Sui-Po Zhang

Antisense oligodeoxynucleotide inhibits D2 dopamine receptor-mediated behavior and D2 messenger RNA.

There are several subtypes of dopamine receptors in the central nervous system which mediate the actions of dopamine in producing its diverse motor and behavioral effects. In this study we determined whether an antisense oligodeoxynucleotide directed to the mRNA encoding one of the subtypes of the dopamine receptor can inhibit a specific dopamine-mediated behavior. Accordingly, the effects of a phosphorothioate-modified antisense oligodeoxynucleotide targeted toward the D2 dopamine receptor mRNA (D2 antisense) was studied in mice with unilateral 6-hydroxydopamine-induced lesions of the corpus striatum. Rotational behavior in response to different agents, and the levels of D2 and D1 dopamine receptors and D2 and D1 dopamine receptor mRNAs in corpus striatum were then measured. In control mice, lesioning resulted in a contralateral rotational behavior in response to the D1 dopamine receptor agonist SKF 38393, the D2 dopamine agonist quinpirole, and the muscarinic cholinergic agonist oxotremorine. Lesioning also caused an increase in D2 dopamine receptor mRNA levels in the dorsolateral striatum. Intraventricular injections of the D2 antisense inhibited rotational behavior induced by quinpirole but not that induced by SKF 38393 or that induced by oxotremorine. Repeated administration of the D2 antisense significantly reduced the levels of the D2 dopamine receptor and D2 dopamine receptor mRNA in the dorsolateral but not the dorsomedial striatum. Similar treatment failed to significantly alter the levels of the D1 dopamine receptor or D1 receptor mRNA in dorsolateral or dorsomedial striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense strategies in neurobiology

The use of antisense oligodeoxynucleotides, targeted to the transcripts encoding biologically active proteins in the nervous system, provides a novel and highly selective means to further our understanding of the function of these proteins. Recent studies of these agents also suggest the possibility of their being used therapeutically for a variety of diseases involving neuronal tissue. In this paper we review studies showing the in vitro and in vivo effects of antisense oligodeoxynucleotides as they relate to neurobiological functions. Particular attention is paid to the behavioral and biochemical effects of antisense oligodeoxynucleotides directed to the various subtypes of receptors for the neurotransmitter dopamine. An example is also provided showing the effects of a plasmid vector expressing an antisense RNA targeted to the calmodulin mRNAs in the PC12 pheochromocytoma cell line. The advantages of antisense oligodeoxynucleotides over traditional pharmacological treatments are assessed, and the advantages of using vectors encoding antisense RNA over the use of antisense oligodeoxynucleotides are also considered. We also describe the criteria that should be used in designing antisense oligodeoxynucleotides and several controls that should be employed to assure their specificity of action.

Dopaminergic and glutamatergic blocking drugs differentially regulate glutamic acid decarboxylase mRNA in mouse brain

Dopaminergic and glutamatergic inputs play an important role in regulating the activity of GABAergic neurons in basal ganglia. To understand more fully the biochemical interactions between these neurotransmitter systems, the effects of blocking dopamine and glutamate (N-methyl-D-aspartate) (NMDA) receptors on the expression of glutamic acid decarboxylase (GAD) mRNA were examined. Persistent blockade of dopamine receptors was achieved by daily injections of EEDQ, a relatively non-selective irreversible D1 and D2 dopamine receptor antagonist, or FNM, a relatively selective irreversible D2 dopamine receptor antagonist. Persistent blockade of NMDA receptors was achieved by continuously infusing dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. The levels of GAD mRNA in mouse brain were measured by in situ hybridization histochemistry following treatment with these agents. Repeated administration of EEDQ increased the levels of GAD mRNA in corpus striatum and frontal and parietal cortex; the first significant effects were seen after 4 days of treatment. Treatment with FNM elicited effects similar to those produced by EEDQ, except FNM also significantly increased GAD mRNA in nucleus accumbens. Neither EEDQ nor FNM produced significant effects on GAD mRNA in olfactory tubercle or septum. Infusion of MK-801 produced a rapid and marked decrease in the levels of GAD mRNA in corpus striatum, nucleus accumbens, olfactory tubercle, septum and frontal and parietal cortex; significant changes were seen as early as 2 days of treatment. No significant effects were seen in globus pallidus. Cellular analysis of emulsion autoradiograms from corpus striatum revealed that MK-801 reduced the amount of GAD mRNA in individual cells as well as the proportion of cells expressing high levels of GAD mRNA. These results suggest that dopamine, though its interaction with D2 dopamine receptors, exerts an inhibitory effect on the expression of GAD mRNA, and that glutamate, though its interaction with NMDA receptors, exerts a stimulatory effect on GAD mRNA expression. They show further that the regulation of gene expression by dopamine receptors or NMDA receptors is different in different regions of the brain.

