Structural features of the 5' upstream regulatory region of the gene encoding rat amyloid precursor protein.

The 5' upstream regulatory region of the gene encoding the rat amyloid precursor protein (APP) was cloned and sequenced. It lacks both a TATA box and a CAAT box, has a high G + C content (68%), is 89% homologous to the corresponding region of the mouse APP gene, and 82% homologous to the corresponding region of the human APP gene. This region contains putative regulatory elements both 5' and 3' to the probable transcription start point (tsp). There are consensus DNA sites for the binding of SP1, AP2, AP4 and GC factor (GCF) proteins, and two GC boxes with the consensus sequence, 5'-GGGYGCRG. Potential regulatory sites with only a single mismatch to the consensus sequences include three SP1, one AP1, five AP2, and two GCF sites, as well as one GC box. There are also six potential stem-loop secondary structures (SSS) near the probable tsp. A consecutive series of elements, consisting of a GC box, AP2 site, three SSS, two SP1 sites, and AP4, AP1 and GCF sites just upstream from the probable tsp, are well-conserved between the rat, mouse and human sequences. An additional AP2 site, two GC boxes, and two additional SSS appear to be conserved between species. However, two possible rat SP1 sites, three possible rat AP2 sites, and two possible rat GCF sites are lacking in the human. On the other hand, the rat sequence is missing four potential SP1 sites, four potential AP2 sites, and nine potential GC boxes which are found in the human sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of gene therapy to treat age-related loss of dopamine D2 receptor.

We have investigated the feasibility of using gene therapy to attenuate the age-related decline in striatal dopamine D2 receptors (D2R) associated with reduced motor control. To this end, we have constructed an adenoviral vector containing the cDNA for the rat D2R. When injected into HeLa and HS24 cells in vitro, the vector induced an abundant message for D2R, as demonstrated by Northern analysis, and produced a membrane-bound protein capable of binding a D2R ligand, [3H]spiperone. When injected into rat striatum in vivo, the vector produced a marked increase in D2R near the site of injection, as evidenced by increased [3H]spiperone binding as well as by another more specific ligand, [125I]iodosulpride. The D2R produced in the striatum were functional, as evidenced by rotational behavior induced by a subcutaneous injection of the dopamine agonist, apomorphine. However, we did not observe any significant improvement in motor performance during preliminary experiments in which aged rats received bilateral striatal injections of the vector. In young rats, vector-induced expression of D2R in striatum was increased markedly three to five days after infection, but then declined to baseline levels by day 21. Loss of expression in aged rats proceeded at a somewhat lower rate. Because of the loss of expression and lack of significant performance enhancement in aged rats following vector injection into the striatum, we are now pursuing other strategies. These include functional assessment of the current vector in D2R null mutant mice as well as construction of new vectors that may yield more long-term expression.

Interaction of nuclear factors from young and old rat brain regions with regulatory sequences of the D2 dopamine receptor gene promoter.

Alterations in the number or functional state of D2 dopamine receptors have been implicated in the decreased motor abilities associated with normal aging, Parkinson's disease and other neurodegenerative diseases. Previous work has demonstrated a substantial decrease in D2 receptor-containing neurons, receptor proteins, steady-state mRNA levels, and the rate of mRNA synthesis with age in the rat striatum in particular and in mammalian brains in general. These observations suggest that one key area of regulatory control is at the level of transcriptional initiation and/or elongation. In the present study gel mobility shift experiments were used to assess the interaction of nuclear proteins from different rat brain regions with DNA containing putative DNA regulatory sites of the transcriptionally active rat D2 receptor gene promoter. Oligonucleotides containing either of the two SP1 binding sites immediately upstream of the primary transcriptional start site were bound by proteins found in nuclear extracts obtained from rat striatum, hippocampus, cortex, and cerebellum. Extracts from striatum and hippocampus formed predominantly low molecular weight complexes which do not contain SP1, as well as a small amount of high molecular weight complexes which may contain SP1 or an SP1-related protein. Cerebellar extracts formed two similar sets of complexes, but they were formed in roughly equal amounts. Extracts from cortex produced a more involved pattern of complexes, but still formed both high molecular weight complexes which contain SP1 and low molecular weight complexes which do not contain SP1. There were differences in the gel mobility as well as the relative amounts of complexes formed with the two SP1-specific oligonucleotides among different brain regions. With respect to possible age-related changes in transcription of the D2 dopamine receptor gene, there appeared to be no statistically significant difference in the DNA-protein complexes formed with striatal nuclear proteins from a population of young rats versus a population of old rats.

