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Dev Dyn. Author manuscript; available in PMC 2016 December 01. Published in final edited form as: Dev Dyn. 2015 December ; 244(12): 1550–1563. doi:10.1002/dvdy.24350.
Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila Svetlana Smith-Trunova1, Ranjini Prithviraj1, Joshua Spurrier1,2, Irina Kuzina1, Qun Gu1, and Edward Giniger1,* 1National
Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg 35, Rm 1C-1002, 35 Convent Dr. Bethesda, MD 20892 USA
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2The
Johns Hopkins University/National Institutes of Health Graduate Partnership Program, National Institutes of Health, Bethesda, MD 20892
Abstract Background—During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors.
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Results—We show here that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. We further show that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, we show that Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and provide evidence that it also acts at additional steps of MB pruning. Conclusions—These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5 we suggest that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, we speculate that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it employs during developmental remodeling. Keywords Axon; dendrite; pruning; microtubules; proteasome; neurodegeneration
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Introduction The structure of the fully formed nervous system depends not just on the construction of neural wiring but also on its dissolution. Neural connections are disassembled through activity-dependent plasticity, through pathological processes including injury and neurodegeneration and also through developmentally-programmed remodeling. Two central issues in the field of neural wiring, therefore, are to identify the molecular mechanisms that disassemble neural connectivity, and to determine whether they are shared by different
*
Corresponding Author: Tel: 301-451-3890, Fax: 301-451-5368, [email protected].
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forms of neural disassembly, or are unique to individual physiological processes (Coleman, 2005, Hoopfer et al., 2006, Tao and Rolls, 2011).
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Developmentally programmed axonal and dendritic remodeling is widespread and is essential to establish neural wiring. In Drosophila, for example, one observes large-scale remodeling of the axonal and dendritic projections of various sensory neurons, motoneurons and central brain interneurons, particularly during the pupal stage of development (Truman, 1990, Watts et al., 2003, Williams et al., 2006). Developmentally programmed remodeling is not limited to species that undergo metamorphosis, however. In vertebrates, the axons of retinal ganglion cells largely overshoot their target zones in the optic tectum or superior colliculus, and topographic mapping is often achieved by programmed remodeling(Yates et al., 2001). This involves back-branching of collaterals to target appropriate A/P positions, and subsequent activity-dependent refinement. An even more striking example is provided by layer 5 projection neurons of the mammalian neocortex. Neurons from both visual and motor areas initially send out axons that branch to multiple regions including the midbrain and spinal cord, with the inappropriate projections pruned away later in development (O'Leary and Koester, 1993).
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On the surface, developmentally programmed remodeling seems to bear similarities to pathological degeneration processes that occur after injury, and in neurodegenerative disease(Luo and O'Leary, 2005, Saxena and Caroni, 2007). For example, expression of the Wlds gene product blocks neurite removal in a variety of experimental paradigms, both in the context of developmental remodeling and after injury or disease (Wang et al., 2012). Similarly, a requirement for the ubiquitin-proteasome system (UPS) is a common feature of many varieties of neurite dissolution (Watts et al., 2003, Zhai et al., 2003, MacInnis and Campenot, 2005, Tao and Rolls, 2011). In contrast, while non-apoptotic activation of effector caspases is an essential element of developmental dendrite disassembly for Class IV sensory neurons of Drosophila (Williams et al., 2006), it is not required for other forms of developmental remodeling, or even for post-injury dissolution of the dendrites of the very same neurons (Tao and Rolls, 2011). Overall, the picture that emerges is that the remodeling process in any specific setting can draw from among an assortment of mechanisms that are collectively competent to perform remodeling, such that some processes will be shared in different cases, and others may act in only a subset of contexts. The rules that determine which mechanisms act in which case remain obscure, but are essential knowledge if one is to predict which mechanism should be activated or repressed to cause or block remodeling in a specific context.
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One outstanding system for investigating CNS remodeling is the Drosophila mushroom body (MB) (Lee et al., 1999, Watts et al., 2003, Yu and Schuldiner, 2014). The mushroom body is the seat of learning and memory in the fly, as well as in other insects (Davis, 2005). Mushroom body projection neurons fall into 3 main classes, α, β and γ (Lee et al., 1999). The γ-neurons are born first, during embryonic and larval development, and carry out MB functions during larval life. Since the lifestyle of the fly changes dramatically between larval and adult stages, however, the MB needs to be substantially rewired at metamorphosis. Consequently, both the axonal and dendritic arbors of the larval MB neurons are disassembled during early pupal development, and regrow in an adult-specific pattern. This
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programmed disassembly of MB γ-neurons has been studied extensively as a model of developmental remodeling. Remodeling is triggered ∼6 hours after the onset of metamorphosis (called “pupariation”) by an organism-wide spike of the steroid hormone ecdysone, which triggers (among other things) a switch in the expression of ecdysone receptor (EcR) isoforms to induce EcR-B in remodeling neurons (Lee et al., 2000). The first overt sign of neuritic disassembly is dissolution of the microtubule cytoskeleton in axons followed by frank fragmentation of dendrites and axons (Watts et al., 2003). Occurrence of fragmentation requires activity of the ubiquitin-proteasome system (UPS), as inhibition of the UPS blocks fragmentation and remodeling (Watts et al., 2003). The fragments are then phagocytosed by glia, phagocytic hemocytes and neighboring neurons (Watts et al., 2004, Williams and Truman, 2005, Hakim et al., 2014, Tasdemir-Yilmaz and Freeman, 2014). However, while these broad outlines of the remodeling process have been described, the detailed molecular machinery that carries out each of these processes, and that coordinates these disparate molecular activities, remains unknown.
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We have shown previously that a late, pathological form of unwiring of the MB in the adult fly occurs in mutants that reduce the activity of the Cdk5 protein kinase (Trunova and Giniger, 2012). Cdk5 is a proline-directed, serine, threonine protein kinase. In sequence Cdk5 is closely related to the cyclin-dependent kinases (Cdks) that drive cell cycle progression, but it has no role in cell division (Dhavan and Tsai, 2001, Lai and Ip, 2009). Rather, Cdk5 is selectively active in postmitotic neurons where it controls cell motility, axon growth and guidance, subcellular compartmentalization of neurons and synaptic homeostasis. The Cdk5 catalytic subunit is expressed ubiquitously, but its kinase activity is strictly dependent on binding to a regulatory subunit called p35 (or, in mammals, its paralog, p39), and p35 expression is limited to postmitotic neurons (Tsai et al., 1994, ConnellCrowley et al., 2000). Remarkably, either gain or loss of function of Cdk5 causes neuronal cell death in cultured mammalian neurons (Tanaka et al., 2001), and adult-onset neurodegeneration in mouse models(Ohshima et al., 1996, Patrick et al., 1999, Takahashi et al., 2010), perhaps because of its central role in neuronal homeostasis(McLinden et al., 2012). Similarly, we found that a Drosophila mutant lacking Cdk5 activity displayed adultonset degeneration of MB projection neurons, characterized by axonal swellings, axonal and dendritic fragmentation, defective proteostasis, and frank tissue loss that was reflected in formation of ‘vacuoles’ in the MB calyx (Trunova and Giniger, 2012). Furthermore, others have shown that Cdk5 mediates photoreceptor degeneration in the Drosophila eye in response to ER stress(Kang et al., 2012).
