Earl R. Shelton

Degeneration of vascular muscle cells in cerebral amyloid angiopathy of Alzheimer disease

In cerebral amyloid angiopathy, the amyloid-beta (A beta) deposits lie primarily in the tunica media suggesting that smooth muscle cells play an important role in A beta deposition. To define this role, we conducted an immunocytochemical study of brain tissue from cases of Alzheimer disease with extensive cerebral amyloid angiopathy and cerebral hemorrhage. Antibodies specific to recombinant beta protein precursor (beta PP) and synthetic peptides homologous to various beta PP sequences from residue 18 to 689 of beta PP695 were used. Antibodies to actin, tropomyosin, alpha-actinin or desmin were used to label muscle cells. Antibodies to A beta sequences intensely recognized the extracellular amyloid deposit. Antibodies raised against beta PP sequences other than the A beta domain recognized smooth muscle cells. beta PP-immunoreactivity was reduced in regions of A beta deposits, since no muscle cells were recognized by cytoskeletal markers or observed ultrastructurally. In order to assess why A beta is deposited in the tunica media, we used biotin-labelled beta PP to determine if beta PP can be locally retained. We found beta PP bound to the tunica media of vessels but not other brain elements. These findings suggest A beta in blood vessels derives from degenerating beta PP-containing smooth muscle cells.

Accumulation of the β amyloid precursor protein at sites of ischemic injury in rat brain

We used various antibodies to the beta amyloid precursor protein (APP) of Alzheimers disease to study changes in the cellular distribution of APP in experimental ischemic brain injury. In contrast to sham operated controls, rats with repeated reversible occlusions of one middle cerebral artery showed striking APP reactivity in astrocytic processes in perifocal regions and white matter tracts. Dystrophic axons and neurons with accumulated APP were also evident in the ipsilateral neocortex and hippocampus. Such changes were also apparent in rats subjected to partial forebrain ischemia by bilateral occlusion of the carotid arteries. Our studies suggest that focal ischemic insults or chronic hypoperfusion leads to increased accumulation of APP in surviving brain cells that may pertain to enhanced beta amyloid deposition in Alzheimers disease.

The cDNA of a human lymphocyte cyclic-AMP phosphodiesterase (PDE IV) reveals a multigene family

Five protein families are needed to encompass the diversity of cyclic-AMP (cAMP) phosphodiesterases (PDE). Family IV PDEs (PDE IV) specifically hydrolyze cAMP with a low Km, and are selectively inhibited by rolipram (Rp) and related drugs. Cloned cDNAs from rat (r) suggest that the PDE IV family comprises four distinct members, designated A, B, C and D. Using RN from a human lymphocytic B-cell line (43D-Cl2), we have isolated a 3.8-kb cDNA by low-stringency screening using a rat PDE IV member B (r-PDE IVB) probe. Expression of the human (h) cDNA in Escherichia coli results in cAMP-specific PDE activity that is Rp sensitive. A single large open reading frame (ORF) predicts a 564-amino-acid protein with 92.9% identity to r-PDE IVB; at the nucleotide level the identity is 86.3%. This h-PDE IVB clone, HPB106, differs from a related cDNA clone isolated by others from h-monocytes [Livi et al., Mol. Cell. Biol. 10 (1990) 2678-2686]. Our analysis identifies the monocyte clone with r-PDE IVA. Southern blots using a 1.2-kb h-PDE IVB probe at low stringency suggest the presence of additional uncloned human PDE IV family members. Analysis of genomic Southern blots using short specific probes from the h-PDE IVA and h-PDE IVB cDNAs indicates that distinct genes encode these two PDE IV family members. RNA from fractionated normal human leukocytes shows major specific messages of 3.0 and 4.6 kb for h-PDE IVA and 3.7 kb for h-PDE IVB.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble derivatives of the β amyloid protein precursor of Alzheimer's disease are labeled by antisera to the β amyloid protein

Abstract The amyloid deposited in Alzheimers disease (AD) is composed primarily of a 39–42 residue polypeptide (βAP) that is derived from a larger β amyloid protein precursor (βAPP). In previous studies, we and others identified full-length, membrane-associated forms of the βAPP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble ∼125 and ∼105 kDa forms of the βAPP found in human cerebrospinal fluid are specifically labeled by several different antisera to the βAP. This finding indicates that both soluble derivatives contain all or part of the βAP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.

Accumulation of amyloid precursor fragment in Alzheimer plaques

Regenerative and degenerative neurites are components of classical senile plaques found in brain tissue of patients with Alzheimers disease (AD). Amyloid beta/A4-protein derived from its precursor, amyloid beta/A4-protein precursor (APP/ABPP), constitutes the major portion of the amyloid core of senile plaques. A large N-terminal portion of APP (approximately Mr 100,000) is released from cells, leaving a minor C-terminal portion (approximately Mr 15,000) behind. A series of antisera against various sequences of APP were prepared and used to study the localization of each sequence in brain tissue. Plaque neurites stained as intensely as neuronal cell bodies with three antisera against the N-terminal portion of APP (N-terminal to a.a. 225), whereas five other antisera directed against the other C-terminal portions of APP (a.a. 284 to C-terminal) and antisera against the Kunitz-type protease inhibitor portion of APP stained plaque neurites less intensely than neuronal cell bodies in the hippocampus. These results suggest that a major part of the APP present in the neuritic component of senile plaques is a fragment representing the N-terminal one-third of the molecule.

Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IVD)

cDNAs encoding human family-IV phosphodiesterase, subtype D (hPDE IVD) were isolated from a human heart cDNA library. The overlapping cDNAs encode a polypeptide of 604 amino acids (aa) with a predicted M(r) of 68,502, which is 91.4% identical to the rat homolog, rPDE IVD. hPDE IVD produced in Escherichia coli was inhibited by rolipram. Expression of the hPDE IVD mRNA is widespread in human tissues and most abundant in skeletal muscle.

Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors.

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the Km for substrate and the EC50 for activation by the cofactor Mg2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11-and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A.

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study.

Increasing recongnition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimers disease (AD) were examined immunihistochemically with antibodies to damin-specific sites of APP (N-terminal, C-terminal, ßA4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with ßA4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labelling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed onlly rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to ßA4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled onlyl cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.

Processing of Alzheimer's disease-associated beta-amyloid precursor protein.

Studies were undertaken on the processing of Alzheimers disease-associated β-amyloid precursor protein in normal cultured human fibroblasts and a human neuroblastoma cell line. Major differences in processing between the secreted and intracellular forms of the precursor were found. The intracellular form appears to undergo amino-terminal processing yielding many smaller fragments, whereas the secreted from does not show any further proteolytic cleavage after its release from the cell surface. In pulse-chase experiments, antibodies to the A4 region immunoprecipitated bands of Mr=92,000−128,000, which represent the intact precursor; several smaller intracellular fragments of Mr=70,000–72,000, 55,000, 33,000 and 6,000 also immunoprecipitated with this antibody. The Mr=6,000 band cleared from the cell very quickly and is postulated to be the A4-carring remnant of the secreted protein. The data show that a fragment of Mr=33,000, which includes the A4 region, is one stable processed end-product of the intracellular precursor protein. It is possible that different posttranslational modifications are the signals responsible for the differences in processing between the secreted and intracellular amyloid precursor protein.