Osamu Yasuhara

Reactive microglia specifically associated with amyloid plaques in Alzheimer's disease brain tissue express melanotransferrin

Several investigations have implicated the involvement of metals in neuropathologies. In particular, the disruption of iron metabolism and iron transport molecules have been demonstrated in Alzheimers disease (AD). We have identified a novel pathway of iron uptake into mammalian cells involving melanotransferrin, or p97, which is independent of the transferrin receptor. Here we investigated whether there is a possible link between this molecule and the pathology of AD. The distributions of melanotransferrin, transferrin and the transferrin receptor were studied immunohistochemically in brain tissues from AD cases. In brain tissues from AD, melanotransferrin and the transferrin receptor were highly localized to capillary endothelium, while transferrin itself was mainly localized to glial cells. In brain tissue derived from AD patients, melanotransferrin was additionally detected in a subset of reactive microglia associated with senile plaques. Our demonstration that melanotransferrin mediates iron uptake through a pathway independent of the transferrin receptor indicates that this mechanism may have a role in AD.

Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium

One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form. We have recently demonstrated that GPI-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain.

Complement C1 inhibitor is produced by brain tissue and is cleaved in Alzheimer disease

C1 inhibitor was identified in human brain tissue by Western blotting and by immunohistochemistry using multiple antibodies to the native protein. The presence of C1 inhibitor mRNA was identified by reverse transcriptase-polymerase chain reaction analysis of brain mRNA extracts. The mRNA was also detected in cultured postmortem human microglia and in the IMR-32 human neuroblastoma cell line. Immunohistochemically, the native protein was detected in residual serum of capillaries and pyramidal neurons of both control and Alzheimer disease cases, as well as in occasional senile plaques of Alzheimer tissue. The reacted protein was detected on dystrophic neurites and neuropil threads in Alzheimer tissue by 4C3 monoclonal antibody, which recognizes a neoepitope following suicide inhibition. These data indicate that C1 inhibitor, a regulatory molecule controlling multiple inflammatory proteolytic cascades, is produced in normal brain. In Alzheimer disease, C1 inhibitor undergoes a prominent reaction in abnormal neuronal processes, such as dystrophic neurites and neuropil threads.

German-Canadian family (family A) with parkinsonism, amyotrophy, and dementia — Longitudinal observations

Etiology of Parkinsons disease (PD), amyotrophy lateral sclerosis (ALS), and Alzheimers disease (AD) remains uncertain. Environmental factors probably play a role, but genetic influences may predispose certain individuals to develop each of these major neurodegenerative disorders. We describe our longitudinal observations concerning a Canadian family traced to Northern Germany. Autosornal dominant inheritance has been established. Affected members present with L-dopa responsive parkinsonism and amyotrophy. In the German portion of the family some individuals displayed only dementia or focal dystonia. Linkage analysis studies performed with polymorphic markers associated with 13 candidate genes provided no significant evidence for linkage with any of the genes examined. Positron emission tomography with [(18)F]-6-fluoro-L-dopa (FD) and [su11C]-raclopride (raclopride) of one affected subject revealed reduced striatal FD uptake particularly in putamen, and an increased raclopride striatum/background ratio. Postmortem levels of dopamine and its metabolites were greatly reduced in caudate and putamen of two patients. There was substantial neuronal loss in the substantia nigra and the presence of abundant eosinophilic granules (different than Lewy bodies) in surviving neurons. One of them also showed mild loss of anterior horn cells, while another showed abundant senile plaques and some neurofibrillary tangles in distribution and intensity typical of mild to moderate AD. Our report further describes this unique family with a combination of clinical features of PD, ALS, and AD. By studying kindreds like this we may learn more about the pathophysiology of sporadic forms of PD, ALS, or even AD.

Expression of cystatin C in rat, monkey and human brains

Expression of cystatin C and its mRNA in brain were investigated by use of immunohistochemical and polymerase chain-reaction techniques. High levels of cystatin C mRNA were detected in every region of rat brain examined, including the cerebral cortex, hippocampus, hypothalamus and cerebellum. Cystatin C-positive astrocytes were found by immunohistochemistry to be distributed throughout the brains of rat, monkey and human. Some neurons were also positive, but the staining was weak and variable. Intensely immunoreactive neurons were abundantly found in the cerebral cortex of some aged human cases and of all Alzheimers disease patients. It is concluded that cystatin C is synthesized and expressed in the central nervous system, especially by astrocytes. Cystatin C might also be involved in the aging process of cortical neurons.

