Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies

Results

Lactate dehydrogenase expression and long-term courtship memory is maintained with age in Canton-S flies

In this study, courtship conditioning was employed to assess long-term memory (Figure 1A). To date, there are no reports examining the effects of age on long-term courtship memory in a reference or control line such as Canton-S (CS). We observed that 24-hour long-term courtship memory was retained in CS up to 30 days of age under standard growth temperature conditions (25°C) (Figure 1B and 1C). These results indicate that long-term courtship memory is not susceptible to age-related impairment, at least up to 30 days of age at 25°C, a time point when 89.7% of the population is still alive.

Figure 1. dLdh protein levels increase in the brain with age while long-term courtship memory is retained in control male flies. (A) Courtship conditioning paradigm used for testing long-term courtship memory throughout this study. (B) Courtship indexes for Canton-S male flies aged 7, 14, or 30 days at 25°C decreased in courtship conditioning rejection trained vs. naïve conditions at all ages. N = 100–117. Naïve and trained flies were compared for each age group using one-sided Mann-Whitney U tests, ^****p < 0.0001. Data presented as violin plot of frequency distribution. (C) Long-term courtship memory indexes for trained flies represented in B do not differ between age groups. Age groups were compared using a Kruskal-Wallis test with Dunn’s multiple comparisons tests. (D) Survival curve of Canton-S male flies singly housed is decreased at 29°C compared with 25°C and ages which were selected for memory testing in control (25°C black) and transgenic (29°C red) flies are highlighted with vertical dotted lines. Curve comparison was made using a Log-rank (Mantel-Cox) test, ^****p < 0.0001. Data presented as violin plot of frequency distribution. (E) Western blot analysis of head extracts from male Canton-S flies aged 7, 14 or 30 days at 29°C showing dLdh protein levels increase with age (F(2, 10) = 36.64, p < 0.0001) n = 4–5. Each sample consists of protein extracts from 20 heads. Comparisons across age were made using one-way ANOVA with Šídák’s multiple comparisons tests between all age groups. ^***p < 0.001, ^****p < 0.0001.

We restricted genetic manipulation of dLdh expression to adulthood by employing a system which utilizes temperature sensitive Gal80 to inhibit Gal4 activity at 18°C, while permitting activity at 29°C [95]. We set up a reference longevity curve, in which CS flies were reared and assayed at 29^oC. At this temperature, flies exhibited approximately 90% survival at 21 days of age, which is similar to CS flies reared at 25°C at 30 days of age (Figure 1D). Thus, to limit survivorship bias we selected 21 days of age raised at 29°C for analysis of aged flies. We also examined endogenous levels of dLdh at this timepoint by western blot analysis. CS flies reared at 29°C exhibited an increase in whole head dLdh protein levels with age (Figure 1E, Supplementary Figure 1), in accordance with our previous evaluation of aged cantonized w¹¹¹⁸ flies maintained at 25°C [45] and with others who have measured dLdh only at the transcript level [78–80, 96].

Both increasing and decreasing neuronal lactate dehydrogenase expression is detrimental to survival and long-term courtship memory in aged flies

In order to assess the effects of altered dLdh expression on memory in an adult context, we used flies containing a temperature sensitive Gal80 driven by a ubiquitously expressing Tubulin driver and Gal4 driven by a pan-neuronal expressing Elav driver (thereafter Elavts-Gal4). These flies were subsequently crossed with flies containing an upstream activation sequence (UAS) driving either the dLdh full-length open reading frame with a triple hemagglutinin (HA) tag on the C-terminus (UAS-dLdh) or an RNA interference construct targeting dLdh (UAS-dLdh-RNAi), to permit either increased or decreased expression of dLdh in adult neurons respectively. Firstly, we verified by western blot that dLdh protein levels decreased in the heads of neuronal dLdh downregulated flies and that there was induction of HA-tagged dLdh production in the heads of neuronal dLdh upregulated flies at 21 days of age (Figure 2A, Supplementary Figures 2 and 3). Using the same courtship conditioning paradigm utilized in CS flies, we tested long-term courtship memory in dLdh transgenic flies. At 7 and 14 days of age, neither neuronal dLdh upregulation nor downregulation elicited a change in long-term courtship memory (Figure 2B and 2C). However, at 21 days of age, flies with either a neuronal upregulation or downregulation of dLdh expression showed a decrease in long-term courtship memory compared to control flies (Figure 2B and 2C). In addition to memory, we tested whether aging affected health in neuronal dLdh transgenic flies by measuring survival and climbing ability. Flies with neuronal dLdh downregulation exhibited a clear decrease in survival compared to control flies, with an earlier onset of death, a pronounced increase in the rate of death, but an unchanged maximum lifespan (Figure 2D). Flies with neuronal dLdh upregulation also exhibited reduced survival compared to control flies, with a similar decrease in time of death onset and rate of death, but also with a decrease in maximum lifespan (Figure 2D). Unhealthy flies tend to exhibit a general inability to maintain locomotor behaviours as assessed using a negative geotaxis assay. We found that climbing ability was decreased in flies with neuronal dLdh upregulation compared to control flies at 21 days of age (Figure 2E). These findings indicate that the rate of age-related decline in climbing ability of flies with neuronal dLdh upregulation was more pronounced than control flies. However, flies with neuronal dLdh downregulation did not exhibit a change in climbing ability compared to control flies at any age (Figure 2E).

