Hidetoshi Ooigawa

L-type amino acid transporter 1 as a potential molecular target in human astrocytic tumors

L‐type amino acid transporter 1 (LAT1) is a Na+‐independent neutral amino acid transport agency and essential for the transport of large neutral amino acids. LAT1 has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells. We have investigated for the first time, the expression of the transporter in the human primary astrocytic tumor tissue from 60 patients. LAT1 is unique because it requires an additional single membrane spanning protein, the heavy chain of 4F2 cell surface antigen (4F2hc), for its functional expression. 4F2hc expression was also determined by immunohistochemistry. Kaplan‐Meier analyses demonstrated that high LAT1 expression correlated with poor survival for the study group as a whole (p < 0.0001) and for those with glioblastoma multiforme in particular (p = 0.0001). Cox regression analyses demonstrated that LAT1 expression was one of significant predictors of outcome, independent of all other variables. On the basis of these findings, we also investigated the effect of the specific inhibitor to LAT1, 2‐aminobicyclo‐2 (2,2,1)‐heptane‐2‐carboxylic acid (BCH), on the survival of C6 glioma cells in vitro and in vivo using a rat C6 glioma model. BCH inhibited the growth of C6 glioma cells in vitro and in vivo in a dose‐dependent manner. Kaplan‐Meier survival data of rats treated with BCH were significant. These findings suggest that LAT1 could be one of the molecular targets in glioma therapy.

Enhanced hippocampal neurodegeneration after traumatic or kainate excitotoxicity in GFAP-null mice

Astrocytes perform a variety of functions in the adult central nervous system. Recent evidence suggests that the upregulation of glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament component, is a biological marker of neurotoxicity after cerebral injury. We herein compared the response to traumatic brain injury or kainic acid (KA)-induced neurotoxicity in GFAP knockout (GFAP-KO) and wild-type (WT) mice. Seventy-two hours after injury, all GFAP-KO mice showed hippocampal CA3 neurodegeneration, whereas WT mice did not show neurodegeneration. Seventy-two hours after KA administration, GFAP-KO mice were more susceptible to KA-induced seizures and had an increased number of pyknotic damaged CA3 neurons than did WT mice. These results indicate that GFAP plays a crucial role in pyramidal neuronal survival after injury or KA-induced neurotoxicity.

High affinity D- and L-serine transporter Asc-1: cloning and dendritic localization in the rat cerebral and cerebellar cortices.

System asc transporter Asc-1, expressed in the brain, transports D- and L-serine with high affinity. To determine the localization of Asc-1 in the rat brain, we isolated a cDNA for the rat orthologue of Asc-1. The encoded protein designated as rAsc-1 (rat Asc-1) exhibited 98% sequence identity to mouse Asc-1 (mAsc-1). Based on amino acid sequences of rAsc-1 and mAsc-1, two polyclonal antibodies against Asc-1 were generated and used for the immunohistochemical analysis on the cerebral and cerebellar cortices of rats and mice. Asc-1 immunoreactivity was detected in neurons, including cerebellar Purkinje neurons and pyramidal neurons in the neocortex and hippocampus. It was clearly localized in dendrites as well as somata. The localization of Asc-1 in brain suggests the significant contribution of Asc-1 to amino acid mobilization in brains including the synaptic clearance of D-serine and the neuronal uptake of L-serine that is essential for survival and dendrite growth of Purkinje neurons in particular.

Nuclear accumulation of basic fibroblast growth factor in human astrocytic tumors.

The authors recently reported that nuclear accumulation of basic fibroblast growth factor (bFGF) demonstrated a significant correlation with recurrence of pituitary adenomas. The current study sought to determine whether nuclear bFGF accumulation was a predictor of survival in patients with astrocytic tumors.

Targeted DNA transfection into the mouse central nervous system using laser- induced stress waves

We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

Temporal and Spatial Profile of Phosphorylated Connexin43 after Traumatic Brain Injury in Rats

Gap junctions are conductive channels formed by membrane proteins termed connexins (Cx), which permit the intercellular exchange of metabolites, ions, and small molecules. Junctional permeability is regulated by pH, membrane potential, and intracellular secondary messengers. The purpose of this study was to elucidate the expression and distribution of astrocytic gap junctions in the hippocampus and the cortex after traumatic brain injury (TBI) in vivo. Adult male Sprague-Dawley rats (300-400 g) were subjected to lateral fluid percussion injury (FPI) at moderate severity (2.6-2.8 atm, 12 msec) using a Dragonfly device model. Phosphorylated gap junction protein levels were quantified using Western blot analysis. Spatial distribution of immunoreactivity for phosphorylated Cx43 (p-Cx43) was analyzed by immunohistochemistry. Our findings showed that p-Cx43 expression in the ipsilateral hippocampus was significantly induced at 1 h after TBI, and remained at a high level until 24 h after injury. The p-Cx43 protein content reached a maximum level at 6 h after injury. In addition, the immunoreactivity for p-Cx43 was localized in the astrocytes surrounding ipsilateral CA3 pyramidal neurons. On the other hand, the protein level in the ipsilateral cortex was not significantly different at any time point after TBI. Double immunostaining using phosphorylated ERK (p-ERK) showed that p-Cx43 and p-ERK immunoreactivities were enhanced in the same astrocytes at 6 h after injury. These findings suggest that astrocytic gap junctions participate in pathophysiological processes in the hippocampus after TBI.

