Dynactin p150Glued–Deficiency in Midbrain Dopaminergic Neurons Leads to Progressive Neurodegeneration and Endoplasmic Reticulum Dysfunction

doi:10.21203/rs.3.rs-1324919/v1

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Dynactin p150^Glued–Deficiency in Midbrain Dopaminergic Neurons Leads to Progressive Neurodegeneration and Endoplasmic Reticulum Dysfunction

https://doi.org/10.21203/rs.3.rs-1324919/v1

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Background

Multiple missense mutations in DCTN1 gene have been linked to Perry syndrome (PS), a rare neurodegenerative disease clinically manifested with parkinsonism, mental depression, and central hypoventilation. DCTN1 encodes dynactin p150^Glued protein, the largest subunit of dynactin motor protein complex. The PS-related mutations are concentrated at the amino-terminal cytoskeleton-associated protein and glycine-rich (CAP-Gly) domain, which mediates the association of p150^Glued with microtubules. Degeneration of nigrostriatal dopaminergic neurons (DANs) was reported in the postmortem brains of PS patients. However, how the dysfunction of p150^Glued affects the function and survival of DANs remains to be determined.

Methods

Using Cre-LoxP genetic manipulation, we crossbred Dctn1 knock-in (KI) mice with Th-Cre mice to generate Dctn1 conditional knockout (cKO) mice for genetic deletion of p150^Glued protein in catecholaminergic neurons, including midbrain DANs. We also crossbred the Dctn1 KI mice with Cre/Esr1 mice to generate pups for primary culture of Dctn1 cKO cells. We then performed series of biochemical, neurochemical, cell biology, behavioral and neuropathological studies on the Dctn1 cKO mice or cells to investigate the underlying pathological mechanisms.

Results

While both Dctn1 control and cKO mice showed deterioration of rotarod motor performance during aging, the process was greatly accelerated in the cKO mice. The loss of p150^Glued led to progressive degeneration of axon fibers and then cell bodies of midbrain DANs. In addition to neurodegeneration, abnormal accumulation of a-synuclein was found in the soma and nuclei of DANs in aged cKO mice. Moreover, before the onset of axon atrophy, both dopamine transporter (DAT) levels and activity were reduced in the DAN axon terminals of cKO mice. Further studies revealed unusually large spheric structures in the axons and dendrites of cKO DANs. Interestingly, endoplasmic reticulum (ER) stress protein BiP was abnormally accumulated in the dendritic spheroids and soma of cKO DANs. Accordingly, the Dctn1 cKO cells displayed impaired ER export and increased unfolded protein response (UPR). As a result, the Dctn1 cKO DANs were more vulnerable to ER stress-induced cell death.

Conclusion

Our findings demonstrate that p150^Glued is important in maintaining the function and survival of midbrain DANs during aging. We particularly highlighted the role of p150^Glued in stabilizing the molecular machinery for ER export, suggesting that the PS-related p150^Glued dysfunction may render the midbrain DANs more susceptible to UPR and ER stress.

Perry syndrome

dynactin p150Glued

GAP-Gly microtubule binding domain

midbrain dopaminergic neuron

neurodegeneration

unfolded protein response

ER stress

Dynein and dynactin motor protein complex mediates the retrograde intracellular transport along microtubule networks in neurons [1]. Genetic mutations in genes encoding components of dynein and dynactin motor protein complex often cause deficits in axonal transport and neurological disorders [2]. A missense mutation in the largest dynactin subunit p150^Glued, encoded by gene, has been linked to a form of slowly progressive autosomal dominant form of lower motor neuron disease, the distal hereditary motor neuropathy 7B (HMN7B) [3]. The mutation, a single-base pair change (957C-T) in the second coding exon of DCTN1 gene, results in an amino acid substitution of G59S in the conserved, cytoskeleton-associated protein and glycine-rich (CAP-Gly) domain of p150^Glued protein. Since then, a number of missense mutations (F52L, K56R, G67D, K68E, G71A/R/E/V, T72P, Q74P, Y78C/H, and Q93H) in the CAP-Gly domain of p150^Glued have been identified as the genetic cause of Perry syndrome (PS) [4], a rare hereditary neurodegenerative disease characterized by parkinsonism with severe mental depression, weight loss, and central hypoventilation [5–7]. Dysfunction of dopamine transmission and degeneration of nigral DANs were reported in PS patients [7], which might contribute to the Parkinson’s disease-like clinical symptoms. However, how these PS-related mutations affect the function and survival of nigrostriatal dopaminergic neurons (DANs) remains to be determined.

The PS-related missense mutations likely alter the conformation of CAP-Gly domain, which thereby interferes with the association of p150^Glued protein with microtubules and other microtubule end-binding proteins [8], resulting in reduced processivity of dynein and dynactin complex with microtubes [9–15]. While genetic deletion of p150^Glued in Dctn1 homozygous knockout mice causes early embryonic lethality [11], the CAP-Gly domain of p150^Glued seems dispensable for dynein/dynactin-mediated retrograde transport in postmitotic cells, like neurons. An alternative splicing variant, p135, which lacks the CAP-Gly domain of p150^Glued, may partially compensate for the loss of full-length p150^Glued protein in retrograde transport [16, 17]. These observations suggest the CAP-Gly domain of p150^Glued may possess some distinct properties in maintaining the function and survival of selective neuron types implicated in PS and other degenerative neurological disorders [7, 15]. In line with this notion, the spinal motor neurons appear to be more vulnerable to the loss of p150^Glued protein than corticospinal motor neurons [17]. Therefore, it would be imperative to understand the selective vulnerability and underlying mechanisms of different neuronal types in PS. Like the homozygous Dctn1 knockout (KO) mice [11], the homozygous G71A KI mice died embryonically [18], suggesting that the PS-related Dctn1 mutation also leads to the dysfunction of p150^Glued protein.

