Comprehensive spatiotemporal mapping of single-cell lineages in developing mouse brain by CRISPR-based barcoding.

A fundamental interest in developmental neuroscience lies in the ability to map the complete single-cell lineages within the brain. To this end, we developed a CRISPR editing-based lineage-specific tracing (CREST) method for clonal tracing in Cre mice. We then used two complementary strategies based on CREST to map single-cell lineages in developing mouse ventral midbrain (vMB). By applying snapshotting CREST (snapCREST), we constructed a spatiotemporal lineage landscape of developing vMB and identified six progenitor archetypes that could represent the principal clonal fates of individual vMB progenitors and three distinct clonal lineages in the floor plate that specified glutamatergic, dopaminergic or both neurons. We further created pandaCREST (progenitor and derivative associating CREST) to associate the transcriptomes of progenitor cells in vivo with their differentiation potentials. We identified multiple origins of dopaminergic neurons and demonstrated that a transcriptome-defined progenitor type comprises heterogeneous progenitors, each with distinct clonal fates and molecular signatures. Therefore, the CREST method and strategies allow comprehensive single-cell lineage analysis that could offer new insights into the molecular programs underlying neural specification.

Functional reconstruction of the basal ganglia neural circuit by human striatal neurons in hypoxic-ischaemic injured brain.

Perinatal hypoxic-ischaemic encephalopathy is the leading cause of neonatal death and permanent neurological deficits, while the basal ganglia is one of the major nuclei that is selectively and greatly affected in the brains of hypoxic-ischaemic encephalopathy patients, especially in severe cases. Human embryonic stem cell-derived neurons have shown great potential in different types of brain disorders in adults. However, it remains unknown whether and how grafted human embryonic stem cell-derived neurons can repair immature brains with hypoxic-ischaemic encephalopathy. Here, by administrating genetically labelled human embryonic stem cell-derived striatal neural progenitors into the ipsilateral striatum of hypoxic-ischaemic encephalopathy-injured mice, we found that the grafted cells gradually matured into GABA spiny projection neurons morphologically and electrophysiologically, and significantly rescued the area loss of hypoxic-ischaemic encephalopathy-injured brains. Intriguingly, using immunohistochemical staining combined with enhanced ascorbate peroxidase-based immunoelectron microscopy and rabies virus-mediated trans-synaptic tracing, we show that the grafts start to extend axonal projections to the endogenous target areas (globus pallidus externa, globus pallidus internus, substantia nigra), form synapses with host striatal, globus pallidus and nigra neurons, and receive extensive and stable synaptic inputs as early as 2 months post-transplantation. Importantly, we further demonstrated functional neural circuits re-established between the grafted neurons and host cortical, striatal and substantial nigra neurons at 3-6 months post-transplantation in the hypoxic-ischaemic encephalopathy-injured brain by optogenetics combined with electrophysiological recording. Finally, the transplanted striatal spiny projection neurons but not spinal GABA neurons restored the motor defects of hypoxic-ischaemic encephalopathy, which were reversed by clozapine-N-oxide-based inhibition of graft function. These findings demonstrate anatomical and functional reconstruction of the basal ganglia neural circuit including multiple loops by striatal spiny projection neurons in hypoxic-ischaemic encephalopathy-injured immature brains, which raises the possibility of such a cell replacement therapeutic strategy for hypoxic-ischaemic encephalopathy in neonates.

Human cerebral organoids establish subcortical projections in the mouse brain after transplantation.

Numerous studies have used human pluripotent stem cell-derived cerebral organoids to elucidate the mystery of human brain development and model neurological diseases in vitro, but the potential for grafted organoid-based therapy in vivo remains unknown. Here, we optimized a culturing protocol capable of efficiently generating small human cerebral organoids. After transplantation into the mouse medial prefrontal cortex, the grafted human cerebral organoids survived and extended projections over 4.5 mm in length to basal brain regions within 1 month. The transplanted cerebral organoids generated human glutamatergic neurons that acquired electrophysiological maturity in the mouse brain. Importantly, the grafted human cerebral organoids functionally integrated into pre-existing neural circuits by forming bidirectional synaptic connections with the mouse host neurons. Furthermore, compared to control mice, the mice transplanted with cerebral organoids showed an increase in freezing time in response to auditory conditioned stimuli, suggesting the potentiation of the startle fear response. Our study showed that subcortical projections can be established by microtransplantation and may provide crucial insights into the therapeutic potential of human cerebral organoids for neurological diseases.