Figure 2. Male flies with either elevated or downregulated neuronal dLdh expression exhibit decreased long-term courtship memory and survival with age. (A) Western blot analysis of head extracts from neuronal transgenic male flies aged 21 days at 29°C showing elevated ectopic HA tagged dLdh expression (upper panel) and decreased endogenous dLdh levels (lower panel) in flies using a Elavts-Gal4 driver to drive UAS-dLDH (with 3xHA tag) and UAS-dLdh-RNAi expression respectively (n = 3), expression was normalized to total protein content (based on Ponceau-S staining). Each sample consisted of protein extracts from 20 heads. Comparison of endogenous dLdh level were made using an unpaired t test, ^*p < 0.05. (B) Courtship indexes for neuronal transgenic male flies aged 7, 14, or 21 days at 29°C post-eclosion showed a decrease in courtship conditioning rejection trained vs. naïve for all genotypes at all ages, except for 21 day upregulation. N = 59–125. Naïve and trained flies were compared within each genotype at each age using one-sided Mann-Whitney U tests, ^****p < 0.0001, ^***p < 0.001, ^*p < 0.05. Data presented as violin plot of frequency distribution. (C) Long-term courtship memory indexes for neuronal transgenic trained flies represented in B. Memory in neuronal transgenic flies only differed from control at 21 days of age (H = 21.33, p < 0.0001), with both flies with dLdh upregulation and downregulation showing reduced memory at 21 day of age compared to control. Genotypes at each age were compared using Kruskal-Wallis tests with Dunn’s multiple comparisons to control, ^****p < 0.0001, ^*p < 0.05. Data presented as violin plot of frequency distribution. (D) In male flies, both neuronal upregulation and downregulation of dLdh resulted in reduced survival compared to control. Shaded area represents the 95% confidence interval. Curve comparisons were made using a Log-rank (Mantel-Cox) test, ^****p < 0.0001. Ages chosen for memory testing are highlighted with vertical dotted lines. (E) Upregulation of neuronal dLdh in male flies decreased climbing ability during negative geotaxis compared to control and dLdh downregulation (genotype effect F(2, 33) = 4.355, p = 0.0210). Comparisons across age and genotype were made using a mixed-effects model with Geisser-Greenhouse correction and Šídák’s multiple comparisons within each age group, ^*p < 0.05, ^****p < 0.0001. Effects of age, genotype, and age by genotype interaction are denoted on the bottom left.

Altered glial lactate dehydrogenase expression promotes differential effects on survival and long-term courtship memory

To restrict manipulation of dLdh expression to glial cells in adults, we employed flies containing a pan-glial expressing Repo driver combined with the same temperature system used for manipulation of neuronal dLdh expression (Repots-Gal4). We verified decreased dLdh protein levels in the heads of glial dLdh downregulated flies and induced HA-tagged dLdh levels in the heads of glial dLdh upregulated flies at 21 days of age by western blot analysis (Figure 3A, Supplementary Figures 3 and 4). We used courtship conditioning to assess memory in these flies. Glial dLdh transgenic flies exhibited no significant differences in memory compared to control flies at 7 and 14 days of age (Figure 3B and 3C), indicating that long-term courtship memory was unimpacted by glial dLdh manipulation at these ages. In contrast, a clear deficit in long-term courtship memory was observed in flies with glial dLdh downregulation compared to control flies at 21 days of age (Figure 3B and 3C). Glial dLdh upregulation had no effect on memory at 21 days of age (Figure 3B and 3C). The effect of altered glial dLdh expression on lifespan was also assessed. We detected a reduction in the survival of flies with glial dLdh upregulation compared to control flies, with an earlier onset of death, a slight increase in the rate of death, and minimal change in maximum lifespan (Figure 3D). Flies with glial dLdh downregulation had indistinguishable survival compared to control flies (Figure 3D). The climbing ability of flies with either glial dLdh upregulation or downregulation did not differ from control flies at all ages (Figure 3E).