Various degrees of neuropathological abnormalities were identified in many different brain regions of PS patients [19]. In this study, we focused on investigating the functional significance of p150^Glued and its CAP-Gly domain in PS-related midbrain DAN degeneration using genetically engineered mouse models. Through crossbreeding Th-Cre transgenic mice [20] with Dctn1 knock-in (KI) mice [17], we generated Dctn1 conditional knockout (cKO) mice. The cKO mice lacked the CAP-Gly domain-containing p150^Glued full-length protein in midbrain DANs. We then performed series of in vitro and in vivo experiments to identify any behavioral, neurochemical, neuropathological, cell biology, and biochemical abnormalities in the cKO mice. Our findings highlighted the importance of CAP-Gly domain in maintaining the ER transport in DANs.

Animals

Dctn1^LoxP KI mice with two LoxP sites in intron 1 and 4 of Dctn1 were created as described previously [17]. Dctn1^LoxP KI mice were crossbred with Th-Cre [20] (MMRRC, Stock Number: 029177-UCD) or Cre/Esr1 [21] (JAX, Stock Number: 004682) mice to obtain Dctn1^LoxP//Cre mice. Dctn1^+/LoxP and Dctn1^+/LoxP/Cre mice were further crossbred to generate Dctn1^LoxP/LoxP/Cre mice in which exons 2 to 4 of Dctn1 and thereby the GAP-Gly domain-containing p150^Glued were deleted from the Cre-expressing cells. In this project, Dctn1^LoxP/LoxP/Th-Cre mice and littermate controls were used for in vivo study, and Dctn1^LoxP/LoxP/Cre/Esr1 pups and littermate controls were used for primary cell culture and for in vitro study. The mice were housed in a 12-hour light/dark cycle and fed regular diet ad libitum. All mouse work followed the guidelines approved by the Institutional Animal Care and Use Committees of the National Institute on Aging, NIH and Beijing Geriatric Hospital.

Genotyping

Animals were genotyped as described previously [17]. 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 Dctn1^LoxP/ 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).

Behavior tests

Open-field test–As described previously [17, 22], male mice were placed in the open-field apparatus with infrared photobeam sensors. Locomotor activities (including ambulatory, rearing and fine movements) and time spent in the center area (around 40% of the total surface of the arena) of 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 minutes.

Rotarod test–As described previously [17, 23], 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.

Immunohistochemistry, light microscopy, and image analysis

Mice were sacrificed and transcardially perfused with 4% paraformaldehyde (PFA) in cold phosphate buffered saline (PBS) as described previously [17, 23]. Mouse brains were collected, post-fixed in 4% PFA/PBS solution overnight, submerged in 30% sucrose in PBS for at least 72 hours, and sectioned at 40 µm thickness using CM1950 cryostat (Leica). Frozen sections were stained with antibodies specific to p150^Glued (amino acid 3-202 at the N-terminus of p150^Glued, 1:200, BD Biosciences), p150^Glued/p135+ (amino acid 1266-1278 at the C-terminus of p150^Glued, 1:500, Abcam), tyrosine hydroxylase (TH, 1:2500, Pel-freez; 1:500, Sigma-Aldrich; 1:500, ImmunoStar; 1:500, Synaptic Systems), dopamine transporter (DAT, 1:500, Millipore), vesicular monoamine transporter 2 (VMAT2, 1:1000, Covance), glial fibrillary acidic protein (GFAP, 1:1000, Abcam), TAR DNA binding protein 43 (TDP-43, 1:500, Proteintech), a-synuclein (1:500, Santa Cruz), binding immunoglobulin protein (BiP, also referred to as GRP78, 1:500, Abcam, ,and p-eIF2α (Ser52) (1:500, Thermo Scientific) as suggested by manufacturers. Alexa Fluor 488-, 546- or 647-conjugated secondary antibody (1:500, Invitrogen) was used to visualize the staining. Fluorescent images were captured using LSM 880 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 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

Unbiased stereological estimation of midbrain dopaminergic neurons was performed as described previously [24, 25]. According to the mouse brain in stereotaxic coordinates, series of 40-µm-thick coronal sections across the midbrain (every fourth section from Bregma −2.54 to −4.24 mm, ten sections per case) were stained with antibody specific to TH (1:2500, Pel-Freez) and subsequently visualized with Vectastain Elite ABC Kit and DAB Kit (Vector Laboratories). Bright field images were captured by Axio microscope Imager A1 (Zeiss). The number of TH-positive neurons was assessed using the optical fractionator function of Stereo Investigator 10 (MicroBrightField). Four or more 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 coefficient of error less than 10% in order to obtain reliable results.

High-performance liquid chromatography (HPLC)

HPLC for dopamine and DOPAC content in striatum was performed as described previously [25, 26]. Mouse dorsal striatum was dissected, weighted and homogenized in 500 µl buffer (0.1 N perchloric acid containing 100 µM EDTA) per 100 mg of tissue. After sonication and centrifugation, the supernatant was collected, frozen and stored at −80°C until assayed for dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) by liquid chromatography with electrochemical detection. Briefly, mobile-phase solution containing octanesulfonic acid as an ion-pairing agent was pumped isocratically through a reversed-phase liquid chromatographic column. Dopamine and DOPAC were quantified by the current produced after exposure of the eluate to a flow-through electrode set to oxidizing and then reducing potentials in series, with recordings from the last electrode reflecting reversibly oxidized species.

