Inhibition of foxo and minibrain in Dopaminergic Neurons Can Model Aspects of Parkinson Disease in Drosophila melanogaster

Symptoms of Parkinson Disease (PD), the second most common neurodegenerative disease, emerge due to degeneration of dopaminergic neurons. Recently, a genome wide study revealed a role for a foxo transcription factor in PD. In the model organism Drosophila melanogaster, we have attempted 1) to inhibit the sole Drosophila homologue of foxo through the directed expression of a stable inducible RNAi transgene and 2) to indirectly increase foxo transcription activity through the inhibition of the kinase minibrain (mnb), a foxo transcriptional inhibitor. To evaluate the lifetime consequences upon the flies, longevity assays and locomotion over time assays were conducted. The inhibition of foxo by foxo-RNAi decreases life span significantly when expressed under the control of Tyrosine Hydroxylase-Gal4 (TH-Gal4). The targeted expression of mnb-RNAi, in the dopaminergic neurons, with an expected loss of suppression of foxo transcriptional activity, results in a significant loss of climbing ability. Thus alteration of foxo activity, both by RNA-inhibition and by down-regulation of an inhibitor of foxo, minibrain, produces novel Drosophila models of Parkinson Disease.


Introduction
Parkinson Disease (PD), only surpassed by Alzheimer Disease, is the second most common human neurodege-nerative disease and the most prevalent neurodegenerative movement disorder [1].Pathologically, PD is characterized by the loss of dopaminergic (DA) neurons in the ventral mesencephalic substantia nigra pars compacta and, usually, by the formation of Lewy bodies (aggregation of proteins including α-synuclein) in the neurons of ventral midbrain and some other regions such as the prefrontal cortex.The associated impairments often include resting tremor, rigidity, bradykinesia and postural instability.Inherited forms (autosomal-dominant and autosomal-recessive) of the disease account for 5% to 15% of all PD cases.Recently, variations in the foxO1 gene among others, have been implicated in PD [2].As part of a widening investigation of the genetic basis of PD, the potential role of foxo in PD has become of interest.
The forkhead box subfamily "o", foxo, is one of the larger family of forkhead genes that encode a class of winged helix-turn-helix proteins which act as transcription factors that control homeostasis in response to external influences including variation in growth factor availability and various stresses (for review see [3]).There are 4 mammalian foxo members (foxO1, foxO3, foxO4 and foxO6 [4], one homologue in C. elegans (daf-16) [5] and one homologue in Drosophila melanogaster (foxo) [6].The behaviour of the foxo proteins is modified through various post-transcriptional modifications such as acetylation, ubiquitination and phosphorylation.The akt kinase can phosphorylate foxo to exclude the transcription factor from the nucleus [7].Another kinase, dualspecificity tyrosine-phosphorylation regulated kinase 1a, Dyrk1a can also phosphorylate foxo [8].Dyrk1a is located within the Down Syndrome Critical Region of human chromosome 21 and its Drosophila homologue, minibrain (mnb), is well conserved [9] [10].Dyrk1a/mnb can phosphorylate foxo to sequester it from the nucleus to suppress transcriptional activity.
Drosophila melanogaster has been proven to be an excellent organism in which to model Parkinson Disease (for reviews see [11] [12]).The first fly model of PD was established through the directed expression of human alpha-synuclein and subsequent loss of dopaminergic neurons [13].Subsequently, a number of genes implicated in PD have been manipulated in this model including Parkin/PARK2 [14] [15] and Pink1/PARK6 [16]- [18].As the role of foxo proteins seemed to be very well conserved and considering the potential role in PD, these experiments were undertaken in the Drosophila model system to take a closer look at foxo modulation in the modeling PD.

Longevity Assay
Longevity assays were performed on critical class males collected under carbon dioxide every 24 hours.Due to the well-established variation in ageing females, only males were analyzed.Approximately 200 male flies were introduced into fresh food without anaesthesia three times a week and kept in numbers of no more than 20 per vial to prevent overcrowding.As they aged, flies were monitored for viability until all perished [19].Results were analyzed with GraphPad Prism 5.For survival results, the statistical test of Mantel-Cox was carried out.

