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Mutations in PTEN-induced kinase 1 (PINK1) cause autosomal recessive and early-onset Parkinson’s disease (PD). PINK1, a kinase involved in a mitochondrial quality control mechanism, acts by promoting the autophagic degradation of damaged mitochondria. Mutations in PINK1 lead to the accumulation of impaired mitochondria and the death of dopaminergic neurons. Folates act as single carbon donors in metabolic reactions such as nucleotide synthesis from purines. Oral folates are available in two forms, folic and folinic acid (FA and FiA, respectively). In Drosophila pink1 mutants, enhancing nucleotide biosynthesis via dietary supplementation with FA during development rescues mitochondrial function and leads to neuroprotection in adults. Orally available FiA bypasses the deconjugation and reduction steps required with FA and is more metabolically active. Here, we investigated the neuroprotective potential of dietary supplementation with FiA in adult pink1 mutant flies. We show that an FiA-enriched diet begun at early to middle stages of adulthood prevents the degeneration of dopaminergic neurons observed in pink1 mutants. An FiA-enriched diet might therefore delay or prevent the neuronal loss in patients with PINK1 mutations and may ameliorate other diseases linked to mitochondrial defects.
It is accepted that mitochondrial defects play an important role in the neuronal demise associated with Parkinson’s disease (PD, reviewed in). Mutations in PINK1, a mitochondrial kinase, lead to an early onset form of autosomal recessive Parkinsonism. PINK1 is involved in mitochondrial quality control (reviewed in). Loss of PINK1 activity results in mitochondrial dysfunction, increased production of toxic reactive oxygen species, and neuronal death (reviewed in). The fruit fly Drosophila melanogaster is a powerful model for exploring the mechanisms of PD-associated neurodegeneration. This invertebrate is also an excellent in vivo platform for testing chemicals with therapeutic potential (reviewed in). Drosophila pink1 mutants show an age dependent loss of dopaminergic neurons that can be blocked via an FA-supplemented diet beginning at early embryogenesis. In humans, FA is generally well absorbed, but the conversion of FA to its metabolically active coenzyme forms is complex (reviewed in). On the other hand, FiA is an immediate precursor of 5,10-methylene-tetrahydrofolate, and oral administration of FiA bypasses the chemical reactions required for the coenzyme conversion of FA. Additionally, unlike FA, dietary FiA might be readily available to the brain (reviewed in).
To determine whether a dietary supplement containing FiA in adult pink1 flies is neuroprotective.
Drosophila pink1 mutants have impaired mitochondria, resulting in pathologies in tissues with high energy requirements, such as skeletal muscles and brain. One of the most easily measurable features of pink1 mutant flies is the degeneration of their indirect flight muscles, which results in a defective (crushed) thorax phenotype in young (3 days) adults (Fig. 1A). The incidence of this crushed thorax in pink1 mutants flies was reported to be significantly decreased by maintaining these flies in a FA-supplemented diet beginning at early embryogenesis. We first evaluated whether FiA can also suppress the crushed thorax phenotype in pink1 mutants. We confirmed that dietary FA led to a reduction in the appearance of crushed thorax phenotype (from 83% to 63%; Fig. 1B) in pink1 mutants. Maintaining pink1 mutants from early embryonic stages on food containing an identical concentration of FiA (4 mM) also led to a significant reduction in the crushed thorax phenotype (from 83% to 61%; Fig. 1B). Taken together, these data show that diets supplemented with either FA or FiA can compensate for muscle degeneration in pink1 mutant flies.
Drosophila pink1 mutants exhibit an age-dependent loss of dopaminergic neurons in the protocerebral posterior lateral 1 (PPL1) cluster, and this loss is first detected in 30 day old adults (Fig. 1C-E). We next investigated whether both FA and FiA could suppress this neurodegeneration. pink1 mutant flies fed on FA since embryonic development are protected from the loss of PPL1 cluster neuronsA. We therefore tested whether an FiA-supplemented diet was also neuroprotective. A diet supplemented with either FA or FiA through embryonic development to adulthood led to significant neuroprotection (Fig. 1F). As the neuronal loss observed in PD is age-dependent, and first reported in 30 day old pink1 mutant flies, we tested how early the degeneration of the PPL1 cluster of neurons occurs. We first detected a significant loss of PPL1 cluster neurons in pink1 mutant flies in 20 day old adults (Fig. 1G). Next, we tested how early during adulthood the dietary supplementation with FiA could achieve neuroprotection in pink1 mutant flies. We began a diet of FiA-supplemented food either on day 1 or day 10 after eclosion of pink1 adults and quantified the loss of PPL1 neurons in 30 day old animals. These results showed that an FiA-supplemented diet when given as late as 10 days post-eclosion is sufficient to prevent neurodegeneration in pink1 mutants (Fig. 1H). Altogether, these findings reveal the neuroprotective potential of an FiA-supplemented diet when fed to Drosophila in a model of PD linked to mitochondrial dysfunction caused by pink1 mutations.
