Probes for INS

Purpose : Mechanisms of axonal insult within the ONH in glaucoma are not fully understood. This study aimed to delineate ONH molecular alterations in chronic stages of glaucoma, in an inherited feline model with ONH structure comparable to humans. Methods : ONH tissues from 10 LTBP2mut/mut cats with glaucoma and 5 wt control cats (age 1-3 yrs) were used to generate cDNA libraries for RNAseq. Weekly intraocular pressure (IOP) data and optic nerve axon counts were available for all subjects. Differentially expressed genes (DEGs) were identified using DESeq2 (false discovery rate < 0.05), and g:Profiler was used for functional enrichment analysis. DEGs in chronic glaucoma were compared to DEGs in an RNA-seq dataset generated by our lab from ONH tissues of LTBP2mut/mut cats prior to axon degeneration. Transcriptomic findings were validated by RNAscope in situ hybridization (ISH) and by immunolabeling (IF) of archived ONH tissue sections. For confirmatory studies, data were compared between groups by two-tailed unpaired t-test or ANOVA (p < 0.05 considered significant). Results : Mean and cumulative IOP over 10mths prior to tissue collection were consistently higher in LTBP2mut/mut than in wt cats. Stratifying subjects by optic nerve damage based on histological evaluation (mild [MLD], moderate [MOD] and severe [SEV] damage), 77, 882 and 1878 DEGs were identified, respectively, in glaucoma relative to age-matched controls. Functional analysis of DEGs in chronic, established glaucoma (MOD and SEV groups) identified upregulated DEGs ascribed to cell adhesion, immune/inflammatory responses, and MAPK cascade, and downregulated DEGs associated with metabolism, fatty acid synthesis, actin cytoskeleton and myelination. Comparing these DEGs in established chronic glaucoma to those in pre-degenerative, early-stage disease, 111 DEGs were shared between stages, including significant upregulation of HP and TNC. ISH confirmed expression of HP and TNC in the ONH, but with sub-regional differences in expression. TNC was highly expressed in the prelaminar - laminar regions and HP in the laminar and retrolaminar regions. ISH and IF identified astrocytes as the predominant ONH cell-type expressing these gene products. Conclusions : Early and chronic stages of glaucoma share a reactive astrocyte molecular signature. Gene expression changes are more complex and enhanced in chronic glaucoma.

Dengue (DENV) and West Nile (WNV) viruses are arthropod-transmitted flaviviruses that respectively cause systemic vascular leakage and encephalitis syndromes in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity of the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. As exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcome. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here we assessed West Nile virus (WNV) NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in circulation facilitate WNV dissemination to the brain and disease outcome. Our findings help understand the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Background About 40 disease genes have been described to date for isolated congenital anomalies of the kidneys and urinary tract (CAKUT), the most common cause of childhood chronic kidney disease. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in such biologic processes as cell migration and focal adhesion, acts downstream of integrin linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva, leading to CAKUT in mice with this variant. Methods To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, as well as the effects of Arhgef6 deficiency in mouse and frog models. Results We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6-but not proband-derived mutant ARHGEF6- increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVAdependent cell spreading. ARHGEF6 mutant proteins showed loss of interaction with PARVA. Three-dimensional MDCK cell cultures expressing ARHGEF6 mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. Conclusions Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvinRAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

