- Research article
- Open Access
Genetic ablation of dynactin p150Glued in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice
© The Author(s). 2018
- Received: 19 September 2017
- Accepted: 19 February 2018
- Published: 1 March 2018
Dynactin p150Glued, the largest subunit of the dynactin macromolecular complex, binds to both microtubules and tubulin dimers through the N-terminal cytoskeleton-associated protein and glycine-rich (CAP-Gly) and basic domains, and serves as an anti-catastrophe factor in stabilizing microtubules in neurons. P150Glued also initiates dynein-mediated axonal retrograde transport. Multiple missense mutations at the CAP-Gly domain of p150Glued are associated with motor neuron diseases and other neurodegenerative disorders, further supporting the importance of microtubule domains (MTBDs) in p150Glued functions. However, most functional studies were performed in vitro. Whether p150Glued is required for neuronal function and survival in vivo is unknown.
Using Cre-loxP genetic manipulation, we first generated a line of p150Glued knock-in mice by inserting two LoxP sites flanking the MTBD-coding exons 2 to 4 of p150Glued–encoding Dctn1 gene (Dctn1LoxP/), and then crossbred the resulting Dctn1LoxP/ mice with Thy1-Cre mice to generate the bigenic p150Glued (Dctn1LoxP/LoxP; Thy1-Cre) conditional knockout (cKO) mice for the downstream motor behavioral and neuropathological studies.
P150Glued expression was completely abolished in Cre-expressing postnatal neurons, including corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs), while the MTBD–truncated forms remained. P150Glued ablation did not affect the formation of dynein/dynactin complex in neurons. The p150Glued cKO mice did not show any obvious developmental phenotypes, but exhibited impairments in motor coordination and rearing after 12 months of age. Around 20% loss of SMNs was found in the lumbar spinal cord of 18-month-old cKO mice, in company with increased gliosis, neuromuscular junction (NMJ) disintegration and muscle atrophy. By contrast, no obvious degeneration of CSMNs, striatal neurons, midbrain dopaminergic neurons, cerebellar granule cells or Purkinje cells was observed. Abnormal accumulation of acetylated α-tubulin, and autophagosome/lysosome proteins was found in the SMNs of aged cKO mice. Additionally, the total and cell surface levels of glutamate receptors were also substantially elevated in the p150Glued-depleted spinal neurons, in correlation with increased vulnerability to excitotoxicity.
Overall, our findings demonstrate that p150Glued is particularly required to maintain the function and survival of SMNs during aging. P150Glued may exert its protective function through regulating the transportation of autophagosomes, lysosomes, and postsynaptic glutamate receptors in neurons.
- Dynactin p150Glued
- Microtubule binding domain
- Motor neuron
- Glutamate receptor
Impairments in intracellular transport are often associated with neurological disorders, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (ALS) . Dynein mediates the retrograde transport of cargos from microtubule plus ends to the minus ends, while the dynactin protein complex is proposed to promote the processivity of dynein motor proteins moving along the microtubules, as well as expand the variety of dynein cargoes [2, 3]. Dynactin consists of more than 20 subunits, with its largest subunit p150Glued to interact with both microtubules and dynein motor protein complex . P150Glued, encoded by the full-length DCTN1 gene, weights around 150 kD and contains the N-terminal CAP-Gly and basic domains, followed by the coiled-coil 1 (CC1) and CC2 domains . Both the CAP-Gly and basic domains exhibit microtubule binding affinity, and together form the tandem MTBDs . On the other hand, the CC1 and CC2 domains mediate the interactions with dynein intermediate chain (DIC) and the other dynactin subunits . The MTBDs of p150Glued are required for cell division by providing essential attachment to the microtubules in spindle formation and chromosome movement , p150Glued inhibition causes cell proliferation arrest  and germline deletion of p150Glued leads to early embryonic lethality . Multiple missense mutations in the CAP-Gly domain of p150Glued have been linked to a slowly progressive, autosomal dominant form of lower motor neuron disease without sensory symptoms ; Perry syndrome, which consists of parkinsonism with severe mental depression and central hypoventilation ; and progressive supranuclear palsy . P150Glued is proposed as an anti-catastrophe factor in maintaining the stability of microtubules in neurons, and facilitate the dynein-mediated axonal retrograde transport in neuronal cultures . These disease-causal mutations seem to weaken the microtubule binding affinity of p150Glued . However, the functional significance of p150Glued has not been critically evaluated in neurons in living animals.
