Age-dependent change in activities of lysosomal enzymes in rat brain.

The age-dependent change in activities of seven lysosomal enzymes (cathepsin D, beta-glucuronidase, acid phosphatase, acid/alkaline DNases and acid/alkaline RNases) was studied in four brain regions (cerebrum, hippocampus, pons and cerebellum) of Wistar rats. The activity of cathepsin D was significantly increased with aging in the four regions. The age-dependent change in activities of acid and alkaline DNases showed the characteristic regional difference, and the ratio of acid to alkaline DNases was increased with aging in all regions. Acid RNase showed the lowest activity in 18-month-old rats, and alkaline RNase activity was decreased with aging. The activity of beta-glucuronidase was higher in 2-month-old rats in all of the regions studied. Acid phosphatase showed no significant age-dependent change except in pons. The study demonstrated that all of the lysosomal enzyme activities do not change in parallel with aging, and that the age-dependent change showed the characteristic regional difference.

Effect of metallic cations and pH on reassembly of glial fibrillary acidic protein.

Glial fibrillary acidic protein (GFAP), which was purified from acetone powder of the bovine spinal cord, was reassembled in 0.1 M imidazole HCl buffer containing metallic cations, Ca(2+), Mg(2+), Na(+) or K(+) at physiological or more acidic pH. An electron microscopy revealed reassembled glial filaments at pH 6.8 without any cations but amorphous aggregates at pH 6.3 which were readily observed as a white precipitate by the naked eye. Under more alkaline pH (pH 7.4) only rod-shaped short filaments were formed. In the presence of mM concentrations of Ca(2+) or Mg(2+), thick bundles of glial filaments, detectable by light microscopy, were formed at acidic pH. At pH 7.4 long reassembled filaments could be formed in the buffer containing divalent cations. Na(+) (0.1 M) made filament-like structures of GFAP but they are rather random compared to the filaments promoted by the divalent cations. K(+) made only amorphous aggregation of the short filaments. These findings indicate that the reassembly of GFAP at physiological pH requires essentially divalent cations but not ionic strength.

Accumulation of amyloid beta-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain.

In aged rat brain, amyloid beta-protein precursor (APP) is accumulated in dendrites and cell bodies of Purkinje cells as full-length or truncated APP, because dendrites and cell bodies are positively stained by antibodies against both the amino- and carboxy-termini of APP. Western blot analysis of homogenates of brains of aged and young rats showed no apparent differences except for an increase in amino-terminal fragments in cerebrum and cerebellum of aged rat. These results indicate that the expression, transport or metabolism of APP in specific regions of brains may be affected by the aging process.

Amyloid beta-protein precursor deposition in rat hippocampus lesioned by ibotenic acid injection.

Ibotenic acid was injected into 3 parts of the lateral rat hippocampus. The animals were sacrificed 100 days after treatment, and studied immunohistochemically. The lesioned side of the hippocampus was highly atrophic with extensive neuronal loss and gliosis. Although silver staining revealed no particular structures such as neurofibrillary tangles or senile plaques, globular and granular depositions of amyloid beta-protein precursor (APP) immunoreactivity was observed by immunostaining in the lesion with the monoclonal antibody (clone 22C11). Increased immunoreactivities of glial fibrillary acidic protein (GFAP) and ubiquitin were found in the lesioned area, while the immunoreactivities of microtubule-associated protein 2 (MAP2) and 200 kDa neurofilament subunit protein (NF-H) were diminished. The results indicate that APP deposition is formed in the lesioned area where neuronal degeneration is produced by ibotenic acid in rat hippocampus.

Neurofilament degradation by bovine brain cathepsin D.

The effect of cathepsin D on bovine neurofilament protein was studied biochemically, immunologically, and morphologically. Degradation products of each neurofilament triplet by bovine brain cathepsin D at neutral pH were identified by electrophoresis and immunoblotting with anti-neurofilament antibodies. The 68-kDa subunit was the most susceptive to cathepsin D proteolysis among the triplet proteins. All of the triplet gave rise to partial degradates of the 50-kDa size. The reconstituted fiber from neurofilament triplet proteins and the 68-kDa subunit protein were attacked by cathepsin D and the mode of disruption of the fiber structure was studied by electronmicroscopy.