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Given that loss of Cdk5 activity destabilizes the wiring of the adult MB, we wondered whether altered Cdk5 activity would also affect MB remodeling during development. We now show that Cdk5 autonomously modulates developmental remodeling of MB γ neurons in Drosophila. Reduction or absence of Cdk5 cell-autonomously retards dendritic and axonal remodeling of MB γ-neurons, while hyperactivation of Cdk5 accelerates remodeling, particularly in dendrites. Genetic experiments demonstrate that the effect of Cdk5 occurs after the ecdysone pulse, and cooperates with the action of the UPS. We further show that Cdk5 controls the earliest known step in the remodeling process, local dissolution of the microtubule cytoskeleton, but that its effects on microtubules alone are not sufficient to
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account completely for Cdk5-dependent regulation of remodeling. Our data therefore identify Cdk5 as a shared regulator of the maintenance of neural connections, acting in normal development as well as pathological degeneration.
Results Cdk5 activity modulates the timecourse of MB remodeling
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In the process of examining morphogenesis of MB γ–neurons in p35 mutants, single-cell analysis revealed an apparent delay in the time course of MB remodeling. We used FLP-ON UAS-mCD8-GFP under control of the γ-neuron specific GAL4 driver 201Y to express the membrane marker mCD8-GFP in single γ-neurons in either wild type (WT) or p35 null flies, and assayed neuronal morphology at various time points in early pupal development (Figure 1; sample sizes for this and all experiments are given in Table 1). In WT animals, consistent with previously published reports (Watts et al., 2003) we observed onset of axonal and dendritic “blebbing” and fragmentation at ∼6 hours after the onset of metamorphosis (after puparium formation, or APF; Fig 1E. See legend for complete description of phenotypic features), with complete loss of dendritic signal by 16 hours and of axonal signal by 20 hours (compare Fig 1M, 18h APF). In contrast, p35 mutants rarely exhibited blebbing and fragmentation at 6 hours (Fig 1F), rather, we typically did not observe extensive fragmentation of axons until 8-10 hours. Moreover, it appeared that the dissolution of embryonic dendrites did not go to completion in the mutants, as at 18 hours, when we saw the (expected) regrowth of dendrites with the appearance of mature, small-caliber adult processes (Fig 1N inset, blue arrowhead), we also still saw processes that resembled the thicker, immature, embryonic dendrites (Fig 1N inset, red arrow). Similar results were obtained when Cdk5 activity was reduced only in γ-neurons by cell-type specific expression of a dominant-negative Cdk5 derivative (Cdk5DN; Fig 1 G, K and O). Conversely, overexpression of p35 just in γ-neurons appeared to accelerate remodeling: compare the nearly complete absence of thick, larval dendrites in OEp35 at 12 h (Fig 1L) with the residual dendrites still present at this stage in the control (Fig 1 I, upper boxed region, red arrow). Moreover, in OEp35 we observed blebbed and fragmenting axons in some brains as early as 4 hours, when they would not be observed at all in wild type (data not shown).
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In order to confirm the apparent delay in axonal remodeling in flies with reduced Cdk5 activity, we expressed tubulin-GFP uniformly in γ–neurons of WT, p35 null mutants, and flies expressing a Cdk5 dominant-negative transgene in the MB, and quantified the time course of pruning compared to control (Figure 2). We also examined animals that overexpressed p35 (OE p35), and consequently had increased Cdk5 activity. To quantify pruning, we isolated control brains at various timepoints during early pupal development and categorized them into three phenotypic classes: those that displayed the typical degree of pruning for that timepoint (“average”), or pruning that was ‘faster’ (more complete) or ‘slower’ (less complete) than average. The “average” condition for each timepoint in our sample was consistent with the degree of pruning reported previously by Watts, et al. for that stage (Watts et al., 2003). (A detailed description of the criteria used for assigning brain lobes to these classes is provided in Experimental Procedures.) For illustrative purposes, examples of ‘fast’, ‘average’ and ‘slow’ control brains are shown for three timepoints (8h,
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12h and 16h APF) in Fig 2 A – C″. We then categorized experimental brains according to the same metric (quantified in Fig 2 D - I) to determine whether the experimental samples fell into the same frequency distribution as wild type or a different one, assessing significance by a chi-squared test that took into account data from the entire timecourse (see Experimental Procedures for details of the statistical analysis). Consistent with the singlecell analysis, we observed clear evidence for delayed and reduced axonal remodeling in p35 mutants (p = .012; chi squared), and a strong trend toward delayed remodeling in flies expressing Cdk5 dn (p=.056). In contrast, this assay did not detect acceleration of the rate of bulk axonal pruning by overexpression of p35 (p=.96). The phenotype of the p35 mutant was not significantly different from that of Cdk5 dn by this metric (p=.68)
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We next extended our analysis to examine dendritic remodeling in flies with or without altered Cdk5 activity (Figure 3). We expressed the membrane marker mCD8-mCherry in MB γ-neurons and measured calyx volume at different time points during remodeling (Fig 3A, C). In control flies, we saw clear reduction in mean calyx volume between ∼8-12 hours APF (quantified in Fig 3B). When we reduced p35 activity, however, we observed two distinct phenotypes (Fig 3 D, E; quantified in Fig 3B). First, consistent with the single-cell analysis, there was a delay in the time course of bulk shrinkage of the calyx, suggestive of a delay in the onset of remodeling, even though the rate of shrinkage once it began was approximately the same in mutant and wild type. Second, the total amount of calyx shrinkage by the end of the pruning period was somewhat less in the loss-of-function conditions than in wild type, consistent with the observation that dendrite dissolution appeared to be incomplete in single-cell labeling of p35 mutants (Fig 1N). Similar effects were also observed with a second membrane marker (mCD8-GFP; data not shown). Finally, we also quantified the effect of p35 overexpression on remodeling and found that shrinkage of the calyx was accelerated and enhanced relative to control flies, opposite to the effect observed in the loss-of-function mutants (Fig 3 F, B). These effects were confirmed by statistical analysis; at mid-remodeling (10hr APF), calyx volume in the p35 mutant is significantly greater than that of control (p= .02; one-tailed Mann-Whitney test) while OEp35 is significantly reduced (p = .04). The hypomorphic loss-of-function condition (Cdk5DN) also shows a trend toward reduced pruning, though it does not achieve formal statistical significance (p = .17). Cdk5 acts downstream of ecdysone in MB remodeling
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The overall process of pruning involves interactions among several cell types, including glia and phagocytic hemocytes, in addition to neurons(Watts et al., 2004, Williams and Truman, 2005, Hakim et al., 2014). Two lines of evidence suggest that the effects of Cdk5 are executed in neurons. First, we verified that p35 expression in adult flies is limited to neurons, as is the case in embryos. We have shown previously that fusion of a myc epitope tag to the C-terminus of p35 does not interfere with its activity (Connell-Crowley et al., 2000). We therefore expressed this fusion protein under control of the native p35 promoter, in the context of a genomic fragment that we have shown previously to rescue the p35 mutant phenotype(Connell-Crowley et al., 2000). Multiple-labeling of adult brains clearly demonstrated that p35-myc is expressed in all neurons. Expression of the tag above background levels was not observed in glial cells, or in any other non-neuronal cells in the
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preparation (Figure 4A). Second, in the phenotypic experiments documented above, expression of a dominant-negative Cdk5 transgene exclusively in neurons produced the same phenotype as did the null mutation (Fig 1 F, J vs G, K; Fig 2 E-I; Fig 3B, D vs E). Given that Cdk5 acts in neurons to control the timing of MB remodeling, we next set out to determine when Cdk5 acts within the remodeling pathway. MB remodeling is triggered by a pulse of the steroid hormone ecdysone. This transient increase in ecdysone signaling can be assayed by the switch it elicits in the identity of the EcR isoform that is expressed in γ– neurons(Lee et al., 2000). We therefore used anti-EcR-B1 antibodies to stain late third instar larvae that were WT, p35 mutant or that overexpress p35 in γ-neurons, and observed that EcR-B1 expression occurred prior to pruning in all three genotypes, as expected (Figure 4B -D). Thus, Cdk5 is not required for the ecdysone trigger.
