Cell specific single viral vector CRISPR/Cas9 editing and genetically encoded tool delivery in the central and peripheral nervous systems.

CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function, and can be combined with genetically-encoded tools such as fluorescent proteins, channelrhodopsins, DREADDs, and various biosensors to more deeply probe the function of these genes in different cell types. However, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 from a genomic locus affords space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus.We validated this strategy with three common tools in neuroscience: ChRonos, a channelrhodopsin, for studying synaptic transmission using optogenetics; GCaMP8f for recording Ca2+ transients using photometry, and mCherry for tracing axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens, glutamatergic neurons projecting from the ventral pallidum to the lateral habenula, dopaminergic neurons in the ventral tegmental area, and proprioceptive neurons in the periphery. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.Significance Statement Our CRISPR/Cas9 approach is the first to use a single vector to both knock-down genes of interest and express tools to monitor, map, and manipulate neurons. We demonstrate its utility in the central nervous system and describe the first systemic CRISPR/Cas9 gene editing with co-expressed reporters in the peripheral nervous system. Our approach fills a significant gap in the neuronal gene editing toolkit, allowing high-throughput study of genes of unknown function in the nervous system, and has broad utility for loss-of-function studies in other biological fields. This tool has great translational potential: it can be used to screen risk factor genes identified through genome-wide association studies, or knock-down native gene expression and reintroduce mutant variants identified in clinical settings.

Cell specific single viral vector CRISPR/Cas9 editing and genetically encoded tool delivery in the central and peripheral nervous systems.

Gene manipulation strategies using germline knockout, conditional knockout, and more recently CRISPR/Cas9 are crucial tools for advancing our understanding of the nervous system. However, traditional gene knockout approaches can be costly and time consuming, may lack cell-type specificity, and can induce germline recombination. Viral gene editing presents and an exciting alternative to more rapidly study genes of unknown function; however, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we have developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 in specific cell types in transgenic mouse lines affords more space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three commonly used tools in neuroscience: ChRonos, a channelrhodopsin, for manipulating synaptic transmission using optogenetics; GCaMP8f for recording Ca2+ transients using fiber photometry, and mCherry for anatomical tracing of axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens (NAc), glutamatergic neurons projecting from the ventral pallidum (VP) to the lateral habenula (LHb), dopaminergic neurons in the ventral tegmental area (VTA), and parvalbumin (PV)-positive proprioceptive neurons in the periphery. This flexible approach should be useful to identify novel genes that affect synaptic transmission, circuit activity, or morphology with a single viral injection.

Development of GLUT4-selective antagonists for multiple myeloma therapy.

Cancer cells consume more glucose to fuel metabolic programs fundamental to sustaining their survival, growth and proliferation. Among the fourteen SLC2A family members, GLUTs 1 and 4 are high-affinity glucose transporters. GLUT4 (SLC2A4) is highly expressed in muscle and adipose tissue. Basally retained within the cell, GLUT4 traffics to the plasma membrane (PM) in response to insulin and exercise-stimulation. The plasma cell malignancy multiple myeloma (MM) exhibits increased constitutive expression of GLUT4 on the PM, co-opting use of GLUT4 for survival and proliferation. GLUT4 inhibition by knockdown or treatment with the FDA-approved HIV protease inhibitor ritonavir leads to cytostatic and/or cytotoxic and chemosensitizing effects in tumor cells both in vitro and in vivo. We recently reported our generation of GLUT4 homology models and virtual high-throughput screening (vHTS) to identify multiple series of novel GLUT4 antagonists. In this report, we describe our initial hit-to-lead optimization to synthesize new analogs with improved potency and selectivity for GLUT4, and the biological characterization of these compounds in a variety of assays. We show that our lead compound (compound 20) decreases glucose uptake and cell proliferation as well as inhibits the expression of pro-survival MCL-1 in MM similar to the effect observed via knockdown of GLUT4 expression. Compound 20 is also effective at chemosensitizing multiple myeloma cell lines and patient samples to venetoclax, dexamethasone and melphalan. In sum, we report development of selective GLUT4 inhibitors lacking inhibitory activity against GLUT1 and GLUT8. We show that selective pharmacological inhibition of GLUT4 is feasible and this may represent a novel strategy for the treatment and chemosensitization of multiple myeloma to standard therapeutics.

Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis.

Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5'-monophosphate-activated protein kinase)-dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.

The role of tumor necrosis factor alpha in regulating the expression of Tamm-Horsfall Protein (uromodulin) in thick ascending limbs during kidney injury.

BACKGROUND: Tamm-Horsfall Protein (THP) is a glycoprotein expressed exclusively by cells of the thick ascending loop (TAL) of Henle. THP has a protective role in acute kidney injury (AKI), and its expression is downregulated in the early stages of injury. Tumor necrosis factor alpha (TNFα) is a cytokine endogenously expressed by the TAL and is also induced by AKI. Therefore, we hypothesized that TNFα is a key regulator of THP expression. METHODS: We used a mouse model of AKI (ischemia-reperfusion injury, IRI) and a cell culture system of a TAL cell line (MKTAL). RESULTS: We show that TNFα is upregulated by TAL cells early after AKI in vivo. The expression of THP and its transcription factor Hepatocyte nuclear factor 1ß (HNF1ß) were concomitantly decreased at the peak of injury. Furthermore, recombinant TNFα inhibits significantly, and in a dose-dependent manner, the expression of THP, but not HNF1ß in MKTAL cells. Interestingly, neither TNFα neutralization nor genetic deletion of TNFα increased THP or HNF levels after injury in vivo. CONCLUSION: Our data suggest that TNFα can inhibit the expression of THP in TAL cells via an HNF1ß-independent mechanism, but the downregulation of THP expression in the early AKI does not depend on TNFα. We propose that TNFα regulates THP expression in a homeostatic setting, but the impact of TNFα on THP during kidney injury is superseded by other factors that could inhibit HNF1ß-mediated expression of THP.

Role of cyclooxygenase-2 in cytokine-induced beta-cell dysfunction and damage by isolated rat and human islets.

Type I diabetes mellitus is an autoimmune disease characterized by the selective destruction of the insulin-secreting beta-cell found in pancreatic islets of Langerhans. Cytokines such as interleukin-1 (IL-1), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mediate beta-cell dysfunction and islet degeneration, in part, through the induction of the inducible isoform of nitric-oxide synthase and the production of nitric oxide by beta-cells. Cytokines also stimulate the expression of the inducible isoform of cyclooxygenase, COX-2, and the production of prostaglandin E(2) (PGE(2)) by rat and human islets; however, the role of increased COX-2 expression and PGE(2) production in mediating cytokine-induced inhibition of islet metabolic function and viability has been incompletely characterized. In this study, we have shown that treatment of rat islets with IL-1beta or human islets with a cytokine mixture containing IL-1beta + IFN-gamma +/- TNF-alpha stimulates COX-2 expression and PGE(2) formation in a time-dependent manner. Co-incubation of rat and human islets with selective COX-2 inhibitors SC-58236 and Celecoxib, respectively, attenuated cytokine-induced PGE(2) formation. However, these inhibitors failed to prevent cytokine-mediated inhibition of insulin secretion or islet degeneration. These findings indicate that selective inhibition of COX-2 activity does not protect rat and human islets from cytokine-induced beta-cell dysfunction and islet degeneration and, furthermore, that islet production of PGE(2) does not mediate these inhibitory and destructive effects.

Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells.

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.