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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Neuron. Author manuscript; available in PMC Oct 26, 2012.
Published in final edited form as:
PMCID: PMC3353876
NIHMSID: NIHMS374245
The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transport at Synaptic Termini
Thomas E. Lloyd,1,2,3,6 James Machamer,2 Kathleen O’Hara,1,3 Ji Han Kim,2 Sarah E. Collins,2 Man Y. Wong,4 Brooke Sahin,1,2 Wendy Imlach,5 Yunpeng Yang,2 Edwin S. Levitan,4 Brian D. McCabe,5 and Alex L. Kolodkin1,3,6
1The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD
2Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD
3Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD
4Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA
5Departments of Pathology & Cell Biology and Neuroscience, Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, NY
6Correspondence to: tlloyd4/at/jhmi.edu and ; kolodkin/at/jhmi.edu
p150Glued is the major subunit of dynactin, a complex that functions with dynein in minus-end directed microtubule transport. Mutations within the p150Glued CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150Glued motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (GlG38S) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the dynein/dynactin complex in neurons causes a specific disruption of vesicle trafficking at terminal boutons (TBs), the distal-most ends of synapses. GlG38S larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150Glued function cooperatively at TBs to coordinate transport. Therefore, the p150Glued CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motor neuron disease.
Disruption of axonal transport is proposed to be a common mechanism in the pathogenesis of neurodegenerative diseases (De Vos et al., 2008; Perlson et al., 2010). Axonal microtubules are polarized with their plus (+) ends at synapses and their minus (−) ends directed towards the soma. Anterograde cargo is transported to the synapse via microtubule (+) end-directed motors of the kinesin family, whereas retrograde transport is mediated via the (−) end-directed motor dynein (Kardon and Vale, 2009). However, it remains unclear how unidirectional transport is regulated at synapses, and how the anterograde and retrograde transport machinery are coordinated.
Dynactin is a protein complex required for dynein-mediated microtubule-based transport. The p150Glued dynactin subunit contains an amino-terminal cytoskeleton-associated protein, Gly-rich (CAP-Gly) domain that is present in several microtubule (+)-end tracking proteins (+TIPs). Interestingly, different missense mutations located within the p150 CAP-Gly domain cause two distinct adult-onset autosomal dominant neurodegenerative diseases: one resulting in motor neuron degeneration, termed Hereditary Motor Neuropathy 7B (HMN7B) or distal spinal and bulbar muscular atrophy (Puls et al., 2003); and the other causing midbrain atrophy and loss of dopaminergic neurons without affecting motor neurons, termed Perry Syndrome (Farrer et al., 2009). HMN7B is caused by a G59S missense mutation that inhibits the ability of dynactin to bind microtubules in vitro (Levy et al., 2006). p150G59S transgenic mice develop progressive motor neuron degeneration with pathological similarities to Amyotrophic Lateral Sclerosis (ALS) (Chevalier-Larsen et al., 2008; Lai et al., 2007; Laird et al., 2008). It is intriguing that different mutations in the same domain of p150Glued cause two dramatically distinct human neurodegeneration syndromes, and the mechanism by which these mutations disrupt p150Glued function in neurons is unknown.
CAP-Gly domains interact with proteins that contain EEY/F motifs in their carboxy termini, including tyrosinated alpha-tubulin (Honnappa et al., 2006; Peris et al., 2006; Weisbrich et al., 2007). While present diffusely along microtubules in most cell types, including neurons, a fraction of p150 protein localizes to microtubule (+) ends in mammalian cells and in Aspergillus (Habermann et al., 2001; Vaughan et al., 1999; Vaughan et al., 2002; Zhang et al., 2003). However, deletion of the p150 microtubule-binding domain does not disrupt cargo transport in S2 cells, and p150G59S transgenic mice do not have apparent defects in axonal transport (Chevalier-Larsen et al., 2008; Kim et al., 2007). Thus, while considerable evidence implicates dynactin in dynein-mediated microtubule-based transport, the function of dynactin and its microtubule-binding domain in regulating axonal transport is unclear.
The dynein/dynactin complex is highly conserved in Drosophila, and the p150 subunit, encoded by the Glued (Gl) gene, genetically interacts with dynein (McGrail et al., 1995; Waterman-Storer and Holzbaur, 1996). The p150 and arp1 dynactin subunits have been proposed to regulate both anterograde and retrograde transport of organelles in Drosophila axons (Haghnia et al., 2007; Pilling et al., 2006), however the mechanism whereby dynactin coordinates bidirectional axonal transport is unknown. Gl1 is a spontaneously isolated Glued allele that causes truncation of the C-terminal third of the protein, and it functions genetically as a dominant-negative allele (referred to here as p150ΔC, see Figure S1A; (Swaroop et al., 1985)). In Drosophila, p150 is enriched at the larval neuromuscular junction (NMJ), and expression of p150ΔC protein in motor neurons causes synapse instability and presynaptic retractions, leading to a reduction in bouton number and impaired neurotransmitter release at the NMJ (Allen et al., 1999; Eaton et al., 2002).
We characterize here disease-associated mutations in p150Glued that reveal a novel function for dynactin at the distal-most ends of synapses. Our data show that p150 and kinesin function synergistically at NMJ terminal boutons (TBs) to regulate dynein-mediated retrograde transport. We show that this function is specifically disrupted by a p150Glued mutation that causes motor neuron disease, but not by p150Glued mutations that cause Perry Syndrome, suggesting that disruption of transport at synaptic termini contributes to the cell type specificity of these diseases.
