The histone demethylase KDM5 is required for synaptic structure and function at the Drosophila neuromuscular junction

Abstract

Mutations in the genes encoding the lysine demethylase 5 (KDM5) family of histone demethylases are observed in individuals with intellectual disability (ID). Despite clear evidence linking KDM5 function to neurodevelopmental pathways, how this family of proteins impacts transcriptional programs to mediate synaptic structure and activity remains unclear. Using the Drosophila larval neuromuscular junction (NMJ), we show that KDM5 is required presynaptically for neuroanatomical development and synaptic function. The Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, which is expected to be diminished by many ID-associated alleles, is required for appropriate synaptic morphology and neurotransmission. The activity of the C5HC2 zinc finger is also required, as an ID-associated mutation in this motif reduces NMJ bouton number, increases bouton size, and alters microtubule dynamics. KDM5 therefore uses demethylase-dependent and independent mechanisms to regulate NMJ structure and activity, highlighting the complex nature by which this chromatin modifier carries out its neuronal gene-regulatory programs.

Keywords: Drosophila; KDM5; glutamatergic signaling; histone demethylase; intellectual disability; microtubule dynamics; motor neuron; neuromuscular junction; transcription.

Figure 1.. kdm5 expression is required in motor neurons for normal NMJ development

(A1–A3) NMJ morphology at muscle 4 of abdominal segment 3 (NMJ4-A3) of control (w¹¹¹⁸) third-instar larvae labeled with the presynaptic marker HRP (magenta; A1) and postsynaptic marker Dlg (green; A2), and merged (A3). (B1–B3) kdm5¹⁴⁰ homozygous larva. (C1–C3) kdm5 knockdown larva (Act5C>kdm5-shRNA). (D1–D3) kdm5¹⁴⁰; elav>UAS-kdm5. (E1–E3) kdm5¹⁴⁰; mef2>UAS-kdm5. (A3′–E3′) Boxed areas in (A3)–(E3) showing higher magnification of type Ib boutons. Scale bars, 10 μm (E3) and 5 μm (E3′). (F) Quantification of type Ib bouton number normalized to muscle surface area. ***p = 0.0001, **p = 0.0041. Control (w¹¹¹⁸) n = 25, kdm5¹⁴⁰ n = 20, Act5C>kdm5-shRNA n = 14, control (+; shRNA) n = 14. Error bars: mean ± SEM (G) Quantification of type Ib bouton number normalized to muscle surface area. ****p < 0.0001, **p = 0.0029, *p = 0.0036 (OK6), *p = 0.0282 (da); ns, not significant. Control (w¹¹¹⁸) n = 16, no driver (kdm5¹⁴⁰; UAS-kdm5) n = 14, kdm5¹⁴⁰; elav>kdm5 n = 12, kdm5¹⁴⁰; OK6>kdm5 n = 10, kdm5¹⁴⁰; mef2>kdm5 n = 10, kdm5¹⁴⁰; repo>kdm5 n = 12, kdm5¹⁴⁰; Act5C>kdm5 n = 10, kdm5¹⁴⁰; da>kdm5 n = 11. Error bars: mean ± SEM (H) Quantification of type Ib bouton size in kdm5¹⁴⁰ larvae and ubiquitous knockdown (Act5C>kdm5-shRNA). Violin plots show the frequency distribution of bouton surface area indicating the median and quartiles. **p = 0.0020. Control (w¹¹¹⁸) n = 327, kdm5¹⁴⁰ n = 280, Act5C>kdm5-shRNA n = 198, control (+; shRNA) n = 281. (I) Quantification of type Ib bouton size in rescue experiments using the genotypes shown. ****p < 0.0001, p = 0.064. Control (w¹¹¹⁸) n = 340, no driver (kdm5¹⁴⁰; UAS.kdm5) n = 149, kdm5¹⁴⁰; elav>kdm5 n = 245, kdm5¹⁴⁰; OK6>kdm5 n = 191, kdm5¹⁴⁰; mef2>kdm5 n = 123, kdm5¹⁴⁰; repo>kdm5 n = 254, kdm5¹⁴⁰; Act5C>kdm5 n = 181, kdm5¹⁴⁰; da>kdm5 n = 201. (J1–K2) Futsch (white) immunolabeling in segments of NMJ4-A3 showing microtubules within synaptic boutons and HRP (magenta) from control (J1 and J2) and kdm5¹⁴⁰ (K1 and K2). Green triangles indicate punctate signal and yellow triangles indicate looped boutons. Scale bar, 10 μm. (L) Quantification of unbundled boutons, punctate and looped, as the percentage of total boutons in NMJ4-A3 of control and kdm5¹⁴⁰. *p = 0.0137, **p = 0.0047. Control n = 20, kdm5¹⁴⁰ n = 21. Error bars: mean ± SEM

