Genetically Engineered Neural Transmission JH Son and TH Joh

PROGRESS Genetically engineered neural transmission JH Son and TH Joh

Laboratory of Molecular Neurobiology, Cornell University Medical College at The WM Burke Medical Research Institute, White Plains, NY 10605, USA

Neural transmission is a communication between neurons and target cells, resulting in behavioral and physiological changes. Defective or altered neural transmission is thought to occur in neuropsychiatric and neurodegenerative illnesses. To probe the biological consequences of defective or altered neural transmission, various genetically engineered transgenic mouse models have been developed, together with conventional pharmacological manipulation. Via genetic manipulation, we are able to engineer speciﬁc neurotransmitters, receptors, inactivation of neurotransmitters or neural innervation density. Moreover, recently developed molecular genetic techniques make it possible to induce either a gene knock out event or transgene expression at a discrete time point in a speciﬁc neuronal population in both embryos and adult animals. In conjunction with pharmacological manipulation, these sophisticated genetic manipulations of neural transmission will provide new tools to control neural transmission in both normal and pathophysiological conditions. Keywords: neural transmission; transgenic animal models; dopamine; serotonin

Introduction of neurotransmitters in genetically engineered animal models. In genetically engineered transgenic mouse Neurons communicate with their target cells in models, we are able to alter neurotransmitter pheno- response to environmental stimuli by releasing a lim- type, either deplete or overproduce a specific neuro- ited quantity of information molecules, neurotransmit- transmitter or a specific neurotransmitter receptor, ters, at their synaptic junctions. Specific receptors on inhibit the inactivation of neurotransmitters or regulate the target cell surface, linked to intracellular secondary innervation density. Since neural transmission occurs messenger systems, transduce extracellular infor- through dynamic synaptic interactions between neuro- mation into the cell for further processing. Through transmitters and a number of specific receptors, the enzymatic inactivation and reuptake mechanisms, the availability of various animal models is essential for signaling role of the neurotransmitter is terminated. the investigation of developmental, physiological and Thus, strict regulation of the flow of information via behavioral consequences of altered neural trans- neural transmission is crucial to the precise control of mission. These animal models may also reveal behavior and physiological functions. Defective or alt- unknown important roles of specific neurotransmitters ered neural transmission affecting normal information and may provide potential clinical implications. processing and storage is thought to occur in neuropsy- In this article we will focus on the monoamine sys- chiatric and neurodegenerative illness.1–3 A number of tem. Catecholamine (CA, dopamine, norepinephrine drugs that modulate neural transmission have been uti- and epinephrine) and serotonin (5-HT) systems are lized as pharmacological tools to probe the biological implicated in the pathophysiology of schizophrenia, consequences of altered neural transmission. While the depression, suicidal behavior, impulsive behavior, pharmacological approach has provided some insights, anxiety, anorexia and drug addiction. Moreover, their the lack of specificity of these pharmacological tools sequential biosynthetic pathways share common has not established conclusively the locus of action of enzymes, which provide a unique opportunity for gen- neurotransmitters and their receptors. The develop- etic alteration of specific neurotransmitter phenotype. ment of appropriate biological models therefore is As summarized in Figure 1, dopamine is synthesized essential to an understanding of neural transmission at from l-tyrosine by tyrosine hydroxylase (TH) and aro- both the molecular and organismal levels. Recent pro- matic l-amino acid decarboxylase (AADC), which are gress in mouse genetics for manipulating the mouse present in all CA neurons. Dopamine ␤-hydroxylase genome4,5 has provided ample opportunity to investi- (DBH) synthesizes norepinephrine with dopamine as gate developmental, physiological and behavioral roles substrate, which is localized in both noradrenergic and adrenergic neurons. The last enzyme phenylethanolamine N-methyltransferase (PNMT), a marker for adre- Correspondence: Dr TH Joh, Laboratory of Molecular Neurobiol- ogy, Cornell University Medical College at the WM Burke Medi- nergic cells, catalyzes the formation of epinephrine cal Research Institute, White Plains, NY 10605, USA from norepinephrine. 5-HT is synthesized by two steps Received and accepted 7 August 1996 catalyzed by tryptophan hydroxylase (TPH) and Genetically engineered neural transmission JH Son and TH Joh 27 tissues of transgenic mice.9 The 6.1 kb of 5′ flanking promoter region was able to direct specific expression in transgenic mouse brain serotonergic cells and prefer- entially pineal gland.10

