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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Recessive mutations in the amyotrophic lateral sclerosis 2 (ALS2) gene have been linked to juvenile-onset ALS2. Although one of the molecular functions of the ALS2 protein is clearly the activation of Rab5, the mechanisms underlying the selective dysfunction and degeneration of motor neurons in vivo remain to be fully understood. Here, we focused on the ALS2 homologue of Drosophila melanogaster, isolated two independent deletions, and systematically compared phenotypes of the mutants with those of animals in which Rab5 function in identified neurons was abrogated. In the dALS2 mutant flies, we found that the stereotypic axonal and dendritic morphologies of neurons shared some features with those in Rab5-deficient flies, but the dALS2 mutant phenotypes were much milder. We also found that the abrogation of Rab5 function in motor neurons strongly depressed the locomotion activity of adults, resembling the behavior of aged dALS2 mutants. Importantly, this age-dependent locomotion deficit of dALS2 mutants was restored to normal by expressing the dALS2 transgene in a wide range of tissues. This finding provided a platform where we could potentially identify particular cell types responsible for the phenotype by tissue-specific rescue experiments. We discuss our results and the future usage of the dALS2 mutant as a new ALS model.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Motor neuron diseases (MNDs) are a group of progressive neurological disorders. They are caused by degeneration of upper motor neurons (UMNs) of the motor cortex in brain and lower motor neurons (LMNs) in brainstem and spinal cord. One of the best-characterized forms of MND is amyotrophic lateral sclerosis (ALS), in which loss of UMNs with or without LMN involvement leads to muscle atrophy, fasciculations and spasticity (Boillee et al. 2006; Pasinelli & Brown 2006; Robberecht & Philips 2013). To elucidate the molecular pathogenesis for these diseases, the functions of the causative genes have been investigated. One of these is ALS2, which was originally discovered through genetic mapping and the identification of recessive mutations in juvenile-onset ALS families, and its clinical features include spasticity of limb and facial muscles accompanying distal amyotrophy of hands and feet (Ben Hamida et al. 1990; Hadano et al. 2007; Cai et al. 2008; Kato 2008).

Human ALS2 protein (often designated as Alsin) comprises several domains that are characteristic of guanine nucleotide exchange factors (GEFs): an RCC1-like domain (RLD), the Dbl homology and pleckstrin homology (DH/PH), and the vacuolar protein sorting 9 (VPS9) domain. The VPS9 domain of ALS2 specifically binds to Rab5 and functions as its GEF (Otomo et al. 2003; Kunita et al. 2004). The majority of ALS2 mutations generate premature termination codons, resulting in protein instability and loss of function of ALS2 (Yamanaka et al. 2003). Cell biological assays suggest that ALS2 dysfunction affects multiple contexts of endosomal trafficking through a Rab5-mediated mechanism (Devon et al. 2006; Hadano et al. 2006; Otomo et al. 2008; Lai et al. 2009), including formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes (Otomo et al. 2011). Although it has been suggested that this loss of GEF function could result in neuronal dysfunction and degeneration, mechanisms that lead to a selective dysfunction and degeneration of motor neurons in vivo remain to be fully understood.

To study the underlying pathologic mechanisms of ALS2 deficiency in vivo, ALS2 knockout mice have been generated and characterized (Cai et al. 2005; Devon et al. 2006; Hadano et al. 2006; Yamanaka et al. 2006). Unexpectedly, the KO mice do not necessarily recapitulate clinical or neuropathological phenotypes consistent with the motor neuron disease. The KO mice do not show apparent overall locomotion deficits in their lifetime, except for a mild motor deficit in the Rotarod test (Deng et al. 2007) and slowed movement (Yamanaka et al. 2006); and no significant difference has been observed in the numbers of cortical or spinal motor neurons when compared to wild-type control mice (Cai et al. 2008). However, the KO mice are more vulnerable to oxidative stress than the wild-type controls (Cai et al. 2005), and expression of a pathogenic form of SOD1 (superoxide dismutase 1) in the ALS2 KO background aggravates the SOD1-associated disease symptoms such as body weight loss, motor dysfunction and the accumulation of insoluble ubiquitinated proteins in the nervous system, leading to earlier death (Hadano et al. 2010).

Here, we attempted to dissect the in vivo roles of ALS2 in the disease by taking advantage of another model organism, Drosophila melanogaster, for two main reasons. First, many of the human genes responsible for neurodegenerative diseases are conserved in the Drosophila genome, including ALS2. Second, a number of versatile Drosophila genetic tools and knowledge of well-characterized stereotypic morphologies of neuronal subtypes can be leveraged to provide mechanistic insights on MND (Lessing & Bonini 2009; Jaiswal et al. 2012). For example, the Drosophila TBPH encodes a human homologue of TDP-43 that is found in cytoplasmic inclusions in ALS (Feiguin et al. 2009), and studies of its tissue-specific knockdown suggest that impaired presynaptic transmission is one of the earliest events in TDP-43-related pathogenesis (Diaper et al. 2013). We focused on the Drosophila homologue of ALS2, isolated its mutants, characterized both anatomical and behavioral phenotypes, and compared these with what has been shown in human patients and KO mice.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

