Potentially amyloidogenic, carboxyl-terminal derivatives of the amyloid protein precursor.

The 39- to 43-amino acid amyloid beta protein (beta AP), which is deposited as amyloid in Alzheimer's disease, is encoded as an internal peptide that begins 99 residues from the carboxyl terminus of a 695- to 770-amino acid glycoprotein referred to as the amyloid beta protein precursor (beta APP). To clarify the processing that produces amyloid, carboxyl-terminal derivatives of the beta APP were analyzed. This analysis showed that the beta APP is normally processed into a complex set of 8- to 12-kilodalton carboxyl-terminal derivatives. The two largest derivatives in human brain have the entire beta AP at or near their amino terminus and are likely to be intermediates in the pathway leading to amyloid deposition.

Interleukin 3 as a trophic factor for central cholinergic neurons in vitro and in vivo.

We have found that interleukin 3 (IL-3), a growth factor for hematopoietic cells, is a novel trophic factor for mouse and rat central cholinergic neurons. It enhanced neurite outgrowth and elevated choline acetyltransferase activity. The effect seems to be specific for cholinergic neurons, since somatostatin release and glutamic acid decarboxylase and 2',3'-cyclic nucleotide 3'-phosphodiesterase activities were not significantly influenced by IL-3. In vivo, IL-3 was infused into the lateral ventricles of rats after unilateral axotomy of the septohippocampal pathways. Two weeks later, the IL-3-treated animals showed significant numbers of acetylcholinesterase-positive neurons remaining in the septal region.

In vivo and in vitro studies of the prevention of proteolipid apoprotein-induced murine experimental allergic encephalomyelitis by monoclonal antibody against L3T4.

The suppressive effect of anti-L3T4 monoclonal antibody (mAb) on murine experimental allergic encephalomyelitis (EAE) induced by sensitization with proteolipid apoprotein (PLP) was examined in vivo and in vitro. This mAb inhibited the antigen-specific proliferation of the encephalitogenic T cell lines but did not block the mitogen-mediated response. Serial injections of the mAb during the pre-effector phase of EAE markedly suppressed the development and relapse of the disease but this treatment initiated after appearance of clinical signs was not effective. In treated animals, L3T4+ T cells in the spleen were profoundly decreased and the antigen-specific proliferative response of spleen cells was completely suppressed. Moreover, adoptive transfer of spleen cells from the treated mice induced resistance against EAE induction in the recipients. However, no obvious evidence for antigen-specific suppressor cells was found in vitro in the L3T4- populations of spleen cells from treated mice.

Modulation of experimental allergic encephalomyelitis (EAE): suppression of active reinduction of EAE in rats recovered from passively transferred disease.

Lewis rats that have recovered from EAE induced by the passive transfer of in vitro activated lymphocytes sensitized to myelin basic protein showed suppression upon subsequent active reinduction of EAE. This suppression was manifested during the early convalescent stage (up to 30 days after the primary cell transfer) and seemed to be acquired partly idiotype-specifically and partly idiotype-nonspecifically. The convalescent rats were fully susceptible to the transfer of sufficient numbers of effector cells, and they could induce pre-effector cells in response to the encephalitogen in vivo as effectively as in naive rats. This suppression was not transferred to naive rats by lymphoid cells from the convalescent rats. The mechanism of this suppression was thought to be a defect in expansion and/or differentiation of pre-effector cells to effector cells in the convalescent rats.

T lymphocyte lines and clones selected against synthetic myelin basic protein 82-102 peptide from Japanese multiple sclerosis patients.

As has been indicated in experimental autoimmune encephalomyelitis (EAE), the application of synthetic peptides for the selection of T cell lines may provide new insights into the pathogenesis of multiple sclerosis (MS). We report here on T cell lines/clones generated from peripheral blood of MS patients against an immunodominant myelin basic protein (MBP) peptide 82-102. This study demonstrates that the selection of T cell lines against the MBP peptide is much more efficient than against whole MBP in generating a large panel of T cell lines/clones, and therefore provides a powerful strategy for studying autoimmune T cell repertoire in individual subjects. The peptide-selected lines and clones recognized MBP 82-102, shorter peptides MBP 89-101, 89-100 and guinea pig whole MBP mainly in the context of HLA-DR, but did not cross-recognize virus-derived peptides homologous to MBP 82-102. Seven out of ten clones were found to recognize MBP 82-102 in the absence of autologous antigen presenting cells (APC), and in three of the seven clones, specificity for MBP 82-102 could be demonstrated only in the absence of APC because of their strong reactivity against autologous APC. Two-color flow cytometry revealed that the clones were heterogeneous with regard to expression of CD4 and CD8 molecules. Overall, the clones selected by the peptide were rather heterogeneous in phenotype and function compared with those selected by whole MBP.

