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C A Marotta

Publications and source records attributed to C A Marotta.

At least 37 records · Page 2Linked to original sources

Overexpression of amyloid precursor protein A4 (beta-amyloid) immunoreactivity in genetically transformed cells: implications for a cellular model of Alzheimer amyloidosis.

Among the major obstacles to clarifying molecular mechanisms involved in amyloid metabolism in Alzheimer disease has been the unavailability of laboratory models for this uniquely human disorder. The present studies were aimed at establishing genetically engineered cell lines that overexpress amyloid immunoreactivity and that may be relevant to amyloid accumulation in the Alzheimer disease brain. We used cloned amyloid cDNA that contains a region encoding A4 (beta-polypeptide) amino acids along with recently developed tumor virus vectors derived from simian virus 40 to prepare transformed cells. After transient and permanent transfection, a variety of cell types overexpressed A4 immunoreactivity that was detected by highly specific monoclonal antibodies. We observed that the use of an amyloid subdomain containing the A4 region, but lacking the sequence of a Kunitz-type protease inhibitor found in amyloid precursor protein variants, was sufficient to obtain cells that overproduced an A4 epitope. The transformed cells were readily propagated in culture and may provide an experimental medium to elucidate aspects of the molecular pathogenesis of Alzheimer disease. The cellular models may also serve as tools for deriving potentially useful therapeutic agents.

Alzheimer Disease↗

Identification of cellular and extracellular sites of amyloid precursor protein extracytoplasmic domain in normal and Alzheimer disease brains.

Information concerning the distribution of various subdomains of the amyloid precursor protein (APP) in brain may illuminate aspects of the normal metabolism of this membrane-associated protein, as well as putative abnormal processing that may occur in Alzheimer disease (AD). We prepared affinity-purified antibody, P2, against an extracytoplasmic APP site and applied it, along with monoclonal antibodies to the beta-peptide, or A4 region, in conjunction with selective cytochemical staining methods, to control and AD tissues. The following was noted: (i) in contrast to A4 epitopes, which are easily demonstrable primarily in extracellular senile plaques of AD patients, the extracytoplasmic P2 antigen was found in association with neurons, glia, and blood vessels in both normal and AD prefrontal cortex; (ii) a subset of senile plaques contained both A4 and P2 antigens; (iii) in some instances, P2 antigen occurred as an extracellular deposit in the absence of A4; (iv) the P2 antigen, but not A4, was also associated with corpora amylacea. In addition to identifying the unique cellular distribution of the APP extracytoplasmic antigen, the results support the view that a segment of this domain undergoes processing and deposition at extracellular sites, including a subset of senile plaques.

Alzheimer Disease↗

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 Alzheimer's 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 neurodegeneration in the Alzheimer brain.

Alzheimer Disease↗

Molecular biology in psychiatric research: Alzheimer's disease as a paradigm.

As illustration of the integration of molecular biological studies into psychiatric research, use of molecular techniques in the study of Alzheimer's disease is described, and the resultant observation of otherwise undetectable pathological changes is noted. Using postmortem Alzheimer brain messenger RNA, recombinant DNA studies led to the ability to clone and characterize the expressed genetic transcript for amyloid. Application of this methodology is discussed in terms of establishing cellular and animal models for a neuropsychiatric disease.

Alzheimer Disease↗

Soluble, phosphorylated forms of the high molecular weight neurofilament protein in perikarya of cultured neuronal cells.

The high molecular weight subunit of neurofilaments (NF-H) in mouse NB2a/d1 neuroblastoma cells is extensively phosphorylated and exhibits an apparent molecular weight of 200 kDa by SDS gel electrophoresis. In this study, we observed that extensively phosphorylated NF-H variants exist as both Triton-soluble and -insoluble forms, which display different cellular distributions. Perikarya and neurites of differentiated NB2a/d1 cells were immunostained by a polyclonal antiserum (anti-NF-H) that specifically recognizes the extensively phosphorylated NF-H forms and a monoclonal antibody (SMI-31) that recognizes phosphorylated epitopes of neurofilament proteins (NFPs). When cells were extracted with Triton X-100 to remove soluble proteins, however, only axonal neurites remained immunoreactive. Immunoblot analyses established the specificity of anti-NF-H and SMI-31 and demonstrated that both Triton-soluble and -insoluble NF-H subunits exhibit an apparent molecular weight of 200 kDa. Incorporation of radiolabeled phosphate into Triton-soluble NF-H following incubation of intact NB2a/d1 cells with 32P-orthophosphate confirmed that the Triton-soluble form of NF-H is a phosphoprotein. Most NF-H subunits in the Triton-soluble fraction sedimented after centrifugation at 100,000 g for 1 h, indicating that they may be present as oligomers. The implications of these data for the development of neurofibrillary pathology are discussed.