Alteration of neurotransmitter phenotype Genetic alteration of noradrenergic neurons to adrenergic phenotype was attempted by simple overexpression of a single PNMT gene in noradrenergic cells of transgenic mice.14,15 The expression of the PNMT gene in brain and sympathetic ganglia induced de novo production of epinephrine and altered the level of norepinephrine and epinephrine in brain and blood. In brain noradrenergic/adrenergic cell bodies of Figure 1 (a) Catecholamine (CA) biosynthetic pathway and the pons-medulla oblongata, the content of both nor- (b) serotonin (5-HT) biosynthetic pathway. epinephrine and epinephrine increased, whereas their brain target areas showed both increased PNMT activity and epinephrine release. In the sympathetic AADC. TPH is known as a specific marker enzyme for target tissues of the transgenic animals the level of epi- 5-HT-producing cells. nephrine was dramatically increased, but their norepinephrine levels were moderately decreased.16 Thus, Qualitative and quantitative manipulation of noradrenergic cells seemed to possess the basic neurotransmitters machinery required for the biosynthesis of epinephrine except PNMT enzyme. However, complete conversion Neuron-specific promoter of norepinephrine to epinephrine did not occur in the To target desired genetic manipulations to phenotype- transgenic animals, probably due to either the pro- specific neurons of transgenic animals, an essential duction of PNMT enzyme under the control of DBH step is to identify cell type-specific gene promoter/ promoter or tightly controlled neural homeostasis of enhancer regions. Identification of such DNA fragment CA levels. Interestingly, these alterations in CA speci- permits neuron-specific genetic manipulations. Several ficity in transgenic mice led to down-regulation of the different groups including ours have characterized the binding sites and the steady-state mRNA level of ␤2- specific promoter regions for genetic manipulation in adrenergic receptor in the target tissues. Although any dopaminergic, noradrenergic, adrenergic, and sero- subtle change in behavior or physiology has not yet tonergic neurons of the brain and peripheral nervous been fully addressed, the animal models used showed system (PNS) as summarized in Table 1. For adult and that agonist-induced regulation of adrenergic receptor embryonic brain CA neurons, 9 kb of rat TH promoter may be one of the mechanisms to maintain neural region was able to direct high level, cell type-specific homeostasis. 6–8 expression of a heterologous gene. In noradrenergic When target cell neurotransmitter phenotype was cells 5.8 kb of human DBH promoter was sufficient to altered in transgenic mice, we observed the marked direct expression in noradrenergic and adrenergic reduction of target cell innervation density.17 In this animal model we want to test the hypothesis that neurotransmitter phenotype of target cells is an important Table 1 Catecholaminergic and serotonergic neuron-spe- factor for the final synaptic linkage and its specificity. cific promoters Thus, ectopic expression of TH enzyme directed by the TPH promoter converted 5-HT/melatonin-producing a Neuronal phenotype Gene Promoter region cells of the pineal gland into cells producing additional dopamine (Figure 1). This phenotypic alteration drasti- 6 11 Dopaminergic neuron TH rat 9 kb or 4.8 kb cally reduced the sympathetic innervation primarily TH human 11 kb of the whole originating from superior cervical ganglia. The sym- gene12 pathetic innervation of the target tissue requires both Noradrenergic neuron TH rat 9 kb6 production of NGF in target tissue and expression of TH human 11 kb of the whole the low affinity NGF receptor (p75) in sympathetic 12 gene neurons during development. Thus, a null mutation of DBH human 5.8 kb9 p75 or ectopic expression of NGF in sympathetic gang- Adrenergic neuron PNMT human 8 kb13 lia showed selective lack or decrease of sympathetic DBH human 5.8 kb9 innervation to pineal gland, respectively.18,19 Interest- 6 TH rat 9 kb ingly, our transgenic mouse model did not show any Serotonergic neuron TPH mouse 6.1 kb10 substantial change of NGF production in the pineal gland. Although the mechanism of the reduced inner- aNote: Some promoter regions have been reported to direct vation is unknown, this animal model supports the minor ectopic expressions in transgenic mice. notion that the target neurotransmitter phenotype Genetically engineered neural transmission JH Son and TH Joh 28 plays some role in the formation of neural connections became nearly normal. Thus, this animal model during development. revealed that the neurotransmitter dopamine seems to be essential for movement, drinking and feeding Overexpression behavior, but may not be critical for the development Overproduction of neurotransmitters or their