Fast-scan cyclic voltammetry (FSCV)

To investigate the kinetics of dopamine release evoked by electrical stimulation, FSCV was performed in 400-µm slices of dorsal striatum as described previously [25, 27, 28]. Striatal slices were bathed in 32°C oxygenated artiଁcial cerebrospinal ଂuid [aCSF: 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 25 mM NaHCO₃, 11 mM glucose, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.4 mM L-ascorbic acid]. Cylindrical carbon-fiber microelectrodes (50–100 µm of exposed fiber) were prepared with T650 fibers (6 µm diameter, Goodfellow) and inserted into a glass pipette. The carbon-fiber electrode was held at −0.4 V, and the potential was increased to 1.2 V and back at 400 V/s every 100 ms using a triangle waveform. Dopamine release was evoked by rectangular, electrical pulse stimulation (100–600 µA, 0.6 ms per phase, biphasic) applied every 5 min. Data collection and analysis were performed using the Demon Voltammetry and Analysis software suite [29]. Ten cyclic voltammograms of charging currents were recorded as background before stimulation, and the average of these responses was subtracted from data collected during and after stimulation. Maximum amplitudes of extracellular dopamine transients were obtained from input/output function (I/O) curves. I/O curves were constructed by plotting stimulus current against the concentration of dopamine response amplitude over a range of stimulus intensities. The time constant of the slope decay (τ) was used for uptake kinetic analysis of evoked dopamine release. Following experiments, electrodes were calibrated using solutions of 1 and 10 µM dopamine in aCSF. Cocaine hydrochloride and other chemicals used in FSCV were purchased from Sigma-Aldrich.

Primary midbrain neuronal culture

As described previously, mouse primary midbrain neuronal cultures were prepared from newborn Dctn1^LoxP/LoxP/Cre/Esr1 pups and littermate controls on postnatal day 0 (P0) [17, 24]. Briefly, midbrain tissues containing SNC and VTA were 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). Dissociated midbrain neurons (~ 3 × 10⁵ cells per coverslip) were plated onto 12-mm round coverslips precoated with poly-d-lysine and laminin (BD Bioscience) in 24-well plate, 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 5 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.

Assessment of dopaminergic neuron survival after treatment with ER stress inducer

The survival rate of dopaminergic neurons after treatment with ER stress inducer was assessed as described previously [24, 27]. 14 DIV primary midbrain neurons were exposed to 0 or 10 nM thapsigargin (Sigma-Aldrich), an ER stress inducing compound. After 48 hours of treatment, neurons were fixed with 4% PFA/PBS and immunostained with TH antibody and secondary antibody. The number of TH-positive neurons on each coverslip was counted under confocal microscope with a ×40 objective. The survival rate of dopaminergic neurons was calculated by dividing the number of TH-positive neurons on each coverslip by the number of TH-positive neurons on the control coverslip (neurons from Dctn1^LoxP/LoxP P0 pups treated with 0 nM thapsigargin).

Primary fibroblast culture

Mouse skin fibroblast cultures were prepared from the dorsal skin of newborn Dctn1^LoxP/LoxP /Cre/Esr1 pups and littermate controls on postnatal day 0 (P0) as previously described [30]. Briefly, skin tissues were collected, rinsed in sterile PBS and minced into small pieces. The minced tissues were triturated with 2 ml medium [DMEM (Invitrogen) supplemented with 10% FBS, 10 U/ml penicillin, and 10 µg/ml streptomycin], sparsely plated into 10-cm culture dish, and maintained at 37°C in a 95% O₂ and 5% CO₂ humidified incubator. After overnight (tissue fragments usually attach firmly to the dish), 8 ml fresh medium was added. After 3-4 days (fibroblasts grown out of tissue fragments usually start to undergo rapid proliferation), medium was changed. On 7 DIV, fibroblasts were trypsinized with TrypLE (Invitrogen), passaged into new dishes, and treated with 1 µM 4-hydroxytamoxifen (4-OHT) to induce CRE recombinase activity. From 11 DIV on, medium was changed every 3-4 days.

Immunocytochemistry

As described previously [24, 31], cultured cells on coverslips were fixed with 4% PFA in PBS for 15 minutes, permeabilized by 0.1% Triton X-100 for 5 minutes and blocked in 10% normal donkey serum (Invitrogen) in PBS for 1 hour at room temperature. Cells were then labeled with primary antibodies against p150^Glued (1:200, BD Biosciences), tyrosine hydroxylase (TH, 1:2000, Pel-freez; 1:500, Sigma-Aldrich; 1:500, ImmunoStar), microtubule-associated protein 2 (MAP2, 1:1000, Abcam), ERGIC53 (1:1000, Sigma-Aldrich), and a-tubulin (1:1000, Abcam) overnight at 4°C in a humidified chamber. After three washes with PBS, secondary antibodies conjugated to Alexa Fluor (Invitrogen; 1:1,000) were applied and incubated for 1 hour at room temperature in the dark. After extensive washes, coverslips were mounted on glass slides with Prolong diamond antifade reagent containing DAPI (Invitrogen), and fluorescence signals were detected using LSM 880 laser scanning confocal microscope (Zeiss). The paired images in all the figures were collected at the same acquisition settings, uniformly processed, presented as either a single optic layer or maximum-intensity projection of confocal stacks, and analyzed with ImageJ.

