ADAM10-Initiated Release of Notch Intracellular Domain Regulates Microtubule Stability and Radial Migration of Cortical Neurons.

Proper neuronal migration is orchestrated by combined membrane signal paradigms, whereas the role and mechanism of regulated intramembrane proteolysis (RIP) remain to be illustrated. We show here that the disintegrin and metalloprotease-domain containing protein 10 (ADAM10) regulates cortical neurons migration by initiating the RIP of Notch. We found that Notch intracellular domain (NICD) significantly rescued the migration defect of ADAM10-deficient neurons. Moreover, ADAM10 deficiency led to reduced neuronal motility and disrupted microtubule (MT) structure, which were associated with downregulated expression of acetylated tubulin and MT-associated proteins. Specifically, the NICD/RBPJ complex bound directly to the promoter, and regulated the neuronal expression level of doublecortin (DCX), a modulator of the MT cytoskeleton. Functionally, DCX overexpression largely restored neuron motility and reversed migration defect caused by ADAM10 knockout. Taken together, these findings demonstrate the direct requirement of ADAM10 in cortical radial migration and reveal the underlying mechanism by linking ADAM10-initiated RIP of Notch to the regulation of MT cytoskeleton through transcriptional control of Dcx expression.

X-linked microtubule-associated protein, Mid1, regulates axon development.

Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS.

The Angelman syndrome protein Ube3a is required for polarized dendrite morphogenesis in pyramidal neurons.

Pyramidal neurons have a highly polarized dendritic morphology, characterized by one long apical dendrite and multiple short basal dendrites. They function as the primary excitatory cells of the mammalian prefrontal cortex and the corticospinal tract. However, the molecular mechanisms underlying the development of polarized dendrite morphology in pyramidal neurons remain poorly understood. Here, we report that the Angelman syndrome (AS) protein ubiquitin-protein ligase E3A (Ube3a) plays an important role in specifying the polarization of pyramidal neuron dendritic arbors in mice. shRNA-mediated downregulation of Ube3a selectively inhibited apical dendrite outgrowth and resulted in impaired dendrite polarity, which could be rescued by coexpressing mouse Ube3a isoform 2, but not isoform 1 or 3. Ube3a knockdown also disrupted the polarized distribution of the Golgi apparatus, a well established cellular mechanism for asymmetric dendritic growth in pyramidal neurons. Furthermore, downregulation of Ube3a completely blocked Reelin-induced rapid deployment of Golgi into dendrite. Consistently, we also observed selective inhibition of apical dendrite outgrowth in pyramidal neurons in a mouse model of AS. Overall, these results show that Ube3a is required for the specification of the apical dendrites and dendrite polarization in pyramidal neurons, and suggest a novel pathological mechanism for AS.

A molecularly defined NAcSh D1 subtype controls feeding and energy homeostasis.

Orchestrating complex behavioral states, such as approach and consumption of food, is critical for survival. In addition to hypothalamus neuronal circuits, the nucleus accumbens (NAc) also plays an important role in controlling appetite and satiety in responses to changing external stimuli. However, the specific neuronal subtypes of NAc involved as well as how the humoral and neuronal signals coordinate to regulate feeding remain incompletely understood. Here, we deciphered the spatial diversity of neuron subtypes of the NAc shell (NAcSh) and defined a dopamine receptor D1(Drd1)- and Serpinb2-expressing subtype located in NAcSh encoding food consumption. Chemogenetics- and optogenetics-mediated regulation of Serpinb2 + neurons bidirectionally regulates food seeking and consumption specifically. Circuitry stimulation revealed the NAcSh Serpinb2 →LH LepR projection controls refeeding and can overcome leptin-mediated feeding suppression. Furthermore, NAcSh Serpinb2 + neuron ablation reduces food intake and upregulates energy expenditure resulting in body weight loss. Together, our study reveals a neural circuit consisted of molecularly distinct neuronal subtype that bidirectionally regulates energy homeostasis, which can serve as a potential therapeutic target for eating disorders.