Locomotor Assay
A standard climbing assay every 6 or 7 days, beginning at day 6 or 7, during the lifespan of the critical class males was carried out by following a standard protocol [20].For the locomotor assay the results were analyzed by GraphPad Prism 5 with an unpaired t-test was carried out to detect any significant differences between means of groups and the slopes of the curves with non-overlapping 95% CI were considered significantly different.

foxo-RNAi Expression under the Direction of ddc-Gal4 Does Not Alter Lifespan or Climbing Ability
To inhibit the expression of foxo, the inducible transgene UAS-foxo-RNAi was placed under the control of the ddc-Gal4 transgene.As a control, the benign responder gene UAS-lacZ resulted in a fairly standard sub-optimal longevity response with a median lifespan of approximately 42 days when directed by ddc-Gal4.When foxo-RNAi was expressed under the control of the ddc-Gal4 transgene, it did not alter the median lifespan nor the overall longevity characteristics compared to the control (Figure 1(a)).Likewise, the measurement of the loss of locomotor function as monitored by the evaluation of climbing ability over time was not altered when foxo-RNAi was directed by ddc-Gal4 (Figure 1(b)).

The Expression of foxo-RNAi Directed by TH-Gal4 Reduces Lifespan Greatly but Alters Climbing Ability over Time Slightly
The inhibition of foxo through expression of the UAS-foxo-RNAi in dopaminergic neurons under the control of the TH-Gal4 transgene reduced lifespan significantly but resulted in a slight alteration in climbing ability over time.The median lifespan of TH-Gal4/UAS-lacZ males was measured to be 58 days whereas the median length of life for the TH-Gal4/UAS-foxo-RNAi male flies was 38 days (Figure 2

Expression of mnb-RNAi Directed by ddc-Gal4 Does Not Alter Lifespan or Climbing Ability
The ddc-Gal4 transgene was used to direct the expression of the interfering mnb-RNAi transgene in the dopaminergic neurons, along with serotonergic neurons and other cells.The UAS-lacZ benign responding transgenics as the control, and ddc-Gal4/mnb-RNAi flies have median lifespans of 42 days and both genotypes (ddc-gal4/UAS-lacZ and ddc-Gal4/UAS-mnb-RNAi) live equally well up to approximately day 70 (Figure 3(a)).The Mantel-Cox statistical test did not reveal any significant difference in their survival pattern.Non-linear regression curves of climbing ability over time for the two genotypes, ddc-Gal4/UAS-lacZ compared to ddc-Gal4/UAS-mnb-RNAi, reveal little difference (Figure 3(b)).Results of statistical analysis of this measurement of locomotion over time did not reveal any significant early loss of climbing ability.

Expression of mnb-RNAi Directed by TH-Gal4 Significantly Diminishes Climbing Ability over Time
With the expression of the inhibitory UAS-mnb-RNAi under the directed control of the TH-Gal4 transgene to the dopaminergic neurons, a very significant decrease in climbing ability over time was observed (Figure 4(a)).
Analysis of ageing of these flies shows that there was no significant difference in lifespan of the two genotypes, and, therefore, inhibition of mnb does not alter greatly the survival of the flies.The median lifespan of flies expressing TH-Gal4 directing the expression of the control responding transgene UAS-lacZ or the UAS-mnb-RNAi both produced a median life span of 58 days with some alive until day 78.However, the climbing ability over time, as illustrated in   of TH-Gal4/UAS-lacZ and TH-Gal4/UAS-mnb-RNAi flies revealed that the decline in climbing ability in day 26, 40 and 47 is significant.

Discussion
In this study, the foxo-RNAi transgene was utilized to directly decrease foxo expression and the mnb-RNAi transgene to induce a slight, indirect elevation in foxo activity, among other effects.The expression of foxo-RNAi in dopaminergic neurons gives two distinct results: 1) expression of foxo-RNAi directed by ddc-Gal4 does not alter life span or climbing ability compared to the control; 2) expression of foxo-RNAi under the control of TH-Gal4 decreases lifespan significantly but does not alter the locomotion of the surviving flies greatly over time.Reduced life span of TH-Gal4/foxo-RNAi flies points to a protective role for foxo against organismal death.This greatly reduced viability may model severe aspects of early onset PD in Drosophila although the survivors maintain the ability to move.Expression of the mnb-RNAi transgene in dopaminergic neurons exhibits in two very different results: 1) directed by ddc-Gal4/mnb-RNAi expression does not alter lifespan or longevity compared to that UAS-lacZ control (Figure 3) and 2) expression of mnb-RNAi directed by the TH-Gal4 transgene, climbing ability was lost over time but the lifespan was not greatly changed (Figure 4).The difference in expression presented by the two transgenes may account for the observed difference in results; the ddc-Gal4 transgene directs the expression of Gal4 and hence the gene under the control of UAS element differently than that of the TH-Gal4 transgene.The dopa decarboxylase enzyme is synthesized in the 150 dopamine and serotonin neurons, in a subset of glial cells and in the most hypodermal cells whereas tyrosine hydroxylase is produced in dopamine synthesizing cells [21] [22].An alternative difference may be that the expression of Gal4 may not be as robust under ddc-Gal4 control compared to the TH-Gal4 transgene.The significant decrease in locomotor activity in these flies may reflect the slight elevation of foxo activity caused by inhibition of mnb in dopaminergic neurons.If so, it is consistent with the results the recent genome wide study [2] indicating a role for a foxo in P.D. Finally, the inhibition of mnb under the control of TH-Gal4 has produced a novel model of Parkinson Disease in Drosophila.

