A screen for genes that regulate synaptic growth reveals mechanisms that stabilize synaptic strength

Synapses grow, prune, and remodel throughout development, experience, and disease. This structural plasticity can destabilize information transfer in the nervous system. However, neural activity remains remarkably stable throughout life, implying that adaptive countermeasures exist to stabilize neurotransmission. Aberrant synaptic structure and function has been associated with a variety of neural diseases including Fragile X syndrome, autism, and intellectual disability. We have screened disruptions in over 300 genes in Drosophila for defects in synaptic growth at the neuromuscular junction. This effort identified 12 mutants with severe reductions or enhancements in synaptic growth. Remarkably, electrophysiological recordings revealed synaptic strength in all but one of these mutants was unchanged compared to wild type. We utilized a combination of genetic, anatomical, and electrophysiological analyses to illuminate three mechanisms that stabilize synaptic strength in the face of alterations in synaptic growth. These include compensatory changes in 1) postsynaptic receptor abundance; 2) presynaptic morphology; and 3) active zone structure. Together, this analysis identifies new genes that regulate synaptic growth and the adaptive strategies that synapses employ to homeostatically stabilize synaptic strength in response. AUTHOR SUMMARY Throughout development, maturation, experience, and disease, synapses undergo dramatic changes in growth and remodeling. Although these processes are necessary for learning and memory, they pose major challenges to stable function in the nervous system. However, neurotransmission is typically constrained within narrow physiological ranges, implying the existence of homeostatic mechanisms that maintain stable functionality despite drastic alterations in synapse number. In this study we investigate the relationship between synaptic growth and function across a variety of mutations in neural and synaptic genes in the fruitfly Drosophila melanogaster. Using the neuromuscular junction as a model system, we reveal three adaptive mechanisms that stabilize synaptic strength when synapses are dramatically under- or over-grown. Together, these findings provide insights into the strategies employed at both pre- and post-synaptic compartments to ensure stable functionality while allowing considerable flexibility in overall synapse number.


INTRODUCTION
7 a single puncta of vGlut intensity to represent a synaptic bouton (Fig 1A and 1B

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We next sought to characterize the relationship between synaptic growth and function in the 226 nine FMRP target mutants in more detail. In particular, we sought to illuminate how, or whether, 227 synaptic scaling or homeostasis was expressed. We first characterized synaptic function and 228 structure in the four undergrowth mutants. Mutations in the first gene, protein kinase C 53E 229 (pkc53E), exhibited reductions in synaptic strength that appeared to scale with synaptic growth 230 ( Fig 3D). Bouton numbers were reduced by ~50% in homozygous mutants of pkc53E (S1 Table) 231 and in pkc53E mutants in trans to a deficiency that removed the entire locus (pkc53E 1 /pkc53E Df ;  Table). Correspondingly, EPSP amplitude was reduced to a similar extent in postsynaptic glutamate receptors in pkc53E mutants. However, we observed no significant 239 difference in mEPSP amplitude in pkc53E mutants compared to wild type ( Fig 4D; S3 Table), Syndrome, a disease resulting in premature aging due to DNA damage [54][55][56]. Null mutations 261 in WRNexo have been generated and characterized in the context of DNA repair in Drosophila 262 [57]. However, roles for WRNexo in synaptic growth or function have not been reported, nor 263 have they been characterized at the NMJ. WRNexo mutants exhibit significant reductions in 264 synaptic growth, with bouton numbers reduced by ~50% compared to wild type controls ( Fig 5A   265 and 5B). However, EPSP amplitude in WRNexo mutants was similar to wild type ( Fig 5C and   266 5D). Quantification of mEPSP amplitude revealed a significant increase in WRNexo mutants 267 compared to wild type, resulting in a corresponding reduction in quantal content (Fig 5C and 268 5D). Together, this suggests that while presynaptic neurotransmitter release is reduced in 269 accordance to reduced synaptic growth in WRNexo mutants, an increase in the postsynaptic 270 responsiveness to neurotransmitter was sufficient to maintain normal synaptic strength.
Gγ30A, the gamma subunit of a heterotrimeric G protein [63]. Interestingly, despite a ~60% 290 reduction in bouton number compared to wild type (Fig 6A and 6B), these two mutants 291 appeared to have no obvious changes in synaptic physiology (Fig 6C and 6D). mEPSP 292 amplitudes were similar to wild type in both mutants, which implies that a presynaptic change in 293 either active zone number and/or release probability likely compensated for reduced bouton 294 number to maintain stable levels of presynaptic neurotransmitter release.