Ia restriction of murine encephalitogenic T-cell lines in vitro and in vivo.

To clarify the functional role of the I region-associated (Ia) antigen in autoimmune central nervous system disorders, we generated long-term cultured lines of encephalitogenic T cells responsive to myelin basic protein from SJL strain mice (H-2s) and investigated genetic restriction in proliferative and encephalitogenic activities of the lines. These cell lines bear a Lyt 1+,2- phenotype, and show antigen-specific and I-As restricted proliferative responses in vitro. These lines induced full-blown experimental allergic encephalomyelitis (EAE) in immuno-compromised recipients carrying the I-As genotype. These data demonstrate that encephalitogenic T lymphocytes recognize the antigen in combination with the Ia antigen to induce EAE.

Experimental allergic encephalomyelitis induced by proteolipid apoprotein in Lewis rats.

Experimental allergic encephalomyelitis (EAE) was induced in inbred Lewis rats by sensitization with bovine white matter proteolipid apoprotein (PLP). 18-61 days after a single injection of 100 micrograms of PLP, 12 of 31 rats (39%) developed clinical EAE and 18 of 23 (78%) showed pathologic EAE with significant demyelination. Lymphocyte proliferative responses and antibodies to PLP were elevated but did not correlate with the clinical or pathologic state. This is the first demonstration of PLP-induced EAE with significant demyelination in rats and will contribute to the study of autoimmune demyelination.

Mild encephalitogenic activity of basic protein--acidic lipid complex from myelin and detection of immune complexes in experimental allergic encephalomyelitis.

Further biochemical and pathological investigation of basic protein--acidic lipid complex from canine cerebral myelin show the presence of sulfatide, phosphatidylserine, ganglioside and basic protein in the molar ratio of approximately 6 : 3 : 1 : 1 and that it is associated with mild encephalitogenic activity in guinea pigs in comparison to intact myelin and basic protein. Circulating immune complexes were detected in the sera of guinea pigs with clinical signs of experimental allergic encephalomyelitis and immunofluorescent staining showed the deposition of immune complexes of immunoglobulin and complement in vessels of white matter and meninges and in the choroid plexus.

Heterogeneous induction of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes.

The expression of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes exposed to heat shock was investigated by double immunolabelling with anti-HSP72 monoclonal antibody (C92F3B-1) and antibodies against galactocerebroside (GalC) or glial fibrillary acidic protein (GFAP). After 3 h recovery from heat shock, an intermediate level of HSP72 immunolabelling was localized in the nucleolus and cytoplasm of astrocytes (less than 25%) and to a lesser extent in oligodendrocytes (less than 2%). After 8-48 h, HSP72 was expressed intensely in the cytoplasm and nuclear matrix of oligodendrocytes (20-40%), while weak/intermediate immunostaining was detectable in astrocytes (5-15%). The levels of HSP72 expressed in oligodendrocytes and astrocytes decreased around 72-120 h, but a few oligodendrocytes (4%) remained intensely immunolabelled. These results indicate that heat shock induces HSP72 in both oligodendrocytes and astrocytes. However, this response is heterogeneous.

Trophic effect of erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in vivo.

In vitro granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), and erythroid differentiation factor (EDF) augmented choline acetyltransferase (ChAT) activity in mouse embryonic primary septal neurons and in cholinergic hybridoma cell line, SN6.10.2.2. This is similar to the effects seen with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, in vivo GM-CSF and EPO promoted survival of septal cholinergic neurons in adult rats which had undergone fimbria-fornix transections. These results suggest that some of the hematopoietic factors act on cholinergic neurons as 'neurotrophic factors' to influence the differentiation, maintenance and regeneration of these neurons.

