2013 — 2017 |
Jackson, F. Rob (co-PI) [⬀] Yang, Yongjie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Astrocyte Heterogeneity and Developmental Maturation in the Cns @ Tufts University Boston
DESCRIPTION (provided by applicant): Astrocytes are now recognized as active components of mature synapses; they structurally ensheath synapses and modulate neurotransmission in the central nervous system (CNS). Astrocyte dysfunction has been implicated in various neurological disorders and has been shown to actively modulate disease progression. Although astrocytes undergo a developmental maturation process in which subtypes form unique and elaborate morphologies and express overlapping but distinct molecular signatures, it is unknown how astrocyte heterogeneity arises during development of the CNS and how astrocyte development is regulated, in part because of a lack of appropriate tools for such studies. We propose to use integrated molecular and genetic approaches in Drosophila and mouse to define factors that distinguish astrocyte subtypes and regulate their developmental maturation. In particular, this project will focus on these aims: 1) Molecularly define astrocyte subtypes within the cortex and in different CNS regions using FACS and TRAP approaches; 2) Perform dEAAT1-based genetic screens to identify regulators of astrocyte development; 3) Develop new cre recombinase mice for studying astrocyte heterogeneity and function in vivo. We have generated a large amount of preliminary data demonstrating feasibility for the three aims summarized above. By characterizing molecular signatures of astrocyte subtypes in the CNS and identifying regulators of astrocyte development, this project will provide markers for astrocyte subtypes in the cortex and novel insights about how astrocyte maturation occurs. The development of a new cre recombinase driver mouse line will facilitate the selective deletion/activation of genes in astrocytes of the cortex, a region for which an existing astrocyte cre driver line is not very effective. Knowledge of astrocyte heterogeneity and new tools for studying it are critical for understanding how astrocytes become dysfunctional and how distinct classes of astrocytes contribute to the pathogenesis of psychiatric disorders.
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0.952 |
2014 — 2015 |
Yang, Yongjie |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Exosomes, Mirnas and Astroglial Glutamate Transporter Eaat2/Glt1 Regulation @ Tufts University Boston
DESCRIPTION (provided by applicant): The astroglial excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) is one of the most important functional synaptic proteins in astroglia and plays important physiological and pathological functions in CNS. The regulation of EAAT2/GLT1 expression has become an excellent model for understanding how neuronal signals coordinate astroglial function at synapses. Exosomes are a class of newly identified membrane vesicles (40-100nm) of endosomal origin that are secreted from cells. They contain various biomolecules, including proteins, lipids, mRNAs and microRNAs (miRNAs). Exosome-mediated communication and its physiological significance in the CNS are essentially unknown. Based on our recently published results, we hypothesize that exosome-mediated transfer of mir-124a from neurons to astroglia increases GLT1 protein expression by suppressing astroglial miRNAs that inhibit GLT1 mRNA 3' UTR function and reduce GLT1 protein expression in astroglia. In this application, we will focus on the following aims: 1) Identify astroglial miRNAs involved in exosome mir-124a-dependent GLT1 expression regulation in astroglia we will identify astroglial miRNAs that are significantly down-regulated by neuronal exosome treatment and miR-124a transfection using miRNA microarrays. We will then validate expression changes of identified astroglial miRNAs using QRT-PCR and determine whether these astroglial miRNAs directly inhibit GLT1 3' UTR function using a luciferase based assay. Finally, we will determine whether these identified astroglial miRNAs reduce GLT1 protein expression levels and GLT1-dependent glutamate uptake in astroglia. 2) Investigate the exosome and mir-124a transfer from neurons to astroglia in vivo we will investigate the in vivo exosome and mir-124 transfer from neurons to astroglia using genetic and labeling approaches. We will generate transgenic mice that selectively express eGFP-tagged CD63 (green fluorescence) in neurons to characterize the temporal and spatial profile of in vivo transfer of neuronal exosomes (indicated by the eGFP-labeled CD63) to astroglia in intact CNS. We will also selectively deliver Alexa 750 (far red fluorescence) labeled mir-124a to motor neurons by femoral nerve injection and retrograde transport, and then examine its transfer from motor neurons to astroglia in vivo. In summary, this project will identify specific miRNAs that modulate GLT1 protein expression. These newly identified miRNAs will provide novel approaches for modulating synaptic function and are promising targets for developing GLT1-based therapeutics for the treatment of neurological diseases/injuries. In addition, this study will characterize a novel exosome-mediated signaling pathway from neurons to astroglia that regulates astroglial GLT1 expression. This intercellular pathway provides new knowledge about how neuronal signals coordinate astroglial functions at synapses.