Figure 3. Male flies with downregulated dLdh expression in glia exhibit decreased long-term courtship memory with age, while flies with upregulated dLdh expression in glia exhibit decreased survival. (A) Western blot analysis of head extracts from glial transgenic male flies aged 21 days at 29°C showing elevated ectopic HA tagged dLdh expression (upper panel) and decreased endogenous dLdh levels (lower panel) in flies using a Repots-Gal4 driver to drive UAS-dLDH (with 3xHA tag) and UAS-dLdh-RNAi expression respectively (n = 3), expression was normalized to total protein content (based on Ponceau-S staining) n = 3. Each sample consisted of head extracts from 20 heads. Comparisons made by unpaired t test, ^**p < 0.01. (B) Courtship indexes for glial transgenic male flies aged 7, 14, or 21 days at 29°C post-eclosion showing courtship conditioning rejection training was decreased compared to naïve flies for all genotypes at 7 and 21 days of age, and control only at 14 days of age. n = 48–72. Naïve and trained flies were compared within each genotype at each age using one-sided Mann-Whitney U tests, ^****p < 0.0001, ^*p < 0.05. Data presented as violin plot of frequency distribution. (C) Long-term courtship memory indexes for glial transgenic trained flies represented in B. Memory was only decreased following glial dLdh downregulation at 21 days of age compared to control (H = 16.9, p = 0.0002). Genotypes at each age were compared using Kruskal-Wallis tests with Dunn’s multiple comparisons to control, ^*p < 0.05. Data presented as violin plot of frequency distribution. (D) In male flies, only glial upregulation of dLdh reduced survival compared to control and dLdh downregulation. Shaded area represents the 95% confidence interval. Curve comparison was made using a Log-rank (Mantel-Cox) test, ^****p < 0.0001. Ages chosen for memory testing are highlighted with vertical dotted lines. (E) Altered glial dLdh expression did not impact climbing ability in male flies during negative geotaxis at any age compared to control. Comparisons across age and genotype were made using a mixed-effects model with Geisser-Greenhouse correction and Tukey’s multiple comparisons within each age group. Effects of age, genotype, and age by genotype interaction are denoted on the bottom left.

Elevated dLdh expression in either neurons or glia promotes neutral lipid accumulation in the brains of aged flies

Glial generated lactate is a substrate for neuronal lipid synthesis [41]. Therefore, we investigated whether lipids were altered in the brains of aged transgenic flies in which either neuronal or glial lactate metabolism was genetically manipulated. We used nile red staining on whole brains dissected from flies at 21 days of age to detect neutral lipids [97, 98]. Brains of flies with dLdh upregulated in either neurons or in glia displayed a pronounced increase in neutral lipids compared to control flies (Figure 4, Supplementary Figure 5). Downregulation of dLdh in either neurons or glia had no apparent impact on neutral lipids detected in the brain, as assessed by nile red staining (Figure 4, Supplementary Figure 5).

Figure 4. Increased glial or neuronal dLdh expression promotes accumulation of neutral lipids in 21 day aged male transgenic fly whole brains. (A) Representative confocal fluorescence microscopy images of neuronal dLdh transgenic male fly brains removed at 21 days of age at 29°C and stained for DNA (Blue = DAPI) and neutral lipids (Red = Nile Red). (B) Quantification of nile red staining mean fluorescence intensity revealed that male flies with neuronal dLdh upregulation have over twice the lipid accumulation as control flies. N = 4–5 (F(2, 10) = 17.69, p = 0.0005). (C) Representative confocal fluorescence microscopy images of glial dLdh transgenic male fly brains dissected out at 21 days of age and stained for DNA (Blue = DAPI) and neutral lipids (Red = Nile Red). (D) Quantification of nile red staining mean fluorescence intensity revealed that male flies with glial dLdh upregulation have over 1.5 times lipid accumulation as control flies. N = 7–8. (F(2, 19) = 19.74, p < 0.0001). Comparisons between genotypes for neuronal and glial dLdh transgenic flies separately were made using one-way ANOVA with Dunnett’s multiple comparisons with control. ^***p < 0.001, ^****p < 0.0001.

