Probes for INS

Purpose : Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. In this study, we characterize the expression pattern of chd7 in the developing zebrafish retina and begin to study its function using two chd7 mutant lines. Methods : All animal procedures were performed in accordance with IACUC and guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wild-type and transgenic zebrafish embryos or larvae were collected at 24, 48, 72 hours post fertilization (hpf) and 4, 5 days post fertilization (dpf). Retinas were sectioned, followed by immunohistochemistry with a Chd7 antibody or RNAscope with a chd7 mRNA probe, and imaged with fluorescent and confocal microscopy. A CRISPR chd7 mutant line was obtained and genotyped by Sanger sequencing to identify a 2 base pair deletion. A second mutant line generated by ENU mutagenesis was outcrossed four times, and genotyped by Sanger sequencing to identify a single point mutation. Results : Chd7 was expressed throughout the developing zebrafish retina at 24 and 48 hpf, when retinal progenitors are actively proliferating and early cells of the retina are beginning to differentiate. At 72 hpf, when most retinal cell types have terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Strong expression of Chd7 was also observed in the photoreceptor cells of the outer nuclear layer (ONL). By 4 and 5 dpf, when the zebrafish larvae display active swimming and visual behaviors, Chd7 expression remained strong in the ONL, where it co-localized with markers of cone and rod photoreceptors. Incrosses of chd7 heterozygous adults yielded homozygous mutant progeny suggesting that the mutations are not early embryonic lethal in zebrafish. Conclusions : Our results demonstrate that Chd7 is expressed throughout development of the zebrafish retina and remains expressed in some newly differentiated retinal cell types, including the photoreceptors. Further work is ongoing to investigate this specific patterning of Chd7 and chd7 mutant fish will be used to understand the function that this factor plays in retinal development.

Purpose : FOXC1 is a transcription factor involved in heart, craniofacial and ocular development in vertebrates. Mutations in FOXC1, along with mutations in PITX2, cause Axenfeld-Rieger syndrome (ARS) and explain approximately half of the ARS cases. However, there is still a significant number of patients with an unknown genetic cause. Expression and activity of transcription factors involved in development are finely controlled by their regulatory elements that are often evolutionarily conserved. It has been shown that mutations in those regulatory elements could be also pathogenic. The goal of this project is to discover regulatory elements for FOXC1 using zebrafish as a vertebrate model and relate this information to human eye development and disease. Methods : BLAST alignments involving regions surrounding human FOXC1 and two orthologous zebrafish genes foxc1a and foxc1b were carried out to identify conserved sequences. CRISPR-Cas9 was used to generate zebrafish lines carrying deletions for the identified conserved elements. The obtained lines were characterized by gross morphology examination, OCT and histology analysis. Expression of foxc1a and foxc1b genes and encoded proteins was assessed by RT-qPCR, in situ hybridization with RNAscope probes and immunohistochemistry. Results : We identified 3 elements downstream of human FOXC1 and 1 element upstream of FOXC1 that were conserved in zebrafish foxc1a or in both foxc1a and foxc1b. The deletion of a 152kb intergenic region comprising all 3 downstream elements (ΔCED1-3) resulted in a downregulation of both the foxc1a transcript and protein in developing zebrafish embryos. Homozygous ΔCED1-3 larvae die at 1 month post fertilization and present with enlarged anterior chambers of the eye and a significant pericardial and other organs’ edema. Deletion of an 82.7Kb region containing only 2 out of the 3 downstream conserved elements (ΔCED2-3) produced a similar but milder phenotype. Deletion of elements upstream either foxc1a (ΔCEU1a) and foxc1b (ΔCEU1b) did not produce a visible phenotype. Conclusions : The identified downstream conserved elements are essential for normal foxc1a expression and their deletion results in a phenotype consistent with foxc1/FOXC1 deficiency in zebrafish and humans. Further studies of these regions in human patients is likely to explain additional ARS cases.