A Motor Neuron Disease-causing Missense Mutation Produces a Partial Loss of Glued Function in Drosophila
The CAP-Gly microtubule-binding domain of p150Glued is phylogenetically conserved, including the residue mutated in HMN7B (Gly59 in human p150, corresponding to Gly38 in Drosophila Glued; Figure S1B). The G59S mutation in human p150 causes a ~50% reduction in binding of purified p150 microtubule-binding domain to microtubules in vitro (Levy et al., 2006). To determine how the corresponding G38S mutation in fly Glued affects microtubule binding in vivo, we purified microtubule-associated proteins from flies conditionally expressing HA-tagged p150. Interestingly, whereas wild type (WT) p150 cosediments with microtubules, as has been described for endogenous p150 (McGrail et al., 1995), the G38S mutant p150 protein exhibits a marked reduction in microtubule association (Figures 1A and S1C).
Figure 1
Figure 1
GlG38S Causes a Partial Loss of Glued Function
To investigate the consequences of the motor neuron disease-associated G59S mutation on Glued function in vivo, we first generated transgenic flies that express WT and mutant human and Drosophila p150 (Figure S1D). Surprisingly, overexpression of human or Drosophila p150WT in multiple independent transgenic lines is extremely toxic, leading to lethality or severe rough eye phenotypes when overexpressed in neurons using the pan-neuronal driver elavC155-GAL4 (Figures 1B and and5C).5C). In contrast, overexpression of human p150G59S or Drosophila p150G38S in neurons causes a mild rough eye phenotype (Figure 1B), suggesting the G59S mutation causes loss-of-function (LOF). Our biochemical data suggest that this LOF is due to a reduction in microtubule binding.
Figure 5
Figure 5
Kinesin Heavy Chain Functions Synergistically with Glued at NMJ Terminal Boutons
While strong overexpression of p150WT is toxic, we found that low-level expression of Drosophila p150WT fully rescues the early larval lethality of Glued null animals (Gl 1– 3/Gl Δ22 (Siller et al., 2005)), demonstrating that these transgenes are fully functional (Figure 1E). Because the toxicity of high-level p150WT overexpression complicates the interpretation of p150G38S phenotypes, we introduced the G38S mutation directly into the endogenous Glued locus in the Drosophila genome using homologous recombination (Figure 1C). This knock-in approach generates an allelic replacement that changes only a single genomic DNA base-pair without introducing exogenous DNA (hereafter referred to as GlG38S), thereby allowing the mutant gene to be expressed under the control of the normal Glued regulatory elements throughout all tissues and stages of development. GlG38S homozygous flies are viable but sterile, whereas GlG38S/Glnull (1–3 or Δ22) flies are late pupal lethal, demonstrating that the GlG38S mutation is a hypomorphic allele of Glued (Figure 1E). The pupal lethality of GlG38S/Glnull animals is fully rescued to adulthood with ubiquitous expression of p150WT, or with a genomic fragment containing the Glued gene (BAC {Gl+}), demonstrating that this lethality is caused by loss of Glued function (Figure 1E). Western analysis shows that the mutant protein is expressed at reduced levels in GlG38S flies compared to controls, suggesting that the mutant protein is unstable (Figure 1D, S1E). A reduced level of mutant protein expression is also seen in mice in which the G59S mutation was introduced into the endogenous p150 locus (Lai et al., 2007).
GlG38S and GlG38S/Gl Δ22 larvae exhibit normal locomotion (Figure 1F); however, GlG38S adult flies have dramatically impaired locomotor activity and are unable to fly (Figure 1G). Adult GlG38S animals develop progressive paralysis with age and have a markedly reduced lifespan (median survival 16 days vs 70 days in WT) (Figure 1H). The GlG38S early adult lethality and locomotor phenotypes are rescued with low-level expression of p150WT (Figure 1G–H), demonstrating that these phenotypes are due to a loss of Glued function. A similar, albiet less severe, locomotor phenotype is seen in the dominant-negative allele of Glued (Gl1/+), confirming that disruption of Glued function in Drosophila causes age-dependent motor deficits and reduced survival (Figure S2A–B). Indeed, a reduction in lifespan is also observed following disruption of Glued function in all neurons, or specifically within motor neurons, by overexpressing either p150 protein lacking its C-terminus (p150ΔC) or dynamitin (Dmn), the p50 subunit of the dynactin complex which disrupts the complex when overexpressed (Burkhardt et al., 1997) (Figure S2B). These data demonstrate that Glued function is required in motor neurons for normal locomotor function and life span.
Endosomes Accumulate at Terminal NMJ Boutons in GlG38S Animals Despite Normal Axonal Transport
The dynactin complex regulates axonal transport in larval axons (Haghnia et al., 2007; Pilling et al., 2006), and disruption of axonal transport may underlie the pathogenesis of dynactin-mediated neurodegenerative diseases. Loss-of-function alleles in genes that encode dynein and dynactin subunits frequently display larval “tail-flip” phenotypes and “axonal jams” that can be labeled with synaptic vesicle markers such as anti-synaptotagmin (Martin et al., 1999). Surprisingly, GlG38S animals do not display either of these phenotypes (Figure S3A and data not shown), suggesting that axonal transport may not be severely disrupted. Since retrograde transport of Rab7(+) signaling endosomes has been proposed to be disrupted in neurodegenerative diseases (Deinhardt et al., 2006; Perlson et al., 2010), we investigated the dynamics of endosomal axonal transport in GlG38S animals by imaging Rab7:GFP in larval segmental nerves (Figure 2A and Movies S1S2). Interestingly, though we see a decrease in the proportion of stationary Rab7:GFP particles in GlG38S animals (Figure 2B), all other axonal transport measures, including flux, velocity, and processivity, are unaffected (Figures 2C–D). We assayed retrograde signaling by the TGF-beta receptor family member Wit, which is blocked in Drosophila overexpressing p150ΔC (McCabe et al., 2003), and observed no reduction in pMad signaling in GlG38S larval motor neuron nuclei (Figure S3B). Taken together, these data suggest that retrograde axonal transport of endosomes occurs normally in GlG38S animals.