LncRNA MALAT1 promotes migration and invasion of non-small-cell lung cancer by targeting miR-206 and activating Akt/mTOR signaling.

Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) functions as a crucial regulator of metastasis in lung cancer. The aim of this study is to unravel the underlying mechanisms of lncRNA MALAT1 in non-small-cell lung cancer (NSCLC). A cohort of 36 NSCLC tumor tissues and adjacent normal tissues was collected postoperatively from patients with NSCLC. qRT-PCR was performed to detect the expression of MALAT1 in both NSCLC tissues and cell lines. Cell migration and invasion were monitored by wound healing assay and transwell invasion assay. Western blot was used to detect the expression levels of epithelial-mesenchymal transition proteins and Akt/mTOR key components after treatment. Dual luciferase reporter assay coupled with qRT-PCR was used to verify the direct interaction between MALAT1 and miR-206. MALAT1 was significantly up-regulated in both NSCLC tissues and cell lines. High expression of MALAT1 correlated positively with tumor size and lymphatic metastasis in NSCLC, whereas no correlation was found between MALAT1 expression and sex, age, clinical stage, and histological grade. We also showed that MALAT1 promoted epithelial-mesenchymal transition, cell migration, and invasion by activating Akt/mTOR signaling in A549 and H1299 cells. miR-206 was a direct downstream target of MALAT1 in NSCLC. MALAT1 promoted cell migration and invasion by sponging miR-206 in NSCLC cells. In addition, miR-206 inhibited MALAT1-mediated activation of Akt/mTOR signaling in A549 and H1299 cells. lncRNA MALAT1 promotes migration and invasion of NSCLC by targeting miR-206 and activating Akt/mTOR signaling.

Identification of early jasmonate-responsive genes in Taxus × media cells by analyzing time series digital gene expression data.

Jasmonate, an effective elicitor, can induce the biosynthesis of paclitaxel, a well-known anticancer drug, in Taxus cell culture. The jasmonate signaling pathway has been well studied in Arabidopsis, and many early jasmonate-responsive genes have been found to be involved in signaling pathway. In Taxus, only a few late jasmonate-responsive genes related to paclitaxel biosynthesis were identified. So, identification of early responsive genes and knowledge of the jasmonate signaling pathway are essential for understanding the effects of jasmonate on paclitaxel biosynthesis and for improving paclitaxel production in Taxus cells. In this study, total RNA of Taxus × media cells cultured in liquid medium was extracted after 0, 0.5, 3, and 24 h of methyl jasmonate treatment. Three biological independent repetitions were performed. The 12 extracted RNA samples were integrated and sequenced on an Illumina HiSeq 2500 platform using the paired-end method. A total of 45,583 transcript clusters were obtained by de novo assembly of the sequenced reads. Based on the transcriptome data, the digital gene expressions of each RNA sample were investigated. We found that after 0.5, 3, and 24 h of methyl jasmonate treatment; 134, 1008, and 987 unigenes were differentially expressed. For the secondary metabolism pathways, phenylalanine pathway unigenes were responsive to jasmonate after 3 h of treatment, while genes related to paclitaxel biosynthesis were induced after 0.5 h of treatment. The digital gene expression levels of candidate genes related to paclitaxel biosynthesis were confirmed by qRT-PCR. Transcriptome sequencing and digital gene expression profiling identified early jasmonate-responsive genes in cultured Taxus × media cells. The comprehensive time series jasmonate-responsive gene expression data have provided transcriptome-wide information about the mechanism of paclitaxel biosynthesis regulation by jasmonate signaling.

