Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Rigor crossbridges are double-headed in fast muscle from crayfish.

The structure of rigor crossbridges was examined by comparing rigor crossbridges in fast muscle fibers from glycerol-extracted abdominal flexor muscle of crayfish with those in "natively decorated" thin filaments from the same muscle. Natively decorated thin filaments were obtained by dissociating the backbone of the myosin filaments of rigor myofibrils in 0.6 M KCl. Intact fibers were freeze-fractured, deep-etched, and rotary shadowed; isolated filaments were either negatively stained or freeze dried and rotary shadowed. The crossbridges on the natively decorated actin maintain the original spacing and the disposition in chevrons and double chevrons for several hours, indicating that no rearrangement of the actomyosin interactions occurs. Thus the crossbridges of the natively decorated filaments were formed within the geometrical constraints of the intact myofibril. The majority of crossbridges in the intact muscle have a triangular shape indicative of double-headed crossbridge. The triangular shape is maintained in the isolated filaments and negative staining resolves two heads in a single crossbridge. In the isolated filaments, crossbridges are attached at uniform acute angles. Unlike those in insect flight muscle (Taylor et al., 1984), lead and rear elements of the double chevron may be both double-headed. Deep-etched images reveal a twisted arrangement of subfilaments in the backbone of the thick filament.

Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters.

We have shown previously that chick muscle cells transformed with Rous sarcoma virus are unable to form clusters of acetylcholine receptors (AChRs) (Anthony, D. T., S. M. Schuetze, and L. L. Rubin. 1984. Proc. Natl. Acad. Sci. USA. 81:2265-2269) and are missing a 37-KD tropomyosin-like protein (TM-2) (Anthony, D. T., R. J. Jacobs-Cohen, G. Marazzi, and L. L. Rubin. 1988. J. Cell Biol. 106:1713-1721). In an attempt to clarify the role of TM-2 in the formation and/or maintenance of AChR clusters, we have microinjected a monoclonal antibody specific for TM-2 (D3-16) into normal chick muscle cells in culture. D3-16 injection blocks the formation of new clusters but does not affect the preexisting ones. In addition, TM-2 is concentrated at rat neuromuscular junctions. These data suggest that TM-2 may play an important role in promoting the formation of AChR clusters.

Functional features of EVI1 and EVI1Δ324 isoforms of MECOM gene in genome-wide transcription regulation and oncogenicity.

The MDS1 and ecotropic viral integration site 1 (EVI1) complex locus (MECOM) gene encodes several transcription factor variants including MDS1-EVI1, EVI1 and EVI1Δ324. Although MDS1-EVI1 has been associated with tumor-suppressing activity, EVI1 is a known oncogene in various cancers, whose expression is associated with poor patient survival. Although EVI1Δ324 is co-transcribed with EVI1, its activity in cancer cells is not fully understood. Previous reports described that unlike EVI1, EVI1Δ324 protein cannot transform fibroblasts because of its disrupted N-terminal zinc finger (ZNF) domain. To better understand EVI1Δ324 biology and function, we obtained genome-wide binding occupancies and expression data in ovarian cancer cells. We characterized its DNA-binding sites, binding motif and target genes. Comparative analyses with previous study show that EVI1 and EVI1Δ324 share similar transcriptional activities linked to their common C-terminus ZNF domain. They bind to an E-twenty-six family (ETS)-like motif, target to a large extent the same genes and cooperate with AP1 transcription factor. EVI1Δ324-occupied genes were 70.7% similar to EVI1-bound genes. More strikingly, EVI1 and EVI1Δ324 differentially expressed genes were 99.87% identical, indicating comparable transcriptional regulatory functions. Consistently with gene ontologies linked to these target genes, EVI1Δ324 expression in HeLa cells could enhance anchorage-independent growth, such as EVI1, showing that EVI1Δ324 expression also lead to pro-oncogenic effects. The main specific feature of EVI1 variant is its N-terminus ZNF domain that binds DNA through GATA-like motif. We found that most GATA-like EVI1 chromatin immunoprecipitation sequencing peaks are far from genes and are not involved in transcriptional regulation. These genomic regions were enriched in simple sequence repeats and displayed high meiotic recombination rates. Overall, our genomics analyses uncovered common and specific features of two major MECOM isoforms. Their influence on transcription and downstream cell proliferation was comparable. However, EVI1-specific GATA-like binding sites, from its N-terminus ZNF domain, associated with high recombination rates, suggesting possible additional oncogenic potential for EVI1 in modulating genomic stability.

Early detection of cryptic memory and glucose uptake deficits in pre-pathological APP mice.

Earlier diagnosis and treatment of Alzheimer's disease would greatly benefit from the identification of biomarkers at the prodromal stage. Using a prominent animal model of aspects of the disease, we here show using clinically relevant methodologies that very young, pre-pathological PDAPP mice, which overexpress mutant human amyloid precursor protein in the brain, exhibit two cryptic deficits that are normally undetected using standard methods of assessment. Despite learning a spatial memory task normally and displaying normal brain glucose uptake, they display faster forgetting after a long delay following performance to a criterion, together with a strong impairment of brain glucose uptake at the time of attempted memory retrieval. Preliminary observations suggest that these deficits, likely caused by an impairment in systems consolidation, could be rescued by immunotherapy with an anti-ß-amyloid antibody. Our data suggest a biomarker strategy for the early detection of ß-amyloid-related abnormalities.

