Browsing by Author "Farris, Shannon"

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    Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic Mice
    Steward, Oswald; Yee, Kelly Matsudaira; Farris, Shannon; Pirbhoy, Patricia S.; Worley, Paul; Okamura, Kohji; Okuno, Hiroyuki; Bito, Haruhiko (Frontiers, 2018-01-31)
    Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized ArcmRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3_UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3_UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3_UTR.
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    Editorial: RNA Localization and Localized Translation in Neurons
    Farris, Shannon; Hacisuleyman, Ezgi; Donlin-Asp, Paul; Cioni, Jean-Michel (Frontiers, 2022-01-12)
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    Hippocampal Subregions Express Distinct Dendritic Transcriptomes that Reveal Differences in Mitochondrial Function in CA2
    Farris, Shannon; Ward, James M.; Carstens, Kelly E.; Samadi, Mahsa; Wang, Yu; Dudek, Serena M. (CellPress, 2019-10-08)
    RNA localization is one mechanism neurons use to spatially and temporally regulate gene expression at synapses. Here, we test the hypothesis that cells exhibiting distinct forms of synaptic plasticity will have differences in dendritically localized RNAs. Indeed, we discover that each major subregion of the adult mouse hippocampus expresses a unique complement of dendritic RNAs. Specifically, we describe more than 1,000 differentially expressed dendritic RNAs, suggesting that RNA localization and local translation are regulated in a cell typespecific manner. Furthermore, by focusing Gene Ontology analyses on the plasticity-resistant CA2, we identify an enrichment of mitochondria-associated pathways in CA2 cell bodies and dendrites, and we provide functional evidence that these pathways differentially influence plasticity and mitochondrial respiration in CA2. These data indicate that differences in dendritic transcriptomes may regulate cell type-specific properties important for learning and memory and may influence region-specific differences in disease pathology.
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    Histone Hypervariants H2A.Z.1 and H2A.Z.2 Play Independent and Context-Specific Roles in Neuronal Activity-Induced Transcription of Arc/ Arg3.1 and Other Immediate Early Genes
    Dunn, Carissa J.; Sarkar, Pushpita; Bailey, Emma R.; Farris, Shannon; Zhao, Meilan; Ward, James M.; Dudek, Serena M.; Saha, Ramendra N. (Society for Neuroscience, 2017)
    The histone variant H2A.Z is an essential and conserved regulator of eukaryotic gene transcription. However, the exact role of this histone in the transcriptional process remains perplexing. In vertebrates, H2A.Z has two hypervariants, H2A.Z.1 and H2A.Z.2, that have almost identical sequences except for three amino acid residues. Due to such similarity, functional specificity of these hypervariants in neurobiological processes, if any, remain largely unknown. In this study with dissociated rat cortical neurons, we asked if H2A.Z hypervariants have distinct functions in regulating basal and activity-induced gene transcription. Hypervariant-specific RNAi and microarray analyses revealed that H2A.Z.1 and H2A.Z.2 regulate basal expression of largely nonoverlapping gene sets, including genes that code for several synaptic proteins. In response to neuronal activity, rapid transcription of our model gene Arc is impaired by depletion of H2A.Z.2, but not H2A.Z.1. This impairment is partially rescued by codepletion of the H2A.Z chaperone, ANP32E. In contrast, under a different context (after 48 h of tetrodotoxin, TTX), rapid transcription of Arc is impaired by depletion of either hypervariant. Such context-dependent roles of H2A.Z hypervariants, as revealed by our multiplexed gene expression assays, are also evident with several other immediate early genes, where regulatory roles of these hypervariants vary from gene to gene under different conditions. Together, our data suggest that H2A.Z hypervariants have context-specific roles that complement each other to mediate activity-induced neuronal gene transcription.
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    Layer-specific mitochondrial diversity across hippocampal CA2 dendrites
    Pannoni, Katy E.; Gil, Daniela; Cawley, Mikel L.; Alsalman, Mayd M.; Campbell, Logan A.; Farris, Shannon (Wiley, 2023-02)
    CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Furthermore, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites, which was confirmed in CA2 with genetically labeled mitochondria and electron microscopy. MCU overexpression in neighboring CA1 led to a preferential localization of MCU in the proximal dendrites of CA1 compared to the distal dendrites, an effect not seen in CA2. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and suggest that MCU can be differentially localized within dendrites, possibly to meet local energy demands.