This study shows that FiA has therapeutic potential for neuroprotection in adult pink1 mutant flies.
We have used an insect model, Drosophila melanogaster, to gain initial insights into whether FiA supplementation has an in vivo neuroprotective potential in a PD model. In contrast to FA, dietary FiA has been proposed to readily cross the blood-brain barrier and might therefore be more capable of counteracting mitochondrial defects in neurons. The blood-brain barrier in Drosophila, an invertebrate, is exclusively formed by glial cells, similar to that present in lower vertebrates (reviewed in). To determine whether dietary FiA is capable of counteracting mitochondrial dysfunction in humans, it would be desirable to determine to what degree FiA can cross the blood-brain barrier in a mammalian model system such as a rodent (mouse or rat) before assessing the neuroprotective potential of FiA for neurons in the human central nervous system.
PD is a disabling disorder for which no cure is yet available; further, after dopaminergic neurons are lost, only a few palliative treatment options for PD symptoms are available. Therefore, treatments that either prevent or delay the onset of the disease at an early stage are needed. There is one active clinical trial of FA in PD (World Health Organization ID NCT01238926). FiA is already approved and used for applications in the clinic as an adjuvant during chemotherapy and can be administered orally, as a dietary supplement, or intravenously. Thus, the drug safety risk is low, and drug development for repurposing FiA as a treatment for PD would be faster than for a novel drug. With this in mind, it seems worthwhile to further test the supplementation of FiA in clinical trials as a potential preventative or palliative therapeutic for PD and to expand the repertoire of treatment options.
Genetics and Drosophila strains
Drosophila melanogaster stocks and crosses were maintained on standard cornmeal agar media at 25°C. The strains used were pink1B9 (a kind gift from A. Whitworth, MRC, Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, UK) and w1118 (Bloomington Stock Centre, Indiana University, Bloomington, IN). All analyses were performed using male flies.
FA (Sigma) and FiA (Sigma) were incorporated into the liquid fly food at 4 mM and left to set. This concentration was used because 4 mM FA was previously shown to have beneficial effects on pink1B9 mutant phenotypes when administered during development. Two different treatment protocols (Fig. 1F) were used based on the objectives of the study: (i) To test the effect of FiA and FA treatment throughout development, crosses between pink1B9 and w1118 were established on normal food and transferred to FiA- or FA-containing food after 2 days. Larvae were treated with FiA or FA throughout development. The adult flies were kept on drug-containing food throughout their lifespan. (ii) To test the effect of FiA treatment beginning at early to middle stages of adulthood, newly hatched adult male flies were collected from normal food and transferred to FiA-containing food at 1 or 10 days after eclosion. In both treatment conditions, adult flies were transferred to vials with fresh food every 2–3 days.
Defective thorax analysis
The depression in the flies’ upper thorax (defective or crushed thorax) was visually assessed as a binary assay on 1 day old males. A chi-squared test was performed to determine whether the degree (percentage) of defective thorax was significant in each population under analysis.
Analysis of dopaminergic neurons
Brains of 30 day old files (unless otherwise stated) were dissected and stained using anti-tyrosine hydroxylase (Immunostar, WI) to detect PPL1 cluster neurons as described previously. Brain samples were placed in PBS plus 0.1% Triton X-100 in a coverslip clamp chamber (ALA Scientific Instruments Inc., NY), positioned using a harp-shaped construction of platinum wire and nylon string and imaged using confocal microscopy. Tyrosine hydroxylase-positive PPL1 cluster neurons were counted in each brain hemisphere. Data acquired for the assessment of each genotype were obtained as a single experimental set before statistical analysis.
Statistical analyses were performed using GraphPad Prism 7 (www.graphpad.com). Data are presented as mean values with error bars indicating ±SD. The number of biological replicates per experimental variable (n) is indicated in the bars. Statistical significance was assessed using one-way ANOVA, two-way ANOVA, or a two-tailed paired t-test. The significance is indicated as * for p <0.05, ** for p <0.01 and *** for p <0.001.
This work was funded by the Medical Research Council.
We thank Amanda Lehmann (MRC Technology) for suggesting FiA as a possible therapeutic agent in a PD model.