Prevention and reduction of AD-type pathology in PDAPP mice immunized with A beta 1-42.

In AD certain brain structures contain a pathological density of A beta protein deposited into plaques. The effect of genetic mutations found in early onset AD patients was an overproduction of A beta 42, strongly suggesting that overproduction of A beta 42 is associated with AD. We hypothesized that an immunological response to A beta 42 might alter its turnover and metabolism. Young PDAPP transgenic mice were immunized with A beta 1-42, which essentially prevented amyloid deposition; astrocytosis was dramatically reduced and there was reduction in A beta-induced inflammatory response as well. A beta 1-42 immunization also appeared to arrest the progression of amyloidosis in older PDAPP mice. A beta immunization appears to increase clearance of amyloid plaques, and may therefore be a novel and effective approach for the treatment of AD.

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.

Abeta vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease.

The authors report a patient with Alzheimer disease (AD) without encephalitis who was immunized with AN-1792 (an adjuvanted formulation of Abeta-42). There were no amyloid plaques in the frontal cortex and abundant Abeta-immunoreactive macrophages, but tangles and amyloid angiopathy were present. The white matter appeared normal and minimal lymphocytic infiltration in the leptomeninges was observed. This case illustrates the effects of an Abeta-based immunization on AD pathogenesis in the absence of overt meningoencephalitis and leukoencephalopathy.

Evidence showing that the nucleolus-envelope region is located on the nor-bearing chromosomes in Aotus trivirgatus.

We investigated whether the existence of the Nucleolus-Envelope Region observed in dividing animal cells is related to the Nucleolus Organizer Region or to other chromosome segments of the NOR-bearing chromosomes, since NORs are usually adjacent to the chromosome segments attached to the nuclear envelope such as the centromere, telomere or heterochromatin region. We used Aotus Trivirgatus fibroblasts whose karyotype is characterized by a single pair of NORs located on the long arm of the third pair of chromosomes, far from the centromere, the telomere and any obvious heterochromatin region. All the nucleoli were seen to be clearly associated with the nuclear envelope but separated from it by the outermost layer of chromatin. These results further support the hypothesis that the NOR is a site of attachment of the chromatin to the nuclear envelope by means of the Nucleolus-Envelope Region. They show that genetically active chromatin is also attached to the nuclear envelope. A model of the arrangement of chromatin during interphase is proposed which provides a functional interpretation of the currently available data.

Rotation of the cell nucleus in living cells: a quantitative analysis.

Nuclear rotation is observed in a variety of cell types. However, few quantitative analyses are reported and the significance of this phenomenon is still unclear. To investigate this type of nuclear movement, we performed a quantitative analysis in mouse L-929 fibroblasts, a cell line chosen since it displays a high nuclear rotational activity. Analyses were performed using time-lapse microcinematography. The relationship between nuclear rotation and other cellular phenomena such as the cell cycle and locomotion were studied. Then, we investigated the rotation in a population of sister cells to study whether it is genetically determined. Finally, we performed a qualitative analysis of nuclear rotation in different cultured cell lines. Results show that nuclear rotations preferentially occur during the phases of the cell cycle which surround mitosis.

In situ arrangement of nor-bearing chromosomes in the interphase nucleus of Aotus trivirgatus.

The spatial organization of the two nucleolus-organizing region (NOR)-bearing chromosomes during interphase was studied in Aotus trivirgatus fibroblasts, using nucleoli as ultrastructural markers. Their distribution was examined by measuring the distances between them in 30 reconstructed nuclei, and comparing these experimental values with the theoretical ones obtained by simulation. Results were as follows: (1) the nucleoli are arranged in a polarized manner inside the nucleus; (2) the nucleoli are tightly bound to the nuclear envelope at two opposites sites; (3) the distance between the two nucleoli is variable, and is shorter than it would be if the two nucleoli were distributed at random. These findings indicate that the NOR-bearing chromosomes are fixed at the nuclear envelope in two opposite areas. They are also consistent with the hypothesis that each chromosome occupies a separate domain inside the nucleus. They can be interpreted according to the model in which chromosome arrangement within the interphase nucleus is based on the separation of the diploid complement into two independent haploid sets.

Analysis of collagen synthesis and assembly in culture by immortalized mouse chondrocytes in the presence or absence of alpha 1(IX) collagen chains.

We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. The cell line obtained (termed MC 615) also expressed some type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit type I collagen synthesis and promote hypertrophy and type X collagen synthesis. The MC 615 cells were grown in agarose in the presence of serum, and GAG accumulation, DNA content, and matrix synthesis rates were monitored after incubation with [35S]sulfate and [3H]- or [14C]proline. SDS-PAGE analysis of pepsin-extracted samples showed that type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density. Type II collagen synthesis was demonstrated by immunoblotting, but type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in proteoglycans and in type II-containing collagen fibrils. To gain insight into the role of type IX collagen in cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX) collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized type II collagen as demonstrated by Western blotting and some type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of pepsin-digested collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in collagen structure and organization. In the absence of alpha 1 (IX) collagen chains, the collagen fibrils seemed to fuse laterally, suggesting that collagen IX acts as a "spacer" between fibrils, to keep them apart.