Figure 2
Figure 2
Terminal NMJ Bouton rather than Axonal Transport Phenotypes in GlG38S Animals
Overexpression of p150ΔC causes a reduction in synaptic bouton number at the NMJ due to presynaptic retractions (Eaton et al., 2002). In contrast, GlG38S animals have a normal number of synaptic boutons in proximal abdominal segments (segments A2 and A3) and a small but significant increase in the number of synaptic boutons in distal segments (segments A5 and A6, Figures 2E–F). Surprisingly, when we look specifically at the distal-most synaptic boutons, called terminal boutons (TBs), we see a ~2-fold increase in bouton volume in distal segments. Interestingly, p150WT-HA expressed in motor neurons is dramatically enriched within NMJ TBs (Figure 2G, arrows), in addition to its expected localization along axons (Figure 2G, asterisk) and in the cytoplasm. We observe that the TB localization of p150WT-HA is apparently greatest within the center of the TB, just distal to where expression of the microtubule-associated protein Futsch becomes undetectable (Figure 2G). p150WT-HA is also enriched at sites of microtubule loops, which are thought to be enriched in microtubule (+) ends (Figure 2G, arrowhead; enlarged in Figure S3C) (Roos et al., 2000). To determine if microtubule (+) ends are also enriched at TBs, we expressed a microtubule (+) end marker, the kinesin motor domain fused to GFP (KhcHead:GFP) (Clark et al., 1994), in motor neurons. Interestingly, we see at the NMJ that KhcHead:GFP is predominantly localized to the TB (Figures 2H and S3D) and, similar to p150WT-HA localization, is enriched within the middle of the TB (Figure 2H, inset). We also observe a similar enrichment of the microtubule (+) end marker EB1:GFP at this location (Movie S3). These data suggest that wild type p150Glued is enriched at microtubule (+) ends of terminal boutons.
Since p150WT-HA is localized within NMJ TBs, we next investigated the morphology of the presynaptic nerve terminal in Glued mutants. Anti-HRP labels the presynaptic membrane at the Drosophila NMJ and binds to neuron-specific transmembrane glycoproteins such as FasII (Desai et al., 1994). Interestingly, we observe intense anti-HRP staining within TBs of GlG38S and GlG38S/GlΔ22 NMJs (Figure 3A), suggesting that neuronal membranes accumulate at these presynaptic termini. Similar to the TB swelling we observed in GlG38S/GlΔ22 mutants, the anti-HRP phenotype is more severe in distal abdominal segments than in proximal segments (Figures 3A and 3D). Approximately 75% of NMJs from distal segments of GlG38S and GlG38S/GlΔ22 larvae display accumulation of anti-HRP staining within TBs, whereas only ~15% of control NMJs have any accumulation of anti-HRP staining within TBs (Figure 3D). Similarly, overexpression of p150G38S in motor neurons (using D42-GAL4) causes a dramatic accumulation of anti-HRP immunoreactivity in large puncta specifically located within the TB, demonstrating that p150G38S can act in a dominant-negative fashion when overexpressed in neurons (Figures 3B–D). Since anti-HRP labels presynaptic transmembrane proteins, these data suggest that membrane-bound vesicles accumulate within TBs of GlG38S NMJs.
Figure 3
Figure 3
Endosomes Accumulate at Terminal NMJ Boutons in GlG38S Mutant Larvae
We next crossed D42-GAL4, UAS-p150G38S (D42 > p150G38S) flies to flies that express fluorescently-tagged markers that label distinct membrane-bound compartments under control of the UAS promoter. Colocalization of the membrane marker mCD8:GFP with anti-HRP in terminal boutons of larvae expressing p150G38S suggests that these anti-HRP positive structures are membrane bound (Figure 3C). Interestingly, the late endosome marker Rab7:GFP colocalizes with anti-HRP in TBs coexpressing both p150G38S and Rab7:GFP, suggesting that these membranous structures include endosomes (Figure 3B). Furthermore, transmembrane proteins known to cycle through endosomes, including synaptotagmin (Takei et al., 1996) and APP (Haass et al., 1992), also accumulate at these TBs and partially colocalize with anti-HRP (Figure 3C). Together, these data show that overexpression of p150G38S causes a marked accumulation of endosomal membranes and proteins at NMJ TBs.
To determine if the accumulation of endosomes at Glued mutant TBs is due to disruption of dynein/dynactin function, we asked if similar phenotypes are present in mutant alleles of genes encoding components of the dynein/dynactin complex. Because most available alleles are early larval or embryonic lethal, we knocked down dynein/dynactin subunits in motor neurons using RNAi (Figure S4A). As expected, knockdown of three dynactin subunits (Gl, cpa, and p62) and three dynein subunits (dhc, dic, and dlic) phenocopies the TB accumulation of anti-HRP immunoreactivity and Syt:GFP we observe in D42 > p150G38S animals (Figures 3C, 3E, and S4B). These data demonstrate that disruption of the dynein/dynactin complex causes an accumulation of endosomes within TBs of the NMJ.
