Janis M. O'Donnell

Interaction of Genetic and Environmental Factors in a Drosophila Parkinsonism Model

Catastrophic loss of dopaminergic neurons is a hallmark of Parkinsons disease. Despite the recent identification of genes associated with familial parkinsonism, the etiology of most Parkinsons disease cases is not understood. Environmental toxins, such as the herbicide paraquat, appear to be risk factors, and it has been proposed that susceptibility is influenced by genetic background. The genetic model organism Drosophila is an advantageous system for the identification of genetic susceptibility factors. Genes that affect dopamine homeostasis are candidate susceptibility factors, because dopamine itself has been implicated in neuron damage. We find that paraquat can replicate a broad spectrum of parkinsonian behavioral symptoms in Drosophila that are associated with loss of specific subsets of dopaminergic neurons. In parallel with epidemiological studies that show an increased incidence of Parkinsons disease in males, male Drosophila exhibit paraquat symptoms earlier than females. We then tested the hypothesis that variation in dopamine-regulating genes, including those that regulate tetrahydrobiopterin, a requisite cofactor in dopamine synthesis, can alter susceptibility to paraquat-induced oxidative damage. Drosophila mutant strains that have increased or decreased dopamine and tetrahydrobiopterin production exhibit variation in susceptibility to paraquat. Surprisingly, protection against the neurotoxicity of paraquat is conferred by mutations that elevate dopamine pathway function, whereas mutations that diminish dopamine pools increase susceptibility. We also find that loss-of-function mutations in a negative regulator of dopamine production, Catecholamines-up, delay the onset of neurological symptoms, dopaminergic neuron death, and morbidity during paraquat exposure but confer sensitivity to hydrogen peroxide.

Functional Interactions Between GTP Cyclohydrolase I and Tyrosine Hydroxylase in Drosophila

Tyrosine hydroxylase requires the regulatory cofactor, tetrahydrobiopterin, for catecholamine biosynthesis. Because guanosine triphosphate cyclohydrolase I is the rate limiting enzyme for the synthesis of this cofactor, it has a key role in catecholamine production. We show that GTP cyclohydrolase and tyrosine hydroxylase (TH) are co-localized in the Drosophila central nervous system. Mutations in the Punch locus, which encodes GTP cyclohydrolase, reduce TH activity; addition of cofactor to crude extracts could not fully rescue this activity in all mutant strains. The decrease in TH activity and the inability to increase it with added cofactor is not due to loss or decreased production of TH protein. We found that TH co-immunoprecipitated with GTP cyclohydrolase when wild type head extracts were incubated with anti-GTP cyclohydrolase antibody. We suggest that regulation of TH by its cofactor may require its association with GTP cyclohydrolase, and that the ability of GTP cyclohydrolase to associate with TH and its role in tetrahydrobiopterin synthesis may be separable functions of this enzyme. These results have important implications for understanding catecholamine-related neural diseases and designing strategies for gene therapy.

DIRECT BINDING OF GTP CYCLOHYDROLASE AND TYROSINE HYDROXYLASE: REGULATORY INTERACTIONS BETWEEN KEY ENZYMES IN DOPAMINE BIOSYNTHESIS*

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.

Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism

The underlying defects in Angelman syndrome (AS) and autism spectrum disorder (ASD) may be in part due to basic defects in synaptic plasticity and function. In some individuals serotonin reuptake inhibitors, which decrease pre-synaptic re-uptake of serotonin, can ameliorate symptoms, as can resperidone, which blocks both dopamine and serotonin receptors. Loss of maternal UBE3A expression causes AS, while maternal duplications of chromosome 15q11.2-q13 that include the UBE3A gene cause ASD, implicating the maternally expressed UBE3A gene in the ASD phenotype. In a Drosophila screen for proteins regulated by UBE3A, we identified a key regulator of monoamine synthesis, the gene Punch, or GCH1, encoding the enzyme GTP cyclohydrolase I. Here we show that Dube3a, the fly UBE3A orthologue, regulates Punch/GCH1 in the fly brain. Over-expression of Dube3a elevates tetrahydrobiopterin (THB), the rate-limiting cofactor in monoamine synthesis while loss of Dube3a has the opposite effect. The fluctuations in dopamine levels were associated with hyper- and hypoactivity, respectively, in flies. We show that changes in Punch/GCH1 and dopamine levels do not depend on the ubiquitin ligase catalytic domain of Dube3a. In addition, both wild type Dube3a and a ubiquitination-defective Dube3a-C/A form were found at high levels in nuclear fractions and appear to be poly-ubiquitinated in vivo by endogenous Dube3a. We propose that the transcriptional co-activation function of Dube3a may regulate GCH1 activity in the brain. These results provide a connection between monoamine synthesis (dopamine/serotonin) and Dube3a expression that may explain why some individuals with ASD or AS respond better to selective serotonin reuptake inhibitors than others.