Preparation of cell fractions

Subcellular fractionation was performed using endoplasmic reticulum isolation kit (Sigma-Aldrich) according to the manufacturer's instruction. All procedures were performed at 4°C. Mouse midbrains were isolated, cut into small pieces, and homogenized in four volumes of ice-cold Isotonic Extraction Buffer (10 mM HEPES, pH 7.8, 250 mM sucrose, 25 mM KCl, and 1 mM EGTA, Protease and Phosphatase Inhibitor Cocktail) with Dounce homogenizer (12 strokes). Cultured cells were harvested, washed with 10 volumes of PBS, and spun down at 600 ⋅ g for 5 minutes. Cell pellet was suspended and incubated in 3 volumes of ice-cold Hypotonic Extraction Buffer (10 mM HEPES, pH 7.8, 25 mM KCl, and 1 mM EGTA, Protease and Phosphatase Inhibitor Cocktail) for 20 minutes to allow the cells to swell. Swollen cells were centrifuged at 600 ⋅ g for 5 minutes. The new cell pellet was homogenized in 2 volumes of ice-cold Isotonic Extraction Buffer with Dounce homogenizer (10 strokes). The homogenate (referred to as total lysate) from brain tissues or cultured cells was spun at 1,000 ⋅ g for 10 min. The supernatant (S1) was collected, and the pellet (P1) was saved as crude nuclei fraction. S1 was centrifuged at 12,000 ⋅ g for 15 min. The supernatant (S2) was collected, and the pellet (P2) was saved as crude mitochondria fraction. S2 was further centrifuged at 100,000 ⋅ g for 60 min. The supernatant (S3) was collected as cytosol fraction, and the pellet (P3) was saved as ER microsomes fraction. Crude nuclei fraction, crude mitochondria fraction and ER microsomes fraction were lysed in 1% SDS buffer. SDS was added into the total lysate and cytosol fraction to 1% final concentration. Equal amount of total protein from total lysate and each fraction were resolved in SDS-PAGE and applied to western blot analysis.

Western blot analysis

Whole-cell lysates were prepared as described previously [32]. Cultured cells or mouse 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 15 000 ⋅ g for 15 min at 4°C. The supernatants were quantified for protein content using the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific). Equal amount of total proteins was separated by NuPage 4-12% Bis-Tris gel (Thermo Scientific) using MES or MOPS running buffer (Thermo Scientific). The separated proteins were then transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer system (Bio-Rad) and incubated with specific primary antibodies. The antibodies used for western blot analysis included p150^Glued N-terminus (specific to p150^Glued, 1:1000, BD Biosciences), p150^Glued C-terminus (specific to both p150^Glued and p135+, 1:1000, Abcam), dynactin subunit p62 (1:1000, Abcam), dynactin subunit p50 (1:1000, BD Biosciences), dynactin subunit actin related protein 1 (ARP1, 1:1000, Sigma-Aldrich), tyrosine hydroxylase (TH, 1:5000, Pel-freez; 1:1000, Sigma-Aldrich; 1:1000, ImmunoStar; 1:5000, Synaptic Systems), dopamine transporter (DAT, 1:1000, Millipore), vesicular monoamine transporter 2 (VMAT2, 1:5000, Covance), VAMP (vesicle-associated membrane protein)-associated protein B (VAPB, 1:1000, Proteintech), calnexin (1:1000, Proteintech), Sec13 [component of the coat protein complex II (COPII), 1:1000, Santa Cruz], Sec23 (component of COPII, 1:1000, Thermo Scientific), Sec31 (component of COPII, 1:1000, BD Biosciences), protein kinase-like endoplasmic reticulum kinase (PERK, 1:1000, Cell Signaling), p-PERK (Thr982) (1:1000, Abcam), eukaryotic initiation factor 2α (eIF2α, 1:1000, Cell Signaling), p-eIF2α (Ser52) (1:1000, Thermo Scientific), activating transcription factor 4 (ATF4, 1:1000, Proteintech), C/EBP-homologous protein (CHOP, 1:1000, Cell Signaling), inositol requiring protein 1α (IRE1α, 1:1000, Abcam), p-IRE1α (Ser724) (1:1000, Abcam), unspliced X-box binding protein 1 (unspliced XBP1, 1:1000, Proteintech), spliced XBP1 (1:1000, Proteintech), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK, 1:1000, Cell Signaling), p-SAPK/JNK (Thr183/Tyr185) (1:1000, Cell Signaling), activating transcription factor (ATF6, 1:1000, Proteintech), cleaved Caspase 3 (1:1000, Cell Signaling), GAPDH (1:1000, Sigma-Adrich), α-tubulin (1:5000, Abcam), 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

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software). Data were presented as mean ± SEM. Statistical significance was determined by comparing means of different groups using unpaired t-test,one-way ANOVA with post-hoc Tukey test, two-way ANOVA with post-hoc Bonferroni test, or two-way ANOVA with Sidak's multiple comparisons test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Generation ofDctn1conditional knockout mice that selectively delete p150^Gluedin midbrain dopaminergic neurons

