Facilitated reduction of beta-amyloid peptide precursor by synthetic oligonucleotides in COS-7 cells expressing a hammerhead ribozyme.

Synthetic deoxyoligonucleotides and phosphorothioate-capped oligonucleotides targeted to bases 112-128 of beta-amyloid peptide precursor (beta APP) mRNA were analyzed for their ability to reduce steady-state beta APP in COS-7 cells and in pMEP4-Rz1 cells that express a hammerhead ribozyme targeted to bases beta APP mRNA 133-148. Cells, incubated in the presence of 10 or 25 microM oligonucleotide, remained viable and morphologically identical to untreated control cells for up to 5 days. Antisense deoxyoligonucleotides beta 112C, beta 114C, and beta 116C specifically lowered beta APP in pMEP4-Rz1 cells compared to noncognate and scrambled oligonucleotide controls. The extent of the beta APP reduction did not depend on oligonucleotide length, although it did depend on the presence and proximity of the ribozyme to the oligonucleotides. beta 117N, a phosphorothioate-capped antisense oligonucleotide, also reduced beta APP levels in pMEP4-Rz1 cells; however, in this case the sense control, beta 117S, affected beta APP similarly, indicating that the observed reduction may be nonspecific. These data imply that deoxyoligonucleotides targeted immediately upstream of a ribozyme binding site can work cooperatively in vivo. Localizing the oligonucleotides and ribozyme and substrate targets to the same cellular pools further confirmed this possibility.

Effects of cadmium, copper, and zinc and beta APP processing and turnover in COS-7 and PC12 cells. Relationship to Alzheimer disease pathology.

The effects of cadmium, copper, and zinc on beta APP metabolism were investigated in COS-7 and PC12 cells. Cadmium chloride (CdCl2) increased beta APP steady-state protein levels and decreased beta APP posttranslational processing. These changes were not accompanied by alterations in beta APP mRNA levels or splicing. In addition, cytosolic alpha-actin and G3PDH levels were not affected. Further, neither zinc (ZnCl2) nor copper (CuSO4) altered beta APP levels or affected its normal processing. Pulse-chase studies revealed that the rate of beta APP maturation decreased twofold in the presence of 25 microM CdCl2 compared to untreated controls. beta APP secretion from the cell also dramatically slowed. These two factors result in the accumulation of partially processed beta APP inside cells. The presence of CdCl2 also decreased the amount of an 8-kDa beta APP C-terminal fragment, indicating that the cellular compartment in which beta APP accumulates is not accessible to alpha-secretase. Studies using Brefeldin A suggest that this compartment may be the cis or medial Golgi. However, A beta production was proportionately increased. These data show that CdCl2 can modulate the beta APP cleavage to favor A beta. Finally, beta APP mis- metabolism was shown to be unrelated to the hsp70 induction elicited by CdCl2; both heat shock and CuSO4 induced hsp70 but had no effect on steady-state levels of beta APP, although heat shock did slow beta APP maturation. These data indicate that hsp70 alone cannot chaperone beta APP through an alternate processing pathway leading to A beta production.

Reduction of histone cytotoxicity by the Alzheimer beta-amyloid peptide precursor.

In a search for Alzheimer beta-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little beta-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD50 = 1.5 microM). Extracellular A beta precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD50 = 5.4 microM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD50 = 3.2 microM) than cells that expressed the vector alone. Histone H4 binds to cell-associated A beta precursor. Cells expressing the A beta precursor-directed ribozyme bound less 125I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface A beta precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein.

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.

Suppression of Alzheimer amyloid precursor protein (APP) expression by exogenous APP mRNA.

To establish a cell line expressing enhanced levels of beta-amyloid precursor protein (beta-APP), we constructed plasmid DNAs expressing beta-APP-751 mRNA and transfected them into COS-1 cells. Using a modified version of the reverse transcriptase polymerase chain reaction which is RNA sensitive to study the beta-APP iso-RNAs, we have made the unexpected observation that enhanced expression of beta-APP-751 mRNA resulted in a significant reduction of beta-APP-770 and -695 mRNA levels. Suppression of beta-APP-770 and -695 was also observed in cells expressing truncated and chimeric beta-App-751 mRNAs. Similar observations were made in P19 cells expressing a chimeric beta-APP-751 mRNA where endogenous beta-APP-751 mRNA levels also were decreased. Also, suppression of beta-APP-770 and -751 mRNAs was observed in human kidney cells expressing exogenous beta-APP-695 mRNA.

Differential activity of trans-acting hammerhead ribozymes targeted to beta amyloid peptide precursor mRNA by altering the symmetry of helices I and III.