CG7158 encodes a homologue of ALS2

Annotated genes in the genome of Drosophila melanogaster include homologues of four human GEFs and two GTPase activating proteins (GAPs) (Fig. 1A and S1 in Supporting Information). Among them, the predicted protein product of a single gene CG7158 shows similarities to ALS2/Alsin. Hereafter, CG7158 and its protein product are designated as dALS2 and dALS2 for simplicity, respectively. Both ALS2/Alsin and dALS2 contain four domains in the same order from their amino-terminals (Fig. 1A), among which the ‘membrane occupation and recognition nexus (MORN)’ motifs and the carboxyl-terminal ‘vacuolar protein sorting 9 (VPS9)’ domain in human ALS2 are necessary for its selective GEF activity for Rab5 in vitro (Otomo et al. 2003). One distinctive difference between dALS2 and ALS2/Alsin is that dALS2 lacks a DH domain, which is adjacent to the PH domain in human ALS2 and provides a basis of its GEF activity for Rac1 (Kunita et al. 2007). Thus, dALS2 could be a more Rab5-specific GEF compared with ALS2.

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Figure 1. Domains of human ALS2 and its Drosophila homologue (dALS2) and an analysis of dALS2 GEF activity. (A) hALS2 has an RCC1-like domain (RLD domain) that is composed of five RCC1-like repeats (amino acids 93–122, 109–165, 170–216, 527–574 and 579–625), a Dbl homology domain (DH domain; amino acids 695–882), a pleckstrin homology domain (PH domain; amino acids 935–1003), seven MORN repeats (amino acids 1049–1071, 1072–1094, 1100–1122, 1123–1143, 1151–1166, 1198–1220 and 1221–1244), and a VPS9 domain (amino acids 1552–1654). dALS2 has an RLD domain that is composed of two RCC1-like repeats (amino acids 258–361 and 308–361), a PH domain (amino acids 617–719), six MORN repeats (amino acids 744–765, 766–786, 789–805, 817–835, 839–855 and 863–885), and a VPS9 domain (amino acids 1373–1481). The Pfam program does not predict a DH domain in dALS2. P1425A and L1439A indicate substitutions of amino acids in the VPS9 domain of dALS2. (B) Analysis of GEF activity of dALS2 in S2 cells (n > 70 cells for each transfection). Each of the dALS2 forms indicated was co-expressed with a FRET probe (Raichu-Rab5[WT]) or its negative control probe (Raichu-Rab5[S34N]) in S2 cells, and the FRET/CFP ratio was determined. See details in the text. Error bars indicate standard deviations. ***P < 0.001 (ANOVA with Bonferroni) and P = 0.852 (Student's t-test). Two other experiments gave results similar to this dataset; in every experiment, the ratio was increased by 40% or more when Raichu-Rab5[WT] was co-expressed with dALS2[WT].

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CG7158/dALS2 has a GEF activity for Rab5

To examine whether dALS2 does possess GEF activity or not, we co-expressed dALS2 and a fluorescence resonance energy transfer (FRET) probe, Raichu-Rab5 in Drosophila S2 cells (Kitano et al. 2008). The probe comprised Venus (a modified YFP), the amino-terminal Rab5-binding domain of EEA1, SECFP (a modified CFP) and Rab5. In this probe design, the increase in the emission ratio reflects an increase in the active GTP-bound form of Rab5 relative to the inactive GDP-bound form in living cells (Kitano et al. 2008). The emission ratio was increased significantly when dALS2 was co-expressed (the blue bar of ‘dALS2[WT]’ in Fig. 1B) compared with the transfection of the vector (the blue bar labeled ‘Control’ in Fig. 1B). This increase in the ratio was indeed dependent on the conversion from the GDP-bound form to the GTP-bound one, as shown by the fact that the emission ratio of Raichu-Rab5[S34N], a constitutive GDP-bound form (Entchev et al. 2000), was unchanged even in the presence of dALS2 (the red bar labeled ‘dALS2[WT]’ in Fig. 1B). We also studied the effects of substitutions of two conserved amino acid residues in the VPS9 domain (Fig. 1A and S2 in Supporting Information), which are necessary for full GEF activity of ALS2 in vitro (Otomo et al. 2003). The two mutant forms of dALS2 (dALS2[P1425A] and dALS2[L1439A]) increased the FRET efficiency of Raichu-Rab5, but not to the same extent as the wild-type form (Fig. 1B). Collectively, these results showed that the wild-type form of dALS2 has GEF activity for Rab5.

dALS2 mutants are viable and fertile

To study the in vivo consequences of dALS2 dysfunction, we mobilized an existing transposable element that was inserted 120-bp upstream of the 1st ATG of the dALS2 coding sequence and isolated two independent alleles (Ex44 and Ex54) that delete approximately 30% of the coding sequence, including the start codon and the entire RLD domain (Fig. 2A). We also obtained precise jumpers, where the transposon excision restored the exact contiguous WT sequence, and used homozygotes of two of these (Ex101/Ex101 and Ex95/Ex95) for subsequent analysis as controls or the wild-type animals. Homozygotes of either Ex44 or Ex54 were viable and fertile; and the adults looked morphologically normal. Thus, dALS2 may be dispensable for viability in flies, as is the case in mice (Cai et al. 2005; Devon et al. 2006; Hadano et al. 2006; Yamanaka et al. 2006). For further phenotypic analysis, we mostly used Ex44 with a larger deletion than Ex54, unless described otherwise, and designated Ex44 homozygotes as dALS2−/−.