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    Localization and local translation of Arc/Arg3.1 mRNA at synapses: some observations and paradoxes
    Steward, Oswald; Farris, Shannon; Pirbhoy, Patricia S.; Darnell, Jennifer; Van Driesche, Sarah J. (Frontiers, 2015-01-12)
    Arc is a unique immediate early gene whose expression is induced as synapses are being modified during learning. The uniqueness comes from the fact that newly synthesized Arc mRNA is rapidly transported throughout dendrites where it localizes near synapses that were recently activated. Here, we summarize aspects of Arc mRNA translation in dendrites in vivo, focusing especially on features of its expression that are paradoxical or that do not fit in with current models of how Arc protein operates. Findings from in vivo studies that donor quite fit include: (1) Following induction of LTP in vivo, Arc mRNA and protein localize near active synapses, but are also distributed throughout dendrites. In contrast, Arc mRNA localizes selectively near active synapses when stimulation is continued as Arc mRNA is transported into dendrites; (2) Strong induction of Arc expression as a result of a seizure does not lead to a rundown of synaptic efficacy in vivo as would be predicted by the hypothesis that high levels of Arc cause glutamate receptor endocytosis and LTD. (3) Arc protein is synthesized in the perinuclear cytoplasm rapidly after transcriptional activation, indicating that at least a pool of Arc mRNA is not translationally repressed to allow for dendritic delivery; (4) Increases in Arc mRNA in dendrites are not paralleled by increases in levels of exon junction complex (EJC) proteins. These results of studies of mRNA trafficking in neurons in vivo provide a new perspective on the possible roles of Arc in activity-dependent synaptic modifications.
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    Mechanisms of mGluR-dependent plasticity in hippocampal area CA2
    Samadi, Mahsa; Hales, Claire A.; Lustberg, Daniel J.; Farris, Shannon; Ross, Madeleine R.; Zhao, Meilan; Hepler, John R.; Harbin, Nicholas H.; Robinson, Emma S. J.; Banks, Paul J.; Bashir, Zafar I.; Dudek, Serena M. (Wiley, 2023-06)
    Pyramidal cells in hippocampal area CA2 have synaptic properties that are distinct from the other CA subregions. Notably, this includes a lack of typical long-term potentiation of stratum radiatum synapses. CA2 neurons express high levels of several known and potential regulators of metabotropic glutamate receptor (mGluR)-dependent signaling including Striatal-Enriched Tyrosine Phosphatase (STEP) and several Regulator of G-protein Signaling (RGS) proteins, yet the functions of these proteins in regulating mGluR-dependent synaptic plasticity in CA2 are completely unknown. Thus, the aim of this study was to examine mGluR-dependent synaptic depression and to determine whether STEP and the RGS proteins RGS4 and RGS14 are involved. Using whole cell voltage-clamp recordings from mouse pyramidal cells, we found that mGluR agonist-induced long-term depression (mGluR-LTD) is more pronounced in CA2 compared with that observed in CA1. This mGluR-LTD in CA2 was found to be protein synthesis and STEP dependent, suggesting that CA2 mGluR-LTD shares mechanistic processes with those seen in CA1, but in addition, RGS14, but not RGS4, was essential for mGluR-LTD in CA2. In addition, we found that exogenous application of STEP could rescue mGluR-LTD in RGS14 KO slices. Supporting a role for CA2 synaptic plasticity in social cognition, we found that RGS14 KO mice had impaired social recognition memory as assessed in a social discrimination task. These results highlight possible roles for mGluRs, RGS14, and STEP in CA2-dependent behaviors, perhaps by biasing the dominant form of synaptic plasticity away from LTP and toward LTD in CA2.