In addition to p150Glued, DCTN1 also encodes p135 and other short splicing variants . P135 lacks the coding exons 2 to 5, resulting in a complete loss of the CAP-Gly domain and a large portion of basic domain. P150Glued is expressed in all types of mammalian cells, while p135 is more abundant in neurons . Dynein/dynactin is required for the mitosis as germline deletion of p150Glued caused early embryonic lethality and apoptosis in p150Glued knockout mice . However, p135 can compensate for the most dynactin activity in p150glued-deficient post-mitotic cells . These observations raise questions about the overall importance of p150Glued in neurons, despite mutations in its CAP-Gly domain are associated with multiple neurological disorders. One possible scenario is that p150Glued proteins are particularly needed to maintain the integrity of microtubule network and the efficiency of axonal retrograde transport in the large projection neurons with long axons, such as the CSMNS and SMNs.
In this study, we utilized Cre-loxP system  to selectively deplete p150Glued but keep p135 expression in CSMNs and SMNs. To our surprise, genetic ablation of p150Glued in postnatal neurons did not cause overt behavioral and neuropathological phenotypes in mice. Only moderate motor deficits and SMN loss were observed in aged p150Glued cKO mice. The p150Glued-lacking neurons appeared to be more susceptible to excitotoxicity in correlation with abnormal augmentations of total and surface expression of glutamate receptors, a potential pathogenic mechanism of SMN degeneration in p150Glued cKO mice.
Generation of Dctn1 LoxP/ knock-in mice and Dctn1 LoxP/LoxP; Cre conditional knockout mice
Genomic DNA was prepared from tail biopsy using DirectPCR Lysis Reagent (Viagen Biotech) and subjected to PCR amplification using specific sets of PCR primers for each genotype, including Dctn1LoxP/ knock-in mice (mDCTN1-Ex4-F, CAG CTG CAA AGA CCA GCA AA; mDCTN1-Ex5-R: CAC ACC ACC TTC TTA GGC TTC A), Cre transgenic mice (CRE-F, CAT TTG GGC CAG CTA AAC AT; CRE-R, TGC ATG ATC TCC GGT ATT GA).
Sucrose density gradient fractionation and sedimentation analysis
Neuronal cells or neural tissues were homogenized in 50 mM Tris-HCl, pH 7.5, containing 150 mM NaC1, 1 mM EDTA and Protease Inhibitor Cocktail (Thermo Scientific). The cytosolic extracts were prepared by centrifugation at 16,000 × g for 15 min at 4 °C, and further clarified at 100,000 × g for 15 min. The resulting supernatant was layered over a 12 ml, 5–20% sucrose density gradient and centrifuged at 120,000 × g for 18 h at 4 °C. 1-ml gradient fractions were collected and equal volumes of all fractions analyzed by SDS-PAGE followed by Western blotting.
Open-field test. As described previously , the ambulatory and rearing activities of male mice were measured by the Flex-Field Activity System (San Diego Instruments). Flex-Field software was used to trace and quantify mouse movement in the unit as the number of beam breaks per 30 min.
Rotarod test. As described previously , male mice were placed onto a rotating rod with auto-acceleration from 0 rpm to 40 rpm for 1 min (San Diego Instruments). The length of time the mouse stayed on the rotating rod was recorded. Three measurements were taken for each animal during each test.
Hindlimb clasping test. In the hindlimb clasping test, male mice were suspended by the tail and observed for 10 s. The severity of hindlimb clasping phenotype was scored as follows: 0 = both hindlimbs consistently spread outward and away from the abdomen, 1 = one hindlimb retracted toward the abdomen, 2 = both hindlimbs partially retracted toward the abdomen, 3 = both hindlimbs completely retracted toward the abdomen. For each mouse, three separate tests were taken over 3 h, and the averaged hindlimb clasping severity score of the three tests was calculated.
