Unconjugated
Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.
Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified "late domain" motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.
In vestibular cerebellum, primary afferents carry signals from single vestibular end organs, whereas secondary afferents from vestibular nucleus carry integrated signals. Selective targeting of distinct mossy fibers determines how the cerebellum processes vestibular signals. We focused on vestibular projections to ON and OFF classes of unipolar brush cells (UBCs), which transform single mossy fiber signals into long-lasting excitation or inhibition respectively, and impact the activity of ensembles of granule cells. To determine whether these contacts are indeed selective, connectivity was traced back from UBC to specific ganglion cell, hair cell and vestibular organ subtypes in mice. We show that a specialized subset of primary afferents contacts ON UBCs, but not OFF UBCs, while secondary afferents contact both subtypes. Striking anatomical differences were observed between primary and secondary afferents, their synapses, and the UBCs they contact. Thus, each class of UBC functions to transform specific signals through distinct anatomical pathways.
© 2019, Balmer and Trussell.
Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.
During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.
To meet the challenge to human health posed by obesity, a better understanding of the regulation of feeding is essential. Medications targeting 5-hydroxytryptamine (5-HT; serotonin) 2C receptors (htr2c; 5-HT2CR) improve obesity. Here we probed the functional significance of 5-HT2CRs specifically within the brainstem nucleus of the solitary tract (5-HT2CRNTS) in feeding behavior. Selective activation of 5-HT2CRNTS decreased feeding and was sufficient to mediate acute food intake reductions elicited by the 5-HT2CR agonist obesity medication lorcaserin. Similar to pro-opiomelanocortin neurons expressed within the hypothalamic arcuate nucleus (POMCARC), a subset of POMCNTS neurons co-expressed 5-HT2CRs and were activated by 5-HT2CR agonists. Knockdown of POMCNTS prevented the acute appetite-suppressive effect of lorcaserin, whereas POMCARC knockdown prevented the full anorectic effect. These data identify 5-HT2CRNTS as a sufficient subpopulation of 5-HT2CRs in reducing food intake when activated and reveal that 5-HT2CR agonist obesity medications require POMC within the NTS and ARC to reduce food intake.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Genetically encoded calcium indicators (GECIs) have gained widespread use for measurement of neuronal activity but their low expression levels in transgenic mice tend to limit sensitivity. We have developed a transgenic mouse line (SyG37) that expresses a ratiometric calcium sensor, SyGCaMP2-mCherry, that is expressed throughout the brain but targeted to presynaptic terminals. Within the CA1 and CA3 regions of hippocampus of male and female mice, SyGaMP2 fluorescence responds linearly up to 10 electrical stimuli at frequencies up to 100 Hz and it can detect responses to a single stimulus. Responses in single boutons can be measured using multiphoton microscopy. The ensemble amplitude of SyGCaMP2 responses is a function of the number of stimuli applied and the number of contributing boutons. The peak responses and initial rates of calcium influx in single boutons in CA1 and CA3 were similar but the rate of calcium clearance from CA3 boutons after stimulation was significantly faster. In CA1, DNQX reduced SyGCaMP2 responses to Schaffer collateral stimulation to 86% of baseline indicating that 14% of the total response originated from presynaptic terminals of neurones synaptically driven via AMPA receptors. Theta burst stimulation induced long-term potentiation (LTP) of both SyGCaMP2 and fEPSP responses in both young and 18-month-old mice. The proportion of postsynaptically connected terminals increased significantly to 76% of the total after LTP induction. The SyG37 mouse allows stable optical detection of synaptic activation and connectivity at the single bouton level and can be used to characterize the contributions of presynaptic calcium to synaptic transmission and plasticity.
Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non-human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC-derived progenitors were grafted into the midbrain of 6-hydroxydopamine-lesioned rats, and analyzed at 6, 18, and 24?weeks for a time-course evaluation of specificity and extent of graft-derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies-based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24?weeks. The timing and extent of graft-derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine-induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC-derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC-derived DA neurons to the midbrain.
© 2018 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.
