Genetic Modeling of Lysosomal Storage Disorders (LSDs), in the Brain-Midgut Axis of Drosophila melanogaster, During Aging

1. Introduction

Lysosomal Storage Disorders (LSDs) are a group of rare diseases comprising more than 70 inherited metabolic disorders that are being characterized by lysosomal dysfunction and subsequent accumulation of undegraded substrates within lysosomes. LSDs are monogenic diseases caused by alterations in genes encoding proteins involved in normal lysosomal function, such as lysosomal enzymes and lysosomal membrane proteins, and their combined prevalence is estimated to be approximately 1 in 8,000 live births [1,2,3,4].

Lysosomal impairment leads to the dysregulation of a diverse range of cellular processes being associated with lysosomes, such as membrane repair, vesicle trafficking, lipid homeostasis, signaling, cell death pathways, autophagic flux and clearance of autophagosomes. Therefore, autophagic impairment has been described as a common mechanism of pathology in an increasing number of LSDs [4,5,6]. Despite their heterogeneity, their major clinical symptoms include hepatosplenomegaly, pulmonary and cardiac disorders, skeletal abnormalities and -often- central nervous system (CNS) dysfunction, with patients being frequently presented with a progressive neurodegenerative clinical course [6,7]. Typically, LSDs are primarily classified according to the biochemical properties of the accumulated undegraded substrate, and include sphingolipidoses, glycogen storage diseases and mucopolysaccharidoses [4,8].

Sphingolipidoses are disorders caused by genetic defects in the catabolism of sphingosine-containing lipids, and their accumulation affects both the CNS and peripheral organs [9]. Gaucher, Fabry, Tay-Sachs and Niemann-Pick are classified among the most common sphingolipid metabolism diseases [8,9,10]. Gaucher disease (GD), which is subdivided into three different types, is the most prevalent form of sphingolipidoses, and is caused by mutations in the GBA gene, which encodes for the lysosomal hydrolase β-Glucocerebrosidase, responsible for the degradation of glucosylceramide into glucose and ceramide [4,8,9,10]. Fabry, an inherited X-linked disease, is the second most common form of sphingolipidoses [2], and is caused by mutations in the GLA gene encoding the enzyme α-Galactosidase A, which catalyzes the lysosomal hydrolysis of globotriaosylceramide [4,7,10]. Tay-Sachs, a type of GM2 gangliosidosis, is presented with severe neurological symptoms and is caused by mutations in the HEXA gene encoding the enzyme β-Hexosaminidase A, which is responsible for breaking down GM2 gangliosides, resulting in their toxic accumulation in neuronal tissues [11]. Niemann-Pick is a group of predominantly neurodegenerative disorders classified in types A and B, caused by mutations in the SMPD1 gene, while type C derives from mutations in the NPC1 or NPC2 genes. In types A and B, the affected enzyme is the Sphingomyelinase (ASM), leading to sphingomyelin buildup, whereas in Niemann-Pick type C, proteins that mediate cholesterol transport from endosomes/lysosomes are seriously affected, causing endo-lysosomal accumulation of cholesterol, glycosphingolipids and sphingomyelin, resulting in severe neurological pathology [1,6,12].

Glycogen storage diseases (GSDs) comprise a group of inherited metabolic disorders caused by mutations in genes encoding enzymes of glycogen metabolism. Among them, Pompe disease, also known as GSD II, is classified as a major LSD family member. Pompe disease results from mutations in the GAA gene encoding α-Glucosidase, which is a key lysosomal enzyme responsible for the hydrolysis of glycogen to glucose. The hallmark of Pompe disease is glycogen accumulation in lysosomes, predominantly in muscle cells, leading to cardiorespiratory failure [4,7,8].

Mucopolysaccharidoses (MPSs) form a group of eleven LSD pathologies, characterized by the cellular accumulation of glycosaminoglycans (GAGs), which are negatively charged polysaccharides essential for several cellular processes, including signaling and development. The classification of MPSs is based on mutations in specific enzymes that catabolize target substrates, with MPS I, II and III being the most common ones [4]. MPS type I (MPS I) is caused by the deficiency of lysosomal hydrolase α-L-Iduronidase (IDUA), leading to the accumulation of dermatan- and heparan-sulfate inside lysosomes of a wide range of tissues. The severe form of MPS I, known as Hurler syndrome, is characterized by early onset, and progressive somatic and neurological impairments [13]. MPS II, also known as Hunter syndrome, is caused by mutations in the IDS gene on the X chromosome and is typically described by neurological deterioration. These mutations result in a critical deficiency of Iduronate-2-sulfatase (IDS), an enzyme responsible for breaking down dermatan- and heparan-sulfate. Finally, Sly disease, also known as MPS VII, is caused by mutations in the GUSB gene, resulting in β-Glucuronidase (GUSB) enzyme deficiency. This leads to the accumulation of dermatan-, heparan- and chondroitin-sulfate GAGs, causing progressive multi-system dysfunctions [4,14].

Neurological dysfunction and progressive neurodegeneration are key symptoms of LSDs [6]. The study of -animal- model organisms is imperative for advancing our understanding of human pathologies, thus enabling the identification of novel disease-related pathways that have the potential to serve as drug targets. Furthermore, recent progress has led to development of more powerful and reliable animal models that can more precisely mirror aberrant phenotypes and pathological processes of human diseases and, in particular, LSDs [15,16,17]. A recently explored therapeutic approach for LSDs is the targeted gene therapy that uses genome-editing technologies, like CRISPR/Cas9. However, these strategies encounter several technical challenges and bioethical considerations, making it essential to study their effects in vivo, using animal models that can closely replicate LSD-specific phenotypes. Given the imminent need for robust in vivo models, we have, herein, generated transgenic Drosophila flies, via exploitation of the -combined- GAL4/UAS and RNAi gene-targeting system, to mechanistically illuminate LSD-associated pathologies, at the genetic level, during aging. This platform provides a dynamic, valuable and versatile tool to deeply investigate systemic pathologies and successfully explore novel therapies for LSD-affected cohorts.

