InteractiveFly: GeneBrief

48 related 2: Biological Overview | References

BIOLOGICAL OVERVIEW

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. This study investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD (Tas, 2018).

This study demonstrates that dFOXO is tissue-autonomously required for the maintenance of DA neurons in the PAM cluster during aging. Evidence is presented that dFOXO and FER2 act in parallel pathways to protect PAM neurons from different forms of cellular stress. However, dFOXO and FER2 partly share downstream pathways leading to the control of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results highlight the notion that multiple genetic and environmental risk factors interact and affect DA neuron survival. Importantly, genome-wide association studies (GWAS) and functional studies in mammals implicated FOXO family TFs, including FOXO1, FOXO3, FOXA1 and FOXA2, in the maintenance of DA neurons and in PD. The current results are in accordance with these studies and further suggest that dfoxo loss of function offers a valuable tool to study the pathogenesis of sporadic PD (Tas, 2018).

In mammals, although constitutive activation of FOXO3 induces loss of DA neurons in the SN, the expression of a dominant negative FOXO3 causes oxidative damage that leads to DA neuron loss. Nevertheless, both the dominant-negative form and mild activation of FOXO3 are neuroprotective in mice overexpressing α-Synuclein. Thus, FOXO3 can be protective or detrimental to DA neurons in the substantia nigra (SN) depending on its activity levels and genetic background. Likewise, in Drosophila, previous studies have shown paradoxical roles for dFOXO in the survival of DA neurons in various PD models. dFOXO overexpression has been shown to ameliorates mitochondrial abnormality and protects DA neurons in Pink1 null mutants. Conversely, dFOXO mediates the death of DA neurons by inducing apoptosis in DJ-1β loss-of-function mutants and in flies overexpressing dLRRK (Tas, 2018).

The apparent paradox concerning the role of FOXOs suggests that the activity of FOXO factors should be tightly regulated in order to exert neuroprotective function, i.e., activity levels of FOXO factors that are too high or too low are both detrimental to DA neurons. Alternatively, the differences in the reagents and experimental conditions used to examine the role of FOXOs in prior studies may have contributed to the differences in the interpretation of the results. A number of previous experiments in Drosophila studies mentioned above used global overexpression of dFOXO and tissues other than DA neurons, such as eyes, muscles and wings, were mainly analyzed to evaluate its effect. Furthermore, for dfoxo loss of function experiments, these studies used dfoxo²¹ or dfoxo²⁵, which contain nucleotide transversions resulting in premature stop codons but nevertheless are not null alleles (Tas, 2018).

The present study used a genuine null allele of dfoxo, dfoxo^Δ94, to examine whether endogenous dFOXO is protective or detrimental to DA neurons. The results demonstrating that dFOXO is protective to DA neurons in the PAM cluster under basal conditions are in accordance with a previous report. Curiously, however, that study observed the loss of DA neurons in the DL1 (dorso lateral 1) cluster, which corresponds to the PPL1 cluster in the nomenclature for adult DA neurons. Additionally, by PAM neuron-targeted constitutive and adult-restricted dfoxo RNAi, the current study shows that dFOXO expression within adult PAM neurons is required for the maintenance of PAM neurons in aged flies (Tas, 2018).

Overexpression of dfoxo in PAM neurons prevents the developmental impairment and age-dependent loss of PAM neurons in Fer2² mutants. Conversely, Fer2 overexpression ameliorates the effect of dfoxo^Δ94 mutation on the development and maintenance of PAM neurons. Since Fer2 and dfoxo do not transcriptionally regulate each other, the reciprocal rescue suggests that their downstream mechanisms partly overlap. In line with this interpretation, this study showed that autophagy and mitochondrial morphology are commonly impaired in PAM neurons of dfoxo^Δ94 and Fer2² mutants (Tas, 2018).

Mounting evidence indicates that FOXO factors regulate autophagy by controlling the expression of Atg genes in flies and mammals. FOXOs also regulate factors controlling mitophagy and mitochondrial remodeling in mammals. Therefore, dFOXO may regulate autophagy and mitophagy in PAM neurons, although dysregulation in autophagy and mitochondrial morphology in dfoxo^Δ94 could be secondary effects of cellular damage. Uncovering genetic pathways downstream of dFOXO and FER2 and how they intersect will yield valuable information, especially because the current results suggest that targeted overexpression of dfoxo or Fer2 in DA neurons may confer protection against DA neuron demise in various genetic models of PD (Tas, 2018).

