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Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties 1-3 . Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain., (© 2023. The Author(s).)

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.

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.

Autophagy has been reported to play protective and pathogenetic roles in cerebral ischemia/reperfusion (I/R)-induced neuronal injury. Our previous studies have shown that TP53-induced glycolysis and apoptosis regulator (TIGAR) ameliorates I/R-induced brain injury and reduces anti-cancer drug-induced autophagy activation. However, if TIGAR plays a regulatory role on autophagy in cerebral I/R injury is still unclear. The purpose of the present study is to investigate the role of TIGAR on I/R-induced autophagy activation and ischemic neuronal injury in vivo and in vitro stroke models using TIGAR-transgenic (tg-TIGAR) mice and TIGAR-knockout (ko-TIGAR) mice. The present study confirmed that autophagy was activated after I/R. Overexpression of TIGAR in tg-TIGAR mice significantly reduced I/R-induced autophagy activation and alleviated brain damage, while knockout of TIGAR in ko-TIGAR mice enhanced I/R-induced autophagy activation and exacerbated brain injury in vivo and in vitro. The different activity of autophagy in tg-TIGAR and ko-TIGAR primary neurons after OGD/R were largely reversed by knockdown or re-expression of TIGAR in these neurons. The autophagy inhibitor 3-methyladenine (3-MA) partly prevented exacerbation of brain damage induced by ko-TIGAR, whereas the autophagy inducer rapamycin partially abolished the neuroprotective effect of tg-TIGAR. Knockout of TIGAR reduced the levels of phosphorylated mTOR and S6KP70, which were blocked by 3-MA and NADPH after I/R and OGD/R in vivo and in vitro, respectively. Overexpression of TIGAR increased the levels of phosphorylated mTOR and S6KP70 under OGD/R condition, this enhancement effect was suppressed by rapamycin. In conclusion, our current data suggest that TIGAR protected against neuronal injury partly through inhibiting autophagy by regulating the mTOR-S6KP70 signaling pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited form of cerebral small vessel disease caused by mutations in conserved residues of NOTCH3. Affected arteries of CADASIL feature fibrosis and accumulation of NOTCH3. A variety of collagen subtypes (types I, III, IV, and VI) have been identified in fibrotic CADASIL vessels. Biglycan (BGN) and decorin (DCN) are class I members of the small leucine-rich proteoglycan (SLRP) family that regulate collagen fibril size. Because DCN has been shown to deposit in arteries in cerebral small vessel disease, we tested whether BGN accumulates in arteries of CADASIL brains. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain. BGN protein was localized to all three layers of arteries (intima, media, and adventitia). Substantially, more immunoreactivity was observed in CADASIL brains compared to controls. Immunoblotting of brain lysates showed a fourfold increase in CADASIL brains (compared to controls). Messenger RNA encoding BGN was also increased in CADASIL and was localized by in situ hybridization to all three vascular layers in CADASIL. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. In conclusion, BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen.

Small penetrating brain artery thickening is a major feature of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Although affected fibrotic arteries of CADASIL have been shown to accumulate collagen, other components that compose pathological arterial walls remain incompletely characterized. We investigated the expression of decorin (DCN), the first collagen-binding small leucine-rich proteoglycan identified, in CADASIL. DCN was markedly upregulated in pathologically affected leptomeningeal and small penetrating arteries in CADASIL and was notably weaker in normal arteries from control brains. DCN protein was localized principally to the media and adventitia and only occasionally expressed in the intima. Immunoblotting of brain lysates showed a three-fold increase of DCN in CADASIL brains (compared with controls). Messenger RNA encoding DCN was five-fold increased in CADASIL. We conclude that DCN is the first identified proteoglycan to be identified in CADASIL arteries and may accumulate through transcriptional mechanisms. Additional studies are warranted to determine whether DCN localizes broadly to pathological small vessels in other cerebrovascular disorders.

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.

Background and Purpose: Leptomeningeal artery abnormalities in Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) have not been extensively characterized. We quantified substructure and diameter of leptomeningeal arteries in CADASIL compared with age-matched controls and the very old; in addition, we characterized intimal thickening in CADASIL using immunohistochemistry., Methods: Frontal and temporal cortex of 6 genetically proven CADASIL brains (average age, 66 years), 6 controls without symptoms of cerebrovascular disease, and 6 very old brains (average age, 89 years) were examined for leptomeningeal artery intimal, medial, and adventitial thickness; inner diameter; and sclerotic index and for smooth muscle markers., Results: The intima of CADASIL arteries was thickened 5-fold compared with controls and the very aged (P<0.0001). Medial thickness was lower in CADASIL compared with controls and the very old (P<0.01). The adventitia was not significantly increased in CADASIL compared with age-matched controls. Arterial diameters were not smaller in CADASIL compared with controls. Sclerotic index was significantly increased in CADASIL compared with other groups (P<0.00001). Intimal cells in CADASIL expressed smooth muscle actin, S100A4, and vimentin but not desmin., Conclusions: Principle changes of leptomeningeal arteries in CADASIL include intimal thickening and medial thinning, but not luminal narrowing. Smooth muscle-like cells participate in neointimal thickening of CADASIL arteries.

Diverse preconditioning (PC) stimuli protect against a wide variety of neuronal insults in animal models, engendering enthusiasm that PC could be used to protect the brain clinically. Candidate clinical applications include cardiac and vascular surgery, after subarachnoid hemorrhage, and prior to conditions in which acute neuronal injury is anticipated. However, disappointments in clinical validation of multiple neuroprotectants suggest potential problems translating animal data into successful human therapies. Thus, despite strong promise of preclinical PC studies, caution should be maintained in translating these findings into clinical applications. The Stroke Therapy Academic Industry Roundtable (STAIR) working group and the National institute of Neurological Diseases and Stroke (NINDS) proposed working guidelines to improve the utility of preclinical studies that form the foundation of therapies for neurological disease. Here, we review the applicability of these consensus criteria to preconditioning studies and discuss additional considerations for PC studies. We propose that special attention should be paid to several areas, including 1) safety and dosage of PC treatments; 2) meticulously matching preclinical modeling to the human condition to be tested; and 3) timing of both the initiation and discontinuation of the PC stimulus relative to injury ictus.

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.

Corpora amylacea (CA) have long been described in aging brains and in patients with neurodegenerative conditions, but their origins have been debated. It has been proposed that CA represent collections of nervous system breakdown products that accumulate within astrocytic cytoplasm. In support of this, studies have shown that CA include glycosylated material, ubiquitin, and an assortment of proteins derived from neuronal cytoplasm. On the other hand, many of these proteins are not specifically localized to neurons or astrocytes; some components of CA, such as complement proteins, are most abundantly expressed outside the central nervous system. The characteristic predilection for CA to accumulate near vessels and ependyma suggests that proteins extravasated from blood or transudated from CSF may form a component of these structures. In this study, we report the immunohistochemical localization of blood and platelet proteins thrombospondin1 and ADAMTS13 in CA from aged individuals and patients with vascular dementia. Thrombospondin1 localized to neurons, but was most prominently localized to CA. An independent serum and platelet expressed protein, ADAMTS13, was found in CA in the same brain regions. In vitro analysis shows that thrombospondin1 and ADAMTS13 form complexes together in cells and in direct protein binding assays. We speculate that CA could result from a conglomeration of interacting proteins from degenerating neurons and from extravasated blood elements released after transient breakdown of the blood-brain barrier.

Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.