Developmental and age-related changes in the D2 dopamine receptor mRNA subtypes in rat brain

The influence of ontogeny and aging on the D2 dopamine receptor mRNA in rat brain were examined using in situ hybridization histochemistry and Northern analysis utilizing oligonucleotide probes complementary to the different D2 mRNA subtypes. At birth, there was a high level of D2 dopamine receptor mRNA in corpus striatum relative to that found in the cerebral cortex and other brain areas. The hybridization signal of striatum (using a probe that hybridizes to both the D2A and D2B mRNA) increased during the first two postnatal weeks, reached a peak at day 16, then declined slightly. The D2A mRNA showed a similar distribution and developmental pattern. Intracisternal injection of 6-hydroxydopamine into neonates did not significantly alter the increase of the D2 dopamine receptor mRNAs, suggesting that neuronal input does not influence the ontogenetic development of this mRNA. In striatum, olfactory tubercule and inferior colliculus, the D2A mRNA declined between 3 and 24 months of age. By contrast, there was an age-related increase in the D2A mRNA in the anterior and intermediate lobes of the pituitary. The mRNA for the D2B dopamine receptor showed very low but nevertheless detectable levels in striatum, olfactory tubercule and pituitary. Like with the D2A mRNA, in 24-month-old rats the D2B mRNA declined in striatum and olfactory tubercule and increased in pituitary. These results show that there are differential tissue-related changes in the mRNAs for the D2 dopamine receptor during both development and aging.

Antisense strategies in dopamine receptor pharmacology

Recent advances in molecular biology have provided pharmacologists the opportunity of developing an entirely new type of agent for studying and treating a variety of biological disorders. These agents, termed antisense oligodeoxynucleotides, have as their target the messenger RNAs encoding specific proteins. They act by binding to selected portions of these mRNAs through complimentary interactions and thereby prevent the synthesis of these proteins. These novel pharmacological tools have the promise of being easier to design and being more selective and predictable in their actions. In addition, insofar as agents targeted to receptors for neurotransmitters are concerned, unlike the classical pharmacological agents, these new compounds may not lead to the upregulation of the very receptors the drugs are designed to inhibit. The present review summarizes briefly studies on the effect of oligodeoxynucleotides antisense to the mRNAs encoding the various subtypes of the dopamine receptor. The studies show that oligodeoxynucleotides antisense to the D2 dopamine receptor when intracerebroventricularly into brains of rodents are rapidly taken up into the brain tissue, distributed to brain cells, and produce effects characteristic of highly selective D2 dopamine antagonists. The compounds also produced specific reductions in the levels of D2 dopamine receptor mRNA and D2 dopamine receptors. Similarly, injecting an antisense oligodeoxynucleotide targeted to the D1 dopamine receptor mRNA produces effects characteristic of D1 dopamine receptor antagonists. Other studies using these agents has produced evidence that there is a small pool of receptors that turn over very rapidly and which constitute the functional pool of these receptors. The evidence suggests further that antisense oligodeoxynucleotides inhibit the synthesis of this small functional pool of dopamine receptors, thereby providing an explanation of why there is often a discordance between changes in dopaminergic function and changes in the levels of dopamine receptors. Studies of antisense oligodeoxynucleotides targeted to the other subtypes of dopamine receptor may help reveal the biological roles that these and other newly discovered subtypes of neurotransmitter receptors have. They may also provide an entirely new and potentially more selective therapeutic regimen for altering the functions of these receptors.

Reduced level of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth

The role calmodulin plays in the growth and differentiation of nerve cells was assessed by altering the levels of calmodulin in the PC12 rat pheochromocytoma cell line and determining the effects of altering these levels on cellular proliferation and differentiation. Calmodulin levels in the PC12 cells were increased or decreased by transfecting the cells with a mammalian expression vector into which the rat calmodulin gene I had been cloned in the sense or antisense orientation, respectively. The cells transfected with the calmodulin sense gene showed increased levels of calmodulin immunoreactivity and increased levels of calmodulin messenger RNA as ascertained by immunocytochemistry and slot-blot analysis, respectively. Cells transfected with the calmodulin antisense construct showed reduced levels of calmodulin immunoreactivity. Reducing the levels of calmodulin by expression of antisense calmodulin messenger RNA resulted in a marked inhibition of cell growth, whereas increasing the levels of calmodulin by overexpressing calmodulin messenger RNA resulted in an acceleration of cell growth. Transfected PC12 cells having reduced levels of calmodulin immunoreactivity exhibited spontaneous outgrowth of long, stable and highly branched neuritic processes. PC12 cells in which calmodulin was overexpressed showed no apparent changes in cell morphology, but did show an altered response to the addition of nerve growth factor. While nerve growth factor slowed cellular proliferation and induced extensive neurite outgrowth, in parental PC12 cells nerve growth factor induced little or no neurite outgrowth and little inhibition of cell proliferation in transfected cells overexpressing calmodulin. These results indicate that calmodulin is essential for the proliferation of nerve cells and for the morphological changes that nerve cells undergo during differentiation. The study also suggests the possibility that a calmodulin antisense approach may be used to inhibit the proliferation of neuronal tumors.