Dynein Heavy Chain Accumulates at GlG38S Terminal Boutons
In filamentus fungi, the dynactin complex is required for MT (+) end localization of dynein and for the interaction between dynein and endosomes (Xiang et al., 2000; Zhang et al., 2010). To determine if dynein is mislocalized in Glued animals, we analyzed the expression of the cytoplasmic dynein heavy chain (cDhc64C, referred to here as Dhc). Surprisingly, GlG38S larvae reveal a striking accumulation of Dhc at NMJ TBs in all segments in 100% of GlG38S and GlG38S/GlΔ22 animals; this phenotype is never observed in wild-type animals (Figures 4A–C and Figures S5A–B). At wild-type synapses, Dhc is localized to small puncta at the periphery of all boutons (Figure 4A), and occasionally small Dhc(+) puncta are observed near the center of the TB (Figure 4E, arrow). In GlG38S animals, however, the mean Dhc signal intensity is increased ~10-fold within TBs, with no significant differences between proximal and distal segments (Figure 4B). Interestingly, in GlG38S larvae, Dhc predominantly accumulates at TBs of the longest branch in synapses with multiple branches (Figures S5A and 5B). These accumulations are not seen in axons or motor neuron cell bodies (Figure S5B and data not shown). Microtubules do not appear to be altered at GlG38S NMJs; however, we did note that mutant TBs with observable microtubule bundles did not accumulate dynein (Figure S5C, arrow), in contrast to those TBs with no significant tubulin staining. These data suggest that dynein accumulates in GlG38S TBs lacking stable microtubules.
Figure 4
Figure 4
Dynein is Mislocalized to the Terminal NMJ Bouton in GlG38S Mutants
The accumulation of Dhc specifically within the TB of GlG38S NMJs is consistent with an inability of the Dhc motor to move retrogradely at TBs. To further investigate dynein function at TBs, we quantified this phenotype in different mutant backgrounds. In GlG38S or GlG38S/GlΔ22 animals, ~ 90% of NMJs have marked TB accumulation of Dhc, whereas 0% of synapses from WT animals exhibit this phenotype (Figure 4C). This phenotype is fully rescued by motor neuron-specific expression of p150WT but not p150G38S (Figure 4D), showing that it is neuron-autonomous and due to a loss of Glued function. A similar phenotype is observed following disruption of the dynactin complex with Glued RNAi or dynamitin overexpression (Figures 4C and 4E), further demonstrating that dynein accumulation is due to loss of dynactin function. Furthermore, overexpression of p150G38S, or an amino-terminal deletion mutant protein lacking the CAP-Gly domain (p150ΔMB), causes a similar dynein mislocalization phenotype; however, this is not observed following overexpression of p150ΔC. These findings demonstrate that disruption of the p150Glued CAP-Gly domain, but not overexpression of p150ΔC (commonly used in Drosophila to disrupt Glued function) causes Dhc accumulation at TBs. Since overexpression of p150G38S phenocopies Glued RNAi, the G38S mutation likely functions in a dominant-negative fashion when overexpressed in motor neurons.
To determine if dynein mislocalization at synaptic termini is specific to motor neurons in Glued animals, we overexpressed p150G38S in sensory neurons and analyzed its effect on dynein localization. Ppk+ multidendritic sensory neuron presynaptic termini (labeled with synaptotagmin:GFP) are distributed in a stereotypical arrangement within the neuropil of the ventral nerve cord (Figure S6A). Interestingly, overexpression of Drosophila p150G38S, or human p150G59S, causes a dramatic accumulation of Dhc within all sensory presynaptic termini (Figure S6B), and a similar phenotype is seen in GlG38S animals (data not shown). Whereas axons in Drosophila and vertebrates are oriented with their microtubule (+) ends distally, in Drosophila dendritic MTs are oriented with their (−) ends distal (Rolls et al., 2007). Consistent with our data suggesting that p150Glued plays a role in dynein-mediated retrograde transport at microtubule (+) ends, Dhc is not mislocalized to ends of sensory dendrites (Figure S5B, arrows). Furthermore, we do not observe Dhc accumulation at the base of dendrites containing microtubule (+) ends (Figure S5B, box). These data demonstrate that loss-of-function mutations in p150Glued that disrupt microtubule-binding lead to an accumulation of dynein at presynaptic termini.
kinesin heavy chain (khc) Functions Synergistically with Glued at Terminal Boutons
To further understand the molecular basis of Glued dysfunction in mutant neurons, we performed a candidate-based screen for modifiers of GlG38S lethality. We identified a null allele of the Kinesin-1 family member kinesin heavy chain (khc8) as a potent enhancer of GlG38S lethality (Figures 5A–B). Conversely, the khc8 allele markedly suppresses the rough eye phenotype we observe following overexpression of wild-type p150 in the Drosophila eye driven by GMR-Gal4 (Figure 5C). These data suggest that Glued and khc function cooperatively, not antagonistically, as would be predicted if they simply regulated microtubule-based transport in opposite directions. A cooperative role for kinesin and dynactin has been proposed (Deacon et al., 2003; Gross et al., 2002; Haghnia et al., 2007; Martin et al., 1999); however, the molecular mechanism of this synergistic interaction is unclear.
One potential mechanism underlying cooperativity between khc and Glued is that kinesin-mediated delivery of p150 to microtubule (+) ends at synaptic termini may be rate-limiting for the initiation of retrograde transport. In Aspergillus, kinesin is required for plus-end localization of dynein and dynactin (Zhang et al., 2003), and the dynein/dynactin complex is anterogradely transported along axons in vertebrates via KIF5, the orthologue of Khc (Hirokawa et al., 2010). Strikingly, whereas Khc is present at very low levels at wild-type NMJs, it accumulates both at TBs in GlG38S animals and also following presynaptic knockdown of dynactin subunits (Figures 5D and S7). This pattern of Khc mislocalization is similar to the Dhc mislocalization we observe in these mutants and, indeed, Khc colocalizes with Dhc at GlG38S TBs (Figure 5E); all TBs with significant Dhc accumulation also show Khc accumulation. We do not see accumulation of Khc or Dhc along motor and sensory axons in larval segmental nerves, (Figure S5A and data not shown), showing that this phenotype specifically occurs at synapses. These data suggest that p150Glued coordinates Khc-mediated anterograde transport with Dhc-mediated retrograde transport at TBs.
