Identifying new players in structural synaptic plasticity through dArc1 interrogation.

The formation, expansion, and pruning of synapses, known as structural synaptic plasticity, is needed for learning and memory, and perturbation of plasticity is associated with many neurological disorders and diseases. Previously, we observed that the Drosophila homolog of Activity-regulated cytoskeleton-associated protein (dArc1), forms a capsid-like structure, associates with its own mRNA, and is transported across synapses. We demonstrated that this transfer is needed for structural synaptic plasticity. To identify mRNAs that are modified by dArc1 in presynaptic neuron and postsynaptic muscle, we disrupted the expression of dArc1 and performed genomic analysis with deep sequencing. We found that dArc1 affects the expression of genes involved in metabolism, phagocytosis, and RNA-splicing. Through immunoprecipitation we also identified potential mRNA cargos of dArc1 capsids. This study suggests that dArc1 acts as a master regulator of plasticity by affecting several distinct and highly conserved cellular processes.

A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy.

BACKGROUND: The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene led us to study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of the well-studied model organism Drosophila melanogaster. The multitude of biological functions attributable to the vitamers catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk and we used RNA interference to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. We further investigated any association between this enhanced disease risk gene with the established PD model induced by expression of α-synuclein in the same neurons. We relied on the pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, to rescue the Pdxk-induced phenotypes. RESULTS: To drive the expression of Pdxk RNA interference in DA neurons of Drosophila, we used Ddc-Gal4 which drives expression in both dopaminergic and serotonergic neurons, to result in decreased longevity and compromised climbing ability, phenotypes that are strongly associated with Drosophila models of PD. The inhibition of Pdxk in the α-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when we inhibited Pdxk in the developing eye using GMR-Gal4, we found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the α-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. CONCLUSIONS: Though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD.

Bax-inhibitor-1 knockdown phenotypes are suppressed by Buffy and exacerbate degeneration in a Drosophila model of Parkinson disease.

BACKGROUND: Bax inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective transmembrane protein that acts as a suppressor of Bax-induced apoptosis by regulation of endoplasmic reticulum stress-induced cell death. We knocked down BI-1 in the sensitive dopa decarboxylase (Ddc) expressing neurons of Drosophila melanogaster to investigate its neuroprotective functions. We additionally sought to rescue the BI-1-induced phenotypes by co-expression with the pro-survival Buffy and determined the effect of BI-1 knockdown on the neurodegenerative α-synuclein-induced Parkinson disease (PD) model. METHODS: We used organismal assays to assess longevity of the flies to determine the effect of the altered expression of BI-1 in the Ddc-Gal4-expressing neurons by employing two RNAi transgenic fly lines. We measured the locomotor ability of these RNAi lines by computing the climbing indices of the climbing ability and compared them to a control line that expresses the lacZ transgene. Finally, we performed biometric analysis of the developing eye, where we counted the number of ommatidia and calculated the area of ommatidial disruption. RESULTS: The knockdown of BI-1 in these neurons was achieved under the direction of the Ddc-Gal4 transgene and resulted in shortened lifespan and precocious loss of locomotor ability. The co-expression of Buffy, the Drosophila anti-apoptotic Bcl-2 homologue, with BI-1-RNAi resulted in suppression of the reduced lifespan and impaired climbing ability. Expression of human α-synuclein in Drosophila dopaminergic neurons results in neuronal degeneration, accompanied by the age-dependent loss in climbing ability. We exploited this neurotoxic system to investigate possible BI-1 neuroprotective function. The co-expression of α-synuclein with BI-1-RNAi results in a slight decrease in lifespan coupled with an impairment in climbing ability. In supportive experiments, we employed the neuron-rich Drosophila compound eye to investigate subtle phenotypes that result from altered gene expression. The knockdown of BI-1 in the Drosophila developing eye under the direction of the GMR-Gal4 transgene results in reduced ommatidia number and increased disruption of the ommatidial array. Similarly, the co-expression of BI-1-RNAi with Buffy results in the suppression of the eye phenotypes. The expression of α-synuclein along with the knockdown of BI-1 resulted in reduction of ommatidia number and more disruption of the ommatidial array. CONCLUSION: Knockdown of BI-1 in the dopaminergic neurons of Drosophila results in a shortened lifespan and premature loss in climbing ability, phenotypes that appear to be strongly associated with models of PD in Drosophila, and which are suppressed upon overexpression of Buffy and worsened by co-expression with α-synuclein. This suggests that BI-1 is neuroprotective and its knockdown can be counteracted by the overexpression of the pro-survival Bcl-2 homologue.

