Whole-brain mapping of histaminergic projections in mouse brain.

Histamine is a conserved neuromodulator in mammalian brains and critically involved in many physiological functions. Understanding the precise structure of the histaminergic network is the cornerstone in elucidating its function. Herein, using histidine decarboxylase (HDC)-CreERT2 mice and genetic labeling strategies, we reconstructed a whole-brain three dimensional (3D) structure of histaminergic neurons and their outputs at 0.32 × 0.32 × 2 µm3 pixel resolution with a cutting-edge fluorescence microoptical sectioning tomography system. We quantified the fluorescence density of all brain areas and found that histaminergic fiber density varied significantly among brain regions. The density of histaminergic fiber was positively correlated with the amount of histamine release induced by optogenetic stimulation or physiological aversive stimulation. Lastly, we reconstructed a fine morphological structure of 60 histaminergic neurons via sparse labeling and uncovered the largely heterogeneous projection pattern of individual histaminergic neurons. Collectively, this study reveals an unprecedented whole-brain quantitative analysis of histaminergic projections at the mesoscopic level, providing a foundation for future functional histaminergic study.

Flexible Atg1/ULK complex composition activates selective autophagy for phosphate starvation.

The molecular basis for bulk autophagy activation due to a deficiency in essential nutrients such as carbohydrates, amino acids, and nitrogen is well understood. Given autophagy functions to reduce surplus to compensate for scarcity, it theoretically possesses the capability to selectively degrade specific substrates to meet distinct metabolic demands. However, direct evidence is still lacking that substantiates the idea that autophagy selectively targets specific substrates (known as selective autophagy) to address particular nutritional needs. Recently, Gross et al. found that during phosphate starvation (P-S), rather than nitrogen starvation (N-S), yeasts selectively eliminate peroxisomes by dynamically altering the composition of the Atg1/ULK kinase complex (AKC) to adapt to P-S. This study elucidates how the metabolite sensor Pho81 flexibly interacts with AKC and guides selective autophagic clearance of peroxisomes during P-S, providing novel insights into the metabolic contribution of autophagy to special nutritional needs.

Dissecting the networks underlying diverse brain disorders after prenatal glucocorticoid overexposure.

New human life begins in the uterus in a period of both extreme plasticity and sensitivity to environmental disturbances. The fetal stage is also a vital period for central nervous system development, with experiences at this point profoundly and permanently shaping brain structure and function. As such, some brain disorders may originate in utero. Glucocorticoids, a class of essential stress hormones, play indispensable roles in fetal development, but overexposure may have lasting impacts on the brain. In this review, we summarize data from recent clinical and non-clinical studies regarding alterations in fetal brains due to prenatal glucocorticoid overexposure that are associated with nervous system disorders. We discuss relevant changes to brain structure and cellular functions and explore the underlying molecular mechanisms. In addition, we summarize factors that may cause differential outcomes between varying brain regions, and outline clinically feasible intervention strategies that are expected to minimize negative consequences arising from fetal glucocorticoid overexposure. Finally, we highlight the need for experimental evidence aided by new technologies to clearly determine the effects of excessive prenatal glucocorticoid exposure. This review consolidates diverse findings to help researchers better understand the relationship between the prenatal glucocorticoid overexposure and the effects it has on various fetal brain regions, promoting further development of critical intervention strategies.

The Emerging Role of Astrocytic Autophagy in Central Nervous System Disorders.

Astrocytes act as "housekeeping cells" for maintaining cerebral homeostasis and play an important role in many disorders. Recent studies further highlight the contribution of autophagy to astrocytic functions, including astrogenesis, the astrocytic removal of neurotoxins or stressors, and astrocytic polarization. More importantly, genetic and pharmacological approaches have provided evidence that outlines the contributions of astrocytic autophagy to several brain disorders, including neurodegeneration, cerebral ischemia, and depression. In this study, we summarize the emerging role of autophagy in regulating astrocytic functions and discuss the contributions of astrocytic autophagy to different CNS disorders.

Monitoring Autophagy by Optical Microscopy.