A subset of dopamine receptor-expressing neurons in the nucleus accumbens controls feeding and energy homeostasis.

Orchestrating complex behaviors, such as approaching and consuming food, is critical for survival. In addition to hypothalamus neuronal circuits, the nucleus accumbens (NAc) also controls appetite and satiety. However, specific neuronal subtypes of the NAc that are involved and how the humoral and neuronal signals coordinate to regulate feeding remain incompletely understood. Here we decipher the spatial diversity of neuron subtypes of the NAc shell (NAcSh) and define a dopamine receptor D1-expressing and Serpinb2-expressing subtype controlling food consumption in male mice. Chemogenetics and optogenetics-mediated regulation of Serpinb2+ neurons bidirectionally regulate food seeking and consumption specifically. Circuitry stimulation reveals that the NAcShSerpinb2→LHLepR projection controls refeeding and can overcome leptin-mediated feeding suppression. Furthermore, NAcSh Serpinb2+ neuron ablation reduces food intake and upregulates energy expenditure, resulting in reduced bodyweight gain. Our study reveals a neural circuit consisting of a molecularly distinct neuronal subtype that bidirectionally regulates energy homeostasis, providing a potential therapeutic target for eating disorders.

Accurate inference of genome-wide spatial expression with iSpatial.

Spatially resolved transcriptomic analyses can reveal molecular insights underlying tissue structure and context-dependent cell-cell or cell-environment interaction. Because of the current technical limitation, obtaining genome-wide spatial transcriptome at single-cell resolution is challenging. Here, we developed a new algorithm named iSpatial to derive the spatial pattern of the entire transcriptome by integrating spatial transcriptomic and single-cell RNA-seq datasets. Compared to other existing methods, iSpatial has higher accuracy in predicting gene expression and spatial distribution. Furthermore, it reduces false-positive and false-negative signals in the original datasets. By testing iSpatial with multiple spatial transcriptomic datasets, we demonstrate its wide applicability to datasets from different tissues and by different techniques. Thus, we provide a computational approach to reveal spatial organization of the entire transcriptome at single-cell resolution. With numerous high-quality datasets available in the public domain, iSpatial provides a unique way to understand the structure and function of complex tissues and disease processes.

A molecularly defined D1 medium spiny neuron subtype negatively regulates cocaine addiction.

The striatum plays a critical role in regulating addiction-related behaviors. The conventional dichotomy model suggests that striatal D1/D2 medium spiny neurons (MSNs) positively/negatively regulate addiction-related behaviors. However, this model does not account for the neuronal heterogeneity and functional diversity of the striatum, and whether MSN subtypes beyond the pan-D1/D2 populations play distinct roles in drug addiction remains unknown. We characterized the role of a tachykinin 2-expressing D1 MSN subtype (Tac2+), present in both rodent and primate striatum, using cocaine addiction mouse models. We found that acute cocaine administration reduces Tac2 neuronal activity, and cocaine conditioning alters neuronal response related to cocaine reward contextual associations. In addition, activation/inhibition of Tac2+ neurons attenuates/promotes cocaine-induced conditioned place preference and cocaine intravenous self-administration. Furthermore, stimulation of the NAc-to-lateral hypothalamic projection of Tac2+ neurons suppresses cocaine reward behavior. Our study reveals an unconventional negative regulatory function of D1 MSNs in drug addiction that operates in a subtype- and projection-specific manner.

Cell type-specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis.