Metabolite levels in the heads of flies fluctuate with age and are readily altered by manipulating glial or neuronal lactate dehydrogenase expression

To determine how manipulation of dLdh in neurons or glia impact age-related changes in metabolism across the brain, we assessed metabolites extracted from transgenic fly heads at 7 and 21 days of age using gas chromatography-mass spectrometry (Figure 5, Supplementary Figure 6). We assumed differences in metabolite levels in transgenic fly head extracts were primarily due to changes in the brain because other structures in the head, such as cuticle and fat bodies, are unaffected by the genetic manipulation we employed to alter lactate metabolism exclusively in CNS cell types. In our metabolite analysis, we initially focused on lactate and pyruvate as these are the metabolites that are directly impacted by dLdh canonical activity (Figure 5A). Surprisingly, brain levels of lactate were unaffected by neuronal dLdh manipulation (Figure 5B). However, the age-related decline in brain pyruvate evident in control flies was prematurely triggered by both downregulation and upregulation of neuronal dLdh, with a clear reduction at 7 days of age that was maintained until 21 days of age in neuronal transgenic flies (Figure 5B). Glial dLdh upregulation caused a pronounced accumulation of lactate at 21 days of age, whereas downregulation had no impact on overall brain lactate levels (Figure 5C). The age-related decline in brain pyruvate levels was unaffected by both glial dLdh upregulation and downregulation (Figure 5C). Next, we examined the effects of altered dLdh expression on mitochondrial metabolism by assessing changes in TCA cycle intermediates, including citrate, alpha-ketoglutarate (αKG), succinate, fumarate, and malate (Figure 5A). We saw no differences in brain citrate level regardless of age or dLdh manipulation (Figure 5D and 5E). In control flies for neuronal dLdh manipulation, brain levels of αKG showed a substantial decline at 21 days of age (Figure 5D), whereas in neuronal dLdh upregulation flies, low levels of αKG in the brain were detected at both 7 and 21 days of age (Figure 5D). In both the glial dLdh upregulation and downregulation lines there were no changes in brain αKG levels at either 7 or 21 days of age (Figure 5E). A small decrease in brain succinate levels was observed in flies with glial dLdh upregulation compared to the control flies, particularly at 21 days of age (Figure 5E). Both upregulation and downregulation of neuronal dLdh promoted an age-related increase in brain fumarate levels compared with the control flies (Figure 5D). An age-related elevation in brain fumarate was only evident in flies with glial dLdh downregulation but not upregulation (Figure 5E). Malate levels in the brain were increased in both neuronal dLdh upregulated and downregulated flies, but this difference was most pronounced at 7 days of age relative to control flies (Figure 5D). Glial dLdh manipulation had no effect on malate levels at any age (Figure 5E). Lastly, we measured brain levels of 2-hydroxyglutarate (2HG), which consists of both the D- and L- enantiomers (Figure 5A). In both control and neuronal dLdh transgenic flies we detected increased 2HG levels in brains with age. (Figure 5F). Interestingly, we found an increase in 2HG levels in flies with glial dLdh upregulation compared with control, with approximately 155-fold higher 2HG levels at 21 days of age compared to 7-day old control flies (Figure 5G). Considering that dLdh catalyzes the formation of L-2HG from αKG, this pronounced increase in 2HG level likely represents increased production of L-2HG.