Overexpression of Buffy enhances the loss of parkin and suppresses the loss of Pink1 phenotypes in Drosophila.

Mutations in parkin (PARK2) and Pink1 (PARK6) are responsible for autosomal recessive forms of early onset Parkinson's disease (PD). Attributed to the failure of neurons to clear dysfunctional mitochondria, loss of gene expression leads to loss of nigrostriatal neurons. The Pink1/parkin pathway plays a role in the quality control mechanism aimed at eliminating defective mitochondria, and the failure of this mechanism results in a reduced lifespan and impaired locomotor ability, among other phenotypes. Inhibition of parkin or Pink1 through the induction of stable RNAi transgene in the Ddc-Gal4-expressing neurons results in such phenotypes to model PD. To further evaluate the effects of the overexpression of the Bcl-2 homologue Buffy, we analysed lifespan and climbing ability in both parkin-RNAi- and Pink1-RNAi-expressing flies. In addition, the effect of Buffy overexpression upon parkin-induced developmental eye defects was examined through GMR-Gal4-dependent expression. Curiously, Buffy overexpression produced very different effects: the parkin-induced phenotypes were enhanced, whereas the Pink1-enhanced phenotypes were suppressed. Interestingly, the overexpression of Buffy along with the inhibition of parkin in the neuron-rich eye results in the suppression of the developmental eye defects.

The HtrA2 Drosophila model of Parkinson's disease is suppressed by the pro-survival Bcl-2 Buffy.

Mutations in High temperature requirement A2 (HtrA2), also designated PARK13, which lead to the loss of its protease activity, have been associated with Parkinson's disease (PD). HtrA2 is a mitochondrial protease that translocates to the cytosol upon the initiation of apoptosis where it participates in the abrogation of inhibitors of apoptosis (IAP) inhibition of caspases. Here, we demonstrate that the loss of the HtrA2 function in the dopaminergic neurons of Drosophila melanogaster results in PD-like phenotypes, and we attempt to restore the age-dependent loss in locomotor ability by co-expressing the sole pro-survival Bcl-2 homologue Buffy. The inhibition of HtrA2 in the dopaminergic neurons of Drosophila resulted in shortened lifespan and impaired climbing ability, and the overexpression of Buffy rescued the reduction in lifespan and the age-dependent loss of locomotor ability. In supportive experiments, the inhibition of HtrA2 in the Drosophila eye results in eye defects, marked by reduction in ommatidia number and increased disruption of the ommatidial array; phenotypes that are suppressed by the overexpression of Buffy.

Loss of porin function in dopaminergic neurons of Drosophila is suppressed by Buffy.

BACKGROUND: Mitochondrial porin, also known as the voltage-dependent anion channel (VDAC), is a multi-functional channel protein that shuttles metabolites between the mitochondria and the cytosol and implicated in cellular life and death decisions. The inhibition of porin under the control of neuronal Ddc-Gal4 result in short lifespan and in an age-dependent loss in locomotor function, phenotypes that are strongly associated with Drosophila models of Parkinson disease. METHODS: Loss of porin function was achieved through exploitation of RNA interference while derivative lines were generated by homologous recombination and tested by PCR. The UAS/Gal4 expression system was exploited with directed expression in neurons achieved with the use of the Dopa decarboxylase and in the developing eye with the Glass multiple reporter transgenes. Statistical analyses for ageing assay employed Log rank (Mantel-Cox) test, climbing indices were fitted with a non-linear curve and confidence intervals compared at 95%. Biometric analysis of the eye phenotypes was obtained by unpaired student T-test. RESULTS: The expression of α-synuclein in neuronal populations that include dopamine producing neurons under the control of Ddc-Gal4 produces a robust Parkinson disease model, and results in severely reduced lifespan and locomotor dysfunction. In addition, the porin-induced phenotypes are greatly suppressed when the pro-survival Bcl-2 homologue Buffy is overexpressed in these neurons and in the developing eye adding to the cellular advantages of altered expression of this anti-apoptotic gene. When we co-expressed α-synuclein along with porin, it results in a decrease in lifespan and impaired climbing ability. This enhancement of the α-synuclein-induced phenotypes observed in neurons was demonstrated in the neuron rich eye, where the simultaneous co-expression of porin-RNAi and α-synuclein resulted in an enhanced eye phenotype, marked by reduced number of ommatidia and increased disarray of the ommatidia. CONCLUSIONS: The inhibition of porin in dopaminergic neurons among others result in reduced lifespan and age-dependent loss in climbing ability, phenotypes that are suppressed by the overexpression of the sole pro-survival Bcl-2 homologue Buffy. The inhibition of porin phenocopies Parkinson disease phenotypes in Drosophila, while the overexpression of Buffy can counteract these phenotypes to improve the overall "healthspan" of the organism.