Thanks to the advances in optical microscope technology and our knowledge of autophagic biomarkers, single-molecule events of autophagy are now accessible to human eyes. Different proteins are involved hierarchically in the biogenesis and maturation of autophagosomes. Detecting these autophagy-related proteins either by immunostaining or fluorescent protein labelling makes the dynamic autophagic process visible. However, low antibody specificity and weak endogenous expression of autophagy-related proteins in certain tissues limit the applicability of immunostaining in autophagy detection. To cope with this, live-cell imaging combined with various fluorescent probes has been developed and employed in monitoring autophagy. As the most widely used autophagic biomarker, LC3 can be used to visualize autophagosomes, and fluorescent probes targeting LC3, i.e., RFP/mCherry-GFP-LC3, and GFP-LC3-RFP-LC3ΔG, can examine autophagy flux dynamically and quantitatively. In addition, the application of novel fluorophores such as Keima helps to detect the temporal and spatial characteristics of autophagy. Furthermore, selective autophagy can be clarified by labelling corresponding substrates and autophagosomes or lysosomes simultaneously. With the help of two-photon microscopy, the process of autophagy in live animals has been uncovered. Here, we summarize the methods for observing autophagy by optical microscopy and the selection of fluorescent markers.

Melatonin receptor agonist ramelteon attenuates mouse acute and chronic ischemic brain injury.

Melatonin receptors (MTs) are potential drug targets for stroke therapy. Ramelteon is a selective melatonin receptor agonist used to treat insomnia. In this study we investigated whether ramelteon could attenuate cerebral ischemia in mice. Acute focal cerebral ischemia was induced in mice via middle cerebral artery occlusion (MCAO). We found oral administration of ramelteon (3.0 mg/kg) significantly attenuated ischemic injury even when it was given 4 h after the onset of ischemia. We showed that administration of ramelteon (3.0 mg/kg) displayed comparable protective efficacy and length of effective time window as administration of edaravone (10 mg/kg, i.p.), which was used in clinic to treat ischemic stroke. Chronic ischemic brain injury was induced in mice using photothrombosis. Oral administration of ramelteon (3.0 mg · kg-1 · d-1) for 7 days after ischemia significantly attenuated functional deficits for at least 15 days. The neuroprotection of ramelteon was blocked by 4-P-PDOT, a specific MT antagonist. We further revealed that ramelteon significantly inhibited autophagy in the peri-infarct cortex in both the mouse ischemia models via regulating AMPK/mTOR signaling pathway. Intracerebroventricular injection of rapamycin, an autophagy activator, compromised the neuroprotection of ramelteon, suggesting ramelteon might attenuate ischemic injury by counteracting autophagic cell death. These data demonstrate for the first time the potential benefits of ramelteon in the treatment of both acute and chronic ischemic brain injury and provide the rationale for the application of ramelteon in stroke therapy.

Autophagy and Mitochondrial Encephalomyopathies.

Mitochondrial encephalomyopathies are a group of disorders affecting skeletal muscles and brain. Although the symptoms vary among these disorders, mitochondrial DNA mutation or loss is the common characteristic. The abnormality of mitochondrial genome usually causes the dysfunction of mitochondrial respiratory and even mitochondrial damage. As a critical way of degradation, attention has been paid to the involvement of autophagy in encephalomyopathies. Autophagy is found activated in these encephalomyopathies-relevant cells as a compensatory manner to eliminate these damaged and dysfunctional mitochondria. However, accumulating evidences indicate that autophagy is incompetent to clear them. The insufficient mitophagy may ultimately accelerate cell death. Here we discuss the involvement of autophagy in encephalomyopathies based on the current evidence. We further look into the future to rescue encephalomyopathies by regulating autophagy. Only five encephalomyopathies are included in this chapter due to the availability of evidence. Nevertheless, these encephalomyopathies share a variety of common features and autophagy may also be regulated in the other encephalomyopathies.

[Research progress on mechanism of Nix-mediated mitophagy].

Autophagy is fundamental to maintain cellular homeostasis. As one kind of the most well-studied selective autophagy, autophagy of mitochondria (mitophagy)is crucial for the clearance of damaged mitochondria. Mitophagy dysfunction has been proved to be closely associated with many human diseases. Nix is a key protein for mitophagy during the maturation of reticulocytes. However, the detailed molecular mechanisms underlying Nix-mediated mitophagy are not fully understood. This article summarizes three possible working models of Nix in mitophagy induction. Firstly, Nix can interplay with Parkin, another important protein for mitophagy, to initiate mitophagy. Secondly, Nix can serve as a receptor for autophagy machinery by interacting with Atg8 family through its LIR motif. Finally, as a BH3-only protein, Nix can compete with Beclin-1 to bind other members of Bcl-2 family resulting in increased free Beclin-1 in cytosol, which further promotes autophagy flux.