Figure 5. Metabolite analysis of transgenic male fly heads aged 7 and 21 days with altered expression of neuronal or glial dLdh revealed alterations in lactate, pyruvate, TCA cycle intermediates, and 2HG levels relative to control flies. (A) Metabolic pathway connections with metabolites produced by dLdh canonical [Pyruvate ↔ Lactate] and non-canonical [αKG→2HG] activity. Metabolites measured by gas chromatography-mass spectrometry (GC-MS) in the heads of transgenic male flies aged 7 and 21 days at 29°C are denoted by a superscript asterisk. (B) Neuronal dLdh manipulation does not impact lactate levels whereas pyruvate levels are reduced at 7 days of age nearly to the level of 21 days of age by both upregulation and downregulation. (C) Glial dLdh upregulation caused an increase in lactate but no effect on pyruvate levels. (D) Neuronal dLdh manipulation promotes alterations in select TCA cycle intermediates. Upregulation of dLdh lowered α-ketoglutarate (αKG) levels. Downregulation and upregulation of dLdh raised fumarate and malate levels. (E) Glial dLdh manipulation caused only slight alterations in TCA cycle intermediates. Citrate, αKG, and malate levels were unaltered. Upregulation of dLdh lowered succinate levels. Downregulation of dLdh raised fumarate levels at 21 days of age. (F) 2-hydroxyglutarate (2HG) levels are generally increased with age without any clear change due to neuronal dLdh manipulation. (G) The age-related increase in 2HG is exacerbated by glial dLdh upregulation but not by downregulation. Comparisons for each metabolite were done between genotype and age groups of neuronal and glial dLdh transgenic flies separately using two-way ANOVAs with Dunnet’s multiple comparisons with control for each age group and with Šídák’s multiple comparisons between age groups within each genotype. Effects of age, genotype, and age by genotype interaction are denoted on the top left of each graph. The structural formula for each metabolite were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) chemical compound database with associated C number below.

Conclusion

In this study we demonstrate the importance of maintaining appropriate levels of dLdh in D. melanogaster glia and neurons for maintenance of long-term courtship memory and survival with age (Figure 6). In addition, our results implicate lipid metabolism, 2HG accumulation, and changes in TCA cycle activity as factors underlying the age-related impacts of perturbed dLdh expression, which likely modifies glia-neuron lactate shuttling in the fly brain. Moreover, we demonstrate that normal features of D. melanogaster aging likely include retention of the ability to form long-term courtship memory, unlike other types of memory, and the accumulation of 2HG in the brain. This study provides a greater understanding of how aging, lactate metabolism, and cognitive processing intersect. Future work is required to determine how lactate metabolism may differ across various D. melanogaster brain regions with age and which subtypes of neurons and glia are most susceptible to glia-neuron lactate shuttle perturbations related to accumulation of neutral lipids and the 2HG enantiomers. Our findings highlight a connection between glia-neuron metabolic coupling and age-related memory impairment in D. melanogaster, a model highly amenable to further exploration of the role of metabolic coupling on memory processes with age.

Figure 6. Graphical summary dLdh level was altered in neurons or glia in the brain of flies. Long-term courtship memory (LTM) was reduced in aged flies by increasing neuronal dLdh and decreasing neuronal or glial dLdh. Survival was reduced by increasing glial dLdh and decreasing neuronal or glial dLdh.

Author Contributions

AKF, JWR, and NHM conducted experiments. AKF performed data analysis and wrote the first draft. AKF, JWR, NHM, JMT, AFS, and RCC contributed to reviewing and editing. AKF, AFS, and RCC contributed to study conceptualization and developed methodology. JMT, AFS, and RCC provided resources, supervision and funding acquisition.

Acknowledgments

We thank the Biotron Facility, Insect suite at Western University, where the flies were reared in specialized climate-controlled walk-in rooms (25°C), or in temperature and humidity-controlled incubators (18°C or 29°C). We thank Amina Kassam, Delina Narendran, Sanjana Arora, Ikjot Mann, Jenan Altoum, Kevin Lee, and Sanjit Sumal for assisting in manual scoring of male fly courtship. We thank Jamie Kramer for assistance in planning the courtship conditioning paradigm and lending us the enclosures to video courtship in. We thank FlyBase for providing information and links which were crucial for this study [173].

Conflicts of Interest

The authors declare that they have no conflicts of interest related to this study.

Ethical Statement

This study involves experiments with non-cephalopod invertebrates which fall under the category of invasiveness A excluded from requiring approval from an institutional review board according to the Canadian Council on Animal Care.

Funding

Funding support for AKF and RCC was provided by the Natural Sciences and Engineering Research Council of Canada under award number RGPIN-2019-355803. RCC was supported by the Helen Battle Professorship. AKF received an Ontario Graduate Scholarship and Postgraduate Scholarship – Doctoral from the Natural Sciences and Engineering Research Council of Canada. Support for JWR and AFS was provided by the Natural Sciences and Engineering Research Council of Canada under award number RGPIN-2015-04275 and RGPIN-2022-05054. JWR received an Ontario Graduate Scholarship and Postgraduate Scholarship – Doctoral from the Natural Sciences and Engineering Research Council of Canada. Support for NHM and JMT was provided by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM119557.

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