To test whether p150Glued and kinesin function cooperatively at synapses, we investigated genetic interactions between khc8 and GlG38S. There is a dramatic enhancement of the GlG38S TB swelling and anti-HRP accumulation phenotypes at all NMJs in all segments when khc gene dosage is reduced by 50% (Figures 5F–G), and the severity of the khc8/+; GlG38S/+ phenotype is similar to the GlG38S homozygous phenotype. Furthermore, the distribution of anti-HRP localization within TBs of khc8/+; GlG38S NMJs is similar to the localization of KhcHead:GFP when it is expressed in wild-type motor neurons (Figure 2B). These data suggest that kinesin functions with p150 in TBs to coordinate bidirectional vesicle transport.
p150G38S Disrupts Retrograde Transport from Terminal Boutons and Synaptic Transmission
To directly investigate p150Glued-mediated regulation of retrograde transport at synaptic termini, we monitored dense core vesicle (DCV) retrograde transport at TBs in larvae overexpressing p150G38S. DCVs are more uniform in size than endosomes, and single vesicles can be imaged at the NMJ in real time using ANF:GFP as a marker (Levitan et al., 2007). Similar to what we observe for Rab7:GFP, overexpression of p150G38S causes a dramatic accumulation of DCVs at TBs (Figure 6A, top panels; and Figure 6B). Following photobleaching of wild-type (D42 driver alone) proximal NMJ boutons, individual DCVs rapidly exit the TB (Movie S4 and Figure 6A, bottom panels). Despite an ~10-fold increase in ANF:GFP fluorescence intensity at the terminal bouton in mutant animals (Figure 6B), ~3 fold fewer vesicles undergo retrograde transport from the terminal bouton (Movie S5 and Figure 6C). Similar effects are seen following overexpression of dominant-negative Glued (p150ΔC) using a novel imaging approach termed SPAIM (simultaneous photobleaching and imaging) (Wong et al., 2012) to specifically visualize retrograde vesicle transport at TBs (Figure 6C). These data directly demonstrate that disruption of dynactin inhibits retrograde transport of DCVs from TBs.
Figure 6
Figure 6
p150G38S Disrupts Retrograde Transport at TBs and Neurotransmitter Release
Terminal NMJ boutons in Drosophila, as compared to proximal boutons, exhibit markedly enhanced synaptic transmission (Guerrero et al., 2005). To determine if the disease-associated GlG38S mutation causes a defect in synaptic transmission, we performed electrophysiological analyses on GlG38S animals at the third-instar larval NMJ. GlG38S animals exhibit a significant reduction in the amplitude of evoked junctional potential (EJP) (Figures 6D–E), and this impairment in evoked synaptic transmission is fully rescued by presynaptic expression of wild-type p150. Therefore, this defect is due to loss of Glued function in motor neurons. We observe no change in the frequency or amplitude of miniature EJPs (mEJPs), showing that spontaneous neurotransmitter release is unaffected in GlG38S animals (Figures 6F–H). These results show that GlG38S animals have a reduction in the quantal content of evoked neurotransmitter release at the NMJ (Figure 6I), despite the presence of a normal number of synaptic boutons at these terminals.
Perry and HMN7B Mutant p150 Proteins Affect Distinct p150 Functions Within Motor Neurons
Perry Syndrome is characterized by degeneration of neurons within the substantia nigra and brainstem, however it does not noticeably affect motor neurons (Farrer et al., 2009). Remarkably, Perry Syndrome, like HMN7B, is also caused by mutations in the CAP-Gly domain of p150 (Figure S1B). Therefore, to gain insight into the cell-type specificity of neurodegeneration caused by different mutations in the p150 CAP-Gly domain, we assessed whether functional differences exist in Drosophila between the HMN7B mutation (G38S) and Perry Syndrome mutations (G50A and G50R). When expressed in Drosophila S2 cells (Figure S8A), both p150G38S and p150G50R form large cytoplasmic puncta (Figure 7A), similar to the protein aggregates seen in patients with these diseases. In contrast, the wild-type protein does not form large puncta in S2 cells and is present diffusely in the cytoplasm. Interestingly, we observe a similar appearance of puncta in Drosophila motor neurons following overexpression of human p150G59S, whereas human p150WT is diffusely expressed in the motor neuron cell body cytoplasm (Figure 7B). To directly compare the effects these different disease mutations have on p150 function in vivo, we utilized transgenic animals that express different HA-tagged p150 proteins at equivalent levels, employing site-specific chromosomal integration (Bischof et al., 2007). When these transgenes are expressed under the control of elav-GAL4, Perry (G50A and G50R) and HMN7B (G38S) mutations cause a reduction in p150 protein expression in vivo compared with wild-type p150 expression, despite equivalent mRNA levels (Figure 7C). These data suggest that both HMN7B and Perry mutations cause the protein to be unstable, as is suggested by the reduced p150 protein levels we observe in GlG38S flies (Figure 1D). We generated a high level expressing transgenic line (G38SHi) that expresses p150G38S mutant protein at levels at least as high, if not higher, than wild-type p150 (Figure 7C), and this line was used to control for protein expression.
