Abstract
The causes of sporadic Parkinson’s Disease (PD) are still unclear, despite its prevalence. By contrast, inherited parkinsonian disorders have a clear genetic basis and have been studied intensively in laboratory organisms, including Drosophila melanogaster. Because inherited parkinsonian disorders clinically resemble sporadic PD, it has been suggested that they may share an underlying etiology. Loss of function mutations in the gene PLA2G6 give rise to inherited neurodegenerative diseases including autosomal recessive early onset parkinsonism (PARK14). Using RNAi to deplete the Drosophila PLA2G6 homolog iPLA2-VIA, we asked whether subsets of neurons, identified by their neurotransmitter usage, were more susceptible to loss of this gene. To model movement disorders connected with PLA2G6-associated neurodegeneration, we used the well-established climbing assay. Our results demonstrated that loss of iPLA2-VIA in GABAergic neurons alone strongly affected locomotor ability in aged flies, similar to pan-neuronal knockdown. Depletion of iPLA2-VIA in both dopaminergic and serotonergic neurons weakly affected locomotor ability as well. Depletion in other neuronal subsets did not disrupt locomotion. Furthermore, reintroducing wild-type iPLA2-VIA into only the dopaminergic neurons of fly knockouts improved climbing performance slightly, while reintroduction into GABAergic neurons rescued climbing performance strikingly, as well as lifespan. Although much research on this gene has focused on the dopaminergic neurons, whose degeneration leads to clinical parkinsonism, our results highlight the importance of GABAergic neurons to PLA2G6-associated neurodegeneration. Because sporadic PD is not thought to impact most GABAergic neurons directly, our data support the idea that sporadic PD and PARK14 have distinct etiologies despite overlapping clinical presentations.
Introduction
Parkinson’s disease (PD) is one of the most prevalent and fastest growing neurodegenerative disorders worldwide (Dorsey and Bloem, 2018; Tanner and Ostrem, 2024). The disease is characterized and diagnosed by its four cardinal motor symptoms: bradykinesia, resting tremor, muscular rigidity, and gait and/or postural disturbances (Jankovic, 2008), which arise from death of the dopamine producing neurons in the substantia nigra pars compacta (SNpc; Dickson, 2012). Available treatments are palliative and typically involve strategies to increase dopamine levels in the brain (Kalia and Lang, 2015; Hayes, 2019; Politis et al., 2012). A major goal for the 21st century is to develop treatments that can slow, halt, or reverse disease progression (Dorsey and Bloem, 2018; Vijiaratnam et al., 2021).
The cytopathological hallmarks of PD are Lewy bodies (LBs) and Lewy neurites (LNs), intraneuronal inclusions that are characterized by the presence of the presynaptic protein α-synuclein (α-syn; Choong and Mochizuki, 2022; Spillantini et al., 1997). Detailed post-mortem analysis has revealed the presence of α-syn+ LBs and LNs in numerous regions of the central and peripheral nervous system beyond the dopaminergic SNpc neurons (Braak et al., 2003; Beach et al., 2009; Braak et al., 2006), consistent with the fact that PD patients usually present with additional non-motor symptoms, including sleep disruption, mood swings, sensory loss (especially smell), dementia, and disturbances in autonomic nervous system function (Jankovic, 2008; Blesa et al., 2022; Horsager et al., 2022). Still, Lewy pathology appears to be limited to select neuronal populations, the mechanistic basis of whose vulnerability is not fully elucidated (Surmeier et al., 2017; Braak et al., 2003; Walsh and Selkoe, 2016). The fact that α-syn is an intrinsically disordered protein that can form oligomers and amyloid fibrils in vitro and in vivo has led to current models suggesting that PD is caused by α-syn aggregation and seeding of LBs and LNs, which then cause neuronal death in the SNpc and elsewhere (Iwai et al., 1995; Weinreb et al., 1996; Spillantini et al., 1998; Goldberg and Lansbury, 2000; Wong and Krainc, 2017; Rietdijk et al., 2017; Jucker and Walker, 2013; Mahul-Mellier et al., 2020). Furthermore, disease progression has been suggested to result from spreading of Lewy pathology between neuron types and brain regions (Braak et al., 2003; Klingelhoefer and Reichmann, 2015). The mechanisms that lead to α-syn aggregation, Lewy pathology, spreading, and neuronal death are still poorly understood (Surmeier et al., 2017; Burke et al., 2008; Jellinger, 2009).