Activities of lysosomal enzymes in rabbit brain with experimental neurofibrillary changes.

Rabbits were injected intracerebrally with aluminum salt leading to experimental neurofibrillary change formation as a model of Alzheimer neurofibrillary change. Eleven days after the injection, the brain tissues were excised from the cortex, hippocampus, and cervical region of spinal cord. Five lysosomal enzymes (cathepsin D, beta-glucuronidase, acid phosphatase, acid DNase, alkaline DNase) were assayed and compared with the control. Cathepsin D, acid DNase and beta-glucuronidase activities increased significantly in all 3 areas of aluminum-injected brain. On the other hand, acid phosphatase and alkaline DNase activities remained at the same level. The results showed the lysosomal enzymes did not change in parallel after aluminum administration, suggesting a role of the increased enzymes in the brain with neurofibrillary changes.

Lysosome instability in aged rat brain.

The study of the age-dependent change in lysosomal enzyme activities of the cerebral tissue showed the significant increase of cathepsin D in the aged rat brain, while those of beta-glucuronidase and acid phosphatase remained unchanged. The subcellular distribution study of cathepsin D and beta-glucuronidase revealed the increased activity of these enzymes in the cytosolic fraction from the aged brain. In vitro incubation of the lysosome fraction from the aged rat brain resulted in more leakage of these two enzymes, indicating the instability of the lysosome in the aged brain, which resembled the effect of L-Leu-methyl ester to the lysosome.

Abnormal distribution of cathepsins in the brain of patients with Alzheimer's disease.

Formalin-fixed paraffin-embedded hippocampal sections of brains with early-onset and late-onset Alzheimer's disease were studied immunohistochemically with antisera against cathepsin D and cathepsin B. In addition to the staining of neuronal perikarya, some of the senile plaques visualized by Bielshowsky silver staining and some of reactive astrocytes were positively stained with the antisera against cathepsin D and cathepsin B in brains with Alzheimer's disease. Abnormal localization of cathepsin D and cathepsin B immunoreactivity in neuronal perikarya was observed in brains with early-onset Alzheimer's disease. These findings demonstrate that the distribution of lysosomal proteases was altered in brains with Alzheimer's disease, suggesting the primary and/or secondary involvement of the lysosomal proteases in the pathological process of Alzheimer's disease.

Involvement of clathrin light chains in the pathology of Pick's disease; implication for impairment of axonal transport.

Clathrin, which constitutes coated vesicles and plays important roles in neuronal functions, has been reported to be involved in the pathology of Alzheimer's disease. In the brains of the patients with Pick's disease, distribution of clathrin was immunohistochemically investigated using monoclonal antibodies binding to different epitopes of clathrin light chain a and b. All the antibodies intensely labeled Pick's body and some perikarya of neurons, indicating impairment of slow axonal transport b (SCb). Antibodies against neurofilament, kinesin and synaptophysin also labeled Pick's body. These observations suggested impairment of axonal transport in the brains with Pick's disease, and might contribute to elucidating the pathology of Pick's body forming. It is implied that common pathological processes might lie in Alzheimer's disease and Pick's disease.

Effects of SDZ ENA 713, novel acetyl cholinesterase inhibitor, on learning of rats with basal forebrain lesions.

1. The effects of SDZ ENA 713, a novel acetyl cholinesterase inhibitor, on rat learning was studied using a step-down avoidance paradigm. 2. Injection of ibotenic acid into the caudolateral part of the basal forebrain (BF) innervating cholinergic neurons to the cerebral cortex, resulted in an increase in the number of trials required to obtain 300-second-latency, and also a decrease in the latency period after attaining 300-second-latency. 3. It is shown that the BF-lesioned rats are impaired in both acquisition and retention of learning. 4. Intraperitoneal injection of 0.10-0.05 mg/kg/day SDZ ENA 713 to the BF-lesioned rats showed amelioration of the learning impairment, with a decreased number of trials required to obtain 300-second-latency as well as an increase in the latency time after repeated training. 5. These results indicate that SDZ ENA 713 improves acquisition and retention impairment in BF-lesioned rats, and that this drug may be useful for demented patients with cholinergic dysfunction, such as Alzheimer's disease.

