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The ortholog of human ssDNA-binding protein SSBP3 influences neurodevelopment and autism-like behaviors in Drosophila melanogaster [1]

['Safa Salim', 'Division Of Biological', 'Biomedical Sciences', 'Bbs', 'College Of Health', 'Life Sciences', 'Chls', 'Hamad Bin Khalifa University', 'Hbku', 'Doha']

Date: 2023-08

1p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism, macro/microcephaly, and dysmorphic features. The 1p32.3 chromosomal region harbors several genes critical for development; however, their validation and characterization remain inadequate. One such gene is the single-stranded DNA-binding protein 3 (SSBP3) and its Drosophila melanogaster ortholog is called sequence-specific single-stranded DNA-binding protein (Ssdp). Here, we investigated consequences of Ssdp manipulations on neurodevelopment, gene expression, physiological function, and autism-associated behaviors using Drosophila models. We found that SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. We observed decreased levels of armadillo in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp functionality. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS), altered neuronal mitochondrial morphology, and up-regulated fission and fusion genes. Flies with elevated levels of Ssdp exhibited heightened anxiety-like behavior, altered decisiveness, defective sensory perception and habituation, abnormal social interaction, and feeding defects, which were phenocopied in the pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Partial rescue of behavioral defects was observed upon normalization of Ssdp levels. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, we show that optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. Collectively, our findings provide evidence that Ssdp, a dosage-sensitive gene in the 1p32.3 chromosomal region, is associated with various anatomical, physiological, and behavioral defects, which may be relevant to neurodevelopmental disorders like autism. Our study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila.

Funding: This study was supported partly by grants from Qatar National Research Fund (QNRF), to F.M.: UREP28-269-1-051, NPRP13S-0121-200130 and NPRP14S-0319-210075. F.M., S.S., S.H., S.B.M.G., A.B., A.S., and F.A. were further supported by the College of Health and Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Qatar Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Genetic tractability and rich behavioral repertoire make Drosophila an excellent model for delineating genetic mechanisms of genes associated with psychiatric and cognition disorders, including ASD [ 17 – 24 ]. To determine the role of CNVs of SSBP3 in neurodevelopment and autism-associated behaviors, we used Ssdp overexpression and pan-neuronal knockdown strategy in male Drosophila fruit flies. We show that Ssdp overexpression modifies the morphology of wing, bristles, and head, and alterations in Ssdp levels affect brain development and glial number. Synaptic density was altered differentially in different brain regions upon Ssdp overexpression. Ssdp overexpression affects brain and wing development potentially via its role in canonical Wnt signaling. Using RNA sequencing, we found that Ssdp overexpression causes differential regulation of numerous genes and affects molecular pathways related to oxidative stress. Ssdp overexpression led to increased reactive oxygen species (ROS) and defective mitochondrial morphology and function. Ssdp overexpression also produced autism-associated behavioral deficits, and most of these features were recapitulated by Ssdp knockdown. Many of these behavioral defects were rescued upon normalization of Ssdp levels. Additionally, behavioral and functional defects were not observed upon temporal Ssdp knockdown in adult flies. We further show that optogenetically manipulating neuronal activity of Ssdp-expressing neurons in Drosophila altered various ASD-associated behavioral phenotypes. Overall, our data suggest that the Ssdp is a dosage-sensitive gene and the defects observed may in part be due to altered gliogenesis and Wnt signaling pathway. Taken together, our study proposes that SSBP3 dosage alterations underlie the various ASD and ID phenotypes associated with the 1p32.3 microdeletion/duplication region.

SSBP3 is an evolutionarily conserved gene, having an ortholog in Drosophila melanogaster [ 13 ]. While humans possess 4 SSBP3 homologs (SSBP1, SSBP2, SSBP3, and SSBP4), the fly genome has only 1; sequence-specific single-stranded DNA-binding protein (Ssdp). The protein sequences of SSBP3 and Ssdp contain highly conserved domains, including a lissencephaly type-1-like homology (LisH) motif and a proline-rich domain [ 13 , 14 ]. These regions in SSBP3 play a significant role in head development [ 14 , 15 ]. Furthermore, Ssdp is a part of the Wnt enhanceosome, which mediates the transcription switches of the Wnt/β-catenin signaling [ 16 ].

