The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

Abstract

Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.

Keywords: Atlastin; ER; Neuron; Reticulon.

Fig. 1.

Chemical fixation disrupts the ER network in motor neurons, muscles, and S2 cells. Representative confocal slices through the center (A) and periphery (A′) of live and fixed wild-type motor neuron (MN) cell bodies in the ventral nerve cord. (B) ER in wild-type larval boutons under live and fixed conditions. (B′) Magnification of boxed regions in B. (C) ER in wild-type muscle 6 from segment A3 under live and fixed conditions. (C′) Magnification of boxed regions in C. (D) ER in S2 cells under live and fixed conditions. (D′) Magnification of boxed regions in D. Chemical fixation was achieved by a 5 min exposure to 4% paraformaldehyde. ER was imaged using BiP–sfGFP–HDEL. Scale bars: 5 µm, except B′ (2 µm).

Fig. 2.

The ER lumen marker BiP–sfGFP–HDEL colocalizes with the ER membrane marker tdTomato–Sec61β. Third-instar larval motor nerve terminals from segment A2 muscle 4. (A–C) Representative confocal z-projections showing the plasma membrane marker myr::tdTomato (A), the ER marker BiP–sfGFP–HDEL (B) and the merged signals (C). (D–F) z-projections showing the ER membrane marker tdTomato–Sec61β (D), the ER lumen marker BiP–sfGFP–HDEL (E) and the merged signals (F). All transgenes were driven by the motor neuron driver OK371-Gal4. Scale bars: 5 µm.

Fig. 3.

Loss of atl disrupts the tubular ER network in motor axons and motor nerve terminals of third-instar larvae. (A) Schematic of the regions of the motor neuron from which images were collected. (B) Representative central confocal slices of motor neuron cell bodies in control [OK371>BiP–sfGFP–HDEL] (Bi) and atl² [atl², OK371>BiP–sfGFP–HDEL] (Bii) larvae. z-projections of motor axons within the ventral nerve cord in wild-type (control) (Biii) and atl² (Biv) larvae. (C) z-projections of neuromuscular junctions from muscle 4 at segment A6 in wild-type (Ci) and atl² (Cii) larvae. Average frequency histograms of pixel intensities taken from regions of interest around boutons for control (Ciii) and atl² (Civ) (w¹¹¹⁸ n=5, atl² n=6; red and blue dashed lines represent the mode and median, respectively, of the histogram and are color coded with the analysis in D). Boxed regions in Ci and Cii are expanded in Ci′ and Cii′, respectively. Dashed lines in Ci′ and Cii′ represent the positions used for the linescans of pixel intensities for wild-type (Cv) and atl² (Cvi). (D) Mode and median of pixel intensity frequency histograms (mean±s.e.m.) are shown for five control and six atl² images. P-values shown represent an unpaired Student's t-test performed with KaleidaGraph. Scale bars: 5 µm, except for Ci′ and Cii′ (2 µm).

Fig. 4.

Expression of UAS-atl^RNAi or UAS-atl^K51A disrupts the tubular ER network in motor axons and presynaptic boutons. (A–C) ER in motor neuron (MN) cell bodies for control [nSyb>BiP-sfGFP-HDEL] (A), atl RNAi [nSyb>BiP-sfGFP-HDEL, atl^RNAi] (B), and dominant-negative Atl [nSyb>BiP-sfGFP-HDEL, atl^K51A] (C). (D–F) ER in motor axons for control (D), atl RNAi (E) and dominant-negative (DN) atl^K51A (F). (G–I) ER in presynaptic boutons for control (G), atl RNAi (H) and dominant-negative atl^K51A (I). (G′–I′) Magnification of boxed regions for wild-type (G′), atl RNAi (H′), and dominant-negative atl^K51A (I′). Scale bars: 5 µm, except for G′,H′ and I′ (2 µm).

Fig. 5.