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Cdk5 interacts genetically with the UPS in remodeling of MB axons
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MB remodeling can be delayed or blocked by inactivation of the ubiquitin-proteasome system (UPS), revealing it as a key component of the remodeling pathway. We therefore performed genetic interaction experiments that suggest that Cdk5 cooperates with the UPS in MB remodeling (Figure 5). Overexpression of the ubiquitin protease UBP2 suppresses proteasome activity(DiAntonio et al., 2001). We therefore reduced UPS activity in the MB by overproducing UBP2 under the control of 201Y-GAL4 and found that, under our conditions, this produces a partial block of the axonal remodeling of γ–neurons, with significant remnants of γ-neuron dorsal lobe axons remaining in the MB at the end of metamorphosis (Fig 5 B, E). As shown above, expression of dominant-negative Cdk5 modestly delays the onset of pruning but the process still progresses essentially to completion (Fig 2). However, when we combine expression of both UBP2 and Cdk5dn, we now see a complete or nearly complete block to remodeling in 100% of individuals, with little or no pruning of γ-neuron dorsal lobe axons (Fig 5 C, E). Conversely, overexpression of p35 partially suppressed the effect of overexpressed UBP2, leading to more complete pruning of MB axons (Figure 5 D, E). Unfortunately, it was not possible to assess interaction between Cdk5 and the UPS in dendritic pruning using this assay since the adult dendrites regrow into the space vacated by the larval dendrites. This gave us no way to assess unambiguously the terminal phenotype of dendritic remodeling under the different genetic conditions. Cdk5 acts prior to MT disassembly in MB remodeling
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One of the first steps in neurite removal during remodeling of MB γ–neurons is local dissolution of the microtubule (MT) cytoskeleton(Watts et al., 2003). Since Cdk5 is known to control the organization and function of the MT cytoskeleton in other contexts(McLinden et al., 2012), we investigated whether Cdk5 acts prior to changes in MT organization in remodeling MB neurons. We focused our attention on dendritic remodeling for three reasons. First, the timing of MT changes in MB dendrite remodeling had not previously been characterized in detail, second, our quantitative assay for calyx volume provided a sensitive assay for investigating the relative timing of remodeling events and third, we have shown previously that we can recapitulate developmental disassembly of MB dendrites in
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organotypic cultures (Prithviraj et al., 2012), providing a unique opportunity for mechanistic dissection of any Cdk5-associated changes in dendritic MTs.
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Two lines of evidence suggested that in dendrites, as in axons, MT dissolution precedes frank dendritic fragmentation in WT MB γ-neurons (Figure 6). We co-expressed the membrane marker mCD8-mCherry with β-tubulin-GFP in γ–neurons (Fig 6G). As remodeling proceeds, we begin to observe patches in the calyx in which tubulin-GFP signal disappears while mCD8-mCherry membrane signal persists (visualized as a local shift in the ratio of the signals in the red and green channels in fixed samples; Figure 6 G vs H). Moreover, we independently quantified the volume of the calyx based on the tubulin channel and the membrane channel in double-labeled samples. Prior to the onset of remodeling, the calyx volume is essentially the same regardless of which label is used (volumeCD8/volumetubulin =1.04+.01; mean + SEM, N = 8 MBs; Fig 6 I). However, as remodeling initiates, this ratio increases to 1.17 by 8hrs APF (p< .01, t-test), and to 1.33 by 10hrs (p≪.0001). As remodeling continues, membrane removal begins to “catch-up” with the loss of tubulin signal and the ratio again begins to equalize, though even at 16h APF, the volumeCD8/volumetubulin is still 1.17 (Fig 6 I).
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We next measured total MT volume in the calyx of flies with a p35 mutation and found a reduction in MT dissolution at mid-pruning (10h APF), much as we found for membrane volume (p=.01, one-tailed Mann-Whitney test; Figure 6B, D). Expression of dominant negative Cdk5 produced a slightly weaker effect in the same direction, though this measurement did not quite reach formal statistical significance (p=.06; Fig 6 B, E). Conversely, hyperactivating Cdk5 by overexpressing p35 enhanced MT dissolution (p=.04; Fig 6 B, F). These data suggested that Cdk5 acts at or prior to the destabilization of the MT cytoskeleton in dendritic remodeling. As expected, the effects of Cdk5 on the initiation and rate of remodeling are also reflected in the membrane:tubulin volume ratio (Fig 6 I). Thus, in Cdk5 loss-of-function genotypes the increase in mCD8/tubulin volume ratio is delayed, not peaking until ∼12h APF; in OEp35 the pruning process reaches completion sooner with the ratio restored almost to baseline by 12h APF.
