Characterization of axonal transport defects inDrosophilaHuntingtin mutants

Abstract

Polyglutamine (polyQ) expansion within Huntingtin (Htt) causes the fatal neurodegenerative disorder Huntington’s Disease (HD). Although Htt is ubiquitously expressed and conserved from Drosophila to humans, its normal biological function is still being elucidated. Here we characterize a role for the Drosophila Htt homolog (dHtt) in fast axonal transport (FAT). Generation and expression of transgenic dHtt-mRFP and human Htt-mRFP fusion proteins in Drosophila revealed co-localization with mitochondria and synaptic vesicles undergoing FAT. However, Htt was not ubiquitously associated with the transport machinery, as it was excluded from dense-core vesicles and APLIP1 containing vesicles. Quantification of cargo movement in dHtt deficient axons revealed that mitochondria and synaptic vesicles show a decrease in the distance and duration of transport, and an increase in the number of pauses. In addition, the ratio of retrograde to anterograde flux was increased in mutant animals. Densecore vesicles did not display similar defects in processivity, but did show altered retrograde to anterograde flux along axons. Given the co-localization with mitochondria and synaptic vesicles, but not dense-core vesicles, the data suggest dHtt likely acts locally at cargo interaction sites to regulate processivity. An increase in dynein heavy chain expression was also observed in dHtt mutants, suggesting that the altered flux observed for all cargo may represent secondary transport changes occurring independent of dHtt’s primary function. Expression of dHtt in a milton (HAP1) mutant background revealed that the protein does not require mitochondria or HAP1 to localize along axons, suggesting Htt has an independent mechanism for coupling with motors to regulate their processivity during axonal transport.

Loss of Huntingtin stimulates capture of retrograde dense-core vesicles to increase synaptic neuropeptide stores

“…1B). Thus, data from the type Is motoneuron terminal differ from findings previously obtained in axons of CCAP-containing peptidergic neurons (Weiss and Littleton, 2016). Furthermore, tracking of DCVs through photobleached proximal boutons showed that Htt KO does not affect DCV transport velocity: for anterograde transport, DCVs moved at 0.89 ± 0.09 μm/s in controls and 0.93 ± 0.10 μm/s in Htt KOs, while for retrograde transport, DCVs moved at 0.81 ± 0.07 μm/s in controls and 0.83 ± 0.08 μm/s in Htt KOs (Control, n = 13; Htt KO, n = 16).…”

“…In addition, Htt scaffolds GAPDH (glyceraldhyde 3-phosphate dehydrogenase) on vesicles and Htt depletion induces detachment of GAPDH from vesicles leading to decreased axonal transport of BDNF and APP (Zala et al, 2013b). Finally, Htt may induce secondary effects on axonal transport by regulating dynein heavy chain expression (Weiss and Littleton, 2016). …”

“…Therefore, Drosophila melanogaster is a powerful model system to study Htt function under physiological and pathological conditions. Recent observations revealed that knockout (KO) of Drosophila Htt leads to subtle changes in axonal transport of dense-core vesicles (DCVs) (i.e., modestly decreased anterograde and increased retrograde axonal flux) in specialized peptidergic neurons (Weiss and Littleton, 2016). DCVs travel by fast anterograde axonal transport to reach nerve terminals where they are either captured in synaptic boutons for many hours to support future neuropeptide release or reverse direction to travel by dynein/dynactin-dependent retrograde transport as part of DCV circulation (Shakiryanova et al, 2006, Wong et al, 2012).…”

Dynein activators and adaptors at a glance

“…HAP1 also interacts with the p150 Glued subunit of dynactin (Li et al, 1998;Engelender et al, 1997), as well as the kinesin heavy chain and light chain (Twelvetrees et al, 2010;McGuire et al, 2006), implicating the two proteins as having a role in intracellular transport (Block-Galarza et al, 1997). While huntingtin has been linked to the axonal transport of several vesicle populations (Wong and Holzbaur, 2014;Gunawardena et al, 2003;Weiss and Littleton, 2016;Colin et al, 2008;Her and Goldstein, 2008), HAP1 is required for the huntingtin-mediated transport of autophagosomes (Wong and Holzbaur, 2014), as well as for APP trafficking (Yang et al, 2012). It has been suggested that huntingtin and HAP1 together act as a platform for both dynein and kinesin attachment to vesicles (Box 1), although the large size of the complex has made it difficult to dissect the underlying mechanisms through in vitro assays.…”

Methodological advances in imaging intravital axonal transport

“…Cargo type should thus be considered and the aims of each individual experiment carefully determined before selecting from the following analysis options, which may also be subdivided into anterograde, retrograde, bi-directional, and combined categories: Speed Frequency of frame-to-frame speeds ( Figure 1Ci ) 11 , 67 , 70 , 71 Individual cargo average velocities ( Figure 1Cii ) 71 Average 52 , 56 , 71 , 92 and maximum cargo speeds ( Figure 1Ciii ) 71 Immobile cargoes can be either included or omitted, and analysed separately 59 , while movement-only speeds (i.e. uninterrupted runs or constant-velocity segments) can be determined 52 , 92 Motility Percentage 14 , 35 , 45 and number 20 , 52 , 56 , 92 of motile cargoes in a given time (also called flux) Percentage of time motile cargoes are moving 12 , 60 Average 56 and longest 45 run distances (run length) Run duration 12 , 13 , 39 Pausing Percentage of cargoes that pause ( Figure 1Civ ) 67 , 71 Percentage 21 , 71 and length 52 , 77 , 92 of time that motile cargoes remain stationary ( Figure 1Cv ) Pause frequency 52 ...…”