Fig 1.
Spastin depletion causes motor impairments in mice.
(A) Gene-targeting strategy. Exons of the spastin gene are indicated by white boxes. The targeted exon is marked in red. The gene-targeting cassette (box) with the 5′- and 3′- homology arms (gradient) is shown in gray. The presence of the LacZ reporter and the neomycin resistance (light gray), frt sites (black), and loxP sites (green) before (KO first allele) and after flp and Cre expression (KO allele) are indicated. (B) PCR genotyping. (C) Western blot analysis of spastin levels in adult hippocampal lysates. NSE = loading control. (D) Accelerating rotarod test using 8-month-old (filled bars) or 14-month-old (open bars) mice. Latency to fall off the rod measures balance and motor learning. Eight-month-old mice: main effect for genotype: F2,61 = 1.07; p = 0.351; followed by pairwise comparisons for (+/+) and (+/−) with p = 0.269 and (+/+) and (−/−) with p = 0.180; 14-month-old mice: main effect for genotype: F2,57 = 3.26; p = 0.046; followed by pairwise comparisons for (+/+) and (+/−) with p = 0.034 and (+/+) and (−/−) with p = 0.035. (E) Pole test using 14-month-old (open bars) mice. Latency to orient downward of the pole measures motor coordination. Main effect for genotype: F2,57 = 2.59; p = 0.084; followed by pairwise comparisons for (+/+) and (−/−) with p = 0.043. (F) Representative pictures of the hind-limb–clasping test using 14-month-old mice. (G) Quantification of the hind-limb–clasping test. Hind-limb extension score measures motor abnormalities. Main effect for genotype: F2,46 = 9.48; p < 0.001; followed by pairwise comparisons for (+/+) and (−/−) with p < 0.001 and (+/−) and (−/−) with p = 0.003. (H-K) Gait analysis using 14-month-old mice. (H) Representative bottom view of the gait analysis test. Stride, base, and gait length as well as angle (α) are indicated. Red rectangle indicates the previous position of the hind paw to display the stride length. (I) Quantified stride velocity. (J) Quantified gait angle, main effect for genotype: F2,46 = 4.90; p = 0.01; followed by pairwise comparisons for (+/+) and (−/−) with p = 0.004. (K) Quantified hind base width, main effect for genotype: F2,46 = 8.78; p = 0.001; followed by pairwise comparisons for (+/+) and (−/−) with p < 0.001 and (+/−) and (−/−) with p = 0.003. (J-N) (+/+) n = 15, (+/−) n = 17, (−/−) n = 14. (L-O) Beam walking test using 14-month-old mice. (M) Latency to cross the beam indicates motor performance. Fourteen-month-old mice: main effect for genotype: F2,53 = 77.839; p < 0.0001; followed by pairwise comparisons for (+/+) and (+/−) with p = 0.001 and (+/+) and (−/−) with p < 0.0001. (N) Number of foot slips while crossing the beam. Fourteen-month-old mice: main effect for genotype: F2,42 = 14.368; p < 0.006; followed by pairwise comparisons for (+/+) and (+/−) with p < 0.006 and (+/+) and (−/−) with p < 0.0001. (O) Percentage of failure to cross the beam. Eight-month-old cohort: (+/+) M = 12 mice, (+/+) F = 11 mice; (+/−) M = 10 mice, (+/−) F = 9 mice; (−/−) M = 11 mice, (−/−) F = 11 mice. Fourteen-month-old cohort: (+/+) M = 11 mice, (+/+) F = 12 mice, (+/−) M = 10 mice, (+/−) F = 12 mice, (−/−) M = 9 mice, (−/−) F = 6 mice. (P) Nissl staining of (+/+) and (−/−) sagittal brains sections. Scale bar, 2 mm. Quantified thickness of (Q) motor cortex areas and (R) somatosensory cortex areas derived from 3- and 14-month-old mice as indicated in (P); (+/+) n = 11–13, (−/−) n = 9–18. (S-U) Immunohistochemistry of cortical sections of (S) 3-month-old and (T) 13-month-old spastin (+/+) and (−/−) mice stained for GFAP to label reactive astrocytes. Note the increase in reactive astrocytes in cortical layer VI (orange region of interest) for spastin (−/−) mice as compared with (+/+) controls; scale bar, 260 μm. (U) Quantification of GFAP signal intensities in cortical layer VI; n = 4 mice per group. ANOVA followed by pairwise comparison was used to assess statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Student t test or ANOVA followed by Tukey’s pairwise comparison was used to assess statistical significance. Data are represented as means ± SEM. Individual quantitative observations that underlie the data presented in this figure are summarized in S1 Data. F, female; flp, recombinase flippase; frt, flippase recognition target; GFAP, glial fibrillary acidic protein; KO, knockout; LacZ, lac operon coding for beta-galactosidase; loxP, locus of X-over P1; M, male; NeoR, neomycin resistance; NSE, neuron specific enolase; WT, wild-type.