Uptake and distribution of fluorescein-labeled D2 dopamine receptor antisense oligodeoxynucleotide in mouse brain.

To determine the uptake and distribution of oligodeoxynucleotides in brain, a 20-mer phosphorothioated oligodeoxynucleotide complementary to a portion of the D2 dopamine receptor mRNA was fluorescently labeled with fluorescein isothiocyanate (FITC) and injected into the lateral cerebral ventricles of mice. At various survival times after the injection, the brains were removed, fixed, sectioned, and viewed under a fluorescent microscope. The results showed that the oligodeoxynucleotide was rapidly taken up into the brain. Initially the label was relatively diffusely spread throughout the interstitial spaces of the brain, then became redistributed to the cellular compartments. The signal extended from those forebrain nuclei located immediately in contact with the ventricles, such as the corpus striatum, septum, and hippocampus, to areas further removed from the ventricles, such as the cerebral cortex, nucleus accumbens, and substantia nigra. When the FITC-labeled D2 antisense oligodeoxynucleotide was given once daily for 4 d, the signal intensity seen 24 h after the last injection appeared to be of greater intensity overall compared to that seen after a single injection. At early time-points the oligodeoxynucleotide signals appeared to be punctuated and were found in cell bodies as well as in proximal dendritic processes. However, not all cells were equally labeled, suggesting an uneven uptake and accumulation of the D2 antisense into the various cell types. At later time-points the fluorescent signal appeared granular; at these times the injected material was largely degraded. These studies show that a D2 dopamine receptor antisense oligodeoxynucleotide is rapidly taken up from cerebral ventricles into brain, becomes widely distributed throughout the brain tissue to areas far removed from direct contact with the ventricles, and appears to accumulate to a different extent in the various brain areas and cell types.

Cholinergic lesions of mouse striatum induced by AF64A alter D2 dopaminergic behavior and reduce D2 dopamine receptors and D2 dopamine receptor mRNA

To determine whether dopamine receptors are expressed in acetylcholine-containing neurons intrinsic to the striatum, and to study further the interactions between the dopaminergic and cholinergic systems, the irreversibly acting cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A), was injected unilaterally into the mouse corpus striatum, and rotational behavior induced by dopamine agonists and certain molecular events associated with this lesion were determined 7 days after lesioning. Brains were analyzed for D2 dopamine receptors by autoradiography, using [3H](-)sulpiride as a ligand, and for D2 dopamine receptor mRNA and glutamic acid decarboxylase mRNA by Northern blot analysis, using selective radiolabelled oligonucleotide probes. Choline uptake sites were determined by binding assays using [3H]hemicholinium-3, a selective choline reuptake blocker, as a ligand. Mice with intrastriatal injections of AF64A showed ipsilateral rotational responses to the non-selective dopamine agonist apomorphine and to the D2 dopamine agonists, pergolide and quinpirole, but not to the D1 dopamine agonist SKF 38393. This was associated with a significant reduction in D2 dopamine receptors in the ipsilateral striatum and a significant decrease in the amount of D2 dopamine receptor mRNA. That AF64A produced a relatively selective cholinergic deficit was supported by the evidence showing that AF64A lesions significantly reduced [3H]hemicholinium-3 binding sites but did not alter glutamic acid decarboxylase (GAD) mRNA. Further, hemicholinium-3, prevented the AF64A-induced changes in rotational behavior. These results suggest that striatal cholinergic interneurons contain D2 dopamine receptors and express the D2 dopamine receptor gene, and that these interneurons are involved in dopamine-mediated rotational behavior.

Localization of the multiple calmodulin messenger RNAs in differentiated PC12 cells.