2. Materials and Methods

2.1. Drosophila Melanogaster Strain Stocks and Culturing Conditions

The Drosophila melanogaster transgenic fly strains w[*]; P{w[+mC]=GAL4-elav.L}3 (RRID:BDSC_8760), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01322}attP2 (RRID:BDSC_34334), y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMJ02101}attP40 (RRID:BDSC_53379), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05804}attP2 (RRID:BDSC_64931), y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC03475}attP40 (RRID:BDSC_51901), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00562}attP2 (RRID:BDSC_33693), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01646}attP40 (RRID:BDSC_37504), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01681}attP40 (RRID:BDSC_38237), y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMJ30222}attP40 (RRID:BDSC_63655), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS05491}attP40 (RRID:BDSC_67025), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC06416}attP40 (RRID:BDSC_67312), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC04581}attP40 (RRID:BDSC_57199), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01984}attP2 (RRID:BDSC_39064) and y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01893}attP40 (RRID:BDSC_38977) were obtained from Bloomington Drosophila Stock Center (NIH P40OD018537) (Indiana, USA). The D. melanogaster transgenic NP1-GAL4 fly strain was kindly provided by Dr. Eric H. Baehrecke [18] (Department of Cancer Biology, University of Massachusetts, Medical School, Worcester, MA, USA).

All fly stocks were maintained at 25 °C, in a relative humidity of 55-65%, under a 12-h light/dark photoperiod, and a laboratory standard Drosophila -nutrition- medium (6.4% rice flour, 5% tomato paste, 3.2% sugar, 0.8% yeast, 0.8% agar, 0.13% Tegosept, 0.4% ethanol and 0.4% propionic acid).

2.2. RNA Extraction and RT-qPCR

Isolation of total cellular RNA from RNAi-targeted fly heads was performed using the PureLink™ RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer’s instructions. Concentration and quality of the isolated RNA were determined using the NanoDrop One UV-Vis. spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), following manufacturer’s protocol.

Relative expression of the, herein, studied genes was examined by Reverse Transcription (real-time) quantitative Polymerase Chain Reaction (RT-qPCR), using specific primers (Table S1), the Fast SYBR^® Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and the Applied Biosystems StepOne^™ (real-time) qPCR System (Thermo Fisher Scientific, Waltham, MA, USA), as described by manufacturer’s guidelines. As a suitable internal control, for normalization of gene expression values, the housekeeping gene Actin 5C was accordingly used. To ensure reproducibility, each assay was performed in technical triplicates, while three negative controls were also included in the analysis. Fold reductions in transcript levels were determined using the comparative 2^−ΔΔCt method [19], which calculates changes in gene expression as a relative fold difference between the gene(s) of interest and the reference gene. Results were presented as a percentage of the relative gene reduction in RNAi-targeted [specifically in neuronal (brain) tissues] flies, compared to control populations. Each experiment was performed three different times, using independent genetic crosses.

2.3. Longevity Measurement

To study viability, newly emerged (∼24 h) female and male -transgenic- flies from each fly cross were collected and maintained in separate vials (~20-25 flies per vial). Flies were kept in a constant temperature and humidity chamber, throughout the experimental period, and periodically provided with fresh food every three days. Survival curves were generated by daily counting the deceased flies. For each viability experiment, the sample size was set to (at least) 100 flies per gender and genetic cross (to ensure statistical significance). All viability experiments were performed at the same time for control and RNAi-overexpressing strains. Each viability experiment was repeated (at least) three times, using independent genetic crosses.

2.4. Negative Geotaxis Assay

The locomotor performance of RNAi-targeted flies was quantified using the negative geotaxis (climbing) assay. Flies of both sexes were initially kept together, and, before the experimental procedure, they were anesthetized and divided into male and female populations (groups of ~20-25 flies, each). Each experimental group was, then, placed in an empty 100 ml cylinder, with a boundary line drawn at the 60 ml mark (10 cm height), and the flies were allowed for 1 min to acclimatize to the environment. To climb simultaneously, flies were gently tapped to the bottom of the cylinder. After time interval of 20 sec, the number of flies that reached or exceeded the 60 ml limit was counted. Five trials with 1 min-time interval were performed for each group. The same populations were tested at different ages, excluding flies that died or flew away. Control and RNAi-targeted fly groups were examined simultaneously. Total sample size for each fly cross and gender was set at (minimum) 100 flies. Three independent biological replicates were used for each fly group examined.

2.5. Structural Alignment

For the structural alignment of Homo sapiens (human) and Drosophila (fly) LSD-related proteins, AlphaFold-specific molecular models were obtained from the neural-network-based AlphaFold2 Protein Structure Database [20,21,22] and the generated protein models were structurally aligned with the PyMOL (v3.1) molecular graphics system [23]. The Root Mean Square Difference (RMSD) value of each alignment was used as an indicator for the reliability of structural alignment [24].