Genome-Wide Analysis of DNA Methylation in Hyperoxia-Exposed Newborn Rat Lung.

PURPOSE: Oxygen therapy is often required to treat newborn infants with respiratory disorders. Prolonged exposure of neonatal rats to hyperoxia reduced alveolar septation, increased terminal air space size, and increased lung fibrosis; these conditions are very similar to those of human bronchopulmonary dysplasia. Epigenetic regulation of gene expression plays a crucial role in bronchopulmonary dysplasia development. METHOD: We reared Sprague-Dawley rat pups in either room air (RA, n = 24) or an atmosphere containing 85% O2 (n = 26) from Postnatal Days 1 to 14. Methylated DNA immunoprecipitation (MeDIP) was used to analyze genome-wide DNA methylation in lung tissues of neonatal rats. Hyperoxia-exposed rats exhibited larger air spaces and thinner septa than RA-exposed rats did on Postnatal Day 14. The rats exposed to hyperoxia exhibited significantly higher mean linear intercepts than did the rats exposed to RA. We applied MeDIP next-generation sequencing for profiling changes in DNA methylation in the rat lungs exposed to hyperoxia and RA. We performed bioinformatics and pathway analyses on the raw sequencing data to identify differentially methylated candidate genes. RESULTS: Our in vivo model revealed that neonatal hyperoxia exposure arrested alveolarization on Postnatal Day 14. We found that the ErbB, actin cytoskeleton, and focal adhesion signaling pathways are epigenetically modulated by exposure to hyperoxia. We demonstrated that hyperoxia exposure contribute in delaying lung development through an epigenetic mechanism by disrupting the expression of genes in lungs that might be involved in alveolarization. CONCLUSIONS: These data indicate that aberrant DNA methylation and deregulation of the actin cytoskeleton and focal adhesion pathways of lung tissues may be involved in the pathophysiology of hyperoxia-induced arrested alveolarization.

Microcomputed tomography assessment of lipopolysaccharide-induced acute lung injury in rat.

PURPOSE: High-resolution microcomputed tomography (micro-CT) is an extremely flexible and accurate technique for three-dimensional examination of biological tissues. The aim was to evaluate the feasibility of micro-CT as a noninvasive tool for analyzing the lung structure during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats. MATERIALS AND METHODS: ALI was induced in rats by intratracheal instillation of LPS (2 mg/kg) in 0.3 ml saline, and the control treatment consisted of intratracheal instillation of an equal volume of normal saline. Morphological changes were assessed by using micro-CT and a light microscope at 24 and 48 hours after LPS instillation. Total volume is the sum of all pixels marked as the whole lung and total air volume (Air V) is the sum volume of all air in the lung. RESULTS: The saline groups exhibited no major histological abnormalities, whereas the LPS groups exhibited patchy areas of haemorrhage and thickened alveolar walls with inflammatory cell infiltration at 24 and 48 hours. The LPS groups had significantly smaller Air V and percent total air volume (Air V/TV) compared with those of the saline groups at 24 and 48 hours. Air V/TV was strongly negatively correlated with the lung injury score (r = -0.641, P = .004). CONCLUSIONS: Micro-CT is a feasible tool for evaluating the lung structure and lung injury progression during LPS-induced ALI.

A simple and efficient system for regulating gene expression in human pluripotent stem cells and derivatives.

Regulatable transgene expression in human pluripotent stem cells (hPSCs) and their progenies is often necessary to dissect gene function in a temporal and spatial manner. However, hPSC lines with inducible transgene expression, especially in differentiated progenies, have not been established due to silencing of randomly inserted genes during stem cell expansion and/or differentiation. Here, we report the use of transcription activator-like effector nucleases-mediated targeting to AAVS1 site to generate versatile conditional hPSC lines. Transgene (both green fluorescent protein and a functional gene) expression in hPSCs and their derivatives was not only sustained but also tightly regulated in response to doxycycline both in vitro and in vivo. We modified the donor construct so that any gene of interest can be readily inserted to produce hPSC lines with conditional transgene expression. This technology will substantially improve the way we study human stem cells.