Stress induces neuronal death in the hippocampus of castrated rats.

Whereas loss of CA3 neurons in the hippocampus of monkeys which died of stress ulcers suggests that some structural changes may occur, there is no direct evidence that shows stress-induced irreversible changes of neurons. When rats were orchidectomized (castrated) and stressed by restraint and immersion in water for 15 min/day for 30 days, significant loss of hippocampal CA3 and CA4 neurons was observed. Furthermore, primary cultured hippocampal neurons survived shorter when treated with corticosterone. This neuronal loss was prevented by simultaneous administration of testosterone in vivo and in vitro. These findings indicate that stress can contribute to neuronal degeneration associated with hypogonadal conditions such as aging.

Production of interleukin-3 by murine central nervous system neurons.

We examined expression and production of interleukin-3 (IL-3) mRNA and IL-3 protein in mouse primary cultured neurons and glia by the reverse transcription and polymerase chain reaction and a bioassay using an IL-3-dependent cell line. IL-3 mRNA was demonstrated mainly in hippocampal neurons but not in glia, while a small but definite production of bioactive IL-3 was detected in septal and hippocampal neuronal cultures. Thus, endogenous IL-3 might be produced by certain neurons in situ.

Demonstration of amyloid beta-protein secretion in a mouse neuronal cell line.

We investigated the secretion of amyloid beta-protein (beta AP) in a mouse neuronal cell line SN49. SN49 cells stably transfected with mouse beta-amyloid precursor protein (APP) 695 cDNA released approximately three times greater amounts of a 4 kDa protein immunoreactive with anti-beta AP antibodies than untransfected and mock-transfected cells. This 4 kDa protein was further identified as mouse beta AP by direct amino acid sequence analysis. These results strongly suggest that the beta AP secretion occurs in mouse neuronal cells as in human cells.

Antibodies to proteolipid apoprotein in chronic relapsing experimental allergic encephalomyelitis.

Titers of serum antibodies to proteolipid apoprotein (PLP) were determined in chronic relapsing experimental allergic encephalomyelitis (EAE) of strain 13 guinea pigs sensitized with whole central nervous system tissue. Levels of the antibodies were slightly higher in the animals with relapse than those without relapse during the early chronic stage (days 40-99 postinoculation). The titers were significantly higher in the relapsed animals during the chronic stage (days 100-199). Although the clinical course was polyphasic, the humoral response to PLP was monophasic. Since PLP alone causes chronic EAE with widespread demyelination in guinea pigs (Yoshimura et al. 1985), the high titers of anti-PLP antibodies seem to have something to do with the immunologic mechanisms of chronic relapsing EAE.

No effect of anti-leprosy drugs in the prevention of Alzheimer's disease and beta-amyloid neurotoxicity.

There is continuing controversy as to whether or not anti-leprosy drugs prevent Alzheimer's disease (AD). Therefore, we examined the effect of anti-leprosy drugs on the prevalence of AD in leprosy patients, and also investigated the effect of anti-leprosy drugs on amyloid beta-protein (Abeta)-induced neurotoxicity in vitro. The present study suggests that anti-leprosy treatments do not prevent the onset of AD. None of our data found anti-leprosy drugs (dapsone, rifampicin, clofazimine, minomycin or ofloxacin) had any effect on Abeta neurotoxicity. It is now important to examine the infection of Mycobacterium leprae in the central nervous system to clarify the reason for the low prevalence of senile dementia, and low frequency of Abeta deposition in leprosy patients.

Immunohistochemical study of Alzheimer's disease using antibodies to synthetic amyloid and fibronectin.

Etiology and source of amyloid deposition in senile plaques of Alzheimer's disease (AD) are still unknown. In order to know whether or not fibronectin (Fn), an adhesive glycoprotein, is related to the amyloid deposition in the senile plaque, we conducted immunohistochemical studies using polyclonal anti-Fn and affinity-purified anti-amyloid component (Affi 28). Affi 28 was made by immunizing a rabbit against the synthetic peptide corresponding to residues 1-28 of the amyloid core protein reported by Masters et al. (1985). According to this study, four points became clear. First, Affi 28 is able to stain the subpial regions of AD as well as cerebrovascular amyloid and amyloid plaque cores. Second, it is suggested either that the etiology and source of neurofibrillary tangles and Pick body is distinct from that of the senile plaque or that any Affi 28 determinants of neurofibrillary tangles and Pick body are obscured sterically. Third, Affi 28 is useful to distinguish the senile plaque from the amyloid plaque of Creutzfeldt-Jakob disease. Last, there is no association between the amyloid in the senile plaque and Fn, at least immunohistochemically. The absence of Fn in the senile plaque suggests that Fn may not be requested for the deposition of amyloid fibrils.