2.6. Statistical Analysis

Statistical analysis, and graphic presentation of the obtained results, were performed using the Statistical Package for Social Sciences (IBM SPSS v25.0 for Windows IBM Corp., New York, NY, USA). Data from the longevity experiments were analyzed with the Kaplan-Meier survival test, using the Log Rank, Breslow test and Τarone-Ware statistical packages. Climbing graphs were plotted as the mean pass rate per genotype/time-point with Sample Standard Deviation (± SSD) value. Statistically significant differences between the compared genotypes, at each time-point, were evaluated by the independent t-test analysis. Significance was accepted at: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

3. Results

3.1. From Humans to Flies: Identification of LSD-Related Gene Orthologs Using DIOPT

The first step for studying human disease genes in animal model organisms, such as Drosophila, is to recognize the putative orthologs that are being associated with the respective diseases. Ortholog genes facilitate functional genomics by allowing hypotheses concerning the functions of genes in one species to be deduced from their orthologs in another species. Therefore, to identify Drosophila orthologs of human LSD-related genes, the DRSC (Drosophila RNAi Screening Center) Integrative Ortholog Prediction Tool (DIOPT) [25] was suitably engaged. DIOPT integrates the results of multiple ortholog-mapping tools based on different algorithms and calculates a simple score reflecting the number of tools that support a given ortholog gene-pair relationship. In version 9, the maximum score for fly-human ortholog relationship is 19, in an 1-19 scale. Based on this score, DIOPT also provides ortholog ranks, being categorized as high, moderate and low, which reflect the confidence level in predicted “orthology” relationships between genes of different species [25,26]. In Table 1, we present a DIOPT-derived extensive list, corresponding to nearly all human LSD-related genes, along with their putative orthologs in Drosophila. In addition, we provide the DIOPT ranking and the associated RNAi strains available from the Bloomington (BDSC) and Vienna (VDRC) [27] Drosophila Stock/Resource Centers, thus offering a useful resource for designing genetic studies on evolutionary conserved disease genes.

Following ortholog prediction, we focused on screening LSDs not previously studied in Drosophila or examined using alternative genetic tools. Our overall objective is to identify novel genotype–phenotype associations between humans and Drosophila, to advance our understanding of the molecular mechanisms underlying LSDs. In an effort to, in vivo, investigate the importance of LSD-related genes at the organ(ism) level, we, next, proceeded to the targeted gene knockdown of their Drosophila orthologs, through employment of the GAL4/UAS genetic system in combination with the RNAi technology [27,28]. Gene silencing was carried out in a tissue-specific manner, along the brain-midgut axis, and the phenotypic assays engaged to quantify gene-function aberrations were life expectancy and climbing capacity. Locomotor impairment was specifically evaluated in Drosophila disease models that exhibited the most pronounced reductions in lifespan along the brain-midgut axis, as these models could most effectively recapitulate key pathological features and were, therefore, deemed the most robust representative systems for the respective human disorders. Negative geotaxis (climbing activity) is a commonly used method for evaluating neuronal dysfunction in flies that results from neuron-specific silencing of selected genes/proteins [29]. For all LSDs, herein examined, the predicted Drosophila orthologs being selected for further investigation were characterized by high or moderate DIOPT rankings (Table 1).

3.2. Structural Conservation of LSD-Associated Proteins Between Homo Sapiens and Drosophila Melanogaster

Three-dimensional structural alignment, via PyMOL [30] engagement, is a valuable tool for revealing structural conservation between ortholog proteins with high sequence similarity and for gaining insights into their structural characteristics. Initially, all selected LSD-related structures of human and Drosophila proteins were retrieved from the neural network-based protein-structure prediction tool AlphaFold2 [20,21,22]. Next, PyMOL alignment was employed, with sequence alignment being followed by structural superposition and subsequent minimization of the Root Mean Square Deviation (RMSD) between the aligned residues through the execution of refinement cycles to reject structural outliers identified during alignment. RMSD value between corresponding atoms of two protein chains is the most common estimator of structural similarity, with RMSD below 3 Å typically indicating close homology [24]. Interestingly, protein comparisons of all Drosophila orthologs derived from DIOPT, carrying high or moderate homologies to their human LSD-related counterparts, yielded low RMSD values, ranging from 0.454 to 1.376 Å, with their majority being measured below 1 Å (Figures S1-S8), thus indicating minor structural variation. Only the comparison of protein structures being produced from the human gene GAA (implicated in Pompe disease) and its Drosophila ortholog Tobi (Figure S5), which shows low homology in DIOPT, resulted in a high RMSD value of 3.291 Å, strongly suggesting a comparatively larger disparity between the human and fly respective gene products. For all the alignments, herein examined, each Drosophila protein structure was aligned to (superimposed with) the human reference, respective, structure. The high degree of structural similarity observed between the aligned (superimposed) ortholog proteins not only reflects their evolutionarily conserved architecture, but also indicates conserved functional properties and roles, thereby strongly supporting the validity of using Drosophila as a powerful, versatile and multifaceted -animal- model system for in vivo investigating human LSD-linked pathologies.

3.3. Modeling of Sphingolipidoses in Drosophila

3.3.1. Gaucher Disease

Sphingolipidoses constitute an essential group of LSDs that are being characterized by the accumulation of sphingolipids [10]. Among them, Gaucher disease is the most common one [2] and is distinguished in three different subtypes, all caused by mutations in the GBA1 gene [4]. A DIOPT analysis to identify Drosophila melanogaster orthologs revealed two candidate genes, Gba1a and Gba1b, both exhibiting high homology scores (Table 1), and encoding protein products with strong structural similarities to their human counterparts (Figure 1A and Figure 2A, for Gba1a and Gba1b, respectively). Therefore, we, next, proceeded to the tissue-specific, RNAi-mediated, downregulation of both Drosophila orthologs, along the brain-midgut axis. To validate the efficiency of gene silencing, and thus the reliability of our genetic models, we quantified Gba1a and Gba1b gene expression levels following neuronal-specific knockdown. The analysis revealed a significant reduction in mRNA expression of ~46% for Gba1a (Figure 1B) and ~53% for Gba1b (Figure 2B), relative to control fly brains. RNAi-mediated downregulation of Gba1a or Gba1b gene, specifically in the nervous system (brain), caused a severe decrease in male fly-life expectancy, with a median survival reduction of ~31 and ~34 days, respectively, compared to control male flies (Figure 1C and Figure 2C). Although female flies also exhibited reduced median survival, in response to neuronal-specific (brain) knockdown of either gene, their pathogenic effects were notably less pronounced as compared to male ones (Figure 1C and Figure 2C). Regarding midgut-specific knockdown models, only male flies demonstrated significantly decreased survival rates (Figure 1D and Figure 2D), with the obtained pathology being comparatively more severe in the Gba1b-specific, RNAi-targeted, flies.