Histology, immunohistochemistry and light microscopy
For neuropathology study, mice were perfused with 4% paraformaldehyde (PFA) in cold phosphate buffered saline (PBS), brains and spinal cords were collected, post-fixed overnight, submerged in 30% sucrose in PBS for at least 72 h, and sectioned at 40–50 μm thickness using CM1950 cryostat (Leica). For muscle pathology analysis, mice were euthanized, gastrocnemius muscles were dissected, snap frozen in 2-methylbutane (Sigma-Aldrich) cooled in dry ice, sectioned at 20 or 40 μm thickness, and fixed in 4% PFA in PBS for 15 min. For Nissl staining, sections were stained with 0.1% Cresyl Violet (Sigma-Aldrich). For HE staining, hematoxylin and eosin stain kit (Vector Laboratories) were used as suggested by manufacturers. For immunostaining, antibodies specific to p150Glued N-terminus (amino acid 3–202, 1:200, BD Biosciences), p150Glued C-terminus (amino acid 1266–1278, 1:500, Abcam), neuron-specific nuclear protein (NeuN, 1:1000, Millipore), CTIP2 (1:200, Abcam), choline acetyltransferase (CHAT, 1:500, Millipore), Glial Fibrillary Acidic Protein (GFAP, 1:1000, Abcam), Ionized calcium-Binding Adaptor Molecule-1 (IBA1, 1:1000, Wako Chemicals), acetylated α-tubulin (ac-TUBA, 1:500, Abcam), Lysosomal Associated Membrane Protein 2 (LAMP2, 1:200, Abcam), Tyrosine Hydroxylase (TH, 1:500, Pel-Freeze) and Calbindin D28K (1:200, Abcam) were used as suggested by manufacturers. For immunofluorescence study, Alexa Fluor 488-, 546- or 647-conjugated secondary antibody (1:500, Invitrogen) was used to visualize the staining. Alexa Fluor 488-conjugated α-bungarotoxin (1:500, Invitrogen) was applied to label postsynaptic acetylcholine receptors of NMJs. Fluorescence images were captured using LSM 780 laser-scanning confocal microscope (Zeiss). The paired images in all the figures were collected at the same gain and offset settings. Post collection processing was applied uniformly to all paired images. The images were presented as either a single optic layer after acquisition in z-series stack scans at 1.0 μm intervals from individual fields or displayed as maximum-intensity projections to represent confocal stacks. For immunohistochemistry study, Vectastain Elite ABC Kit and DAB Kit (Vector Laboratories) were used to visualize the staining. Bright field images were captured by Axio microscope Imager A1 (Zeiss).
For the quantitative assessment of various marker protein distributions, images were taken using identical settings and exported to ImageJ (NIH) for imaging analysis. Images were converted to an 8-bit color scale (fluorescence intensity from 0 to 255) using ImageJ. The areas of interest were first selected with Polygon or Freehand selection tools and then subjected to measurement by mean optical intensities or area fractions. The mean intensity for the background area was subtracted from the selected area to determine the net mean intensity.
Stereology for CSMNs, striatal neurons, SMNs and midbrain dopaminergic neurons was performed as described previously [19, 20]. To examine the number of CSMNs and striatal neurons, series of sagittal brain sections (40 μm per section thickness, every ninth section, nine sections per case) were processed for Nissl staining or CTIP2 immunohistochemistry. The motor cortex was designated as layer V neurons in the anteromedial cortex, and outlined according to the mouse brain in stereotaxic coordinates. To examine the number of SMNs, series of coronal spinal cord sections (50 μm per section, every tenth section from L3 to L5, ten sections per case) were processed for Nissl staining or CHAT immunohistochemistry. To examine the number of midbrain dopaminergic neurons, series of coronal sections across the midbrain (40 μm per section, every fourth section from Bregma − 2.54 to − 4.24 mm, ten sections per case) were processed for TH immunohistochemistry. For the unbiased stereological estimation of CSMNs, striatal neurons, SMNs and midbrain dopaminergic neurons, the number of CTIP2 positive or Nissl-stained large (>15 μm in diameter) cortical neurons in layer V, Nissl-stained neurons in stratum, CHAT positive or Nissl-stained large (>35 μm in diameter) spinal neurons in ventral horn, and TH-positive neurons in substantia nigra pars compacta (SNpc) were assessed using the Optical Fractionator function of Stereo Investigator 10 (MicroBrightField). Six mice were used per genotype at each time point. Counters were blinded to the genotypes of the samples. The sampling scheme was designed to have a coefficient of error less than 10% in order to obtain reliable results.
