- Open Access
Blocking meningeal lymphatic drainage aggravates Parkinson’s disease-like pathology in mice overexpressing mutated α-synuclein
© The Author(s). 2019
- Received: 7 September 2018
- Accepted: 15 February 2019
- Published: 1 March 2019
Abnormal aggregation of brain α-synuclein is a central step in the pathogenesis of Parkinson’s disease (PD), thus, it is reliable to promote the clearance of α-synuclein to prevent and treat PD. Recent studies have revealed an essential role of glymphatic system and meningeal lymphatic vessels in the clearance of brain macromolecules, however, their pathophysiological aspects remain elusive.
Meningeal lymphatic drainage of 18-week-old A53T mice was blocked via ligating the deep cervical lymph nodes. Six weeks later, glymphatic functions and PD-like phenotypes were systemically analyzed.
Glymphatic influx of cerebrospinal fluid tracer was reduced in A53T mice, accompanied with perivascular aggregation of α-synuclein and impaired polarization of aquaporin 4 expression in substantia nigra. Cervical lymphatic ligation aggravated glymphatic dysfunction of A53T mice, causing more severe accumulation of α-synuclein, glial activation, inflammation, dopaminergic neuronal loss and motor deficits.
The results suggest that brain lymphatic clearance dysfunction may be an aggravating factor in PD pathology.
- A53T transgenic mice
- Glymphatic clearance
- Parkinson’s disease
Parkinson’s disease (PD) is an age-dependent neurodegenerative disease, characterized by progressive loss of midbrain dopaminergic neurons and formation of Lewy bodies [1, 2]. Although the pathogenesis of PD has not been completely elucidated, excessive accumulation of toxic forms of α-synuclein (α-syn) with a series of secondary pathological cascades plays crucial roles in the onset of PD. This is mainly due to an imbalance between production and clearance of α-syn in the brain [3, 4]. A small part of patients with early-onset PD show increased α-syn production because of mutations in the α-syn gene, while the vast majority of PD patients suffer from decreased removal of α-syn from the brain [5, 6]. Previous studies have reported that α-syn is degraded through various intracellular clearance mechanisms including autophagy lysosomal pathways [7–9]. However, it is unclear whether there is an extracellular route to remove soluble α-syn directly from the brain. Exploring this issue will help to discover potentially new strategies for the prevention and treatment of PD.
Recent findings have suggested that brain lymphatic drainage system including a brain-wide network of paravascular channels, termed the ‘glymphatic system’ and meningeal lymphatic vessels drain macromolecules from the brain parenchyma to the deep cervical lymph nodes (Dclns) [10, 11]. Further evidence suggests that glymphatic function depends on astroglial aquaporin-4 (AQP4), contributing to a major portion of brain soluble amyloid-β (Aβ) clearance . Impaired glymphatic clearance exacerbates Aβ deposition in the brain of a mouse model of Alzheimer’s disease (AD) . In addition, the glymphatic system facilitates convective exchange of various interstitial solutes including glucose, lipids, amino acids, lactic acid and neurotransmitters between cerebrospinal fluid (CSF) and interstitial fluid (ISF) [14, 15]. Furthermore, disruption of meningeal lymphatic drainage aggravates parenchymal Aβ accumulation and cognitive decline of AD transgenic mice [16, 17]. However, it remains unclear whether glymphatic clearance dysfunction is involved in brain α-syn aggregation and related pathology.
To address this question, we investigated glymphatic clearance function of A53T mice overexpressing mutated human α-syn. The consequence of blocking meningeal lymphatic drainage on α-syn-related pathophysiology was also observed in the PD animal model. The finding suggests that impaired brain lymphatic clearance is involved in PD pathology.