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    Mechanisms underlying neural circuit remodeling in Toxoplasma gondii infection
    Carrillo, Gabriela Lizana (Virginia Tech, 2022-09-20)
    The central nervous system (CNS) is protected by a vascular blood-brain barrier that prevents many types of pathogens from entering the brain. Still, some pathogens have evolved mechanisms to traverse this barrier and establish a long-term infection. The apicomplexan parasite, Toxoplasma gondii, is one such pathogen with the ability to infect the CNS in virtually all warm-blooded animals, including humans. Across the globe, an estimated 30% of the human population is infected with Toxoplasma, an infection for which mounting evidence suggests increases the risk for developing neurological and neuropsychiatric disorders, like seizures and schizophrenia. In my dissertation, I investigate the telencephalic neural circuit changes induced by long-term Toxoplasma infection in the mouse brain and the neuroimmune signaling role of the complement system in mediating microglial remodeling of neural circuits following parasitic infection. While there has been keen interest in investigating neural circuit changes in the amygdala – a region of the brain involved in fear response and which Toxoplasma infection alters in many species of infected hosts – the hippocampus and cortex have been less explored. These are brain regions for which Toxoplasma also has tropism, and moreover, are rich with fast-spiking parvalbumin perisomatic synapses, a type of GABAergic synapse whose dysfunction has been implicated in epilepsy and schizophrenia. By employing a range of visualization techniques to assess cell-to-cell connectivity and neuron-glia interactions (including immunohistochemistry, ultrastructural microscopy, and microglia-specific reporter mouse lines), I discovered that longterm Toxoplasma infection causes microglia to target and ensheath neuronal somata in these regions and subsequently phagocytose their perisomatic inhibitory synapses. These findings provide a novel model by which Toxoplasma infection within the brain can lead to seizure susceptibility and a wider range of behavioral and cognitive changes unrelated to fear response. In the Toxoplasma infected brain, microglia, along with monocytes recruited to the brain from the periphery, coordinate a neuroinflammatory response against pathogenic invasion. This is characterized by a widespread activation of these cells and their increased interaction with neurons and their synaptic inputs. Yet, whether T. gondii infection triggers microglia and monocytes (i.e. phagocytes) to target, ensheath, and remove perisomatic inhibitory synapses on neuronal somata indiscriminately, or whether specificity exists in this type of circuit remodeling, remained unclear. Through a comprehensive assessment of phagocyte interactions with cortical neuron subtypes, I demonstrate that phagocytes selectively target and ensheath excitatory pyramidal cells in long-term infection. Moreover, coupling of in situ hybridization with transgenic reporter lines and immunolabeling revealed that in addition to phagocytes, excitatory neurons also express complement component C3 following infection (while inhibitory interneurons do not). Lastly, by employing targeted deletion of complement components, C1q and C3, I show that complement is required for phagocyte ensheathment of excitatory cells and the subsequent removal of perisomatic inhibitory synapses on these cells (albeit not through the classical pathway). Together, these studies highlight a novel role for complement in mediating synapse-type and cell-type specific circuit remodeling in the Toxoplasma infected brain.
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    Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq
    Farris, Shannon; Wang, Yu; Ward, James M.; Dudek, Serena M. (Frontiers, 2017-06-17)
    Obtaining high quality RNA from complex biological tissues, such as the brain, is needed for establishing high-fidelity cell-type specific transcriptomes. Although combining genetic labeling techniques with laser capture microdissection (LCM) is generally sufficient, concerns over RNA degradation and limited yields call into question results of many sequencing studies. Here we set out to address both of these issues by: (1) developing a fluorescence-assisted LCM protocol that yields high quality RNA from fresh-frozen tissues; and (2) determining a suitable RNA-Seq library generation method for limited amounts of RNA (1–5 ng total RNA). The latter focused on comparing commercially available kits able to produce libraries of sufficient concentration and complexity while limiting PCR amplification biases. We find that high quality RNA (RNA integrity number, RIN, >9) of sufficient concentration can be isolated from lasercaptured material from thinly-sectioned tissues when digestion time and temperature are minimized. Furthermore, we found that library generation approaches that retain ribosomal RNA (rRNA) through cDNA library generation required fewer cycles of PCR, minimizing bias in the resulting libraries. Lastly, end stage depletion of rRNA prior to sequencing enriches for target RNAs, thereby increasing read depth and level of gene detection while decreasing sequencing costs. Here we describe our protocol for generating robust RNA-Seq libraries from laser-captured tissue and demonstrate that with this method, we obtain samples with RNA quality superior to the current standard in the LCM field, and show that low-input RNA-Seq kits that minimize PCR bias produce high fidelity sequencing metrics with less variability compared to current practices.