Adenovirus-mediated gene transfer of dopamine D2 receptor cDNA into rat striatum.

A robust feature of mammalian aging associated with diminished motor control is the loss of dopamine D2 receptors from the neostriatum. Decline in this neurotransmitter receptor is also observed in neurodegenerative disorders, such as Huntington's disease and late-stage Parkinson's disease. We have constructed a replication-deficient adenoviral vector to transfer rat dopamine D2 receptor cDNA to brain as a possible therapeutic strategy. Using tissue culture cells infected with this vector, we detected dopamine D2 receptor mRNA by Northern analysis and functional receptor protein in membrane preparations as specific binding of the dopamine D2 receptor ligand, [3H]spiperone. In vivo demonstration involved autoradiographic analysis of [3H]spiperone binding in rat striatum following injection of the adenoviral vector. Dopamine D2 receptor expression was amplified markedly above normal concentrations in the injection site, whereas no increased expression was observed in sites receiving control treatments. These results demonstrate the potential of gene therapy using adenoviral vectors to transfer neurotransmitter receptor proteins to the brain to reverse deficiencies in specific neurodegenerative disorders.

Rotational behavior produced by adenovirus-mediated gene transfer of dopamine D2 receptor into rat striatum.

We investigated the expression and functionality of a previously developed adenoviral vector carrying the rat cDNA for the dopamine D2 receptor (D2R), AdCMV.DopD2R. Comparative analysis of the autoradiographic images from the striatum injected with AdCMV.DopD2R and the contralateral striatum injected with a control vector, AdCMV.Null, in male rats indicated that D2R binding was increased by 40-60% on days 3 and 5 after injection, but then declined to baseline levels by day 21. When injected with apomorphine on days 3 and 7 after vector injection, experimental groups that had received unilateral striatal injections of AdCMV.DopD2R exhibited a distinct and significant laterality in rotational behavior. These results provide the first demonstration of an adenovirally mediated, intracerebral delivery of a functional neurotransmitter receptor.

The rat amyloid precursor protein promoter contains two DNA regulatory elements which influence high level gene expression.

We have investigated the transcriptional activity and regulatory elements of the rat amyloid precursor protein promoter. A DNA fragment containing 375 base pairs upstream of the start codon drives transcription in rat PC12 cells at a level greater than five-fold that of the SV40 promoter. This fragment contains a predominant transcription start point and several additional start points which are similar to those found in the human promoter. The strong promoter activity appears to be dependent upon two small DNA regulatory elements. Deletion of one element at positions -260 through -248 reduces activity by 85%. This is the first report of a positive regulatory element at this location. Deletion of a second element at positions -223 through -192 reduces activity by 30%. Gel mobility shift assays with nuclear extracts from whole rat brain suggest that nuclear proteins interact directly with the second element but not with the first one.

Use of synthetic ribosome binding site for overproduction of the 5B protein of insertion sequence IS5.

Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Although there was no detectable expression from the 5B open reading frame when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. beta-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.

DNA binding and regulatory effects of transcription factors SP1 and USF at the rat amyloid precursor protein gene promoter.

Two DNA elements which we have termed SAA and GAG have been shown to control expression of the rat amyloid precursor protein (APP) gene, and the region containing the SAA element has been shown to interact with nuclear proteins [Hoffman and Chernak (1994) Biochem. Biophys. Res. Commun. 201, 610-617]. In this report we study DNA sequences and proteins which influence the activity of the SAA element. An oligonucleotide containing the SAA element is specifically bound by nuclear proteins derived from rat PC12 cells, consistently forming four complexes designated C25, C30, C35 and C40 in electrophoretic mobility shift assays (EMSAs). We demonstrate that the C25, C30 and C40 complexes involve the binding of nuclear proteins to an SP1 consensus sequence located within the SAA element and that the C25 complex contains a protein antigenically related to the human SP1 protein. We establish further that the C35 complex requires a USF recognition site located within the SAA element and contains a protein antigenically related to the human upstream stimulatory factor (USF) protein. Using APP promoter/luciferase reporter gene constructs, we demonstrate that both the SP1 and the USF sites can play a role in the transcriptional activity of the SAA element. Finally, we show that complexes similar to the C25, C30 and C35 complexes are formed by rat cortex nuclear extracts and the SAA element in EMSA experiments, suggesting the relevance of our in vitro observations to the in vivo functioning of the rat APP promoter.

Synthesis and overproduction of the 5A protein of insertion sequence IS5.

We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence IS5. Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA. When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.