In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.
Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.
Copyright © 2018 Elsevier Inc. All rights reserved.
Friedreich's ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the FXN gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart. Transplanted HSPCs engrafted and then differentiated into microglia in the brain and spinal cord and into macrophages in the DRGs, heart, and muscle of YG8R FRDA mice. We observed the transfer of wild-type frataxin and Cox8 mitochondrial proteins from HSPC-derived microglia/macrophages to FRDA mouse neurons and muscle myocytes in vivo. Our results show the HSPC-mediated phenotypic rescue of FRDA in YG8R mice and suggest that this approach should be investigated further as a strategy for treating FRDA.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Emotionally salient situations usually trigger arousal along with autonomic and neuroendocrine reactions. To determine whether the extended amygdala plays a role in sleep-wakefulness regulation, we examined the effects of optogenetic and pharmacogenetic excitation of GABAergic neurons in the bed nucleus of the stria terminalis (GABABNST neurons). Acute optogenetic excitation of these cells during nonrapid eye movement (NREM) sleep resulted in an immediate state transition to wakefulness, whereas stimulation during REM sleep showed no effect on sleep-wakefulness states in male mice. An anterograde tracing study suggested GABABNST neurons send axonal projections to several brain regions implicated in arousal, including the preoptic area, lateral hypothalamus, periaqueductal gray, deep mesencephalic nucleus, and parabrachial nucleus. A dual orexin receptor antagonist, DORA-22, did not affect the optogenetic transition from NREM sleep to wakefulness. Chemogenetic excitation of GABABNST neurons evoked a sustained wakefulness state, but this arousal effect was markedly attenuated by DORA-22. These observations suggest that GABABNST neurons play an important role in transition from NREM sleep to wakefulness without the function of orexin neurons, but prolonged excitation of these cells mobilizes the orexin system to sustain wakefulness.SIGNIFICANCE STATEMENT We examined the role of the bed nucleus of the stria terminalis (BNST) in the regulation of wakefulness. Optogenetic excitation of GABAergic neurons in the BNST (GABABNST neurons) during nonrapid eye movement (NREM) sleep in mice resulted in immediate transition to a wakefulness state without function of orexins. Prolonged excitation of GABABNST neurons by a chemogenetic method evoked a longer-lasting, sustained wakefulness state, which was abolished by preadministration of a dual orexin receptor antagonist, DORA-22. This study revealed a role of the BNST GABAergic system in sleep-wakefulness control, especially in shifting animals' behavioral states from NREM sleep to wakefulness, and provides an important insight into the pathophysiology of insomnia and the role of orexin in arousal regulation.
Copyright © 2017 the authors 0270-6474/17/377164-13$15.00/0.
The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.
© 2017 The Authors.
In addition to motor function, the cerebellum has been implicated in cognitive and social behaviors. Various structural and functional abnormalities of Purkinje cells (PCs) have been observed in schizophrenia and autism. As PCs express the gene Disrupted-In-Schizophrenia-1 (DISC1), and DISC1 variants have been associated with neurodevelopmental disorders, we evaluated the role of DISC1 in cerebellar physiology and associated behaviors using a mouse model of inducible and selective expression of a dominant-negative, C-terminus truncated human DISC1 (mutant DISC1) in PCs. Mutant DISC1 male mice demonstrated impaired social and novel placement recognition. No group differences were found in novelty-induced hyperactivity, elevated plus maze test, spontaneous alternation, spatial recognition in Y maze, sociability or accelerated rotarod. Expression of mutant DISC1 was associated with a decreased number of large somata PCs (volume: 3000-5000µm3) and an increased number of smaller somata PCs (volume: 750-1000µm3) without affecting the total number of PCs or the volume of the cerebellum. Compared to control mice, attached loose patch recordings of PCs in mutant DISC1 mice revealed increased spontaneous firing of PCs; and whole cell recordings showed increased amplitude and frequency of mEPSCs without significant changes in either Rinput or parallel fiber EPSC paired-pulse ratio. Our findings indicate that mutant DISC1 alters the physiology of PCs, possibly leading to abnormal recognition memory in mice.