Although PD etiology is obscure, a genetic contribution is suggested by the existence of familial parkinsonian disorders, several of which are clinically indistinguishable from sporadic PD (Guadagnolo et al., 2021; Trinh and Farrer, 2013; Miki et al., 2017). Genetic analysis of these disorders has led to the identification of more than 20 causative “PARK” genes (Guadagnolo et al., 2021; Ayajuddin et al., 2018; Deng et al., 2018). The fact that human variants in the α-syn gene SNCA (PARK1), or multiplications of the SNCA locus, underpin autosomal dominant parkinsonism supports a common etiology between sporadic PD and inherited parkinsonian disorders, and there is mounting evidence that other PARK gene variants also contribute to sporadic PD (Jankovic, 2008; Kim et al., 2024; Nalls et al., 2011; Do et al., 2011). Thus, it is expected that investigating the PARK genes will be relevant to understanding the development and progression of sporadic PD as well as inherited parkinsonism (Miki et al., 2017). The presence of PARK gene orthologs in other animal species has permitted development of numerous laboratory parkinsonism models, including in Drosophila melanogaster, which have yielded important insights into various cellular and molecular aspects of parkinsonian diseases (Ayajuddin et al., 2018; Hewitt and Whitworth, 2017). Nonetheless, the precise relationship between the various inherited parkinsonian disorders and sporadic PD still is unclear.
Mutations in the group 6 phospholipase A2 gene PLA2G6 were identified in 2009 as the underlying cause of a familial inherited dystonia-parkinsonism disorder (Paisan-Ruiz et al., 2009; Sina et al., 2009). Since then, this gene also has been associated with autosomal recessive early onset parkinsonism and has been designated PARK14 (Paisán-Ruiz et al., 2010; Shi et al., 2011; Guo et al., 2018; Chu et al., 2020). Five groups have independently generated Drosophila models of PLA2G6-associated neurodegeneration with loss of function mutations in the fly ortholog iPLA2-VIA (Mori et al., 2019; Banerjee et al., 2021; Lin et al., 2018; Kinghorn et al., 2015; Iliadi et al., 2018). Loss of iPLA2-VIA leads to a constellation of neurodegenerative signs and symptoms, including several that have been documented in other Drosophila parkinsonism models such as loss of the dopaminergic neurons (Mori et al., 2019) and age-dependent loss of locomotor ability, observable in negative geotaxis climbing assays (Mori et al., 2019; Banerjee et al., 2021; Kinghorn et al., 2015; Iliadi et al., 2018). Furthermore, loss of iPLA2-VIA in Drosophila has been proposed to exacerbate α-syn aggregation, apparently linking this gene to development of sporadic PD (Mori et al., 2019).
Neurodegeneration in iPLA2-VIA mutants is not limited to the dopaminergic neurons. Prior reports have documented cell death throughout the brain (Iliadi et al., 2018) and degeneration of photoreceptor neurons in the retinas (Lin et al., 2018) of Drosophila iPLA2-VIA mutants. In order to better understand the extent of neuronal sensitivity to loss of iPLA2-VIA, we used RNAi to knock down the gene in subsets of neurons, defined by their neurotransmitter usage (Deng et al., 2019). To monitor the effect of our manipulations, we used the climbing assay, which is a well-established behavioral read-out of neurodegeneration (Barone and Bohmann, 2013). After confirming prior reports that pan-neuronal knockdown of iPLA2-VIA can phenocopy the climbing defect of the knockout mutant (Banerjee et al., 2021; Iliadi et al., 2018), we demonstrated that knockdown in GABAergic neurons alone can fully phenocopy the strong age-dependent climbing defect. By contrast, knockdown in octopaminergic-tyraminergic, cholinergic, or glutamatergic neurons did not affect climbing ability up to 30 days of age. Knockdown in dopaminergic and serotonergic neurons produced a small age-dependent climbing defect that was much weaker than that produced by knockdown in GABAergic neurons. Moreover, reintroducing wild-type iPLA2-VIA into GABAergic neurons of the mutant strongly rescued defective climbing, while reintroduction in dopaminergic neurons rescued weakly. Reintroducing wild-type iPLA2-VIA into GABAergic neurons also rescued the lifespan of the mutants. Altogether, our data indicate that GABAergic neurons are an important site of iPLA2-VIA neuroprotective activity, that severe locomotor defects arise when the gene is lost from these cells, and that restoring wild-type iPLA2-VIA in these cells can slow age-dependent loss of locomotor ability and death. This is consistent with clinical reports of patients suffering from PLA2G6-associated neurodegeneration (PLAN), who consistently show high penetrance degeneration of GABAergic cerebellar tissue (Guo et al., 2018; Gregory et al., 2008; Salih et al., 2013). However, this stands in contrast to sporadic PD, in which predominantly GABAergic regions such as the cerebellum and globus pallidus are largely spared from degeneration (Dickson, 2012; Braak et al., 2003).