Immunohistochemical localization of hyaluronic acid in rat and human brain

The immunohistochemical localization of hyaluronic acid (HA) was studied in rat and human brain using the monoclonal antibody NDOG1, which specifically recognizes HA. In both rat and human brain, HA-like immunoreactivity formed characteristic coats around neurons in highly selective areas. The staining was abolished by pretreatment of sections with testicular and Streptomyces hyaluronidases, indicating that the staining was specific for HA. In rat brain, positive neurons were located in the cerebral cortex, subiculum, amygdala, thalamic reticular nucleus, nuclei of the inferior colliculus, nuclei of the trapezoid body, and vestibular nuclei. They were also scattered in the hypothalamus, substantia nigra pars reticularis, red nucleus, parabrachial nuclei, brainstem reticular nuclear group, ventral cochlear nucleus, nuclei of lateral lemniscus, and deep cerebellar nuclei. Double immunohistochemical studies showed that many neurons staining for HA were positive for parvalbumin, with minor exceptions in the amygdala and piriform cortex, where some HA-positive neurons were also positive for calbindin-D28k. In the areas studied in human brain, the distribution of HA-positive neurons was virtually identical to that in rat brain. HA-positive neurons were not significantly altered in Alzheimer disease (AD) brain, suggesting that these neurons are resistant to the pathological process of AD.

Expression of chromogranin A in lesions in the central nervous system from patients with neurological diseases.

Expression of chromogranin A in various neurological diseases was examined immunohistochemically using purified anti-human chromogranin A antiserum. The antibody stained dystrophic neurites in senile plaques in Alzheimer disease brain, Pick bodies and ballooned neurons in Picks disease brain, some Lewy bodies in the substantia nigra of Parkinsons disease, and axonal swellings in various neurological conditions including Parkinsons disease, striatonigral degeneration, Shy-Drager syndrome, amyotrophic lateral sclerosis and cerebral infarction. The present study shows that expression of chromogranin A is not an exclusive feature of Alzheimer disease or Picks disease, and indicates that it could be a useful marker for various neurological diseases.

Hageman factor and its binding sites are present in senile plaques of Alzheimer's disease

Hageman factor (HF) or factor XII participates in several defense systems of the body. These include coagulation, fibrinolysis and complement activation. We investigated the expression of HF and its mRNA in control and Alzheimers disease (AD) brain, using immunohistochemistry and polymerase-chain reaction (PCR) techniques. HF mRNA was detected in control and AD brain extracts, indicating that HF can be produced by endogenous brain cells. HF-like immunoreactivity was present in residual serum of capillaries in both control and AD brain, consistent with its known presence in the circulation. In addition, AD senile plaques were stained. The staining was dramatically enhanced when AD sections were incubated with solutions containing HF, indicating that plaques contain not only HF but also binding sites for HF. The enhanced staining was eliminated by pretreatment of solutions with the HF-binding agent kaolin. It was also eliminated by pretreatment of sections with protamine, an agent which strongly binds to negative surfaces. These data suggest that negatively charged surfaces in plaques might bind HF in vivo. Since HF can be activated by contact with negative surfaces, locally released HF could be playing a role in initiating a variety of inflammatory responses in AD brain.

Expression of the complement membrane attack complex and its inhibitors in Pick disease brain

The immunohistochemical localization of the complement membrane attack complex (MAC) was examined in Pick disease brain and compared with the distribution of three of its inhibitors, vitronectin, protectin and clusterin. Pick bodies were stained intensely for both the MAC and protectin, weakly for vitronectin, but negatively for clusterin. However, the clusterin antibody intensely stained some pyramidal neurons in affected cortical areas, including ballooned neurons. The present study indicates that a complement-mediated attack is associated with the formation of Pick bodies, and provides further suggestive evidence that clusterin may be a marker for active neuronal degeneration.

Production of antisera to acidic fibroblast growth factor and their application to immunohistochemical study in rat brain

Antisera against acidic fibroblast growth factor purified from bovine brain were produced in rabbits and used for immunohistochemical study of the rat brain. When examined in an immunospot assay using a nitrocellulose membrane, the best antibody was capable of detecting 80 fmol of acidic fibroblast growth factor but failed to react even with up to 5 pmol of basic fibroblast growth factor. Using this antiserum, the immunohistochemical distribution of acidic fibroblast growth factor was examined in rat brain. Acidic fibroblast growth factor-like immunoreactivity was localized mainly in a subpopulation of ependymal cells and tanycytes, as well as in some glial cells. Positive ependymal cells were observed throughout the walls of ventricles, including the third ventricle and cerebral aqueduct. Immunoreactive processes of tanycytes were found extending from the ventral wall of the third ventricle to the brain parenchyma and surface. The most intense immunostaining was observed in circumventricular organs such as the organum vasculosum laminalis terminalis and the subfornical organ. Particularly in the latter organ, there was an extremely dense plexus of immunoreactive fibers and processes around the wall of capillaries. The present results suggest that the effects of acidic fibroblast growth factor on brain functions may be exerted through the circumventricular organs and/or ependymal cells.