Phosphorylated neurofilament accumulation in neuronal perikarya by cyclosporin A injection in rat brain.

Cyclosporin A, a calcineurin inhibitor, was administered into the rat hippocampus. Seven days after drug administration the effects of phosphatase activity suppression in the brain to changes in neuronal cytoskeletal proteins were studied. Rat brain homogenates injected with cyclosporin A showed 16-38% suppression of Ca/calmodulin-dependent phosphatase activity measured by 32P released from 32P-labeled histone, indicating that cyclosporin A acts as an inhibitor of calcineurin after binding with cyclophilin in the brain. Hematoxylin-eosin staining revealed many basophilic neurons in the pyramidal layer of hippocampus, cerebellum, thalamus and cerebral cortex. Immunohistochemical study with anti-phosphorylated neurofilament 200kDa antibody showed positive staining of neuronal perikarya of these basophilic neurons. The number of immunopositive neurons with anti-phosphorylated neurofilament 200KDa increased with the concentration of cyclosporin A injected into the brain, indicating a dose-dependent effect of the compound. Staining with anti-dephosphorylated neurofilament 200KDa (SMI-32) was decreased in neuronal perikarya of cyclosporin A injected brains compared with that of control brains injected with dimethyl sulfoxide alone, suggesting an increased phosphorylation of neurofilament 200KDa subunit protein. The perikarya of these basophilic neurons were not stained with anti-PHF (paired helical filaments) or anti-tau antibodies. Immunostaining with anti-ubiquitin was not increased in these cells. Immunostaining with anti-calcineurin A and anti-calcineurin B showed positive staining of neurons in hippocampus, cerebellum and caudate nucleus both in experimental and control brains. Calcineurin B immunoreactivity was more intense in pyramidal cells in hippocampus injected with cyclosporin A than in controls. Western blot study with antibody against calcineurin A revealed significantly more degradation products of calcineurin A in cyclosporin A injected brains. The present data suggest that cyclosporin A inhibits calcineurin activity in the brain, which results in increased phosphorylation of perikaryal neurofilaments. Since abnormal phosphorylation is speculated in the pathological process of Alzheimer's disease, the results will help elucidate the participation of phosphatase in the pathology of cytoskeletal proteins in Alzheimer's disease.

Brain histamine in Alzheimer's disease.

The concentration of histamine (HA) has been determined by high-performance liquid chromatography (HPLC) with fluorometric detection in 21 different regions of brains from patients with senile dementia of the Alzheimer type (SDAT) and subjects (CB) whose causes of death were not related to neuropsychiatric, neurological and/or neurodegenerative diseases. The highest levels of HA in the central nervous system (CNS) of both control (CB) and SDAT samples were found in the posterior hypothalamus (CB = 3.13 +/- 0.63 pmol/mg; SDAT = 7.75 +/- 1.43 pmol/mg, p less than 0.005), where the HA neurons are located, and in the anterior hypothalamus (CB = 1.77 +/- 0.33 pmol/mg; SDAT = 2.82 +/- 0.45 pmol/mg, p less than 0.005). The lowest HA levels were detected in the cerebellum (CB = 0.12 +/- 0.04 pmol/mg; SDAT = 0.24 +/- 0.09 pmol/mg, p less than 0.01) and medulla oblongata. HA levels were significantly higher in SDAT than in CB in the following areas: motor cortex (Brodmann's area 4) (A4), premotor cortex (A6), postcentral gyrus (A1,2), posterior parietal cortex (A5,7), superior temporal gyrus (A41,42), temporal pole (A38), primary and secondary visual cortices (A17,18), anterior and posterior regions of the hypothalamus, putamen, caudate nucleus, nucleus accumbens, thalamus, hippocampus, pons, medulla oblongata and cerebellum. No changes were seen in globus pallidus and corpus callosum. Since the origin of HA in the brain is dependent upon three main compartments (neuronal, mast cell, vascular smooth muscle), with approximately 60-80% of the total HA belonging to the neuronal pool, on the basis of neurochemical data we postulate that the increase in the levels of HA in SDAT might account for or be associated with alterations in neuroendocrine, cognitive, neurovascular and sleep-wakefulness functions.