Figure 7
Figure 7
Aggregates within Motor Neurons and Dynein Mislocalization is Specific to p150 Mutations that cause HMN7B but not Perry Syndrome
Similar to what we observed in S2 cells and motor neurons following expression of human p150G59S, when p150G38S-HA is expressed in motor neurons using the OK371-GAL4 driver, we find large puncta within motor neuron cell bodies, whereas p150WT-HA is diffusely present in the motor neuron cytoplasm (Figures 7D and S8B). In the G38SHi line, all motor neurons show p150-HA(+) puncta, and many are very large (Figures 7D and S8B); in the low-expressing line, however, large puncta are rarely observed (seen in at least two motor neurons in 5 out of 6 animals). In contrast, large puncta are not detected in p150G50A-HA or p150G50R-HA animals (no puncta seen in 6 animals each, Figures 7D and S8B). Because large puncta were detected in p150G38S-HA animals that express lower levels of p150 protein (but equivalent mRNA levels) compared with p150G50A-HA or p150G50R-HA animals, we conclude that within motor neurons the HMN7B mutation makes p150 more aggregate-prone than the Perry mutations.
To determine if Perry Syndrome mutations cause dynein-mislocalization phenotypes similar to those we observe in HMN7B (GlG38S) mutant animals, we overexpressed wild-type and mutant p150HA proteins in motor neurons. Overexpression of p150HA causes significant toxicity, similar to what we observed with high-level overexpression of untagged p150, as evidenced by a reduction in bouton number, abnormal synapse morphology, appearance of axonal swellings (data not shown), and accumulation of anti-HRP within the terminal bouton (Figure 7E). However, we only observed TB accumulation of Dhc following p150G38S-HA overexpression (Figure 6E–F); there was no difference in Dhc distribution among larvae overexpressing p150WT-HA, p150G50A-HA, or p150G50R-HA. Furthermore, motor neuron-specific expression of p150WT-HA, p150G50A-HA, and p150G50R-HA, but not p150G38S-HA, rescued the Dhc mislocalization phenotype observed in GlG38S/GlΔ22 animals (Figure 7F). These data show that TB Dhc accumulation is specific to the motor-neuron disease-associated mutation in p150, and therefore these results identify a key distinction between different mutations, both within the p150 CAP-Gly domain, that cause two distinct human neurodegenerative diseases.
To better understand the underlying cause of neurodegenerative diseases resulting from mutations in the CAP-Gly domain of the dynactin subunit p150, we introduced disease-associated p150Glued mutations into Drosophila using homologous recombination and transgenesis. Interestingly, p150 is enriched at MT (+) ends of NMJ terminal boutons (TBs), and GlG38S larvae develop TB swellings and accumulation of the retrograde motor dynein. We find strong synergistic genetic interactions between khc and glued that produce phenotypes at TBs, suggesting that p150-mediated coordination of bidirectional axonal transport occurs at synaptic termini. Our data suggest that the CAP-Gly domain of p150 is required for initiation of dynein-mediated retrograde transport at terminal boutons.
p150 Regulates Dynein-mediated Retrograde Transport at Synaptic Termini
We demonstrate here that p150 is enriched at TB microtubule (+) ends, consistent with the known function of CAP-Gly domain-containing proteins. Localization of p150 at (+) ends has been observed in nonneuronal cells (Habermann et al., 2001; Vaughan et al., 1999; Vaughan et al., 2002; Zhang et al., 2003), and dynein localization to (+) ends in Aspergillus requires p150 (Xiang et al., 2000). The p150 microtubule-binding domain has been proposed to regulate the processivity of retrograde microtubule transport via a “skating” mechanism (Culver-Hanlon et al., 2006). However, analysis of microtubule transport in S2 cells lacking the MT-binding domain demonstrates normal (−) end-directed transport (Kim et al., 2007). Furthermore, in budding yeast, the G59S mutation or CAP-Gly deletion mutants disrupt nuclear migration but not other dynein-dependent transport events (Moore et al., 2009). Our analysis of endosomal axon transport in GlG38S animals further suggests that loss of p150 microtubule binding ability does not affect (−) end-directed transport in axons.
The accumulation of dynein and kinesin motor proteins, and also endosomal vesicles, specifically within the TB of Glued mutants suggests that dynactin may function to coordinate retrograde transport at TBs. Indeed, we show using live imaging at the NMJ that disruption of dynactin causes accumulation of dense core vesicles at TBs, and these DCVs fail to undergo retrograde transport out of this distal-most synaptic bouton. These data directly demonstrate that dynactin plays a critical role in regulating retrograde transport at TBs.
Why are retrograde transport defects seen specifically at GlG38S TBs and not along axons, which also have MT plus ends? There are at least two (non-mutually exclusive) explanations for this observation: (1) TBs have dynamic MTs, but lack stabilized MT bundles (Pawson et al., 2008). In this model, MAP-stabilized MTs may recruit motors and cargo independent of dynactin and compensate for the disruption of MT binding by p150 along the axon and in proximal boutons; indeed, MAPs can directly associate with dynein and kinesin (Jimenez-Mateos et al., 2005; Sung et al., 2008). (2) Retrograde transport rates are much higher at TBs than in proximal boutons or axons (Wong et al., 2012). In this model, continuous anterograde transport of vesicles to TBs may overwhelm the ability of cargo to undergo p150-independent capture for subsequent retrograde transport at GlG38S TBs. Since retrograde endosomal transport may occur normally in GlG38S mutants from proximal boutons (which comprise the overwhelming majority of boutons at the NMJ), this may explain why we do not observe a disruption of retrograde transport along axons.