Fig 2.
Spastin regulates working memory and associative memory.
(A, B) Spontaneous alternation in the Y-maze using 8-month-old (filled bars) or 14-month-old (open bars) mice. Percent alternation between arm entries measures short-term spatial recognition. Eight-month-old mice: F2,58 = 12.56; p < 0.0001; followed by pairwise comparisons for (+/+) and (+/−) with p < 0.001 and (+/+) and (−/−) with p < 0.0001; 14-month old mice: main effect for genotype: F2,55 = 2.46; p = 0.09; followed by pairwise comparisons for (+/+) and (+/−) with p = 0.249 and (+/+) and (−/−) with p = 0.033. ANOVA was used to assess statistical significance. (C) Explorative behavior, revealed by overall number of arm entries in the Y-maze using 8-month-old (filled) or 14-month-old (striped) mice. Two-way ANOVA (genotype × arm); main effect for genotype: F2,119 = 1.649; p = 0.197; pairwise comparisons: 8-month-old mice (+/+) and (+/−) with p = 0.639 and (+/+) and (−/−) with p = 0.615. Fourteen-month-old mice (+/+) and (+/−) with p = 0.914 and (+/+) and (−/−) with p = 0.090. (D) Experimental design of T-maze experiment. (E) Working and reference memory in the T-maze using 8-month-old (filled bars) or 14-month-old (open bars) mice. Dotted line represents 50% chance level. Confined alternation scores above 50% indicate reference memory of the confined arm. Eight-month-old mice: ANOVA, main effect for genotype: F1,61 = 3.04; p = 0.055; followed by pairwise comparisons for (+/+) and (+/−) with p = 0.066 and (+/+) and (−/−) with p = 0.025; 14-month-old mice: ANOVA, main effect for genotype: F2,46 = 4.17; p = 0.021; followed by pairwise comparisons for (+/+) and (+/−) with p = 0.1 and (+/+) and (−/−) with p = 0.006. Additional analysis showed that only the (+/+) group alternated at levels significantly above the chance. (+/−) and (−/−) mice performed at 50% chance level or below, respectively; one-sample t test against 50% chance level: 8-month-old mice (+/+): p < 0.0001; (+/−) p < 0.379; (−/−): p = 0.321. Fourteen-month-old mice (+/+): p < 0.033; (+/−): p = 0.727; (−/−): p = 0.044. (F) Experimental design of contextual fear-conditioning experiment. (G-H) Percentage time of freezing, indicative of context/shock association, during acquisition at day 1 for 8-month-old mice (G) and 14-month-old mice (H). Two-way ANOVA (genotype × age): F2,118 = 0.328; p = 0.721; main effect for genotype: F2,118 = 1.779; p = 0.173. (I) Percentage time of freezing, indicative of conditioned fear memories, at day 2, context A. Eight-month-old mice: ANOVA, main effect for genotype: F2,61 = 2.09; p = 0.132; pairwise comparisons for (+/+) and (+/−) with p = 0.104 and (+/+) and (−/−) with p = 0.070; 14-month-old mice: ANOVA, main effect for genotype: F2,57 = 5.5524; p = 0.006; pairwise comparisons for (+/+) and (+/−) with p = 0.038 and (+/+) and (−/−) with p = 0.002. (J) Percentage time of freezing at day 3, context B. Eight-month-old mice: ANOVA, main effect for genotype: F2,61 = 0.429; p = 0.653; pairwise comparisons for (+/+) and (+/−) with p = 0.565 and (+/+) and (−/−) with p = 0.716; 14-month-old mice: ANOVA, main effect for genotype: F2,57 = 1.611; p = 0.209; pairwise comparisons for (+/+) and (+/−) with p = 0.771 and (+/+) and (−/−) with p = 0.091. (K, L) Percentage time of freezing at days 4–7, context A, indicative of extinction learning in 8-month-old mice (K) and 14-month-old mice (L). Three-way ANOVA (day × genotype × age): F6,354 = 2.021; p < 0.062; 8-month-old mice: two-way ANOVA (day × genotype): F6,183 = 0.862; p = 0.524; main effect for genotype: F2,61 = 0.305; p = 0.738. Fourteen-month-old mice: two-way ANOVA (day × genotype): F6,171 = 2.845; p = 0.011; main effect for genotype: F1,57 = 35.484: p < 0.0001; pairwise comparisons (+/+) and (+/−): p < 0.0001 and for (+/+) and (−/−) p < 0.0001. Eight-month-old cohort: (+/+) M = 12 mice, (+/+) F = 11 mice; (+/−) M = 10 mice, (+/−) F = 9 mice; (−/−) M = 11 mice, (−/−) F = 11 mice. Fourteen-month-old cohort: (+/+) M = 11 mice, (+/+) F = 12 mice, (+/−) M = 10 mice, (+/−) F = 12 mice, (−/−) M = 9 mice, (−/−) F = 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as means ± SEM. Individual quantitative observations that underlie the data presented in this figure are summarized in S2 Data. F, female; M, male.