Copyright © 2017 Elsevier Inc. All rights reserved.
Astrocytes extend highly branched processes that form functionally isolated microdomains, facilitating local homeostasis by redistributing ions, removing neurotransmitters, and releasing factors to influence blood flow and neuronal activity. Microdomains exhibit spontaneous increases in calcium (Ca2+), but the mechanisms and functional significance of this localized signaling are unknown. By developing conditional, membrane-anchored GCaMP3 mice, we found that microdomain activity that occurs in the absence of inositol triphosphate (IP3)-dependent release from endoplasmic reticulum arises through Ca2+ efflux from mitochondria during brief openings of the mitochondrial permeability transition pore. These microdomain Ca2+ transients were facilitated by the production of reactive oxygen species during oxidative phosphorylation and were enhanced by expression of a mutant form of superoxide dismutase 1 (SOD1 G93A) that causes astrocyte dysfunction and neurodegeneration in amyotrophic lateral sclerosis (ALS). By localizing mitochondria to microdomains, astrocytes ensure local metabolic support for energetically demanding processes and enable coupling between metabolic demand and Ca2+ signaling events.
Copyright © 2017 Elsevier Inc. All rights reserved.
The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.
Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.
© 2016. Published by The Company of Biologists Ltd.
Tactile stimuli are integrated and processed by neuronal circuits in the deep dorsal horn of the spinal cord. Several spinal interneuron populations have been implicated in tactile information processing. However, dorsal horn projection neurons that contribute to the postsynaptic dorsal column (PSDC) pathway transmitting tactile information to the brain are poorly characterized. Here, we show that spinal neurons marked by the expression of Zic2creER mediate light touch sensitivity and textural discrimination. A subset of Zic2creER neurons are PSDC neurons that project to brainstem dorsal column nuclei, and chemogenetic activation of Zic2 PSDC neurons increases sensitivity to light touch stimuli. Zic2 neurons receive direct input from the cortex and brainstem motor nuclei and are required for corrective motor movements. These results suggest that Zic2 neurons integrate sensory input from cutaneous afferents with descending signals from the brain to promote corrective movements and transmit processed touch information back to the brain. VIDEO ABSTRACT.
Copyright © 2019 Elsevier Inc. All rights reserved.
Cell replacement is currently being explored as a therapeutic approach for neurodegenerative disease. Using stem cells as a source, transplantable progenitors can now be generated under conditions compliant with clinical application in patients. In this study, we elucidate factors controlling target-appropriate innervation and circuitry integration of human embryonic stem cell (hESC)-derived grafts after transplantation to the adult brain. We show that cell-intrinsic factors determine graft-derived axonal innervation, whereas synaptic inputs from host neurons primarily reflect the graft location. Furthermore, we provide evidence that hESC-derived dopaminergic grafts transplanted in a long-term preclinical rat model of Parkinson's disease (PD) receive synaptic input from subtypes of host cortical, striatal, and pallidal neurons that are known to regulate the function of endogenous nigral dopamine neurons. This refined understanding of how graft neurons integrate with host circuitry will be important for the design of clinical stem-cell-based replacement therapies for PD, as well as for other neurodegenerative diseases.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
Brain tumor stem cells (BTSCs) are a chemoresistant population that can drive tumor growth and relapse, but the lack of BTSC-specific markers prevents selective targeting that spares resident stem cells. Through a ribosome-profiling analysis of mouse neural stem cells (NSCs) and BTSCs, we find glycerol-3-phosphate dehydrogenase 1 (GPD1) expression specifically in BTSCs and not in NSCs. GPD1 expression is present in the dormant BTSC population, which is enriched at tumor borders and drives tumor relapse after chemotherapy. GPD1 inhibition prolongs survival in mouse models of glioblastoma in part through altering cellular metabolism and protein translation, compromising BTSC maintenance. Metabolomic and lipidomic analyses confirm that GPD1+ BTSCs have a profile distinct from that of NSCs, which is dependent on GPD1 expression. Similar GPD1 expression patterns and prognostic associations are observed in human gliomas. This study provides an attractive therapeutic target for treating brain tumors and new insights into mechanisms regulating BTSC dormancy.