Background Opioid discontinuation generates a withdrawal syndrome marked by increased negative affect. Increased symptoms of anxiety and dysphoria during opioid discontinuation are a significant barrier to achieving long-term abstinence in opioid-dependent individuals. While adaptations in the nucleus accumbens are implicated in the opioid abstinence syndrome, the precise neural mechanisms are poorly understood. Additionally, our current knowledge is limited to changes following natural and semi-synthetic opioids, despite recent increases in synthetic opioid use and overdose. Methods We used a combination of cell subtype specific viral-labeling and electrophysiology in male and female mice to investigate structural and functional plasticity in nucleus accumbens medium spiny neuron (MSNs) subtypes after fentanyl abstinence. We characterized molecular adaptations after fentanyl abstinence with subtype specific RNAseq and Weighted Gene Co-expression Network Analysis. We used viral-mediated gene transfer to manipulate the molecular signature of fentanyl abstinence in D1-MSNs. Results Here we show fentanyl abstinence increases anxiety-like behavior, decreases social interaction, and engenders MSN subtype-specific plasticity in both sexes. D1, but not D2-MSNs exhibit dendritic atrophy and an increase in excitatory drive. We identified a cluster of co-expressed dendritic morphology genes downregulated selectively in D1-MSNs that are transcriptionally co-regulated by E2F1. E2f1 expression in D1-MSNs protects against loss of dendritic complexity, altered physiology, and negative affect-like behaviors caused by fentanyl abstinence. Conclusion Our findings indicate fentanyl abstinence causes unique structural, functional, and molecular changes in nucleus accumbens D1-MSNs that can be targeted to alleviate negative affective symptoms during abstinence.

Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 mM NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE (p < 0.01), and raised VLDL contents in the supernatants (p < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE (p < 0.01), and increased VLDL contents in the supernatants (p < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with schizophrenia risk. Integration of RNA-sequencing data from postmortem human brains with these risk SNPs identified transcripts associated with increased schizophrenia susceptibility, including a class of exon 9-spliced isoforms of Sorting nexin-19 (SNX19d9) and an isoform of Arsenic methyltransferase (AS3MT) splicing out exons 2 and 3 (AS3MTd2d3). However, the biological function of these transcript variants is unclear. Defining the cell types where these risk transcripts are dominantly expressed is an important step to understand function, in prioritizing specific cell types and/or neural pathways in subsequent studies. To identify the cell type-specific localization of SNX19 and AS3MT in the human dorsolateral prefrontal cortex (DLPFC), we used single-molecule in situ hybridization techniques combined with automated quantification and machine learning approaches to analyze 10 postmortem brains of neurotypical individuals. These analyses revealed that both pan-SNX19 and pan-AS3MT were more highly expressed in neurons than non-neurons in layers II/III and VI of DLPFC. Furthermore, pan-SNX19 was preferentially expressed in glutamatergic neurons, while pan-AS3MT was preferentially expressed in GABAergic neurons. Finally, we utilized duplex BaseScope technology, to delineate the localization of SNX19d9 and AS3MTd2d3 splice variants, revealing consistent trends in spatial gene expression among pan-transcripts and schizophrenia risk-related transcript variants. These findings demonstrate that schizophrenia risk transcripts have distinct localization patterns in the healthy human brains, and suggest that SNX19 transcripts might disrupt the normal function of glutamatergic neurons, while AS3MT may lead to disturbances in the GABAergic system in the pathophysiology of schizophrenia.

Central oxytocin receptor (OTR) expression is extremely sensitive to circulating steroid hormones and OTRs influence many of the neurobehavioural adaptations associated with female reproduction (e.g., postpartum caregiving, aggression, cognition, affective responses). Changes in central OTR expression across female reproduction have often been studied, but almost all of such research has focused on the forebrain, ignoring hormone-sensitive midbrain sites such as the serotonergic dorsal raphe (DR) that are also critical for postpartum behaviours. To investigate the effects of female reproductive state on OTRs in the DR, we first used autoradiography to examine OTR binding across four female reproductive states in laboratory rats: dioestrous virgin, pregnancy day 10, the day of parturition and postpartum day 7. OTR binding in the rostral DR (but not other DR subregions) was approximately 250% higher in parturient rats compared to dioestrous virgins and dropped back down to virgin levels by postpartum day 7. Given the chemical heterogeneity of the DR, we then examined OTR expression in the three most abundant neuronal phenotypes of the DR (i.e., serotonin, GABA and dopamine) in dioestrous virgins and recently parturient females. Using dual-label immunohistochemistry and in situ hybridisation, we found that twice as many dopaminergic cells in the parturient rostral DR contained OTR immunoreactivity compared to that found in virgins. On the other hand, mothers had fewer rostral DR GABAergic cells expressing OTRs than did virgins. OTR expression in serotonin cells did not differ between the two groups. Overall, these results suggest that the rostral subregion of the midbrain DR is uniquely sensitive to oxytocin around the time of parturition, with subpopulations of cells that become more sensitive (i.e., dopamine), less sensitive (i.e., GABA) and show no change (i.e., serotonin) to this neuropeptide. This dynamic OTR signalling in the female DR may help drive the numerous behavioural changes across female reproduction that are necessary for successful motherhood.

