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

Mutant human torsinA, responsible for early-onset dystonia, dominantly suppresses GTPCH expression, dopamine levels and locomotion in Drosophila melanogaster

Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG) deletion that results in the loss of a glutamic acid residue (ΔE302/303) in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH) proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine. Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4) levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the wild type and the mutant torsin proteins

Data from: 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

Identification and characterization of a novel receptor-like kinase involved in the initiation and regulation of Arabidopsis innate immunity

Receptor-like kinases (RLKs) are known to be involved in the recognition of pathogen-associated molecular patterns (PAMPs) and subsequently activate resistance pathways against broad classes of pathogens. While initiation and maintenance of defense pathways is critical for survival, mechanisms to damp down these responses are just as necessary though currently not as well understood. We have identified CRLK1, an Arabidopsis RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knock-out mutants in crlk1 are more susceptible to both biotrophic and necrotrophic fungal pathogens though the response of the mutants to bacterial pathogens is unaffected. Interestingly expression of MAPK3, an important positive regulator of innate immunity, is increased in crlk1 mutants. Our data show that CRLK1 is essential for the establishment of defense against biotrophic and necrotrophic fungi and that the mutation in CRLK1 does not fully block chitin-enhanced Arabidopsis resistance. We show that CRLK1 is a functional kinase in vitro and its kinase activity required the presence of manganese. Overexpression of a 35S:CRLK1: GUS fusion protein in Arabidopsis confers enhanced resistance to the powdery mildew pathogen Golovinomycetes cichoracearum. In addition, CRLK1 induction by chitin is increased in mapk3 and several wrky mutants indicating that CRLK1 may be repressed by MAPK3 and WRKY transcription factors in planta. The results presented provide important information about the function and regulation of CRLK1 in Arabidopsis. (Published By University of Alabama Libraries

The development and regulation of adult abdominal segment reduction in drosophila

A central aim of evolutionary research is to understand the genetic and molecular mechanisms underlying the morphological divergence of species. A growing body of evidence has been accumulated showing mutations in cis-regulatory elements that affect the regulation of gene expression are a common source of morphological evolution. A number of fundamental studies have been done on several rapidly evolving traits, including Drosophila pigmentation patterns and larval trichome patterns, and skeletal structures of stickleback fish. However, the evolution of conserved traits is not well understood. For my dissertation, I focused on understanding the genetic mechanisms that lead to the robustness and maintenance of conserved traits. Specifically, I use abdominal segment reduction as a genetic model to address my question. Adult female Drosophila develops seven abdominal segments, while adult males only have six segments. In the following studies, we investigated 1) When and how male A7 segment is reduced during development. We found male A7 segment is reduced between 24 h APF and 40 h APF. 2) The mechanisms responsible for the loss of male A7. We found male A7 is eliminated by the combination of suppression of cell proliferation and differentiation, segment compartmental transformation and apoptosis. 3) The genetic regulation of male A7 reduction. I found male A7 reduction is controlled by the Hox protein Abd-B and sex-determination factor Dsx through negative regulation of Wg and DER activity. 4) Gene interactions within the regulatory network that controls male A7 reduction. I found the regulation of wg is the key node in this highly interactive regulatory network. 5) The regulatory control of wg during pupal development. I identified two CREs (wg8 and wg9) regulating wg expression in pupal abdomen. These findings show that the regulation wg is at the center of a highly interactive regulatory network, which may contribute to the robustness and long-term maintenance of segment reduction in Cyclorrhapha. (Published By University of Alabama Libraries

Dopamine homeostasis and environmental risk factors in Parkinson's Disease model

The neurotransmitter dopamine (DA) is an important factor in the regulation of many biological processes, from pleasure and addiction to balance and locomotion. Therefore, understanding and defining the mechanisms and factors that are required for proper DA homeostasis is an integral component in managing and elucidating the causes of DA related diseases. Among these diseases, Parkinson's disease (PD) is the most notable and remains one of the most researched yet puzzling motor system associated neurological disorders. PD is characterized by a preferential loss of DA neurons in the substantia nigra. Though the cause and exact mechanism of this disease remains undefined, numerous environmental factors such as metals and pesticides have been associated with the etiology of the disease process. In the following studies, the genetic components of DA homeostasis and environmental risk factors in a Drosophila model of PD are investigated. The implication of metals as a component in the pathology of PD is examined in relationship to zinc toxicity. Catecholamines's up (Catsup), which plays a crucial role in regulating DA homeostasis and is proposed to be a member of the mammalian KE4 ZIP transporter family, demonstrates zinc sensitivity, with the proposed underlying factors being a dysregulation of DA synthesis and DA transport. The findings of this report demonstrate that loss of dopamine transporter (DAT) function, results in a more robust sensitivity to zinc than that seen in Catsup mutants. In addition exogenous DA increases sensitivity of wild type flies to zinc, similar to that which is seen Catsup mutants. Interestingly, LiCl ameliorates the toxic effects of zinc. The results also demonstrate a functional relationship between paraquat toxicity and DAT, which affects DA transport. To determine the consequences of early exposure to paraquat on lifespan and mobility, the effect of a one time exposure to young adult flies was observed. The results of this experiment show that a brief exposure to paraquat illicts long term detrimental affects on survival as well as parkinsonian type phenotypes. (Published By University of Alabama Libraries