Conclusion
In conclusion, the inhibition of foxo through directed RNA-inhibition directed by TH-Gal4 in the dopaminergic neurons can significantly reduce lifespan in Drosophila melanogaster.Most importantly, the inhibition of mnb, which encodes a kinase that negatively regulates foxo transcriptional activity in the dopaminergic neurons can model aspects of Parkinson Disease in flies by a significant premature reduction in locomotor ability over time.

Figure 1 .
Figure 1.The expression of foxo-RNAi under the control of ddc-Gal4 does not alter lifespan or climbing ability in D. melanogaster males at 25˚C.(a) Longevity assays were performed on ddc-Gal4/UAS-lacZ and ddc-Gal4/UAS-foxo-RNAi critical male flies (initial n = 200 for both) and the pairwise comparison does not reveal any significant difference.Both genotypes displayed a median lifespan of 42 days; (b) Critical class males of the two genotypes (ddc-Gal4/UAS-lacZ and ddc-Gal4/ UAS-foxo-RNAi) were analyzed by a locomotion assay over time (initial n = 70 was to reduced to a minimum of 5 through lethality) and non-linear regression curve was fitted to best demonstrate the climbing over time pattern.The 95% CI slopes overlap indicating any difference caused is likely due to chance.

Figure 2 .
Figure 2. The expression of foxo-RNAi directed by TH-Gal4 reduces lifespan greatly and climbing ability slightly in D. melanogaster males at 25˚C.(a) The foxo-RNAi transgene induced in dopaminergic neurons under the control of TH-Gal4 transgene (TH-Gal4/UAS-foxo-RNAi) reduces lifespan of critical class males significantly compared to the control (TH-Gal4/ UAS-lacZ) as supported by Mantel-Cox test results (initial n = 200 for both); (b) Comparison of climbing ability over time for TH-Gal4/UAS-lacZ and TH-Gal4/UAS-foxo-RNAi shows that the pattern of their climbing ability is slightly different, notably at days 26 and 40 (initial n = 70 was to reduced to a minimum of 5 through lethality).Error bars represent standard error of mean and an asterisk indicates a significant difference.

Figure 4 (Figure 3 .
Figure 3.The expression of mnb-RNAi directed by ddc-Gal4 did not alter lifespan or climbing ability in D. melanogaster raised at 25˚C.(a) Longevity assay were performed on ddc-Gal4/UAS-lacZ and ddc-Gal4/UAS-mnb-RNAi critical male flies (initial n = 200 for both) and do not reveal significant differences in lifespan when analysed by Mantel-Cox to detect significant differences in survival pattern.Both genotypes gave a median lifespan of 42 days; (b) The expression of mnb-RNAi directed by ddc-Gal4 did not change climbing ability over time compared to the ddc-Gal4/UAS-lacZ control (initial n = 70 was to reduced to a minimum of 5 through lethality).

Figure 4 .
Figure 4.The expression of mnb-RNAi in dopaminergic neurons using TH-Gal4 transgene does not alter lifespan but decreases climbing ability significantly in D. melanogaster raised at 25˚C.(a) Lifespan was not reduced or increased when mnb-RNAi is expressed under the control of TH-Gal4 transgene (initial n = 200); (b) Climbing ability was significantly reduced at days 26, 40 and 47 when mnb-RNAi was induced in dopaminergic neurons by the TH-Gal4 transgene (initial n value = 70).Error bars represent standard error of mean and asterisk indicates significant difference.