Bcl-2 homologue Debcl enhances α-synuclein-induced phenotypes in Drosophila.

BACKGROUND: Parkinson disease (PD) is a debilitating movement disorder that afflicts 1-2% of the population over 50 years of age. The common hallmark for both sporadic and familial forms of PD is mitochondrial dysfunction. Mammals have at least twenty proapoptotic and antiapoptotic Bcl-2 family members, in contrast, only two Bcl-2 family genes have been identified in Drosophila melanogaster, the proapoptotic mitochondrial localized Debcl and the antiapoptotic Buffy. The expression of the human transgene α-synuclein, a gene that is strongly associated with inherited forms of PD, in dopaminergic neurons (DA) of Drosophila, results in loss of neurons and locomotor dysfunction to model PD in flies. The altered expression of Debcl in the DA neurons and neuron-rich eye and along with the expression of α-synuclein offers an opportunity to highlight the role of Debcl in mitochondrial-dependent neuronal degeneration and death. RESULTS: The directed overexpression of Debcl using the Ddc-Gal4 transgene in the DA of Drosophila resulted in flies with severely decreased survival and a premature age-dependent loss in climbing ability. The inhibition of Debcl resulted in enhanced survival and improved climbing ability whereas the overexpression of Debcl in the α-synuclein-induced Drosophila model of PD resulted in more severe phenotypes. In addition, the co-expression of Debcl along with Buffy partially counteracts the Debcl-induced phenotypes, to improve the lifespan and the associated loss of locomotor ability observed. In complementary experiments, the overexpression of Debcl along with the expression of α-synuclein in the eye, enhanced the eye ablation that results from the overexpression of Debcl. The co-expression of Buffy along with Debcl overexpression results in the rescue of the moderate developmental eye defects. The co-expression of Buffy along with inhibition of Debcl partially restores the eye to a roughened eye phenotype. DISCUSSION: The overexpression of Debcl in DA neurons produces flies with shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with models of PD in Drosophila. The co-expression of Debcl along with α-synuclein enhanced the PD-like phenotypes. The co-expression of Debcl along with Buffy suppresses these phenotypes. Complementary experiments in the Drosophila eye show similar trends during development. Taken all together these results suggest a role for Debcl in neurodegenerative disorders.

The Bcl-2 homologue Buffy rescues α-synuclein-induced Parkinson disease-like phenotypes in Drosophila.

BACKGROUND: In contrast to the complexity found in mammals, only two Bcl-2 family genes have been found in Drosophila melanogaster including the pro-cell survival, human Bok-related orthologue, Buffy. The directed expression of α-synuclein, the first gene identified to contribute to inherited forms of Parkinson disease (PD), in the dopaminergic neurons (DA) of flies has provided a robust and well-studied Drosophila model of PD complete with the loss of neurons and accompanying motor defects. To more fully understand the biological basis of Bcl-2 genes in PD, we altered the expression of Buffy in the dopamine producing neurons with and without the expression of α-synuclein, and in the developing neuron-rich eye. RESULTS: To alter the expression of Buffy in the dopaminergic neurons of Drosophila, the Ddc-Gal4 transgene was used. The directed expression of Buffy in the dopamine producing neurons resulted in flies with increased climbing ability and enhanced survival, while the inhibition of Buffy in the dopaminergic neurons reduced climbing ability over time prematurely, similar to the phenotype observed in the α-synuclein-induced Drosophila model of PD. Subsequently, the expression of Buffy was altered in the α-synuclein-induced Drosophila model of PD. Analysis revealed that Buffy acted to rescue the associated loss of locomotor ability observed in the α-synuclein-induced model of PD, while Buffy RNA interference resulted in an enhanced α-synuclein-induced loss of climbing ability. In complementary experiments the overexpression of Buffy in the developing eye suppressed the mild rough eye phenotype that results from Gal4 expression and from α-synuclein expression. When Buffy is inhibited the roughened eye phenotype is enhanced. CONCLUSIONS: The inhibition of Buffy in DA neurons produces a novel model of PD in Drosophila. The directed expression of Buffy in DA neurons provide protection and counteracts the α-synuclein-induced Parkinson disease-like phenotypes. Taken all together this demonstrates a role for Buffy, a Bcl-2 pro-cell survival gene, in neuroprotection.