Figure 2.. The demethylase activity of KDM5 is required to regulate NMJ bouton number

(A) Schematic of Drosophila KDM5 showing the position of the mutations in the JmjC domain that abolish enzymatic activity. (B1–C3) NMJ4-A3 of wild-type (kdm5^WT; B1–B3) and demethylase-inactive (kdm5^JmjC*; C1–C3) third-instar larvae stained with HRP (B1 and C1) and Dlg (B2 and C2), and merged (B3 and C3). Scale bar, 10 μm. (D) Quantification of type Ib bouton number normalized to muscle surface area in kdm5^WT and kdm5^JmjC*. ****p < 0.0001. kdm5^WT n = 15, kdm5^JmjC* n = 31. Error bars: mean ± SEM (E) Quantification of type Ib bouton size in kdm5^WT and kdm5^JmjC*. kdm5^WT n = 234, kdm5^JmjC* n = 388. ns, not significant.

Figure 3.. Fly strains harboring ID-associated mutations are viable and developmentally similar to controls

(A) Schematic of Drosophila KDM5 showing missense mutations that are equivalent ID-associated variants in human KDM5C. (B) Western blot of larval CNS from kdm5^WT, kdm5^R873W, kdm5^Y874C, and kdm5^L854F showing expression of HA-tagged KDM5 (top), H3K4me3 (middle), and total histone H3 (bottom). (C) Triplicate quantification of H3K4me3 levels relative to total histone H3 in kdm5^R873W, kdm5^Y874C, and kdm5^L854F compared to the ratio observed in kdm5^WT. ns, not significant. Error bars: mean ± SEM (D) Developmental timing of control (w¹¹¹⁸), kdm5^WT, kdm5¹⁴⁰, kdm5^R873W, kdm5^Y874C, and kdm5^L854F. The dashed line indicates 50% pupariation. Control n = 88, kdm5¹⁴⁰ n = 52, kdm5^WT n = 398, kdm5^R873W n = 216, kdm5^Y874C n = 294, kdm5^L854F n = 137. Error bars: SEM (E) Adult survival of homozygous kdm5^WT, kdm5^R873W, kdm5^Y874C, and kdm5^L854 adult flies from intercrossed heterozygous parents as a percentage of the number expected based on Mendelian expectations. ***p = 0.0001, **p = 0.0057. Total scored flies: kdm5^WT n = 359, kdm5^R873W n = 1,164, kdm5^Y874C n = 1,193, kdm5^L854F n = 544. Error bars: mean ± SEM (F–I) kdm5^WT (F), kdm5^R873W (G), kdm5^Y874C (H), and kdm5^L854F (I) female flies. Scale bar, 50 μm.

Figure 4.. The L854F mutation in the C5HC2 domain of KDM5 affects NMJ bouton size and number