Preparation of postsynaptic density (PSD) fraction
PSD fractions were prepared from mouse brains as described previously . All procedures were performed at 4 °C. Mouse brains were isolated and homogenized in 10 volumes of ice-cold Buffer A (0.32 M sucrose, 5 mM HEPES, pH 7.4, 1 mM MgCl2, 0.5 mM CaCl2, Protease and Phosphatase Inhibitor Cocktail) with Teflon homogenizer (12 strokes). The homogenate was spun at 1400 × g for 10 min. Supernatant (S1’) was saved and pellet (P1’) was homogenized again with Teflon homogenizer in another 10 volumes of Buffer A (5 strokes). After centrifugation at 700 × g for 10 min, the supernatant (S1’) was collected and pooled with S1, and the pellet (P1’) were saved as crude nuclear fraction. Pooled S1 and S1’ were centrifuged at 13,800 × g for 10 min to collect the crude synaptosomal pellet (P2) and the supernatant (S2), which contains the cytosol and light membranes. S2 was centrifuged at 100,000 × g for 15 min to separate the cytosolic fraction (Cyt) and the light membrane fraction (LM). P2 was resuspended in 10 volumes of Buffer B (0.32 M sucrose, 6 mM Tris, pH 8.0, supplemented with Protease and Phosphatase Inhibitor Cocktail) with Teflon homogenizer (5 strokes). The P2 suspension was loaded onto a discontinuous sucrose gradient (0.85 M/1 M/1.15 M sucrose solution in 6 mM Tris, pH 8.0), and centrifuged at 82,500 × g for 2 h. Synaptosome fraction (Syn) was collected from the layer between 1 M and 1.15 M sucrose, supplemented with 0.5% Triton X-100 and mixed for 15 min. The suspension was spun at 32,800 × g for 20 min, and the resulting pellet was saved as PSD fraction (PSD). P1’, LM and PSD fractions were lysed in 1% SDS buffer. SDS were added into Cyt and Syn fraction to 1% final concentration. Equal amount of total protein from each fraction were resolved in SDS-PAGE and applied to western blot analysis.
Primary neuron culture
Mouse primary neuron cultures were prepared from the cortex or spinal cord of newborn (postnatal day 0, P0) pups, as described previously [21, 22]. Briefly, individual cortex or spinal cord was dissected and subjected to papain digestion (5 U/ml, Worthington Biochemicals) for 40 min at 37 °C. The digested tissue was carefully triturated into single cells using increasingly smaller pipette tips. The cells were then centrifuged at 250 × g for 5 min and resuspended in warm Basal Medium Eagle (BME, Sigma-Aldrich) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Invitrogen), 1× N2/B27 supplement (100× stock, Invitrogen), 1× GlutaMax (100× stock, Invitrogen), 0.45% D-glucose (Sigma-Aldrich), 10 U/ml penicillin (Invitrogen), and 10 μg/ml streptomycin (Invitrogen). The dissociated neurons were seeded in plated in Biocoat Poly-D-Lysine Cellware plate (BD Biosciences), and maintained at 37 °C in a 95% O2 and 5% CO2 humidified incubator. Twenty-four hours after seeding, the cultures were switched to serum-free medium supplemented with 1 μM cytosine β-D-arabinofuranoside (Sigma-Aldrich) to suppress the proliferation of glia and 1 μM 4-hydroxytamoxifen (4-OHT, Sigma-Aldrich) to induce CRE recombinase activity. Starting from 5 days in vitro (DIV), culture medium was changed twice every week.
Biotinylation of neuronal surface proteins
Spinal neurons were cultured at the density of 1.2 × 106 per well in Poly-D-Lysine coated 6-Well plate (BD Biosciences) and treated with 0 or 10 μM glutamate (Sigma-Aldrich) on 20 DIV. After 24-h treatment, neurons were washed with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS/CM), and then incubated with 1 ml biotin solution (0.5 mg/ml Sulfo-NHS-SS-Biotin in cold PBS/CM, Thermo Fisher Scientific) for 20 min at 4 °C and then washed subsequently with PBS/CM, 0.1 M glycine solution, and TBS (25 mM Tris-HCl, pH 7.4, containing 137 mM NaCl) buffer. Cells were harvested in 250 μl RIPA buffer (Sigma) supplemented with protease inhibitor and phosphatase inhibitor cocktail, lysed on ice for 30 min, and centrifuged at 16,000 × g for 15 min at 4 °C. Resulting supernatant containing equal amount of total protein was incubated with 50 μl neutral-avidin agarose (Pierce) at 4 °C for 2 h with gently rotating. After washing in RIPA buffer for 5 times, the biotin-labeled surface protein was eluted with SDS sample buffer by heating at 70 °C for 10 min. Total proteins and isolated biotinylated surface proteins were analyzed by western blotting.
