The neuroprotective effect of human uncoupling protein 2 (hUCP2) requires cAMP-dependent protein kinase in a toxin model of Parkinson's disease.

Parkinson's disease (PD), caused by selective loss of dopaminergic (DA) neurons in the substantia nigra, is the most common movement disorder with no cure or effective treatment. Exposure to the mitochondrial complex I inhibitor rotenone recapitulates pathological hallmarks of PD in rodents and selective loss of DA neurons in Drosophila. However, mechanisms underlying rotenone toxicity are not completely resolved. We previously reported a neuroprotective effect of human uncoupling protein 2 (hUCP2) against rotenone toxicity in adult fly DA neurons. In the current study, we show that increased mitochondrial fusion is protective from rotenone toxicity whereas increased fission sensitizes the neurons to rotenone-induced cell loss in vivo. In primary DA neurons, rotenone-induced mitochondrial fragmentation and lethality is attenuated as the result of hucp2 expression. To test the idea that the neuroprotective mechanism of hUCP2 involves modulation of mitochondrial dynamics, we detect preserved mitochondrial network, mobility and fusion events in hucp2 expressing DA neurons exposed to rotenone. hucp2 expression also increases intracellular cAMP levels. Thus, we hypothesize that cAMP-dependent protein kinase (PKA) might be an effector that mediates hUCP2-associated neuroprotection against rotenone. Indeed, PKA inhibitors block preserved mitochondrial integrity, movement and cell survival in hucp2 expressing DA neurons exposed to rotenone. Taken together, we present strong evidence identifying a hUCP2-PKA axis that controls mitochondrial dynamics and survival in DA neurons exposed to rotenone implicating a novel therapeutic strategy in modifying the progression of PD pathogenesis.

A neuroprotective role of the human uncoupling protein 2 (hUCP2) in a Drosophila Parkinson's disease model.

Parkinson's disease (PD), caused by selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta, is the most common movement disorder. While its etiology remains unknown, mitochondrial dysfunction is recognized as one of the major cellular defects contributing to PD pathogenesis. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in neuroprotection in several neuronal injury models. Here we show that hucp2 expression in Drosophila DA neurons under the control of the tyrosine hydroxylase (TH) promoter protects those flies against the mitochondrial toxin rotenone-induced DA neuron death, head dopamine depletion, impaired locomotor activity and energy deficiency. Under normal conditions, hUCP2 flies maintain an enhanced locomotor activity and have higher steady-state ATP levels suggesting improved energy homeostasis. We show that while no increased mitochondrial DNA content or volume fraction is measured in hUCP2 flies, augmented mitochondrial complex I activity is detected. Those results suggest that it is increased mitochondrial function but not mitochondrial biogenesis that appears responsible for higher ATP levels in hUCP2 flies. Consistent with this notion, an up-regulation of Spargel, the Drosophila peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) homologue is detected in hUCP2 flies. Furthermore, a Spargel target gene Tfam, the mitochondrial transcription factor A is up-regulated in hUCP2 flies. Taken together, our results demonstrate a neuroprotective effect of hUCP2 in DA neurons in a Drosophila sporadic PD model. Moreover, as the TH promoter activity is present in both DA neurons and epidermis, our results reveal that hucp2 expression in those tissues may act as a stress signal to trigger Spargel activation resulting in enhanced mitochondrial function and increased energy metabolism.

Increased energy metabolism rescues glia-induced pathology in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by an expanded CAG tract within the coding region of Huntingtin protein. Mutant Huntingtin (mHtt) is ubiquitously expressed, abundantly in neurons but also significantly in glial cells. Neuron-intrinsic mechanism and alterations in glia-to-neuron communication both contribute to the neuronal dysfunction and death in HD pathology. However, it remains to be determined the role of glial cells in HD pathogenesis. In recent years, development of Drosophila models facilitated the dissection of the cellular and molecular events in polyQ-related diseases. By using genetic approaches in Drosophila, we manipulated the expression levels of mitochondrial uncoupling proteins (UCPs) that regulate production of both ATP and reactive oxygen species in mitochondria. We discovered that enhanced levels of UCPs alleviated the HD phenotype when mHtt was selectively expressed in glia, including defects in locomotor behavior and early death of Drosophila. In contrast, UCPs failed to prevent the HD toxicity in neurons. Increased oxidative stress defense was found to rescue neuron but not glia-induced pathology. Evidence is now emerging that UCPs are fundamental to adapt the energy metabolism in order to meet the metabolic demand. Thus, we propose that UCPs are glioprotective by rescuing energy-dependent functions in glia that are challenged by mHtt. In support of this, increasing glucose entry in glia was found to alleviate glia-induced pathology. Altogether, our data emphasize the importance of energy metabolism in the glial alterations in HD and may lead to a new therapeutic avenue.

Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly.

The oxidative stress hypothesis of aging predicts that a reduction in the generation of mitochondrial reactive oxygen species (ROS) will decrease oxidative damage and extend life span. Increasing mitochondrial proton leak-dependent state 4 respiration by increasing mitochondrial uncoupling is an intervention postulated to decrease mitochondrial ROS production. When human UCP2 (hUCP2) is targeted to the mitochondria of adult fly neurons, we find an increase in state 4 respiration, a decrease in ROS production, a decrease in oxidative damage, heightened resistance to the free radical generator paraquat, and an extension in life span without compromising fertility or physical activity. Our results demonstrate that neuronal-specific expression of hUCP2 in adult flies decreases cellular oxidative damage and is sufficient to extend life span.

Involvement of Drosophila uncoupling protein 5 in metabolism and aging.

A novel uncoupling protein, UCP5, has recently been characterized as a functional mitochondrial uncoupler in Drosophila. Here we demonstrate that UCP5 knockout (UCP5KO) flies are highly sensitive to starvation stress, a phenotype that can be reversed by ectopic neuronal expression of UCP5. UCP5KO flies live longer than controls on low-calorie diets, have a decreased level of fertility, and gain less weight than controls on high-calorie diets. However, isolated mitochondria from UCP5KO flies display the same respiration patterns as controls. Furthermore, total ATP levels in both UCP5KO and control flies are comparable. UCP5KO flies have a lower body composition of sugars, and during starvation stress their triglyceride reserves are depleted more rapidly than controls. Taken together, these data indicate that UCP5 is important to maintain metabolic homeostasis in the fly. We hypothesize that UCP5 influences hormonal control of metabolism.

Functional implications of Drosophila insulin-like peptides in metabolism, aging, and dietary restriction.

The neuroendocrine architecture and insulin/insulin-like signaling (IIS) events in Drosophila are remarkably conserved. As IIS pathway governs growth and development, metabolism, reproduction, stress response, and longevity; temporal, spatial, and nutrient regulation of dilps encoding Drosophila insulin-like peptides (DILPs) provides potential mechanisms in modulating IIS. Of eight DILPs (DILP1-8) identified, recent studies have furthered our understanding of physiological roles of DILP2, DILP3, DILP5, and DILP6 in metabolism, aging, and responses to dietary restriction (DR), which will be the focus of this review. While the DILP producing IPCs of the brain secrete DILP2, 3, and 5, fat body produces DILP6. Identification of factors that influence dilp expression and DILP secretion has provided insight into the intricate regulatory mechanisms underlying transcriptional regulation of those genes and the activity of each peptide. Studies involving loss-of-function dilp mutations have defined the roles of DILP2 and DILP6 in carbohydrate and lipid metabolism, respectively. While DILP3 has been implicated to modulate lipid metabolism, a metabolic role for DILP5 is yet to be determined. Loss of dilp2 or adult fat body specific expression of dilp6 has been shown to extend lifespan, establishing their roles in longevity regulation. The exact role of DILP3 in aging awaits further clarification. While DILP5 has been shown associated with DR-mediated lifespan extension, contradictory evidence that precludes a direct involvement of DILP5 in DR exists. This review highlights recent findings on the importance of conserved DILPs in metabolic homeostasis, DR, and aging, providing strong evidence for the use of DILPs in modeling metabolic disorders such as diabetes and hyperinsulinemia in the fly that could further our understanding of the underlying processes and identify therapeutic strategies to treat them.

Insulin injection and hemolymph extraction to measure insulin sensitivity in adult Drosophila melanogaster.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs). Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis. Here, we have modified a previously described procedure for insulin injection and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Adult Drosophila melanogaster as a model for the study of glucose homeostasis.

