inducible-and-reversible-dominant-negative-dn-protein-inhibition

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

Creighton University School of Dentistry

Fasudil (HA-1077), a specific Rho kinase II (ROCKII) inhibitor, is in clinical trials for recovery from spinal cord injury (SCI). The primary role of Fasudil is in axonal regeneration, as it inhibits ROCKII, the key signaling molecule involved in collapse of axon growth cone. Astrogliosis, due to the activation of astrocytes is an indicator of CNS injury. In early stages of injury, GFAP expression increases, helping to restore the integrity of the CNS. An increase in GFAP expression is also a marker of astrogliosis. Thus, reducing GFAP and hence astrogliosis at later stages of SCI is important for neuroregeneration and functional recovery. CoCl2 was used to induce hypoxic injury in astrocytic cell lines A172 (24h) and in spinal cord dorsal column white matter (8h). Several different techniques were used to study the changes in GFAP expression such as real-time PCR, western blotting and immunofluorescence staining with confocal microscopy. Hypoxia increased the expression of GFAP in A172 cells and in the spinal cord dorsal column after CoCl2 (100μM) treatment for 24h and 8h, respectively. We observed 11 folds increase in protein expression in A172 cells (24h) and 4.5 folds in spinal cord dorsal column (8h). The RNA expression was increased 3 folds in A172 cells after 24h of treatment and 4 folds in spinal cord dorsal column after 8h of treatment with 100μM CoCl2. Treatment with fasudil (20μM) significantly reduces the expression of GFAP in A172 cells and in spinal cord dorsal column. Fasudil also decreased activation of NF-κB in A172 cells after hypoxic injury. In the present study, we observed that fasudil reduces the expression of GFAP (consequently, astrogliosis) after hypoxic injury to A172 cells and spinal cord dorsal column. Our studies demonstrate that fasudil also plays a role in GFAP expression by reducing NF-κB activation at the injury site which could further help in axonal regeneration. 

Fasudil reduces GFAP expression after hypoxic injury.

Retinoblastoma 1 (Rb1) is an essential gene regulating cellular proliferation, differentiation, and homeostasis. To exert these functions, Rb1 is recruited and physically interacts with a growing variety of signaling pathways. While Rb1 does not appear to be ubiquitously expressed, its expression has been confirmed in a variety of hematopoietic and neuronal-derived cells, including the inner ear hair cells (HCs). Studies in transgenic mice demonstrate that complete germline or conditional Rb1 deletion leads to abnormal cell proliferation, followed by massive apoptosis; making it difficult to fully address Rb1's biochemical activities. To overcome these limitations, we developed a tetracycline-inducible TetO-CB-myc6-Rb1 (CBRb) mouse model to achieve transient and inducible dominant-negative (DN) inhibition of the endogenous RB1 protein. Our strategy involved fusing the Rb1 gene to the lysosomal protease pre-procathepsin B (CB), thus allowing for further routing of the DN-CBRb fusion protein and its interacting complexes for proteolytic degradation. Moreover, reversibility of the system is achieved upon suppression of doxycycline (Dox) administration. Preliminary characterization of DN-CBRb mice bred to a ubiquitous rtTA mouse line demonstrated a significant inhibition of the endogenous RB1 protein in the inner ear and in a number of other organs where RB1 is expressed. Examination of the postnatal (P) DN-CBRb mice inner ear at P10 and P28 showed the presence of supernumerary inner HCs (IHCs) in the lower turns of the cochleae, which corresponds to the described expression domain of the endogenous Rb1 gene. Selective and reversible suppression of gene expression is both an experimental tool for defining function and a potential means to medical therapy. Given the limitations associated with Rb1-null mice lethality, this model provides a valuable resource for understanding RB1 activity, relative contribution to HC regeneration and its potential therapeutic application. 

Shikha Tarang

Department of Oral Biology

Creighton University School of Dentistry

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Shikha Tarang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Oral Biology

Creighton University School of Dentistry

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Department of Oral Biology