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Finally, we wished to test whether the effects of Cdk5 on remodeling are mediated entirely through its effects on MT stability, or whether Cdk5 acts at multiple steps in the process. We reasoned that if Cdk5 regulates remodeling solely by destabilizing MTs, then direct pharmacological destabilization of MTs should have the same effect as activating Cdk5. We therefore took advantage of our system for MB remodeling in culture (Prithviraj et al., 2012) to perform an epistasis experiment to interrogate the interaction between Cdk5 activity and direct, pharmacological destabilization of microtubules. We isolated brains from early pupal flies (1.5 – 3h APF) that were either WT, or had gain or loss of function of p35, and cultured them for ∼10 hours in the presence or absence of the microtubule destabilizing agent vinblastine (see Experimental Procedures for details). We then fixed the brains and measured the ratio of calyx volume as assayed with mCD8-mCherry (membrane-limited) vs that with tubulin-GFP (MT-limited) in each brain, using the same method applied in the in vivo experiment of Fig 6, above. Control experiments show that dendritic remodeling is delayed upon Cdk5 inactivation in cultured brains, just as in vivo (RP and EG, unpublished observations). Dev Dyn. Author manuscript; available in PMC 2016 December 01.
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While increase or decrease of Cdk5 activity changed the latency to MB remodeling (Figures 3 and 6, above), it had no effect on the ratio of membrane:MT volume in snapshots taken relatively late during MB pruning ex vivo: both reporters were affected equally and the ratio of volumeCD8/volumetubulin was not changed (Figure 7). In contrast, treatment of wild type brains with vinblastine increases that ratio, in other words, it increases microtubule loss relative to overall dendrite loss at that time point (p < .02; t-test). Moreover, when a treatment that delays remodeling (p35 mutant or Cdk5dn) was combined with one that destabilizes MTs directly (vinblastine), the membrane:MT volume ratio was the same as that in wild type brains treated with vinblastine alone. In contrast, when we both enhanced MT dissolution by application of vinblastine, and accelerated pruning by overexpression of p35, the combination now allowed membrane loss to keep pace with MT removal, resulting in a significant decrease in the membrane:MT ratio at the time of assay (volumeCD8/ volumetubulin = 1.04+.01, mean vs SEM; p 20 MB for all genotypes; see Table 1 for details). Statistical significance was assessed by chi-square, comparing the occupancy of the 3 bins (normal pruning, partial pruning, no pruning) for each genotypic comparison. Genotypes: Control: w;201Y-Gal4,UAS-mCD8-GFP/+ UBP2: w;201Y-Gal4,UAS-mCD8-GFP/ UAS-UBP2/+ Cdk5DN: w;201Y-Gal4,UAS-mCD8-GFP/UAS-UBP2; UAS-Cdk5-DN-FLAG/+ OEp35: w;201Y-Gal4,UAS-mCD8-GFP/UAS-UBP2; UAS-p35-myc/+
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Fig 6. Time course of tubulin dissolution during calyx remodeling in flies with altered Cdk5 activity
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Tubulin-GFP and mCD8-mCherry were expressed in γ-neurons of flies of the indicated genotypes. Brains were dissected at the indicated times, fixed and visualized by confocal microscopy. (A), (C – F) Fluorescent images showing tubulin-GFP signal in MB of the indicated genotypes and ages. In each pair of images, the upper panel is a maximum intensity projection of the entire MB, with the calyx highlighted by dotted, blue outline; lower panel is a single optical section taken through the center of the MB. (B) The calyx was manually outlined in optical sections, calyx volume calculated and normalized to the pre-pruning average, as for the experiment of Fig 3. Graph displays the change in average calyx volume at different times; error bars show SEM. Note the delay and reduction of pruning in Cdk5 loss-of-function conditions, and the acceleration/increase of pruning in p35 overexpression. Note: tubulin quantification in this experiment employed the same samples for which membrane quantification was presented in Fig 3, but analysis was done independently. (G, H) Separated mCD8-mCherry and tubulin-GFP channels, and overlay, in wild type brain at WPP stage (before pruning), and 10h APF (during pruning). Note near-perfect overlap of
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membrane and tubulin signal prior to pruning, but emergence of red tissue (membrane+, tubulin-; white arrows) in calyx as pruning proceeds. (I) The ratio of the calyx volume as measured with mCD8-mCherry/tubulin-GFP was calculated for each MB, averaged for each timepoint and plotted as a function of age for each indicated genotype. In all genotypes, both markers give the same absolute volume before pruning (ratio ∼1), then the ratio rises as pruning initiates with MT dissolution ∼ 6-8h APF, and then declines back toward baseline as membrane fragmentation and removal occur at later stages (∼12 – 18h APF). Error bars indicate SEM.
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Author Manuscript Author Manuscript Fig 7. Interaction of altered Cdk5 with tubulin destabilization during pruning in culture
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WPP of the indicated genotypes were generated that expressed both β–tubulin-GFP and mCD8-mCherry in MB gamma neurons. Brains were explanted and allowed to undergo remodeling in culture for 10 hours in growth medium with or without the MT destabilizing drug, vinblastine, fixed, and examined by confocal microscopy. Calyx volume was measured separately using each probe, and a ratio determined for residual membrane volume vs residual dendritic MT volume. The Cdk5/p35 genotype did not affect the ratio of membrane:MT volume: neither gain nor loss of function produced a statistically significant change in this ratio (p ≫ .05). In contrast, vinblastine treatment preferentially destabilized MTs leading to an increase in the membrane:MT ratio both in wild type and in flies with reduced Cdk5 activity. Surprisingly, combining p35 overexpression with vinblastine treatment led to a significant decrease in the membrane:MT ratio.
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Table 1
Sample sizes for all experiments
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The number of samples examined is indicated for each genotype in each experiment included in this study. See individual figures for experimental details. Control
p35 mutant
Cdk5 DN
OE p35
WPP
12
15
7
10
6h APF
26
17
13
19
12h
14
14
7
9
18h
16
10
12
5
6h
13
6
14
12
8h
25
24
7
20
10h
16
21
11
15
12h
12
15
7
7
16h
9
8
8
4
20h
10
22
5
6
WPP
8
8
8
10
4h
4
7
7
7
6h
10
4
11
10
8h
10
6
3
6
10h
7
9
6
9
12h
9
11
7
6
16h
7
6
6
4
A
9
N/A
N/A
N/A
B-D
9
9
N/A
9
Fig 5 (MB scored)
control
UBP2
UBP2 + Cdk5 DN
UBP2 + OE p35
48h APF
22
39
29
39
Fig 7 (MB quantified)
Control
p35 mutant
Cdk5 DN
OE p35
w/o drug
10
11
8
11
+ drug
8
12
8
11
Fig 1 (hemispheres with small clones)
Fig 2 (MB scored)
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Fig 3 and 6 (MB quantified)
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Fig 4 (Brains examined)
Fig 6 (see above)
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Dev Dyn. Author manuscript; available in PMC 2016 December 01. Published in final edited form as: Dev Dyn. 2015 December ; 244(12): 1550–1563. doi:10.1002/dvdy.24350.
Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila Svetlana Smith-Trunova1, Ranjini Prithviraj1, Joshua Spurrier1,2, Irina Kuzina1, Qun Gu1, and Edward Giniger1,* 1National
Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg 35, Rm 1C-1002, 35 Convent Dr. Bethesda, MD 20892 USA
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2The
Johns Hopkins University/National Institutes of Health Graduate Partnership Program, National Institutes of Health, Bethesda, MD 20892
Abstract Background—During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors.
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Results—We show here that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. We further show that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, we show that Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and provide evidence that it also acts at additional steps of MB pruning. Conclusions—These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5 we suggest that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, we speculate that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it employs during developmental remodeling. Keywords Axon; dendrite; pruning; microtubules; proteasome; neurodegeneration
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Introduction The structure of the fully formed nervous system depends not just on the construction of neural wiring but also on its dissolution. Neural connections are disassembled through activity-dependent plasticity, through pathological processes including injury and neurodegeneration and also through developmentally-programmed remodeling. Two central issues in the field of neural wiring, therefore, are to identify the molecular mechanisms that disassemble neural connectivity, and to determine whether they are shared by different
*
Corresponding Author: Tel: 301-451-3890, Fax: 301-451-5368, [email protected].
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forms of neural disassembly, or are unique to individual physiological processes (Coleman, 2005, Hoopfer et al., 2006, Tao and Rolls, 2011).
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Developmentally programmed axonal and dendritic remodeling is widespread and is essential to establish neural wiring. In Drosophila, for example, one observes large-scale remodeling of the axonal and dendritic projections of various sensory neurons, motoneurons and central brain interneurons, particularly during the pupal stage of development (Truman, 1990, Watts et al., 2003, Williams et al., 2006). Developmentally programmed remodeling is not limited to species that undergo metamorphosis, however. In vertebrates, the axons of retinal ganglion cells largely overshoot their target zones in the optic tectum or superior colliculus, and topographic mapping is often achieved by programmed remodeling(Yates et al., 2001). This involves back-branching of collaterals to target appropriate A/P positions, and subsequent activity-dependent refinement. An even more striking example is provided by layer 5 projection neurons of the mammalian neocortex. Neurons from both visual and motor areas initially send out axons that branch to multiple regions including the midbrain and spinal cord, with the inappropriate projections pruned away later in development (O'Leary and Koester, 1993).
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On the surface, developmentally programmed remodeling seems to bear similarities to pathological degeneration processes that occur after injury, and in neurodegenerative disease(Luo and O'Leary, 2005, Saxena and Caroni, 2007). For example, expression of the Wlds gene product blocks neurite removal in a variety of experimental paradigms, both in the context of developmental remodeling and after injury or disease (Wang et al., 2012). Similarly, a requirement for the ubiquitin-proteasome system (UPS) is a common feature of many varieties of neurite dissolution (Watts et al., 2003, Zhai et al., 2003, MacInnis and Campenot, 2005, Tao and Rolls, 2011). In contrast, while non-apoptotic activation of effector caspases is an essential element of developmental dendrite disassembly for Class IV sensory neurons of Drosophila (Williams et al., 2006), it is not required for other forms of developmental remodeling, or even for post-injury dissolution of the dendrites of the very same neurons (Tao and Rolls, 2011). Overall, the picture that emerges is that the remodeling process in any specific setting can draw from among an assortment of mechanisms that are collectively competent to perform remodeling, such that some processes will be shared in different cases, and others may act in only a subset of contexts. The rules that determine which mechanisms act in which case remain obscure, but are essential knowledge if one is to predict which mechanism should be activated or repressed to cause or block remodeling in a specific context.
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One outstanding system for investigating CNS remodeling is the Drosophila mushroom body (MB) (Lee et al., 1999, Watts et al., 2003, Yu and Schuldiner, 2014). The mushroom body is the seat of learning and memory in the fly, as well as in other insects (Davis, 2005). Mushroom body projection neurons fall into 3 main classes, α, β and γ (Lee et al., 1999). The γ-neurons are born first, during embryonic and larval development, and carry out MB functions during larval life. Since the lifestyle of the fly changes dramatically between larval and adult stages, however, the MB needs to be substantially rewired at metamorphosis. Consequently, both the axonal and dendritic arbors of the larval MB neurons are disassembled during early pupal development, and regrow in an adult-specific pattern. This
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programmed disassembly of MB γ-neurons has been studied extensively as a model of developmental remodeling. Remodeling is triggered ∼6 hours after the onset of metamorphosis (called “pupariation”) by an organism-wide spike of the steroid hormone ecdysone, which triggers (among other things) a switch in the expression of ecdysone receptor (EcR) isoforms to induce EcR-B in remodeling neurons (Lee et al., 2000). The first overt sign of neuritic disassembly is dissolution of the microtubule cytoskeleton in axons followed by frank fragmentation of dendrites and axons (Watts et al., 2003). Occurrence of fragmentation requires activity of the ubiquitin-proteasome system (UPS), as inhibition of the UPS blocks fragmentation and remodeling (Watts et al., 2003). The fragments are then phagocytosed by glia, phagocytic hemocytes and neighboring neurons (Watts et al., 2004, Williams and Truman, 2005, Hakim et al., 2014, Tasdemir-Yilmaz and Freeman, 2014). However, while these broad outlines of the remodeling process have been described, the detailed molecular machinery that carries out each of these processes, and that coordinates these disparate molecular activities, remains unknown.
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We have shown previously that a late, pathological form of unwiring of the MB in the adult fly occurs in mutants that reduce the activity of the Cdk5 protein kinase (Trunova and Giniger, 2012). Cdk5 is a proline-directed, serine, threonine protein kinase. In sequence Cdk5 is closely related to the cyclin-dependent kinases (Cdks) that drive cell cycle progression, but it has no role in cell division (Dhavan and Tsai, 2001, Lai and Ip, 2009). Rather, Cdk5 is selectively active in postmitotic neurons where it controls cell motility, axon growth and guidance, subcellular compartmentalization of neurons and synaptic homeostasis. The Cdk5 catalytic subunit is expressed ubiquitously, but its kinase activity is strictly dependent on binding to a regulatory subunit called p35 (or, in mammals, its paralog, p39), and p35 expression is limited to postmitotic neurons (Tsai et al., 1994, ConnellCrowley et al., 2000). Remarkably, either gain or loss of function of Cdk5 causes neuronal cell death in cultured mammalian neurons (Tanaka et al., 2001), and adult-onset neurodegeneration in mouse models(Ohshima et al., 1996, Patrick et al., 1999, Takahashi et al., 2010), perhaps because of its central role in neuronal homeostasis(McLinden et al., 2012). Similarly, we found that a Drosophila mutant lacking Cdk5 activity displayed adultonset degeneration of MB projection neurons, characterized by axonal swellings, axonal and dendritic fragmentation, defective proteostasis, and frank tissue loss that was reflected in formation of ‘vacuoles’ in the MB calyx (Trunova and Giniger, 2012). Furthermore, others have shown that Cdk5 mediates photoreceptor degeneration in the Drosophila eye in response to ER stress(Kang et al., 2012).