Materials and methods
Fly stocks and culture
Drosophila were raised on standard media. Experimental crosses and F1 cohorts were kept at 26 °C under 12 h L:D cycle. Fly strains were w1118 (BDSC_5905), HMS01544 (BDSC_36129),yw (BDSC_6599), yv; attP2 (BDSC 36303), elav-GAL4 (BDSC_458), ple-GAL4 (BDSC_8848), Vesicular GABA Transporter (VGAT)-GAL4 (BDSC_58980), Dopa decarboxylase (Ddc)-GAL4 (BDSC_7010), Tryptophan hydroxylase (Trh)-GAL4 (BDSC_38388), Tyrosine decarboxylase 2 (Tdc2)-GAL4 (BDSC_9313), Choline Acetyltransferase (ChAT)-GAL4 (BDSC_56500), and Vesicular glutamate transporter (VGluT)-GAL4 (BDSC_26160) from Bloomington Drosophila Stock Center. We used FlyBase to find information on the stocks listed above. The iPLA2-VIA knockout mutant ∆23 and the UAS-iPLA2-VIA-PB line were described previously (Banerjee et al., 2021).
Climbing assays
F1 flies were collected every 2–3 days, males were grouped into cohorts of up to 12 flies, and they were transferred to new food every 5 days during aging. Climbing assays were conducted on each group at 10, 20, and 30 days from eclosion and were performed at room temperature as described in Banerjee et al. (2021). Briefly, each fly cohort was transferred to a fresh food vial and a clean empty vial was placed on top of the food vial. Flies were tapped to the bottom and given 20 s to climb 6 cm. Each assay consisted of five climbing trials. Each fly earned one point for successfully climbing to or past the 6 cm mark within 20 s. The total number of points for the group was divided by the number of flies in the group to determine the climbing index. Climbing indices were assessed for normality using Shapiro–Wilk test, and statistical comparisons were made using two-way ANOVA and Tukey’s post-hoc comparisons, performed in R. Graphs were made in R with ggplot2. Graphs show average climbing index per condition, error bars are SEM. At least 8 groups were sampled per condition. Genotypes and sample sizes are given in Table 1.
Table 1
| 10 days | 20 days | 30 days | ||||
|---|---|---|---|---|---|---|
| Genotype | Groups | Flies | Groups | Flies | Groups | Flies |
| Knockdown climbing | ||||||
| VGAT-GAL4/+ | 8 | 71 | 11 | 90 | 11 | 90 |
| VGAT-GAL4/+; UAS-iPLA2-VIA-RNAi/+ | 8 | 79 | 9 | 86 | 10 | 91 |
| Ddc-GAL4/+ | 15 | 133 | 15 | 128 | 14 | 97 |
| Ddc-GAL4/+; UAS-iPLA2-VIA-RNAi/+ | 15 | 133 | 15 | 128 | 15 | 114 |
| elav-GAL4/Y | 20 | 193 | 17 | 164 | 16 | 153 |
| elav-GAL4/Y; UAS-iPLA2-VIA-RNAi/+ | 17 | 186 | 17 | 182 | 16 | 170 |
| ple-GAL4/+ | 10 | 99 | 10 | 98 | 10 | 98 |
| ple-GAL4/UAS-iPLA2-VIA-RNAi | 10 | 95 | 9 | 84 | 9 | 73 |
| Trh-GAL4/+ | 10 | 102 | 10 | 93 | 10 | 97 |
| Trh-GAL4/UAS-iPLA2-VIA-RNAi | 12 | 127 | 12 | 115 | 12 | 112 |
| Tdc2-GAL4/+ | 16 | 125 | 16 | 110 | 12 | 88 |
| Tdc2-GAL4/+; UAS-iPLA2-VIA-RNAi/+ | 15 | 136 | 15 | 129 | 15 | 114 |
| ChAT-GAL4/+ | 12 | 114 | 12 | 105 | 11 | 96 |
| ChAT-GAL4/+; UAS-iPLA2-VIA-RNAi/+ | 12 | 119 | 11 | 104 | 10 | 96 |
| VGlut-GAL4/+ | 10 | 85 | 9 | 63 | 9 | 60 |
| VGlut-GAL4/+; UAS-iPLA2-VIA-RNAi/+ | 17 | 147 | 16 | 127 | 16 | 122 |
| 15 days | 20 days | |||
|---|---|---|---|---|
| Groups | Flies | Groups | Flies | |
| Climbing rescue | ||||
| VGAT-GAL4/+; iPLA2-VIA∆23/∆23 | 12 | 84 | 11 | 76 |
| VGAT-GAL4/UAS-iPLA2-VIA-PB; iPLA2-VIA∆23/∆23 | 13 | 85 | 13 | 83 |
| ple-GAL4, iPLA2-VIA∆23/+, iPLA2-VIA∆23 | 14 | 113 | 14 | 111 |
| UAS-iPLA2-VIA-PB/+; ple-GAL4, iPLA2-VIA∆23/+, iPLA2-VIA∆23 | 14 | 113 | 14 | 102 |
| Flies | ||||
| Lifespan rescue | ||||
| VGAT-GAL4/+ | 109 | |||
| VGAT-GAL4/+; iPLA2-VIA∆23/∆23 | 66 | |||
| VGAT-GAL4/UAS-iPLA2-VIA-PB; iPLA2-VIA∆23/∆23 | 104 | |||
Genotypes and samples sizes.