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We therefore quantified the number of BRP puncta per NMJ in cont and Gγ30A mutants.
296 Surprisingly, immunostaining of BRP revealed that total puncta number per NMJ were similar in 297 both cont and Gγ30A mutants to wild type (Fig 6E and 6F). Further analysis found that while 298 bouton numbers were indeed reduced, individual boutons were significantly enlarged in area in 299 these mutants (Fig 6E and 6F). Thus, although cont and Gγ30A were defined as synaptic 300 undergrowth mutants based on our bouton counting assay, increased bouton area conserved 301 total neuronal membrane area ( Fig 6F). Consistently, quantification of BRP puncta per bouton 302 revealed a significant increase in both cont and Gγ30A (Fig 6E and 6F), demonstrating that 303 active zone number scaled with the enhanced NMJ membrane and area of individual boutons.

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Thus, despite a reduction in overall bouton number, increased synapse number per bouton was 305 sufficient to maintain total synapse number per NMJ, and synaptic strength, in both cont and stress gene receptor expression enhancing protein (reep). Despite the diverse functions of 315 these genes (S2 Table), they shared a common 40-50% increase in the number of synaptic 316 boutons per NMJ but stable synaptic strength (Fig 7A and 7C). Electrophysiological analysis 317 revealed no significant changes in mEPSP amplitude, EPSP amplitude, or quantal content ( Fig   318   7B and 7E; S1 Table). This suggests the postsynaptic sensitivity to neurotransmitter was not 319 impacted in these mutants, and implies a change in synapse number and/or release probability 320 likely compensated for the increased bouton number shared in these mutants.

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Next, we quantified the total number of BRP puncta per NMJ in these overgrowth 322 mutants. We found an increase in total BRP puncta number per NMJ that correlated with the 323 enhanced synaptic growth observed in each overgrowth mutant (Fig 7A and 7E).

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Correspondingly, we observed no major differences in bouton size, leading to a parallel 325 increase in total neuronal membrane surface area per NMJ and no change in BRP puncta 326 density (S3 Table). Hence, BRP puncta number essentially scales with bouton number in the 327 overgrowth mutants, in contrast to the undergrowth mutants detailed in Fig 6. This suggests that 328 a reduction in release probability per active zone likely stabilized synaptic strength in these 329 mutants.

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The size and abundance of material at individual active zones can vary considerably,

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and several studies have found that these properties can correlate with release probability [64-  Table). Indeed, the average 341 BRP puncta area scaled with total BRP puncta number per NMJ in wild type and in the synaptic 342 overgrowth mutants ( Fig 8C; R 2 =0.27, p-value=0.0006). While we did observe a significant 343 inverse correlation (R 2 value) between BRP puncta number and area, the curve fit of these data total abundance of BRP per NMJ, reflected in the sum fluorescence intensity of BRP puncta 346 across an entire NMJ, was not significantly different between wild type and the five overgrowth 347 mutants ( Fig 8D; S3 Table). Thus, an apparent tuning of active zone size may have 348 compensated for increased number to reduce release probability per active zone and maintain 349 synaptic strength in the overgrowth mutants isolated from the genetic screen.

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Through a forward genetic screen of ~300 mutants, we have identified genes required for 353 property regulation of synaptic growth and neurotransmission. This approach has revealed 354 several new mutations and RNAi lines that disrupt synaptic growth and function, while also 355 demonstrating that these processes are regulated through distinct pathways. This data implies 356 the existence of a homeostat that stabilizes global synaptic strength while permitting substantial 357 flexibility in synaptic growth. Our analysis has defined three adaptive mechanisms that operate 358 to maintain synaptic strength when synaptic growth is dramatically altered.
glutamate release via modulation of potassium channels, calcium influx, short-term plasticity,

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There is emerging evidence that both homeostatic and Hebbian forms of plasticity share 374 common genes and signaling networks [8,[87][88][89]. While the Drosophila NMJ is built for stability 375 and has proven to be a powerful model to investigate glutamatergic transmission and 376 homeostatic plasticity, contrasting forms of Hebbian plasticity are less obvious at this synapse.

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Hence, mutations of genes with specialized functions in non-glutamatergic synaptic 378 transmission or Hebbian plasticity are unlikely to reveal phenotypes using the screening 379 strategy we employed. However, a variety of genes were identified with significant and more 380 subtle roles in regulating synaptic growth and baseline function (S1 Table). Mutations in one    analyses of synaptic terminals and active zones) is considered an n of 1 since each presynaptic 515 motor neuron terminal is confined to its own muscular hemisegment. For these experiments, 516 muscles 4 or 6 were analyzed from hemisegments A3 for each larvae, and thus each larvae 517 contributes 2 NMJs per experiment. To control for variability between larvae within a genotype,

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for immunostaining experiments involving BRP and GluRIII, NMJs were analyzed from no less 519 than 6 individual larvae.

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Statistical analysis was performed using GraphPad Prism software. Data were tested for 521 normality using a D'Agostino-Pearson omnibus normality test. Normally distributed data were 522 analyzed for statistical significance using a t-test (pairwise comparison), or an analysis of 523 variance (ANOVA) and Tukey's test for multiple comparisons. For non-normally distributed data,

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Wilcoxon rank-sum test or Dunn's multiple comparisons after nonparametric ANOVA were used.