Background: The Psychiatric Ratings using Intermediate Stratified Markers (PRISM) project focuses on understanding the biological background behind social deficits, specifically social withdrawal irrespective of diagnosis. Reduced connectional integrity in fiber tracts such as Forceps minor has been indicated in low social individuals as a part of the PRISM 1 project. These fiber tracts are also involved in the Default Mode Network (DMN) and the Social network and they share a common region, the Orbitofrontal Cortex (OFC).This study aims to back-translate the clinical data to preclinical studies and associate social dysfunction in rodents with DMN and particularly OFC. Parvalbumin interneurons are targeted based on their fundamental role in maintaining Excitatory Inhibitory (E/I) balance in brain circuits. Numerous studies indicate behavioral impairment in rodents by increasing excitability of PV+ interneurons. Methods: As an initial step, we characterized the population of projection neurons within OFCs by combining Cholera Toxin subunit B (CTB) as a retrograde tracer and In situ hybridization (ISH) technique (RNAscope). We identified the expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) by using Slc17a7 and Slc32a1 probes. CTB was injected unilaterally in the left OFC (AP=2.68, ML=-0.8, DV=2.2). after 10 days mice were perfused and RNAscope assay was performed using RNAscope™ Multiplex Fluorescent kit (ACDBio™).For inducing hypoactivation of OFC, we introduced an excitatory DREADD (designer receptors exclusively activated by designer drugs) to PV+ interneurons by using a PV-Cre mouse line. Mice were injected either AAV-hSyn-DIO-hM3D(Gq)-mCherry virus (n=12) or AAV-hSyn-DIO-mCherry (n=12) as control virus. As a novel behavioral tool, Radiofrequency identification (RFID)-assisted SocialScan combined with video tracking has been used, which provides a long-term observation of social behaviors. Monitoring the behavior in groups of four was performed for 7 days in total. After two pre-application days, Clozapine-N-oxide (CNO) was injected three times on consecutive days intraperitoneally (5mg/kg) as an activator of hM3D. application days were followed by two post-application days. Mice were perfused and RNAscope was performed to visualize c-fos mRNA expression as neuronal activity marker, and PV expression to validate our virus and mouse line efficacy. Results: ISH results indicated VGLUT1 has the highest expression within projection neurons (81%). 6% are VGAT+ and only 3% are both VGLUT1/VGAT positive neurons. Despite demonstrating the GABAergic projection neurons as a minority, their crucial role as local interneurons to moderate the excitatory neurons is indisputable.In in vivo study, CNO administration induced social dysregulation in DREAAD mice, demonstrated by a reduction in different social parameters (approach, fight, etc.) in terms of duration. During post-application days, DREAAD mice showed significantly higher social interaction in all definedparameters (Social Approach: p=0.0009, unpaired T-test) and locomotion as a non-social parameter (p= 0.0207).Results from ISH support our hypothesis that DREADD activation of PV+ interneurons is followed by high expression of neuronal activity markers in these targeted interneurons. Conclusion: This study indicates that manipulation of PV+ interneurons using artificially engineered activating protein receptors, generates in effect activation of these interneurons, and this manipulation particularly in OFC could cause social dysfunction in mice.

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.

Sex differences occur in the structure and function of the rat cerebral cortex and hippocampus, which can change from the juvenile period through old age. Although the evidence is incomplete, it appears that in at least some portions of the cortex these differences develop due to the rise of ovarian hormones at puberty and are potentially not dependent on the perinatal rise in testosterone, which is essential for sexual differentiation of the hypothalamus and sexual behavior. During aging of female rats, the presence of continued ovarian hormone secretion after cessation of the estrous cycle also influences sex differences in neuroanatomical structure and cognitive behavior, resulting in nullification or reversal of sex differences seen in younger adults. Sex differences can be altered by experience in a stimulating environment during the juvenile/adolescent period, and sex differences in performance even can be affected by the parameters of a task. Thus, broad generalizations about differences such as “spatial ability” are to be avoided. It is clear that to understand how the brain produces behavior, sex and hormones have to be taken into account.

Early life stress (ELS) is a well-characterized risk factor for mood and anxiety disorders. GABAergic microcircuits in the amygdala are critically implicated in anxiety; however, whether their function is altered after ELS is not known. Here we identify a novel mechanism by which kainate receptors (KARs) modulate feedforward inhibition in the lateral amygdala (LA) and show that this mechanism is downregulated after ELS induced by maternal separation (MS). Specifically, we show that in control rats but not after MS, endogenous activity of GluK1 subunit containing KARs disinhibit LA principal neurons during activation of cortical afferents. GluK1 antagonism attenuated excitability of parvalbumin (PV)-expressing interneurons, resulting in loss of PV-dependent inhibitory control and an increase in firing of somatostatin-expressing interneurons. Inactivation of Grik1 expression locally in the adult amygdala reduced ongoing GABAergic transmission and was sufficient to produce a mild anxiety-like behavioral phenotype. Interestingly, MS and GluK1-dependent phenotypes showed similar gender specificity, being detectable in male but not female rodents. Our data identify a novel KAR-dependent mechanism for cell-type and projection-specific functional modulation of the LA GABAergic microcircuit and suggest that the loss of GluK1 KAR function contributes to anxiogenesis after ELS.