Assessment of cell survival by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay
Spinal neurons were cultured at the density of 1.0 × 105 per well in Poly-D-Lysine coated 96-Well plate (BD Biosciences) and treated with 0 or 10 μM glutamate (Sigma-Aldrich) on 20 DIV. After 24-h treatment, the cells were incubated with 0.5 mg/ml MTT (Sigma-Aldrich) at 37 °C for 4 h. After the media were removed, dimethyl sulfoxide (DMSO, 100 μl) was added to each well to solubilize the formazan crystals generated by viable mitochondrial succinate dehydrogenase from MTT. The absorbance at 570 nm was measured using a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices) as the MTT reducing activity of the cells.
Western blot analysis
Neurons or tissues were homogenized and sonicated in RIPA buffer (Sigma-Aldrich) or 1% SDS lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, pH 7.5, and 1% SDS) supplemented with Protease Inhibitor Cocktails (Thermo Scientific). Lysates were clarified by centrifugation at 15000 × g for 15 min at 4 °C. The supernatants were quantified for protein content using the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) and separated by 4–12% NuPage BisTris-PAGE (Invitrogen) using MES or MOPS running buffer (Invitrogen). The separated proteins were then transferred to nitrocellulose membranes using the iBlot Dry Blotting system (Invitrogen) and incubated with specific primary antibodies. The antibodies used for western blot analysis included p150Glued N-terminus (amino acid 3–202, 1:1000, BD Biosciences), p150Glued C-terminus (amino acid 1266–1278, 1:1000, Abcam), dynactin subunit p62 (1:1000, Abcam), dynactin subunit p50 (1:5000, BD Biosciences), dynactin subunit Actin Related Protein 1 (ARP1, 1:1000, Sigma-Aldrich), dynein heavy chain (DHC, 1:500, Santa Cruz Biotechnology), dynein intermediate chain (DIC, 1:1000, Sigma-Aldrich), dynein light chain (DLC, 1:500, Santa Cruz Biotechnology), α-tubulin (TUBA, 1:10000, Abcam), acetylated α-tubulin (ac-TUBA, 1:10000, Abcam), tyrosinated α-tubulin (tyro-TUBA, 1:10000, Abcam), detyrosinated α-tubulin (detyro-TUBA, 1:10000, Abcam), Autophagy Related Protein ATG3 (ATG3, 1:1000, Cell Signaling), ATG5 (1:1000, Cell Signaling), ATG7 (1:1000, Cell Signaling), autophagy related protein LC3 (1:1000, Abcam), Lysosomal Associated Membrane Protein 1 (LAMP1, 1:1000, BD Biosciences), LAMP2 (1:1000, Abcam), synaptophysin (SYP, 1:10000, Millipore), Postsynaptic Density Protein 95 (PSD95, 1:1000, Sigma-Aldrich), AMPA receptor subunit GLUR1 (1:1000, Abcam), GLUR2 (1:1000, BD Biosciences), NMDA receptor subunit NR2A (1:250, BD Biosciences), NR2B ((1:500, BD Biosciences), and β-actin (1:5000, Sigma-Aldrich). Protein signals were visualized by IRDye secondary antibodies and Odyssey system (LI-COR Biosciences), and quantified with NIH ImageJ software.
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software). Data were presented as mean ± SEM. Statistical significance was determined by comparing means of different groups using unpaired t-test, one-way or two-way ANOVA followed by the post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Selective depletion of p150Glued expression in neurons
To selectively disrupt the expression of p150Glued, we initially generated Dctn1LoxP knock-in mice by inserting two LoxP sites in the first and fourth introns of mouse Dctn1 gene locus, respectively, resulting in a complete loss CAP-Gly domain and partial loss of basic domain (Fig. 1a-c). The Dctn1LoxP knock-in mice were then crossbred with Cre/Esr1 mice to generate Dctn1LoxP/LoxP; Cre/Esr1 pups and littermate controls for neuronal cultures. The administration of tamoxifen activated the estrogen receptor (ESR) tagged-Cre and removed the floxed exons, resulting in a selective ablation of p150Glued expression in cortical neurons cultured from postnatal day 0 (P0) Dctn1LoxP/LoxP; Cre/Esr1 pups (Fig. 1d, e). P150Glued proteins were identified by an antibody against the N-terminal 200 amino acids of p150Glued. By contrast, the levels of p135 and other N-terminal truncated p150Glued, called collectively as p135+, were substantially increased (Fig. 1d, e). The p135+ proteins were recognized by an antibody against the C-terminus of p150Glued. On the other hand, the other dynactin subunits–p62, p50, and ARP1 showed comparable expression levels in Dctn1LoxP/LoxP; Cre/Esr1 and control neuronal cultures (Fig. 1d, e). Therefore, genetic depletion of p150Glued increases p135+ expression, but does not affect the other dynactin subunit levels in neurons.