Furthermore, compared to control flies, adult males with either Gba1a or Gba1b gene-knockdown profile, specifically in the nervous system (brain), displayed progressive, age-dependent, locomotor defects (Figure 1E and Figure 2E), which comparatively proved more detrimental after 10 days of age, post-eclosion. Of note, due to the high mortality levels of male Gba1a- and Gba1b-targeted (via RNAi) flies, specifically in neuronal tissues (brain), we were unable to collect -statistically- sufficient numbers of fly individuals, for reliably conducting climbing experiments at the 30^th day of age, post-eclosion. In contrast, female flies with neuronal-specific (brain) knockdown of either gene retained relatively normal climbing performance compared to their control counterparts (Figure 1E and Figure 2E for Gba1a-RNAi and Gba1b-RNAi flies, respectively).

Taken together, the RNAi-mediated knockdown of either Gba1a or Gba1b Drosophila orthologs of human GBA1 gene proved able to strikingly recapitulate key pathogenic features of Gaucher disease, strongly supporting the in vivo power and value of the fly brain-midgut axis, for reliably modeling and illuminating disease mechanisms, and promptly discovering therapeutic regimens.

3.3.2. Fabry Disease

Fabry disease is an X-linked monogenic disorder and it has been reported as the second most common LSD [10]. In humans, the disease is caused by mutations in the GLA gene, which encodes the Lysosomal α-Galactosidase A, an enzyme responsible for the hydrolysis of globotriaosylceramide. In Drosophila, two orthologs (CG7997 and CG5731) of the human GLA gene have been identified. Although both genes receive moderate rankings and modest DIOPT scores (Table 1), the structural comparisons of their cognate protein products (CG7997 and CG5731) to the human counterpart (GLA) reveal very low RMSD values (below 0.5 Å; Figure 3A and Figure 4A), thus indicating the high degree of structural conservation (during species evolution). RNAi-mediated, neuronal (brain)-specific, knockdown of the CG7997 and CG5731 genes led to a decrease in mRNA expression of ~8% and ~61%, respectively, compared to control flies (Figure 3B and Figure 4B). As expected from the low knockdown efficiency of CG7997, the lifespan of flies being subjected to RNAi-mediated silencing of this gene, along the brain-midgut axis, did not significantly differ from that of control fly population (Figure 3C,D). In contrast, knockdown of CG5731, in either the nervous system (brain) or midgut tissues, resulted in flies with significantly shortened life expectancy (Figure 4C,D). Interestingly, female flies were more severely affected, exhibiting a median lifespan reduction of~28 days following neuronal (brain)-specific knockdown and of ~40 days after midgut-specific silencing of the CG5731 gene (Figure 4C,D).

Furthermore, flies of both sexes presented locomotor deficiencies, as early as day 10 post-eclosion, after neuronal (brain)-specific knockdown of the CG5731 gene, as demonstrated by the negative-geotaxis (climbing-activity) assay (Figure 4E). The observation that female flies, compared to male populations, are more severely affected by the CG5731 downregulation, may be mechanistically associated with differences in sex-dependent metabolic demand and hormonal regulation, and/or sexually dimorphic tissue-specific gene expression patterns.

3.3.3. Niemann-Pick Disease

Niemann-Pick disease types C1 and C2 are caused by mutations in the NPC1 and NPC2 gene loci, leading to impaired intracellular cholesterol trafficking, and subsequent accumulation of cholesterol and sphingolipids in lysosomes [4,12]. In Drosophila, the DIOPT tool recognized Npc1a and Npc2a, as high confidence orthologs of human NPC1 and NPC2 genes, respectively (Table 1). Furthermore, structural alignment analysis yielded RMSD values below 1 Å, for both the NPC1-Npc1a and NPC2-Npc2a protein product comparisons, thus indicating a high degree of structural similarity between the human and Drosophila proteins (during species evolution) (Figure 5A and Figure 6A). Neuronal (brain)-specific downregulation of Npc1a and Npc2a genes resulted in ~14% and ~58% reductions in mRNA expression levels (Figure 5B and Figure 6B), respectively. Interestingly, albeit the moderate knockdown efficiency in the Npc1a-targeted flies, both male Drosophila Niemann-Pick models exhibited a significant decrease in lifespan, along the brain-midgut axis, with their median life expectancy being reduced by ~29 (Figure 5C; Npc1α targeting) and ~26 (Figure 6C; Npc2α targeting) days, following neuronal (brain)-specific downregulation, and ~20 (Figure 5D; Npc1α targeting) and ~17 (Figure 6D; Npc2α targeting) days, after midgut-specific targeting of the fly ortholog -respective- genes.

In contrast, female flies presented only a modest reduction in their lifespan, which could be detected only after ~50 days of age (post-eclosion), following neuronal (brain)-specific knockdown of either gene (Figure 5C and Figure 6C), whereas their -respective- silencing, specifically in the midgut tissues, resulted in similar to control survival profiles (Figure 5D and Figure 6D).

RNAi-mediated knockdown of Npc1a gene in the nervous system (brain) caused an early and significant decline in climbing ability of male flies, which could be readily observed from day 10, post-eclosion (Figure 5E). Intriguingly, neuronal (brain)-specific silencing of Npc2a gene in males did not affect their climbing performance, strongly suggesting that Npc2a may play a secondary, or redundant, role, compared to Npc1a gene, in locomotor function(s) (Figure 6E). Of note, female -transgenic- flies of both genotypes exhibited climbing activities similar to control ones (Figure 5E and Figure 6E). Strikingly, our findings indicate that even moderate reductions of Npc1a gene expression are sufficient to impair locomotor performance, in a sex-specific manner, during Drosophila aging.

