|
|
| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
|
MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 613 |
The
Use Of Herpes Simplex Virus-1 Vectors In Nociceptive Biology |
Rahul
Srinivasan |
chronic
pain, herpes simplex virus, vanilloid/capsaicin receptor (TRPV1), protein kinase C epsilon (PKCe)-mediated
receptor phosphorylation |
The pellet was then resuspended
in DMEM containing 10% heat inactivated horse serum and 5% fetal bovine serum (Sigma), and plated
on…35 mm Petri dishes with 10 mm glass bottom microwells (Mattek Corporation, Ashland, MA) for
imaging experiments.
|
inverted
fluorescence microscopy |
rat |
neuronal |
DRG |
| Abstract |
| |
The United States has 80 million employees with chronic
pain resulting in annual losses of 61.2 billion dollars due to pain-related productive time
lost. In addition, pain-related depression and inactivity reduce the quality of life. The development
of effective analgesics is therefore important from a public health perspective. In this dissertation,
the natural properties of herpes simplex virus (HSV-1) vectors are exploited to (i) develop
an HSV-1 vector-based selection system that can potentially identify natural or chemical inhibitors
of chronic pain and (ii) to test HSV-1 vector-expressed dominant negative PKCå (DNP) as
a strategy to treat chronic pain. The vanilloid/capsaicin receptor (TRPV1) is a pro-nociceptive
calcium ion channel that is upregulated in chronic pain. This occurs partly due to protein kinase
C epsilon (PKCå)?mediated receptor phosphorylation. An HSV-1 vector expressing TRPV1 (vTT)
was engineered and vTT-expressed TRPV1 functionality was confirmed. Treatment of vTT-infected
cells with capsaicin or resiniferatoxin caused concentration-dependent Ca+2 influx, leading
to cell-death and a dramatic reduction in infectious particle yield. TRPV1 antagonists, ruthenium
red and SB-366791 reversed agonist-induced cell-death and rescued vTT growth, providing a basis
for selection. Selection for antagonists was modeled using a mixed infection of vTT and vHG
(capsaicin resistant control vector) and virus passage in the presence capsaicin. These experiments
demonstrated that a single control vector particle was readily isolated from a population of
105 vTT particles. This approach can be used to identify antagonists from chemical or gene libraries
and offers advantages of (i) a platform assay applicable to other ion channels and (ii) adaptability
to high throughput formats. Dominant negative PKCå (DNP) was engineered into HSV-1 to
create the vector, vHDNP. Following functional confirmation of vHDNP in U2OS, Vero cells and
neurons, cobalt uptake showed a reduction of capsaicin sensitive vHDNP-transduced neurons. Electrophysiology
confirmed this and also demonstrated a knockdown of TRPV1-PKCå coupling in nociceptive
neurons. In-vivo studies of noxious heat-induced nocisponsive behavior in vHDNP-inoculated rats
showed a subtle inhibition of withdrawal responses when compared with controls. In conclusion,
HSV-1 expressed dominant negative PKCå is a viable strategy to specifically inhibit TRPV1
function in order to treat chronic pain. |
|
|
|
|
