Eric C. Larsen

G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis

The activation of G protein–coupled receptors (GPCRs) can result in an inhibition of Ca2+-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca2+ influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca2+ entry. Here we characterize direct inhibitory actions of G protein βγ (Gβγ) on Ca2+-triggered vesicle exocytosis in permeable PC12 cells. Gβγ inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gβγ bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca2+-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gβγ interactions with the Ca2+-dependent fusion machinery.

11C-(R)-PK11195 PET Imaging of Microglial Activation and Response to Minocycline in Zymosan-Treated Rats

We sought to advance methodology for studying microglial activation and putative therapeutic downregulation in response to minocycline by means of noninvasive in vivo imaging. A reproducible focal white matter lesion was used to reliably compare treatment conditions. Methods: The corpus callosum of female Sprague Dawley rats was injected with zymosan to promote microglial activation as confirmed by hematoxylin and eosin staining, 3H-PK11195 autoradiography, and CD11b immunohistochemistry. A subset of subjects was treated systemically with minocycline to potentially alter microglial activation. Seven days after zymosan injection, subjects were imaged with PET using the radiotracer 11C-(R)-PK11195. In vivo binding was evaluated using the distribution volume ratio (DVR) with respect to a reference region. Results: At the lesion site, the observed 11C-(R)-PK11195 DVR for each treatment was as follows: mean saline DVR ± SD, 1.17 ± 0.05 (n = 5); zymosan-only DVR, 1.96 ± 0.33 (n = 10); and zymosan with minocycline DVR, 1.58 ± 0.12 (n = 9). Therefore, compared with controls, zymosan increased binding (P = 0.0001, 2-tailed t test) and minocycline treatment reduced zymosan-induced binding by 46% (P = 0.004, 2-tailed t test). Conclusion: Zymosan-induced microglial activation and its response to minocycline can be quantitatively imaged in the rat brain using 11C-(R)-PK11195 PET. The ability to detect a treatment effect in a focal white-matter lesion may be of use in studying therapies for multiple sclerosis (MS).

Migration and remyelination by oligodendrocyte progenitor cells transplanted adjacent to focal areas of spinal cord inflammation

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Exogenous cell replacement in MS lesions has been proposed as a means of achieving remyelination when endogenous remyelination has failed. However, the ability of exogenous cells to remyelinate axons in the presence of inflammation remains uncertain. We have explored the remyelinating capacity of an oligodendrocyte progenitor cell line CG‐4 transduced with the GFP gene and transplanted adjacent to a zymosan‐induced focal demyelination model in the rat spinal cord. The resulting zymosan‐induced lesions were characterized by persistent macrophage/microglia activation, focal demyelination, degeneration of axons, and reactive astrogliosis. GFP+ CG‐4 cells were found to migrate preferentially toward the inflammatory lesion and survive inside the lesion. A proportion of GFP+ CG‐4 cells differentiated into mature oligodendrocytes and remyelinated axons within the lesion. These findings suggest that grafted oligodendrocyte progenitors may migrate toward areas of inflammation in the adult rat spinal cord, where they can survive and differentiate into myelinating oligodendrocytes.

Characterization of a PLP-overexpressing transgenic rat, a model for the connatal form of Pelizaeus–Merzbacher disease

Pelizaeus-Merzbacher disease (PMD) most frequently results from duplication of the Plp1 gene with a correlation between disease severity and increasing copy number of the gene. Animal models of PMD, in particular those overexpressing the Plp1 gene, have been sought in attempts to provide systems in which potential therapies can be tested. Here we describe a rat model of the severe connatal form of PMD and provide a detailed characterization of its pathology and molecular biology, prior to testing therapeutic approaches. We determined the exact copy number of Plp1, and the resulting effects on RNA and protein expression. Distinct differences in myelin and disparate distributions of myelin protein markers in comparison to wild-type controls were observed. Altered expression of Plp1 also caused an increase in the apoptotic cell death of oligodendrocytes. These results provide the platform from which to test the effectiveness of in vivo therapies.

Generation of Cultured Oligodendrocyte Progenitor Cells from Rat Neonatal Brains

The oligodendrocyte progenitor cell (OPC) is one of the most studied progenitor cells of the body. It has been extensively researched in tissue culture and more recently in vivo using a wide range of markers that recognize transcription factors and cell surface markers and identify its earliest development from neural stem cells onward. Isolation of OPCs in large numbers and in purified preparations has been sought after as a source of cells for the repair of human myelin disorders. It has been proposed that such cells could be used as an exogenous source of cells for the treatment of lesions in multiple sclerosis and the less common genetic myelin disorders such as Pelizaeus-Merzbacher disease. Prior to clinical trials, such approaches can be tested in animal models. Here, we describe the isolation of rat OPCs in culture conditions that provide large numbers of purified populations of cells.

Transplantation of Stem Cells and Their Derivatives in the Treatment of Multiple Sclerosis

Multiple sclerosis (MS) is a debilitating disorder of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal degeneration. Chronic demyelination is believed to result in axon degeneration, leading to long-term disability in the majority of MS patients. However, there are currently no therapies available that will promote myelination. The therapeutic potential of exogenous stem cells in the treatment of a MS is the subject of intensive investigation. The pluripotency and self-renewal properties exhibited by stem cells offer a potentially limitless source of cells that can differentiate into myelinating oligodendrocytes following transplantation into the damaged CNS. Transplanted precursor cells derived from stem cells have been shown to myelinate axons in genetic models of myelin disease and in models of chemically-induced demyelination. However, studies are still ongoing to determine whether stem cell-derived precursor cells are capable of myelinating axons in animal models of MS, an important step in demonstrating the therapeutic potential of transplanted cells in MS. In addition, the method of cell delivery to patients and the selection of MS patients for cell-based repair therapy are issues that are being explored.