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Given that loss of Cdk5 activity destabilizes the wiring of the adult MB, we wondered whether altered Cdk5 activity would also affect MB remodeling during development. We now show that Cdk5 autonomously modulates developmental remodeling of MB γ neurons in Drosophila. Reduction or absence of Cdk5 cell-autonomously retards dendritic and axonal remodeling of MB γ-neurons, while hyperactivation of Cdk5 accelerates remodeling, particularly in dendrites. Genetic experiments demonstrate that the effect of Cdk5 occurs after the ecdysone pulse, and cooperates with the action of the UPS. We further show that Cdk5 controls the earliest known step in the remodeling process, local dissolution of the microtubule cytoskeleton, but that its effects on microtubules alone are not sufficient to
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account completely for Cdk5-dependent regulation of remodeling. Our data therefore identify Cdk5 as a shared regulator of the maintenance of neural connections, acting in normal development as well as pathological degeneration.
Results Cdk5 activity modulates the timecourse of MB remodeling
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In the process of examining morphogenesis of MB γ–neurons in p35 mutants, single-cell analysis revealed an apparent delay in the time course of MB remodeling. We used FLP-ON UAS-mCD8-GFP under control of the γ-neuron specific GAL4 driver 201Y to express the membrane marker mCD8-GFP in single γ-neurons in either wild type (WT) or p35 null flies, and assayed neuronal morphology at various time points in early pupal development (Figure 1; sample sizes for this and all experiments are given in Table 1). In WT animals, consistent with previously published reports (Watts et al., 2003) we observed onset of axonal and dendritic “blebbing” and fragmentation at ∼6 hours after the onset of metamorphosis (after puparium formation, or APF; Fig 1E. See legend for complete description of phenotypic features), with complete loss of dendritic signal by 16 hours and of axonal signal by 20 hours (compare Fig 1M, 18h APF). In contrast, p35 mutants rarely exhibited blebbing and fragmentation at 6 hours (Fig 1F), rather, we typically did not observe extensive fragmentation of axons until 8-10 hours. Moreover, it appeared that the dissolution of embryonic dendrites did not go to completion in the mutants, as at 18 hours, when we saw the (expected) regrowth of dendrites with the appearance of mature, small-caliber adult processes (Fig 1N inset, blue arrowhead), we also still saw processes that resembled the thicker, immature, embryonic dendrites (Fig 1N inset, red arrow). Similar results were obtained when Cdk5 activity was reduced only in γ-neurons by cell-type specific expression of a dominant-negative Cdk5 derivative (Cdk5DN; Fig 1 G, K and O). Conversely, overexpression of p35 just in γ-neurons appeared to accelerate remodeling: compare the nearly complete absence of thick, larval dendrites in OEp35 at 12 h (Fig 1L) with the residual dendrites still present at this stage in the control (Fig 1 I, upper boxed region, red arrow). Moreover, in OEp35 we observed blebbed and fragmenting axons in some brains as early as 4 hours, when they would not be observed at all in wild type (data not shown).
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In order to confirm the apparent delay in axonal remodeling in flies with reduced Cdk5 activity, we expressed tubulin-GFP uniformly in γ–neurons of WT, p35 null mutants, and flies expressing a Cdk5 dominant-negative transgene in the MB, and quantified the time course of pruning compared to control (Figure 2). We also examined animals that overexpressed p35 (OE p35), and consequently had increased Cdk5 activity. To quantify pruning, we isolated control brains at various timepoints during early pupal development and categorized them into three phenotypic classes: those that displayed the typical degree of pruning for that timepoint (“average”), or pruning that was ‘faster’ (more complete) or ‘slower’ (less complete) than average. The “average” condition for each timepoint in our sample was consistent with the degree of pruning reported previously by Watts, et al. for that stage (Watts et al., 2003). (A detailed description of the criteria used for assigning brain lobes to these classes is provided in Experimental Procedures.) For illustrative purposes, examples of ‘fast’, ‘average’ and ‘slow’ control brains are shown for three timepoints (8h,
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12h and 16h APF) in Fig 2 A – C″. We then categorized experimental brains according to the same metric (quantified in Fig 2 D - I) to determine whether the experimental samples fell into the same frequency distribution as wild type or a different one, assessing significance by a chi-squared test that took into account data from the entire timecourse (see Experimental Procedures for details of the statistical analysis). Consistent with the singlecell analysis, we observed clear evidence for delayed and reduced axonal remodeling in p35 mutants (p = .012; chi squared), and a strong trend toward delayed remodeling in flies expressing Cdk5 dn (p=.056). In contrast, this assay did not detect acceleration of the rate of bulk axonal pruning by overexpression of p35 (p=.96). The phenotype of the p35 mutant was not significantly different from that of Cdk5 dn by this metric (p=.68)
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We next extended our analysis to examine dendritic remodeling in flies with or without altered Cdk5 activity (Figure 3). We expressed the membrane marker mCD8-mCherry in MB γ-neurons and measured calyx volume at different time points during remodeling (Fig 3A, C). In control flies, we saw clear reduction in mean calyx volume between ∼8-12 hours APF (quantified in Fig 3B). When we reduced p35 activity, however, we observed two distinct phenotypes (Fig 3 D, E; quantified in Fig 3B). First, consistent with the single-cell analysis, there was a delay in the time course of bulk shrinkage of the calyx, suggestive of a delay in the onset of remodeling, even though the rate of shrinkage once it began was approximately the same in mutant and wild type. Second, the total amount of calyx shrinkage by the end of the pruning period was somewhat less in the loss-of-function conditions than in wild type, consistent with the observation that dendrite dissolution appeared to be incomplete in single-cell labeling of p35 mutants (Fig 1N). Similar effects were also observed with a second membrane marker (mCD8-GFP; data not shown). Finally, we also quantified the effect of p35 overexpression on remodeling and found that shrinkage of the calyx was accelerated and enhanced relative to control flies, opposite to the effect observed in the loss-of-function mutants (Fig 3 F, B). These effects were confirmed by statistical analysis; at mid-remodeling (10hr APF), calyx volume in the p35 mutant is significantly greater than that of control (p= .02; one-tailed Mann-Whitney test) while OEp35 is significantly reduced (p = .04). The hypomorphic loss-of-function condition (Cdk5DN) also shows a trend toward reduced pruning, though it does not achieve formal statistical significance (p = .17). Cdk5 acts downstream of ecdysone in MB remodeling
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The overall process of pruning involves interactions among several cell types, including glia and phagocytic hemocytes, in addition to neurons(Watts et al., 2004, Williams and Truman, 2005, Hakim et al., 2014). Two lines of evidence suggest that the effects of Cdk5 are executed in neurons. First, we verified that p35 expression in adult flies is limited to neurons, as is the case in embryos. We have shown previously that fusion of a myc epitope tag to the C-terminus of p35 does not interfere with its activity (Connell-Crowley et al., 2000). We therefore expressed this fusion protein under control of the native p35 promoter, in the context of a genomic fragment that we have shown previously to rescue the p35 mutant phenotype(Connell-Crowley et al., 2000). Multiple-labeling of adult brains clearly demonstrated that p35-myc is expressed in all neurons. Expression of the tag above background levels was not observed in glial cells, or in any other non-neuronal cells in the
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preparation (Figure 4A). Second, in the phenotypic experiments documented above, expression of a dominant-negative Cdk5 transgene exclusively in neurons produced the same phenotype as did the null mutation (Fig 1 F, J vs G, K; Fig 2 E-I; Fig 3B, D vs E). Given that Cdk5 acts in neurons to control the timing of MB remodeling, we next set out to determine when Cdk5 acts within the remodeling pathway. MB remodeling is triggered by a pulse of the steroid hormone ecdysone. This transient increase in ecdysone signaling can be assayed by the switch it elicits in the identity of the EcR isoform that is expressed in γ– neurons(Lee et al., 2000). We therefore used anti-EcR-B1 antibodies to stain late third instar larvae that were WT, p35 mutant or that overexpress p35 in γ-neurons, and observed that EcR-B1 expression occurred prior to pruning in all three genotypes, as expected (Figure 4B -D). Thus, Cdk5 is not required for the ecdysone trigger.