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Figure 2. dALS2 locus and its protein. (A) Exon/intron organization of the dALS2 locus and genomic structures of mutant alleles. Gray and orange boxes are UTRs and CDSs, respectively. A blue arrow on the wild-type genome indicates the position of the transposon insertion, and black arrows indicate primers that were used for genomic PCR. Deleted regions in individual alleles are shown with allele numbers. Green lines indicate primers that were used in RT-PCR. (B) RT-PCR detection of dALS2 mRNA from adults of one of the precise jumpers (Ex101/Ex101) and from those of dALS2−/− (Ex44/Ex44). (C) In situ hybridization analysis of dALS2 expression in embryos at stage 13 (top) or stage 16 (bottom). Ventral views. (Left) Embryo hybridized with a sense probe. (Middle and right) Embryo hybridized with an antisense probe. The boxed regions in the middle panels are magnified in the right. dALS2 is expressed in a wide range of tissues including the epidermis (arrowheads) and the ventral nerve cord (boxed regions). Arrows mark unidentified cell types in the ventral nerve cord that express dALS2 at a high level.

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RT-PCR analysis confirmed the deletion of the amino-terminal coding sequence of dALS2 in adult dALS2−/− flies (Fig. 2B). To address whether truncated polypeptides might be made by translation initiation from internal ATG codons downstream of the deletion in dALS2−/−, we generated antibodies to the carboxyl-terminal VPS9 domain. Unfortunately however, our antibodies failed to detect endogenous dALS2 with high sensitivity (data not shown); thus, we could not exclude the possibility of the generation of the truncated polypeptides. Nonetheless, it is known that ALS2 without the RLD domain no longer associates with endosomes (Yamanaka et al. 2003), so the truncated dALS2 polypeptides, if synthesized from Ex44 and Ex54 alleles, would most likely not be functional.

It is shown that Rab5 is widely expressed, albeit with different expression levels in different tissues including larval and adult nervous systems and imaginal disks (Chan et al. 2011). dALS2 is also expressed in a broad range of tissues including the nervous system and the epidermis in the embryo (Fig. 2C), larval central nervous system, adult brains and larval and adult guts (St Pierre et al. 2014).

Morphological analysis of axon terminals and dendritic arborization of identified neurons in the dALS2 mutant and in wild-type flies expressing a dominant negative form of Rab5

We searched for neurological disorders in the dALS2−/− mutant. In parallel, we addressed how far we could explain the detected phenotypes of the mutants as the disregulation of Rab5 activity. To establish a basis for comparison, we expressed a dominant negative form of Rab5[S34N] (Entchev et al. 2000) in wild-type neurons and addressed whether this caused effects reminiscent of the mutant phenotypes (Fig. 3).

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Figure 3. Phenotypes of NMJs in the dALS2 mutant and those elicited by Rab5[S34N] expression in motor neurons. (A-D) Phenotypes in wandering third instar larvae. (A and C) NMJs at muscles 6/7 (A) and muscle 4 (C) in segment A2 that were stained for HRP (green) and Brp (magenta merged with green look white). (B and D) Quantitative analysis. The numbers of synaptic boutons were normalized by the surface areas of individual muscles. Bars indicate means. **P < 0.01 ***P < 0.001 (ANOVA with Tukey's HSD post hoc test). Relevant genotypes in A and B are Ex101/Ex101 (wild type) and Ex44/Ex44 (dALS2−/−); and those in C and D are [OK371-Gal4/UAS-mmRFP] (Control), [OK371-Gal4/+; UAS-Rab5[S34N]/+] (Rab5S34N), [OK371-Gal4/+; UAS-GFP:Rab5[WT]/+] (Rab5WT), [OK371-Gal4/UAS-3HA:dALS2[WT]] (dALS2WT). Scale bars: 20 μm. (E-K) Phenotypes of motor axons in abdomens of 3- to 4-day-old adult females (E, J, K) and 2-week-old adult females (F-I). (E-I) A low-power view of the lateral and ventral plates of the wild type (E). The boxed region in E indicates an example of motor axon terminals, and blue arrows mark spiracles. (F) High-power images of axon terminals that innervate ventral abdominal muscles of segment A5 of the wild type and the mutant at 2 week old. Note that neither of the images in F is a magnification of the boxed region in E. The area of each presynaptic arbor was defined as a polygon outlined with a magenta broken line, which was quantitatively analyzed in G-I. Quantifications of the area size (G), the number of boutons in each area (H) and the number of normalized boutons (I). **P < 0.01 (Student's t-test). (J and K) Low-power ventral views along the midlines (magenta broken lines). The boxed regions highlight distinct growth of axon terminals between the two genotypes. Scale bars: 50 μm (E, J, K) and 20 μm (F). Relevant genotypes in E-I are [OK371-Gal4 UAS-mCD8:3xEGFP/OK371-Gal4 UAS-mCD8:3xEGFP] (E), [OK371-Gal4 UAS-mCD8:3xEGFP/OK371-Gal4 UAS-mCD8:3xEGFP; Ex101/Ex101] (wild type in F-I), [OK371-Gal4 UAS-mCD8:3xEGFP/OK371-Gal4 UAS-mCD8:3xEGFP; Ex44/Ex44] (dALS2−/− in F-I); and those in J and K are [OK371-Gal4 UAS-mCD8:3xEGFP/UAS-mmRFP] (J), and [OK371-Gal4 UAS-mCD8:3xEGFP/+; UAS-Rab5[S34N]/+] (K).

