Kazue Kasai

Voxel-Based Morphometric Analysis of Gray Matter in First Episode Schizophrenia

Voxel-based morphometry (VBM) may afford a more rapid and extensive survey of gray matter abnormalities in schizophrenia than manually drawn region of interest (ROI) analysis, the current gold standard in structural MRI. Unfortunately, VBM has not been validated by comparison with ROI analyses, nor used in first-episode patients with schizophrenia or affective psychosis, who lack structural changes associated with chronicity. An SPM99-based implementation of VBM was used to compare a group of 16 patients with first-episode schizophrenia and a group of 18 normal controls and, as a further comparison, 16 first-episode patients with affective psychosis. All groups were matched for age and handedness. High spatial resolution structural images were normalized to the SPM99 template and then segmented, smoothed, and subjected to an ANCOVA. Schizophrenia vs control group comparisons: Voxel-by-voxel comparison of gray matter densities showed that only the left STG region was significantly different when corrected for multiple comparisons (P <.05), consistent with our previously reported manual ROI results. Analysis of the extent of voxel clusters, replicated with permutation analyses, revealed group differences in bilateral anterior cingulate gyri and insula (not previously examined by us with manually drawn ROI) and unilateral parietal lobe, but not in medial temporal lobe (where our ROI analysis had shown differences). However, use of a smaller smoothing kernel and a small volume correction revealed left-sided hippocampal group differences. Affective psychosis comparisons: When the same statistical thresholding criteria were used, no significant differences between affective psychosis patients and controls were noted. Since a major interest was whether patients with affective psychosis shared some anatomical abnormalities with schizophrenia, we applied a small volume correction and searched within the regions that were significantly less dense in schizophrenia compared to control subjects. With this statistical correction, the insula showed, bilaterally, the same pattern of differences in affective disorder subjects as that in schizophrenic subjects, whereas both left STG and left hippocampus showed statistical differences between affectives and schizophrenics, indicating the abnormalities specific to first-episode schizophrenia. These findings suggest both the promise and utility of VBM in evaluating gray matter abnormalities. They further suggest the importance of comparing VBM findings with more traditional ROI analyses until the reasons for the differences between methods are determined.

Histone Deacetylase Inhibitors Augment Antitumor Efficacy of Herpes-based Oncolytic Viruses

Replication-conditional (oncolytic) mutants of herpes simplex virus (HSV), are considered promising therapeutic alternatives for human malignancies, and chemotherapeutic adjuvants are increasingly sought to augment their efficacy. Histone deacetylase (HDAC) inhibitors are a new class of antineoplastic agents because of their potent activity in growth arrest, differentiation, and apoptotic death of cancer cells. The ability of the HDAC inhibitors to upregulate exogenous transgene expression and inhibit interferon (IFN) responses prompted our exploration of their use in improving the antitumor efficacy of oncolytic HSV. We discovered that the yield of viral progeny increased significantly when cultured glioma cells were treated with HDAC inhibitors before viral infection. Valproic acid (VPA), a commonly used antiepileptic agent with HDAC inhibitory activity, proved most effective when used to treat glioma cells before viral infection, but not concomitantly with viral infection. Pretreatment with VPA inhibited the induction of several IFN-responsive antiviral genes, augmented the transcriptional level of viral genes, and improved viral propagation, even in the presence of type I IFNs. Moreover, VPA pretreatment improved the propagation and therapeutic efficacy of oncolytic HSV in a human glioma xenograft model in vivo. These findings indicate that HDAC inhibitors can improve the efficacy of tumor virotherapies.Replication-conditional (oncolytic) mutants of herpes simplex virus (HSV), are considered promising therapeutic alternatives for human malignancies, and chemotherapeutic adjuvants are increasingly sought to augment their efficacy. Histone deacetylase (HDAC) inhibitors are a new class of antineoplastic agents because of their potent activity in growth arrest, differentiation, and apoptotic death of cancer cells. The ability of the HDAC inhibitors to upregulate exogenous transgene expression and inhibit interferon (IFN) responses prompted our exploration of their use in improving the antitumor efficacy of oncolytic HSV. We discovered that the yield of viral progeny increased significantly when cultured glioma cells were treated with HDAC inhibitors before viral infection. Valproic acid (VPA), a commonly used antiepileptic agent with HDAC inhibitory activity, proved most effective when used to treat glioma cells before viral infection, but not concomitantly with viral infection. Pretreatment with VPA inhibited the induction of several IFN-responsive antiviral genes, augmented the transcriptional level of viral genes, and improved viral propagation, even in the presence of type I IFNs. Moreover, VPA pretreatment improved the propagation and therapeutic efficacy of oncolytic HSV in a human glioma xenograft model in vivo. These findings indicate that HDAC inhibitors can improve the efficacy of tumor virotherapies.

