Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains

学位記番号：医博甲1670

Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling

Endotoxin, a bacterial lipopolysaccharide (LPS), causes fatal septic shock via Toll-like receptor (TLR)4 on effector cells of innate immunity like macrophages, where it activates nuclear factor κB (NF-κB) and mitogen-activated protein (MAP) kinases to induce proinflammatory cytokines such as tumor necrosis factor (TNF)-α. Dok-1 and Dok-2 are adaptor proteins that negatively regulate Ras–Erk signaling downstream of protein tyrosine kinases (PTKs). Here, we demonstrate that LPS rapidly induced the tyrosine phosphorylation and adaptor function of these proteins. The stimulation with LPS of macrophages from mice lacking Dok-1 or Dok-2 induced elevated Erk activation, but not the other MAP kinases or NF-κB, resulting in hyperproduction of TNF-α and nitric oxide. Furthermore, the mutant mice showed hyperproduction of TNF-α and hypersensitivity to LPS. However, macrophages from these mutant mice reacted normally to other pathogenic molecules, CpG oligodeoxynucleotides, poly(I:C) ribonucleotides, or Pam3CSK4 lipopeptide, which activated cognate TLRs but induced no tyrosine phosphorylation of Dok-1 or Dok-2. Forced expression of either adaptor, but not a mutant having a Tyr/Phe substitution, in macrophages inhibited LPS-induced Erk activation and TNF-α production. Thus, Dok-1 and Dok-2 are essential negative regulators downstream of TLR4, implying a novel PTK-dependent pathway in innate immunity

Role of Dok-1 and Dok-2 in Myeloid Homeostasis and Suppression of Leukemia

Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia

Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains.

Adeno-associated virus (AAV) vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb) were obtained by progressively deleting the original 2.0-kb promoter from the 5' end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength) and 0.2-kb (70% astrocyte specificity) promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity

Evaluation of Fundus Blood Flow in Normal Individuals and Patients with Internal Carotid Artery Obstruction Using Laser Speckle Flowgraphy

<div><p>Purpose</p><p>We investigated whether laser speckle flowgraphy (LSFG) results are comparable in both eyes and whether it is useful in the diagnosis of disparity in ocular ischemic syndrome (OIS) patients.</p><p>Methods</p><p>We compared the mean blur rate (MBR) value for various fundus regions in both eyes of 41 healthy subjects and 15 internal carotid artery occlusion (ICAO) cases. We calculated the standard value of the Laterality Index (LI), which was the MBR comparison of both eyes in each of the regions, in the control subjects. We then investigated the correlation between both eyes for the LIs in the entire fundus, the degree of ICAO and visual function.</p><p>Results</p><p>The disparity of the LIs in both eyes was least in the entire area of the fundus in control subjects and there was a significant correlation between both eyes of the 41 healthy individuals (P = 0.019). Significant correlations were found for the LI, visual acuity and degree of ICAO. The specificity and sensitivity of LI in the entire area was 93.8% and 100%, respectively.</p><p>Conclusions</p><p>LSFG revealed normal individuals have symmetrical fundus blood flow. LSFG could detect OIS and might be a useful tool for detecting disparities in fundus blood flow.</p></div

Case 1. The false color maps of a patient with ocular ischemic syndrome.

<p>Top, right eye; bottom, left eye. Although the color map of the right eye is normal, that of the left eye shows that the choroidal vasculature has disappeared and that there are faint retinal vessels present, suggesting decreased retinal and choroidal blood flows.</p

Absence of astrocyte specificity for the marmoset- and human-derived GFAP promoters in the mouse cerebrum.

<p>(A-C) Cerebral slices lentivirally expressing GFP under the control of the 0.3-kb marmoset-derived cjGFAP (A), 0.3-kb mouse-derived mGFAP (B) or 0.3-kb human-derived hGFAP (C) promoter were triple-immunostained for GFP (green), GFAP (magenta) and NeuN (a neuronal marker, cyan). Note the predominant expression of GFP in neurons (arrow) by the marmoset- and human-derived promoter, which is in sharp contrast to the astrocyte-specific expression (arrowhead) by the mouse-derived promoter. Scale bars, 50 μm. (D) Schema depicting morphology of neuron and astrocyte in the cerebral cortex. (E) Quantitative analysis of the astrocyte specificity for the cjGFAP, mGFAP and hGFAP promoters. More than 300 GFP-positive cells from 3 mice (3 slices/mouse) were randomly selected, and the ratio of GFAP-labeled astrocytes were determined in these random selections. Asterisks indicate statistically significant differences between the mouse promoter and the marmoset or human promoter, as determined by one-way ANOVA followed by Tukey’s post hoc test, ***<i>p</i><0.001.</p

AAV9 vector-mediated GFP expression in the marmoset brain.

<p>AAV9 vectors expressing GFP under the control of the 0.3-kb cjGFAP promoter were injected into the cerebral and cerebellar cortices. (A and B) Bright field images of the marmoset whole brain overlaid with GFP fluorescence. (C and D) Bright field images of the sagittal sections of the cerebellar (C) and cerebral (D) hemispheres are presented with GFP fluorescence. Scale bars, 2 mm.</p

Bilateral comparison of the mean blur ratios in the entire image in normal individuals.

<p>Correlation coefficient of the linear fit r = 0.8594 (P = 0.019).</p

Summary of MBR values from 41 healthy individuals.

<p>Summary of MBR values from 41 healthy individuals.</p