Masafumi Onodera

Possibility for neurogenesis in substantia nigra of parkinsonian brain.

Recent studies of enhanced hippocampal neurogenesis by antidepressants suggest enhancement of neurogenesis is a potentially effective therapy in neurodegenerative diseases. In this study, we evaluated nigral neurogenesis in animals and autopsy brains including patients with Parkinsons disease (PD). First, proliferating cells in substantia nigra were labeled with retroviral transduction of green fluorescent protein, which is an efficient method to label neuronal stem cells. Subsequent differentiation of labeled cells was followed; many transduced cells became microglia, but no differentiation into tyrosine hydroxylase–positive neurons was detected at 4 weeks after injection, in both intact rodents and those treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. Second, polysialic acid (PSA)–like immunoreactivity, indicative of newly differentiated neurons, was detected in the substantia nigra of rodent, primate, and human midbrains. A large number of PSA‐positive cells were detected in the substantia nigra pars reticulata of some patients with PD. In rats and a macaque monkey, the dopamine‐depleted hemispheres showed more PSA staining than the intact side. A small number of tyrosine hydroxylase–positive cells were PSA‐positive. Our results suggest enhanced neural reconstruction in PD, which may be important in the design of new therapies against the progression of PD. Ann Neurol 2005

The fate of neural progenitor cells expressing astrocytic and radial glial markers in the postnatal rat dentate gyrus

In the dentate gyrus neurons continue to be generated from late embryonic to adult stage. Recent extensive studies have unveiled several key aspects of the adult neurogenesis, but only few attempts have so far been made on the analysis of the early postnatal neurogenenesis, a transition state between the embryonic and adult neurogenesis. Here, we focus on the early postnatal neurogenesis and examine the nature and development of neural progenitor cells in Wistar rats. Immunohistochemistry for Ki67, a cell cycle marker, and 5‐bromo‐2‐deoxyuridine (BrdU) labelling show that cell proliferation occurs mainly in the hilus and partly in the subgranular zone. A majority of the proliferating cells express S100β and astrocyte‐specific glutamate transporter (GLAST) and the subpopulation are also positive for glial fibrillary acidic protein (GFAP) and nestin. Tracing with BrdU and our modified retrovirus vector carrying enhanced green fluorescent protein (GFP) indicate that a substantial population of the proliferating cells differentiate into proliferative neuroblasts and immature neurons in the hilus, which then migrate to the granule cell layer (66.8%), leaving a long axon‐like process behind in the hilus, and the others mainly become star‐shaped astrocytes (12.0%) and radial glia‐like cells (4.7%) in the subgranular zone. These results suggest that the progenitors of the granule cells expressing astrocytic and radial glial markers, proliferate and differentiate into neurons mainly in the hilus during the early postnatal period.

Clustering, Migration, and Neurite Formation of Neural Precursor Cells in the Adult Rat Hippocampus

Adult neurogenesis occurs in the subgranular zone and innermost part of the dentate granule cell layer. To examine how neural precursor cells proliferate, migrate, and extend their neurites, we performed BrdU‐ and improved retrovirus‐green fluorescence protein (GFP)‐labeling analyses. Soon after labeling the majority of BrdU+ cells and GFP+ cells expressed Ki67, a cell cycle marker, and formed clusters together with PSA+ neuroblasts. Most of the Ki67+ proliferating cells expressed Hu, an immature and mature neuronal marker, and the subpopulation expressed both Hu+ and GFAP+. In the clusters, Ki67+ and PSA+ cells strongly expressed β‐catenin and N‐cadherin, but PSA+ cells outside the clusters did not. Therefore, it was mainly Hu+ neuronal precursor cells that proliferated within clusters in which the cluster cells are closely associated via cell adhesion molecules, such as N‐cadherin/β‐cateninIn and PSA. The newly generated cells appeared to stay in the clusters for a few days and then disperse around the clusters. The findings of this in vivo analysis and in vitro time‐lapse imaging of early postnatal hippocampal slices support the notion that most postmitotic neuroblasts migrate tangentially from clusters, extending tangentially oriented processes, one of which often retains close contact with the clusters, and finally extend radial processes, or prospective apical dendrites. These results suggest that the clustering cells and tangentially migrating cells have a systematic cellular arrangement and intercellular interaction. J. Comp. Neurol. 502:275–290, 2007.

Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.