receptors of neural circuits controlling these behaviors. In the in transgenic mice was investigated to explore their case of norepinephrine-deficient mice, most homo- therapeutic potential. For instance, overexpression of zygous embryos died in utero, and only a small number tyrosine hydroxylase (TH), the rate-limiting enzyme of reached adulthood. Cardiovascular failure was the CA biosynthesis12 demonstrated potential application main cause of the death of the norepinephrine- in mechanistic and physiological studies of neuro- deficient mutant. Together with TH-deficient mice, transmitter homeostasis. When human TH gene was these animal models lacking specific CA provide new overexpressed in the brain and adrenal gland of trans- means to examine the role of dopamine, norepi- genic mice, the level of TH mRNA was 50-fold higher nephrine and epinephrine in mammalian develop- in brain and 5-fold higher in adrenal gland than that ment, physiology and behavior. Those important data of endogenous mouse TH mRNA. Surprisingly, in this cannot be clearly obtained from classical pharmaco- transgenic mouse brain TH enzyme activity was only logical studies. 2- to 5-fold higher than that of nontransgenic brain. Dopamine and norepinephrine levels in the transgenic mouse brain were not significantly changed from those in nontransgenic mice. Thus, TH enzyme activity Manipulation of neurotransmitter receptors seemed to be strictly regulated to maintain the neural homeostasis in transgenic brain. It is possible to regu- As described earlier, the animal model overexpressing late the intracellular second messenger system by over- the ␤2-adrenergic receptor20 opened a unique way of expression of its receptor. For instance, cardiac-specific regulating the second messenger system linked to a overexpression of the ␤2-adrenergic receptor in trans- specific receptor, which has important implications for genic mice increased atrial contractility and left ven- various neuropsychiatric illnesses. Another way to tricular function by the high concentration of cAMP, manipulate neural transmission is the targeted inacti- which was normally augmented by sympathetic norep- vation of CA receptors or 5-HT receptors characterized inephrine or adrenal epinephrine.20 During stress it is by pharmacological studies. Often information essential to increase catecholamine secretion and obtained from gene-targeted mutant mice is either con- stimulate myocardial ␤-adrenergic receptors for inten- firmatory or contradicts our prediction based on phar- sification of cardiac function. This animal model will macological studies. For example, to investigate the be clinically useful for the mechanistic study of physiological role of the most abundant dopamine chronic heart failure in humans, which is characterized receptors, D1 and D2, in dopaminergic neural trans- by a marked reduction of myocardial ␤-adrenergic mission, transgenic mouse models lacking either D1 or receptors.21 Also, the model implies that regulation of D2 receptor were generated using homologous recom- a second messenger system linked to a specific neuro- bination.25–28 The animals lacking D1 receptor exhib- transmitter receptor can be achieved by a controlled ited either normal behavior or locomotor hyperactivity, expression of the receptor molecules in specific cells in contrast to pharmacological studies using D1 recep- of the brain and periphery. tor antagonists exerting suppressive effect. This approach revealed the essential role of the D1 receptor Depletion by gene knock out in the locomotor stimulant effects of cocaine. The D2 The knock out of a specific CA biosynthesis enzyme receptor-deficient mouse demonstrated akinetic and gene, in conjunction with gene knock in, enabled the bradykinetic behaviors and lack of spontaneous move- generation of mutant mice lacking only dopamine,22 ment as predicted from D2 antagonist treatment. norepinephrine and epinephrine23 or all three CA.24 The serotonergic system modulates key physiologi- The dopaminergic system in brain has major roles in cal functions including appetite, motor activity, sleep, the regulation of movement, cognitive, reward and sexual activity, learning and memory and has been emotional behavior and is implicated in the pathophy- associated with mood disorders. Recent studies have siology of Parkinson’s disease, schizophrenia, identified more than 14 mammalian 5-HT receptors depression and drug addiction. The dopamine- and several isoforms of known 5-HT receptors.29 Thus, deficient mice were generated by both targeted disrup- gene targeting technology became a major player to tion of TH gene and restoring TH activity only in nor- investigate the physiological role of serotonergic neural adrenergic and adrenergic cells. The brains of newborn transmission, revealing some unexpected functions of