A lack of p150Glued does not affect the formation of dynein and dynactin complexes
Generation of p150Glued conditional knockout mice that lack p150Glued expression in the postnatal forebrain and spinal neurons
Our previous study suggests the G59S missense mutation in the MTBDs of p150Glued might cause the autosomal dominant motor neuron disease through a dominant-negative mechanism . On the one hand, the G59S substitution disrupts the normal folding and destabilizes the mutant p150Glued proteins. On the other hand, the residual mutant proteins might dimerize with the wild-type proteins and thereby compromises the overall function of p150Glued. However, the exact pathogenic mechanisms of G59S mutation remain controversial . To test the hypothesis that p150Glued is required for the survival of motor neurons, we decided to remove p150Glued from both CSMNs in the frontal cortex and SMNs in the ventral spinal cords by generating Dctn1LoxP/LoxP; Thy1-Cre cKO mice. In line with the early study , Cre was widely expressed by neurons in the forebrain regions (Additional file 1: Figure S1A), including olfactory bulb, frontal cortex, striatum, and hippocampus, as well as in the spinal cord (Additional file 1: Figure S1B). Although the expression of Cre was detectable in the brain extracts of P0 pups, a substantial reduction of p150Glued expression became apparent in 2-month-old Dctn1LoxP/LoxP; Thy1-Cre mice (Additional file 1: Figure S1C). The depletion of p150Glued restrictive to adult neurons may avoid any potential developmental defects in the p150Glued cKO mice.
P150Glued conditional knockout mice develop late-onset motor impairments
P150Glued conditional knockout mice develop late-onset, selective degeneration of SMNs
P150Glued depletion induces astrogliosis and microgliosis in the spinal cord of aged p150Glued cKO mice
Given that astrogliosis and microgliosis are often in accompany with motor neurodegeneration in the spinal cord , we stained the lumbar spinal sections with antibodies against astrogliosis marker GFAP and microgliosis marker IBA1. In line with previous findings , we observed a substantial induction of astrogliosis and microgliosis in the spinal cord of 18-month-old cKO mice (Fig. 5e, f).
P150Glued ablation causes severe neuromuscular defects in aged p150Glued cKO mice
A loss of p150Glued increases α-tubulin acetylation
A loss of p150Glued promotes autophagosome and lysosome protein accumulation
P150Glued and other dynactin subunits are more enriched in the postsynaptic sites compared to dynein motor proteins
P150Glued depletion leads to increased cell surface targeting of glutamate receptors and increased vulnerability to glutamate-induced excitotoxicity
In the current study, we generated and characterized a line of p150Glued cKO mice to critically evaluate the roles of CAP-Gly domain-containing dynactin p150Glued in postnatal neurons. A loss of p150Glued in postnatal neurons did not affect the assembly of dynein/dynactin motor protein complex in neurons, but caused rather selective SMN degeneration and movement impairments in aged mice. The depletion of p150Glued also led to substantial elevation of acetylated α-tubulin, glutamate receptors, and proteins involved in autophagosome and lysosome pathways in the cKO neurons. Finally, p150Glued ablation rendered spinal neurons more susceptible to excitotoxicity. Considering all the disease-causative mutations compromise the MTBD functions of p150Glued, increased excitotoxicity may underlie a common pathogenic mechanism of p150Glued mutations in diverse neurodegenerative diseases.