Consistent with the known role of PAM neurons in controlling locomotion, startle-induced climbing ability in dfoxo^Δ94 and Fer2² mutants is significantly improved by the expression of dfoxo with R58E02-GAL4. However, R58E02>dfoxo does not rescue the shortened lifespan of dfoxo^Δ94 and even further reduces the lifespan of Fer2². Therefore, neuroprotective role of dFOXO is independent of its role in longevity regulation. Many fly models of PD show lifespan shortening, which is likely caused by the systemic effect of mitochondrial impairment and/or elevated oxidative stress levels rather than DA neuron demise. Lifespan shortening of dfoxo^Δ94 and Fer2² mutants may be similarly attributed to the impairment in mitochondrial biology or (in the case of Fer2²) oxidative stress regulation in cells other than PAM neurons (Tas, 2018).

Given that mitochondrial dysfunction and oxidative stress are tightly linked and both implicated in neurodegeneration, it is surprising that no evidence was found that PAM degeneration in dfoxo^Δ94 is associated with chronic or acute oxidative stress, unlike Fer2² mutants. The results also show no evidence that amino acid intake during adulthood is relevant for survival of PAM neurons. Then, how is dFOXO signaling activated during adulthood to promote PAM neuron survival in aged flies? Aging is associated with loss of proteostasis and FOXOs play a key role in cellular proteostasis. Consistent with the findings in other tissues, autophagy levels in PAM neurons decrease with age, and this is accelerated in dfoxo^Δ94. Thus, age-dependent decrease in basal activity of autophagy might be an intracellular stress signal that leads to the activation of dFOXO in PAM neurons (Tas, 2018).

This study reveals an unexpected crosstalk between two pathways mediated by two TFs, dFOXO and FER2, in the development and maintenance of DA neurons in the PAM cluster. Importantly, both genes are also required for the proper development of PAM neurons. This is in line with the fact that several mammalian TFs required for DA neuron development play critical roles in the maintenance of adult midbrain DA neurons. dFOXO homologs FOXA1 and A2 fall within this category, suggesting that TFs having dual roles in the development and maintenance of DA neurons is an evolutionarily conserved mechanism of neuroprotection. Furthermore, the data suggest that loss of dfoxo expression before adulthood has lasting detrimental effect on the survival of PAM neurons in aging flies, which may be partly regulated non-cell-autonomously by dFOXO in the larval fat body or in other tissues. In conclusion, this study provides a starting point to investigate TF networks underlying the link between aberrant neural development and neurodegeneration, which will present new opportunities to better understand the etiology of sporadic PD (Tas, 2018).

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. This study investigated whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, these results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival (Bou Dib, 2014).

Dopaminergic (DA) neurons play critical roles in motor control, cognition and motivation and are affected in many neurological and psychiatric disorders. The progressive degeneration of DA neurons in the substantia nigra pars compacta (SNc) is a principal pathological feature of Parkinson's disease (PD). PD is the most prevalent neurodegenerative movement disorder, for which no preventive or restorative therapies are available. The discovery of the genes associated with the rare familial forms of PD has led to the development of many animal models and advanced the understanding of PD pathogenesis. However, the majority of PD cases are sporadic and likely caused by a combination of environmental factors, such as pesticide exposure, and endogenous risk factors. These endogenous risk factors remain largely unknown. A recent meta-analysis on genome-wide association studies (GWAS) for PD showed that SNPs in the genes involved in multiple aspects of neural development are highly represented in sporadic PD patients, suggesting that genetic variations in these pathways may contribute to PD susceptibility. Indeed, several studies in mammals have shown the critical roles of developmental genes, such as Engrailed1, foxa2 and Nurr1, in the survival of DA neurons in old age. The identification and characterization of such genes may yield a better molecular understanding of adult-onset neurodegeneration in PD (Bou Dib, 2014).

The nervous system in invertebrate model organisms such as Drosophila and C.elegans shares many features with its mammalian counterpart and offers a powerful tool to study neural development and neurodegeneration. Drosophila DA neurons comprise multiple subclasses, some of which play roles similar to those played by the DA neurons in mammals, such as reward signaling and sleep regulation. The nematode C.elegans has 8 DA neurons, which are thought to have mechanosensory functions and have been shown to play a role in the modulation of locomotion. Despite advances in anatomical and functional characterization, the mechanisms underlying the development and maintenance of DA neurons in flies and worms are poorly understood. Drosophila Fer2, a homolog of mammalian p48/ptf1a, belongs to the bHLH-transcription factor family, which is often involved in neurogenesis and neural subtype specification. The mammalian p48 gene is a critical regulator for neural tube development (Meredith, 2009), in which a candidate causal SNP for PD has been detected. Previously, studies have shown that Fer2 is required for the development of a subclass of circadian clock neurons, ventral Lateral Neurons (LNvs) (Nagoshi, 2010). This study characterized additional roles of Fer2 to better understand the genetic mechanisms of neuronal subtype development and maintenance. Unexpectedly it was found that Fer2 is required for the development and maintenance of a subclass of DA neurons important for locomotion. Fer2 exerts its neuroprotective role in adulthood in the oxidative stress response, and loss of Fer2 expression in adulthood causes adult-onset progressive degeneration of these DA neurons. It was further demonstrated that the C. elegans homolog of p48, hlh-13, is also required for the survival of DA neurons in adult worms under oxidative stress. Collectively, these results established a conserved role of p48 homologs in protecting DA neurons from oxidative stress and degeneration (Bou Dib, 2014).