Pulpitis refers to inflammation of the inner pulp by invading microbes, and tissue repair occurs due to odontogenic differentiation of human dental pulp cells (hDPCs) with multidifferentiation potential. Long noncoding RNAs (lncRNAs) can modulate numerous pathological and biological processes; however, the role of lncRNAs in the inflammation and regeneration of the dentin-pulp complex in pulpitis is unclear. Here, we performed high-throughput sequencing to identify differentially expressed lncRNAs between human normal and inflamed pulp and concluded that lncMEG3 (lncRNA maternally expressed gene 3, MEG3) was significantly upregulated in both inflamed pulp and LPS-treated hDPCs. MEG3 expression in the pulp tissue was detected using the RNAscope™ technique. RNA pulldown assays identified the MEG3-interacting proteins and the potential mechanisms. With MEG3 knockdown, we investigated the role of MEG3 in the secretion of inflammatory cytokines in LPS-treated hDPCs and odontogenic differentiation of hDPCs. MEG3 downregulation inhibited the secretion of TNF-α, IL-1β and IL-6 in LPS-treated hDPCs, and the p38/MAPK signaling pathway may be related to this effect. MEG3 knockdown promoted odontogenic differentiation of hDPCs by regulating the Wnt/β-catenin signaling pathway. Our study suggested that MEG3 has a negative effect on inflammation and regeneration of the dentin-pulp complex in pulpitis.

Adult hippocampal neurogenesis is implicated in antidepressant action in rodents and primates, yet relationships to human major depression (MDD) and antidepressant effects are unclear. In postmortem human hippocampus, we found doublecortin (DCX) protein and mRNA+ cells co-expressing neuronal but not astroglial or microglial markers. We defined neuroblasts as DCX+ cells located in the subgranular zone that co-expressed neuron-specific beta-tubulin (TUJ1) or lacked co-expression with neuronal nuclear marker (NeuN). Untreated MDD, regardless of clinical state, had fewer DCX-positive cells and neuroblasts in the rostral dentate gyrus compared with non-psychiatric controls. High-functioning, but not low-functioning, antidepressant-treated MDD, exhibited more DCX/TUJ1+ neuroblasts than untreated MDD. Groups did not differ in number of immature neurons, defined as DCX/NeuN+ cells in the inner granule cell layer. Deficient neuroblasts may be linked to hippocampal-dependent cognitive deficits in MDD. Similar neuroblast number between controls and higher-functioning antidepressant treated subjects warrants evaluation of neuroblasts as a treatment target.

The maturation and function of osteoblasts (OBs) rely heavily on the reversible phosphorylation of signaling proteins. To date, most of the work in OBs has focused on phosphorylation by tyrosyl kinases, but little has been revealed about dephosphorylation by protein tyrosine phosphatases (PTPases). SHP2 (encoded by PTPN11) is a ubiquitously expressed PTPase. PTPN11 mutations are associated with both bone and cartilage manifestations in patients with Noonan syndrome (NS) and metachondromatosis (MC), although the underlying mechanisms remain elusive. Here, we report that SHP2 deletion in bone gamma-carboxyglutamate protein-expressing (Bglap+) bone cells leads to massive osteopenia in both trabecular and cortical bones due to the failure of bone cell maturation and enhanced osteoclast activity, and its deletion in Bglap+ chondrocytes results in the onset of enchondroma and osteochondroma in aged mice with increased tubular bone length. Mechanistically, SHP2 was found to be required for osteoblastic differentiation by promoting RUNX2/OSTERIX signaling and for the suppression of osteoclastogenesis by inhibiting STAT3-mediated RANKL production by osteoblasts and osteocytes. These findings are likely to explain the compromised skeletal system in NS and MC patients and to inform the development of novel therapeutics to combat skeletal disorders.

Parkinson's disease is a progressive neurodegenerative disorder characterised by the accumulation of misfolded ?-synuclein in selected brain regions, including the substantia nigra pars compacta (SNpc), where marked loss of dopaminergic neurons is also observed. Yet, the relationship between misfolded ?-synuclein and neurotoxicity currently remains unclear. As the principal route for degradation of misfolded proteins in mammalian cells, the ubiquitin-proteasome system (UPS) is critical for maintenance of cellular proteostasis. Misfolded ?-synuclein impairs UPS function and contributes to neuronal death in vitro. Here, we examine its effects in vivo using adeno-associated viruses to co-express A53T ?-synuclein and the ubiquitinated reporter protein UbG76V-GFP in rat SNpc. We found that ?-synuclein over-expression leads to early-onset catalytic impairment of the 26S proteasome with associated UPS dysfunction, preceding the onset of behavioural deficits and dopaminergic neurodegeneration. UPS failure in dopaminergic neurons was also associated with selective accumulation of ?-synuclein phosphorylated at the serine 129 residue, which has previously been linked to increased neurotoxicity. Our study highlights a role for ?-synuclein in disturbing proteostasis which may contribute to neurodegeneration in vivo