In humans, single-stranded DNA-binding protein 3 (SSBP3) is located in the 1p32.3 chromosomal region. A recent genome-wide assessment of the population frequency of deletion and duplication of CNVs determined SSBP3 to be one of the genes showing significant evidence of both haploinsufficiency and triplosensitivity [ 12 ]. Suggesting that a microdeletion of SSBP3 or a duplication that contributes an additional copy of the entire gene may alter the dosage of SSBP3 and produce deleterious phenotypes.

Autism spectrum disorders (ASD) are a group of heterogeneous neurodevelopmental disorders (NDDs) caused by multiple genetic/genomic dysfunctions, including chromosomal rearrangements, microdeletions, copy number variations (CNVs), and point mutations [ 1 ]. Autism is reported to be 4 times more prevalent in males than in females [ 2 – 4 ]. Around 15% to 20% of patients with NDDs, such as ASD, harbor chromosomal microdeletions [ 5 – 7 ]. As many as 23 patients with 1p32.3 microdeletion and 34 patients with 1p32.3 duplication are listed in the DECIPHER database [ 8 ], with many reported to have developmental delay, macrocephaly, intellectual disability (ID), and autism [ 9 ]. Notably, DECIPHER database has a record of 98 autistic patients who have micro/macrocephaly and many of these patients have 1p32.3 microdeletion or duplication [ 8 ]. Changes in the dosage of some genes by deletion or duplication can cause NDDs including ASD and ID [ 10 ]. Further, dosage sensitivity has been proposed to be a predominant causative factor underlying CNV pathogenicity [ 11 ]. Although the link between gene dosage sensitivity and diseases is well established, the mechanism of the pathogenicity remains unclear.

Results

Ssdp overexpression leads to alterations in Drosophila wing, mechanosensory bristles, and head size Minor physical anomalies (MPAs) are subtle morphological abnormalities of the face, head, and limbs, which are suggested to represent external markers of atypical brain development in ASD [42–44]. Studies have suggested a link between autistic traits, overall level of functioning, and MPAs [43,44]. Of note, craniofacial anomalies are a recurring feature of a subpopulation of ASD children with distinctive morphologies, including decreased facial midline height and long width of mouth, with ID and increased severity of ASD symptoms being comorbidities [45,46]. This comes as no surprise since both brain and face have common origins from neuroectodermal tissue and their development is closely coordinated due to their physical proximity and mutual molecular coordination [47]. Concomitantly, we asked whether overexpression of Ssdp produces any morphological abnormalities that phenocopy MPAs in ASD children. We performed scanning electron microscopy (SEM) with high magnification to detect structural changes in the wing, head, thorax, and eyes of 3- to 4-day-old Ssdp[2082-G4] adult flies compared to w1118 controls. We observed a novel phenotype in the wings of Ssdp[2082-G4] flies, with enhanced and deeper indentations on the surface of the wing in comparison to controls (Fig 3A). An increase in the number of interocellar bristles was observed in Ssdp[2082-G4] flies compared to controls (Fig 3B). There was also loss of bristles on the surface of pedicel on the antenna of Ssdp[2082-G4] flies compared to controls (Fig 3E). Lastly, overall, the Ssdp[2082-G4] heads appeared larger than the heads of w1118 controls (Fig 3E). No phenotypic differences were observed in the thorax, eyes, and proboscis of Ssdp[2082-G4] flies and controls (Fig 3C–3E, respectively). Our data suggest that Ssdp functions early in development and regulates the morphogenesis of wings, mechanosensory bristles, and head. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Ssdp overexpression alters morphology of wing, mechanosensory bristles, and head. High-resolution SEM images showing wing, head, thorax, and eyes of heterozygous Ssdp[2082-G4]/+ flies compared to w1118 controls at high magnification. (A) Deeper indentations are observed on the surface of the wings of Ssdp[2082-G4] /+ flies compared to controls. Red arrows point towards the difference of phenotype. (B) The number of interocellar bristles is increased in Ssdp[2082-G4]/+ flies compared to controls. Numbers in green mark the increased number of interocellar bristles. (C) No phenotypic differences are observed in the bristles present on the thorax of Ssdp[2082-G4]/+ flies and controls. (D) No phenotypic differences are observed in the eyes of Ssdp[2082-G4]/+ flies and controls. (E) Overall, the head of Ssdp[2082-G4]/+ fly is larger than that of control. Some bristles on the surface of the pedicel on the antenna of Ssdp[2082-G4]/+ flies are lost compared to controls. No phenotypic differences are observed in proboscis of both Ssdp[2082-G4]/+ flies and controls. Red lines highlight increase in head size. Green squares depict loss of bristles on the pedicel. Scale bars are depicted at the bottom right of each image. aOR, anterior orbital bristles; mOR, middle orbital bristles; pOR, posterior orbital bristles; pVT, posterior verticel bristles; aVT, anterior vertical bristles; OC, ocellar bristles; PV, postvertical bristles; SEM, scanning electron microscope; Ssdp, sequence-specific single-stranded DNA-binding protein. https://doi.org/10.1371/journal.pbio.3002210.g003