atl² and Rtnl1¹ decrease evoked neurotransmitter release. Average EJP traces for wild-type (w¹¹¹⁸) (A) and atl² (B) larvae at 0.4, 0.6, 1.0, and 1.5 mM Ca²⁺. In ascending [Ca²⁺], for w¹¹¹⁸ n=7, 17, 7, and 7 and for atl² n=7, 23, 7, and 7. (C) Mean±s.e.m. EJP amplitude for w¹¹¹⁸, atl², Rtnl1¹, and Rtnl1¹; atl². P-values shown represent a one-way ANOVA using a Fisher's LSD post hoc test performed with KaleidaGraph. (D) Mean±s.e.m. EJP amplitude for atl²; arm>UAS-atl⁺, Rtnl1¹; arm>UAS-Rtnl1⁺, and their respective controls using the ubiquitous arm-Gal4 driver. P-values shown represent an unpaired Student's t-test performed with KaleidaGraph. Recordings for C and D were performed at 0.6 mM Ca²⁺. (E) Mean±s.e.m. corrected quantal content for w¹¹¹⁸, Rtnl1¹, atl², and Rtnl1¹; atl². In ascending [Ca²⁺], w¹¹¹⁸ n=7, 17, 7, and 7; atl² n=7, 23, 7, and 7; Rtnl1¹ n=7, 10, 7, and 7; Rtnl1¹; atl² n=7, 7, 7, and 7. Resting membrane potentials (RMPs) were not significantly different among the four genotypes at bath [Ca²⁺] of 0.4 mM or 1.5 mM. At bath [Ca²⁺] of 0.6 mM or 1.0 mM, RMPs of wild-type and atl² were significantly different (P=0.0266, wild-type more hyperpolarized, at 0.6 mM [Ca²⁺], and P=0.0316, atl² more hyperpolarized, at 1.0 mM [Ca²⁺]). RMP of Rtnl1¹; atl² was significantly different from other genotypes at a bath [Ca²⁺] of 1.0 mM, Rtnl1¹; atl² more hyperpolarized. (F) Mean±s.e.m. corrected quantal content for elav>dcr, elav>dcr, atl^RNAi, and elav>dcr, Rtnl1^RNAi at 1.5 mM [Ca²⁺]. (G) Mean±s.e.m. mEJP amplitudes for w¹¹¹⁸, atl², Rtnl1¹, and Rtnl1¹; atl², pooled from all four [Ca²⁺]. (H) Mean±s.e.m. mEJP amplitudes for the indicated genotypes collected at 1.5 mM bath [Ca²⁺]. P-values in D,F,G represent an unpaired Student's t-test performed with KaleidaGraph. Recordings were performed in HL3 from muscle 6 in segment A6. Each scattergram data point is the average of the first 20 EJP responses (successes and failures) recorded for each larva.

Fig. 6.

Neuronal expression of UAS-atl^RNAi and UAS-atl^K51A decreases evoked neurotransmitter release. (A) Mean±s.e.m. EJP amplitude and corresponding percentage success for wild-type (w¹¹¹⁸), atl², UAS-atl^RNAi and UAS-atl^K51A. Drivers include the pan-neuronal elav-Gal4 and nSyb-Gal4 and the motor neuronal D42-Gal4. (B) Mean±s.e.m. EJP amplitude and percentage success for muscle (Mef2-Gal4) and glial (Gli-Gal4) expression of UAS-atl^RNAi and UAS-atl^K51A. Recordings for A and B were made in HL3.1 at 0.1 mM Ca²⁺ from muscle 6 in segment A6. Each scattergram data point is the average of the first 20 EJP responses (successes and failures) recorded for each larva. P-values shown represent an unpaired Student's t-test performed with KaleidaGraph.

Fig. 7.

Rtnl1 affects neurotransmitter release from multiple tissues at the NMJ. (A) Mean±s.e.m. EJP amplitude and corresponding percentage success for wild-type (w¹¹¹⁸), Rtnl1¹, and UAS-Rtnl1^RNAi expression in neurons, muscles and glia using elav-Gal4, Mef2-Gal4 and Gli-Gal4, respectively. (B) Mean±s.e.m. EJP amplitude and corresponding percentage success for w¹¹¹⁸, Rtnl1¹ and expression of UAS-Rtnl1⁺ in Rtnl1¹ mutants. UAS-Rtnl1⁺ was expressed ubiquitously, in motor neurons and in muscles using da-Gal4, D42-Gal4 and Mef2-Gal4, respectively. OK371, Mef2 and Gli-Gal4 denote concurrent expression of UAS-Rtnl1⁺ from motor neurons, muscles and glia. Recordings for A and B were made in HL3.1 at 0.1 mM Ca²⁺ from muscle 6 in segment A6. Each scattergram data point is the average of the first 20 EJP responses (successes and failures) recorded for each larva. P-values shown represent an unpaired Student's t-test performed with KaleidaGraph.

Fig. 8.

atl² increases BMP signaling in motor neurons. Representative confocal images of w¹¹¹⁸ (A) and atl² (B) motor neuron nuclei from third-instar larvae labeled with anti-pMad antibody. Scale bar: 10 µm. (C) Mean±s.e.m. pMad levels for w¹¹¹⁸, atl², atl²; arm>+, atl²; arm>atl⁺, atl²; OK6>+, and atl²; OK6>atl⁺ in motor neuron nuclei. Each scattergram data point is the average nuclear signal intensity per area for a single larva. P-values shown represent an unpaired Student's t-test performed with KaleidaGraph.