Schizophrenia (SCZ) is a chronic, serious mental disorder. Although more than 200 SCZ-associated genes have been identified, the underlying molecular and cellular mechanisms remain largely unknown. Here, we generated a Setd1a (SET domain containing 1A) haploinsufficiency mouse model to understand how this SCZ-associated epigenetic factor affects gene expression in brain regions highly relevant to SCZ. Single-cell RNA sequencing revealed that Setd1a heterozygosity causes highly variable transcriptional adaptations across different cell types in prefrontal cortex (PFC) and striatum. The Foxp2+ neurons exhibit the most prominent gene expression changes among the different neuron subtypes in PFC, which correlate with changes in histone H3 lysine 4 trimethylation. Many of the genes dysregulated in Setd1a+/- mice are involved in neuron morphogenesis and synaptic function. Consistently, Setd1a+/- mice exhibit certain behavioral features of patients with SCZ. Collectively, our study establishes Setd1a+/- mice as a model for understanding SCZ and uncovers a complex brain region- and cell type-specific dysregulation that potentially underlies SCZ pathogenesis.

Decoding molecular and cellular heterogeneity of mouse nucleus accumbens.

The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. In this study, we generated a cell census of the mouse NAc using single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization, revealing a high level of cell heterogeneity in this brain region. Here we show that the transcriptional and spatial diversity of neuron subtypes underlie the NAc's anatomic and functional heterogeneity. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the structure and function of the NAc, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.

Air pollution and hospital outpatient visits for conjunctivitis: a time-series analysis in Tai'an, China.

Conjunctivitis is one of the most common eye-related health problems and significantly influences patients' quality of life. Whether air pollution increased the risks of conjunctivitis is still unclear. Daily counts of outpatient visits for conjunctivitis, air pollution, and meteorological data during January 1, 2015-December 31, 2019 were collected from Tai'an, China. Generalized additive model with Poisson distribution was used to estimate the relationship between air pollution and visits for conjunctivitis, after controlling for the long-term and seasonal trends, weather variables, and day of the week. The effect of air pollution on visits for conjunctivitis was generally acute and significant at the current day and disappeared after 2 days. The relative risk of conjunctivitis visits associated with per 10 µg/m3 increases in PM2.5, PM10, SO2, and NO2 at lag 0-2 days was 1.006 (95% CI: 1.001-1.011), 1.003 (95% CI: 1.000-1.0107), 1.023 (95% CI: 1.009-1.037), and 1.025 (95% CI: 1.010-1.040), respectively. The impact of air pollution on visits for conjunctivitis varied greatly by individual characteristics. The impact of NO2 was higher in males than in females, with the opposite trend for SO2 and PM2.5. Effect estimates of air pollutants were higher among return visits for conjunctivitis, the elderly, and white-collar workers. Our study highlights that the vulnerable subpopulations should pay more attention to protect themselves from air pollution.

Effect modification of the short-term effects of air pollution on morbidity by season: A systematic review and meta-analysis.

Studies of the health effects of air pollution have traditionally controlled for ambient temperature as a confounder, and vice versa. However, season might be an important factor contributing to adverse health effects of air pollution. Given the current inconsistencies in results of previous studies on the effect modification of air pollution on morbidity by season, a systematic review and meta-analysis was conducted to synthesize the current evidence on effects of season on air pollution and morbidity. The electronic databases including PubMed, Web of Science, Embase, CNKI, and Wanfang were used to identify papers published up to the 30st of November in 2019. We identified 4284 articles, after screening, eighty papers met the inclusion criteria. Significant effect modification of CO, O3, SO2 and NO2 on morbidity by season was observed, with corresponding ratio of relative risk of 1.0009 (95% CI: 1.0001-1.0018), 1.0080 (95% CI: 1.0021-1.0138), 0.9828 (95% CI: 0.9697-0.9962) and 0.9896 (95% CI: 0.9824-0.9968), respectively. Season significantly modified the effect of CO on pneumonia, the effect of SO2 on cardiovascular disease, the effect of PM10 on stroke, and the effect of O3 on stroke, asthma and pneumonia. The effect modifications of air pollution by season were similar among males and females, while the effect estimates seem to be higher among children under 18 years old and the elderly aged 75 or over. Further research is needed to better understand the mechanisms underlying the seasonal variance of the effect of air pollutants on morbidity.

Cell type-specific transcriptional programs in mouse prefrontal cortex during adolescence and addiction.

