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. 2019 Aug 15;86(4):294-305.
doi: 10.1016/j.biopsych.2019.04.029. Epub 2019 May 9.

Habituation Learning Is a Widely Affected Mechanism in Drosophila Models of Intellectual Disability and Autism Spectrum Disorders

Michaela Fenckova  1 , Laura E R Blok  1 , Lenke Asztalos  2 , David P Goodman  3 , Pavel Cizek  4 , Euginia L Singgih  5 , Jeffrey C Glennon  6 , Joanna IntHout  7 , Christiane Zweier  8 , Evan E Eichler  9 , Catherine R von Reyn  10 , Raphael A Bernier  11 , Zoltan Asztalos  12 , Annette Schenck  13
Affiliations

Habituation Learning Is a Widely Affected Mechanism in Drosophila Models of Intellectual Disability and Autism Spectrum Disorders

Michaela Fenckova et al. Biol Psychiatry. .

Abstract

Background: Although habituation is one of the most ancient and fundamental forms of learning, its regulators and its relevance for human disease are poorly understood.

Methods: We manipulated the orthologs of 286 genes implicated in intellectual disability (ID) with or without comorbid autism spectrum disorder (ASD) specifically in Drosophila neurons, and we tested these models in light-off jump habituation. We dissected neuronal substrates underlying the identified habituation deficits and integrated genotype-phenotype annotations, gene ontologies, and interaction networks to determine the clinical features and molecular processes that are associated with habituation deficits.

Results: We identified >100 genes required for habituation learning. For 93 of these genes, a role in habituation learning was previously unknown. These genes characterize ID disorders with macrocephaly and/or overgrowth and comorbid ASD. Moreover, individuals with ASD from the Simons Simplex Collection carrying damaging de novo mutations in these genes exhibit increased aberrant behaviors associated with inappropriate, stereotypic speech. At the molecular level, ID genes required for normal habituation are enriched in synaptic function and converge on Ras/mitogen-activated protein kinase (Ras/MAPK) signaling. Both increased Ras/MAPK signaling in gamma-aminobutyric acidergic (GABAergic) neurons and decreased Ras/MAPK signaling in cholinergic neurons specifically inhibit the adaptive habituation response.

Conclusions: Our work supports the relevance of habituation learning to ASD, identifies an unprecedented number of novel habituation players, supports an emerging role for inhibitory neurons in habituation, and reveals an opposing, circuit-level-based mechanism for Ras/MAPK signaling. These findings establish habituation as a possible, widely applicable functional readout and target for pharmacologic intervention in ID/ASD.

Keywords: Autism spectrum disorder; Drosophila; GABAergic neurons; Habituation learning; Intellectual disability; Ras/MAPK.

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Conflict of interest statement

Financial disclosures:

In the past 3 years, J.C.G. has acted as a consultant to Boehringer Ingelheim GmbH but is not an employee, stock- or share-holder of this company. E.E.E. is on the scientific advisory board (SAB) of DNAnexus, Inc.. Z.A. is a director and shareholder of Aktogen Ltd.. L.A. is a director of Aktogen Ltd.. The commercial light-off jump habituation system was purchased from Aktogen Ltd.. Aktogen Ltd. provided training of the personnel and ~ 150 experiments from the initial screen were performed at Aktogen Ltd. by M.F. and L.A.. All other authors report no biomedical financial interests or potential conflicts of interest.