Pre-stroke Metformin Treatment is Neuroprotective Involving AMPK Reduction.

Long-term metformin treatment reduces the risk of stroke. However, the effective administration pattern and indications of metformin on acute cerebral ischemia are unclear. To investigate the neuroprotective treatment duration and dosage of metformin on focal ischemia mice and the association of neuroprotection with 5'-adenosine monophosphate-activated protein kinase (AMPK) regulations, male C57BL/6 mice were subjected to permanent or transient middle cerebral artery occlusion (MCAO) and metformin of 3, 10 and 30 mg/kg was intraperitoneally injected 1, 3 or 7 days prior to MCAO, or at the onset, or 1, 3 or 6 h after reperfusion, respectively. Infarct volumes, neurological deficit score, cell apoptosis, both total and phosphorylated AMPK expressions were assessed. Results showed that prolonged pretreatment to 7 days of metformin (10 mg/kg) significantly ameliorated brain infarct, neurological scores and cell apoptosis in permanent MCAO mice. Shorter (3 days or 1 day) or without pretreatment of metformin was not effective, suggesting a pretreatment time window. In transient MCAO mice, metformin showed no neuroprotection even with pretreatment. The expressions of total and phosphorylated AMPK were sharply decreased with effective metformin pretreatments in ischemic brains. Our data provided the first evidence that in acute ischemic injury, a 7-days pretreatment duration of 10 mg/kg metformin is necessary for its neuroprotection, and metformin may not be beneficial in the cases of blood reperfusion.

Targeting Histamine and Histamine Receptors for Memory Regulation: An Emotional Perspective.

Histamine has long been accepted as a pro-cognitive agent. However, lines of evidence have suggested that the roles of histamine in learning and memory processes are much more complex than previously thought. When explained by the spatial perspectives, there are many contradictory results. However, using emotional memory perspectives, we suspect that the histaminergic system may interplay with stress, reward inhibition, and attention to modulate emotional memory formation. The functional diversity of histamine makes it a viable target for clinical management of neuropsychiatric disorders. Here, we update the current knowledge about the functions of histamine in emotional memory and summarize the underlying molecular and neural circuit mechanisms. Finally, we review the main clinical studies about the impacts of histamine-related compounds on memory and discuss insights into future research on the roles of histamine in emotional memory. Despite the recent progress in histamine research, the histaminergic emotional memory circuits are poorly understood, and it is also worth verifying the functions of histamine receptors in a more spatiotemporally specific manner.

Histamine H1 receptors in dentate gyrus-projecting cholinergic neurons of the medial septum suppress contextual fear retrieval in mice.

Fear memory is essential for survival and adaptation, yet excessive fear memories can lead to emotional disabilities and mental disorders. Despite previous researches have indicated that histamine H1 receptor (H1R) exerts critical and intricate effects on fear memory, the role of H1R is still not clarified. Here, we show that deletion of H1R gene in medial septum (MS) but not other cholinergic neurons selectively enhances contextual fear memory without affecting cued memory by differentially activating the dentate gyrus (DG) neurons in mice. H1R in cholinergic neurons mediates the contextual fear retrieval rather than consolidation by decreasing acetylcholine release pattern in DG. Furthermore, selective knockdown of H1R in the MS is sufficient to enhance contextual fear memory by manipulating the retrieval-induced neurons in DG. Our results suggest that H1R in MS cholinergic neurons is critical for contextual fear retrieval, and could be a potential therapeutic target for individuals with fear-related disorders.

Molecular Determinant Underlying Selective Coupling of Primary G-Protein by Class A GPCRs.

G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H2R and H3R couple with Gs- or Gi-proteins respectively. Here, three cryo-EM structures of H2R-Gs and H3R-Gi complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H3R, wherein the amino group interacts with E2065.46 of H3R instead of the conserved D1143.32 of other aminergic receptors. Furthermore, comparative analysis of the H2R-Gs and H3R-Gi complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the Gs and Gi/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary Gs- and Gi/o-coupling selectivity.

Post-Stroke Neuropsychiatric Complications: Types, Pathogenesis, and Therapeutic Intervention.