Calmodulin, a ubiquitous calcium-binding protein which is involved in many biological processes, including cell proliferation and differentiation, has been shown to be encoded by three genes from which five calmodulin messenger RNAs are transcribed. In our previous studies, using the PC12 pheochromocytoma cell line as a model system for neuronal differentiation, all five calmodulin messenger RNAs were found to be present, and treatment with both nerve growth factor and dibutyryl cyclic AMP, which induce neurite outgrowth in these cells, increased the level of calmodulin and differentially increased the levels of the various calmodulin messenger RNAs. In an attempt to uncover the nature of the differential increase in the calmodulin messenger RNAs during neuronal differentiation, we examined here the subcellular distribution of the individual calmodulin messenger RNAs in PC12 cells treated with nerve growth factor and dibutyryl cyclic AMP by in situ hybridization cytochemistry, using radiolabeled oligodeoxynucleotide probes. Using an oligodeoxynucleotide probe which detects all of the calmodulin transcripts, the calmodulin messenger RNAs were found to be distributed throughout the cell bodies of differentiated PC12 cells; significant amounts of calmodulin messenger RNAs were also found in most neurites (approximately 70% of the total number). Using specific probes for the calmodulin messenger RNAs derived from each calmodulin gene, distinct patterns of localization of the different calmodulin messenger RNAs were revealed. The messenger RNAs from calmodulin genes I and II were readily detected in all cell bodies and in about one-half of the neurites. In contrast, a weak signal for the messenger RNAs from calmodulin gene III was associated with cell bodies, while no significant signal was found in neurites. A population distribution analysis of the labeling of individual PC12 cell bodies, as determined by counting autoradiographic grains, revealed differences in the relative abundance of each group of messenger RNAs derived from each of the three calmodulin genes. The order of relative abundance of the messenger RNAs in cell bodies was found to be: calmodulin gene II messenger RNA > calmodulin gene I messenger RNAs >> calmodulin gene III messenger RNAs. An analysis of the labeling density along neurites indicated a similar density of neuritic messenger RNAs from calmodulin gene I and calmodulin gene II, whereas there was no significant signal for the messenger RNAs from calmodulin gene III.(ABSTRACT TRUNCATED AT 400 WORDS)

INTRASTRIATAL ADMINISTRATION OF AN OLIGODEOXYNUCLEOTIDE ANTISENSE TO THE D2 DOPAMINE RECEPTOR mRNA INHIBITS D2 DOPAMINE RECEPTOR-MEDIATED BEHAVIOR AND D2 DOPAMINE RECEPTORS IN NORMAL MICE AND IN MICE LESIONED WITH 6-HYDROXYDOPAMINE

Previous studies have shown that the intracerebroventricular injection of antisense oligodeoxynucleotides targeted to the mRNAs encoding the different subtypes of dopamine receptors inhibited behaviors mediated by these receptors. The present studies were designed to determine whether such antisense oligodeoxynucleotides could produce similar effects when injected into a discrete brain area. A D2 dopamine receptor antisense oligodeoxynucleotide (D2 antisense) was repeatedly injected into one corpus striatum of either normal mice or mice with unilateral lesions of the striatum induced by 6-hydroxydopamine. In the latter, intrastriatal injection of D2 antisense blocked the contralateral rotational behavior induced by the parenteral administration of the D2 dopamine receptor agonist quinpirole. The inhibitory effect of D2 antisense was dose- and time-related and was reversed upon cessation of D2 antisense treatment. This inhibitory effect was also selective in that D2 antisense treatment inhibited the rotational behavior induced by quinpirole but not that induced by the D1 dopamine receptor agonist SKF 38393 or by the muscarinic cholinergic agonist oxotremorine. Following repeated intrastriatal injections of D2 antisense into normal mice, parenteral administration of quinpirole caused rotational behavior ipsilateral to the side in which the D2 antisense was injected. No such rotational behavior was seen when similarly treated mice were challenged with SKF 38393 or oxotremorine. The quinpirole-induced rotational behavior in mice given intrastriatal injections of D2 antisense disappeared upon cessation of D2 antisense treatment. Repeated intrastriatal administration of D2 antisense also caused a significant reduction in the levels of D2, but not D1, dopamine receptors in striatum, as determined by receptor autoradiography. The levels of D2 dopamine receptors returned to normal upon cessation of D2 antisense treatment. Intrastriatal administration of an oligodeoxynucleotide with randomly placed nucleotides failed to alter the rotational response to quinpirole in either 6-hydroxydopamine-lesioned or normal mice and failed to alter the levels of D2 dopamine receptors in striatum. These results show that selective inhibition of behavioral responses mediated by D2 dopamine receptors can be achieved by the direct injection of a D2 antisense oligodeoxynucleotide into a discrete brain area.