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0.952 |
2016 — 2020 |
Yang, Yongjie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Astrolglia-Mediated Pathogenic Mechanisms in Fragile X Syndrome (Fxs) @ Tufts University Boston
? DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) is a developmental intellectual disability caused by the loss of fragile X mental retardation protein (FMRP) function. Altered synaptic plasticity and downstream protein synthesis has been implicated in the pathogenesis of FXS, contributing to typical FXS phenotypes. Although the loss of FMRP in neurons abolishes its repressive function on protein synthesis, how synaptic activation and plasticity (especially in cortex) is altered in FXS remains largely unclear. Interestingly, previou in vivo experiments to selectively delete neuronal FMRP showed partial FXS-related phenotypes. This implies that the selective loss of neuronal FMRP may not be sufficient to induce full FXS pathology and therefore that the loss of FMRP in other brain cells may also contribute to pathogenesis of FXS. Previous studies have found FMRP is expressed in astroglial cells. However, pathogenic roles of the in vivo loss of the astroglial FMRP in FXS remain essentially unexplored. We found significantly reduced glutamate transporter GLT1 and EAAT2 (human analog of rodent GLT1) expression in the cortex of the mouse model (fmr1 knock-out, KO mice) of FXS and human FXS post-mortem samples, and decreased glutamate uptake in fmr1 KO mice. We recently generated inducible astroglia-specific fmr1 conditional knock-out (i-astro-fmr1-cKO) and restoration (i-astro-fmr1-cON) mouse models in which the fmr1 allele is selectively disrupted or restored in astroglia, respectively. We showed that selective deletion of the astroglial FMRP plays a primary role in GLT1 reduction in FXS in vitro and in vivo. Selective deletion of the astroglial FMRP also leads to decreased synaptic AMPA/NMDA current ratio and FXS-like behavior phenotypes (hyperactivity and exaggerated memory extinction). Based on previous observations and our preliminary results, we propose to investigate astroglial dysfunctions in FXS. Specifically, we will 1) Test if the loss of astroglial FMRP contributes to th pathogenesis of FXS in vivo; 2) Investigate mechanisms for GLT1 dysregulation in FXS mouse models; 3) Test if GLT1 up-regulation attenuates FXS phenotypes in FXS mouse models We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. Results from this project will determine if the selective loss of astroglial FMRP contributes to the FXS pathogenesis by potentially reducing GLT1 expression and impairing extracellular glutamate uptake. These results may demonstrate a conceptually novel astroglia-mediated pathogenic pathway in FXS. In addition, the effects of GLT1 up-regulation on FXS-related phenotypes in mouse models of FXS will potentially validate astroglial GLT1 as a new therapeutic target for treating FXS.
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0.952 |
2016 — 2017 |
Yang, Yongjie |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Exosomal Mirna Signaling in Cocaine Addiction @ Tufts University Boston
Abstract This application is responsive to the PAR-15-284 (Extracellular Vesicles and Substance Abuse). Cocaine self-administration significantly reduces glutamate transporter GLT1 protein expression and impairs uptake of extracellular glutamate. Glutamate transporter GLT1 is the physiologically dominant glutamate transporter in the mammalian central nervous system (CNS). GLT1 is selectively and abundantly expressed in astrocytes after postnatal development. They are typically concentrated on the plasma membranes of peri-synaptic astroglial processes where they tightly control extracellular glutamate levels to limit the ?spill-out?