds examined, including exact mass measurements. The excellent limits of detection and broad scope make it a valuable research tool for examining the endogenous hallucinogen pathways in the central nervous system. We report here, for the first time, the presence of N,N-dimethyltryptamine in pineal gland microdialysate obtained from the rat.

ABO blood antigens define human cerebral endothelial diversity.

Cerebral endothelial cells participate in the blood-brain barrier and regulate activity-dependent changes in brain blood flow. It has been assumed that all cerebral endothelial cells are similar, but genetic studies in mice suggest that there are heterogeneous populations of endothelial cells in rodent brain. In this study, we tested for molecular heterogeneity of endothelial cells in the human brain. Human brains (five A and five O blood type patients) from autopsies were analyzed by immunohistochemistry and immunofluorescence using antibodies against von Willebrand factor (vWF) and A and H blood group antigens. vWF and ABO antigens were confined to the endothelium. Although all endothelial cells expressed vWF, capillary endothelial cells from A blood type brains showed a heterogeneous expression of A and H antigens, with individual cells expressing either one or both antigens. There were no differences between the gray and the white matter in the percentage of A-reactive or H-reactive capillaries. We conclude that ABO antigen expression in the human brain is modulated at the level of the individual endothelial cell. Future studies are warranted to determine whether differences in capillary permeability and cerebral autoregulation vary over short distances within the brain.