Figures

Figure 1.
Figure 1.. Habituation screen of intellectual disability genes, phenotype distribution and proof of principle
(A) Procedure, phenotype categories and phenotype distribution of the light-off jump habituation screen. Knockdowns that resulted in lethality, no jumper phenotype (defined as less than 50% flies jumping in at least one of the first five light-off trials) or premature habituation plus increased fatigue were assigned to the category “non-performers” and their habituation was not further analyzed. Other phenotype categories are “habituation deficient”, “not affected”, and “premature habituation” (the latter if no fatigue was detected in secondary assay, see example in Figure S4). Drosophila orthologs of 34% of the investigated human ID genes were associated with defects in habituation learning. See also Table S2, S3. (B, C, D) Defective habituation upon neuron-specific RNAi-mediated knockdown of G9a, Synapsin (syn), and dunce (dnc) (2xGMR-wIR/+; UAS-RNAi/elav-Gal4, UAS-Dicer-2, in red) compared to their respective genetic background controls (2xGMR-wIR/+; elav-Gal4, UAS-Dicer-2/+, in gray). Jump response curves show the average jump response (% of jumping flies) over 100 light-off trials at 1 s inter-trial interval). Mean TTC: the mean number of trials that flies needed to reach the no-jump criterion (see Methods and Materials) presented as Mean TTC ± SEM. *** padj<0.001, ** padj<0.01, based on FDR-corrected lm analysis. A complete list of ID genes with previously identified habituation defects is provided as Table S8, adding further proof of principle.
Figure 2.
Figure 2.. Habituation deficits in Drosophila characterize ID genes with synapse-related functions
Of 25 gene ontology (GO)-based processes, “habituation deficient” genes are specifically and significantly enriched in processes related to synapse (E=1.59, p=0.024). Genes with no effect on habituation do not show significant enrichment in any GO process. * p<0.05, based on Fisher’s exact test. All enrichment scores, p-values and enriched genes are listed in Table S4.
Figure 3.
Figure 3.. Habituation deficits in Drosophila characterize ID genes associated with macrocephaly in humans
Enrichment of Drosophila phenotype categories across 27 ID-accompanying clinical features (41). “Habituation deficient” genes show specificity for macrocephaly and/or overgrowth (E=2.19, p=0.018) ** p<0.01, * p<0.05, based on Fisher’s Exact test. For enrichment among the “non-performers” category, see Figure S5. Enrichment scores, p-values and enriched genes are listed in Table S4.
Figure 4.
Figure 4.. Habituation deficits in Drosophila characterize ID genes associated with ASD and deficits in specific behavioral domains
(A,B) Enrichment of Drosophila phenotype categories “habituation deficient” and “not affected” in ID plus ASD-associated genes identified in SFARI database (ASD SFARI, E=1.65, p=0.016, (A)) and SSC cohort (ASD SSC, E=1.64, p=0.029 (B)). Circles represent total number of tested ID plus ASD-associated genes. (C) Genes associated with “habituation deficient” versus “not affected” phenotype categories in Drosophila show tendency for more aberrant behaviors on the ABC (p=0.04) in the ASD SSC cohort. Data presented as mean score ± SEM. * p<0.05, based on MANOVA. See also Table S5 (list of ASD SSC and ASD SFARI genes) and Table S6 (complete MANOVA results).
Figure 5.
Figure 5.. A central role for Ras-MAPK signaling in habituation learning