Lifespan assay
F1 flies were collected every 2–3 days without gas. After 2–3 days, F1 males of the appropriate genotype were separated over light gas and grouped into cohorts of up to 15 flies per vial. Thereafter, F1 cohorts were passed without gas to fresh vials every 2–3 days. Censors (escapers) and deaths were recorded at each pass. Adapted from Delventhal et al. (2022) and Piper and Partridge (2016). Kaplan–Meier curves were generated in R. Statistical comparison by log-rank test. Genotypes and sample sizes are given in Table 1.
Results
Knocking down iPLA2-VIA in GABAergic or dopaminergic and serotonergic neurons leads to age-dependent climbing defects
iPLA2-VIA loss of function leads to reduced climbing ability after 20 days of age post-eclosion (Banerjee et al., 2021; Kinghorn et al., 2015; Iliadi et al., 2018). To determine which neurons are sensitive to the loss of iPLA2-VIA, we knocked down the gene in discrete neuronal subsets according to their neurotransmitter production. We used well-characterized GAL4 drivers that target distinct neuronal populations in the adult fly nervous system using regulatory sequences from genes involved in neurotransmitter synthesis or transport (Deng et al., 2019; Chen et al., 2013). Climbing assays were conducted at 10, 20, and 30 days post-eclosion.
We confirmed that pan-neuronal knockdown of iPLA2-VIA using the elav-GAL4 driver produced a strong climbing defect at 30 days of age, as demonstrated previously (Figure 1A, gray bars; Banerjee et al., 2021; Kinghorn et al., 2015; Iliadi et al., 2018). This effect was age-dependent, as it is for the iPLA2-VIA knockout mutant. To knock down iPLA2-VIA in only GABAergic neurons, we used the Vesicular GABA Transporter (VGAT)-GAL4 driver (Fei et al., 2010), which resulted in a strong age-dependent climbing defect that phenocopied the effect observed with pan-neuronal knockdown (Figure 1B, light blue bars).
Figure 1
Knocking down iPLA2-VIA in GABAergic or dopaminergic and serotonergic neurons leads to age-dependent climbing defects. (A) Climbing ability was dramatically reduced in flies expressing iPLA2-VIA RNAi with the pan-neuronal driver elav-GAL4 (gray) at day 30, while climbing ability was maintained in age-matched control flies expressing elav-GAL4 alone (black). p = 3.77 × 10−10 for 30 day knockdown (KD) compared to 30 day control. (B) Climbing ability was dramatically reduced in flies expressing iPLA2-VIA RNAi with the GABAergic driver VGAT-GAL4 (light blue) at day 30 compared to age-matched control flies carrying VGAT-GAL4 alone (dark blue), phenocopying the effect seen with pan-neuronal knockdown. p = 2.49 × 10−11 for 30 day KD compared to 30 day control. Note that 30 day VGAT-GAL4 control flies had slightly reduced climbing ability compared to 10 day VGAT-GAL4 control flies, p = 0.00727, but neither age point was significantly different from 20 day VGAT-GAL4 control flies. (C) Flies expressing iPLA2-VIA RNAi in dopaminergic and serotonergic neurons with Ddc-GAL4 showed reduced climbing ability at day 30 (light red), while age-matched control flies carrying Ddc-GAL4 alone did not lose climbing ability (dark red). p = 2.12 × 10−4 for 30 day KD compared to 30 day control. (D) Flies expressing iPLA2-VIA RNAi in serotonergic neurons with Trh-GAL4 had reduced climbing ability at day 30 (light purple), while age-matched control flies carrying Trh-GAL4 alone retained climbing ability (dark purple). p = 2.57 × 10−5 for 30 day KD compared to 30 day control.