Study of cytoskeletal proteins in fibroblasts cultured from familial Alzheimer's disease.

Cytoskeletal proteins of the cultured fibroblasts obtained from Alzheimer's disease patients were studied. Western blotting studies of tubulin, actin, and vimentin showed no difference between Alzheimer and the control fibroblasts. Western blotting studies of vimentin revealed five partial degradation products in 50 K-57 K Da. molecular size region, but no difference in the degradation pattern was noticed between Alzheimer and the control fibroblasts. The size of fodrin molecule, however, was quite different between Alzheimer and the control fibroblasts. Comparing the molecular size of fodrin purified from the bovine brain, it is concluded that fodrin in Alzheimer fibroblasts is not degraded, while significant amount of fodrin in the control fibroblasts is partially degraded resulting in the smaller size of the 160 K and 200 K Da. molecular weight products.

Assembly, disassembly, and exchange of glial fibrillary acidic protein.

The kinetics and dynamics of glial fibrillary acidic protein (GFAP) assembly was explored by a fluorescence energy transfer assay method. Purified GFAP was stoichiometrically labeled at a single cysteine residue with fluorescein-maleimide. Soluble labeled GFAP in a low ionic strength buffer was assembled into 10 nm filaments by rapidly increasing the ionic strength, and the kinetics of GFAP assembly was monitored by the reduction in fluorescence due to self-quenching of fluorescein. The extent of fluorescence quench correlated with both the formation of 10 nm filament morphology and the amount of protein pelleted at 12,000g. The assembly of GFAP is critically dependent upon both protein and magnesium ion concentration, and at the critical concentration for GFAP assembly is approximately 40 micrograms/ml. Disassembly of GFAP filaments was also observed as a relief of fluorescence quenching after dilution of labeled GFAP filaments. When labeled GFAP filaments were mixed with an excess of unlabeled filaments, a rapid increase of fluorescence was observed, which is due to an exchange of subunits between labeled and unlabeled GFAP filaments. These results indicate that GFAP filaments are dynamic structures and that a small pool of kinetically active unassembled GFAP subunits are in a dynamic equilibrium with assembled GFAP filaments. The ability of GFAP to assemble, disassemble, and undergo subunit exchange has important implications for the organization and dynamics of astroglia cell cytoskeleton during development and in response to injury.

Histaminergic neuromodulation of the release of vasopressin.

In an attempt to clarify the nature of histaminergic neuromodulation of the vasopressinergic system, several studies under different experimental paradigms were carried out. L-Histidine loads (8 mmol/kg, i.p.) induced a marked increase in histamine (HA) in the anterior (AHR) and posterior (PHR) hypothalamic regions, the median eminence (ME) and adenohypophysis (Ah) with no apparent effect on the concentration of HA in the neurohypophysis (Nh), as measured by high-performance liquid chromatography. These findings correlated with decreases in vasopressin (VP) levels in the AHR and ME, accompanied by increases of the neuropeptide in the PHR and Ah. Intraperitoneal injections of HA (6 mumol/kg), resulted in a significant (p less than 0.005) rise in VP levels in the PHR, ME and Ah. HA induced an elevation of VP in the prefrontal cortex (PFC) from 6.23 +/- 2.02 to 43 +/- 4.05 microU/mg, as well as a 60% reduction in neurohypophyseal VP. These HA-induced VP responses were abolished by both mepyramine (3 mumol/kg) and famotidine (4 mumol/kg) in the PHR and PFC. Mepyramine suppressed the HA-induced VP response in the Ah and enhanced it in the Nh, while famotidine did the opposite. When alpha-fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase, was administered at doses of 100 mg/kg/day (i.p.), hypothalamic HA levels fell by 40-45% after 1 h, by 50% after 3 h, and by 65-80% after 24 h in adrenalectomized rats. In the same conditions, but after a week of treatment with FMH, the VP response to adrenalectomy was clearly impaired.(ABSTRACT TRUNCATED AT 250 WORDS)