Ssdp affects mitochondrial morphology and fission/fusion machinery in Drosophila brain Mitochondria are dynamic and continuously undergo fission and fusion processes to maintain their shape, quality, and function. However, this machinery may be impaired in cells under stress [58]. Given the alterations in genes in RNA-seq data were associated with oxidative stress regulation (catalase, sepia, GstE11, GstE1, GstD5. CG9920), antioxidant function (Dhrs4, CG40486), and mitochondrial dynamics and function (Gdap1 and Hsp23) and increase in oxidative stress in specific regions of the brain in Ssdp[2082-G4] flies, we further investigated whether these are associated with abnormalities in the morphology of mitochondria in neurons. We assessed the area, circularity, and length of mitochondria in the brains of 3- to 4-day-old flies expressing UAS-Mito-Red in Ssdp[2082-G4]-Gal4 and Elav-Gal4 (serving as controls) (Fig 8A). Notably, we observed a difference in the pattern of Mito-Red expression using the 2 drivers, with the distribution being more spread and diffuse in the control brains. We suggest that this is an artifact of the Elav-Gal4 driver rather than a representation of mitochondrial morphology [59,60]. Hence, the SEZ region, presenting a more comparable mitochondrial morphology, was used for analysis in the controls. We observed a significant decrease in the area and circularity of mitochondria in both SLP and SEZ (Fig 8B). However, the length was significantly increased (Fig 8B). This data suggests an enhanced mitochondrial fusion or defective fission process [51] in Ssdp overexpressing flies. We then performed qRT-PCR to investigate genes involved in the fission/fusion process in the heads of Ssdp[2082-G4] and controls and observed the up-regulation of 1 fission (Drp1) and 1 fusion gene (Marf) (Fig 8C). The expression levels of Fis1 and Opa1 were not affected. We further confirmed the up-regulation of Drp1 and Marf by performing qRT-PCR on the brains of 3- to 4-day-old Ssdp[2082-G4] and controls (S7 Fig). Overall, our data suggests that Ssdp overexpression results in disbalance of mitochondrial fission/fusion machinery, with an enhancement in fusion events. PPT PowerPoint slide