In order to determine whether distributing the pairing bases in helices I and III of a hammerhead ribozyme asymmetrically would enhance the cleavage of trans-acting ribozymes designed to degrade cellular mRNAs, we measured the cleavage properties of symmetric and asymmetric ribozymes targeted to the amyloid peptide precursor (beta APP) mRNA. Five ribozymes were formed from three beta APP synthetic mRNA analogs and two ribozyme RNA core sequences. Symmetric ribozymes beta 133/Rz133 and beta 125/Rz133 contained 8 bp in helix I and 7 bp in helix III. Asymmetric ribozyme beta 125/Rz125 had 13 bp in helix I and 4 bp in helix III, asymmetric ribozyme beta 123/Rz125 had 11 bp in helix I and 4 bp in helix III, while asymmetric ribozyme beta 133/Rz125 contained 8 bp in helix I and 4 bp in helix III. The ability of each ribozyme to cleave its substrate RNA was first assessed under single-turnover conditions at 37 degrees C. These studies revealed that only symmetric ribozyme, beta 133/Rz133, and asymmetric ribozyme beta 123/Rz125 effectively cleaved their substrates. Further studies using a 80 degrees C, 1-min-->37 degrees C, 1-min temperature cycling paradigm were performed to increase the cleavage efficiency of the ribozymes. Under these conditions ribozymes beta 133/Rz133, beta 125/Rz125, and beta 123/Rz125 were kinetically well behaved. Therefore, the fact that the symmetric ribozyme beta 133/Rz133 was more active than its asymmetric counterparts indicates that symmetrically distributing the pairing bases in helices I and III around this cleavage site is preferred.

Ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.

Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and trans-acting hairpin ribozymes were constructed. In one set of vectors ribozyme RNA transcription was placed under the control of a mouse mammary tumor virus long terminal repeat (MMTV-LTR). In the other set ribozyme expression was controlled by a metallothionein IIA (Mt-IIA) promoter. Each ribozyme was directed to the first target sequence in the Alzheimer amyloid peptide precursor mRNA (beta APP mRNA), 5' decreases GUC decreases 3'. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of beta APP mRNA as well as beta APP pre-mRNA, was shown to cleave a beta APP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of beta APP mRNA were reduced 25-30% in cells containing either active or mutant hammerhead ribozyme vectors driven by the MMTV-LTR promoter grown in the presence of glucocorticoids. In cell lines bearing Mt-IIA driven ribozymes steady-state levels of beta APP mRNA were reduced 67-80% in both hammerhead and hairpin ribozyme containing cell lines following promoter induction by glucocorticoids. These levels correlate with the appearance of low levels of induced ribozyme RNA. In contrast, steady-state alpha-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of beta APP were correspondingly reduced. Neither the RNA nor protein decreases observed in ribozyme transfected cell lines were observed in stably transfected control cells bearing the vector alone. These results suggest that ribozyme-mediated degradation of beta APP mRNA in COS-7 cells does not depend on ribozyme cleavage.

A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus.

The presynaptic terminal and axon of neurons can undergo structural changes in response to environmental signals. Since these changes require protein synthesis in the cell body, the needs of the periphery must somehow be communicated to the cell soma. To look for such a mechanism, we used artificial protein constructs with properties expected of a signal that is transported from the axon to the nucleus. One construct consisted of the nuclear import signal peptide (sp) of the SV40 large T antigen, coupled to human serum albumin (HSA) and rhodamine (r). When injected into the axoplasm of Aplysia californica neurons in vitro, the rHSA-sp was transported in the retrograde direction through the axon to the cell body and then into the nucleus. Little, if any, moved in the anterograde direction toward growth cones. The retrograde movement of injected rHSA-sp was rapid (greater than 25 mm/d) and depended upon intact microtubules. The sp portion of rHSA-sp provided access to both the retrograde transport system and the nuclear import apparatus. Thus, rHSA was not transported at all, but accumulated in organelles near the injection site. Also, rHSA-sp containing an sp with a Lys to Thr substitution, which is known to reduce nuclear import markedly, was transported only poorly. To look for endogenous molecules that use this system, we affinity-purified a rabbit polyclonal antibody to the signal sequence. The antibody recognized an 83 kDa polypeptide on Western blots of Aplysia nervous tissue. These data indicate that Aplysia neurons contain the machinery to convey macromolecules from the axon periphery to the nucleus.

Cytochrome oxidase activity as a measure of synaptogenesis by multifunctional neuron L7 of Aplysia.

Neuron L7 of the marine mollusc, Aplysia californica, is unique in that it innervates five different target tissues in the animal. We show that when L7 is grown in vitro with two of these targets, that is, muscle cells isolated from the auricle or the gill vein, newly formed L7 neurites contact the muscle cells. Chemical synapses are formed since intracellular stimulation of L7 elicits contraction of individual muscle cells. Interestingly, auricle muscles are also innervated by neuron RBhe and co-cultures of RBhe and auricle muscle cells also exhibit synapse formation. To explore the molecular basis for synaptogenesis between L7 and its targets, it would be useful to quantify the extent of synapse formation in vitro, that is, to determine how many muscle cells can be innervated by a single L7. We show that this can be attained by staining for cytochrome oxidase activity. Cultures of auricle and gill vein muscles were exposed to the appropriate neurotransmitter in order to elicit contraction. The cells were then fixed and stained. In both cases, only cells that contracted were stained and electron microscopy showed reaction product associated with the cristae of mitochondria. When this procedure was applied to cultures of L7 and muscle cells, 38 +/- 2.8% (S.E.M.; n = 7) of the cells on the neurites were stained and therefore responded to L7 stimulation. Thus, part of the L7-RBhe circuit can be assembled in vitro and the extent of synaptogenesis can be accurately quantitated.