Reports from multiple model systems have documented the sensitivity of dopaminergic neurons to iPLA2-VIA loss of function (Mori et al., 2019; Sanchez et al., 2018; Chiu et al., 2019). We therefore used the Dopa decarboxylase (Ddc)-GAL4 driver to knock down iPLA2-VIA (Li et al., 2000), which caused a small but significant age-dependent loss of climbing ability as expected (Figure 1C, light red bars). Because Ddc-GAL4 is expressed in both dopaminergic and serotonergic neurons, we established two separate sets of crosses using either pale (ple)-GAL4 to target iPLA2-VIA in dopaminergic neurons only (Friggi-Grelin et al., 2003) or the Tryptophan hydroxylase (Trh)-GAL4 driver for serotonergic neurons only (Alekseyenko et al., 2010). Knockdown in serotonergic neurons caused a similar age-dependent loss of climbing ability (Figure 1D, light purple bars), but surprisingly, knockdown in dopaminergic neurons alone did not reduce climbing ability (Supplementary Figure S1A). Thus, our data suggest that loss of iPLA2-VIA in serotonergic neurons, and likely also dopaminergic neurons, leads to age-dependent climbing defects.
Knocking down iPLA2-VIA in octopaminergic and tyraminergic, cholinergic, or glutamatergic neurons does not lead to age-dependent climbing defects
In the same study, we tested an additional three neuronal subsets. We knocked down iPLA2-VIA in octopaminergic and tyraminergic neurons using Tyrosine decarboxylase 2 (Tdc2)-GAL4 (Cole et al., 2005), in cholinergic neurons using Choline Acetyltransferase (ChAT)-GAL4 (Salvaterra and Kitamoto, 2001), or in glutamatergic neurons using Vesicular Glutamate Transporter (VGluT)-GAL4 (Daniels et al., 2008). None of these manipulations caused climbing defects (Supplementary Figures S1B–D).
Restoring iPLA2-VIA in GABAergic or dopaminergic neurons rescues climbing defects
The above experiments showed that iPLA2-VIA is necessary in GABAergic neurons, and to a lesser extent in dopaminergic and serotonergic neurons, to maintain normal climbing ability with age. To assess whether iPLA2-VIA is sufficient in these neurons, we used GAL4 to express a UAS-driven wild-type iPLA2-VIA-PB transgene in the iPLA2-VIA∆23 mutant, as described previously (Banerjee et al., 2021). Note that in wild-type flies with iPLA2-VIA knockdown, climbing defects were not evident until after 20 days of age. By contrast, iPLA2-VIA∆23 mutant flies showed strong climbing defects at 20 days of age at 26 °C. This discrepancy likely is due to the pleiotropic effect of losing iPLA2-VIA from multiple vulnerable tissue types in the mutant (Banerjee et al., 2021). Nevertheless, despite the requirement for iPLA2-VIA in multiple tissue types, wild-type iPLA2-VIA expressed in the GABAergic neurons of mutant flies using the VGAT-GAL4 driver demonstrated a striking rescue of climbing ability at 20 days of age (Figure 2A, dark blue bars). Expressing wild-type iPLA2-VIA in dopaminergic neurons using the ple-GAL4 driver also improved deficient climbing of the mutant at both 15 and 20 days of age, albeit to a weaker extent than expression in GABAergic neurons (Figure 2B, dark red bars). Together, our knockdown and rescue results show that iPLA2-VIA is necessary and sufficient in GABAergic neurons for protection from age-dependent loss of climbing ability.
Figure 2
iPLA2-VIA knockout mutants are rescued by expression of wild-type iPLA2-VIA in GABAergic or dopaminergic neurons. (A)iPLA2-VIA∆23 flies carrying the GABAergic driver VGAT-GAL4 showed dramatically reduced climbing ability at 20 days of age at 26 °C (light blue). This was rescued strongly by expression of UAS-iPLA2-VIA-PB (dark blue), p = 2.22 × 10−9. For 15 day rescue versus 15 day control, p = 0.260 (not significant). (B)iPLA2-VIA∆23 flies carrying the dopaminergic driver ple-GAL4 showed reduced climbing ability at 20 days of age at 26 °C (light red), which was rescued weakly by expression of UAS-iPLA2-VIA-PB (dark red), p = 0.0597. iPLA2-VIA∆23 flies carrying ple-GAL4 also showed slightly reduced climbing ability at 15 days of age, which was improved by expression of UAS-iPLA2-VIA-PB, p = 0.00221. (C)iPLA2-VIA∆23 mutant flies (light blue, carrying VGAT-GAL4) had severely reduced lifespans compared to control flies (black, VGAT-GAL4/+) at 26 °C. iPLA2-VIA∆23 mutants expressing UAS-iPLA2-VIA-PB with VGAT-GAL4 (dark blue) showed significantly improved survival, p = 7.82 × 10−7 for rescued flies compared to mutant flies by log-rank test. Age in days post-eclosion. Censors indicated by “|”.