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TIFF original image Download: Fig 8. Ssdp overexpression causes abnormalities in mitochondrial morphology and dynamics. (A) Immunohistochemistry images of brains expressing UAS-Mito-Red in the Elav-Gal4 and Ssdp[2082-G4]-Gal4 labeled neurons. White squares highlight the SEZ and SLP in Ssdp[2082-G4]-Gal4 image. Magnified images of SEZ and SLP regions are also shown. (B) Mitochondrial area in SEZ (Cohen’s d = −2.0 [95 CI −2.84, −1.13], p < 0.0001, n = 10) and SLP (Cohen’s d = −3.8 [95 CI −5.39, −2.55], p < 0.0001, n = 10), and circularity in SEZ (Cohen’s d = −2.6 [95 CI −3.57, −1.65], p < 0.0001, n = 10) and SLP (Cohen’s d = −2.6 [95 CI −3.4, −1.6], p < 0.0001, n = 10) are significantly decreased, while length in SEZ (Cohen’s d = 1.5 [95 CI 0.822, 2.19], p = 8.0 × 10−04, n = 9–10) and SLP (Cohen’s d = 2.1 [95 CI 1.29, 2.86], p < 0.0001, n = 10) is significantly increased in Ssdp[2082-G4] in comparison to Elav controls. Quantification was performed per hemisphere. (C) Genes associated with mitochondrial fission (Drp1, p = 0.048, n = 7) and mitochondrial fusion (Marf1, p = 0.020, n = 7) are up-regulated in Ssdp[2082-G4]/+ in comparison to w1118 controls. Fis1 (p = 0.54, n = 7) and Opa1 (p = 0.16, n = 7) mRNA levels were unaltered in Ssdp[2082-G4]/+ flies compared to controls. P-values were obtained by Student’s t test. For all qRT-PCR experiments, expression was normalized to Rpl32. Horizontal red line represents the mean value. Each black dot is a data point representing an independent biological replicate. For B, p-values were obtained by permutation t test. P-values less than 0.05 were considered significant. The raw data underlying panels 8B and C can be found in S1 Data file. SEZ, subesophageal zone; SLP, superior lateral protocerebrum; Ssdp, sequence-specific single-stranded DNA-binding protein. https://doi.org/10.1371/journal.pbio.3002210.g008

Alteration in Ssdp expression affects anxiety-like behavior and decision-making Anxiety and difficulty in decision-making are common phenotypes observed in autistic individuals [49,50,52]. Multiple high-confidence ASD risk genes associated with anxiety were dysregulated in our RNA-seq data including TMLHE and GRID2 [61], DNAH10 [62], GRIA1 [63], SLC6A4 [64], and IGF1 [65]. Further, genes associated with motor defects were also dysregulated including GRIA1 [66], SLC6A4 [67], and GRID2 [68]. To investigate the effects of Ssdp CNVs on fly locomotor behavior, we recorded the activity of 3- to 4-day-old flies using the Drosophila arousal tracking (DART) system in an open field arena [69]. In this assay, apart from measuring fly locomotor output like average walking speed, we also measured wall-following, an anxiety-like behavior [19,69]. We observed that Ssdp manipulations did not affect average locomotor speed (Fig 9A). However, Ssdp[2082-G4] flies but not the knockdown flies, exhibited a significant increase in the percentage of time spent near the wall edge compared to the controls, suggesting higher anxiety (Figs 9B and S8A). PPT PowerPoint slide