Semple rabies vaccine: presence of myelin basic protein and proteolipid protein and its activity in experimental allergic encephalomyelitis.

Myelin basic protein (MBP) as a cause of postvaccinal encephalomyelitis (PVE) due to Semple rabies vaccine (SRV) has been suggested in previous reports. No actual measurement of MBP in SRV was done. In this study we detected MBP and PLP in the vaccine using immunological methods. The vaccine was found to contain 28 micrograms MBP per ml vaccine. Inoculation with SRV plus adjuvant resulted in the development of experimental allergic encephalomyelitis (EAE) in 2 of 3 guinea pigs. For control, chick embryo vaccine (CEV) was used and MBP was not detected. EAE was not induced in animals inoculated with it. These results suggest that MBP in vaccines may play a decisive role in the production of PVE.

Passive transfer of experimental allergic encephalomyelitis induced by proteolipid apoprotein.

In an attempt to obtain insight into the pathogenesis of proteolipid apoprotein (PLP)-induced experimental allergic encephalomyelitis (EAE) in Lewis rats (Yamamura et al. 1986), PLP-sensitized lymph node cells or spleen cells were passively transferred into normal or irradiated (400 rad) recipients after incubation with concanavalin A or PLP. Clinical EAE manifested by paraparesis was successfully transferred into irradiated recipients with 2 - 2.5 X 10(8) of the primary cultured cells and histologic EAE could be transferred with as few as 5 X 10(7) cells into naive recipients. This is the first demonstration of passive EAE induced with PLP-sensitized lymphoid cells and suggests the pathogenetic importance of cell-mediated immunity to PLP.

Chronic experimental allergic encephalomyelitis in guinea pigs induced by proteolipid protein.

A chronic experimental allergic encephalomyelitis (EAE) was produced in Hartley guinea pigs with bovine white matter proteolipid protein (PLP), in which the levels of myelin basic protein (MBP) and galactocerebroside (GC) were less than 0.014% and 0.13%, respectively, by our method of purification. Cells of an MBP-specific T-cell line did not proliferate in the presence of 100 micrograms of PLP and antigen-presenting cells. Eleven animals were sensitized with 250 micrograms of PLP in Freund's complete adjuvant. Three guinea pigs developed paraplegia about 45 days after sensitization. Histological examination of the three animals revealed marked demyelinating lesions in the spinal cord, particularly in the dorsal columns and subpial regions of the lateral and anterior columns. Another guinea pig without apparent clinical symptoms had demyelinating plaques in the dorsal columns of the spinal cord and periventricular white matter of the brain. Antibodies to PLP were highly elevated in the animals with demyelinating plaques but antibodies to MBP and GC were not elevated in the serum samples. Skin response to PLP was positive in sensitized animals, but was not related to the clinical state. Since none of four strain 13 guinea pigs developed chronic EAE, it seems to be strain specific. These results suggest that PLP is encephalitogenic and produces demyelination in the central nervous system without contamination by MBP or GC in Hartley guinea pigs.

Developmental and aging changes in the expression patterns of beta-amyloid in the brains of normal and Down syndrome cases.

Immunohistochemical staining with polyclonal antibodies to synthetic amyloid (residues 1-28 of A4) was performed on normal and Down syndrome brains from fetuses to adults. Positive staining appeared in the cytoplasmic processes of astrocytes in the subpial layer and white matter of developing brains, and reappeared in astrocytic fibers of the subpial layer as well as in cerebrovascular and plaque core amyloid in elderly brains. The reappearance of positively stained astrocytes and amyloid occurred earlier in adult Down syndrome patients. The results indicate that the A4 protein is a developmental protein, and its reappearance in Alzheimer and adult Down syndrome brains may be related to the regeneration process.