Almost all stroke survivors suffer physical disabilities and neuropsychiatric disturbances, which can be briefly divided into post-stroke neurological diseases and post-stroke psychiatric disorders. The former type mainly includes post-stroke pain, post-stroke epilepsy, and post-stroke dementia while the latter one includes post-stroke depression, post-stroke anxiety, post-stroke apathy and post-stroke fatigue. Multiple risk factors are related to these post-stroke neuropsychiatric complications, such as age, gender, lifestyle, stroke type, medication, lesion location, and comorbidities. Recent studies have revealed several critical mechanisms underlying these complications, namely inflammatory response, dysregulation of the hypothalamic pituitary adrenal axis, cholinergic dysfunction, reduced level of 5-hydroxytryptamine, glutamate-mediated excitotoxicity and mitochondrial dysfunction. Moreover, clinical efforts have successfully given birth to many practical pharmaceutic strategies, such as anti-inflammatory medications, acetylcholinesterase inhibitors, and selective serotonin reuptake inhibitors, as well as diverse rehabilitative modalities to help patients physically and mentally. However, the efficacy of these interventions is still under debate. Further investigations into these post-stroke neuropsychiatric complications, from both basic and clinical perspectives, are urgent for the development of effective treatment strategies.

Umbelliferone protects against cerebral ischemic injury through selective autophagy of mitochondria.

Effective therapeutic treatments for ischemic stroke are limited. Previous studies suggest selective activation of mitophagy alleviates cerebral ischemic injury while excessive autophagy is detrimental. However, few compounds are available to selectively activate mitophagy without affecting autophagy flux. Here, we found that acute administration of Umbelliferone (UMB) upon reperfusion exerted neuroprotective effects against ischemic injury in mice subjected to transient middle cerebral artery occlusion (tMCAO) and suppressed oxygen-glucose deprivation reperfusion (OGD-R)-induced apoptosis in SH-SY5Y cells. Interestingly, UMB promoted the translocation of mitophagy adaptor SQSTM1 to mitochondria and further reduced the mitochondrial content as well as the expression of SQSTM1 in SHSY5Y cells after OGD-R. Importantly, both the mitochondrial loss and reduction of SQSTM1 expression after UMB incubation can be reversed by autophagy inhibitor chloroquine and wortmannin, proving the mitophagy activation by UMB. Nevertheless, UMB failed to further affect neither LC3 lipidation nor the number of autophagosomes after cerebral ischemia in vivo and in vitro. Furthermore, UMB facilitated OGD-R-induced mitophagy in a Parkin-dependent manner. Inhibition of autophagy/mitophagy either pharmaceutically or genetically abolished the neuroprotective effects of UMB. Taken all, these results suggest that UMB protects against cerebral ischemic injury, both in vivo and in vitro, via promoting mitophagy without increasing the autophagic flux. UMB might serve as a potential leading compound for selectively activating mitophagy and the treatment of ischemic stroke.

Dissecting the brain with spatially resolved multi-omics.

Recent studies have highlighted spatially resolved multi-omics technologies, including spatial genomics, transcriptomics, proteomics, and metabolomics, as powerful tools to decipher the spatial heterogeneity of the brain. Here, we focus on two major approaches in spatial transcriptomics (next-generation sequencing-based technologies and image-based technologies), and mass spectrometry imaging technologies used in spatial proteomics and spatial metabolomics. Furthermore, we discuss their applications in neuroscience, including building the brain atlas, uncovering gene expression patterns of neurons for special behaviors, deciphering the molecular basis of neuronal communication, and providing a more comprehensive explanation of the molecular mechanisms underlying central nervous system disorders. However, further efforts are still needed toward the integrative application of multi-omics technologies, including the real-time spatial multi-omics analysis in living cells, the detailed gene profile in a whole-brain view, and the combination of functional verification.

Postsynaptic histamine H3 receptors in ventral basal forebrain cholinergic neurons modulate contextual fear memory.