What is the mechanism whereby p150 regulates retrograde transport at terminal boutons? Growing microtubules are dynamically unstable, and (−) end-directed microtubule transport of Golgi membranes is initiated upon contact with microtubule (+) ends, a process that requires p150 (Vaughan et al., 2002). We propose that a similar “search and capture” mechanism occurs at synaptic termini, whereby growing microtubules explore the terminal bouton and, upon contact with the dynactin/dynein complex, cargo are recruited for retrograde transport (Figure 8). A similar model has been proposed for dynactin +TIP function in nonneuronal cells (Vaughan, 2004; Wu et al., 2006). Though dynamic MT (+) ends are observed throughout axons and the NMJ (Pawson et al., 2008), we propose they are uniquely required for retrograde transport at synaptic termini, which lack stable microtubule bundles.
Figure 8
Figure 8
Model for p150Glued Function at Synaptic Termini
Kinesin and p150Glued Function Cooperatively at Synaptic Termini
Our genetic analyses demonstrate a strong synergistic interaction between kinesin and dynactin at NMJ synapses, the opposite of what one would predict if these proteins solely functioned in unidirectional anterograde or retrograde axonal transport, respectively. The dynein/dynactin complex requires kinesin for anterograde transport along axons, and the interaction between dynein at (+) ends and early endosomes in Aspergillus requires kinesin (Zhang et al., 2010). Thus, kinesin may be required to localize the dynactin/dynein complex to microtubule (+) ends at synapses, where it captures vesicular cargo for the initiation of retrograde transport (Figure 8). Therefore, kinesin-mediated delivery of dynein/dynactin to (+) ends likely allows for coordination of kinesin-mediated anterograde transport and dynein-mediated retrograde transport at synapses.
How Does Disruption of p150 Affect Synapse Function?
We show here that loss of dynactin in Drosophila motor neurons causes a robust accumulation of endosomal membranes specifically within swollen NMJ TBs. Interestingly, these phenotypes are most severe in distal abdominal larval segments, similar to the distal-predominant symptoms observed in patients. Our live imaging of DCV transport at TBs suggests that these phenotypes are due to a defect in retrograde transport from the TB. In GlG38S animals, we see a reduction in evoked neurotransmitter release, despite normal spontaneous release. This physiologic phenotype is reminiscent of Rab5 mutants, which are defective in endosomal trafficking at synapses (Wucherpfennig et al., 2003). Overexpression of p150ΔC causes a reduction in evoked neurotransmitter release due to presynaptic retractions (Eaton et al., 2002). In contrast, our GlG38S mutants do not exhibt a decrease in the number of synaptic boutons at the larval NMJ; rather, GlG38S mutants develop TB swelling and accumulation of endosomal membranes. NMJ TBs exhibit greater neurotransmitter exocytosis than proximal boutons (Guerrero et al., 2005), and TBs are critical for DCV circulation to proximal boutons (Wong et al., 2012). Since endosomes are hypothesized to be important sorting stations for synaptic vesicle proteins (Bonanomi et al., 2006; Hoopmann et al., 2010; Uytterhoeven et al., 2011), we suggest that the impairment in neurotransmitter release is due to a disruption of synaptic vesicle sorting or release at TBs.
Mechanism Underlying Motor Neuron Degeneration in HMN7B
A central hypothesis that explains how mutations in the dynein/dynactin complex cause motor neuron degenerative disease postulates that disruption of dynein-mediated retrograde axonal transport underlies these diseases (Perlson et al., 2010). However, no biochemical evidence of axonal transport disruption was observed in transgenic mice expressing p150G59S (Chevalier-Larsen et al., 2008). Similarly, we show here that axonal transport of endosomes, and also retrograde endosomal signaling, is not disrupted in GlG38S animals. Thus, mutations in the CAP-Gly domain of p150 do not apparently affect cargo transport along MTs; our data suggest that the HMN7B mutation specifically disrupts p150 function at MT (+) ends of synapses.
How does disruption of MT(+) end binding lead to neurodegeneration? Presynaptic retractions that occur early in the pathogenesis of motor neuron degenerative disease (Fischer et al., 2004) start at the distal-most end of synapses and are observed in larvae with severe disruption of p150Glued (Eaton et al., 2002). Therefore, one potential mechanism is that the terminal bouton phenotypes we observe here lead to synapse instability and retraction with aging. Alternatively, the dynactin/dynein complex is believed to mediate (−) end-directed microtubule based transport of multiple cargos throughout neurons, including ER, Golgi, and mitochondria. We cannot exclude the possibility that a function of p150Glued not assayed in this study is also disrupted by these mutations and is more relevant to the pathogenesis of disease.
Both HMN7B and Perry Syndrome are caused by dominant mutations in p150, and the mechanisms by which these mutations cause disease are unknown. Most dominantly-inherited neurodegenerative diseases (including SOD-mediated ALS and polyglutamine-expansion diseases) are caused by gain of a toxic function. Our analysis of the HMN7B mutation in flies does not provide evidence for a gain of function. Instead, we demonstrate that the disease-causing mutations in p150 result in a partial loss of Glued function, and they may also function in a dominant-negative fashion when overexpressed. This is consistent with the severe phenotypes seen in transgenic mice when p150G59S is overexpressed (Chevalier-Larsen et al., 2008; Laird et al., 2008). Thus, our data suggest that a loss-of-function and/or dominant-negative mechanism causes HMN7B motor neuron disease.