3.3.4. Tay-Sachs/Sandhoff Disease(s)

The GM2 gangliosidoses are caused by defects in the degradation of GM2 ganglioside, leading to its accumulation primarily within neuronal cells. The degradation of GM2 ganglioside requires the lysosomal isoform Hex A, a β-Hexosaminidase enzyme composed of α- and β-subunits being encoded by the HEXA and HEXB genes, respectively. Mutations in these genes result in the development of Tay-Sachs (HEXA) and Sandhoff (HEXB) diseases [10,11]. DIOPT analysis of the Drosophila genome identified three putative orthologs encoding β-N-Acetylhexosaminidase-like enzymes; the Hexo1, Hexo2 and fdl genes (Table 1). We focused on Hexo1 and Hexo2, as they present the highest sequence homology and significant structural similarity to their human counterparts (Figure 7A,E).

RNAi-mediated knockdown of these genes in the nervous system (brain tissues) caused significant reduction in mRNA expression of ~46% for Hexo1 (Figure 7B) and ~45% for Hexo2 (Figure 7F) genes. Neuronal tissue-specific silencing of Hexo1 ortholog gene led to an age-dependent reduction in the median lifespan of male flies by ~8 days, whereas, in females, it caused a mild increase in early-life survival, followed by a decline in longevity during late(r)-life stages (Figure 7C). In contrast, (pan-)neuronal silencing of Hexo2 caused a -comparatively- more pronounced decrease in median lifespan by ~26 days, for male flies, and by ~11 days, for female populations (Figure 7G). Notably, RNAi-mediated downregulation of each gene in the midgut tissues proved to induce no significant impact on the lifespan profile of either gender (Figure 7D,H), thereby suggesting a limited, or redundant, functional role for Hexo1 and Hexo2 genes in Drosophila midgut environment, during aging.

Taken together, the RNAi transgenic lines used in this study proved capable to recapitulate key pathological features of sphingolipidoses, thus reinforcing the exploitation of Drosophila as a reliable and powerful model organism, for unmasking the molecular underpinnings of sphingolipidoses and related LSD pathologies, during aging.

3.4. Modeling of Pompe Disease in Drosophila

3.4.1. Pompe Disease

Pompe disease is a glycogen storage disorder caused by a deficiency in Lysosomal acid-a-Glucosidase, which is encoded by the GAA gene, and leads to intra-lysosomal glycogen accumulation [6]. In Drosophila, there is no single direct ortholog of the human GAA gene, hitherto, pinpointed. However, three homolog genes can be, in silico, identified; GCS2alpha, which has a moderate DIOPT score, and tobi and CG33080, which show low homology values (Table 1). For our study, we selected GCS2alpha gene, as its cognate protein is being presented with the -comparatively- highest (predicted) homology and structural similarity to its human counterpart (Figure 8A). Furthermore, we also included tobi gene (Figure 8E), due to its relatively higher DIOPT score, compared to the CG33080 respective one. The gene-silencing efficiency of our RNAi-based strategy, using a neuronal-specific driver, proved significantly strong for both ortholog genes, as the reduction in mRNA expression level was measured at ~63% for GCS2alpha (Figure 8B) and at ~64% for tobi (Figure 8F) gene, versus control conditions. Neuronal (brain)-specific knockdown of GCS2alpha caused a significant decrease of lifespan in male flies, a major pathology that was clearly detected from early adulthood (Figure 8C). Regarding tobi, its downregulation in males was linked to an age-dependent phenotype typified by reduced viability being observed after ~40 days from hatching (Figure 8G). Intriguingly, female flies did not present any negative effect on lifespan, after silencing of either gene in the nervous system (Figure 8C,G). In fact, tobi knockdown was shown to rather improve than deteriorate female viability (Figure 8G). Our data strongly suggest for the sexually dimorphic contribution of GCS2alpha and tobi genes to tightly controlling lifespan in Drosophila. Nevertheless, midgut-specific silencing of GCS2alpha or tobi gene did not seem to cause any significant effect on life expectancy in either gender, strongly supporting their (GCS2alpha and tobi) predominant functional involvement in the nervous (brain), but not midgut, system, during Drosophila aging (Figure 8D,H).

3.5. Modeling of Mucopolysaccharidoses in Drosophila

Mucopolysaccharidoses (MPSs) are caused by deficiencies in specific lysosomal hydrolases responsible for the sequential degradation of one or more glycosaminoglycans (GAGs), thus resulting in their lysosomal accumulation and ultimately cellular dysfunction [8].

3.5.1. Hurler Syndrome

Hurler syndrome, or MPS type I, is caused by a deficiency in α-L-Iduronidase (encoded by the IDUA gene), leading to the pathological storage of dermatan and heparan sulfate inside the lysosomes of a wide range of tissues [13]. In Drosophila, DIOPT analysis identified Idua as the single ortholog of human IDUA gene, with a high-confidence homology score (Table 1) and an RMSD value of 1.123 Å (Figure 9A). Neuronal-specific RNAi-mediated knockdown of Idua gene caused a ~30% reduction in gene expression (Figure 9B), which proved sufficient to induce a pathological phenotype in male flies. These transgenic males exhibited a severely shortened lifespan (Figure 9C), with their median survival being reduced by ~24 days, and impaired climbing ability commencing as early as day one of their adult life (Figure 9E). Strikingly, high mortality rates prevented the inclusion of 30-day-old male flies in the climbing assay, due to insufficient sample size. In contrast, female transgenic flies were characterized by absence of statistically significant changes in either life expectancy (Figure 9C) or climbing activity, apart from a slight improvement in locomotor performance observed at -approximately- day 30 (post-eclosion) (Figure 9E). Of note, midgut-specific knockdown of the Idua gene resulted in mildly reduced survival, for both transgenic fly sexes, compared to controls (Figure 9D).