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Cdk5 interacts genetically with the UPS in remodeling of MB axons
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MB remodeling can be delayed or blocked by inactivation of the ubiquitin-proteasome system (UPS), revealing it as a key component of the remodeling pathway. We therefore performed genetic interaction experiments that suggest that Cdk5 cooperates with the UPS in MB remodeling (Figure 5). Overexpression of the ubiquitin protease UBP2 suppresses proteasome activity(DiAntonio et al., 2001). We therefore reduced UPS activity in the MB by overproducing UBP2 under the control of 201Y-GAL4 and found that, under our conditions, this produces a partial block of the axonal remodeling of γ–neurons, with significant remnants of γ-neuron dorsal lobe axons remaining in the MB at the end of metamorphosis (Fig 5 B, E). As shown above, expression of dominant-negative Cdk5 modestly delays the onset of pruning but the process still progresses essentially to completion (Fig 2). However, when we combine expression of both UBP2 and Cdk5dn, we now see a complete or nearly complete block to remodeling in 100% of individuals, with little or no pruning of γ-neuron dorsal lobe axons (Fig 5 C, E). Conversely, overexpression of p35 partially suppressed the effect of overexpressed UBP2, leading to more complete pruning of MB axons (Figure 5 D, E). Unfortunately, it was not possible to assess interaction between Cdk5 and the UPS in dendritic pruning using this assay since the adult dendrites regrow into the space vacated by the larval dendrites. This gave us no way to assess unambiguously the terminal phenotype of dendritic remodeling under the different genetic conditions. Cdk5 acts prior to MT disassembly in MB remodeling
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One of the first steps in neurite removal during remodeling of MB γ–neurons is local dissolution of the microtubule (MT) cytoskeleton(Watts et al., 2003). Since Cdk5 is known to control the organization and function of the MT cytoskeleton in other contexts(McLinden et al., 2012), we investigated whether Cdk5 acts prior to changes in MT organization in remodeling MB neurons. We focused our attention on dendritic remodeling for three reasons. First, the timing of MT changes in MB dendrite remodeling had not previously been characterized in detail, second, our quantitative assay for calyx volume provided a sensitive assay for investigating the relative timing of remodeling events and third, we have shown previously that we can recapitulate developmental disassembly of MB dendrites in
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organotypic cultures (Prithviraj et al., 2012), providing a unique opportunity for mechanistic dissection of any Cdk5-associated changes in dendritic MTs.
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Two lines of evidence suggested that in dendrites, as in axons, MT dissolution precedes frank dendritic fragmentation in WT MB γ-neurons (Figure 6). We co-expressed the membrane marker mCD8-mCherry with β-tubulin-GFP in γ–neurons (Fig 6G). As remodeling proceeds, we begin to observe patches in the calyx in which tubulin-GFP signal disappears while mCD8-mCherry membrane signal persists (visualized as a local shift in the ratio of the signals in the red and green channels in fixed samples; Figure 6 G vs H). Moreover, we independently quantified the volume of the calyx based on the tubulin channel and the membrane channel in double-labeled samples. Prior to the onset of remodeling, the calyx volume is essentially the same regardless of which label is used (volumeCD8/volumetubulin =1.04+.01; mean + SEM, N = 8 MBs; Fig 6 I). However, as remodeling initiates, this ratio increases to 1.17 by 8hrs APF (p< .01, t-test), and to 1.33 by 10hrs (p≪.0001). As remodeling continues, membrane removal begins to “catch-up” with the loss of tubulin signal and the ratio again begins to equalize, though even at 16h APF, the volumeCD8/volumetubulin is still 1.17 (Fig 6 I).
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We next measured total MT volume in the calyx of flies with a p35 mutation and found a reduction in MT dissolution at mid-pruning (10h APF), much as we found for membrane volume (p=.01, one-tailed Mann-Whitney test; Figure 6B, D). Expression of dominant negative Cdk5 produced a slightly weaker effect in the same direction, though this measurement did not quite reach formal statistical significance (p=.06; Fig 6 B, E). Conversely, hyperactivating Cdk5 by overexpressing p35 enhanced MT dissolution (p=.04; Fig 6 B, F). These data suggested that Cdk5 acts at or prior to the destabilization of the MT cytoskeleton in dendritic remodeling. As expected, the effects of Cdk5 on the initiation and rate of remodeling are also reflected in the membrane:tubulin volume ratio (Fig 6 I). Thus, in Cdk5 loss-of-function genotypes the increase in mCD8/tubulin volume ratio is delayed, not peaking until ∼12h APF; in OEp35 the pruning process reaches completion sooner with the ratio restored almost to baseline by 12h APF.