Coordinated activity-induced transcriptional changes across multiple neuron subtypes of the prefrontal cortex (PFC) play a pivotal role in encoding and regulating major cognitive behaviors. Yet, the specific transcriptional programs in each neuron subtype remain unknown. Using single-cell RNA sequencing (scRNA-seq), here we comprehensively classify all unique cell subtypes in the PFC. We analyze transcriptional dynamics of each cell subtype under a naturally adaptive and an induced condition. Adaptive changes during adolescence (between P21 and P60), a highly dynamic phase of postnatal neuroplasticity, profoundly impacted transcription in each neuron subtype, including cell type-specific regulation of genes implicated in major neuropsychiatric disorders. On the other hand, an induced plasticity evoked by chronic cocaine addiction resulted in progressive transcriptional changes in multiple neuron subtypes and became most pronounced upon prolonged drug withdrawal. Our findings lay a foundation for understanding cell type-specific postnatal transcriptional dynamics under normal PFC function and in neuropsychiatric disease states.

Global Associations of Air Pollution and Conjunctivitis Diseases: A Systematic Review and Meta-Analysis.

(1) Background: As the most common eye disease diagnosed in emergency departments, conjunctivitis has caused serious health and economic burdens worldwide. However, whether air pollution may be a risk factor for conjunctivitis is still inconsistent among current evidence. (2) Methods: We searched the literature on the relationship between air pollution and conjunctivitis in multiple English databases before 18 March 2019. Meta-analysis, meta-regression, and funnel plots were used to integrate the data, identify the sources of bias, and determine the publication bias, respectively. (3) Results: A total of 2450 papers were found, 12 of which were finally included. The pooled relative risk for each 10 µg/m3 increase of air pollution on conjunctivitis was 1.0006 (95%CI: 0.9993-1.0019) for CO, 1.0287 (1.0120-1.0457) for NO2, 1.0089 (1.0030-1.0149) for O3, 1.0004 (0.9976-1.0032) for PM2.5, 1.0033 (0.9982-1.0083) for PM10, and 1.0045 (0.9908-1.0185) for SO2. In the subgroup, PM2.5 and O3 had a greater impact on conjunctivitis risk in women than in men, and people <18 years old than those ≥18 years old. Relative humidity significantly modified the risk of O3 on conjunctivitis (p = 0.023), explaining 45% of the between-study heterogeneity. (4) Conclusion: Globally, air pollution has considerable health risks for conjunctivitis. Females and the youth were more vulnerable to PM2.5, NO2, and O3. Reductions of air pollution levels are still warranted to protect the vulnerable populations.

In vivo nuclear capture and molecular profiling identifies Gmeb1 as a transcriptional regulator essential for dopamine neuron function.

Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators.

PRRT2 deficiency induces paroxysmal kinesigenic dyskinesia by regulating synaptic transmission in cerebellum.

Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.

Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity.

The hypothalamus is one of the most complex brain structures involved in homeostatic regulation. Defining cell composition and identifying cell-type-specific transcriptional features of the hypothalamus is essential for understanding its functions and related disorders. Here, we report single-cell RNA sequencing results of adult mouse hypothalamus, which defines 11 non-neuronal and 34 neuronal cell clusters with distinct transcriptional signatures. Analyses of cell-type-specific transcriptomes reveal gene expression dynamics underlying oligodendrocyte differentiation and tanycyte subtypes. Additionally, data analysis provides a comprehensive view of neuropeptide expression across hypothalamic neuronal subtypes and uncover Crabp1+ and Pax6+ neuronal populations in specific hypothalamic sub-regions. Furthermore, we found food deprivation exhibited differential transcriptional effects among the different neuronal subtypes, suggesting functional specification of various neuronal subtypes. Thus, the work provides a comprehensive transcriptional perspective of adult hypothalamus, which serves as a valuable resource for dissecting cell-type-specific functions of this complex brain region.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.