Many neurodegenerative disorders are multi-factorial, in which interactions between environmental and genetic factors play important causal roles. Oxidative stress has emerged as a major pathogenic factor for common neurodegenerative diseases, yet how such a ubiquitous phenomenon leads to the loss of selective neuronal populations remains unclear. This study presentd evidence that loss-of-function in p48 homologs in Drosophila and C. elegans renders DA neurons susceptible to degeneration under oxidative stress in adult animals. Interestingly, genome-wide association studies for PD have identified candidate causal SNPs in p48/ptf1a, suggesting the possibility that p48 loss-of-function may represent an as-yet-unknown genetic risk factor that increases susceptibility of DA neurons to environmental toxins also in mammals. Many familial PD-associated genes are widely expressed; nevertheless, mutations in these genes result in a selective loss of SNc DA neurons, suggesting that cell-type-specific factors, those similar to Fer2 and hlh-13, might contribute to the DA neuron vulnerability even in the familial PD cases. The identification of Fer2 and hlh-13 upstream and downstream pathways may thus shed light on the common mechanisms underlying the selective loss of DA neurons in diverse PD cases (Bou Dib, 2014).

The major cellular phenotype in Fer2¹ mutants was the developmental defects in 2 subsets of DA neurons, in addition to the developmental loss of LNvs, although the possibility cannot be excluded that other neuronal types are also affected. Judging from the results of the lineage-tracing experiments and the observation of DA neurons in the pupal brain, Fer2 is not a selector gene for dopaminergic phenotype in PAM/PAL neurons but is required for neurogenesis or survival of postmitotic neurons before phenotypic maturation. The notion that genes required for the development of DA neurons confer important roles in adult DA neuron survival has been postulated by several studies in mammals. Although the molecular mechanisms underlying their roles in adult neurons remain elusive, these developmental genes may actively control the genetic programs required for the maintenance of cell identity in adults. Findings on the Fer2's dual roles extend this notion to invertebrate nervous systems and underscore its significance (Bou Dib, 2014).

PAM neuron-targeted Fer2 knockdown induces PAM neuron degeneration and mitochondrial dysfunction within PAM neurons. These results indicate that mitochondrial dysfunction and cell death can be induced by a cell-autonomous reduction of Fer2 expression within the PAM cluster. On the other hand, ROS levels are increased brain-wide in the Fer2² flies, despite the fact that Fer2 expression is restricted to several clusters of cells in the brain. Thus, loss of Fer2 expression leads to both cell-autonomous and non-cell-autonomous consequences to the animal's well-being. How does the brain-wide ROS increase occur by Fer2 mutation although Fer2 is not expressed ubiquitously? An intriguing recent study in C. elegans demonstrated that mitochondrial perturbation in neuronal cells modulates mitochondrial stress response in distal tissues non-cell-autonomously (Durieux, 2011). Flies might exhibit similar non-cell-autonomous mitochondrial stress response that causes systemic ROS production. Systemic increase in oxidative stress is a clinical feature common to many aging-related neurological diseases including PD. Studies in mammals have documented that inflammation is a major factor mediating excessive ROS production and PD pathology. Activated microglia produces ROS and mediates DA neuron death. Dying DA neurons stimulate microglia, exacerbating the ROS production and DA neurodegeneration. As CNS glia in Drosophila are thought to possess immune-like function, similar mechanisms via inflammatory responses might mediate global elevation of ROS production in Fer2 mutants (Bou Dib, 2014).