Altogether, the, herein, obtained results demonstrate that, following Idua suppression, Drosophila manifests key neurological and viability-related pathologies, which represent phenotypes indicative of human Hurler syndrome, thereby highlighting the strong potential of “fly-Idua^RNAi” genetic platform to serve as a reliable and effective in vivo -animal- disease model, for mechanistically investigating Hurler syndrome pathogenesis, and pre-clinically supporting high-throughput drug-screening systems, towards the discovery of novel therapeutic schemes and regimens.

3.5.2. Hunter Syndrome

Hunter syndrome, or MPS type II, is a rare X-linked recessive disorder caused by functional deficiency of the lysosomal enzyme Iduronate-2-sulfatase (encoded by the IDS gene, in humans), which is critical for the catabolism of certain glycosaminoglycans (GAGs); the dermatan- and heparan-sulfate -GAG- species [14]. In Drosophila, a single ortholog gene, the Ids, can be identified in the 3^rd chromosome, with a high DIOPT score (Table 1) and a strong structural similarity of its protein product to the human protein counterpart (RMSD value as low as 0.473 Å; Figure 10A). Targeted, RNAi-mediated, knockdown of the Ids gene, specifically, in neuronal (brain) cells led to ~26% reduction in mRNA expression levels (Figure 10B). Despite this modest downregulation, male flies were presented with an age-dependent decrease in life expectancy, with their median survival being reduced by ~18 days, compared to control males (Figure 10C). In contrast, female flies were largely unaffected, producing survival curves comparable to those of the control populations (Figure 10C).

Similarly, tissue-specific knockdown of the Ids gene in Drosophila midgut tissues did not seem to significantly affect the lifespan profile of either fly-sex setting (Figure 10D), further emphasizing the functional importance of Ids gene product, specifically, in the central nervous system (CNS). It may be the remaining Ids activity, of ~74%, that lies near a functional threshold being capable to sufficiently maintaining viability in female, but not male, fly populations, during aging.

3.5.3. Sly Disease

Sly disease, or MPS type VII, is an autosomal recessive LSD caused by mutations in the human GUSB gene, which encodes the β-Glucuronidase enzyme. Loss of this enzyme leads to the accumulation of undegraded, or partially degraded, glycosaminoglycans (GAGs), ultimately resulting in widespread cellular dysfunction [14]. In Drosophila, three orthologs of the human GUSB gene have been identified. Among them, CG15117 exhibits the highest DIOPT score (Table 1) and a notably low RMSD value of 0.598 Å (Figure 11A), indicative of their strong structural similarity. In the, herein, developed Drosophila model of Sly disease, RNAi-mediated knockdown of the CG15117 gene, specifically, in the nervous system (brain) revealed a modest reduction, of ~39%, in mRNA expression levels (Figure 11B). Male transgenic flies with neuronal (brain)-specific downregulation of CG15117 gene were characterized by an age-dependent decline in lifespan, with a median reduction of ~16 days, compared to control males (Figure 11C). In contrast, female transgenic flies were presented with similar-to-control survival curves, under the same growth conditions (Figure 11C).

Interestingly, midgut-specific knockdown of the CG15117 gene, also, caused a pronounced, sex-dependent, lifespan impairment pattern. Male transgenic flies presented a significantly shortened lifespan, as clearly indicated by their reduced median and maximum lifespan of ~25 and ~40 days, respectively (Figure 11D). Female transgenic flies were (comparatively) less affected, with only a slight decrease in maximum lifespan being observed, thereby suggesting a male-specific vulnerability to CG15117 loss in both neuronal (brain) and midgut tissues. Negative-geotaxis (climbing-activity) assay of RNAi-mediated, CG15117-targeted, flies, specifically in the nervous system (brain), unveiled a progressive, age-dependent, decline in locomotor activity, for both sexes (Figure 11E). Although CG15117-downregulated flies presented near to normal climbing activity during the initial days of adult life, with only a mild early decline being observed in females, their motor performance deteriorated significantly from day 10 onward. By this stage, their climbing efficiency closely resembled that of 20-day-old control flies, thereby indicating an accelerated onset of age-dependent locomotor impairment (Figure 11E).

Taken together, the majority of the, herein, developed Drosophila models of Mucopolysaccharidoses (MPSs) successfully recapitulate key pathological features of MPS disorders, such as shortened lifespan and progressive motor decline. These invertebrate models provide a powerful platform for conducting genetic screens, in vivo, to: (a) mechanistically illuminating MPSs, (b) identifying genetic modifiers, and (c) conducting rapid, reliable, comprehensive and cost-effective drug-screening trials that are unfeasible to be implemented in -typical- vertebrate (e.g., zebrafish and mouse) model organisms.

4. Discussion

Most Lysosomal Storage Disorders (LSDs) lack effective treatments, rendering genome editing one of the most promising therapeutic strategies. However, before these genome editing tools can be applied in humans, several critical steps must precede, ranging from in vitro testing to clinical trials. To maximize safety and gather extensive preliminary data, in vivo modeling, using invertebrates, has gained major attention in the recent years [15,17]. These organisms offer a wide array of genetic tools and allow the in vivo study of various biological pathways and therapeutic approaches, in shorter times and with fewer ethical concerns than those in mammals. Drosophila melanogaster is a well-established invertebrate model system that offers an ideal background for genetic and biological studies of different human pathologies, as it contains functional orthologs for ~75% of the human disease-related genes [31]. Drosophila also features a plethora of genetic tools, including the GAL4/UAS, CRISPR/Cas9 and RNAi molecular platforms, which allow cell/tissue-specific gene targeting/downregulation [32,33].