Fig 3.
Spastin depletion reduces spine and synapse density.
(A) Representative images of rhodamine-phalloidin–labeled neurons at DIV18. EGFP used as volume marker transfected at DIV10. (B) Quantification of total number of spines per 10 μm dendrite length. (+/+), n = 30; (−/−), n = 35. (C) Quantification of spine types per 10 μm dendrite length. Scale bar, 10 μm; magnification, scale bar, 5 μm. (+/+), n = 30; (−/−), n = 35. (D) EM micrographs of CA1 region from spastin (+/+) and (−/−) mice (3 months). n = 3 mice per group. Scale bar, 1 μm; magnification, scale bar, 100 nm. (E) Quantification of synapse numbers per ROI (18 μm2), (+/+), n = 17; (−/−), n = 21. (F) Coimmunostaining of Syp and PSD-95 at DIV14 to visualize excitatory synaptic contacts (magenta signals), following expression of different GFP-Spastin constructs as indicated. Representative dendritic regions of hippocampal neurons are depicted; scale bar, 2 μm. (G-I) Quantification of synapses per 10 μm dendrite length at DIV10, 14 and 18; n = 80–98 dendritic regions per group. *p < 0.05, **p < 0.01, ***p < 0.001. Student t test and ANOVA followed by pairwise comparison were used to assess statistical significance. Data are represented as means ± SEM. Individual quantitative observations that underlie the data presented in this figure are summarized in S3 Data. DIV, days in vitro; EGFP, enhanced green fluorescent protein; EM, electron microscopy; GFP, green fluorescent protein; ns, not significant; PSD-95, postsynaptic density protein 95; ROI, region of interest; Syp, synaptophysin.
Fig 4.
Spastin depletion affects LFPs and EPSC frequency.
(A-C) LFPs upon single-pulse stimulation in CA1 before (pre) and 20 minutes after (post) theta burst stimulation (time point: 0 minutes, black arrow) of CA3 Schaffer collaterals in (+/+) and (−/−) mice. The gap in the recording traces results from elimination of the stimulation artifact immediately before the LFP response. Less pronounced increase in LFP slope in spastin-deficient mice (−/−, circles). Green circles indicate significantly different values (p < 0.05) during the first 5 minutes following stimulation (black arrow) compared with (+/+) littermates (filled squares). (+/+) n = 7, (−/−) n = 9. (D-F) EPSC amplitudes at mossy-fiber-to-CA3 pyramidal cell synapses using whole-cell patch-clamp recordings. Synaptic currents evoked by mossy-fiber stimulation in CA3 pyramidal cells. Black: controls; orange: 50 μM D-APV; green: D-APV plus 10 μM NBQX. (+/+) n = 8, (−/−) n = 9. (G) Equal D-APV–sensitive and D-APV–insensitive synaptic currents indicate unaltered contributions of NMDARs and AMPARs in both genotypes. (+/+) n = 7, (−/−) n = 5. (H, I) Whole-cell patch-clamp recordings using DIV14–18 neurons. (J-M) Cumulative probability histograms for mEPSC inter-event intervals and amplitudes. (+/+) n = 8, (−/−) n = 14. *p < 0.05. Student t test was used to assess statistical significance. Data are represented as means ± SEM. Individual quantitative observations that underlie the data presented in this figure are summarized in S4 Data. D-APV, d-2-amino-5-phosphonovaleric acid; DIV, days in vitro; EPSC, excitatory postsynaptic current; LFP, local field potential; mEPSC, miniature EPSC; NBQX, 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline; NMDAR, N-methyl-d-aspartate receptor.