The absence of more drastic morphological and functional phenotypes in p150Glued cKO neurons suggests that p135 and other MTBD-lacking p150Glued splicing variants may largely substitute the functions of p150Glued in neurons. The CC1 domain of p150Glued mediates the interaction with DIC and other dynein motor protein complex . Since p135 and other p150Glued short forms contain the CC1 domain, it is not surprising that dynein and dynactin complex remains intact and appears mostly functional in neurons. P150Glued thereby is likely devoted to some special tasks in neurons. Neurons, especially the large projection neurons like CSMNs and SMNs, contain long axons. Microtubules form the backbone of these axons. P150Glued is proposed to play a critical role in maintaining the stability of microtubules by serving as an anti-catastrophe factor in neuronal cultures . However, we did not observe any substantial loss of axons in brain and spinal cord of presyptomatic p150Glued cKO mice. Instead, we found a substantial augmentation of acetylated α-tubulin in neural tissues, including the axons of SMNs. It has been well documented that the acetylation of α-tubulin improves the stability of microtubules . Therefore, the increased acetylation of α-tubulin may serve as a compensatory mechanism to maintain the integrity of microtubules in the absence of p150Glued. It will be interesting to delineate the signaling pathways leading to the increased α-tubulin acetylation in p150Glued cKO neurons.
In addition to maintaining microtubule stability, p150Glued is also proposed as a key player in uploading various cargos to the dynein motor complex in the axon terminals before being transported to the soma . For example, live imaging studies show p150Glued captures the cargos and loads to the microtubule plus ends for the minus-end-directed transport . Accordingly, we found a lack of p150Glued caused substantial disruption of NMJ structures in p150Glued cKO mice. Moreover, our studies suggest that p150Glued also plays a role in uploading postsynaptic glutamate receptors to dynein-mediated transport in dendrites. As results, p150Glued depletion led to increased accumulation of glutamate receptors in the postsynaptic sites, which makes the p150Glued cKO neurons more vulnerable to glutamate stimulation, suggesting increased excitotoxicity may contribute to the eventual loss of SMNs in p150Glued cKO mice. Future studies will be needed to investigate whether the disease-causative mutations also make neurons subject to excitotoxicity.
The dynein/dynactin complex is critical for organelle transport from cell periphery to the perinuclear sites along the microtubule networks, such as the centripetal movement of autophagosomes, lysosomes and endosomes . It has been shown in the previous studies that overexpressing the motor neuron disease–causative G59S mutant p150Glued impairs the autophagosome and lysosome pathways [1, 30]. In line with these earlier findings, we observed similar defects in abnormal accumulation of autophagosome and lysosome proteins in p150Glued-lacking SMNs. The disruption of autophagosome and lysosome-mediated protein turnover thereby could also lead to the SMN loss in aging.
While in this study we focused on the roles of p150Glued in CSMNs and SMNs, the floxed MTBD of p150Glued can also be removed from the other neuron and cell types at different time points based on the spatial and temporal expression pattern of Cre DNA recombinase. The availability of p150Glued cKO mice therefore provides a useful tool to study the functional significance of the CAP-Gly domain of p150Glued in vivo.
Multiple missense mutations in the CAP-Gly domain of dynactin p150Glued have been linked to lower motor neuron disease, and other degenerative neurological disorders. These disease-causative mutations seem to weaken the microtubule binding affinity of p150Glued. However, the functional significance of p150Glued has not been critically evaluated in vivo. In the present study, we generated and characterized a line of p150Glued cKO mice to critically evaluate the roles of CAP-Gly domain-containing dynactin p150Glued in postnatal neurons. The depletion of p150Glued caused selective spinal motor neuron degeneration and movement impairments in aged mice. Substantial elevation of acetylated α-tubulin, proteins involved in autophagosome and lysosome pathways, and glutamate receptors were observed in the cKO neurons, while higher glutamate receptor levels were correlated with increase susceptibility of spinal neurons to excitotoxicity. Increased excitotoxicity may underlie a common pathogenic mechanism of p150Glued mutations in diverse neurodegenerative diseases.
The authors thank Cai lab members for their various supports, China Scholarship Council (CSC) for its International Exchange Program, and Beijing Municipal Administration of Hospitals for its High-level Talents Program.
This work was supported in part by the intramural research programs of National Institute on Aging (AG000946), National Natural Science Foundation of China (No. 81601117), Beijing Natural Science Foundation (No. 7152077 and 7184221), Beijing Hundreds and Thousands of Talents Project (No. 2017A14), and Beijing Nova Program (No. xx2018099).
HC, JY, CL. HS conceived the research and designed the experiments. JY, CL, HS, CX, LS, C-XL, JD, and YL performed experiments. HC and JY analyzed data and wrote manuscript. CL critically reviewed manuscript. All authors read and approved the final manuscript.
All animal procedures conformed to the NIH guide for the ethical care and use of laboratory animals. Animal protocols were approved by the Institutional Animal Care and Use Committee of National Institute on Aging.
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