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We initially focused on presynaptic terminals of motor axons that innervate abdominal body wall muscles and form neuromuscular junctions (NMJs) with stereotyped connectivity (Kohsaka et al. 2012; Menon et al. 2013). To visualize presynaptic terminals in larvae, we double-labeled presynaptic membranes and active zones (anti-HRP and anti-Bruchpilot (Brp), respectively; Coyle et al. 2004) and quantified the number of Brp-positive boutons (Fig. 3A). The number of the boutons that was normalized by the area of the innervated muscle was not significantly different between the wild type and the mutant (Fig. 3B). We did not find a noticeable difference in the number of postsynaptic marker (Mhc-CD8:YFP:Sh; Zito et al. 1999)-positive boutons; neither did we find a significant difference in the size of the muscles imaged in Fig. 3A and 3C (data not shown).

We also imaged motor axons in whole-mount adults using a transgenic marker (Fig. 3E; Hebbar et al. 2006) and found that there was no noticeable defect in axonal growth in mutants at the ages of 3–4 days and 3 weeks. We further measured the area of each presynaptic arbor and counted the number of boutons at the ventral NMJ in 2-week-old adults (the magenta-line polygons of Fig. 3F, and Fig. 3G-I). Although the normalized bouton number was not significantly different between the two genotypes (Fig. 3I), the absolute value of the bouton number per terminal was increased in the mutant (Fig. 3H).

To express Rab5[S34N] in the motor neuron, we used OK371-Gal4, an enhancer trap line where the Gal4 gene is inserted in the gene encoding a vesicular glutamate transporter promoter (Mahr & Aberle 2006). Rab5[S34N] expression resulted in an increase in the bouton number of presynaptic terminals of motor axons in larvae and adults (Fig. 3C,D,J and K), and the Rab5[S34N] effect was more dramatic than the phenotype in the dALS2 mutant. In addition to the increase in the number of boutons, each bouton became smaller than that of the control axon terminals at larval NMJs (Fig. 3C). From earlier experiments with Rab5[S34N], it is known that Rab5 controls synaptic transmission at larval NMJs (Wucherpfennig et al. 2003). In that study, Rab5[S34N] expression was kept low during embryonic and early larval stages so that it did not affect morphological development of NMJs.

We used dendritic arborization (da) neurons as another assay system (Figure S3A; Shimono et al. 2009; Jan & Jan 2010). Previously, we showed that Rab5[S34N] expression or a rab5 null mutation strongly downsizes and simplifies dendritic arbors of class IV da neurons (Satoh et al. 2008). Our quantifications of the da neurons in the dALS2 mutant showed mild defects in dendrite morphogenesis (Figure S3B,C in Supporting Information): the total dendritic length and the number of branch endings per cell decreased by 11% and 19%, respectively, relative to wild type, whereas the coverage index was not altered (Figure S3C in Supporting Information). The over-expression of dALS2[WT] did not cause a significant effect on morphological development of the NMJs or the dendritic arbors (Fig. 3D and data not shown).

Collectively, the dALS2 mutant exhibited much milder phenotypes compared with the severe effects of the abrogation of Rab5 function. This might be explained by a contribution of at least one of the other Rab5 GEFs, Rabex5, (Figure S1 in Supporting Information) to regulation of synaptic vesicles and dendrite morphogenesis (Sann et al. 2012; Mori et al. 2013).

Mutant adults showed a lowered locomotion activity

In addition to the cell morphological phenotypes, we found that Rab5[S34N] expression in the motor neurons strongly depressed the climbing ability of adults (Figure S4A and Movies S1 and S2 in Supporting Information). This prompted us to perform the climbing assay with the dALS2−/− mutant adults, which showed the most prominent phenotype, and it was an age-dependent locomotion deficit that was causally related to loss of dALS2 function (Figs 4 and 5).

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Figure 4. dALS2 mutant exhibited age-dependent locomotor dysfunction. Wild type and dALS2−/− were assayed for negative geotaxis at the indicated ages, scoring each gender separately. The genotypes of the wild type in A and B are Ex101/Ex101 and Ex95/Ex95, respectively, while the genotype of the mutants is Ex44/Ex44. ***P < 0.001 (Student's t-test).