An MRI study of temporal lobe abnormalities and negative symptoms in chronic schizophrenia

Previous magnetic resonance imaging (MRI) studies have reported various subtle brain abnormalities in schizophrenic patients, including temporal lobe abnormalities, which are of particular interest given the role of this brain region in auditory and language processing, and the characteristic deficits in these processes in schizophrenia. Subjects in this study were 16 male patients diagnosed with chronic schizophrenia and 15 healthy male comparison subjects. These patients were characterized by negative symptoms. High spatial resolution coronal MRI 1.5-mm-thick slices were used to measure the gray matter volume of the superior temporal gyrus, anterior and posterior amygdala/hippocampal complex, and parahippocampal gyrus. Patients, relative to normal comparison subjects, evinced a reduction of gray matter volume in bilateral superior temporal gyri and anterior amygdala/hippocampal complex. The reduction in gray matter of the superior temporal gyrus in patients with schizophrenia is consistent with previous findings, and is noteworthy in that it was found in this group of patients with predominantly negative symptoms. The reduction in the anterior amygdala/hippocampal complex was an additional temporal lobe finding. These results underscore the role of temporal lobe structures in the pathophysiology of schizophrenia.

Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth

Cyclic AMP regulates multiple neuronal functions, including neurite outgrowth and axonal regeneration. GPR3, GPR6, and GPR12 make up a family of constitutively active G protein-coupled receptors (GPCRs) that share greater than 50% identity and 65% similarity at the amino acid level. They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production. When the constitutively active GPCRs were overexpressed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by myelin-associated glycoprotein. GPR12-mediated neurite outgrowth was the most prominent and was shown to depend on Gs and cAMP-dependent protein kinase. Moreover, the GPR12-mediated rescue from myelin-associated glycoprotein inhibition was attributable to cAMP-dependent protein kinase-mediated inhibition of the small GTPase, RhoA. Among the three receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons. When the endogenous GPR3 was knocked down, significant reduction of neurite growth was observed, which was reversed by expression of either GPR3 or GPR12. Taken together, our results indicate that expression of the constitutively active GPCRs up-regulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibition. Further characterization of the GPCRs in developing and injured mammalian neurons should provide insights into how basal cAMP levels are regulated in neurons and could establish a firm scientific foundation for applying receptor biology to treatment of various neurological disorders.

Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin

ABSTRACT The herpes simplex virus (HSV)-based amplicon vector, a bacterial-viral-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle [MC] amplicon). Although the same dose of the minicircle amplicon vector in normal human fibroblasts resulted in an expression of luciferase approximately 20 times higher than that caused by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Finally, nude mice injected with the MC amplicon exhibited higher and more sustained expression of luciferase than those injected with the conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. Although additional improvements are absolutely required, these findings are a significant first step toward developing a novel HSV amplicon vector that can achieve enhanced long-term transgene expression.

Histone deacetylase 6 inhibition enhances oncolytic viral replication in glioma

Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem-like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells.

