In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber

Die neuromuskuläre Verbindung in Drosophila als ein Modelsystem zur Untersuchung molekularer Mechanismen der Synapsenentwicklung und Degeneration

More than a century ago the fruit fly Drosophila melanogaster was established as a genetic model system. The Drosophila neuromuscular junction (NMJ) has proven as an adequate model system to study molecular mechanisms of development of glutamatergic synapses. Discoveries first made in flies had a brought impact on neuroscience research in vertebrates, as many genes and fundamental aspects of cell biology are conserved between Drosophila and vertebrates. In addition, Drosophila has established as an expedient model system in the study of human neurodegeneration during the last two decades. The first chapter of this thesis focuses on the role of the protein phosphatase 2A (PP2A) in the development of individual synapses at the Drosophila NMJ. The Drosophila NMJ is organized in a chain of boutons, each bouton containing 10-50 individual synapses. Within each synapse the presynaptic active zone protein Bruchpilot (Brp) is directly apposed to a cluster of postsynaptic glutamate receptors (GluR). The postsynaptic clustering of GluR precedes the presynaptic localization of Brp, but within ten hours after GluR clustering Brp is localized at the apposing active zone. When PP2A was inhibited, about 30% of the GluR clusters were found to be unapposed to the presynaptic Brp. These GluR clusters were almost threefold smaller than GluR clusters apposed to Brp and were more prevalent in the distal boutons of the NMJ. In vivo imaging of GluR clustering further revealed that within six hours of development almost no new GluR clusters formed when PP2A was inhibited. Thus neuronal inhibition of PP2A impairs presynaptic maturation of synapses and causes additional defects in postsynaptic development. The second and central part of this thesis focuses on the establishment of a Drosophila model of the degenerative motor-neuron disorder hereditary spastic paraplegia (HSP). The common pathological feature, of this group of clinically and genetically heterogeneous disorders, is a progressive retrograde axonopathy of the longest corticospinal motor-neurons. Here we establish a Drosophila model for the HSP subtype SPG10. The SPG10 gene encodes the neuron specific kinesin heavy chain KIF5A of vertebrate kinesin-1. To model SPG10 in Drosophila, the HSP-associated mutation N256S in human KIF5A was introduced into Drosophila Khc at the corresponding site (N262S) and the mutated KhcN262S was expressed in a Drosophila wild-type background. This Drosophila SPG10 model recapitulates key disease features of HSP such as impairments in locomotion, axonal swellings, degeneration of synapses and a more severe affliction of longer axons. We show that KhcN262S acts as a dominant-negative allele in vivo. Furthermore our data suggest that the pathology in the Drosophila SPG10 model establishes due to impaired localization of cargo necessary for maintenance of structure and/or function of the synapses or the axon. As possible mechanisms contributing to pathology in the Drosophila SPG10 model reduced mitochondrial density at the NMJ, a reduction in bone morphogenetic protein (BMP) signaling and alterations in axonal as well as neuromuscular cytoskeleton were identified. All of these findings have been linked to neurodegenerative disorders before. The presented Drosophila SPG10 model provides a valuable tool for continuative studies of the mechanisms of initiation and progression of pathology in the HSP subtype SPG10.Vor über einem Jahrhundert hat sich die Fruchtfliege Drosophila melanogaster als genetisches Modelsystem etabliert. Die neuromuskuläre Verbindung in Drosophila hat sich als geeignetes Modelsystem zur Untersuchung molekularer Mechanismen der Entwicklung glutamaterger Synapsen erwiesen. In Drosophila gemachte Entdeckungen waren von großer Bedeutung für die neurowissenschaftliche Forschung in Vertebraten, da