Animals↗

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 Alzheimer's 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 Alzheimer's 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.

Alzheimer Disease↗

Laminar-specific distribution and infrastructural detail of amyloid in the Alzheimer disease cortex visualized by computer-enhanced imaging of epitopes recognized by monoclonal antibodies.

Monoclonal antibodies to the A4 amyloid polypeptide were used in immunocytochemical staining of the Alzheimer disease prefrontal cortex. Analysis of the resulting staining patterns allowed us to evaluate the amounts and distribution of amyloid-protein deposits exclusive of other senile-plaque components. Previously unappreciated infra-structural details of amyloid in the Alzheimer disease brain became accessible through computer-enhanced imaging procedures. Four discrete morphologic classes of amyloid deposits were observed and classified as punctate, macular, ring, and ring-with-core configurations. Computer imaging indicated that all four classes of immunostained deposits contain internal gradients of density. The classes were nonuniformly distributed with regard to size and location within cortical laminae. Our results support two separate but complementary hypotheses concerning the molecular neuropathology of Alzheimer disease in the prefrontal cortex. (i) Irrespective of cortical layer or morphology, density-gradient analyses suggest that amyloid deposits are elaborated through molecular and cellular events that may involve diffusion or coalescence of the A4 polypeptide. (ii) The distribution and morphology of prefrontal cortical amyloid deposits may be dependent upon underlying laminar-specific structures of the neocortex.

Alzheimer Disease↗

Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex.

To gain insight into factors associated with the excessive accumulation of beta-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, we cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)+ RNA from AD cortices was used for the preparation of lambda gt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the beta-amyloid domain and corresponded to 75% of the coding region and approximately equal to 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, we conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

Aged↗

A modular arrangement of neuronal processes in human cortex: disruption with aging and in Alzheimer's disease.

Studies were undertaken to assess whether or not neuron-specific immunostaining of the human brain can reveal unique cytoarchitectural features that may be affected by healthy aging and Alzheimer's disease (AD). Human prefrontal cortex (PFC) and anterior cingulate cortex (ACC) were stained with an antibody raised against the neurofilament protein 200,000 molecular weight subunit (NFP-200) using an avidin-biotin immunolocalization procedure. Immunostaining of cortex was neuron-specific and highlighted axons in particular. This staining has revealed an orderly arrangement of horizontal and vertical axon bundles which form latticelike compartments or modules throughout most of the matrix of the two cortical areas studied. The ACC shows this pattern to be intact in individuals through the ninth decade, while the PFC there was blurring of the modularity beyond the fifth decade. Irrespective of the age-related blurring of the latticelike arrangement of axons in PFC, neurologically normal elderly individuals nevertheless showed a highly organized appearance to the cortical matrix. By contrast, patients with AD showed a marked disruption of the modular arrangement of fibers in PFC, but not ACC. Differences between PFC fibers in controls and AD patients were also demonstrated with an axon-specific monoclonal antibody that reacted with phosphorylated epitopes of the NFP-200. The chaotic appearance of fiber staining in PFC seen in patients with AD was noted to be present in one subject who died in an early stage of the disease. The possible significance of this previously unknown aspect of cortical cytoarchitecture for normal cognitive functioning in humans is discussed.

Adolescent↗

Increased vertical axon numbers in cingulate cortex of schizophrenics.

Data generated from an earlier study have suggested a model in which greater numbers of long, vertical, associative axons may occur in the anterior cingulate cortex of schizophrenic patients relative to control subjects. This hypothesis has now been tested using neuron-specific antibodies raised against the 200-kilodalton neurofilament subunit, a component of neuronal cytoskeleton, to immunostain axons of human postmortem cingulate cortex. A manual method for counting axons in the region of layer II and sublamina IIIA has been designed and applied blindly to parallel control and schizophrenic immunoprocessed specimens. The results show that there are 25% more vertical axons in the schizophrenic than in the control specimens. Preferentially higher numbers of both long vertical axons (62%) and axons associated with blood vessels (52%) have also been noted in the schizophrenic specimens. By contrast, the number of large-caliber horizontal axons was the same in the two groups; therefore, the greater number of vertical axons in schizophrenic specimens does not appear to represent a nonspecific effect. When these data are corrected for the effects of several confounding variables using analysis of covariance, the overall pattern of the results persists. These findings suggest the possibility that there might be an increase of associative inputs into the anterior cingulate cortex of schizophrenic patients, although it is not clear at present whether the differences noted, if replicative, may be primarily or perhaps only secondarily related to the disorder.