We crossbred Th-Cre mice [20] with Dctn1 LoxP (Dctn1^+/LoxP) KI mice [17] to generate Dctn1^+/+ [referred as wild-type (WT)], Th-Cre/Dctn1^+/+ (referred as Cre), Dctn1^Loxp/LoxP [referred as control (Ctrl)], and Th-Cre/Dctn1^LoxP/LoxP conditional knockout (referred as cKO) mice. The composition of offspring with different genotypes followed the Mendelian ratio, indicating a normal embryonic development of cKO mice. Western blot analyses revealed substantially reduced Dctn1 full-length p150^Glued protein, but markedly increased the levels of alternatively spliced p135 and related forms (p135+) in the midbrain tissues dissected from 1-month-old cKO mice compared to the age-matched WT, Cre and Ctrl mice (Fig. 1a, b). The Dctn1 p135+ forms lack the CAP-Gly domain [17]. By contrast, the levels of dynactin p62, p50, ARP1 subunits were not affected in the cKO mice (Fig. 1a). Additionally, the levels of p150^Glued and other dynactin components were comparable in the olfactory bulb, cerebral cortex, hippocampus, striatum, cerebellum and brainstem of Ctrl and cKO mice (Supplementary Fig. S1). The residual p150^Glued protein detected in the cKO samples is likely derived from the non-dopaminergic cells in the tissue preparations (Fig. 1a, b). Indeed, immunostaining of midbrain sections with an antibody against the CAP-Gly domain-containing N-terminal of p150^Glued demonstrated a complete loss of p150^Glued staining in the tyrosine hydrogenase (TH)-positive DANs (Fig. 1c). Therefore, we generated a line of Dctn1 cKO mice that disrupted the expression of p150^Glued in midbrain DANs.

In a parallel study, we crossbred the tamoxifen inducible Cre/Esr1 transgenic mice [21] with Dctn1^Loxp/+ KI mice and isolated the midbrain tissues from postnatal day 1 (P1) pups for neuronal cultures. Western blot and immunocytochemistry confirmed the loss of p150^Glued in the Cre/Esr1/ Dctn1^Loxp/LoxP cKO cells, including the DANs (Supplementary Fig. S2). The neuronal cultures allowed for in-depth cell biology and biochemical studies on the role of p150^Glued in intracellular transport.

Dctn1cKO mice exhibit more profound deterioration of rotarod performance than the controls during aging

The Dctn1 cKO mice developed normally and displayed no overt behavior phenotypes. Although weight loss is a typical clinical manifestation of PS patients [7], the cKO mice weighted similarly as the littermate Ctrl mice at 1, 3, 6, 12, and 18 months of age (Fig. 2a). The cKO mice also displayed normal locomotion, rearing, fine movement, and time spent in the center of arena in the Open-field test when examined at 1, 3, 6, 12, and 18 months of age (Fig. 2b-e). Since mouse models with deficiency in dopamine transmission are very sensitive to rotarod test [33, 34], we also tested the performance of cKO mice in accelerating rotarod at different time points during aging. While the cKO mice performed as well as the Ctrl mice at 1 and 3 months of age in the rotarod test, the performance of cKO mice deteriorated more profoundly than the Ctrl mice starting at 6 months of age (Fig. 2f). Considering that the nigral DANs are essential in rotarod motor skill learning [33, 34], this accelerated deterioration of rotarod performance could be a consequence of the progressive loss of DANs and dysfunction of dopamine transmission in the Dctn1 cKO mice during aging.

Dctn1cKO mice display progressive degeneration of DANs and other neuropathological abnormalities in the midbrain.

Using unbiased stereological cell counting, we found that while the numbers of TH-positive midbrain DANs in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) were comparable between 12-month-old Ctrl and Dctn1 cKO mice, the cell numbers were substantially reduced in the 24-month-old Dctn1 cKO mice compared to the age-matched Ctrl mice (Fig. 3a, b). Therefore, genetic deletion of p150^Glued in midbrain DANs leads to progressive neurodegeneration in both SNc and VTA regions.

The dendrites of DANs located in the ventral SNc often protrude perpendicularly into the underneath substantia nigra par reticulata (SNr) region (Fig. 3c) and form synaptic connections with the incoming axon fibers [35]. Interestingly, TH staining revealed some remarkably large sphere-like structures often at the tips of DAN dendrites in the SNr of 6- and 24-month-old Dctn1 cKO mice compared to the age-matched Ctrl mice (Fig. 3c, d). The diameters of those dendritic spheroids were often larger than 20mm, and the numbers were substantially increased during the aging of Dctn1 cKO mice (Fig. 3c, d). In addition to the neuronal loss and dendritic morphological changes, increased astrogliosis visualized by glial fibrillary acidic protein (GFAP) staining was also observed in the SNc and SNr of 24-month-old Dctn1 cKO mice (Supplementary Fig. 3a, b). Interestingly, a loss of nigral DANs and the appearance of sphere-like neurite swellings and gliosis were also observed in the substantia nigra (SN) and globus pallidus of PS patients [6, 19, 36]. Therefore, the Dctn1 cKO mice recapitulate some key neuropathological features of PS.

Abnormal somatic accumulation of a-synuclein but not TDP-43 in the midbrain DANs of agedDctn1cKO mice.

Since abnormal TAR DNA-binding protein 43 (TDP-43)-positive cytoplasmic inclusions were often identified in the SN and other brain regions of PS patients [6, 19, 36], we examined the subcellular location of TDP-43 in the midbrain DANs of 24-month-old Ctrl and Dctn1 cKO mice. However, we did not detect any apparent accumulation of TDP-43 in the cytosol of either Ctrl or Dctn1 cKO DANs (Fig. 4a, b). By contrast, although very sparse Lewy bodies were identified in the brain of PS patients [4, 5], we observed a substantial increase of cytoplasmic and nuclear accumulation of a-synuclein in the midbrain DANs of 24-month-old Dctn1 cKO mice compared to the Ctrl mice (Fig. 4c, d). While a-synuclein normally is enriched in the axon terminals, the abnormal cumulation of a-synuclein in cytosol and nucleus is implicated for neurodegeneration [23, 37, 38].

Progressive degeneration of DAN axon terminals in the dorsal striatum ofDctn1cKO mice.