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TIFF original image Download: Fig 9. Ssdp knockdown and overexpression cause autism-like behavioral deficits in Drosophila. (A) Active average speed in an open field arena is unaffected in Elav-Gal4>UAS-Ssdp-RNAi flies compared to the genotypic controls w1118>Elav-Gal4 and w1118>Ssdp-RNAi (n = 45–50, Cliff’s Δ = 0.16 [95 CI −0.0242, 0.335], p = 0.12), and in heterozygous Ssdp[2082-G4]/+ flies compared to w1118 controls (n = 45–50, Cliff’s Δ = −0.20 [95 CI −0.427, 0.0407], p = 0.0938). (B) Ssdp[2082-G4]/+ (n = 45–50, Cliff’s Δ = 0.32 [95 CI 0.0787, 0.527], p = 0.0098) but not knockdown flies (n = 45–50, Cliff’s Δ = 0.12 [95 CI −0.0702, 0.293], p = 0.256) show a significantly higher wall-following compared to controls. (C) Ssdp knockdown and Ssdp[2082-G4] flies are impaired in approach angle bimodal distribution. Fly representations created with BioRender.com. (D) Ssdp knockdown flies show increased startle to the first blue light pulse (genotypic controls, n = 38, Cohen’s d = 0.48 [95 CI 0.28, 0.69], p < 0.0001; Elav>Ssdp-RNAi, n = 38, Cohen’s d = 0.8 [95 CI 0.53, 1.1], p < 0.0001) and defective habituation to the fifth pulse, compared to their genotypic controls (n = 37–39, Cohen’s d = 0.69 [95 CI 0.31, 1.1], p = 2.0 × 10−04). (E) Ssdp[2082-G4]/+ also show increased startle to the first pulse (w1118, n = 39, Cohen’s d = −0.09 [95 CI −0.43, 0.37], p = 0.7; Ssdp[2082-G4]/+, n = 38, Cohen’s d = 0.3 [95 CI 0.07, 0.55], p = 0.025) and defective habituation to the fifth pulse compared to controls (n = 37–38, Cohen’s d = 0.62 [95 CI 0.23, 0.96], p = 0.008). (F) Sample frames of interactions among males of different genotypes (top) and heatmap occupancy plots for all flies in each group (bottom). (G) Interaction time was reduced in both Ssdp knockdown (n = 36–41, Cliff’s Δ = −0.64 [95 CI −0.79, −0.44], p < 0.0001) and Ssdp[2082-G4]/+ flies (n = 45–48, Cliff’s Δ = −0.41 [95 CI −0.62, −0.17], p = 0.001). (H) Schematic of FlyPad assay. Created with BioRender.com. (I) Ssdp knockdown (n = 42–43, Cohen’s d = −1.07 [95 CI −1.39, −0.73], p < 0.0001) and Ssdp[2082-G4]/+ (n = 35–36, Cohen’s d = −1.02 [95 CI −1.5, −0.57], p < 0.0001) flies exhibit reduced duration of interaction with food. (J) Heatmap depicts effect size (Cliff’s Δ) for all the feeding parameters between controls and experimental groups. Asterisks indicate level of statistical significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In scatter plots, each dot represents the mean value for a single fly. Horizontal red line represents the mean value. P-values are from a two-sided permutation t test. The raw data underlying panels 9A–E, G, and I can be found in S1 Data file. Ssdp, sequence-specific single-stranded DNA-binding protein. https://doi.org/10.1371/journal.pbio.3002210.g009 We next assessed wall approach-avoidance behavior in an open field [69], by analyzing the proportional distribution of the wall approach angles of 3- to 4-day-old flies. A fly approaching the wall avoids a head-on collision [69]; however, a fly defective in quick decision-making might collide with the wall more often. As reported earlier [69], control flies exhibited a bimodal distribution in wall approach angles, with the most preferred angles being around ±45° (Fig 9C). In contrast to the bimodal distribution of control flies, both Ssdp knockdown and Ssdp overexpressing flies showed flattened approach-angle distribution, with a peak around 0° (Fig 9C). These results suggest that flies with altered Ssdp expression experience indecisiveness when approaching the wall and walk head-on, which might cause head-butting. Overall, our data indicates that Ssdp regulates mental processes including anxiety-like behaviors and decision-making.

Ssdp affects sensory perception and habituation Adult Drosophila startle in response to odor [70] and light-on-light-off-stimuli [71]. Repeated presentation of the same stimuli leads to a decrease in behavioral response, known as habituation learning. Habituation learning represents a higher cognitive function of filtering and processing sensory information to navigate a dynamic environment [71]. Defective learning, or ID, is a core behavioral feature of individuals with NDDs like ASD [72] and has previously been used to characterize autism genes in Drosophila [71]. To investigate the functional role of Ssdp in startle and habituation behavior, we subjected 3- to 4-day-old flies to a blue light-on after-dark period paradigm, measuring startle response to a visual stimulus and habituation to repeated stimuli. Ssdp knockdown and Ssdp[2082-G4] flies displayed enhanced baseline speed compared to controls (S8B and S8C Fig, respectively) and heightened startle response to the first blue light pulse (Fig 9D and 9E, respectively). Ssdp knockdown and Ssdp[2082-G4] flies also exhibited defective habituation to the fifth light pulse compared to the controls (Fig 9D and 9E, respectively). This data provides the evidence that Ssdp is required by Drosophila for visual filtering mechanisms and processing sensory information. From our RNA-Seq data, among the 11 differentially regulated high-confidence ASD risk genes, there are few, which display learning disability phenotypes, which include GRIA1 [73], GRID2 [74,75], and IGF1 [76].