Axons↗

Posttranslational modification of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons.

The progressive modification of newly synthesized neurofilament proteins (NFPs) during axoplasmic transport in mouse retinal ganglion cell (RGC) neurons was studied after RGC perikarya were pulse-labeled with 32P-orthophosphate or radiolabeled amino acids. The 3 NFP subunits, H(igh), M(iddle), and L(ow), were among a group of axonally transported proteins that incorporated high levels of 32P. Covalent addition of phosphate slowed the electrophoretic mobility of H and M on SDS polyacrylamide gels and shifted the charge of all 3 subunits toward more acidic pH values, thereby providing an index of the phosphorylation state of this radiolabeled population of NFPs. NFPs were extensively phosphorylated before they entered axons at the optic nerve level, and continued to be modified during transport along RGC axons at the optic nerve and tract level. H and M exhibited charge shifts of 0.2-0.6 units toward a more acidic pH during axoplasmic transport. The charge modifications became more prominent when NFPs reached distal axonal levels, which may indicate regional differences in the activity of this modification process along axons. By contrast, the L subunit became more basic in charge, consistent with decreases in the phosphorylation state during transport. Additional observations (Nixon and Lewis, 1986) that a considerable proportion of phosphate groups initially added to L and M were later removed as neurofilaments advanced along RGC axons support the notion that the changing phosphorylation state of NFP subunits during axoplasmic transport reflects a dynamic equilibrium between phosphorylation and dephosphorylation events. Topographical remodeling of phosphate groups on NFPs during axoplasmic transport is proposed as a possible mechanism for coordinating interactions between neurofilaments and other constituents, as these elements are transported and integrated into the axonal cytoskeleton.

Animals↗

Immunostaining of neurofilament protein in human postmortem cortex: a sensitive and specific approach to the pattern analysis of human cortical cytoarchitecture.

Antibodies raised against the 200 000 neurofilament protein (NFP) of rat brain have been successfully applied to the staining of human postmortem cortex using a modified immunoperoxidase procedure. Human anterior cingulate cortex which has been in formaldehyde fixative for as long as 4 years shows extensive and reliable staining of axons and, to a lesser degree, apical dendrites of cortical neurons. The immunostaining of 200 000 NFP in human cortex has revealed cytoarchitectural details not generally visible with other more conventional neuroanatomical stains, particularly when counterstaining with cresyl violet is employed. Potential applications of this approach to the pattern analysis of human cortical cytoarchitecture in both health and disease are discussed.

Animals↗

Alzheimer's disease brain: alterations in RNA levels and in a ribonuclease-inhibitor complex.

A macromolecular alteration occurs at the posttranscriptional level in the Alzheimer's disease (AD) brain. Compared with age-matched controls, total cellular RNA and polyadenylated RNA were substantially reduced in the AD cortex with many neuritic plaques and neurofibrillary tangles. RNA changes are associated with a significant increase in alkaline ribonuclease activity due to an abnormality in the ribonuclease-inhibitor complex. The decrease in protein synthesis in the AD brain, previously observed in patients severely affected with AD, and in translation systems in vitro with AD cortical messenger RNA, may be partly related to an enzyme-inhibitor alteration that affects RNA levels and activity. Decreased protein synthesis therefore may contribute to the characteristic decline in certain neurotransmitter enzymes and to the loss of neurons in the AD brain.

Aged↗

Degradation of neurofilament proteins by purified human brain cathepsin D.

Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were two- to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP break-down products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFPs by CD comigrated on two-dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskeletal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.

Brain↗

Limited proteolytic modification of a neurofilament protein involves a proteinase activated by endogenous levels of calcium.

Posttranslational modification of a structural protein by limited proteolysis is demonstrated for the first time in the nervous system. The 145,000 dalton subunit of neurofilaments in mouse retinal ganglion cell (RGC) axons is selectively converted in vitro to the major 143,000 and 140,000 dalton neurofilament subunits by a neutral proteinase that is activated by endogenous levels of calcium and is distinguishable from other known brain proteinases. The close similarities between this in vitro process and the previously observed modification of the 145,000 dalton neurofilament protein during axoplasmic transport in vivo suggest that the same enzymatic mechanism is involved. These findings imply that limited proteolysis is an active process along central axons in vivo and that this enzyme may play a specific role in the function of the neuronal cytoskeleton.

Animals↗