Hitherto, the recognition of the microbiota role in tumorigenesis and clinical studies mostly focused on the intestinal flora. In contrast to the gut microbiome, microorganisms resident in tumor tissue are in close contact with cancer cells and therefore have the potential to have similar or even different functional patterns to the gut flora. Some investigations have shown intratumoral bacteria, which might come from commensal microbiota in mucosal areas including the gastrointestinal tract and oral cavity, or from nearby normal tissues. The existence, origin, and interactions of intratumoral bacteria with the tumor microenvironment all contribute to intratumoral microorganism heterogeneity. Intratumoral bacteria have a significant role in tumor formation. They can contribute to cancer at the genetic level by secreting poisons that directly damage DNA and also intimately related to immune system response at the systemic level. Intratumoral bacteria have an impact on chemotherapy and immunotherapy in cancer. Importantly, various properties of bacteria such as targeting and ease of modification make them powerful candidates for precision therapy, and combining microbial therapies with other therapies is expected to improve the effectiveness of cancer treatment. In this review, we mainly described the heterogeneity and potential sources of intratumoral bacteria, overviewed the important mechanisms by which they were involved in tumor progression, and summarized their potential value in oncology therapy. At last, we highlight the problems of research in this field, and look forward to a new wave of studies using the various applications of intratumoral microorganisms in cancer therapy.

GPR37 is an orphan GPCR and expressed in different brain regions. However, its biological function in pain regulation remains poorly understood. Recently, we identified Neuroprotectin D1 (NPD1) as a novel ligand of GPR37. NPD1 is a specialized pro-resolving mediator (SPM) and bio-synthesized from fish oil DHA (docosahexaenoic acid) . Here we reported a protective role of GRP37/NPD1 signaling in traumatic brain injury (TBI)-induced neuropathic pain. Mild TBI was induced by closed-head impact and the neuropathic pain was assessed by periorbital and cutaneous mechanical allodynia. In contrast to DHA (300 μg) showing no effects, peri-surgical treatment of NPD1 via intravenous injection (300 ng) effectively prevented TBI-induced locomotor deficiency and mechanical hypersensitivity in mice. Intraperitoneal post-treatment of NPD1 also significantly reduced established neuropathic pain in TBI mice. We also found that NPD1 treatment could inhibit TBI-induced neuroinflammation, characterized by microglia and astrocyte activation in the cortex and hippocampus. Furthermore, demyelination occurred after TBI, which was prevented by peri-surgical treatment of NPD1. RNAscope in situ hybridization revealed high Gpr37 mRNA expression in oligodendrocytes of the motor cortex and hippocampus . TBI resulted in a significant decrease in GPR37 expression, which could be restored by NPD1 treatment. GPR37 is protective and mice lacking Gpr37 (Gpr37-/-) exhibited prolonged pain symptoms after TBI. Furthermore, the protective effect of NPD1 was abolished in Gpr37-/- mice. All these findings suggest that activation of NPD1/GPR37 signaling pathway is a promising therapeutic strategy for preventing and treating neuropathic pain and its comorbidities after TBI. Funding: DoD grant W81XWH2110885.