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Figure 5. The locomotion deficit of the mutant was rescued by widely expressing the dALS2 transgene. Rescue experiment of the locomotion deficit of the dALS2−/− mutant using Ubi-Gal4. Females and males of the four genotypes were assayed for negative geotaxis at 1 week (A) or 2 weeks (B) after eclosion. Relevant detailed genotypes (from left to right in each graph) were [Ubi-Gal4/UAS-mmRFP; Ex101/Ex101] (Ubi>RFP; WT), [Ubi-Gal4/UAS-dALS2; Ex101/Ex101] (Ubi>dALS2; WT), [Ubi-Gal4/UAS-mmRFP; Ex44/Ex44] (Ubi>RFP; dALS2−/−), and [Ubi-Gal4/UAS-dALS2; Ex44/Ex44] (Ubi>dALS2; dALS2−/−). Magenta asterisks indicate statistically significant differences of the cohort from the wild type (Ubi>RFP; WT); and blue asterisks from the mutant (Ubi>RFP; dALS2−/−). *P < 0.05; **P < 0.01; ***P < 0.001; NS: Statistically not significant (> 0.05). ANOVA with Tukey's HSD post hoc test. In the top right graph, the significance of the difference between Ubi>RFP; dALS2−/− and Ubi>dALS2; dALS2−/− is marginal, so the exact P value is provided.

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The locomotion assay used was the negative geotaxis assay, which is an established method to test the climbing ability of Drosophila (Figure S4B–D in Supporting Information; Ali et al. 2011; see details in 'Experimental procedures'). We reared the dALS2−/− mutants with either of the controls (Ex101/Ex101 or Ex95/Ex95) at the same time and examined the climbing ability of the mutant flies (Fig. 4). We found that the adult mutants showed a locomotion defect at 2 weeks after eclosion, but not at 1 week or younger ages. At 4 weeks after eclosion, most of the mutant females failed to reach a fixed height within the allowed time (Figure S4C,D in Supporting Information).

Locomotion deficit of the mutant was rescued by dALS2 transgene expression

We addressed whether or not this adult-onset climbing disability could be restored by tissue-specific expression of the wild-type dALS2 cDNA. We started with broad expression, because dALS2 is also expressed outside the nervous system (St Pierre et al. 2014) and because it has been suggested, but not rigorously demonstrated, that the motor neuron is the primary locus of ALS2 action in mice.

To realize broad expression, we used Ubiquitin (Ubi)-Gal4 to drive expression of the wild-type dALS2 transgene in a wide range of tissues (Fig. 5). As a negative control for the transgene expression, we tested the effect of mmRFP expression on the climbing ability. Two-week-old dALS2−/− adults showed lowered climbing ability, compared with wild type, and this phenotype was restored to normal by dALS2 transgene expression in both females and males (compare blue and green data points in Fig. 5B). These results showed that the age-dependent locomotion deficit is indeed a loss-of-function phenotype of dALS2. This phenotype is reminiscent of the moderate, age-dependent deficit in motor coordination in ALS2-null mice (Cai et al. 2005).

We next attempted to address whether the cell-type-specific dALS2 transgene expression in motor neurons could rescue the mutant phenotype (Fig. 6). In this experiment, we used OK371-Gal4. This rescue strategy, however, made the assessment of the recovery difficult. This is primarily because the dALS2 expression in the wild-type motor neurons caused a deleterious effect on the climbing activity (compare red data points with orange ones in Fig. 6). The dALS2-expressing aged mutants of both females and males tended to improve the climbing ability when compared to mRFP-expressing age-matched adults (compare blue points with green ones in Fig. 6B), although our results did not reach statistical significance.

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Figure 6. Locomotion deficit rescue experiment using a motor neuron-specific Gal4 driver. Females and males of the four genotypes were assayed for negative geotaxis at 1 week (A) or 2 weeks (B) after eclosion. Relevant detailed genotypes (from left to right in each graph) were [OK371-Gal4/UAS-mmRFP; Ex101/Ex101] (OK371 > RFP; WT), [OK371-Gal4/UAS-dALS2; Ex101/Ex101] (OK371 > dALS2; WT), [OK371-Gal4/UAS-mmRFP; Ex44/Ex44] (OK371 > RFP; dALS2−/−) and [OK371-Gal4/UAS-dALS2; Ex44/Ex44] (OK371 > dALS2; dALS2−/−). Magenta asterisks indicate statistically significant differences of the cohort from the wild type (OK371 > RFP; WT); and blue asterisks from the mutant (OK371 > RFP; dALS2−/−). *P < 0.05; ***P < 0.001 NS, Statistically not significant (> 0.05). ANOVA with Tukey's HSD post-hoc test.