Copyright © 2019 Elsevier Inc. All rights reserved.
Time locking between neocortical sleep slow oscillations, thalamo-cortical spindles, and hippocampal sharp-wave ripples has convincingly been shown to be a key element of systems consolidation. Here we investigate the role of monosynaptic projections from ventral/intermediate hippocampus to medial prefrontal cortex (mPFC) in sleep-dependent memory consolidation in male mice. Following acquisition learning in the Barnes maze, we optogenetically silenced the axonal terminals of hippocampal projections within mPFC during slow-wave sleep. This silencing during SWS selectively impaired recent but not remote memory in the absence of effects on error rate and escape latencies. Furthermore, it prevented the development of the most efficient search strategy and sleep spindle time-locking to slow oscillation. An increase in post-learning sleep sharp-wave ripple (SPWR) density and reduced time locking of learning-associated SPWR activity to sleep spindles may be a less specific response. Our results demonstrate that monosynaptic projections from hippocampus to mPFC contribute to sleep-dependent memory consolidation, potentially by affecting the temporal coupling of sleep-associated electrophysiological events.SIGNIFICANCE STATEMENT Convincing evidence supports the role of slow-wave sleep (SWS), and the relevance of close temporal coupling of neuronal activity between brain regions for systems consolidation. Less attention has been paid so far to the specific neuronal pathways underlying these processes. Here, we optogenetically silenced the direct monosynaptic projection from ventral/intermediate hippocampus (HC) to medial prefrontal cortex (mPFC) during SWS in male mice following repeated learning trials in a weakly aversive spatial task. Our results confirm the concept that the monosynaptic projection between HC and mPFC contributes to memory consolidation and support an important functional role of this pathway in shaping the temporal precision among sleep-associated electrophysiological events.
Copyright © 2019 the authors.
Dorsal root ganglion (DRG) sensory neuron subtypes defined by their in vivo properties display distinct intrinsic electrical properties. We used bulk RNA sequencing of genetically labeled neurons and electrophysiological analyses to define ion channel contributions to the intrinsic electrical properties of DRG neuron subtypes. The transcriptome profiles of eight DRG neuron subtypes revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by these data, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties. The distinct ensembles of voltage-gated ion channels predicted to underlie the unique intrinsic physiological properties of eight DRG neuron subtypes are presented.
Copyright © 2019 Elsevier Inc. All rights reserved.
Grasping an object or crossing a trench requires the integration of information on the operating distance of our limbs with precise distance estimation. The reach of our hands and step size of our legs are learned by the visual feedback we get during our actions. This implicit knowledge of our peripersonal space is first acquired during infancy but will be continuously updated throughout our whole life [1]. In contrast, body size of holometabolous insects does not change after metamorphosis; nevertheless, they do have to learn their body reaches at least once. The body size of Drosophila imagines can vary by about 15% depending on environmental factors like food quality and temperature [2]. To investigate how flies acquire knowledge about and memorize their body size, we studied their decisions to either refrain from or initiate climbing over gaps exceeding their body size [3]. Naive (dark-reared) flies overestimate their size and have to learn it from the parallax motion of the retinal images of objects in their environment while walking. Naive flies can be trained in a striped arena and manipulated to underestimate their size, but once consolidated, this memory seems to last for a lifetime. Consolidation of this memory is stress sensitive only in the first 2 h after training but cannot be retrieved for the next 12 h. We have identified a set of intrinsic, lateral neurons of the protocerebral bridge of the central complex [4, 5] that depend on dCREB2 transcriptional activity for long-term memory consolidation and maintenance.
Copyright © 2019 Elsevier Ltd. All rights reserved.
The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasis and after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.
Copyright © 2019 Elsevier Inc. All rights reserved.
Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE.SIGNIFICANCE STATEMENT Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOn-inducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/macular degenerative diseases.
Copyright © 2019 the authors.
Neural progenitor cell grafts form new relays across sites of spinal cord injury (SCI). Using a panel of neuronal markers, we demonstrate that spinal neural progenitor grafts to sites of rodent SCI adopt diverse spinal motor and sensory interneuronal fates, representing most neuronal subtypes of the intact spinal cord, and spontaneously segregate into domains of distinct cell clusters. Host corticospinal motor axons regenerating into neural progenitor grafts innervate appropriate pre-motor interneurons, based on trans-synaptic tracing with herpes simplex virus. A human spinal neural progenitor cell graft to a non-human primate also received topographically appropriate corticospinal axon regeneration. Thus, grafted spinal neural progenitor cells give rise to a variety of neuronal progeny that are typical of the normal spinal cord; remarkably, regenerating injured adult corticospinal motor axons spontaneously locate appropriate motor domains in the heterogeneous, developing graft environment, without a need for additional exogenous guidance.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
Sleep and wakefulness are controlled by a wide range of neuronal populations in the mammalian brain. Activation of adenosine A2A receptor (A2AR)-expressing neurons in the nucleus accumbens (NAc) core promotes slow-wave sleep (SWS). The neuronal mechanism by which activation of NAc A2AR neurons induces SWS, however, is unknown. We hypothesized that the ability of NAc activation to induce sleep is mediated by the classic somnogen adenosine, which can be formed by various processes in all types of cells. Here, to investigate whether astrocytes are involved in the ability of the NAc to regulate SWS, we ablated glial fibrillary acidic protein (GFAP)-positive cells in the NAc core of mice by virus-mediated expression of diphtheria toxin (DT) receptors and intraperitoneal administration of DT. Analysis of electroencephalogram and electromyogram recordings of DT-treated wild-type mice revealed that SWS was remarkably increased at 1 week after DT treatment, whereas sleep-wake behavior was unchanged in DT-treated A2AR knockout mice. Cell ablation was associated with an increased number of GFAP-positive cells and activation of microglia in the NAc. In-vivo microdialysis revealed significantly increased levels of extracellular adenosine in the NAc at 1 week after DT treatment. Our findings suggest that elevated adenosine levels in the NAc core promote SWS by acting on A2ARs and provide the first evidence that adenosine is an endogenous candidate for activating NAc A2AR neurons that have the ability to induce SWS.
Copyright © 2019 Elsevier Ltd. All rights reserved.
Functional mosaic analysis allows for the direct comparison of mutant cells with differentially marked control cells in the same organism. While this offers a powerful approach for elucidating the role of specific genes or signalling pathways in cell populations of interest, genetic strategies for generating functional mosaicism remain challenging. We describe a novel and streamlined approach for functional mosaic analysis, which combines stochastic Cre/lox recombination with gene targeting in the ROSA26 locus. With the RoMo strategy a cell population of interest is randomly split into a cyan fluorescent and red fluorescent subset, of which the latter overexpresses a chosen transgene. To integrate this approach into high-throughput gene targeting initiatives, we developed a procedure that utilizes Gateway cloning for the generation of new targeting vectors. RoMo can be used for gain-of-function experiments or for altering signaling pathways in a mosaic fashion. To demonstrate this, we developed RoMo-dnGs mice, in which Cre-recombined red fluorescent cells co-express a dominant-negative Gs protein. RoMo-dnGs mice allowed us to inhibit G protein-coupled receptor activation in a fraction of cells, which could then be directly compared to differentially marked control cells in the same animal. We demonstrate how RoMo-dnGs mice can be used to obtain mosaicism in the brain and in peripheral organs for various cell types. RoMo offers an efficient new approach for functional mosaic analysis that extends the current toolbox and may reveal important new insights into in vivo gene function.
© 2018 Wiley Periodicals, Inc.
