Ronald E. Majocha

Development of an anti-Aβ monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer's disease

We evaluated the efficacy of murine monoclonal antibodies (MAbs) targeted to the Aβ amyloid of Alzheimers disease for development of procedures for the in vivo identification of amyloid angiopathy (AA). MAbs to Aβ were prepared and screened for effectiveness in visualizing AA and neuritic plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with technetium-99m (99mTc) using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity toward amyloid-laden blood vessels and neuritic plaques. A highly specific murine MAb, 10H3, was identified and characterized that fulfills criteria necessary for the development of an in vivo diagnostic imaging agent. Toxicity studies in rats showed the MAb to be safe. Biodistribution studies in mice demonstrated desirable properties for use as an imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Monoclonal Antibody to Embryonic CNS Antigen A2B5 Provides Evidence for the Involvement of Membrane Components at Sites of Alzheimer Degeneration and Detects Sulfatides as Well as Gangliosides

Immunohistological and biochemical studies were initiated to determine whether or not neural membrane components were associated with degenerative changes characteristic of Alzheimers disease (AD). Monoclonal antibody A2B5, developed against embryonic chick retinal cells and previously shown to react with neural surface gangliosides, was applied to formalin‐fixed sections of control and AD brain tissue. Frontal cortex and hippocampus of AD cases exhibited high levels of A2B5 immunoreactivity within those neurons undergoing neurofibrillary degeneration. Neuritic processes associated with senile plaques were also highly reactive with the A2B5 antibody. The amount of gangliosides and their pattern after HPTLC were the same in control and AD cases. However, the unexpected observation was made that the A2B5 antibody reacted with human brain sulfatides in addition to the expected reactivity with minor gangliosides. The average level of sulfatides in AD brain was significantly higher than in normal controls. The data support the involvement of one or more membrane components with neu‐rodegeneration in the Alzheimer brain.

Axonal Polypeptides Cross-Reactive with Antibodies to Neurofilament Proteins

Abstract: Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross‐reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000 and 70,000 NFPs (200K, 140K and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase‐conjugated indirect antibody procedure, however, differences were detected in the cross‐reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and thus, was suitable for the detection of cross‐reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti‐200K, anti‐140K, or anti‐70K was applied to overloaded two‐dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross‐reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Tilers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a variety of conditions.

Molecular and cellular biology of Alzheimer amyloid.

Alzheimer’s Disease (AD), a disorder of unknown etiology, is the most common form of adult-onset dementia and is characterized by severe intellectual deterioration. The definitive diagnosis of AD is made by postmortem examination of the brain, which reveals large quantities of neurofibrillary tangles (NFT) and senile plaques within the parenchyma. The NFT are composed of paired helical filaments associated with several cytoskeletal proteins. The primary protein component of senile plaques is β/A4 amyloid, a 42–43 amino acid peptide derived from a much larger molecule, the amyloid precursor protein (APP). Vascular β/A4 amyloidosis is also prevalent in the disease. The mechanism by which β/A4 amyloid accumulates in the AD brain is unknown. Recent research has demonstrated that the precursor molecule, APP, is a transmembrane protein with a large extracytoplasmic domain, a membrane spanning region that includes the portion that gives rise to β/A4 amyloid, and a short intracytoplasmic domain. The precursor has multiple forms among which are those that differ by a variable length insert within the extracytoplasmic domain. The insert has sequence homology to the family of Kunitz protease inhibitor proteins. Cellular and animal models have been developed to study the nature of APP processing and the biological and behavioral consequences of β/A4 amyloidosis. The results of such studies indicate that the normal processing of APP involves enzymatic cleavage of the molecule within the β/A4 amyloid region, thus preventing the accumulation of β/A4 in the normal brain. The factors leading to abnormal processing of APP, and consequent β/A4 amyloid accumulation within the AD brain, have yet to be identified. In cell culture, the biological effects associated with β/A4 amyloid include neurotrophic and neurotoxic activities, while the peptide has also been shown to have dramatic behavioral effects in animal models.

Evidence for axonal loss in regions occupied by senile plaques in Alzheimer cortex.

The studies described have sought to determine what, if any, relationship exists between axons and the senile plaque, a hallmark histopathological feature of Alzheimers disease. A double stain was performed on both early and late Alzheimer frontal cortex tissues in order to examine the interaction between axons stained with antibodies against the 200,000 mol. wt neurofilament subunit (NFP-200) of the axon cytoskeleton and Thioflavin-S, a fluorescent dye that stains plaques. Serial photomicrographs of plaques were taken and axon and plaque profiles were three-dimensionally reconstructed. Analysis of computer-processed images revealed that there were fewer axons within plaques than in regions lying one and two plaque distances away. When axons were observed passing through plaques, swelling and disruption of normal morphology was frequently present. Statistical analyses of axon counts within and around placques showed a gradient of axon density, with increased numbers occurring at progressive distances from the placque. Similar patterns were seen for early and late stages of the disease. The results of this study indicate that disruption of the axonal cytoskeleton may occur within the regions occupied by plaques.