Are the abnormal mitochondria in PAM neurons a cause or a consequence of the ROS upregulation? Because mitochondrial defects and excessive ROS production are inter-dependent, it is not possible to clarify the causality in the current study. However, because Fer2 expression is upregulated upon H₂O₂ treatment and the same acute H₂O₂ treatment triggers PAM neuron death in the absence of Fer2, the hypothesis is favored that Fer2 provides protection against oxidative stress rather than directly acting on mitochondria. These phenomena are remarkably similar in C. elegans; an acute H₂O₂ treatment upregulates hlh-13 expression and triggers DA neuron degeneration in hlh-13 null mutants. These data suggest that the oxidative stress response is an ancestral role of p48 homologs. Alternatively, hlh-13's roles in neural development in worms might have been taken over by other genes. Either way, these findings suggest that loss-of-function in Fer2 and hlh-13 can be used to study pathophysiology of DA neuron degeneration under oxidative stress. Interestingly, Fer2 mRNA levels remain upregulated at least up to 12 hr after the 24-hr H₂O₂ treatment, whereas hlh-13 mRNA levels return to the non-treated levels 2 hr after a brief H₂O₂ treatment. This difference in gene expression kinetics may reflect the duration of the H₂O₂ treatment, RNA stability, or difference in signal transduction mechanisms. Various stress response genes show highly restricted temporal expression upon stress, as the continuous activation of these genes are often detrimental to the cell. Initial upregulation of hlh-13 immediately after an acute oxidative stress might be necessary and sufficient to trigger the downstream genetic programs that continue to scavenge ROS and repair the cellular damages during the following days. Identification of the downstream genetic programs controlled by Fer2 and hlh-13 will be a key toward understanding the evolutionarily conserved mechanisms of neuroprotection (Bou Dib, 2014).

Dissecting differential gene expression within the circadian neuronal circuit of Drosophila

Behavioral circadian rhythms are controlled by a neuronal circuit consisting of diverse neuronal subgroups. To understand the molecular mechanisms underlying the roles of neuronal subgroups within the Drosophila circadian circuit, this study used cell-type specific gene-expression profiling and identified a large number of genes specifically expressed in all clock neurons or in two important subgroups. Moreover, two circadian genes, which are expressed specifically in subsets of clock cells and affect different aspects of rhythms, were identified and characterized. The transcription factor Fer2 is expressed in ventral lateral neurons; it is required for the specification of lateral neurons and therefore their ability to drive locomotor rhythms. The Drosophila melanogaster homolog of the vertebrate circadian gene nocturnin is expressed in a subset of dorsal neurons and mediates the circadian light response. The approach should also enable the molecular dissection of many different Drosophila neuronal circuits (Nogoshi, 2010).

Search PubMed for articles about Drosophila Fer2

Bou Dib, P., Gnagi, B., Daly, F., Sabado, V., Tas, D., Glauser, D. A., Meister, P. and Nagoshi, E. (2014). A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress. PLoS Genet 10(10): e1004718. PubMed ID: 25340742

Durieux, J., Wolff, S. and Dillin, A. (2011). The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell 144(1): 79-91. PubMed ID: 21215371

Iskusnykh, I. Y., Steshina, E. Y. and Chizhikov, V. V. (2016). Loss of Ptf1a leads to a widespread cell-fate misspecification in the brainstem, affecting the development of somatosensory and viscerosensory nuclei. J Neurosci 36(9): 2691-2710. PubMed ID: 26937009

Meredith, D. M., Masui, T., Swift, G. H., MacDonald, R. J. and Johnson, J. E. (2009). Multiple transcriptional mechanisms control Ptf1a levels during neural development including autoregulation by the PTF1-J complex. J Neurosci 29(36): 11139-11148. PubMed ID: 19741120

Mona, B., Avila, J. M., Meredith, D. M., Kollipara, R. K. and Johnson, J. E. (2016). Regulating the dorsal neural tube expression of Ptf1a through a distal 3' enhancer. Dev Biol 418(1): 216-225. PubMed ID: 27350561

Nagoshi, E., Sugino, K., Kula, E., Okazaki, E., Tachibana, T., Nelson, S. and Rosbash, M. (2010). Dissecting differential gene expression within the circadian neuronal circuit of Drosophila. Nat Neurosci 13(1): 60-68. PubMed ID: 19966839

Russ, J. B., Borromeo, M. D., Kollipara, R. K., Bommareddy, P. K., Johnson, J. E. and Kaltschmidt, J. A. (2015). Misexpression of ptf1a in cortical pyramidal cells in vivo promotes an inhibitory peptidergic identity. J Neurosci 35(15): 6028-6037. PubMed ID: 25878276

Tas, D., Stickley, L., Miozzo, F., Koch, R., Loncle, N., Sabado, V., Gnagi, B. and Nagoshi, E. (2018). Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons. PLoS Genet 14(3): e1007271. PubMed ID: 29529025

Willet, S. G., Hale, M. A., Grapin-Botton, A., Magnuson, M. A., MacDonald, R. J. and Wright, C. V. (2014). Dominant and context-specific control of endodermal organ allocation by Ptf1a. Development 141(22): 4385-4394. PubMed ID: 25371369

date revised: 25 July 2022

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