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Finally, we wished to test whether the effects of Cdk5 on remodeling are mediated entirely through its effects on MT stability, or whether Cdk5 acts at multiple steps in the process. We reasoned that if Cdk5 regulates remodeling solely by destabilizing MTs, then direct pharmacological destabilization of MTs should have the same effect as activating Cdk5. We therefore took advantage of our system for MB remodeling in culture (Prithviraj et al., 2012) to perform an epistasis experiment to interrogate the interaction between Cdk5 activity and direct, pharmacological destabilization of microtubules. We isolated brains from early pupal flies (1.5 – 3h APF) that were either WT, or had gain or loss of function of p35, and cultured them for ∼10 hours in the presence or absence of the microtubule destabilizing agent vinblastine (see Experimental Procedures for details). We then fixed the brains and measured the ratio of calyx volume as assayed with mCD8-mCherry (membrane-limited) vs that with tubulin-GFP (MT-limited) in each brain, using the same method applied in the in vivo experiment of Fig 6, above. Control experiments show that dendritic remodeling is delayed upon Cdk5 inactivation in cultured brains, just as in vivo (RP and EG, unpublished observations). Dev Dyn. Author manuscript; available in PMC 2016 December 01.
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While increase or decrease of Cdk5 activity changed the latency to MB remodeling (Figures 3 and 6, above), it had no effect on the ratio of membrane:MT volume in snapshots taken relatively late during MB pruning ex vivo: both reporters were affected equally and the ratio of volumeCD8/volumetubulin was not changed (Figure 7). In contrast, treatment of wild type brains with vinblastine increases that ratio, in other words, it increases microtubule loss relative to overall dendrite loss at that time point (p < .02; t-test). Moreover, when a treatment that delays remodeling (p35 mutant or Cdk5dn) was combined with one that destabilizes MTs directly (vinblastine), the membrane:MT volume ratio was the same as that in wild type brains treated with vinblastine alone. In contrast, when we both enhanced MT dissolution by application of vinblastine, and accelerated pruning by overexpression of p35, the combination now allowed membrane loss to keep pace with MT removal, resulting in a significant decrease in the membrane:MT ratio at the time of assay (volumeCD8/ volumetubulin = 1.04+.01, mean vs SEM; p 20 MB for all genotypes; see Table 1 for details). Statistical significance was assessed by chi-square, comparing the occupancy of the 3 bins (normal pruning, partial pruning, no pruning) for each genotypic comparison. Genotypes: Control: w;201Y-Gal4,UAS-mCD8-GFP/+ UBP2: w;201Y-Gal4,UAS-mCD8-GFP/ UAS-UBP2/+ Cdk5DN: w;201Y-Gal4,UAS-mCD8-GFP/UAS-UBP2; UAS-Cdk5-DN-FLAG/+ OEp35: w;201Y-Gal4,UAS-mCD8-GFP/UAS-UBP2; UAS-p35-myc/+
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Fig 6. Time course of tubulin dissolution during calyx remodeling in flies with altered Cdk5 activity
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Tubulin-GFP and mCD8-mCherry were expressed in γ-neurons of flies of the indicated genotypes. Brains were dissected at the indicated times, fixed and visualized by confocal microscopy. (A), (C – F) Fluorescent images showing tubulin-GFP signal in MB of the indicated genotypes and ages. In each pair of images, the upper panel is a maximum intensity projection of the entire MB, with the calyx highlighted by dotted, blue outline; lower panel is a single optical section taken through the center of the MB. (B) The calyx was manually outlined in optical sections, calyx volume calculated and normalized to the pre-pruning average, as for the experiment of Fig 3. Graph displays the change in average calyx volume at different times; error bars show SEM. Note the delay and reduction of pruning in Cdk5 loss-of-function conditions, and the acceleration/increase of pruning in p35 overexpression. Note: tubulin quantification in this experiment employed the same samples for which membrane quantification was presented in Fig 3, but analysis was done independently. (G, H) Separated mCD8-mCherry and tubulin-GFP channels, and overlay, in wild type brain at WPP stage (before pruning), and 10h APF (during pruning). Note near-perfect overlap of
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membrane and tubulin signal prior to pruning, but emergence of red tissue (membrane+, tubulin-; white arrows) in calyx as pruning proceeds. (I) The ratio of the calyx volume as measured with mCD8-mCherry/tubulin-GFP was calculated for each MB, averaged for each timepoint and plotted as a function of age for each indicated genotype. In all genotypes, both markers give the same absolute volume before pruning (ratio ∼1), then the ratio rises as pruning initiates with MT dissolution ∼ 6-8h APF, and then declines back toward baseline as membrane fragmentation and removal occur at later stages (∼12 – 18h APF). Error bars indicate SEM.
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Author Manuscript Author Manuscript Fig 7. Interaction of altered Cdk5 with tubulin destabilization during pruning in culture
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WPP of the indicated genotypes were generated that expressed both β–tubulin-GFP and mCD8-mCherry in MB gamma neurons. Brains were explanted and allowed to undergo remodeling in culture for 10 hours in growth medium with or without the MT destabilizing drug, vinblastine, fixed, and examined by confocal microscopy. Calyx volume was measured separately using each probe, and a ratio determined for residual membrane volume vs residual dendritic MT volume. The Cdk5/p35 genotype did not affect the ratio of membrane:MT volume: neither gain nor loss of function produced a statistically significant change in this ratio (p ≫ .05). In contrast, vinblastine treatment preferentially destabilized MTs leading to an increase in the membrane:MT ratio both in wild type and in flies with reduced Cdk5 activity. Surprisingly, combining p35 overexpression with vinblastine treatment led to a significant decrease in the membrane:MT ratio.
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Table 1
Sample sizes for all experiments
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The number of samples examined is indicated for each genotype in each experiment included in this study. See individual figures for experimental details. Control
p35 mutant
Cdk5 DN
OE p35
WPP
12
15
7
10
6h APF
26
17
13
19
12h
14
14
7
9
18h
16
10
12
5
6h
13
6
14
12
8h
25
24
7
20
10h
16
21
11
15
12h
12
15
7
7
16h
9
8
8
4
20h
10
22
5
6
WPP
8
8
8
10
4h
4
7
7
7
6h
10
4
11
10
8h
10
6
3
6
10h
7
9
6
9
12h
9
11
7
6
16h
7
6
6
4
A
9
N/A
N/A
N/A
B-D
9
9
N/A
9
Fig 5 (MB scored)
control
UBP2
UBP2 + Cdk5 DN
UBP2 + OE p35
48h APF
22
39
29
39
Fig 7 (MB quantified)
Control
p35 mutant
Cdk5 DN
OE p35
w/o drug
10
11
8
11
+ drug
8
12
8
11
Fig 1 (hemispheres with small clones)
Fig 2 (MB scored)
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Fig 3 and 6 (MB quantified)
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Fig 4 (Brains examined)
Fig 6 (see above)
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