Adult hippocampal neurogenesis (AHN) is a well-studied phenomenon that involves the derivation of new neurons from neural progenitor cells in the dentate gyrus region of the hippocampus, an area responsible for cognitive functions such as learning and memory storage. Moreover, the hippocampus is known to be implicated in neurological conditions such as Alzheimer's disease. Although AHN has been extensively observed in animal models for twenty years, its existence and persistence in humans have been widely debated in academia, heavily based on post-mortem immunohistochemical markers. Using the search engines PubMed and Google Scholar for “Adult Human Neurogenesis,” 143 articles that were most relevant to the history of AHN discovery, detection in rodents, immunohistochemical studies on post-mortem human sections, and therapeutic development targeting AHN were reviewed. This review article highlights the current understanding of AHN in rodents and humans, its implications in neurodegenerative diseases and therapeutics, and the inconsistencies and methodological variabilities encountered in studying AHN in humans. Furthermore, the correlation between AHN and diseases such as mood disorders and Alzheimer's disease is still not well established, with conflicting findings reported. Standardization of transcriptomic methodologies and increased availability of post-mortem human brain samples are crucial in advancing AHN research. This review article attempts to discover the fascinating and controversial world of adult human neurogenesis and its potential implications in treating neurological disorders. Apart from the discussion on AHN existence, tackling devastating diseases with this supplemental knowledge can lead to therapeutic advancements which greatly rely on understanding not only the presence of AHN but the mechanisms mediating its availability.

SELENON-related congenital myopathy is characterized by proximal weakness starting in infancy, early respiratory insufficiency, and early development of severe scoliosis. While changes in the SELENON gene, which encodes the protein SelN, are known to cause this disease the mechanisms through which loss of SelN lead to myopathy are not well understood. Previous studies suggest that SelN may have multiple roles in muscle, including regulating development of Type II muscle fibers, modulating excitation-contraction coupling through interactions with RYR1 and other muscle calcium channels, and possibly supporting satellite cell activation and proliferation following muscle injury. One particular challenge to understanding the role of SelN in skeletal muscle has been the inability to directly visualize SelN expression within muscle fibers and supporting cells due to a lack of robust antibodies for immunohistochemistry. Studies of mRNA expression and Western blot analysis of protein expression suggest significant post-transcriptional regulation of protein expression with an overall pattern of high expression in developing muscle and other developing tissues and low-level ubitquitous expression in mature tissues but evaluation of SelN expression in more limited sub-populations of cells has not been possible. Experiments in mouse suggest that loss of SelN expression results in decreased satellite cell proliferation following muscle injury. Here, I use a newly developed zebrafish model with mNeonGreen-tagged SelN to directly visualize SelN expression in satellite cells following muscle injury and show that SelN expression increases in activated satellite cells following mechanical muscle injury. This provides support SelN playing a role in satellite cell activation and proliferation during muscle repair following injury.

Although best known for their involvement in modulating nociception, Neuropeptide FF (NPFF) group peptides have been suggested to fulfil a variety of biological functions such as feeding, anxiety behaviors and thermogenesis. However, evidence supporting these functions of NPFF is mostly pharmacological, leaving the physiological relevance unaddressed. Here we examined the physiological impact of lack of NPFF signalling in both genders using a Npff-/- mouse model. NPFF expression in the mouse is restricted to the spinal cord and brainstem while its cognate receptor NPFFR2 has wider distribution throughout the brain. Both male and female Npff-/- mice showed reduced repetitive behaviors evidenced in the marble burying test and self-grooming test. A decrease in anxiety-related behaviors in the Npff-/- mice was also observe in the open field test and to a lesser degree in an elevated plus maze test. Moreover, both male and female Npff-/- mice exhibited increased water intake resulting from increases in drinking size, rather than number of drinking events. During a fasting-refeeding challenge, Npff-/- mice of both genders displayed alterations in reparatory exchange ratio that reflect a greater fuel type flexibility. Npff-/- mice were otherwise wild-type-like regarding body weight, body composition, feeding behaviors, locomotion or energy expenditure. Together, these findings reveal the important physiological roles of NPFF signalling in the regulation of anxiety-related and repetitive behaviors, fluid homeostasis and oxidative fuel selection, highlighting the therapeutical potential of the NPFF system in a number of behavioral and metabolic disorders.