dopamine-deﬁcient mice displayed no more obvious these receptors. The mutant mouse lacking the 5-HT1B anatomical deﬁciencies except reduced brain size, showed enhanced aggressive behavior, which sug-

possibly as a result of neuronal compaction, than those gested the involvement of 5-HT1B receptor in aggressive of wild-type mice.22 Dopamine-deﬁcient animals failed behavior.30 Of interest, the mutant mouse lacking 5-

to thrive and died during postnatal week 3 due to HT2C was overweight due to abnormal feeding behavior hypoactivity and aphagia. When l-DOPA was adminis- and might have been prone to spontaneous death tered continuously, locomotion and food consumption from seizures.31 Genetically engineered neural transmission JH Son and TH Joh 29 Manipulation of neurotransmitter inactivation density as well as the regulation of synaptic efficacy.39– mechanisms 41 Therefore, appropriate genetic manipulation of the expression of neurotrophic factors will be another way To alter the termination of neural transmission, mice to modulate neural transmission in animal models. lacking monoamine oxidase A (MAOA) were generated by insertional mutation.32 Enhanced aggression was manifested by adult male mice. In pup brain 5-HT and Future direction and implications norepinephrine concentrations were increased up to 9- Pharmacological manipulation of neural transmission fold and 2-fold, respectively. Thus, the dramatically has been an important tool to probe neuronal com- altered 5-HT metabolism seemed to be linked to severe munication in the nervous system. It has unraveled the behavioral abnormalities. In fact, a point mutation in defective neural transmission underlying various neur- the eighth exon of human MAOA gene, which abol- opsychiatric and neurodegenerative illnesses which ished MAOA catalytic activity, is known to be associa- 33 led to the development of new drugs to improve neuro- ted with impulsive aggression in affected men. pathological conditions. However, the inherent experi- The dopamine transporter has an important role in mental limitation of pharmacological manipulation locomotion, cognition, affect and neuroendocrine func- such as lack of drugs with sufficient specificity and dif- tions by controlling rapid uptake of released dopamine ficulty in localized drug delivery, necessitated the molecules. Thus, it is a target site for antidepressants, employment of complementary approaches, such as cocaine and amphetamines. In the mouse model lack- the recently developed genetic approach to study neu- ing the dopamine transporter, the released dopamine ral transmission in vivo. For instance, pharmacological lasted 100 times longer, resulting in spontaneous hyp- 34 treatment with a D1 receptor antagonist produces an erlocomotion. Moreover, psycostimulants, cocaine acute and partial inhibition, in contrast to the genetic and amphetamines, have no effect in this animal knock out of D1 receptor, clearly resulting in a perma- model. Therefore, the genetic regulation of either MAO nent and complete inhibition. Another cause of the enzymes’ activities or monoamine transporter level can functional discrepancy between pharmacological be an essential tool to modulate neural transmission. manipulation and genetic manipulation is that transgenic mouse mutants, carrying either new transgenes Manipulation of neural innervation or a targeted gene knock out, can be often compensated by up- or down-regulation of the related gene family Neonatal cell death of nigrostriatal dopaminergic neu- during embryonic development. Therefore, the pheno- rons produced by 6-hydroxydopamine administration type of transgenic mouse models could be a mere resulted in serotonergic hyperinnervation of striatum consequence of developmental abnormality, instead of from the dorsal raphe nucleus.35 The serotonergic hyp- the physiological role of a targeted gene product per se. erinnervation increased the capacity of 5-HT release by Recent development of molecular genetic techniques 3-fold in the striatum of the neonatally dopamine- makes it possible to circumvent this problem by spati- depleted rat. But the resting level of 5-HT in extracellu- otemporal regulation of a gene-targeting or transgene lar fluid of the striatum was not different from that in expression. For instance, an inducible and/or tissue- intact rats due to the concomitant increase in high- specific knock out is technically possible to initiate a affinity 5-HT uptake. Our characterized neurodevelop- gene-targeting event at a discrete time point in a spe- mental mouse mutant, Anorexia (anx), exhibited anor- cific neuronal population in adult animals, which exic starvation and abnormal behaviors (body tremors, experience no specific mutation during develop- hyperactivity, abnormal gait and headweaving), which ment.42,43 Moreover, using the inducible tetracycline resemble phenomena induced by pharmacological binary system, specific gene expression can be induced overstimulation of 5-HT receptors.36 Immunohisto- with spatiotemporal control in transgenic mice.44 chemical analysis of anx brain revealed extensive sero- These powerful genetic tools will allow us to generate tonergic hyperinnervation in its normal target fields, more sophisticated animal models, which will provide such as hippocampus, cortex, olfactory bulb and cere- unprecedented insights into neural transmission bellum, which seemed to increase 5-HT release. 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