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The above results can be interpreted in several ways. First, dALS2 is indeed required in the motor neuron; however, the motor neuron GAL4 driver (OK371-Gal4) failed to correct the phenotype significantly because this Gal4-driven expression of dALS2 far exceeds the physiological range and disturbs precise spatial-temporal regulation of Rab5 activity. Second, dALS2 is supplied in the motor neuron at larval stages by Ubi-GAL4 and a portion of the proteins persist and function at the adult stage (we found that Ubi-GAL4 is expressed in the motor neuron in larvae, but not in adults). Third, dALS2 is critically required in cell types other than the motor neuron to prevent the deterioration of locomotion during aging (e.g., the presumptive ‘upper’ motor neuron in flies); and Ubi-GAL4, not OK371-Gal4, is expressed in that cell type. Use of the rich resource of GAL4 stocks (Jenett et al. 2012) and searches for the stocks that realize appropriate expression levels of dALS2 would allow us to distinguish these possibilities.

Aging, longevity and sensitivity to oxidative stress of the dALS2 mutant

It is known that even wild-type flies decline in climbing performance over time (Ganetzky & Flanagan 1978; Le Bourg & Lints 1992). Therefore, we suspected that aging might be accelerated in the dALS2 mutant and/or that the mutant might be short-lived. As a part of normal aging, the expression of several essential autophagy genes declines in fly neural tissues, which is causative of accumulations of ubiquitinated proteins and p62/Ref(2)P (Simonsen et al. 2008; Tonoki et al. 2009; Bartlett et al. 2011). This has been shown by the facts that ubiquitin-positive puncta precociously appear in atg8 mutants, which are defective in autophagy, and that enhanced neuronal Atg8a expression suppresses the accumulation of insoluble ubiquitinated proteins (Simonsen et al. 2008). Therefore, we examined levels of ubiquitinated proteins and p62 in the wild type and the dALS2 mutant adults at distinct ages (Figure S5 in Supporting Information).

At the age of 3–4 days after eclosion, ubiquitin-positive puncta were barely detected immunohistochemically in any regions of the brains of the wild type or the dALS2 mutant, whereas they were found in 2-week-old adults irrespective of the genotypes (Figure S5A in Supporting Information). Via Western blot analysis of whole lysates, we did not find overt differences in the levels of ubiquitinated proteins and p62, between the genotypes, at either of the two ages investigated (Figure S5B in Supporting Information); and we did not find a noticeable difference in the amount of insoluble ubiquitinated proteins (IUP), either (data not shown). In contrast to the dALS2 mutant, ubiquitin-positive puncta precociously appeared in atg8 mutants as previously reported (data not shown). Thus, we did not find evidence for accelerated aging in the dALS2 mutant and our results suggest that dALS2 plays a relatively minor role in protein homeostasis, compared with the core machinery of autophagy.

Unexpectedly, the mutant (Ex44/Ex44) exhibited a significantly longer life span than either of the two wild-type stocks (Ex101/Ex101 or Ex95/Ex95) under our experimental protocol (Figure S6A–D in Supporting Information). However, the Ubi-Gal4-dependent dALS2 transgene expression failed to normalize the life span of Ex44/Ex44 (data not shown); and homozygous females of another deletion allele (Ex54/Ex54) did not exhibit a similarly significant longevity phenotype (Figure S6E–F in Supporting Information). Thus, we could not conclude that the longevity phenotype of Ex44/Ex44 was due to the loss of dALS2 function. Nonetheless, it is fairly safe to conclude that the loss of dALS2 function does not reduce the life span.

Intrigued by the fact that the ALS2 KO mice are more sensitive to oxidative stress than the wild-type controls (Cai et al. 2005), we recorded survival of the wild-type and dALS2 mutant adult flies (Ex44/Ex44 or Ex54/Ex54) when they were exposed to hydrogen peroxide-containing food (Vrailas-Mortimer et al. 2011). Under this condition, Ex44/Ex44 females, but not Ex44/Ex44 males, were more sensitive to the stress than the wild-type females, whereas we did not obtain reproducible results when Ex54/Ex54 adults were tested. Again, we were unable to restore the supersensitive phenotype of Ex44/Ex44 females by the Ubi-Gal4-dependent dALS2 rescue experiment (data not shown).

Future utility of the Drosophila ALS models

In this study, we pursued the possibility that the Drosophila dALS2 mutant could become a new animal model for investigating mechanistic insights into the disease. Our discovery of the age-dependent locomotion deficit has provided a platform, where we could potentially specify the responsible cell types, verify the common belief that the primary focus of dALS2 action is the UMN, and screen genetic backgrounds or drugs that could alleviate or possibly prevent the onset of the symptoms.