Neurogenesis continues throughout life in the hippocampus. To study postnatal neurogenesis in vitro, hippocampal slices from rats on postnatal day 5 (P5) were cultured on a porous membrane for 14 or 21 days. In the initial experiments, precursor cells were labeled with bromodeoxyuridine (BrdU) after 7 days in culture because hippocampal slices are generally used in experiments after 1–2 weeks in culture. Fourteen days after labeling, however, only about 10% of BrdU‐labeled cells expressed neuronal markers, although in living rats, about 80% of cells labeled with BrdU on P5 had become neurons by P19. Next, rats were injected with BrdU 30 min before culture, after which hippocampal slices were cultured for 14 days to examine the capacity of in vivo–labeled neural precursors to differentiate into neurons in vitro. In this case, more than two‐thirds of BrdU‐labeled cells expressed neuronal markers, such as Hu, NeuN, and PSA‐NCAM. Furthermore, precursor cells underwent early in vitro labeling by incubation with BrdU or a modified retrovirus vector carrying EGFP for 30 min from the beginning of the culture. This procedure resulted in a similar high rate of neuronal differentiation and normal development into granule cells. In addition, time‐lapse imaging with retrovirus‐EGFP revealed migration of neural precursors from the hilus to the granule cell layer. These results indicate that in vivo– and early in vitro–labeled cultures are readily available ex vivo models for studying postnatal neurogenesis and suggest that the capacity of neural precursors to differentiate into neurons is reduced during the culture period.

Parvovirus Nonstructural Proteins Induce an Epigenetic Modification through Histone Acetylation in Host Genes and Revert Tumor Malignancy to Benignancy

ABSTRACT Several malignant tumor cells become apoptotic and revert to the benign phenotype upon parvovirus infection. Recently, we demonstrated that the rat parvovirus RPV/UT also induces apoptosis in the rat thymic lymphoma cell line C58(NT)D. However, a minority of cells that escaped apoptosis showed properties different from the parental cells, such as resistance to apoptosis, enhanced cell adherence, and suppressed tumorigenicity. The present study was performed to determine the molecular mechanism of parvovirus-induced phenotypic modification, including oncosuppression. We demonstrated that the nonstructural (NS) proteins of RPV/UT induced apoptosis in C58(NT)D cells and suppressed tumor growth in vivo. Interestingly, NS proteins induced the expression of ciliary neurotrophic factor receptor alpha, which is up-regulated in revertant cell clones, and enhanced histone acetylation of its gene. These results indicate that parvoviral NS regulate host gene expression through histone acetylation, suggesting a possible mechanism of oncosuppression.

Gene and cell therapy for relapsed leukemia after allo-stem cell transplantation.

To control severe GvHD while maintaining strong GvL effects in the context of allo-stem cell transplantation (allo-SCT), a phase I/II clinical trial of infusions of donor lymphocytes transduced with the herpes simplex virus thymidine kinase (TK-DLI) started at the Tsukuba University Hospital. To date, five (2 AML, 2 ALL, and 1 MDS) out of eight patients enrolled in the trial received approximately 7x10(7) transduced cells per kilogram of body weight and four patients showed some clinical responses such inhibition of the leukemic cell proliferation or mitigation of lymph node swelling. Especially, one MDS patient achieved complete remission and has remained in CR for 2 years after the treatment. GvHD developed in two patients (1 acute and 1 chronic) and the acute (grade III) was successfully controlled by administration of ganciclovir without any immunosuppressive drugs. Since HSV-TK as a strong antigen induced CTLs against transduced cells in patients, however, TK-DLI is expected to provide a more effective adoptive immune cell therapy by performance just after allo-SCT where the patients immune function is severely damaged.

Antitumor activity of chimeric immunoreceptor gene‐modified Tc1 and Th1 cells against autologous carcinoembryonic antigen‐expressing colon cancer cells

To generate tumor‐specific and interferon (IFN)‐γ‐producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)‐specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T‐cell receptor (cIgTCR) gene composed of single‐chain variable fragments from an anti‐CEA‐specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3ζ. These cells, designated Tc1‐T and Th1‐T bodies, respectively, showed strong antitumor activity against CEA‐expressing tumor cells in RAG2–/– mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1‐T and Th1‐T bodies from cancer patients with defective T‐cell function because of significant immunosuppression. Here, we prepared Tc1‐T and Th1‐T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T‐cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN‐γ in response to CEA‐expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1‐T bodies supported the survival of Tc1‐T bodies through cell‐to‐cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus‐mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1‐T and Th1‐T bodies will become a promising strategy for adoptive immunotherapy of human cancer. (Cancer Sci 2006; 97: 920–927)