(A1–D3) NMJ morphology at muscle 4-A3 of third-instar larvae labeled with HRP (magenta; A1, B1, C1, and D1) and Dlg (green; A2, B2, C2, and D2), and merged (A3, B3, C3, and D3). (A1–A3) kdm5^WT. (B1–B3) kdm5^R873W. (C1–CC3) kdm5^Y874C. (D1–D3) kdm5^L854F. Scale bar, 10 μm. (E) Quantification of type Ib bouton number normalized to muscle surface area from kdm5^WT, kdm5^R873W, kdm5^Y874C, and kdm5^L854 larvae. ****p < 0.0001. kdm5^WT n = 15, kdm5^R873W n = 25, kdm5^Y874C n = 16, kdm5^L854F n = 22. ns, not significant. Error bars: mean ± SEM (F) Quantification of type Ib bouton size from kdm5^WT, kdm5^R873W, kdm5^Y874C, and kdm5^L854 larvae. ****p<0.0001. kdm5^WT n = 309, kdm5^R873W n = 214, kdm5^Y874C n = 97, kdm5^L854F n = 176. (G–I) Futsch (white) immunolabeling and HRP (magenta) in NMJ4-A3 from kdm5^WT (G1 and G2), kdm5^JmjC* (H1 and H2), and kdm5^L854 (I1 and I2) larvae. Green triangles indicate boutons with punctate signal and yellow triangles indicate looped boutons. Scale bar, 10 μm. (J) Quantification of unbundled boutons, punctate (green) and looped (yellow), as the percentage of total boutons in NMJ4-A3 of kdm5^WT, kdm5^JmjC*, and kdm5^L854 larvae. ***p = 0.0007. kdm5^WT n = 19, kdm5^JmjC* = 12, kdm5^L854F n = 14. Error bars: mean ± SEM

Figure 5.. KDM5 mutations in the JmjC and C5HC2 domains affect larval locomotion

(A–E) Movement of third-instar kdm5^WT, kdm5^JmjC*, and kdm5^L854F larvae. kdm5^WT n = 45, kdm5^JmjC* n = 45, kdm5^L854F n = 45. (A) Traveled distance. *p = 0.0114, ****p < 0.0001. Error bars: mean ± SEM (B) Average speed. *p = 0.0105, ****p < 0.0001. Error bars: mean ± SEM (C) Maximum speed. ****p < 0.0001. Error bars: mean ± SEM (D) Average speed normalized to larval size. *p = 0.0248, ****p < 0.0001. Error bars: mean ± SEM (E) Representative path trajectories of 10 larvae from each genotype. (F) Representative evoked excitatory junctional currents (eEJCs) recorded from muscle 6 in kdm5^WT, kdm5^JmjC*, and kdm5^L854F larvae. (G) Quantification of eEJC amplitudes. *p = 0.0368; ns, not significant. Error bars: mean ± SEM (H and I) Quantification of miniature excitatory junctional current (mEJC) amplitude (H) and frequency (I). Error bars: mean ± SEM (J) Quantification of quantal content (eEJC area (nA*ms)/mEJC area (nA*ms)).Error bars: mean ± SEM (K) Paired-pulse ratio (eEJC amplitude second response/eEJC amplitude first response) across multiple interstimulus intervals. Error bars: SEM (L) eEJC amplitudes, normalized to the first response, during HFS (20 Hz × 60 s) followed by a recovery period of stimulation (0.2 Hz × 50 s). kdm5^WT n = 12, kdm5^JmjC n = 14, kdm5^L854F n = 13. Error bars representing SEM are not displayed here to facilitate visualization.

Figure 6.. kdm5^JmjC* and kdm5^L854F affect gene expression programs in the larval VNC

(A and B) Volcano plots showing dysregulated genes (5% FDR; red) in the VNC of kdm5^JmjC* (A) and kdm5^L854F (B). (C) Venn diagram showing overlap between kdm5^JmjC* and kdm5^L854F RNA-seq data. p = 2.15e-37. (D) Correlation between overlapping dysregulated genes from kdm5^L854F and kdm5^JmjC*. (E) Networks from enrichedGOcategories affected in kdm5^JmjC* (GO:0005856), kdm5^JmjC* and kdm5^L854F (GO:0005576), and kdm5^L854F (GO:0065008). Red color indicates upregulation and blue indicates downregulation compared to kdm5^WT. See also Tables S1 and S2.

Figure 7.. Model of KDM5 function at the NMJ

KDM5 regulates gene expression programs in motor neurons that are required for normal morphology and synaptic function of the NMJ.