In addition to animal behaviors and neuronal cell morphologies, the absence of dALS2 function could impact synaptic transmission. Control of Rab5 activity is required for normal development of NMJ as described above; in addition, Rab5 regulates the efficacy of the evoked neurotransmitter release once the NMJ is formed (Wucherpfennig et al. 2003). So NMJs in the dALS2 mutant could be a target of physiological and ultrastructural investigations. Other future targets are premotor interneurons that control the neurotransmitter release at NMJ and further upstream neural circuits, which are functional counterparts of UMNs in mammals. Identification of such neurons and technical accessibility to those would allow us to readdress whether the markers of neuronal aging and/or the Drosophila homologue of TDP-43 are accumulated in those particular neuronal classes, and this approach may validate Drosophila as a tractable model of not only ALS2 but also other genetic causes of ALS.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Fly stocks

We used the Gal4-UAS system (Brand & Perrimon 1993) to express transgenes and to visualize motor neurons and da neurons. Gal4 driver strains used were Gr-Gal4 lines (Weiss et al. 2011), OK371-Gal4 (Mahr & Aberle 2006), and Ubi-Gal4 (the Bloomington Stock Center). UAS stocks and others were UAS-mCD8:GFP and UAS-myr-mRFP (mmRFP; the Bloomington Stock Center), UAS-mCD8:3xEGFP (Kakihara et al. 2008), UAS-Rab5[S43N] (Entchev et al. 2000), Mhc-CD8:YFP:Sh (Zito et al. 1999) and atg82 (the Bloomington Stock Center). The P element of CG7158G4607 (the Bloomington Stock Center) was mobilized, and a total of one hundred and fifty excision lines were established. Genomic deletions within the CG7158 locus were identified by genomic PCR using primers (sequences of the two primers in Fig. 2A are 5′-GCGTATGCAAACCAGCAGTA-3′ and 5′-CCCGAAATCTTTGAGCTGAG-3′) and subsequent direct sequencing. Transgenic flies were generated by a standard method.

Molecular cloning

The FRET probes, Raichu-Rab5[WT] and Raichu-Rab5[S34N], are described (Kitano et al. 2008), and the DNA fragment encoding Raichu-Rab5 was inserted into a pUAST-based vector. The full-length coding sequence (CDS) of dALS2/CG7158 was amplified using pOT2-LD33266 (Berkeley Drosophila Genome Project) as a template and inserted into pUAST-3xHA to generate pUAST-3xHA:dALS2[WT]. CDSs of dALS2[P1425A] and dALS2[L1439A] were generated by site-directed mutagenesis using two single-primer reactions. The primers were 5′-GGTGAGCGCTATCATGTTGT-3′ and 5′-ACAACATGATAGCGCTCACC-3′ for dALS2[P1425A], and 5′-ACCAGCGTGGGGAACAGCA-3′ and 5′-TGCTGTTCCCCACGCTGGT-3′ for dALS2 [L1439A]. The CDSs of dALS2[P1425A] and dALS2[L1439A] were also inserted into pUAST-3xHA.

Drosophila S2 cells and FRET imaging

S2 cells were cultured at 25 °C in Schneider's Drosophila medium (Gibco) containing 10% fetal bovine serum (Gibco) and penicillin/streptomycin, and passaged every 3 to 4 days. S2 cells were cotransfected with pDA-Gal4 (actin-promoter Gal4; a gift from Fumio Matsuzaki's lab), pUAST-Raichu-Rab5, and either of pUAST-3xHA:dALS2[WT], pUAST-3xHA:dALS2[P1425A] or pUAST-3xHA:dALS2[L1439A] using HilyMax (Dojindo), and used for microscopic analysis 2 days after transfection. FRET imaging was carried out as described (Tsuyama et al. 2013).

RT-PCR and in situ hybridization

We isolated total RNA from wild-type and dALS2−/− adults with an RNeasy kit (QIAGEN). After cDNA was synthesized using Ready-To-GO RT-PCR Beads (GE Healthcare UK), expression of dALS2 and Actin5c was detected using the following primers: 5′-TCTGCAGCAGTCCAGCACATAC-3′ and 5′-CATTTTGCCGAAACCTTCAATG-3′ for dALS2, and 5′-GTGCTGTGTGGATACTCC-3′ and 5′-TGTGGTGCCAGATCTTCTCC-3′ for Actin5c. RNA probes for in situ hybridization were made using a cDNA clone LD33266 (Berkeley Drosophila Genome Project) as a template and hybridized with fixed embryos essentially as described (Yamamoto et al. 2006; Hattori et al. 2007; Matsubara et al. 2011).

Production of antibodies and Western blotting

Guinea pigs were immunized with a GST protein that had been fused to the N-terminal 410 amino acids of dALS2 to generate anti-N1 antibody, and the C-terminal 196 amino acids to generate anti-C1 antibody. We solubilized dALS2-expressing S2 cells by sonication in the sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antibodies recognized dALS2 that was endogenously expressed in S2 cells and the one over-expressed by Ubi-Gal4 in transgenic flies (data not shown). Adult heads were homogenized in the sample solution, and lysates were boiled immediately. The lysates were separated on 8% polyacrylamide gels in WIDE RANGE Gel Preparation Buffer for SDS–PAGE (Nacalai) and transferred to PVDF membranes (Millipore). To detect ubiquitinated proteins and p62 proteins, lysates equivalent to 7.5 fly heads were run per lane (Figure S5B). The primary antibodies were used at the following concentrations: guinea pig anti-dALS2 (1:1000), rabbit anti-p62 (1:4000; Pircs et al. 2012), mouse anti-polyubiquitin FK2 (1:1000; COSMO BIO) and mouse anti-actin MAB1501 (1:1000; Millipore). Signals were detected with Chemi-Lumi One Super (Nacalai).

