Akinori Matsuo

The 4F2hc/LAT1 complex transports L-DOPA across the blood-brain barrier

L-DOPA is transported across the blood-brain barrier (BBB) by an amino acid transporter, system L. Recently, it has been demonstrated that system L consists of two subunits, 4F2hc and either LAT1 or LAT2. 4F2hc/LAT1 and 4F2hc/LAT2 show different transport characteristics, while their distribution in the brain has not been determined. To clarify whether 4F2hc/LAT1 participates in L-DOPA transport across the BBB, we first examined the expression of 4F2hc/LAT1 in the mouse brain capillary endothelial cell line, MBEC4, as an in vitro BBB model. Northern hybridization and immunoblotting revealed that both 4F2hc and LAT1 are expressed and form a heterodimer in MBEC4 cells. To confirm whether 4F2hc/LAT1 acts as system L to transport L-DOPA, we characterized L-DOPA uptake into the cells. The uptake process was time-dependent, temperature-sensitive, and Na(+)-independent. Neutral amino acids with bulky side chains and a bicyclic amino acid, 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid (BCH), inhibited L-DOPA uptake into MBEC4 cells to a great extent, while an acidic amino acid, basic amino acids, and glycine had no effect. Other neutral amino acids, such as alanine, asparagine, glutamine, serine, and threonine inhibited L-DOPA uptake by 40-70% at most. These characteristics are more compatible with those of 4F2hc/LAT1, rather than those of 4F2hc/LAT2. Finally, immunohistochemistry with anti-LAT1 antibody demonstrated that LAT1 is predominantly expressed in the microvessels of the central nervous system. This is the first report showing that the 4F2hc/LAT1 complex participates in L-DOPA transport across the BBB.

Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat.

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.

Myelin degeneration in multiple system atrophy detected by unique antibodies.

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.

The endoplasmic reticulum-Golgi pathway is a target for translocation and aggregation of mutant superoxide dismutase linked to ALS

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% cases of familial amyotrophic lateral sclerosis (ALS). However, the mechanism of motor neuron degeneration caused by ALS‐linked SOD1 mutants is not fully understood. Here, we used novel live cell imaging techniques to demonstrate the subcellular localization of EGFP‐fused SOD1 of both wild‐type (WT) and ALS‐linked mutant forms in the endoplasmic reticulum (ER) and Golgi. The presence of WT and mutant SOD1 species in luminal structures was further confirmed by immunoblotting analysis of microsomal fractions from spinal cord lysates of SOD1 transgenic mice prepared by sucrose density‐gradient ultracentrifugation. Chemical cross‐linking studies also revealed an age‐dependent aggregation of mutant SOD1, but not of WT SOD1, prominently in the microsomal fraction. Cell‐free translocation assays provided evidence that monomeric SOD1 is a molecular form that can be translocated into luminal structures in the presence of ATP. Our finding that the ER‐Golgi pathway is a predominant cellular site of aggregation of mutant SOD1 suggests that secretion could play a key role in pathogenesis, which is in line with the view that the disease is non‐cell autonomous.—Urushitani, M., Ezzi, S. A., Matsuo, A., Tooyama, I., Julien, J.‐P. The endoplasmic reticulum‐Golgi pathway is a target for translocation and aggregation of mutant superoxide dismutase linked to ALS. FASEB J. 22, 2476–2487 (2008)

Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction.

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Immunohistochemical localization in the rat brain of an epitope corresponding to the fibroblast growth factor receptor-1.

The localization of fibroblast growth factor receptor-1 was investigated in rat brain by immunohistochemistry using a polyclonal antibody against an acidic peptide sequence of chicken fibroblast growth factor receptor-1. For raising the antisera in rabbits, we synthesized the oligopeptide EDDDDEDDSSSEEKEAD which is a highly acidic region of chicken fibroblast growth factor receptor-1. The oligopeptide was used as a haptenic antigen by conjugating with poly-L-glutamate as a carrier protein. On immunospot assay, the best antiserum was capable of detecting 15.7 pmols of both the chicken and its analogous human oligopeptides but failed to react even with up to 1 nmol of poly-L-glutamate. When rat brain homogenate was examined by Western blots, the antiserum revealed two bands with molecular weights of 145,000 and 75,000 corresponding to known sizes of the membrane-bound and secreted forms of the rat receptor, respectively. Immunohistochemistry in rat brain demonstrated that putative fibroblast growth factor receptor-1 immunoreactivity sites were present mainly in neurons but also in tanycytes and ependymal cells. Positive neurons were distributed widely in various brain regions, but were particularly abundant in such regions as the lateral hypothalamus, substantia nigra, locus coeruleus and raphe nuclei. The present study suggests that fibroblast growth factor receptor-1 is expressed preferentially in certain neuronal systems that appear to be under the influence of fibroblast growth factors in the normal brain. The result should facilitate study of the functional significance of fibroblast growth factors in these brain neurons.

Human neural stem cells overexpressing choline acetyltransferase restore cognitive function of kainic acid-induced learning and memory deficit animals.

Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.