In the present study, we suitably employed the -binary- GAL4/UAS and RNAi genetic systems, to selectively knockdown fly orthologs of human LSD-related genes, along the brain-midgut axis, during aging. Employment of commercially available transgenic strains, directly obtained from Bloomington Drosophila Stock Center (BDSC; Indiana, USA), enabled us to systemically investigate their morbid effects, in vivo. These findings provide a powerful invertebrate model for future studies, to broadly explore and deeply comprehend the molecular mechanisms that control LSD-pathology (initiation and progression), and to successfully develop novel genetic- and drug-based strategies for LSD-targeting therapies.

Sphingolipidoses represent a sub-category of LSDs being developed by deficiencies in enzymes responsible for the catabolism of sphingolipids, and they mainly affect nervous-system and peripheral-organ tissues. Gaucher disease (GD) is the most prevalent form and derives from deficiencies in the β-Glucocerebrosidase (GBA1) enzyme, leading to toxic accumulation of glucosylceramide [10]. Utilization of mouse models for Gaucher disease (GD) has proven challenging and limited, due to the elevated perinatal lethality associated with GBA1 gene mutations [10,34].

Hence, towards the establishment of a new in vivo model for the disease (GD), we, herein, investigated the impact of downregulating the Drosophila Gba1a and Gba1b orthologs of human GBA1 gene, along the brain-midgut axis, during aging. Our results revealed a marked reduction in lifespan and climbing ability, with females being more severely affected, compared to male populations. A previous study of Drosophila Minos-insertion mutants of the GBA1 orthologs reported that Gba1b mutants exhibited shortened lifespan and impaired climbing ability, whereas Gba1a mutants did not present significant pathologies [35]. Although Drosophila Gba1a and Gba1b fly orthologs show differential tissue-expression patterns, with Gba1a being primarily expressed in the midgut, and Gba1b being detected in the adult head and fat body [36], both genes seem to affect fly longevity and kinetic ability in a similar pattern, when downregulated in brain and midgut tissues.

Therefore, our approach indicates that both genes are essential for motor performance and survival in Drosophila. The progressive loss of neuronal cells and the resulting neurotoxicity in Gaucher disease (GD) [10] likely underlie the observed pathologies in locomotor activity and lifespan. The, herein, identified sex-specific differences in our Drosophila Gaucher disease (GD) model system may arise from multiple factors, including hormonal regulation, metabolic programs, immune responses and reproductive properties [37,38,39]. Taken together, our findings strongly support Drosophila as a powerful and versatile in vivo model for Gaucher disease (GD), providing insights into its genetic and pathophysiological mechanisms, including sex-specific disease manifestations.

Fabry is an X-linked recessive sphingolipidosis caused by a deficiency in the lysosomal enzyme α-Galactosidase A, due to mutations in the human GLA gene [7]. Interestingly, in our model system, herein being investigated, genetic downregulation of the fly GLA ortholog, CG5731, proved capable to more severely affect female flies, in both life expectancy and climbing capacity, compared to male populations. Of note, the sex-linked inheritance pattern having been observed in humans cannot be directly applied in Drosophila, since male flies upregulate their single X chromosome via dosage compensation [40] and, additionally, CG5731 is not an X-linked gene. A mechanistic explanation for the -comparatively- increased sensitivity detected in female flies may be associated with gender- and/or tissue-specific gene-expression programs, and differences in metabolic/nutritional demands and/or hormonal pathway/network activities. Moderate homologies might also reflect redundant or compensatory functions by other enzymes, or alternative mechanisms, in Drosophila that are absent or less efficient in humans. In a mouse model of the disease (FD), both male and female mice, deficient in α-Gal A (α-Galactosidase A), manifested a clinically normal phenotype at the 10^th-14^th weeks of age, thus rendering Fabry-disease (FD) modeling, in this vertebrate/mammalian system, challenging and limited [41]. However, in a study of Drosophila transgenic populations, expressing the human mutant GLA (variant) forms A156V and A285D, significant locomotor dysfunction and reduced lifespan were observed, compared to control flies (expressing the human wild-type enzyme). Strikingly, these phenotypes could be ameliorated with Migalastat (Fabry disease -FD- medication) treatment [42].

Altogether, our RNAi-based genetic platform, which targets the endogenous expression of CG5731 fly gene (human GLA homolog), specifically in the brain-midgut axis, during aging, may offer a powerful, reliable, multifaceted, dynamic and sensitive in vivo model system, for comprehensively studying Fabry disease (FD), to enabling efficient drug screening and to illuminating underlying disease mechanisms.

Niemann-Pick type C disease (NPC) is a neurodegenerative disorder that is sub-divided into types C1 and C2, depending on the respective -human- gene (NPC1 or NPC2) that is mutated. It is characterized by abnormalities in the intracellular transport of endocytosed cholesterol, which leads to the accumulation of cholesterol and sphingolipids within endo-lysosomes [6,12]. In the present study, we investigated the consequences of RNAi-mediated knockdown of Drosophila Npc1a and Npc2a gene orthologs, suitably engaging the GAL4/UAS genetic system, along the brain-midgut axis, during aging. The obtained male -transgenic- flies were characterized by reduced lifespan and locomotor dysfunction, for either organ-specific (brain, or midgut) targeting, in contrast to the female flies, which exhibited near-to-normal phenotypes.

A previous study in Drosophila, using loss-of-function mutants of the Npc1a gene, revealed developmental arrest at the first larval stage [43], thus rendering age-dependent pathologies during adulthood impossible to be profiled. Strikingly, in our model, although the viability patterns for the two genes are largely similar, the relative expression of Npc1a gene was detected less markedly reduced, compared to the Npc2a respective one (Figure 5B and Figure 6B). This indicates that even a modest decrease in the Npc1a gene-expression levels, within the nervous system (brain) setting, is sufficient to trigger a pathological phenotype, thereby highlighting the Npc1a essential role(s) in Drosophila well-being, during aging.