/?spill-over? of glutamate from excitatory synapses. The mechanisms for GLT1 dysregulation in cocaine (and other addictive substance) self-administration are currently unknown. Exosomes are a class of newly identified membrane vesicles (40-100nm) of endosomal origin that are secreted from cells; they contain various biomolecules, including proteins, lipids, mRNAs and microRNAs (miRNAs). Exosome-mediated intercellular signaling from neuron to glia and its physiological significance in the CNS are essentially unknown. Based on our previously published and additional preliminary results, we hypothesize that exosome- mediated transfer of mir-124 from neurons to astrocytes is altered, which underlie GLT1 dysregulation in the cocaine addiction model. In this application, we will focus on the following aims: 1) Investigate exosome and mir-124 transfer from neurons to astrocytes in the cocaine addiction model we will first examine exosome secretion dynamics from striatum neuronal cultures. we will also breed CD63-eGFPf/f with dopamine receptor D1 or D2 (Drd1 or Drd2) Cre mice that allow selective labeling of exosomes in D1+ or D2+ medium spiny neurons (MSNs). We will then examine the transfer of labeled exosomes from D1+ or D2+ MSNs to neighboring astrocytes in nucleus accumbens (NAc) during different stages of cocaine addiction in situ. We will also examine changes of mir-124 levels in astrocytes by mir-124 in situ hybridization in NAc in the cocaine model. 2) Determine whether mir-124-mediated up-regulation of GLT1 attenuates cocaine relapse- associated synaptic activation we will perform stereotaxic injection to deliver mir-124 into NAc core during cocaine self-administration and test whether exogenously delivered mir-124 is able to prevent GLT1 loss and attenuate enhanced synaptic activation on MSNs in NAc during cocaine relapse. In summary, this study will investigate alterations of exosome and mi-124 transfer from neurons to astrocytes in the cocaine addiction model. This study will provide novel insights about the patho(physiological) significance of exosome-mediated miRNA transfer in mammalian CNS, especially in understanding how dysregulation of neuron to glial signaling contributes to drug addiction and relapse. Lastly, the test of mir-124's effects on cocaine relapse-associated synaptic activation may provide a new approach to intervene cocaine relapse.
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0.952 |
2018 |
Trotti, Davide [⬀] Yang, Yongjie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Exosome-Mediated Propagation of Disease Linked Poly-Dipeptides in C9orf72-Ftd/Als @ Thomas Jefferson University
A growing body of evidence uncovered a propensity for frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) pathogenic proteins to propagate from cell-to-cell. Although few other mechanisms have been proposed, secretion of exosomes has been reported to occur from different neural cell types, including neurons, and to potentially serve as a new intercellular communication route within the CNS. Interestingly, based on the evidence of focality and neuroanatomical propagation of clinical symptoms, it was also hypothesized that the cerebro-spinal fluid (CSF) could serve as vehicle for pathogenic proteins spread, at least in ALS. Utilizing different in vitro cell culture platforms, including spinal motor neurons derived from iPSCs of C9orf72 patients, we recently learnt that C9orf72-linked dipeptide proteins (DPRs) spread between neural cells via the exosome-dependent pathway. By analyzing a newly generated exosome-reporter transgenic mouse, we also found that exosomes are capable of migrating extensive distance in vivo. These observations led us to postulate that an exosome-mediated propagation of DPRs could be a modality by which toxic insults spread in disease-afflicted CNS areas in C9orf72-FTD/ALS. We will be testing using complementary in vitro and in vivo approaches the novel hypothesis that transmitted DPRs transfer injury via exosomes to both neighboring cells, but also to neurons downstream in synaptic circuits. We propose: (1) To investigate exosome-mediated mechanisms of DPRs transmission in CNS cells; (2) To examine the modalities of cell-to-cell propagation of DPRs in vivo; (3) To examine whether cell transfer of DPRs propagates toxicity. The proposed work has the potential to open up an entirely new field of C9orf72 FTD/ALS research, at the same time, providing important clues to the fundamental biological processes in brain cellular communications relevant to brain diseases. Thus, the results are expected to have a significant impact for understanding C9orf72-linked FTD/ALS pathogenesis and eventually treating patients.