A typical N-terminal Extensions Confer Novel Regulatory Properties on GTP Cyclohydrolase Isoforms in Drosophila melanogaster

The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.

The Protective Effect of Minocycline in a Paraquat-Induced Parkinson's Disease Model in Drosophila is Modified in Altered Genetic Backgrounds

Epidemiological studies link the herbicide paraquat to increased incidence of Parkinsons disease (PD). We previously reported that Drosophila exposed to paraquat recapitulate PD symptoms, including region-specific degeneration of dopaminergic neurons. Minocycline, a tetracycline derivative, exerts ameliorative effects in neurodegenerative disease models, including Drosophila. We investigated whether our environmental toxin-based PD model could contribute to an understanding of cellular and genetic mechanisms of minocycline action and whether we could assess potential interference with these drug effects in altered genetic backgrounds. Cofeeding of minocycline with paraquat prolonged survival, rescued mobility defects, blocked generation of reactive oxygen species, and extended dopaminergic neuron survival, as has been reported previously for a genetic model of PD in Drosophila. We then extended this study to identify potential interactions of minocycline with genes regulating dopamine homeostasis that might modify protection against paraquat and found that deficits in GTP cyclohydrolase adversely affect minocycline rescue. We further performed genetic studies to identify signaling pathways that are necessary for minocycline protection against paraquat toxicity and found that mutations in the Drosophila genes that encode c-Jun N-terminal kinase (JNK) and Akt/Protein kinase B block minocycline rescue.

dtorsin, the Drosophila Ortholog of the Early-Onset Dystonia TOR1A (DYT1), Plays a Novel Role in Dopamine Metabolism

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.

Novel whole-tissue quantitative assay of nitric oxide levels in Drosophila neuroinflammatory response.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinsons disease (PD), Alzheimers disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Modulation of social space by dopamine in Drosophila melanogaster, but no effect on the avoidance of the Drosophila stress odorant

Appropriate response to others is necessary for social interactions. Yet little is known about how neurotransmitters regulate attractive and repulsive social cues. Using genetic and pharmacological manipulations in Drosophila melanogaster, we show that dopamine is contributing the response to others in a social group, specifically, social spacing, but not the avoidance of odours released by stressed flies (dSO). Interestingly, this dopamine-mediated behaviour is prominent only in the day-time, and its effect varies depending on tissue, sex and type of manipulation. Furthermore, alteration of dopamine levels has no effect on dSO avoidance regardless of sex, which suggests that a different neurotransmitter regulates this response.

Invertebrate Models of Dystonia

The neurological movement disorder dystonia is an umbrella term for a heterogeneous group of related conditions where at least 20 monogenic forms have been identified. Despite the substantial advances resulting from the identification of these loci, the function of many DYT gene products remains unclear. Comparative genomics using simple animal models to examine the evolutionarily conserved functional relationships with monogenic dystonias represents a rapid route toward a comprehensive understanding of these movement disorders. Current studies using the invertebrate animal models Caenorhabditis elegans and Drosophila melanogaster are uncovering cellular functions and mechanisms associated with mutant forms of the well-conserved gene products corresponding to DYT1, DYT5a, DYT5b, and DYT12 dystonias. Here we review recent findings from the invertebrate literature pertaining to molecular mechanisms of these gene products, torsinA, GTP cyclohydrolase I, tyrosine hydroxylase, and the alpha subunit of Na+/K ATPase, respectively. In each study, the application of powerful genetic tools developed over decades of intensive work with both of these invertebrate systems has led to mechanistic insights into these human disorders. These models are particularly amenable to large-scale genetic screens for modifiers or additional alleles, which are bolstering our understanding of the molecular functions associated with these gene products. Moreover, the use of invertebrate models for the evaluation of DYT genetic loci and their genetic interaction networks has predictive value and can provide a path forward for therapeutic intervention.