Restoring iPLA2-VIA in GABAergic neurons rescues lifespan
Loss of function iPLA2-VIA mutants have severely shortened lifespans, a common symptom of neurodegeneration (Lin et al., 2018; Kinghorn et al., 2015; Iliadi et al., 2018). Therefore, as an additional test for the importance of GABAergic neurons, we assessed whether restoring wild-type iPLA2-VIA-PB in GABAergic neurons only could rescue the lifespan of iPLA2-VIA∆23 mutants. Notably, rescued mutant flies carrying VGAT-GAL4 and the wild-type iPLA2-VIA-PB transgene had significantly improved survival compared to mutant flies carrying just VGAT-GAL4 (Figure 2C), affirming that GABAergic neurons are a critical cell type in PLAN.
Discussion
In this study, we examined the effect of knocking down iPLA2-VIA in different neuron types, classified by their neurotransmitter usage, on age-dependent climbing ability. Our results identify GABAergic neurons as a key neuronal population in PLA2G6-associated neurodegeneration. Depletion of iPLA2-VIA expression in GABAergic neurons using RNAi led to severe age-dependent climbing defects similar to those caused by depleting iPLA2-VIA pan-neuronally (Figures 1A,B), and restoration of a wild-type iPLA2-VIA allele in GABAergic neurons led to robust rescue of both climbing ability and lifespan in the iPLA2-VIA knockout mutant (Figures 2A,C). Age-dependent loss of climbing ability also was observed when iPLA2-VIA was knocked down in dopaminergic and serotonergic neurons with Ddc-GAL4 (Figure 1C), and restoring the wild-type allele in dopaminergic neurons alone with ple-GAL4 modestly rescued climbing in the mutant (Figure 2B). These data are consistent with previous studies indicating a requirement for iPLA2-VIA in dopaminergic neurons (Mori et al., 2019; Sanchez et al., 2018; Chiu et al., 2019). Furthermore, our results showing that iPLA2-VIA knockdown with Trh-GAL4 reduced climbing ability suggest that serotonergic neurons are important as well (Figure 1D). Knockdown in other neuronal populations, i.e., octopaminergic and tyraminergic, cholinergic, and glutamatergic neurons, did not affect climbing (Supplementary Figures S1B–D).
Limitations of the study
The negative geotaxis climbing assay has been a mainstay of Drosophila behavioral genetics for decades and has been used extensively to monitor neurodegeneration (Barone and Bohmann, 2013; Inagaki et al., 2010). Although numerous modifications to the assay have been developed over the years, the current study relied on a simple protocol, on account of its cost and equipment efficiencies, its utility for rapid assimilation by many experimenters, and its sufficiency in detecting age-dependent locomotor defects in iPLA2-VIA mutants (Banerjee et al., 2021). Still, subtle motor defects may have been missed in our study. It is also possible that in those conditions that failed to show an effect in our climbing assays, lower GAL4 expression resulted in lower knockdown efficacy, although the drivers we used are well-characterized and commonly used (e.g., Deng et al., 2019; Chen et al., 2013; Howard et al., 2019). Lower GAL4 expression likely explains the fact that knockdown in dopaminergic neurons only with ple-GAL4 failed to affect climbing (Supplementary Figure S1A), despite much evidence that dopaminergic neurons are sensitive to loss of iPLA2-VIA. Higher resolution techniques, preferably with computerized video tracking and image processing, should be used in the future to improve detection of altered locomotion under iPLA2-VIA loss of function conditions (Aggarwal et al., 2019; Wu et al., 2019). Furthermore, our study did not account for other types of neurological defects aside from those affecting locomotion. Of note, although survival of iPLA2-VIA mutants was improved by reintroducing the wild-type allele into GABAergic neurons only, the lifespans of the rescued flies were still markedly attenuated compared to control flies, indicating the involvement of other cell types in overall survival (Figure 2C). Single cell RNA-seq has revealed widespread iPLA2-VIA expression in the fly brain, coincident with most neurotransmitters (Davie et al., 2018), and prior studies have shown cell death throughout the fly brain in iPLA2-VIA loss of function mutants (Iliadi et al., 2018). Because our study did not examine cell death histologically, it remains to be seen precisely which brain areas and/or neuron types succumb to cell death, and over what time frame.