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0.948 |
2018 |
Tesco, Giuseppina Yang, Yongjie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neuronal/Glial Exosome Signaling and Beta-Amyloid Propagation in Alzheimer's Disease (Ad) @ Tufts University Boston
Abstract Alzheimer's disease (AD) is a progressive brain disease that severely affects memory, reasoning, and behaviors. Currently, there is no cure available for AD, making it one of the most unmet medical challenges in our society. In the AD, the formation and toxicity of ?-amyloid plaque and tau-positive neurofibrillary tangles are central to the AD pathology. Especially, both ?-amyloid plaque and tau positive neurofibrillary tangles propagate from entorhinal cortex (EC) to hippocampal regions, significantly spreading the toxicity and exacerbate the disease pathology. Recently, exosome-mediated secretion and intercellular communication has been implicated in disease initiation and propagation in AD. Exosomes (size 40-150 nm), a major type of secreted extracellular vesicles (EVs), are derived from intraluminal vesicles (ILV) that are budded inwardly from the early endosomal compartment, and are released from multiple vesicular bodies (MVBs), during endosome maturation. However, very little is currently known about whether and how exosome dynamics (ILV trafficking, exosome secretion and uptake) is altered by the expression of the mutant APP in CNS cells in the rodent (APPNL-F/NL-F mice) and human (fAD iPSC-derived neurons) AD models. Especially, the dynamic changes of the secretion and migration of cell-type specifically secreted exosomes in vivo in APPNL-F/NL-F mice and their potential toxicity to synapses has not been investigated. We have previously published that both neurons and glia secrete exosomes. We have recently generated a knock-in CD63-GFPf/f mouse line from which the copGFP (a variant of GFP)-fused CD63 (membrane marker of ILV/exosomes) can be induced in a Cre-dependent manner, allowing cell-type specific labeling and tracing of exosomes in situ in different (patho)physiological conditions. By employing this new tool, we further found that exosomes are abundantly present in vivo and secreted exosomes can migrate extensive distance from the initial secreted site. Based on previous studies and our preliminary results, we propose to investigate the following aims: 1) Determine the effect of mutant APP on exosome dynamics in CNS cells in mouse and human AD models; 2) Investigate cell-type specific exosome-mediated A? plaque propagation and toxicity mechanisms in the AD model. We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. We will employ mouse genetics, virus injections, imaging, and biochemical approaches to investigate these two aims. Outcomes from this project will provide much needed new insights about exosome secretion/uptake in various CNS cells in APPNL-F/NL-F mice. Importantly, our in vivo investigation of cell-type specifically secreted exosomes and their association to the A? pathology in AD models, -by employing our newly generated exosome reporter (CD63-GFP cKI) mice, will be a significant step forward in understanding the in vivo roles of cell-type specific exosomes in AD pathogenesis, which will ultimately lead to new therapeutic opportunities.
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0.952 |
2020 — 2021 |
Yang, Yongjie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Exosomal Mirna in Neuron to Astroglial Communication in the Cns @ Tufts University Boston
Abstract Neuron to (astro)glia communication is essential for functional synaptic transmission and physiology in the CNS. Despite the important modulatory roles of astroglia in synapse function, molecular pathways that regulate the neuron-astroglia functional unit are largely undefined. Exosomes (50-150 nm in diameter), a major type of secreted extracellular vesicles (EVs), are derived from intraluminal vesicles (ILVs) in the early endosomal compartment and are released from cells during endosome maturation. EVs and exosomes secreted from various CNS cell types have emerged as a novel and important intercellular communication pathway in the CNS. In particular, miRNAs (miRs) are often found in exosomes to shuttle between cells for intercellular signaling. Intercellular transfers of miRs have been observed in CNS cells to regulate glutamate transporter function, promote myelination, and maintain brain vascular integrity. Exosomal signaling has also been implicated in pathological conditions of the CNS, including neurological injury, neurodegenerative diseases, and glioblastoma. Despite the strong rigor in prior studies to suggest the importance of the exosomal pathway in CNS cell communication, these studies are largely based on culture models or human CSF samples, exosome signaling in situ in the CNS remains essentially unexplored. In addition, fundamentally important cell biology aspects of this pathway, such as neuronal activity's influence, exosome internalization mechanisms, and downstream regulation in recipient CNS cells also remain unknown. This is particularly important to address as CNS cell types are highly distinct from cancer/immune cells where most of exosome knowledge is currently gained and exosome signaling mechanisms can be very cell-type heterogeneous. Based on our published study and additional preliminary results, we propose the following aims in this project: Aim 1: Determine the effect of neuronal activity on the subcellular localization of ILVs and neuronal exosome secretion; Aim 2: Dissect recognition pathways and entry mechanisms involved in astroglial internalization of neuronal exosomes; Aim 3: Investigate genetic regulation of neuronal exosomal miR-124 in astroglia; We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. We will employ mouse genetics, molecular biology, virus injections, various imaging, and biochemical approaches to complete these aims. Outcomes from this project will present in vivo evidence to support a previously unrecognized mode of communication from neurons to glia in the CNS. It will also provide much-needed cell biological knowledge and insights for understanding exosome signaling in neuron to glia communication, especially about miR-124-3p's non-cell autonomous genetic regulation in astroglia following its internalization. As altered neuron to (astro)glia communication is clearly implicated in many neurological diseases, this knowledge and insights can significantly help understand how this pathway is involved in CNS diseases.
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0.952 |