Gene und wesentliche Aspekte der Zellbiologie konserviert sind zwischen Drosophila und Vertebraten. Innerhalb der letzten 20 Jahre hat sich Drosophila zudem als ein nützliches Modelsystem zur Untersuchung humaner neurodegenerativer Erkrankungen etabliert. Der erste Teil der vorliegenden Arbeit beschäftigt sich mit der Rolle der Proteinphosphatase 2A (PP2A) in der Entwicklung einzelner Synapsen an der neuromuskulären Verbindung in Drosophila. Die Drosophila neuromuskuläre Verbindung besteht aus einer Kette von Boutons, wobei jeder Bouton 10-50 individuelle Synapsen enthält. An den einzelnen Synapsen liegen sich die präsynaptischen aktiven Zonen, markiert durch das Protein Bruchpilot (Brp), und die postsynaptischen Glutamatrezeptorfelder (GluR) gegenüber. Daher ist jede einzelne Synapse gekennzeichnet durch die präsynaptische Lokalisation von Brp und die gegenüberliegende postsynaptische Lokalisation von GluR. Bei der Entstehung einer neuen Synapse geht das Clustern der postsynaptischen GluR-Felder der präsynaptischen Lokalisation von Brp voraus, die spätestens innerhalb von zehn Stunden nach dem Clustern der GluR erfolgt. Wurde die Funktion von PP2A inhibiert, waren dagegen etwa 30% der GluR-Felder nicht von präsynaptischem Brp opponiert. Diese GluR-Felder befanden sich vor allem in den distalen Boutons der neuromuskulären Verbindung und waren etwa dreimal kleiner als GluR-Felder welche präsynaptischem Brp gegenüberlagen. Untersuchung der Dynamik von GluR-Feldern in vivo über einen Zeitraum von sechs Stunden zeigte dass fast keine neuen GluR-Felder gebildet wurden wenn die Funktion von PP2A inhibiert war. Unsere Ergebnisse zeigen dass die neuronale Inhibierung von PP2A die präsynaptische Reifung einzelner Synapsen negativ beeinflusst und zusätzlich einen Einfluss auf die Entwicklung der Postsynapse hat. Der zweite und zentrale Teil der vorliegenden Arbeit befasst sich mit der Etablierung eines Drosophila Models für die degenerative Motorneuronen Erkrankung Hereditäre Spastische Spinalparalyse (HSP). Diese Gruppe von Erkrankungen ist gekennzeichnet durch eine progressive retrograde Degeneration der längsten Axone des corticospinalen Traktes im Menschen. Im Verlauf der vorliegenden Arbeit haben wir ein Drosophila Model für den HSP Subtyp SPG10 etabliert. Das SPG10 Gen codiert für eine neuronale Form der schweren Kette des Kinesin-1 in Vertebraten, KIF5A. Für das Drosophila SPG10 Model wurde die im humanen KIF5A vorkommende, HSP assoziierte Mutation N256S in die schwere Kette (Khc) des Drosophila Kinesin-1 an korrespondierender Stelle (N262S) eingeführt und das mutierte KhcN262S in einem wild-type Hintergrund in Drosophila exprimiert. Das Drosophila SPG10 Model rekapituliert charakteristische HSP Merkmale, wie eine Beeinträchtigung der Lokomotion, axonale Schwellungen, synaptische Degeneration sowie eine schwerwiegendere Beeinträchtigung längerer Axone. Zudem konnten wir zeigen dass KhcN262S dominant negativ auf wild-type Khc in vivo wirkt. Des weiteren deuten unsere Daten an, dass die im Drosophila Model beobachtete Symptomatik durch das Fehlen eines bestimmten Cargos an der Synapse verursacht wird, welches möglicherweise für die Aufrechterhaltung der Funktion und/oder Struktur von Synapse und Axon notwendig ist. Zudem wurden weitere Mechanismen identifiziert welche eine mögliche Rolle in der Entwicklung der Pathologie im Drosophila Model spielen. So wurden eine stark reduzierte Mitochondriendichte an der neuromuskulären Verbindung, eine Beeinträchtigung des neuronalen BMP Signalweges sowie Veränderungen des neuromuskulären und axonalen Zytoskelettes beobachtet. Diese Mechanismen sind in anderen Studien als mögliche Ursachen mit neurodegenerativen Erkrankungen in Verbindung gebracht worden. Das hier vorgestellte SPG10 Drosophila Model stellt ein wertvolles Hilfsmittel dar, um in weiteren Untersuchungen weiterführende Einsichten in die Mechanismen des Beginns und der Weiterentwicklung der SPG10 Pathologie zu gewinne

Establishment of a <i>Drosophila</i> SPG 10 model.