Myo9b and RICS modulate dendritic morphology of cortical neurons.

Regulated growth and branching of dendritic processes is critical for the establishment of neuronal circuitry and normal brain functions. Rho family GTPases, including RhoA, Rac1, and Cdc42, play a prominent role in dendritic development. RhoA inhibits dendritic branching and growth, whereas Rac1/Cdc42 does the opposite. It has been suggested that the activity of RhoA must be kept low to allow dendritic growth. However, how neurons restrict the activation of RhoA for proper dendritic development is not clear. In the present study, we undertook a comprehensive loss-of-function analysis of putative RhoA GTPase-activating proteins (RhoA GAPs) in the cortical neurons. The expression of 16 RhoA GAPs was detected in the developing rat brain, and RNA interference experiments suggest that 2 of them, Myo9b and RICS, are critical regulators of dendritic morphogenesis. Knockdown of either Myo9b or RICS in cultured cortical neurons or developing cortex resulted in decreased dendrite length and number. Inhibition of RhoA/ROCK signaling restores the defects of dendritic morphology induced by knockdown of Myo9b or RICS. These data demonstrate that Myo9b and RICS repress RhoA/Rock signaling and modulate dendritic morphogenesis in cortical neurons, providing evidence for critical physiological function of RhoA GAPs in regulation of dendritic development.

[Neurons in NAc core and BLA are activated during cocaine context-associated reward memory retrieval in mice].

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli make addiction hard to cure by contributing to cocaine seeking and relapse. So it's of great importance to examine the neurobiological basis of addiction memory. Cocaine conditioned place preference (CPP) used in this study is a form of Pavlovian conditioning which can establish associations between drug and contextual factors. c-Fos and Zif268 are commonly used immediate early gene (IEG) makers to identify neurons that are activated after a stimulus or behavioral conditioning. This study was designed to reveal neuronal c-Fos, Zif268 expression pattern in 10 brain regions following cocaine context-associated reward memory retrieval in mice, combining animal behavioral study and immunofluorescence method. C57BL/6 mice were randomly divided into 3 groups: Saline retrieval, Cocaine retrieval, and No retrieval of cocaine groups. Cocaine retrieval and No retrieval of cocaine underwent CPP training (one side paired with cocaine, and the other side with saline) except that No retrieval of cocaine group didn't undergo CPP test. Saline retrieval group received saline injections (i.p) on both sides. The results showed that: Neuronal c-Fos, Zif268 protein expression levels in nucleus accumbens (NAc) core both were elevated in Cocaine retrieval group compared with those in Saline retrieval (Control) group during cocaine context-associated reward memory retrieval. Zif268 protein expression level in basolateral amygdala (BLA) was also elevated in Cocaine retrieval group compared with that in control mice. Elevation was not seen in other regions such as hippocampus, prefrontal cortex (PFC). Thus, NAc core and BLA were activated during cocaine context-associated reward memory retrieval. The results suggest that neurons that are activated in NAc core and BLA are crucial basis of cocaine context-associated reward memory.

The role of cardiac microenvironment in cardiovascular diseases: implications for therapy.

Due to aging populations and changes in lifestyle, cardiovascular diseases including cardiomyopathy, hypertension, and atherosclerosis, are the leading causes of death worldwide. The heart is a complicated organ composed of multicellular types, including cardiomyocytes, fibroblasts, endothelial cells, vascular smooth muscle cells, and immune cells. Cellular specialization and complex interplay between different cell types are crucial for the cardiac tissue homeostasis and coordinated function of the heart. Mounting studies have demonstrated that dysfunctional cells and disordered cardiac microenvironment are closely associated with the pathogenesis of various cardiovascular diseases. In this paper, we discuss the composition and the homeostasis of cardiac tissues, and focus on the role of cardiac environment and underlying molecular mechanisms in various cardiovascular diseases. Besides, we elucidate the novel treatment for cardiovascular diseases, including stem cell therapy and targeted therapy. Clarification of these issues may provide novel insights into the prevention and potential targets for cardiovascular diseases.