Four Distinct Phases of Basket/Stellate Cell Migration after Entering their Final Destination (the Molecular Layer) in the Developing Cerebellum

In the adult cerebellum, basket/stellate cells are scattered throughout the ML, but little is known about the process underlying the cell dispersion. To determine the allocation of stellate/basket cells within the ML, we examined their migration in the early postnatal mouse cerebellum. We found that after entering the ML, basket/stellate cells sequentially exhibit four distinct phases of migration. First, the cells migrated radially from the bottom to the top while exhibiting saltatory movement with a single leading process (Phase I). Second, the cells turned at the top and migrated tangentially in a rostro-caudal direction, with an occasional reversal of the direction of migration (Phase II). Third, the cells turned and migrated radially within the ML at a significantly reduced speed while repeatedly extending and withdrawing the leading processes (Phase III). Fourth, the cells turned at the middle and migrated tangentially at their slowest speed, while extending several dendrite-like processes after having completely withdrawn the leading process (Phase IV). Finally, the cells stopped and completed their migration. These results suggest that the dispersion of basket/stellate cells in the ML is controlled by the orchestrated activity of external guidance cues, cell-cell contact and intrinsic programs in a position- and time-dependent manner.

DNA Demethylating Agents Synergize with Oncolytic HSV1 against Malignant Gliomas

Purpose: Oncolytic viruses (OV) based on herpes simplex virus type 1 (HSV1) are being used in clinical trials for a variety of cancers. The OV, rQNestin34.5, uses a nestin promoter/enhancer to selectively drive robust viral replication in malignant glioma cells. We have discovered that this promoter becomes extensively methylated in infected glioma cells, reducing OV efficacy. Experimental Design: We used demethylating drugs [5-azacytidine (5-Aza)], decitabine, or valproic acid (VPA) in both in vitro and in vivo malignant glioma models to determine if they improved the efficacy of rQNestin34.5 therapy. Results: The use of demethylating agents, such as 5-Aza, improved OV replication and tumor cell lysis in vitro and, in fact, synergized pharmacologically on Chou–Talalay analysis. In vivo, the combination of the demethylating agents, 5-Aza or decitabine, with rQNestin34.5 significantly prolonged the survivorship of athymic mice harboring intracranial human glioma xenografts over single agent alone. Conclusion: These results, thus, provide further justification for the exploration of demethylating agents when combined with the OV, rQNestin34.5, in preclinical therapeutics and, possibly, clinical trials for malignant glioma. Clin Cancer Res; 19(21); 5952–9. ©2013 AACR.

DNA-Based Methods to Prepare Helper Virus-Free Herpes Amplicon Vectors and Versatile Design of Amplicon Vector Plasmids

The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.

Toxicology and Biodistribution Studies for MGH2.1, an Oncolytic Virus that Expresses Two Prodrug-activating Genes, in Combination with Prodrugs

MGH2.1 is a herpes simplex virus type 1 (HSV1) oncolytic virus that expresses two prodrug-activating transgenes: the cyclophosphamide (CPA)-activating cytochrome P4502B1 (CYP2B1) and the CPT11-activating secreted human intestinal carboxylesterase (shiCE). Toxicology and biodistribution of MGH2.1 in the presence/absence of prodrugs was evaluated in mice. MGH2.1 ± prodrugs was cytotoxic to human glioma cells, but not to normal cells. Pharmacokinetically, intracranial MGH2.1 did not significantly alter the metabolism of intraperitoneally (i.p.) administered prodrugs in mouse plasma, brain, or liver. MGH2.1 did not induce an acute inflammatory reaction. MGH2.1 DNA was detected in brains of mice inoculated with 108 pfus for up to 60 days. However, only one animal showed evidence of viral gene expression at this time. Expression of virally encoded genes was restricted to brain. Intracranial inoculation of MGH2.1 did not induce lethality at 108 pfus in the absence of prodrugs and at 106 pfus in the presence of prodrugs. This study provides safety and toxicology data justifying a possible clinical trial of intratumoral injection of MGH2.1 with peripheral administration of CPA and/or CPT11 prodrugs in humans with malignant gliomas.