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    Role of retinal inputs and astrocytes for the development of visual thalamus
    Somaiya, Rachana Deven (Virginia Tech, 2022-06-01)
    Axons of retinal ganglion cells (RGCs) send visual information to a number of retinorecipient regions in the brain. In rodents, visual thalamus receives dense innervations from RGC axons and is important for both image-forming and nonimage-forming visual functions. Retinal inputs invade visual thalamus during embryonic development, before the arrival of non-retinal inputs (such as local interneurons and axonal inputs from other brain regions). In this dissertation, I explore how early innervation of RGC axons affects circuitry in visual thalamus and the role of visual experience, neural activity, and molecular cues in the development. While the development of astrocytes in cortex has been well-described, they have been largely overlooked in visual thalamus. Using immunohistochemical, functional, and ultrastructural analysis, I show that astrocytes in visual thalamus reach adult-like morphological properties and functionality at retinogeniculate synapses early in development, by eye-opening and before visual experience. These studies reveal that while experience-dependent visual activity from RGC axons is critical for many aspects of visual thalamus development, astrocytic maturation occurs independent of that information about our visual environment. As with astrocytes, little progress has been made in understanding the development of interneurons in the visual thalamus. Here, I show that retinal inputs interact with thalamic astrocytes to influence the recruitment of GABAergic interneurons into visual thalamus. I found that this interaction between RGC axons and astrocytes is not dependent on neural activity of RGCs. Using transcriptomic analysis, in situ hybridization, and reporter lines, I observed thalamus-projecting RGCs express SHH and astrocytes in visual thalamus express SHH signaling molecules. My results reveal that SHH signaling between RGC axons and astrocytes is critical for astrocytic fibroblast growth factor 15 (FGF15) expression in developing visual thalamus. Ultimately, FGF15 serves as a potent motogen that is essential for thalamic interneuron migration. These data identify a novel morphogen-dependent and activity-independent mechanism that mediates crosstalk between RGCs and astrocytes to facilitate the recruitment of interneurons into the developing visual thalamus.
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    Social and novel contexts modify hippocampal CA2 representations of space
    Alexander, Georgia M.; Farris, Shannon; Pirone, Jason R.; Zheng, Chenguang; Colgin, Laura L.; Dudek, Serena M. (Springer Communications, 2016-01-25)
    The hippocampus supports a cognitive map of space and is critical for encoding declarative memory (who, what, when and where). Recent studies have implicated hippocampal subfield CA2 in social and contextual memory but how it does so remains unknown. Here we find that in adult male rats, presentation of a social stimulus (novel or familiar rat) or a novel object induces global remapping of place fields in CA2 with no effect on neuronal firing rate or immediate early gene expression. This remapping did not occur in CA1, suggesting this effect is specific for CA2. Thus, modification of existing spatial representations might be a potential mechanism by which CA2 encodes social and novel contextual information.
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    Spatiotemporal Regulation of Transcript Isoform Expression in the Hippocampus
    Park, Joun; Farris, Shannon (Frontiers, 2021-07-08)
    Proper development and plasticity of hippocampal neurons require specific RNA isoforms to be expressed in the right place at the right time. Precise spatiotemporal transcript regulation requires the incorporation of essential regulatory RNA sequences into expressed isoforms. In this review, we describe several RNA processing strategies utilized by hippocampal neurons to regulate the spatiotemporal expression of genes critical to development and plasticity. The works described here demonstrate how the hippocampus is an ideal investigative model for uncovering alternate isoform-specific mechanisms that restrict the expression of transcripts in space and time.