Single-Cell Transcriptome Landscape and Cell Fate Decoding in Human Brain Organoids after Transplantation.

Human stem cells and derivatives transplantation are widely used to treat nervous system diseases, while the fate determination of transplanted cells is not well elucidated. To explore cell fate changes of human brain organoids before and after transplantation, human brain organoids are transplanted into prefrontal cortex (PFC) and hippocampus (HIP), respectively. Single-cell sequencing is then performed. According to time-series sample comparison, transplanted cells mainly undergo neural development at 2 months post-transplantation (MPT) and then glial development at 4MPT, respectively. A different brain region sample comparison shows that organoids grafted to PFC have obtained cell fate close to those of host cells in PFC, other than HIP, which may be regulated by the abundant expression of dopamine (DA) and acetylcholine (Ach) in PFC. Meanwhile, morphological complexity of human astrocyte grafts is greater in PFC than in HIP. DA and Ach both activate the calcium activity and increase morphological complexity of astrocytes in vitro. This study demonstrates that human brain organoids receive host niche factor regulation after transplantation, resulting in the alignment of grafted cell fate with implanted brain regions, which may contribute to a better understanding of cell transplantation and regenerative medicine.

High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.

Deciphering patterns of connectivity between neurons in the brain is a critical step toward understanding brain function. Imaging-based neuroanatomical tracing identifies area-to-area or sparse neuron-to-neuron connectivity patterns, but with limited throughput. Barcode-based connectomics maps large numbers of single-neuron projections, but remains a challenge for jointly analyzing single-cell transcriptomics. Here, we established a rAAV2-retro barcode-based multiplexed tracing method that simultaneously characterizes the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets and found that projection-defined vmPFC neurons are molecularly heterogeneous. We identified transcriptional signatures of projection-specific vmPFC neurons, and verified Pou3f1 as a marker gene enriched in neurons projecting to the lateral hypothalamus, denoting a distinct subset with collateral projections to both dorsomedial striatum and lateral hypothalamus. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.

Human midbrain dopaminergic progenitors.

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.

Human neural stem cells.

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

Mechanism of carbon nanotube growth in expanded graphite via catalytic pyrolysis reaction using carbores P as a carbon source.

Carbon nanotubes (CNTs) had potential applications in energy conversion and storage devices, and it could be prepared by expanded graphite loaded with catalyst at high temperature, however, the mechanism of carbon nanotube growth in expanded graphite need further confirmation. In this work, carbon nanotubes' in situ growth in expanded graphite (EG) were prepared via catalytic pyrolysis reaction using carbores P as a carbon source and Co(NO3)3•6H2O as a catalyst. The results of X-ray diffraction (XRD), scanning electron microscope (SEM) and energy dispersive X-ray spectroscope (EDS) indicated the carbon nanotubes could generate in, EG with the presence of carbores P as a carbon source and cobalt nitrate as a catalyst. More interestingly, the growth mechanism of carbon nanotubes could be concluded by the results of differential thermal analysis-thermogravimetry-mass spectrometry (DTA-TG-MS) and X-ray photoelectron spectroscopy (XPS) analysis. The pyrolysis products of carbores P were mainly hydrocarbon gas such as CH4 gas, which reacts with Co(NO3)3·6H2O catalyst to reduces CoOx to Co particles, then the carbon form pyrolysis was deposited the on the surface catalyst Co particles and, after continuous solid dissolution and precipitation, carbon nanotubes were at last generated in EG at last.

Mapping of clonal lineages across developmental stages in human neural differentiation.