Overly strong fear memories can cause pathological conditions. Histamine H3 receptor (H3R) has been viewed as an optimal drug target for CNS disorders, but its role in fear memory remains elusive. We find that a selective deficit of H3R in cholinergic neurons, but not in glutamatergic neurons, enhances freezing level during contextual fear memory retrieval without affecting cued memory. Consistently, genetically knocking down H3R or chemogenetically activating cholinergic neurons in the ventral basal forebrain (vBF) mimics this enhanced fear memory, whereas the freezing augmentation is rescued by re-expressing H3R or chemogenetic inhibition of vBF cholinergic neurons. Spatiotemporal regulation of H3R by a light-sensitive rhodopsin-H3R fusion protein suggests that postsynaptic H3Rs in vBF cholinergic neurons, but not presynaptic H3Rs of cholinergic projections in the dorsal hippocampus, are responsible for modulating contextual fear memory. Therefore, precise modulation of H3R in a cell-type- and subcellular-location-specific manner should be explored for pathological fear memory.

Pathogenesis-adaptive polydopamine nanosystem for sequential therapy of ischemic stroke.

Ischemic stroke is lethal cerebrovascular disease, and reperfusion as the main strategy of blood supply restoration can cause severe ischemic brain damage. Considered as the major obstacle in medication for stroke, neuroinflammation after reperfusion undergoes dynamic progression, making precision treatment for stroke a Herculean task. In this work, we report a pathogenesis-adaptive polydopamine nanosystem for sequential therapy of ischemic stroke. Intrinsic free radical scavenging and tailored mesostructure of the nanosystem can attenuate oxidative stress at the initial stage. Upon microglial overactivation at the later stage, minocycline-loaded nanosystem can timely reverse the pro-inflammatory transition in response to activated matrix metalloproteinase-2, providing on-demand regulation. Further in vivo stroke study demonstrates a higher survival rate and improved brain recovery of the sequential strategy, compared with mono-therapy and combined therapy. Complemented with satisfactory biosafety results, this adaptive nanosystem for sequential and on-demand regulation of post-stroke neuroinflammation is a promising approach to ischemic stroke therapy.

Reconstruction of the cell pseudo-space from single-cell RNA sequencing data with scSpace.

Tissues are highly complicated with spatial heterogeneity in gene expression. However, the cutting-edge single-cell RNA-seq technology eliminates the spatial information of individual cells, which contributes to the characterization of cell identities. Herein, we propose single-cell spatial position associated co-embeddings (scSpace), an integrative method to identify spatially variable cell subpopulations by reconstructing cells onto a pseudo-space with spatial transcriptome references (Visium, STARmap, Slide-seq, etc.). We benchmark scSpace with both simulated and biological datasets, and demonstrate that scSpace can accurately and robustly identify spatially variated cell subpopulations. When employed to reconstruct the spatial architectures of complex tissue such as the brain cortex, the small intestinal villus, the liver lobule, the kidney, the embryonic heart, and others, scSpace shows promising performance on revealing the pairwise cellular spatial association within single-cell data. The application of scSpace in melanoma and COVID-19 exhibits a broad prospect in the discovery of spatial therapeutic markers.

Targeting Histamine and Histamine Receptors for the Precise Regulation of Feeding.

Histamine has long been accepted as an anorexigenic agent. However, lines of evidence have suggested that the roles of histamine in feeding behaviors are much more complex than previously thought, being involved in satiety, satiation, feeding motivation, feeding circadian rhythm, and taste perception and memory. The functional diversity of histamine makes it a viable target for clinical management of obesity and other feeding-related disorders. Here, we update the current knowledge about the functions of histamine in feeding and summarize the underlying molecular and neural circuit mechanisms. Finally, we review the main clinical studies about the impacts of histamine-related compounds on weight control and discuss insights into future research on the roles of histamine in feeding. Despite the recent progress in histamine research, the histaminergic feeding circuits are poorly understood, and it is also worth verifying the functions of histamine receptors in a more spatiotemporally specific manner.

The Diverse Network of Brain Histamine in Feeding: Dissect its Functions in a Circuit-specific Way.

Feeding is an intrinsic and important behavior regulated by complex molecular, cellular and circuit-level mechanisms, one of which is the brain histaminergic network. In the past decades, many studies have provided a foundation of knowledge about the relationship between feeding and histamine receptors, which are deemed to have therapeutic potential but are not successful in treating feeding- related diseases. Indeed, the histaminergic circuits underlying feeding are poorly understood and characterized. This review describes current knowledge of histamine in feeding at the receptor level. Further, we provide insight into putative histamine- involved feeding circuits based on the classic feeding circuits. Understanding the histaminergic network in a circuit-specific way may be therapeutically relevant for increasing the drug specificity and precise treatment in feeding-related diseases.