A motor neuron disease–associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death

Dynactin Subunit p150^Glued Is a Neuron-Specific Anti-Catastrophe Factor

<div><p>Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150^Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150^Glued may also modulate the dynamics of microtubules, we hypothesized that p150^Glued might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate <i>in vitro</i> using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150^Glued is a potent anti-catastrophe factor for microtubules. p150^Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150^Glued are required in tandem for this activity. p150^Glued is alternatively spliced <i>in vivo</i>, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150^Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150^Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150^Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease.</p></div

p150^Glued is a neuron-specific microtubule anti-catastrophe factor.

<p>p150^Glued inhibits microtubule catastrophe by binding both to microtubules and to soluble tubulin, leading to enhanced microtubule stability along the axon. Depletion of endogenous p150^Glued by RNAi leads to more frequent microtubule catastrophe in neurons.</p

p150 catalyzes microtubules nucleation.

<p>(A) Light scattering traces for increasing concentrations of p150 Nt-GCN4. At right, the scale was magnified to illustrate the initial increase in light scattering observed with increasing concentrations of p150 Nt-GCN4. Error bars represent SEM of three or more independent experiments and are omitted for clarity at right. (B) p150 Nt-GCN4 decreases nucleation time, defined as initial appearance of signal over background. Fit is to a double exponential decay. Note that error bars are smaller than symbols. (C) Schematic of the TIRF nucleation assay. p150 constructs are immobilized and the chamber is then perfused with rhodamine-labeled tubulin. (D) Montage from TIRF nucleation assay shows that dimeric p150 Nt-GCN4 catalyzes microtubule nucleation in contrast to monomeric p150 Nt. Scale bar, 12.5 µm. (E) Montage of representative microtubules nucleated in the presence of a low concentration of p150 dimer. Yellow arrows identify the site of nucleation. Red arrows identify the growing plus- and minus-ends. Scale bar, 5 µm.</p

p150^Glued stabilizes microtubules in neurons.

<p>(A) Depletion of p150^Glued in COS7 cells by siRNA (KD) relative to control cells treated with scrambled (Scram) oligonucleotides; upper panel is a Western blot probed with a monoclonal antibody to p150^Glued, and lower panel is Coomassie staining to show equal protein loading. (B) Representative rainbow-coded maximum intensity projections show that the overall microtubule architecture and dynamics are not perturbed in COS7 cells depleted of p150^Glued relative to control cells. (C) PlusTipTracker quantitation of EB3-GFP comet velocities shows that knockdown does not alter parameters of microtubule dynamics, including distance to catastrophe, a measure of the catastrophe frequency (<i>p</i>>0.1). (D) Western blot showing knockdown of p150^Glued in DRG neurons (KD), relative to control neurons or COS7 cells treated with scrambled oligonucleotide. Note the differential splice forms of p150^Glued expressed in neurons relative to COS7 cells. (E) Space-filling fluorescence in representative neurites used to investigate microtubule dynamics in primary DRG neurons. (F) Rainbow-coded maximum intensity projections of selected EB3-GFP comets demonstrate microtubule dynamics in DRG neurons. (G) Kymographs of GFP-EB3 comets in cultured DRG neurons treated with either scrambled or p150^Glued RNAi reveal that in neurons, p150^Glued inhibits catastrophe. (H) Camera lucida tracing of the kymographs in panel G to indicate polymerization events persisting greater than 5 µm (black) and less than 2 µm (magenta). (I) Kymographs of GFP-EB3 comets in neurons depleted of endogenous p150^Glued by RNAi and rescued with either full-length p150, Δ5–7, or the neuron-specific alternative splice form p135. (J) Analysis of microtubule dynamics in scrambled control neurons, and neurons depleted of endogenous p150^Glued with or without rescue with resistant constructs of p150 or p135 and Δ5–7. Bars represent mean of comet parameters from multiple cells on multiple days ± SEM. Statistical testing was performed via <i>t</i> test with correction for multiple comparisons. *** <i>p</i><0.001. (K) In vitro analysis of catastrophe rates demonstrates that the Perry syndrome-associated mutation Q74P p150 Nt-GCN4 does not inhibit microtubule catastrophe (<i>p</i>>0.5), as compared to the wild-type p150Nt-GCN4 construct. (L, M) Kymographs and quantitation of GFP-EB3 comets in cultured DRG neurons depleted of endogenous p150^Glued and rescued with either wild-type or Q74P p150^Glued reveal that mutant p150 is defective in inhibiting microtubule catastrophe in neurons.</p