The Atoh7 transcription factor catalyzes the rate-limiting step in the specification of retinal ganglion cells (RGCs). As a tool to study vertebrate retinal development, we validate an antibody that recognizes human and mouse Atoh7 polypeptide, using informative knockout and transgenic mouse tissues and overexpression experiments. The transient features of Atoh7 protein expression during retinal neurogenesis match the expected pattern at the tissue and cellular level. Further, we compare endogenous Atoh7 to established RGC markers, reporter mouse lines and cell cycle markers, demonstrating the utility of the antibody to investigate molecular mechanisms of retinal histogenesis.
Copyright © 2017 Elsevier B.V. All rights reserved.
Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.
Copyright © 2017 Elsevier Inc. All rights reserved.
Adult neurogenesis in the olfactory epithelium is often depicted as a unidirectional pathway during homeostasis and repair. We challenge the unidirectionality of this model by showing that epithelial injury unlocks the potential for Ascl1+ progenitors and Neurog1+ specified neuronal precursors to dedifferentiate into multipotent stem/progenitor cells that contribute significantly to tissue regeneration in the murine olfactory epithelium (OE). We characterize these dedifferentiating cells using several lineage-tracing strains and single-cell mRNA-seq, and we show that Sox2 is required for initiating dedifferentiation and that inhibition of Ezh2 promotes multipotent progenitor expansion. These results suggest that the apparent hierarchy of neuronal differentiation is not irreversible and that lineage commitment can be overridden following severe tissue injury. We elucidate a previously unappreciated pathway for endogenous tissue repair by a highly regenerative neuroepithelium and introduce a system to study the mechanisms underlying plasticity in the OE that can be adapted for other tissues.
Copyright © 2017 Elsevier Inc. All rights reserved.
Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.
A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.
Copyright © 2016, American Association for the Advancement of Science.
The ability of the adult mammalian brain to compensate for neuronal loss caused by injury or disease is very limited. Transplantation aims to replace lost neurons, but the extent to which new neurons can integrate into existing circuits is unknown. Here, using chronic in vivo two-photon imaging, we show that embryonic neurons transplanted into the visual cortex of adult mice mature into bona fide pyramidal cells with selective pruning of basal dendrites, achieving adult-like densities of dendritic spines and axonal boutons within 4-8 weeks. Monosynaptic tracing experiments reveal that grafted neurons receive area-specific, afferent inputs matching those of pyramidal neurons in the normal visual cortex, including topographically organized geniculo-cortical connections. Furthermore, stimulus-selective responses refine over the course of many weeks and finally become indistinguishable from those of host neurons. Thus, grafted neurons can integrate with great specificity into neocortical circuits that normally never incorporate new neurons in the adult brain.
Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.
Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.
X-linked Charcot-Marie-Tooth disease (CMTX1) results from numerous mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32) and is one of the commonest forms of inherited neuropathy. Owing to the expression of Cx32 not only in Schwann cells but also in oligodendrocytes, a subset of CMT1X patients develops central nervous system (CNS) clinical manifestations in addition to peripheral neuropathy. While most GJB1 mutations appear to cause peripheral neuropathy through loss of Cx32 function, the cellular mechanisms underlying the CNS manifestations remain controversial. A novel start codon GJB1 mutation (p.Met1Ile) has been found in a CMT1X patient presenting with recurrent episodes of transient encephalomyelitis without apparent signs of peripheral neuropathy. In order to clarify the functional consequences of this mutation, we examined the cellular expression of two different constructs cloned from genomic DNA including the mutated start codon. None of the cloned constructs resulted in detectable expression of Cx32 by immunocytochemistry or immunoblot, although mRNA was produced at normal levels. Furthermore, co-expression with the other major oligodendrocyte connexin, Cx47, had no negative effect on GJ formation by Cx47. Finally, lysosomal and proteasomal inhibition in cells expressing the start codon mutant constructs failed to recover any detection of Cx32 as a result of impaired protein degradation. Our results indicate that the Cx32 start codon mutation is equivalent to a complete loss of the protein with failure of translation, although transcription is not impaired. Thus, complete loss of Cx32 function is sufficient to produce CNS dysfunction with clinical manifestations.