As axon dystrophy was reported in the brains of PS patients [6, 19, 36], we examined the density and morphology of DAN axon terminals in the dorsal striatum of Ctrl and Dctn1 cKO mice. We found a markedly reduction of axon density in the 24-month-old cKO mice compared to the age-matched controls (Fig. 5a, b). Furthermore, increasing numbers of abnormal axon swelling were observed in the 6- and 24-month-old cKO mice compared to the age-matched controls (Fig. 5c, d). Interestingly, the swellings were packed with vesicular monoamine transporter 2 (VMAT2)-positive puncta (Fig. 5c), suggesting potential abnormalities in dopamine transmission in the dorsal striatum of Dctn1 cKO mice.

Alterations of dopamine release and uptake in the dorsal striatum ofDctn1cKO mice.

Using HPLC, we did not detect any changes of dopamine (DA), dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxytryptamine (5-HT) content in the dorsal striatum of 12-month-old Dctn1 cKO mice compared to controls (Fig. 6a). Correlatively, no apparent loss of DAN axon fibers was found in the same age group of Dctn1 cKO mice (Fig. 5b). We then quantified the evoked dopamine release by fast-scan cyclic voltammetry (FSCV) in striatal slices prepared from 12-month-old Dctn1 Ctrl and cKO mice. In response to either single- or burst (25Hz)-pulse stimulation, the peak evoked dopamine release was substantially higher from the cKO axon terminals compared to the controls (Fig. 6b, c). Additionally, the time constant of the slope delay, which measures the kinetics of dopamine uptake, was also increased in cKO axon terminals (Fig. 6d). Together, these data suggest that the loss of p150^Glued might reduce dopamine uptake at the Dctn1 cKO axon terminals, resulting in elevated dopamine concentration in the extrasynaptic space upon stimulation.

Reduced dopamine transporter expression and activity in the dorsal striatum of youngDctn1cKO mice.

Since dopamine transporter (DAT) mediates the uptake of dopamine [39], we examined the protein levels of DAT, VMAT2, and TH in the striatal homogenates of 6-month-old Dctn1 Ctrl and cKO mice. The expression of DAT, but not VMAT2 or TH, was substantially reduced in the cKO samples (Fig. 7a, b). Immunostaining further confirmed a marked reduction of DAT-positive puncta in the dorsal striatum of cKO mice (Fig. 7c, d). In correlation with the reduced DAT expression, when pharmacologically blocked with DAT inhibitor cocaine, the increase of dopamine release was less robust from the cKO axon terminals compared to the controls (Fig. 7e). Together, these results demonstrate that the lack of p150^Glued leads to reduced expression and activity of DAT in DANs. Interestingly, reduced DAT ligand binding and activity were also observed in the striatum of PS patients [5].

Abnormal accumulation of endoplasmic reticulum stress proteins in the dendritic spheroids and soma ofDctn1cKO midbrain dopaminergic neurons.

To investigate the composition of neurite spheroids, we co-stained the midbrain sections with DAT, TH and various intracellular organelle markers and found that binding immunoglobulin protein (BiP), a residential protein in the lumen of endoplasmic reticulum (ER), was enriched in the dendritic spheroids of nigral DANs of 6- and 24-month-old Dctn1 cKO mice (Fig. 8a, b). Furthermore, we also observed abnormal accumulation of BiP in the soma of cKO DANs during aging (Fig. 8c, d). BiP functions as molecular chaperon in protein folding, translocation, ER-associated degradation, and ER stress response [40, 41]. The abnormal accumulation of BiP in the soma and neurite of DANs indicates impairments of ER functions in the Dctn1 cKO DANs.

P150^Glueddeficiency compromises ER export and protein maturation.

A previous study demonstrates that p150^Glued stabilizes the coat protein complex II (COPII) at ER exit site (ERES) through direct interaction with COPII component Sec23 [42, 43]. Consistent with the early findings, we found that the levels of Sec23 and other COPII protein Sec13 and Sec31 were markedly reduced in the ER microsomes isolated from cultured Dctn1 cKO fibroblasts (Fig. 9a, b). Furthermore, while the ERES is normally concentrated at one side of nucleus in the control fibroblasts as visualized by the ER-Golgi intermediate compartment 53 kDa protein (ERGIC-53) immunostaining, the ERES was dispersed surrounding the nucleus of Dctn1 cKO fibroblasts (Fig. 9c). The reduction of Sec13 and Sec31 levels was also observed in the ER microsomes isolated from the midbrain of 4-month-old Dctn1^LoxP/LoxP/CreEsr1 inducible conditional knockout (icKO) mice compared to the control Dctn1^LoxP/LoxP (Ctrl) mice (Fig. 9d, e). The concentration of COPII at ERES is critical for ER export of secretory and membrane proteins [44]. The relative levels of mature verse immature forms can be used as an indicator to estimate the efficiency of ER export in cells [31]. Accordingly, western blots showed substantial increase of immature DAT in the ER microsome fraction extracted from the midbrain of Dctn1 icKO mice compared to the controls (Fig. 9d, e), indicating that more immature DAT protein is trapped in the ER of p150^Glued-deficient DANs. The retention of immature DAT in ER may explain the reduction of DAT protein in the axon terminals of Dctn1 cKO mice (Fig. 7). Therefore, our studies in Dctn1 cKO fibroblasts and icKO midbrain tissues further support the notion that the lack of p150^Glued destabilizes the COPII complex at ERES and compromises the ER export [42, 43].

P150^Glued-deficiency activates unfolded protein response and exacerbates ER stress-induced dopaminergic neuron death.