Genetic ablation of Drosophila melanogaster insulin-like peptide (DILP) and adipokinetic hormone-producing cells accompanied by cell biological and metabolic measurements have revealed functional conservation in nutrient sensing and the underlying signaling mechanisms between mammal and fruit fly. Despite significant advances gained in understanding the neuroendocrine responses to nutrient changes during developmental larval stages, we discuss here the need for investigating glucose homeostasis in the post-mitotic adult stage as the result of ablation of DILP producing cells (IPCs). Our recent studies demonstrate that while both constitutive and adult-specific partial ablation of IPCs renders those flies hyperglycemic and glucose intolerant, flies with adult-specific IPC ablation remain insulin sensitive. Our results substantiate a role of adult IPCs in modulating aspects of glucose homeostasis and highlight the complexity in DILP action in the adult fly.

Glucose increases activity and Ca2+ in insulin-producing cells of adult Drosophila.

We sought to understand the mechanisms underlying glucose sensing in Drosophila melanogaster. We found that insulin-producing cells (IPCs) of adult Drosophila respond to glucose and glibenclamide with a burst-like pattern of activity. Under controlled conditions IPCs have a resting membrane potential of -62+/-4 mV. In response to glucose or glibenclamide, IPCs generate action potentials at a threshold of -36+/-1.4 mV with an amplitude of 46+/-4 mV and width of 9.3+/-1.8 ms. Real-time Ca imaging confirms that IPCs respond to glucose and glibenclamide with increased intracellular Ca. These results provide the first detailed characterization of electrical properties of IPCs of adult Drosophila and suggest that these cells sense glucose by a mechanism similar to mammalian pancreatic ß cells.

Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance.

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic beta cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.

Increased uncoupling protein (UCP) activity in Drosophila insulin-producing neurons attenuates insulin signaling and extends lifespan.

To understand the role of mitochondrial uncoupling protein (UCP) in regulating insulin signaling and glucose homeostasis, we created transgenicDrosophila lines with targeted UCP expression in insulin producing cells (IPCs). Increased UCP activity in IPCs results in decreased steady state Ca(2+) levels in IPCs as well as decreased PI3K activity and increased FoxO nuclear localization in periphery. This reduced systemic insulin signaling is accompanied by a mild hyperglycemia and extended life span. To test the hypothesis that ATP-sensitive potassium (K(ATP)) channels may link changes in metabolic activity (e.g., glucose mediated ATP production or UCP-mediated ATP reduction) with insulin secretion, we characterized the effects of glucose and a specific K(ATP) channel blocker, glibenclamide on membrane potential in adult IPCs. Exposure to glucose depolarizes membrane potential of IPCs and this effect is mimicked with glibenclamide, suggesting that K(ATP) channels contribute to the mechanism whereby IPCs sense changes in circulating sugar. Further, as demonstrated in mammalian beta-pancreatic cells, high glucose initiates a robust Ca(2+) influx in adult IPCs. The presence of functional K(ATP) channels in adult IPCs is further substantiated by in situ hybridization detecting the transcript for the sulfonylurea receptor (Sur) subunit of the K(ATP) channel in those cells. Quantitative expression analysis demon-strates a reduction in transcripts for both Sur and the inward rectifying potassium channel (Kir) subunits when IPCs are partially ablated. In summary, we have demonstrated a role for UCP in adult Drosophila IPCs in influencing systemic insulin signaling and longevity by a mechanism that may involve K(ATP) channels.

Functional characterization of a Drosophila mitochondrial uncoupling protein.

Sequence alignment of conserved signature motifs predicts the existence of the uncoupling protein 5 (UCP5)/brain mitochondrial carrier protein (BMCP1) homologue in Drosophila melanogaster. Here we demonstrate the functional characterization of the Drosophila melanogaster UCP5 protein (DmUCP5) in the heterologous yeast system, the first insect UCP reported to date. We show that physiological levels of DmUCP5 expression are responsible for an increase in state 4 respiration rates and a decrease in mitochondrial membrane potential. Furthermore, similar to UCP1, UCP2, and UCP3, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by the purine nucleotide GDP. Thus, DmUCP5 shares the mechanisms known to regulate the UCPs characterized to date. A lack of growth inhibition observed in DmUCP5 expressing yeast is consistent with the notion that physiological uncoupling has a minimal effect on cell growth. Finally, semiquantitative RT-PCR analysis shows a distinctive pattern of DmUCP5 expression predominantly localized in the adult head, similar to the expression pattern of its mammalian homologues. The conserved regulation of the expression of this gene from mammals to fruit flies suggests a role for UCP5 in the brain.