In toto, our genetic approach provides a powerful, trustworthy and manageable model system, for mechanistically illuminating and therapeutically advancing Niemann-Pick type C disease (NPC), in vivo.

GM2 gangliosidoses are characterized by excessive accumulation of ganglioside -GM2- species and related glycolipids in the lysosomes. The main forms include Tay-Sachs disease (TSD), caused by mutations in the HEXA gene, and Sandhoff disease (SD), caused by mutations in the HEXB gene [9,10,11]. In Drosophila, three genes (Hexo1, Hexo2 and fdl) have been identified, as encoding β-Hexosaminidase-like enzymes, based on sequence homologies to human Hexosaminidases [44,45]. Strikingly, RNAi-mediated downregulation of the Hexo2 (but not Hexo1) gene, specifically in the brain, revealed a remarkable reduction in life expectancy of Drosophila -transgenic- male flies (Figure 7G). Given that GM2 gangliosidoses are known to predominantly affect the central nervous system (CNS) [10,11], our results point to the essential contribution of -certain- β-Hexosaminidases to neuronal development and CNS/brain functionality in Drosophila, during aging, thereby validating model’s relevance to molecularly investigating GM2 gangliosidoses-induced neuro-pathologies, in vivo.

Mucopolysaccharidoses (MPSs) comprise a class of 11 lysosomal storage disorders, with each one being derived from -driver- deficiency in the activity of a distinct lysosomal hydrolase; they all belong to a family of enzymes that are critically involved in the sequential degradation of glycosaminoglycans (GAGs). MPS I and MPS II sub-types were typically classified among the first syndromes identified within this group [8]. In our Hurler syndrome (HLS) (MPS I; α-L-Iduronidase deficiency) in vivo model, although the expression of IDUA gene is not severely downregulated, reduced lifespan and locomotor deficiency, along the brain-midgut axis, were observed. Of note, a distinct study, regarding Hurler-syndrome (HLS) modeling, using a similar strategy, but different RNAi strains, which can still target the same Drosophila IDUA ortholog (CG6201) gene, has been previously reported by Filippis et al. [46]. However, in their set of experiments, although flies with reduced expression of the IDUA gene, in neuronal and glial cells, were presented with locomotion deficiencies, they, unexpectedly, manifested a longer lifespan, compared to controls [46].

Hence, our Drosophila Hurler-syndrome (HLS) model represents an invaluable, powerful, informative, constructive, manageable, novel, and, also, complementary (to the existing) -biological- tool, for genetically dissecting disease mechanisms and systemically expanding the repertoire of -experimental- in vivo models, hitherto available, to deeper investigating Hurler-syndrome (HLS) pathology, both mechanistically and therapeutically, for human’s maximum benefit.

Hunter syndrome (HNS) (MPS II; Iduronate-2-sulphatase deficiency) is an X-linked recessive LSD. Remarkably, in our -invertebrate- model system, only males exhibited a notable reduction in life expectancy, a pathological phenotype that is genetically associated with the sex-dependent nature of the (HNS) disease, in humans. The genetic modeling of Hunter syndrome (HNS) in Drosophila has been previously described, using the same (“RNAi”) strains, with the authors concluding that residual Ids/Ids activity(ties) may be sufficient to rescue MPS II-related pathologies, since, in their lethality assays, the survival from larva to pupa and the metamorphosis to the adult phase were not affected [47]. In contrast to their argument that engagement of RNAi-dependent -transgenic- technology for MPS II knockdown is not an effective strategy, our data strongly suggest that, under certain circumstances and specific settings, the exploitation of male flies, as a novel and reliable model system, for Hunter syndrome (HNS)-pathology research, in vivo, should not be ignored, or disregarded.

Employment of our Sly-disease (SLD) (MPS VII; β-Glucuronidase deficiency) -LSD- model demonstrated a remarkable reduction in the viability of male flies, along the brain-midgut axis, together with a progressive decline in locomotor activity for both sexes, during aging. A Drosophila model of MPS VII, developed by knocking-out the CG2135 gene, the fly ortholog of human GUSB, has been previously established, by Bar et al., successfully recapitulating key features of the Sly disease (SLD), such as shortened lifespan, motor deficiencies and neurological abnormalities [48]. Notably, Drosophila possesses two orthologs of the human GUSB gene; the CG2135 (βGlu) and the CG15117, with the latter exhibiting a slightly higher similarity score in DIOPT [25]. Although Bar et al. found that CG15117 was 6-fold less active than CG2135, our results clearly demonstrate that targeted downregulation of CG15117, in either brain or midgut tissues, during aging, critically compromises male fly viability, thereby strongly suggesting its (CG15117) beneficial utilization as an additional, but important and powerful, screening tool, for Sly disease (SLD) research, in vivo.

Altogether, we have, herein, identified the Drosophila orthologs of genes that are responsible for the most common Lysosomal Storage Disorders (LSDs), in humans, and systematically screened them for “patho-phenotypic” effects on life expectancy and climbing proficiency, specifically within the brain-midgut axis, during aging, suitably engaging the GAL4/UAS -binary- transgenic system, in combination with the RNAi-mediated gene-silencing platform. Most of these, in vivo, LSD models in Drosophila, herein, proved capable to successfully recapitulate key-disease phenotypes being identified in humans, including significantly reduced lifespan and progressive climbing deficiency, which serve as proxy for neuro-muscular disintegration, in age- and sex-dependent manners.

These -consistent- phenotypic parallels undoubtedly underline the value and importance of Drosophila as a robust, reliable, powerful, rapid, multifaceted, versatile and manageable, invertebrate, model system, ideally suitable and exploitable, for high-throughput genetic and pharmacological, in vivo, screenings, aiming at pathological-phenotype(s) rescue(s), while, also, providing invaluable insights into the underlying molecular and neurological mechanisms, tightly controlling LSD-specific pathologies and therapeutic-treatment responses.