Fig 5.
Spastin depletion alters growth and structure of MTs.
(A) Electron micrograph of MT in DIV14 neurons. Note that because of ultrathin sectioning, MTs appear as fragments in three-dimensional dendrites growing across other neurites. Scale bar, 200 nm. (B-D) Quantification of MT mean fragment length, total fragment length, and inter-microtubule fragment distance. (+/+) n = 20, (−/−) n = 14 based on three independent neuronal cultures per group. (E-H) Live imaging of EB3-tdTomato in DIV12 neurons following coexpression of different GFP-Spastin constructs, as indicated. (E) Upper panel depicts GFP expression. Lower panels: representative kymographs of EB3-tdTomato acquired from secondary dendrites (white rectangle). Scale bar, 10 μm. Quantification of (F) comet number, (G) velocity, and (H) growth distance; (+/+_GFP) n = 36 (12 cells), (−/−_GFP) n = 80 (27 cells), (−/−_Spast-WT) n = 42 (14 cells), (−/−_Spast-K388R) n = 48 (16 cells). (I) Immunostaining of polyglutamylated tubulin (GT335 antibody) in CA1. (+/+), n = 9 mice; (−/−), n = 9 mice. Scale bar, 100 μm. (J) Quantification of (I). (K, L) Quantitative western blot analysis of Poly-Glu Tub, total α-Tub, and NSE (loading control). (+/+) n = 4, (−/−) n = 4, **p < 0.01, ***p < 0.001. Student t test (B-D and J), ANOVA followed by pairwise comparison (F-H), and one-way ANOVA (L) were used to assess statistical significance. Data are represented as means ± SEM. Individual quantitative observations that underlie the data presented in this figure are summarized in S5 Data. DIV, days in vitro; EB3, end-binding protein 3; GFP, green fluorescent protein MT, microtubule; NSE, neuron specific enolase; Poly-Glu, polyglutamylation; PTM, posttranslational modification; ROI, region of interest; Tub, tubulin; WT, wild-type.
Fig 6.
Impaired kinesin processivity is rescued by reducing tubulin polyglutamylation in spastin KO neurons.
(A) Representative western blot depicting total KIF5C expression levels from brain homogenate of spastin (+/+) and (−/−) mice. (B) Quantification of data shown in (A). (C) Representative western blot depicting binding of KIF5C to MTs from brain homogenate of spastin (+/+) and (−/−) mice in the presence of nonhydrolyzable AMP-PNP (promotes kinesin–MT binding). (D) Quantification of (C). Data were normalized to Tubβ3. The percentage of the mutant signal relative to the WT signal is plotted. (+/+) n = 3, (−/−) n = 3 (MT preparations from independent mouse brains). One-way ANOVA was used to assess statistical significance. (E-G) KIF5C motility in dendrites at DIV12 using KIF5C without a cargo-binding domain fused to tdTomato and pex26 (peroxisome binding domain) KIF5C-tdTomato-pex26 is coexpressed with different GFP-Spastin constructs as indicated. (E) Representative kymographs of KIF5C. (F) Quantification of mean KIF5C velocity (G) Quantification of mean traveled distances; (+/+_control) n = 60 (20 cells), (−/−_control) n = 51 (17 cells), (−/−_Spast-WT) n = 59 (20 cells), (−/−_Spast-K388R) n = 51 (17 cells). (H) Representative immunostaining of polyglutamylated tubulin (GT335 antibody) using (+/+) or (−/−) DIV14 hippocampal neurons 3 days after transfection of control or shRNA-PGs1 constructs. Scale bar, 10 μm. (I) Quantification of GT335 signal intensities as shown in (H) (+/+_control) n = 26 cells, (−/−_control) n = 46, (−/−_shRNA-PGs1) n = 46. (J-L) KIF5C-tdTomato-pex26 motility in dendrites at DIV14 following cotransfection of control or shRNA-PGs1 constructs, both encoding a GFP reporter (hippocampal neurons derived from spastin [+/+] and [−/−] mice). (J) Representative kymographs of KIF5C. (K) Quantification of mean KIF5C velocity. (L) Quantification of mean travel distances; (+/+_control) n = 40 (11 cells), (−/−_control) n = 50 (18 cells), (−/−_shRNA-PGs1) n = 45 (16 cells). Time-lapse images were taken at 0.5 frames per second. (M) Coimmunostaining of Syp and PSD-95 at DIV14 to visualize excitatory synaptic contacts (magenta signals) following cotransfection of control or shRNA-PGs1 constructs, both encoding a GFP reporter (hippocampal neurons derived from spastin [+/+] and [−/−] mice). Representative dendritic regions of hippocampal neurons are depicted; scale bar, 2 μm. (N) Quantification of synapses per 10 μm dendrite length as shown in (M); (+/+_control) n = 56 (28 cells), (−/−_control) n = 65 (33 cells), (−/−_shRNA-PGs1) n = 64 (32 cells). *p < 0.05, **p < 0.01, ***p < 0.001. Student t test and ANOVA followed by pairwise comparison were used to assess statistical significance. Data are represented as means ± SEM. Individual quantitative observations that underlie the data presented in this figure are summarized in S6 Data. AMP-PNP, adenylyl-imidodiphosphate; DIV, days in vitro; GFP, green fluorescent protein; KIF, kinesin family protein; KO, knockout; MT, microtubule; ns, not significant; pex26, peroxisome assembly protein 26; PGs1, polyglutamylase subunit 1; PSD-95, postsynaptic density protein 95; shRNA, short hairpin RNA; Syp, synaptophysin; Tubβ3, tubulin β3; WT, wild-type.
Fig 7.
Spastin depletion impairs synaptic cargo transport and cell surface delivery.
(A-D) Live imaging of Synaptophysin-EGFP transport. (A) Kymograph. (B) Travel distance. (C) Velocity. (D) Percent of stationary particles in axons. (+/+) n = 152 (15 cells), (−/−) n = 130 (12 cells). Images were taken at one frame per second. (E-H) Presynaptic vesicle analysis using EM. (E) Electron micrograph from CA1. Scale bar, 200 nm. (F) Undocked vesicle number. (G) Docked vesicle number. (H) Vesicles per synapse terminal. (+/+) n = 3 mice, (−/−) n = 3 mice. Student t test was used to assess statistical significance. (I-K) AMPAR neuronal transport detecting pFusionRed-GluA2 particle mobility over time. (I) Kymograph. (J) Velocity. (K) Travel distance. (+/+), n = 17 (12 cells) particles; (−/−), n = 16 (12 cells). (L-N) GluA2 subcellular expression levels analyzed using western blotting, following subcellular fractionation. (L) Representative western blots. (M) Quantification of GluA2 in total protein fraction (S1). (+/+), n = 7; (−/−), n = 6. (N) Quantification of GluA2 in plasma membrane–enriched fraction (P2). (+/+) n = 4, (−/−) n = 4. NSE used as loading control. (O, P) GluA2-AMPAR cell surface staining using DIV18 neurons. (O) Cell surface GluA2 (red) expression in dendrites; scale bar, 10 μm; magnification, 5 μm. EGFP (green) used as volume marker. (P) Cell surface GluA2 signal intensity. (+/+), n = 19; (−/−), n = 19. Student t test was used to assess statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as means ± SEM. (Q) Model. Loss of spastin-mediated MT severing leads to longer MTs characterized by an accumulation of tubulin polyglutamyl side chains. The resulting increase in negative charge at the MT surface affects the binding and mobility of motors (e.g., kinesins) and their respective cargo proteins (e.g., vesicular AMPARs). The observed deficits in synaptic cargo delivery are in agreement with the cognitive deficits in spastin KO mice and patients with SPG4-type HSP. Individual quantitative observations that underlie the data presented in this figure are summarized in S7 Data. AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; DIV, days in vitro; EGFP, enhanced green fluorescent protein; EM, electron microscopy; GluA2, Glutamate receptor AMPA type subunit 2; KO, knockout; MT, microtubule; NSE, neuron specific enolase; SPG, spastic paraplegia.