Since the accumulation of unfolded proteins in ER and deficiency in ER export trigger unfolded protein response (UPR) and ER stress [45], we examined the activation of a series of proteins implicated in the UPR pathway by western blot analyses. We found substantially increased phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor-2α (eIF2a), inositol-requiring enzyme 1 α (IRE1a), and stress-activated protein kinase (SAPK) in the cKO cell lysates compared to the controls (Fig. 10a, b). We also observed upregulation of activating transcription factor 4 and 6 (AF4 and ATF6), CCAAT-enhancer-binding protein homologous protein (CHOP), spliced X-Box Binding Protein 1 (XBP1) and cleaved Caspase 3 in the cultured Dctn1 cKO fibroblasts (Fig. 10a, b). We further detected a markedly increase of phosphorylated eIF2a (p- eIF2a) in the midbrain DANs of 18-month-old Dctn1 cKO mice (Fig. 10c, d). Finally, we found that the cultured Dctn1 cKO midbrain DANs were more susceptible to thapsigargin-induced ER stress and cell death (Fig. 10e). Together, these findings suggest an important function of p150^Glued in protecting DANs against ER stress.

In the present study we generated and characterized Dctn1 cKO mice that genetically delete the CAP-Gly domain-containing p150^Glued protein in midbrain DANs. Neuropathologically, the lack of p150^Glued led to progressive loss of midbrain DANs, formation of large neurite spheroids, axon atrophy, and abnormal accumulation of a-synuclein in the soma and nuclei. Behaviorally, the cKO mice showed accelerated deterioration in rotarod motor performance compared to the control mice during aging. Furthermore, we found that the loss of p150^Glued particularly affected the expression and subcellular localization of ERES proteins. The impairment of COPII-mediated ER export likely contributes to deficiency in ER-dependent protein maturation and export and increase of UPR and ER stress. Finally, we provide evidence that a lack of p150^Glued makes DANs more susceptible to ER stress, suggesting that ER stress may contribute to the loss of DANs in PS.

P150^Glued, the largest subunit of dynactin complex, exists as a dimer that binds directly to both microtubules and cytoplasmic dynein intermediate chain [13]. Previous in vitro studies demonstrate that p150^Glued is critical for movement of mitotic spindle and nucleus during cell proliferation [12]. Accordingly, we found that genetic deletion of p150^Glued causes early embryonic lethality of Dctn1 homozygous KO mice [11]. However, genetic deletion of p150^Glued in postmitotic neurons does not lead to overt and widespread neuronal loss in mouse brains [17], supporting the notion that the cellular functions of p150^Glued can be largely replaced with its CAP-Gly domain-lacking alternative splicing variants in neurons [16]. Nonetheless, since the HMN7B and PS-linked missense mutations all reside in the CAP-Gly domain of p150^Glued [3, 4], the CAP-Gly domain-containing p150^Glued protein must participate in some distinct cellular processes important in maintaining the function and survival of spinal motor neurons and midbrain DANs during aging. Given that both Dctn1 homozygous G59S and G71A KI mice show the same early embryonic lethality as the Dctn1 homozygous KO mice [11, 18], the HMN7B and PS-linked missense mutations must compromise the function of p150^Glued. Dynein/dynactin complex is critical for an array of cellular activities, including ER-to-Golgi transport, the centripetal movement of lysosomes and endosomes, spindle formation, chromosome movement, nuclear positioning, and axonogenesis [12]. Therefore, selective deletion of p150^Glued in the disease-affected neuronal populations may provide a useful experimental platform to identify the key intracellular pathways responsible for their differential vulnerabilities in HMN7B and PS [17].

In a recent study, we genetically deleted p150^Glued in the postnatal forebrain and spinal neurons, including both corticospinal and spinal motor neurons [17]. The aged p150^Glued-deficient mice showed impairments in rearing and rotarod tests, as well as a preferential degeneration of spinal motor neurons. Further histological and biochemical assays suggested that p150^Glued may exert its protective function through regulating the transportation of autophagosomes, lysosomes, and postsynaptic glutamate receptors in spinal motor neurons. By contrast, when we genetically deleted p150^Glued in midbrain DANs, we did not observe overt upregulations of autophagosome and lysosome markers (data not shown). Instead, we found that the lack of p150^Glued particularly affected the ER function as documented with excessive accumulation of ER chaperone BiP in dendritic spheroids and soma, reduced levels of COPII components, deficiency in ER export, and increase of UPR and ER stress. These findings further support an important role of p150^Glued in the functional coupling of ER export to microtubules [43]. Since the carboxyl terminal of DAT protein also binds to COPII component SEC24 and genetic knockdown of SEC24 impairs the cell surface targeting of DAT [46–48], the impaired ER export likely also contributes to the increased accumulation of immature DAT protein in the ER and reduced DAT level in the axon terminals of p150^Glued–deficient DANs.

While we focused the present study on the role of p150^Glued in midbrain DANs and PS-related parkinsonism, it remains to be determined the underlying pathological mechanisms of PS-related psychiatric symptoms such as depression and apathy, which appeared as an early clinical feature prior to parkinsonism [4]. Except for the loss midbrain DANs, the postmortem brains of PS patients also displayed variable degrees of neurodegeneration in other nuclei in basal ganglia, as well as in hypothalamus, locus coeruleus (LC), periaqueductal gray matter, ventrolateral medulla, dorsal raphe nucleus, and pontine reticular formation [19]. Although the norepinephrine neurons in LC also express TH, the Th-Cre transgenic mice used in the present study showed a mosaic expression pattern of Cre DNA recombinase in the LC, in which around 30% of LC neurons contained Cre but no obvious loss of LC neurons was found (data not shown). Future studies are needed to crossbreed the Dctn1 KI mice with different Cre lines to continue exploring the role of p150^Glued in other cell types. For example, the loss serotonergic neurons in the medullary raphe and ventrolateral medulla [49] may be responsible for the respiratory failure seen in PS patients. It would be interesting to test this hypothesis with a selective deletion of p150^Glued in those serotonergic neurons using a suitable Cre line.