Changes in Ssdp expression impairs social interactions Drosophila provides a range of behavioral repertoires, including social interactions, which may be reminiscent of human social interaction behavior [77,78]. Impaired social interactions have previously been shown in multiple Drosophila ASD models [17,20,79,80]. Here, we studied social behaviors in a group of four 3- to 4-day-old male flies having reduced or elevated Ssdp expression. We analyzed parameters such as the sitting interaction time of each fly with another fly and the number of sitting contacts in 20 min. We observed that Ssdp knockdown and Ssdp[2082-G4] flies have significantly decreased interaction time (Fig 9F and 9G) and decreased the number of contacts (S8D Fig) with each other compared to their respective controls. Our data strongly suggests that both decreased and elevated levels of Ssdp cause impairment in social interactions. We inspected our RNA-seq data and found that among the 11 differentially regulated high-confidence ASD risk genes, GRIA1 [81], GRID1 [82], and IGF1 [83] have been shown to modulate social behavior.

Ssdp regulates feeding behavior Feeding-related problems are very common among individuals affected with ASD [84,85]. In the Drosophila brain, Ssdp expresses in many SEZ neurons, a region known to affect feeding behavior [86]. We determined if the altered Ssdp expression might influence feeding behavior in 3- to 4-day-old flies. We utilized the Fly Proboscis and Activity detector (FlyPAD) [87] (Fig 9H) and observed that Ssdp knockdown and Ssdp[2082-G4] flies have significantly fewer sips and meals, with decreased duration of interaction with food and increased intervals between sips and meals (Figs 9I and 9J and S8E). Our data suggests that Ssdp regulates feeding behavior in a dose-dependent manner. Closer inspection of RNA-seq data revealed that hunger and satiety controlling factors were among the dysregulated genes in heads of Ssdp[2082-G4] flies. Neuropeptides such as insulin-like peptides (ILPs) 3 and 5 were up-regulated, and the satiety controlling neurohormone, female-specific independent of transformer (fit) was down-regulated. These ILPs and the neurohormone, fit, are known to be secreted from a specific set of neurons in the fly brain and control satiety signals that negatively affect motivation for feeding [88]. It is worth noting here that in the Drosophila brain, Insulin Receptor (InR) is known to suppress the neurohormone fit [88].

Partial rescue of behavioral defects upon knocking down Ssdp expression in Ssdp-overexpressing cells Given that overexpression of Ssdp in Ssdp-positive cells produced multiple behavioral defects, we next asked whether knockdown of Ssdp in these cells would rescue the behavioral defects observed. We first performed qRT-PCR to analyze Ssdp mRNA expression in the heads of 3- to 4-day-old Ssdp[2082-G4] flies and Ssdp-RNAi-expressing Ssdp[2082-G4] flies and compared it to the relative mRNA expression in heads of w1118 controls. Consistent with our previous finding, Ssdp mRNA levels were increased in the heads of Ssdp[2082-G4] flies; however, the Ssdp mRNA level in the heads of Ssdp-RNAi-expressing Ssdp[2082-G4] flies were reduced by 12.5% compared to Ssdp[2082-G4] flies but were not statistically significant (S9A Fig). Furthermore, the mRNA levels in Ssdp-RNAi-expressing Ssdp[2082-G4] flies were still higher compared to w1118 controls but they were not statistically different (S9A Fig). We then performed multiple behavioral analyses on 3- to 4-day-old Ssdp[2082-G4] and Ssdp-RNAi-expressing Ssdp[2082-G4] flies to determine the implications of this reduction in Ssdp. As shown before (Figs 9 and S8), in Ssdp[2082-G4] flies, locomotor activity was unchanged (S9B Fig), wall-following was enhanced (S9C Fig), habituation speed was decreased (S9D Fig), and interaction time and number of contacts were decreased (S9E Fig) compared to controls. However, in Ssdp-RNAi-expressing Ssdp[2082-G4] flies, locomotor activity was reduced (S9B Fig), wall-following behavior was unchanged (S9C Fig), habituation speed was unchanged (S9D Fig), and sociation interaction defects were still observed (S9E Fig), in comparison to w1118 controls. Compared to Ssdp[2082-G4] flies, Ssdp-RNAi-expressing Ssdp[2082-G4] flies showed decreased average speed (S9B Fig), anxiety (S9C Fig), and habituation speed (S9D Fig), but showed no change in social interaction (S9E Fig). Our data suggests that knocking down Ssdp in Ssdp-positive cells that overexpress Ssdp, normalizes Ssdp mRNA levels and rescues anxiety and habituation learning behavioral deficits. We rationalize that even miniscule alterations in Ssdp levels manifest as alterations in the social interaction of the flies.

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