Although further analysis of adult GlG38S flies will be required to determine how well they model HMN7B pathologically, several of our findings indicate that this model does share features with human motor neuron diseases, including: aggregation of mutant protein within motor neurons, adult onset locomotor impairment, and a deficit in synaptic transmission at the NMJ. How mutations in ubiquitously expressed proteins cause degeneration of specific neuronal subtypes is a fundamental question that must be addressed if we are to understand the etiology of neurodegenerative diseases. In inherited neuropathies, the long axonal length of motor neurons that innervate distal limb muscles is believed to underlie the length-dependent pathology (Hirokawa et al., 2010); however in most neurodegenerative diseases, including HMN7B and Perry Syndrome, the reason that specific neurons are affected is unknown. The identification of mutations within the same domain of the same protein that cause two distinct neurodegenerative syndromes provides a unique opportunity to understand how these mutations differentially affect protein function, and our data lend insight into the molecular mechanisms underlying the cell type specificity of distinct neurodegeneration syndromes.
The G59S mutation is predicted to destabilize the CAP-Gly domain, whereas the Perry mutations all lie on the surface of this domain. Destabilization of the CAP-Gly domain by the G59S mutation may make it more susceptible to aggregation, as we observe here in Drosophila motor neurons. Furthermore, it is likely that distinct protein-protein interactions are disrupted by these different mutations. We only observe an accumulation of dynein at synaptic termini following overexpression of the HMN7B mutant forms of p150, and not the Perry mutations. We propose that specific disruption of the interaction between p150 and microtubule (+) ends at synaptic termini underlies the motor neuron specificity of neurodegeneration in HMN7B.
Fly Strains
All crosses were performed at 25 °C. Canton-S and w1118 were used as wild-type control lines. The human p150WT and p150G59S constructs were generated by cloning C-terminal flag-tagged p150 cDNA obtained from P. Wong (Laird et al., 2008) into pUAST. The G38S mutation was generated in the Drosophila p150 cDNA (RE24170) using the Stratagene Quick-change mutagenesis kit. The GlG38S knock-in allele was generated as described (Rong et al., 2002, and Supplemental Methods). The Gl1 and Gl1-3 alleles were provided by Tom Hays (Martin et al., 1999); GlΔ22 (Siller et al., 2005) and UAS-GFP:Gl (full length (aa1-1265) and ΔMB (aa201-1265)) were generously provided by Chris Doe. Additional lines are described in Supplemental Methods.
Biochemistry, Immunohistochemistry, and Imaging
Microtubule-associated proteins from adult flies collected 16 hours after a 1 hr 37 °C heat shock to induce GAL4 expression were purified from fly extracts as described (McGrail et al., 1995). NMJ analysis was limited to muscle 4 unless stated otherwise. Antibodies used are detailed in Supplemental Methods. A synapse was considered to have TB anti-HRP or anti-Dhc accumulation if the fluorescence intensity within the TB was clearly much higher than in proximal boutons. Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope using a PLAN-APO 63x, 1.4 NA oil immersion objective. Maximum-intensity Z projections of confocal stacks were generated and processed using Adobe Photoshop. Intensity measurements and NMJ TB volume were obtained by thresholding with Imaris software. For scanning electron microscopy, fly heads were coated with gold:palladium using a vacuum evaporator and imaged immediately using a LEO/Zeiss Field-emission SEM.
SPAIM experiments were performed as described (Wong et al., 2012). For other live imaging experiments, wandering third-instar larvae were rinsed and pinned in Ca2+-free HL3 on the sylgard insert of a custom made imaging mount, and a coverslip was placed over the preparation and secured. Imaging of axonal transport was performed on a Zeiss Axio Observer with a 40X oil objective (EC Plan-Neofluar 1.3 NA) and collected on an AxioCAM CCD camera. Movies were analyzed as described (Louie et al., 2008). For ANF:GFP FRAP experiments, spinning disc confocal images of dense core vesicles were acquired at muscle 6/7 NMJs using a Zeiss Axio Imager Z1 microscope and 63X 1.4 NA oil immersion objective and collected on a QuantEM 512SC camera (Photometrics). ANF:GFP in proximal boutons was bleached using a 488 nm laser controlled by a Mosaic Digital Illumination System (Photonic Instruments, INC).
Electrophysiology and Statistics
Electrophysiological recordings from muscle 6, segment A3, were performed as described (Imlach and McCabe, 2009). Data are expressed as mean +/− standard error of the mean. A Student’s t-test was performed for pair-wise comparisons between each genotype and its wild-type control using GraphPad Prism.
Highlights
  • p150 and kinesin coordinate bidirectional transport at NMJ Terminal Boutons (TBs)
  • p150 regulates initiation of retrograde transport at TBs
  • A Motor neuron disease mutation in p150 inhibits dynein-mediated transport at TBs
  • A Perry Syndrome mutant p150 does not aggregate or block dynein in motor neurons
Supplementary Material
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Acknowledgments
We are grateful to Chris Doe, Vladimir Gelfand, Tom Hays, Phil Wong, Sangyun Jeong, and Herman Aberle for reagents. We thank Ben Choi for pMad work, and Manish Jaiswal and Vafa Bayat for helpful comments. We thank Erik Griffin, Geraldine Seydoux, Norm Haughey, Terry Shelley, Michele Pucak and the NINDS Multi-photon Core Facility (MH084020) at JHMI for assistance with imaging. We also thank the BDSC, VDRC, and DGRC for fly stocks and reagents. This work was funded by the Packard Center for ALS Research (A. L. K. and T. E. L.), P2ALS (B. D. M.), NINDS K08-NS062890 to T. E. L., R01-NS35165 to A. L. K, and RO1-NS32385 to M. Y. W. and E. S. L.
A. L. K. is an Investigator of the Howard Hughes Medical Institute.
Footnotes
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