<p>(A) Khc protein alignment across species (<i>Danio rerio</i> (Dr), <i>Tribolium castaneum</i> (Tc), <i>Xenopus tropicalis</i> (Xt), <i>Drosophila melanogaster</i> (Dm), <i>Homo sapiens</i> (Hs), <i>Rattus norvegicus</i> (Rn)) shows unique conservation of the amino acids spanning from the β-sheet 7 (β7) to the α-helix 4 (α4). Pathological mutations (red dots) are highly enriched in the latter half of loop 11 (L11) that links β7 to α4. The human mutation at position 256 (red star) was selected for further analysis. Accession numbers for the proteins used in the alignment are: Dr: GenBank_CAQ15489.1; Tc: EFA10675.1; Xt: NCBI_NP_001096215.1; Dm: GenBank_AAF58029.1, Hs: NCBI_NP_004975.2; Rn: EDM 16486.1 (B) 3D protein structure of rat Khc (3KIN) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066-Kozielski1" target="_blank">[8]</a>. The microtubule binding site is shown in yellow and green. L11 (brown cylinder), which was not resolved in the crystal structure, was added between β7 (yellow) and α4 (pink). (C) Analysis of larval locomotion. Larvae overexpressing D42>Khc^N262S (25°C) are almost completely (excluding the head) paralyzed (red arrow), whereas D42>Khc^wt+N262S larvae are still crawling, but display a tail-flipping phenotype (green arrowhead). (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s007" target="_blank">Videos S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s008" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s009" target="_blank">S3</a>) (D) Kaplan-Meier survival curve recorded at 18°C (Genotypes: black: D42>w¹¹¹⁸; Green: D42>Khc^wt; Blue: D42>Khc^N262S; Red: D42>Khc^wt+N262S) (E) Summary of the data derived from the survival analysis shown in (D). Statistical significance of data was determined by a series of Mantel-Cox tests. *** p<0.001.</p

Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a <em>Drosophila</em> HSP Model

<div><p>Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a <em>Drosophila</em> model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of <em>Drosophila</em> Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in <em>Drosophila</em>. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.</p> </div

Neuromuscular function.

<p>Evoked junctional potentials (EJPs), the half width of EJPs and mini excitatory junctional potentials (mEJPs) were determined in D42>w¹¹¹⁸, D42>khc^N262S and D42>khc^wt+N262S larvae. (A) Averaged traces of EJPs. Stimulation artifacts were removed. (B) Quantification of averaged EJP size. (C) Half width of EJPs. (D) Averaged amplitudes of mEJPs from control and mutant larvae. (E) Quantification of averaged mEJP size. (F) Quantification of the number of vesicles released per action potential. Statistics n = 8–9 larvae per genotype for B,C,E,F. One-way ANOVA followed by Tukey-Kramer post-test ** p<0.01; *** p<0.001. The standard error of the mean (s.e.m) is shown as a box, the standard deviation (s.d.) as a black line.</p

Analysis of larval locomotion and morphometric analysis of segmental nerves.