Chapter 19 Transcriptional and translational regulatory mechanisms during normal aging of the mammalian brain and in Alzheimer's disease

Publisher Summary Irrespective of the etiology of Alzheimers disease (AD), increasing evidence supports the hypothesis that basic defects at the level of transcription or translation, or both, may be involved in the pathogenesis of this illness. Decreased levels of protein synthesis can be demonstrated in living patients with AD and the AD post-mortem brain messenger RNA has diminished capacity to stimulate high levels of protein synthesis in vitro. Although numerous factors may contribute to the observed decline in in vivo functional activity, the concentration of the brain RNA appears to be particularly important for regulating the rate of translation. During normal aging, the level of the brain RNA decreases. In AD, there appears to be an accelerated general decline in neuronal RNA and a further decrease within neurons containing neurofibrillary tangles. RNA extracted from affected regions supports the limited synthesis of proteins. Although the overall levels of in vitro synthesis tend to be low, the specific activity of certain proteins, such as glial fibrillary acidic protein, remains high. These observations may be related, in part, to increased degradation of neuronal RNA with sparing of glial RNA. The level of AD brain alkaline ribonuclease activity is increased due to the loss of inhibitor activity.

The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA

Although the precise role of astrocytes in the pathogenesis of Alzheimers disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimers disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.

Evidence for a diffusional model of Alzheimer amyloid A4 (β-amyloid) deposition during neuritic plaque formation

A recent study reported that Alzheimer senile plaques immunostained with monoclonal antibodies against the A4 (beta-amyloid) region of the amyloid precursor protein show gradients of density (Majocha R. E., Benes F. M., Reifel R. L., Rodenrys A. M. and Marotta C. A., Proc. natn. Acad. Sci. U.S.A. 85, 6182-6186, 1988). Although more than one explanation was suggested for this observation, the possible involvement of a diffusional process during plaque maturation was considered. In order to examine this hypothesis, specimens from prefrontal cortex, entorhinal area and hippocampal formation were immunoprocessed in a similar fashion and subjected to quantitative microdensitometric analyses of A4 amyloid reaction product. All plaques in the three brain areas examined showed a curvilinear relationship between the area of amyloid reaction product (expressed in pixel counts) and optical density (expressed as each of six grey scale levels). There was an increase in the area of amyloid at progressively lower density levels. When the area of amyloid reaction product at each density level was correlated with the overall size of individual plaques, it was found that there was a striking increase in the correlation coefficients at progressively lower grey scale levels, with r = 0.853 at the lowest level examined. When a second order derivation of these correlations was performed by expressing individual r-values with respect to an optical density index, an asymptotic relationship resulted with the lowest density levels showing an increasingly sharp rise toward unity. These data are consistent overall with a model for plaque maturation that involves diffusion of amyloid protein through the extracellular space from focal regions of high density where synthesis and/or release may occur.

Conditioned media from the injured lower vertebrate CNS promote neurite outgrowth from mammalian brain neurons in vitro

Unlike most pathways of the mature mammalian central nervous system (CNS), the CNS of lower vertebrates can regenerate after jury, a capacity that may be due to the secretion of neurite-promoting factors from the injured CNS. We report that conditioned media (CM) from the injured optic nerve of the mature goldfish promoted marked neurite outgrowth from dissociated embryonic rat cortical and hindbrain neurons in serum-free, neuron-enriched culture. This property was not shared by CM from intact goldfish optic nerve, or from intact or injured optic nerve of mature rats. Neurite-promoting activity was obtained at concentrations as low as 100 ng total protein/ml of CM from injured goldfish optic nerve, and was associated with a distinctive morphology of neurite outgrowth. Due its properties of non-dialyzability, heat lability, and trypsin sensitivity, the neurite-promoting factor(s) appeared to be one or more protein species of MW greater than 12,000. Factors secreted by the regenerating CNS of lower vertebrates can directly promote outgrowth of mammalian CNS neurons.

Membrane surface ruffling in cells that over-express Alzheimer amyloid β/A4 C-terminal peptide

Deposition of beta/A4 amyloid in brain is a defining characteristic of Alzheimer disease (AD); however, the extent to which amyloid deposits may interfere with normal cellular processes is incompletely understood. We examined this issue by means of PC12 cells. After transfection with DNA coding for 97 amino acids of the beta/A4 C-terminal region of the amyloid precursor protein, beta/A4 antigen was visible at the cell membrane. We report that normal unstimulated PC12 cells exhibit ruffling activity at the cell surface when plated on a plastic substrate. Relative to control cells, however, those that over-expressed the beta/A4 C-terminal peptide had significantly higher levels of ruffling activity, suggesting a structural and/or functional membrane modification. Similar cellular alterations, if present, in Alzheimer brain cells, may indicate disturbances in membrane-associated functions, including intercellular communication.