(A) Highly connected communities identified by unbiased community clustering, colored by their functional proximity (Figure S6). Red circles and gene names highlight nodes representing “habituation deficient” genes. For complete list of communities and genes see Table S7. (B) Nodes connecting four communities from the central module represent the core components of Ras-MAPK signaling. (C) Schematic representation of Ras-MAPK signaling and associated mechanisms in ID disorders called ‘Rasopathies’. (D) Increasing Ras signaling by inducing either loss of function of negative Ras regulators (left side of pathway scheme) or by constitutively activating Ras (right side) disrupts habituation learning. Left: Defective habituation upon neuron-specific knockdown of negative Ras regulators, Nf1 (2xGMR-wIR/+; Nf1-RNAivdrc35877/elav-Gal4, UAS-Dicer-2, N=72, in red) and Spred (2xGMR-wIR/+; Spred-RNAivdrc18024/elav-Gal4, UAS-Dicer-2, N=73, in red), compared to their corresponding genetic background controls (2xGMR-wIR/+; elav-Gal4, UAS-Dicer-2/+. N: 55, 20, in gray). *** padj<0.001, based on lm analysis and FDR correction in the screen (see Methods and Materials). Right: Defects in habituation learning in a heterozygous, constitutively active Ras mutant (Ras1R68Q/+, N=55, in green) compared to its genetic background control (N=43 in gray), and upon neuron-specific expression of Ras1R68Q (elav>Ras1R68Q: UAS-Ras1R68Q/2xGMR-wIR; elav-Gal4, UAS-Dicer-2/+, N=52, in green) compared to its genetic background control (2xGMR-wIR/+; elav-Gal4, UAS-Dicer-2/+, N=34, in gray). *** p<0.001, based on lm analysis. Data presented as Mean TTC ± SEM.
Figure 6.
Figure 6.. Dual, opposing role of Ras-MAPK signaling in GABAergic and cholinergic neurons in the regulation of habituation learning
(A) No effect on habituation of Ras1R68Q (N=51, in green), Nf1-RNAi (N=38, in red), and Spred-RNAi (N=55, in red) upon expression in cholinergic neurons compared to their respective genetic background controls (Cha-Gal4/+; 2xGMR-wIR/+, N: 54, 45, 54 in gray). Expression of RafGOF in cholinergic neurons resulted in lethality. (B) Defective habituation of Ras1R68Q (N=52, in green), RafGOF (N=57, in green), Nf1-RNAi (N=55, in red), and Spred-RNAi (N=37, in red) on habituation upon expression in GABAergic neurons compared to their respective genetic background controls (Gad1-Gal4/+; 2xGMR-wIR/+, N: 50, 50, 39, 58 in gray). (C) Defective habituation of Csw-RNAi (UAS-Csw-RNAivdrc21756/Y; Cha-Gal4/+; 2xGMR-wIR/+, N=58), Sos1-RNAi (UAS-Sos1-RNAivdrc42848/Cha-Gal4; 2xGMR-wIR/+, N=56), Ras1-RNAi (UAS-Ras1-RNAivdrc106642/Cha-Gal4; 2xGMR-wIR/+, N=55), Raf-RNAi (UAS-Raf-RNAivdrc20909/Cha-Gal4; 2xGMR-wIR/+, N=59) and Mek-RNAi (Cha-Gal4/+; UAS-Mek-RNAivdrc40026/2xGMR-wIR, N=58) in cholinergic neurons (in green) compared to their respective genetic background controls (Cha-Gal4/+; 2xGMR-wIR/+, N: 62, 54, 34, 46, 46, in gray). (D) No effect on habituation of Csw-RNAi (N=58), Sos1-RNAi (N=51), Ras1-RNAi (N=53), Raf-RNAi (N=52) and Mek-RNAi (N=54) in GABAergic neurons (in green) compared to their respective genetic background controls (Gad1-Gal4/+; 2xGMR-wIR/+, N: 60, 46, 54, 39, 39, in gray). Data presented as Mean TTC ± SEM. *** p<0.001, ** p<0.01, * p<0.05, based on lm analysis.
Figure 7.
Figure 7.. Connections between “habituation deficient” genes
Connections between “habituation deficient” genes, including Ras, identified in the reference network used for community clustering (See SM) with significantly increased connectivity (PIE score=1.89, p<0.001). Nodes are colored based on the community to which they belong. Nodes that represent “habituation deficient” genes but are not members of a community are labeled in black.
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References

    1. Peeke H (1973): Habituation: Behavioral studies. (Vol. 1), Academic Press.
    1. Colombo J, Mitchell DW (2009): Infant visual habituation. Neurobiol Learn Mem 92: 225–234. - PMC - PubMed
    1. Rankin CH, Abrams T, Barry RJ, Bhatnagar S, Clayton D, Colombo J, et al. (2009): Habituation Revisited: An Updated and Revised Description of the Behavioral Characteristics of Habituation. Neurobiol Learn Mem 92: 135–138. - PMC - PubMed
    1. Barron HC, Vogels TP, Behrens TE, Ramaswami M (2017): Inhibitory engrams in perception and memory. Proc Natl Acad Sci 201701812. - PMC - PubMed
    1. Kavšek M (2004): Predicting later IQ from infant visual habituation and dishabituation: A meta-analysis. J Appl Dev Psychol 25.

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