Relation to human PLA2G6-associated neurodegeneration
Drosophila and mammals share most of the major neurotransmitters, including GABA, dopamine, serotonin, acetylcholine, and glutamate, with replacement of the vertebrate epinephrine and norepinephrine with the functionally analogous octopamine and tyramine in insects (Deng et al., 2019; Deshpande et al., 2020). A wealth of studies also suggest many similar functions and behavioral outputs, including in learning and memory, circadian rhythms, reward sensing, etc. (Kasture et al., 2018). Nevertheless, the limited similarities in neuroanatomical organization between insects and vertebrates and the coexistence of multiple neuron types within each neuroanatomical region may impede direct extrapolation of findings between species. Moreover, the fact that many neurons produce multiple neurotransmitters may confound a simple classification system based on this property (Deng et al., 2019). With these caveats in mind, we turn our attention to human PLA2G6-associated neurodegeneration (PLAN).
PLAN was first described in 2006 as a collection of neurodegenerative disorders affecting children and young adults (i.e., infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, and neurodegeneration with brain iron accumulation) with severe loss of motor coordination (Gregory et al., 2008; Morgan et al., 2006). Since then, loss of PLA2G6 in human patients has been associated also with inherited parkinsonism, as well as with dystonia and ataxia (Paisan-Ruiz et al., 2009; Guo et al., 2018; Xue et al., 2023; Erro et al., 2017). Thus, while PLA2G6 loss of function clearly causes movement disorders, it is less clear whether specific subsets of neurons are more vulnerable than others. Parkinsonism results from perturbations in the dopaminergic circuit of the basal ganglia, while perturbations in other areas, including the cerebellum, can lead to dystonia and ataxia (Dickson, 2012; Sharma, 2019). Consistently with the clinical symptoms, MRI studies of human PLAN patients have revealed abnormalities in multiple brain regions, and importantly, the most commonly affected brain region is the cerebellum, which is largely GABAergic (Gregory et al., 2008; Salih et al., 2013). Our results showing that age-dependent climbing behavior in Drosophila is strongly dependent on iPLA2-VIA in GABAergic neurons are in line with human clinical and MRI data, as well as with mouse PLA2G6 knockouts showing loss of GABAergic Purkinje cells in the cerebellum (Zhao et al., 2011). So far, Drosophila studies have utilized straightforward amorphic or hypomorphic iPLA2-VIA conditions. It has been speculated that specific molecular lesions in the gene may result in distinct clinical symptoms, but evidence has been inconclusive (Guo et al., 2018; Xue et al., 2023; Engel et al., 2010). Furthermore, it is unclear how mutations in PLA2G6 lead to neurodegeneration, although abnormalities in mitochondria, endolysosomal pathways, Ca+2 handling, ER and presynaptic membranes, and phospholipid acyl chain composition have been noted (Mori et al., 2019; Lin et al., 2018; Kinghorn et al., 2015; Chiu et al., 2019; Zhou et al., 2016; Beck et al., 2011).
Pathologic process and relation to sporadic PD
Mutations in any of the more than 20 “PARK” genes can cause parkinsonism and, in some cases, mimic sporadic PD. However, it remains unclear how closely the underlying pathological sequence of events matches between each inherited parkinsonian disorder and sporadic PD. Loss of function mutations in PLA2G6/PARK14 have been suggested to induce cellular conditions similar to those occurring in sporadic PD, e.g., by causing lysosomal dysfunction (Lin et al., 2018) or by promoting α-syn aggregation (Mori et al., 2019). Indeed, dopaminergic neurons degenerate in both PLAN and sporadic PD (Miki et al., 2017; Mori et al., 2019; Sanchez et al., 2018; Chiu et al., 2019). Serotonergic neurons also succumb in sporadic PD (Politis et al., 2012; Braak et al., 2003), and although their status in PLAN is not well-described, this study suggests they are relevant to PLAN as well. However, in sporadic PD, GABAergic regions including the globus pallidus and cerebellum largely are spared from Lewy pathology and neurodegeneration (Dickson, 2012; Braak et al., 2003). By contrast, PLAN patients often experience degeneration in the cerebellum and iron accumulation in the globus pallidus (Guo et al., 2018; Gregory et al., 2008). Our experiments indicate that GABAergic neurons are extremely sensitive to loss of iPLA2-VIA, and that this can cause severe locomotor impairment. Together, our results along with the clinical and human pathology observations may suggest that the cytopathological stimuli in PLA2G6-associated neurodegeneration are different from those in sporadic PD, although they likely eventually converge on similar or identical pathways as neurodegeneration proceeds. This is consistent with recent large scale association studies that have failed to find a link between PLA2G6 and sporadic PD (Kim et al., 2024; Tomiyama et al., 2011; Liu et al., 2020). To date, it is still unknown what determines the specific neuronal sensitivities in PD and other neurodegenerative conditions (Braak et al., 2003; Paß et al., 2021).