The cell lineages across developmental stages remain to be elucidated. Here, we developed single-cell split barcoding (SISBAR) that allows clonal tracking of single-cell transcriptomes across stages in an in vitro model of human ventral midbrain-hindbrain differentiation. We developed "potential-spective" and "origin-spective" analyses to investigate the cross-stage lineage relationships and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. We uncovered many previously uncharacterized converging and diverging trajectories. Furthermore, we demonstrate that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multilineage fates of a progenitor cell-type represent the collective results of distinct rather than similar clonal fates of individual progenitors, each with distinct molecular signatures. Specifically, we uncovered a ventral midbrain progenitor cluster as the common clonal origin of midbrain dopaminergic (mDA) neurons, midbrain glutamatergic neurons, and vascular and leptomeningeal cells and identified a surface marker that can improve graft outcomes.

microRNA-660 Enhances Cisplatin Sensitivity via Decreasing SATB2 Expression in Lung Adenocarcinoma.

Increasing evidence suggests that microRNAs' (miRNAs) abnormal expression is one of the main factors of chemotherapy resistance in various cancers. However, the role of miRNAs in lung adenocarcinoma (LUAD) resistance to cisplatin is still unclear. In this study, we analyzed a microarray dataset to investigate miRNAs related to cisplatin resistance in LUAD. The expression of miRNAs in LUAD tissues and cell lines was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Special AT-Rich Sequence-Binding Protein 2 (SATB2) in LUAD cell lines was detected using RT-qPCR and Western blot. Cell proliferation was measured by CCK8 and colony formation assays, while cell cycle and apoptosis were measured by flow cytometry. A dual-luciferase reporter assay was performed to confirm that SATB2 is a target gene of microRNA-660 (miR-660). We showed that the expression of miR-660 was not only decreased in LUAD cells and tissues but also further decreased in the cisplatin-resistant A549 cell line. The overexpression of miR-660 increased cisplatin sensitivity in LUAD cells. In addition, we identified SATB2 as a direct target gene of miR-660. We also revealed that miR-660 increased cisplatin sensitivity in LUAD cells via targeting SATB2. In conclusion, miR-660/SATB2 axis is a key regulator of cisplatin resistance in LUAD.

Noncoding transcripts are linked to brain resting-state activity in non-human primates.

Brain-derived transcriptomes are known to correlate with resting-state brain activity in humans. Whether this association holds in nonhuman primates remains uncertain. Here, we search for such molecular correlates by integrating 757 transcriptomes derived from 100 macaque cortical regions with resting-state activity in separate conspecifics. We observe that 150 noncoding genes explain variations in resting-state activity at a comparable level with protein-coding genes. In-depth analysis of these noncoding genes reveals that they are connected to the function of nonneuronal cells such as oligodendrocytes. Co-expression network analysis finds that the modules of noncoding genes are linked to both autism and schizophrenia risk genes. Moreover, genes associated with resting-state noncoding genes are highly enriched in human resting-state functional genes and memory-effect genes, and their links with resting-state functional magnetic resonance imaging (fMRI) signals are altered in the brains of patients with autism. Our results highlight the potential for noncoding RNAs to explain resting-state activity in the nonhuman primate brain.

Serine 363 of the {delta}-opioid receptor is crucial for adopting distinct pathways to activate ERK1/2 in response to stimulation with different ligands.

Distinct opioid receptor agonists have been proved to induce differential patterns of ERK activation, but the underlying mechanisms remain unclear. Here, we report that Ser363 in the δ-opioid receptor (δOR) determines the different abilities of the δOR agonists DPDPE and TIPP to activate ERK by G-protein- or ß-arrestin-dependent pathways. Although both DPDPE and TIPP activated ERK1/2, they showed different temporal, spatial and desensitization patterns of ERK activation. We show that that DPDPE employed G protein as the primary mediator to activate the ERK cascade in an Src-dependent manner, whereas TIPP mainly adopted a ß-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the ß-arrestin1/2-mediated pathway upon Ser363 mutation, accompanied by the same pattern of ERK activation as that induced by TIPP. Additionally, we found that TIPP- but not DPDPE-activated ERK could phosphorylate G-protein-coupled receptor kinase-2 and ß-arrestin1. However, such functional differences of ERK disappeared with the mutation of Ser363. Therefore, the present study reveals a crucial role for Ser363 in agonist-specific regulation of ERK activation patterns and functions.