Different mechanisms of corpus callosum atrophy in Alzheimer’s disease and vascular dementia

Abstract.Previous neuroimaging studies have indicated that corpus callosum atrophy in Alzheimer’s disease (AD) and large vessel occlusive disease (LVOD) is caused by interhemispheric disconnection, namely Wallerian degeneration of interhemispheric commissural nerve fibers originating from pyramidal neurons in the cerebral cortex. However, this hypothesis has not been tested from a neuropathological viewpoint. In the present study, 22 brains with AD (presenile onset, 9; senile onset, 13), 6 brains with Binswanger’s disease (BD), a form of vascular dementia and 3 brains with LVOD were compared with 6 non-neurological control brains.White matter lesions in the deep white matter and corpus callosum were quantified as a fiber density score by image analysis of myelin-stained sections. Axonal damage and astrogliosis were assessed by immunohistochemistry for amyloid precursor protein and glial fibrillary acidic protein, respectively.The corpus callosum thickness at the anterior part of the body was decreased in AD and LVOD,but not in BD significantly, as compared with the controls. The corpus callosum thickness correlated roughly with brain weight in AD (R = 0.50),and with the severity of deep white matter lesions in BD (R = 0.81). Atrophy of the brain and corpus callosum was more marked in presenile onset AD than in senile onset AD. With immunohistochemistry, the corpus callosum showed axonal damage and gliosis with a decreased fiber density score in BD and LVOD, but not in AD. Thus, corpus callosum atrophy was correlated with brain atrophy in AD, which is relevant to the mechanism of interhemispheric disconnection,whereas corpus callosum lesions in BD were secondary to deep white matter lesions. Corpus callosum atrophy in LVOD may indicate interhemispheric disconnection, but focal ischemic injuries may also be involved.

Immunohistochemical localization of glial cell line-derived neurotrophic factor family receptor α-1 in the rat brain: confirmation of expression in various neuronal systems

The localization of glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1 (GFRalpha-1) was investigated in rat brain by immunohistochemistry using a polyclonal antibody against a specific sequence of the rat protein. For raising the antisera in rabbits, we synthesized the oligopeptide SDVFQQVEHISKGN that corresponds to residues 139 to 152 of rat GFRalpha-1. On immunospot assay, 0.5 microg/ml of an affinity-purified antibody was capable of detecting 7.8 pmol of the rat GFRalpha-1 oligopeptides. When rat brain homogenates were examined by Western blots, the antibody revealed two main bands with molecular weights of approximately 47 kDa and 53 kDa, corresponding to the known sizes of GFRalpha-1. Immunohistochemistry in rat brain demonstrated that GFRalpha-1-like immunoreactivity was present in neurons but not in glial cells. The localization of GFRalpha-1-like immunoreactivity was largely consistent with that of the corresponding GFRalpha-1 mRNA. Positive neurons were distributed widely in various brain regions, but were particularly abundant in such regions as the olfactory bulb, diagonal band, substantia innominata, zona incerta, substantia nigra, cerebellar cortex, nuclei of the cranial nerves including auditory system and spinal motoneurons. The present study showed that GFRalpha-1 in the normal central nervous system is expressed preferentially in certain multiple neuronal systems that include cholinergic system as well as dopaminergic system and motor neurons. As GFRalpha-1 protein was found in numerous brain structures, GDNF family ligands may have therapeutic application not only in degenerative diseases affecting in specific nervous systems, such as Parkinsons disease, amyotrophic lateral sclerosis and multiple system atrophy, but in diffusely damaging diseases like cerebrovascular diseases.

Immunohistochemical localization of glial cell line-derived neurotrophic factor in the human central nervous system

Glial cell line-derived neurotrophic factor, initially purified from the rat glial cell line B49, has the ability to promote the survival and differentiation of various types of neurons in the central and peripheral nervous systems. In the present study, to evaluate the physiological role of glial cell line-derived neurotrophic factor in the central nervous system, we investigated the cellular and regional distribution of glial cell line-derived neurotrophic factor immunoreactivity in autopsied control human brains and spinal cords using a polyclonal glial cell line-derived neurotrophic factor-specific antibody. On western blot analysis, the antibody reacted with recombinant human glial cell line-derived neurotrophic factor, and recognized a single band at a molecular weight of approximately 34,000 in human brain homogenates. Glial cell line-derived neurotrophic factor immunoreactivity was observed mainly in the neuronal somata, dendrites and axons. In the telencephalon, diencephalon and brainstem, the cell bodies and proximal processes of several neuronal subtypes were immunostained with punctate dots. Furthermore, immunopositive nerve fibers were also observed, and numerous axons were intensely immunolabeled in the internal segment of the globus pallidus and the pars reticulata of the substantia nigra. In the cerebellum, the most conspicuous immunostaining was found in the Purkinje cells, in which the somata and dendrites were strongly immunolabeled. Intense immunoreactivity was also detected in the posterior horn of the spinal cord. In addition to the neuronal elements, immunopositive glial cell bodies and processes were observed in various regions. Our results suggest that glial cell line-derived neurotrophic factor is widely localized, but can be found selectively in certain neuronal subpopulations of the human central nervous system. Glial cell line-derived neurotrophic factor may regulate the maintenance of neuronal functions under normal circumstances.