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The manuscript presents research on animals that do not require ethical approval for their study.
Author contributions
NM: Investigation, Writing – original draft. SB: Investigation, Writing – review & editing. WB: Investigation, Writing – review & editing. YC: Investigation, Writing – review & editing. EC: Investigation, Writing – review & editing. BC: Investigation, Writing – review & editing. MD: Investigation, Writing – review & editing. MG: Investigation, Writing – review & editing. PH: Investigation, Writing – review & editing. EJ: Investigation, Writing – review & editing. ALL: Investigation, Writing – review & editing. DL: Investigation, Writing – review & editing. AL: Investigation, Writing – review & editing. YM: Investigation, Writing – review & editing. AM: Investigation, Writing – review & editing. JP: Investigation, Writing – review & editing. AR: Investigation, Writing – review & editing. JR: Investigation, Writing – review & editing. ES: Investigation, Writing – review & editing. RS: Investigation, Writing – review & editing. YYS: Investigation, Writing – review & editing. YDS: Investigation, Writing – review & editing. MS: Investigation, Writing – review & editing. YFS: Investigation, Writing – review & editing. YYW: Investigation, Writing – review & editing. JS: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. Funding from NIH grant # R15-HD080511 supported lab supplies, fly stocks, fly food, and personnel. Funding from the Yeshiva University Provost’s Office supported personnel and publication costs.
Acknowledgments
We are grateful to the Bloomington Drosophila Stock Center for fly stocks (NIH P40OD018537), to Flybase for information on GAL4 lines, to Becky Delventhal for advice on study design, and to Rebecca Spokony and Jessica Treisman for critical comments on the manuscript. Berta Chalouh-Hara, Yael Laks, Nathan Hill, Liam Eliach, Joshua Levieddin, Joshua Feigin, and Aryeh Lejtman helped keep research operations running smoothly. Moshe Carroll, Yaakov Tzvi Cantor, Eitan Edinger, Arie Barkats, Jeremy Bassali, Yedidya Blau, Adin Blumofe, Moshe Carroll, Yaakov Tzvi Cantor, Eitan Edinger, Ben Epstein, Joshua Feigin, Yechezkal Freundlich, Elan Goldstein, Joshua Hamburger, David Hanan, Jason Hirschprung, Zev Hirt, Jaden Jubas, Charles Kleinman, Natan Levin, Noah Mogyoros, Ezra Mokhtar, Phillip Nagler, Joshua Peyman, Ariel Raskin, Jonah Rocheeld, Yehuda Spivak, and Aaron Stolarov helped with experimental replicates.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins.2025.1534243/full#supplementary-material
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Summary
Keywords
PLA2G6 , PLA2G6-associated neurodegeneration, PARK14 , Parkinson’s disease, locomotor decline
Citation
Meimoun NS, Benji S, Besharim WZ, Cantor YY, Carroll ES, Coplin BI, Davidovics MB, Gerber M, Hirschprung PM, Jacobson EI, Levenbrown AL, Levitt DT, Levy A, Mazin YZ, Moskowitz AD, Purow JI, Rimberg A, Rothstein JE, Saks EY, Saperstein R, Scher YY, Schwarcz YD, Silver M, Stein YF, Wiener YY and Steinhauer J (2025) GABAergic neurons are a key cell type in a Drosophila model of PARK14/PLA2G6-associated neurodegeneration. Front. Neurosci. 19:1534243. doi: 10.3389/fnins.2025.1534243
Received
25 November 2024
Accepted
16 October 2025
Published
01 December 2025
Volume
19 - 2025
Edited by
Michael Goard, University of California, Santa Barbara, United States
Reviewed by
Francesco Liguori, Santa Lucia Foundation (IRCCS), Italy
Samir Ranjan Panda, University of California, San Francisco, United States
Updates
Copyright
© 2025 Meimoun, Benji, Besharim, Cantor, Carroll, Coplin, Davidovics, Gerber, Hirschprung, Jacobson, Levenbrown, Levitt, Levy, Mazin, Moskowitz, Purow, Rimberg, Rothstein, Saks, Saperstein, Scher, Schwarcz, Silver, Stein, Wiener and Steinhauer.
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*Correspondence: Josefa Steinhauer, jsteinha@yu.edu
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.