While the TDP-43–positive cytoplasmic inclusions were often identified in the SN and other brain regions of PS patients [5, 6, 19, 36], we did not detect any apparent accumulation of TDP-43 in the cytosol of Dctn1 cKO DANs, suggesting that TDP-43 pathology might not contribute to the loss of midbrain DANs during aging. Nonetheless, future studies would be needed to determine whether the TDP-43 pathology contributes to the degeneration of other neuronal subtypes. In contrast, although the a-synuclein–containing Lewy bodies were rarely spotted in the postmortem brain of PS patients [4, 5], we observed a substantial increase of cytoplasmic and nuclear accumulation of a-synuclein in the midbrain DANs of aged Dctn1 cKO mice. Our findings are in line with previous studies which suggest that the abnormal somatic and nuclear cumulation of a-synuclein contributes to neurodegeneration [23, 37, 38]. However, it remains to determine how the lack of p150^Glued in the DANs leads to the retention of a-synuclein in cell bodies

To understand how the dysfunction of p150^Glued protein contributes to PS-related parkinsonism and DAN loss, we genetically deleted p150^Glued in the midbrain DANs of Dctn1 cKO mice. The cKO mice recapitulated some key neuropathological features of PS, such as the degeneration of midbrain DANs, dysfunction of dopamine transporter DAT, axonal atrophy, appearance of large neurite spheroids and gliosis. We further revealed an important role of p150^Glued in regulating COPII vesicles for ER export, suggesting that the lack of p150^Glued may subject the midbrain DANs to UPR and ER stress-related cell death. Our studies raise the significance of ER stress in the degeneration of DANs. The Dctn1 cKO mice may serve as a useful mouse model to further investigate the pathogenic mechanism of midbrain DAN loss in Parkinson’s disease.

HMN7B

hereditary motor neuropathy 7B

DCTN1

dynactin 1

CAP-Gly

cytoskeleton-associated protein and glycine-rich

Perry syndrome

DAN

dopaminergic neuron

knockout

cKO

conditional knockout

knock-in

PFA

paraformaldehyde

DAT

dopamine transporter

tyrosine hydroxylase

VMAT2

vesicular monoamine transporter 2

GFAP

glial fibrillary acidic protein

TDP-43

TAR DNA binding protein 43

BiP

binding immunoglobulin protein

HPLC

high-performance liquid chromatography

DOPAC

3,4-dihydroxyphenylacetic acid

FSCV

fast-scan cyclic voltammetry

aCSF

artificial cerebrospinal fluid

BCA

bicinchoninic acid

VAPB

VAMP (vesicle-associated membrane protein)-associated protein B

COPII

component of the coat protein complex II

Sec13, 23, and 31

components of COPII

PERK

protein kinase-like endoplasmic reticulum kinase

eIF2a

eukaryotic initiation factor 2a

p-eIF2a

phosphorylated eIF2a

ATF4 and 6

activating transcription factor 4 and 6

CHOP

CCAAT-enhancer-binding protein homologous protein

IRE1a

inositol requiring protein 1a

XBP1

X-box binding protein 1

SPARK/JNK

stress-activated protein kinase/Jun-amino-terminal kinase

Ctrl

control

SNc

substantia nigra pars compacta

VTA

ventral tegmental area

SNr

substantia nigra par reticulata

5-HT

5-hydroxytryptamine

endoplasmic reticulum

ERES

ER exit site

ERGIC-53

ER-Golgi intermediate compartment 53 kDa protein

UPR

unfolded protein response

locus coeruleus

Ethics approval

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 and Beijing Geriatric Hospital.

Consent for publication

All authors have read the manuscript and indicated consent for publication.

Availability of data and materials

All reagents and animal models will be available upon request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported in part by the intramural research programs of National Institute on Aging (AG000946), National Natural Science Foundation of China (No. 82071438 and 81601117), Beijing Natural Science Foundation (No. 7202077 and 7152077), Beijing BaiQianWan Talents Project (No. 2017002), and Beijing Nova Program (No. xx2018099).

Authors’ contributions

HC, JY, and CS conceived the research and designed the experiments. JY, CS, XY, BS, JZ, KL, and LS performed experiments. HC, CS and JY analyzed data and wrote manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank Dr. David M Lovinger of National Institute on Alcohol Abuse and Alcoholism for assisting the FSCV recording, members of Cai and Yu Labs for various supports, China Scholarship Council (CSC) for its International Exchange Program, and Beijing Hospital Authority for its Science & Technology Innovation Program and High-level Talents Program.

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Dynactin p150^Glued–Deficiency in Midbrain Dopaminergic Neurons Leads to Progressive Neurodegeneration and Endoplasmic Reticulum Dysfunction

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Abstract

Background

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Introduction

Methods

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Genotyping

Behavior tests

Immunohistochemistry, light microscopy, and image analysis

Stereology

High-performance liquid chromatography (HPLC)

Fast-scan cyclic voltammetry (FSCV)

Primary midbrain neuronal culture

Assessment of dopaminergic neuron survival after treatment with ER stress inducer

Primary fibroblast culture

Immunocytochemistry

Preparation of cell fractions

Western blot analysis

Statistical analysis

Results

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