Alteration of gap junction proteins (connexins) following lateral fluid percussion injury in rats

Gap junctions are intercellular channels that mediate the cytoplasmic exchange of small hydrophilic molecules and are formed by a family of integral membrane proteins called connexins (Cxs). Cx43 is expressed predominantly in astrocytes, while Cx36 is expressed in neurons. In this study, we show alteration of Cx43 and Cx36 in the hippocampus after traumatic brain injury in rats. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion injury of moderate severity. Brain coronal sections were used for immunohistochemistry with Cx43 and Cx36 antibodies. Cx43 immunoreactivity was increased in reactive astrocytes in the damaged hippocampus 24 hours after injury, and persisted for 72 hours. On the other hand, Cx36 immunoreactivity increased in CA3 neurons 1 hour after injury, and decreased later. These results indicate that gap junctions might participate in the pathophysiological process after traumatic brain injury.

Focal brain edema and natriuretic peptides in patients with subarachnoid hemorrhage

Central salt wasting syndrome may be caused by pathological increases in serum natriuretic peptides after subarachnoid hemorrhage (SAH). However, it is unclear as to why the serum concentration of atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) increases in the subacute phase of SAH. The present study was designed to assess the correlation between focal brain edema and serum concentration of ANP or BNP in patients with SAH. Focal brain edema was found in 8 SAH-patients and peaked between days 4 and 7 of SAH. The mean serum ANP and BNP levels in patients with focal brain edema were significantly higher than those in patients without focal brain edema between days 4 and 14 of SAH. These results suggest that focal brain edema might correlate with increased levels of ANP and BNP in the subacute phase of SAH.

Simultaneous measurement of changes in light absorption due to the reduction of cytochrome c oxidase and light scattering in rat brains during loss of tissue viability

We performed the simultaneous measurement of intrinsic optical signals (IOSs) related to metabolic activity and cellular and subcellular morphological characteristics, i.e., light scattering for a rat global ischemic brain model made by rapidly removing blood by saline infusion. The signals were measured on the basis of multiwavelength diffuse reflectances in which 605 and 830 nm were used to detect the IOSs that are thought to be dominantly affected by redox changes of heme aa(3) and CuA in cytochrome c oxidase (CcO), respectively. For measuring the scattering signal, the wavelength that was found to be most insensitive to the absorption changes, e.g., approximately 620 nm, was used. The measurements suggested that an increase in the absorption due to reduction of heme aa(3) occurred soon after blood clearance, and this was followed by a large triphasic change in light scattering, during which time a decrease in the absorption due to reduction of CuA occurred. Through the triphasic scattering change, scattering signals increased by 5.2 +/- 1.5% (n = 5), and the increase in light scattering showed significant correlation with both the reflectance intensity changes at 605 and 830 nm. This suggests that morphological changes in cells correlate with reductions of heme aa(3) and CuA. Histological analysis of tissue after the triphasic scattering change showed no alteration in either the nuclei or the cytoskeleton, but electron microscopic observation revealed deformed, enlarged mitochondria and expanded dendrites. These findings suggest that the simultaneous measurement of absorption signals related to the redox changes in the CcO and the scattering signal is useful for monitoring tissue viability in the brain.

High expression of L-type amino acid transporter 1 in infiltrating glioma cells

L-type amino acid transporter 1 (LAT1), a neutral amino acid transport agent, is essential for the transport of large neutral amino acids. LAT1 also corresponds to tumor-associated gene-1 (TA1), an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells such as glioma cells. We have investigated the expression of the transporter in the human primary glioma tissue from 68 patients. Among these patients, we could see the border zone between tumors and normal bain tissues in 10 patients. By WHO criteria, two of the specimens were diagnosed as grade 2, three as grade 3, and five as grade 4 [glioblastoma multiforme (GBM)]. In 9 of 10 cases, we could identify the infiltrating glioma cells associated with stronger immunoreactivity for LAT1. These tumor cells aggregated around the neurons in the border zone and were often found in the perivascular space. In one GBM case, the tumors seemed to develop expansively and separated from the normal brain with a border of arachnoid membrane. The expression of LAT1 was always higher in infiltrating glioma cells than in cells located in the center of the tumor. These findings suggest that LAT1 is one of the molecular targets for glioma therapy.