Immunohistochemistry

Wandering third instar larvae were dissected in PBS, fixed in PBS containing 3.7% formaldehyde for 10 min, incubated in primary antibodies overnight at 4 °C and in secondary antibodies for 1–2 days at 4 °C. Adult brains were fixed in PBS containing 3.7% formaldehyde for 1 h. The primary antibodies were used at the following concentrations: rabbit anti-HRP (1:300; Jackson ImmunoResearch), mouse NC82 (1: 100; Developmental Studies Hybridoma Bank [DSHB] at the University of Iowa), mouse anti-polyubiquitin FK2 (1:500; COSMO BIO) and rabbit polyclonal antibody against Drosophila p62 protein (1:2000; Pircs et al. 2012).

Imaging and quantification

In fillet preparations of wandering 3rd instar larvae, we counted the numbers of boutons of motor nerve endings at muscles 6/7 in segment A2 as previously described (Coyle et al. 2004). To acquire images of adult abdominal NMJs, we mounted abdomens as described (Shimono et al. 2009) and imaged motor nerve endings in the ventral plate. We quantified the area size of the nerve endings and the total number of boutons in segments A4 and A5 as described by Hebbar et al. (2006). Images of larval da neurons were acquired and quantitatively analyzed essentially as described (Hattori et al. 2007; Parrish et al. 2009; Matsubara et al. 2011).

Negative geotaxis assay, longevity and sensitivity to oxidant exposure

Flies of each genotype were collected 1 day after eclosion and mated for 1 day. After this mating, males and females were separated, and sorted into ten groups of ten individuals of each gender. These groups of individual genotypes were reared at the same time in the same incubator and used for the assays. In the negative geotaxis assay, we transferred each group of ten flies into an empty test vial, gently tapped the flies to the bottom of the vial and scored the number of flies that could climb above the 8-cm mark by 10 s after the tap (Figure S4B). This test was repeated ten times for each group, and the mean (average climbing pass rate) was plotted as a single data point (Figs 4-6). We basically followed published protocols of assays for negative geotaxis (Ali et al. 2011), longevity (Linford et al. 2013) and sensitivity to oxidant exposure, (Vrailas-Mortimer et al. 2011).

Databases

For homology and domain searches, we used Homologene in the NCBI BioSystems Database (Geer et al. 2010) and Pfam27.0 (Punta et al. 2012), respectively. To search for genomic information of Drosophila melanogaster, we used FlyBase (St Pierre et al. 2014).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

The fly stocks were provided by the Drosophila Genetic Resource Center at Kyoto Institute of Technology, the NIG stock center and the Bloomington Stock Center. We thank John Carlson, Gabor Juhász, Akinao Nose, Hiroshi Kohsaka, Chihiro Hama, Pernille Rørth and Dirk Bohmann for other stocks and related information. We also thank the Developmental Studies Hybridoma Bank (DSHB), Michiyuki Matsuda, Shinji Hadano, Joh-E Ikeda, Mitsunori Fukuda, Shigeki Yoshiura, and Ayako Tonoki very much for antibodies, plasmids and/or their technical advice; Uemura laboratory members for critical discussion and encouragements; and Mayumi Futamata, Kumiko Shimizu, Junko Mizukoshi, Kanae Oki and Yuriko Ito for their technical assistance. This work was supported by a grant from the programs Grants-in-Aid for Scientific Research on Innovative Areas ‘Mesoscopic neurocircuitry’ (22115006 to T. Uemura) and on ‘Comprehensive Brain Science Network’ from the Ministry of Education, Science, Sports and Culture of Japan. R. I. and T. T. were recipients of fellowships of the Japan Society for the Promotion of Science.

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  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
gtc12146-sup-0001-FigureS1.pdfapplication/PDF149KFigure S1 Human and Drosophila Rab5-GEFs and GAPs.
gtc12146-sup-0002-FigureS2.pdfapplication/PDF149KFigure S2 Multiple sequence alignment of VPS9 domains of Rab5 GEFs.
gtc12146-sup-0003-FigureS3.pdfapplication/PDF491KFigure S3 Phenotypes of class IV da neurons in the dALS2 mutant.
gtc12146-sup-0004-FigureS4.pdfapplication/PDF295KFigure S4 Negative geotaxis assay.
gtc12146-sup-0005-FigureS5.pdfapplication/PDF300KFigure S5 Aging was not accelerated in the dALS2 mutant.
gtc12146-sup-0006-FigureS6.pdfapplication/PDF178KFigure S6 Life span assay.
gtc12146-sup-0007-Movie1.movvideo/mov3077KMovie S1 Climbing ability of the control adults (0-3 days old). The genotype is OK371-Gal4/UAS-mmRFP.
gtc12146-sup-0008-Movie2.movvideo/mov5525KMovie S2 Climbing ability of Rab5[S34N]-expressing adults (0-3 days old). After the tap, the flies were recorded longer than 10 seconds. The genotype is OK371-Gal4/+; UAS-Rab5[S34N]/+.
gtc12146-sup-0009-DataS1.docxWord document15K 

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