Author: Triboulet, Robinson ; Brendel, Christian ; Guda, Swaroopa ; Bentler, Martin ; Du, Peng ; Xu, Haiming ; Williams, David A ; Gregory, Richard I ; Park, Hee Ho

RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNA^miR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNA^miR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNA^miR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNA^miR-containing vectors for RNAi-based therapy.

01 Jul 2015·Journal of NeurochemistryQ2 · MEDICINE

p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing

Q2 · MEDICINE

Article

Author: Posada‐Duque, Rafael Andres ; Piedrahita, Diego ; González‐Billault, Christian ; López‐Tobón, Alejandro ; Cardona‐Gomez, Gloria Patricia

AbstractCDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate‐mediated excitotoxicity using primary hippocampal neurons transduced with adeno‐associated virus 2.5 viral vector eGFP‐tagged scrambled or CDK5 shRNA‐miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down‐stream Rho GTPase activity. Furthermore, p35 over‐expression and constitutively active Rac1 mimicked CDK5 silencing‐induced neuroprotection. In addition, 3xTg‐Alzheimer's disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA‐miR, and spatial learning and memory were performed 3 weeks post‐injection using ‘Morris’ water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi.

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CDK5 plays an important role in neurotransmission in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. Our findings suggest that p35/Rac1 signaling is critical in the CDK5 shRNAmiR‐induced neuroprotection against glutamate neurotoxicity and is also correlated with the recovery of cognitive function in 3xTg‐AD mice. CDK5 shRNAmiR blocks calpain activation, and the cleavage of p35 to p25 produced by glutamate, which generates neuroprotection in a p35 up‐regulation dependent mode and its down‐stream control of Rho GTPases, such as Rac1 and RhoA.

01 Dec 2012·BMC ImmunologyQ4 · MEDICINE

Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes

Q4 · MEDICINE

ArticleOA

Author: Dustin N D Lippert ; John A Wilkins

BACKGROUNDLymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration.RESULTSMicroscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1.CONCLUSIONSThe present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.

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Anemia, Sickle Cell	Phase 1	-	The Children's Hospital Corp.	-
Anemia, Sickle Cell	Phase 1	-	bluebird bio, Inc.	-

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