<p>Larvae used to assay locomotion were raised at 25°C. Larvae used for analysis of axonal cargo accumulations were raised at 29°C. All larvae (except khc^−/− and khc ^+/−) carried one copy of the motoneuron-specific driver D42-Gal4. (A) Analysis of larval locomotion velocities. (B–I) Analysis of axonal cargo accumulation was performed by staining segmental nerves of third instar larva for the membrane marker anti-HRP and for various cargos. Axonal cargo accumulations (arrowheads in B,D,F,H) were defined as segments of the nerve characterized by a bright anti-HRP staining and the simultaneous accumulation of cargo. Both the area fraction of the nerve filled with cargo accumulations and the number of cargo accumulations per 1000 µm² of the nerve were significantly increased in larvae expressing khc^N262S, either alone, or in combination with Khc^wt. (B,C) Cysteine-string protein (CSP) and (D,E) the vesicular glutamate transporter (DV-Glut) were selected as markers for synaptic vesicles. The kinesin-3 cargos (F,G) ANF-GFP and (H,I) Brp were used as markers for dense core vesicles and active zone precursor vesicles, respectively. Scale bars in B–I: 10 µm. For all quantifications, n = 7–10 axons per genotype were used. Statistical significance (A,C,E,G,I) was determined by using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line. * p<0.05; ** p<0.01, *** p<0.001.</p

Characterization of adult <i>Drosophila</i> SPG 10 model flies.

<p>(A) Analysis of adult wing posture. While control flies have a normal wing posture (green arrowheads), Khc^N262S expressing flies frequently display abnormal wing postures such as holding their wings up (red arrow), suggesting either the degeneration or functional impairment of the indirect flight muscles. 2-day old male flies raised at 18°C are shown. All flies contained a copy of the motoneuron specific driver D42-Gal4. (B) Analysis of thorax muscle integrity provided no evidence for the degeneration of the indirect flight muscles. Scale bar: 100 µm. (C) Western blot of whole fly head extracts after adult-onset pan-neuronal (elav^C155-Gal4/tub-gal80ts) expression of Khc^N262S and Khc^wt at 29°C for 13 days. Levels of endogenous Khc from 10 heads of control flies (w¹¹¹⁸) are comparable to Khc levels in 2.5 heads from flies overexpressing Khc (Khc^N262S and Khc^wt). The number of heads loaded per lane is indicated. β-Tubulin was used as loading control. (D) Climbing assay of adult flies 16 days after adult-onset pan-neuronal expression of Khc (white bars). Although Khc^wt expression caused no adverse effects on climbing scores compared with control (grey bars), Khc^N262S expression significantly lowered the climbing score. Statistical significance was tested using an unpaired, two-tailed student's t-test (*** p<0.001). The standard error of the mean (s.e.m) is shown as a box, standard deviation (s.d.) as a black line.</p

Light microscopic analysis of NMJ degeneration.

<p>Degeneration was revealed and scored by immunofluorescent staining. All larvae carried one copy of the motoneuron-specific driver D42-Gal4 and were raised at 29°C. (A–D) Confocal images of immunofluorescent staining showing NMJs 6/7, segment A5 of mid-third-instar <i>Drosophila</i> larvae. To visualize the subsynaptic reticulum, we used a GFP insertion in the discs-large locus. Neuronal membranes were visualized with an antibody against horseradish peroxidase (HRP). Synaptic vesicles were stained using m-α-Synapsin (Syn) antibody. For Khc^N262S- and Khc^wt+N262S-expressing larvae, which showed a strong reduction in Syn intensity, an additional, false-colored panel (high exposure) is shown. In this panel, the brightness was adjusted for better visibility of weak signals. Scale bar: 10 µm; right panels 5 µm. Genotypes: (A) D42>w¹¹¹⁸; (B) D42>Khc^wt; (C) D42>Khc^N262S; (D) D42>Khc^wt+N262S. (E) To integrate the frequency of retractions, we used a neurodegenerative scoring system to combine the occurrence of dystrophic boutons and minor pathological alterations at the NMJs into a single measure for the degree of pathological alterations (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s001" target="_blank">Figure S1A</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s001" target="_blank">S1F</a>). Using this scoring system, we detected a significant degree of neurodegenerative alterations in larvae expressing Khc^N262S either alone or in combination with Khc^wt. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line. ** p<0.01.</p