A53T mice were constructed by Nanjing University . Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-fold higher than that in the controls as determined by quantitative PCR. Genotyped male mice were housed under standard conditions (room temperature 18 ~ 22 °C, humidity 30 ~ 50%, well-ventilated, a 12-h light-dark cycle). In addition, three-month old male AQP4 knockout (AQP4−/−) mice in a CD1 genetic background  were used to evaluate the effect of Aqp4 gene deletion on removal of injected exogenous α-syn clearance from the brain parenchyma. All efforts were made to minimize and reduce number of animals used. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Ligation of the deep cervical lymph nodes (LDclns)
The procedure of LDclns was performed according to published literature . Briefly, after anesthetized with 4% chloral hydrate, 18-week-old A53T mice and WT mice were fixed on a stereotaxic apparatus in a supine position, and longitudinally incised along the midline of the neck (approximately 1 cm). Under a microscope, fat and soft tissue were separated with a blunt forceps. Then, the sternocleidomastoid muscles were retracted and both sides of the dCLNs were exposed. Their afferent vessels were carefully ligated using 8–0 nylon suture. The mouse skin was sutured and disinfected with iodophor. Sham-operated mice were only surgically exposed the dcLNs without ligation. The animals were returned to their cages after recovery from anesthesia and continued to be fed for 6 weeks, followed by behavioral testing or CSF tracer experiments.
The experiment was used to assess autonomic motor ability in mice . Mice were placed in an open box (length: 60 cm, width: 60 cm; height: 25 cm) and allowed to move freely for 5 min. The total distance traveled was recorded by an open field software (Clever Sys Inc., VA, USA). After each test, the box was cleaned for avoiding the odors that might affect testing results of the next animal.
Rotarod test was used to detect the ability of motor coordination on a rotating rod (diameter 3.2 cm) . Mice were trained for 5 min, 3 times a day for 3 days. The speed started at 4 rpm and accelerated at 0.1 rpm/s. The test was performed three times per mouse, and the time of mouse falling from the rotarod was recorded. The mean time for 3 independent experiments was then calculated.
Pole test was used to evaluate autonomic ability of mice . A straight wooden bar of 1 cm in diameter and 50 cm in length was made. The top of the stick was fixed with a wooden ball (about 1.2 cm in diameter). The ball was wrapped around the gauze to prevent the animal from slipping off. The mouse was placed on the top of the rod, the time for completing turn of the head (T-turn) and the total time from the top to the bottom of the rod (T-total) were recorded. If the mouse failed to flip or slip completely, the time was recorded as 120 s. All mice were trained three times per day for three days prior to the formal testing.
Intracisternal infusion of fluorescent tracer
Intracisternal infusion of fluorescent tracer was performed as previously described . Anesthetized mouse was fixed in a stereotaxic instrument, and the posterior atlanto-occipital membrane was exposed. Five microliter of Texas Red-dextran-3 (TR-d3, MW 3 kD; Invitrogen; Cat # D3328) at 0.5 mg ml− 1 in artificial CSF was infused into the cisterna magna through a 50 μl syringe mounted with a 27-gauge needle, connected to a constant current syringe pump. The intracisternal infusion was carried out at a rate of 2 μl/min. Thirty min after the start of infusion, the mouse was anesthetized again and perfused trans-cardially with 4% paraformaldehyde (PFA). The brains and dcLNs were removed and post-fixed in the same fixative for 24 h.
Stereotaxic injection of α-syn
AQP4−/− mice and WT mice were given stereotaxic injection of soluble recombinant human α-syn. An injection cannula (27 gauge) was inserted stereotaxically into substantia nigra (SN) (anteroposterior: − 3.0 mm; mediolateral: ±1.3 mm; dorsoventral: − 4.2 mm) . The infusion was performed at a rate of 0.25 μl/min, and 2.5 μl of recombinant human α-syn Ala30-Pro (A30P) or α-syn A53T (Sigma-Aldrich; Cat # S1196; Cat # S1071; 3 μg/ml in phosphate buffer solution (PBS)), or the same volume of PBS were injected into the mouse. At 30, 60 or 120 min after injection, mice were sacrificed for enzyme-linked immunosorbent assay (ELISA) to detect injected α-syn residue in the brain. Western blotting and immunofluorescence were also performed on ventral midbrain samples at the time point of 120 min.
Brain water content measurement
Mice were anesthetized and brains were removed, weighed, heated in an oven at 70 °C for 72 h and reweighed. Percentage water content was calculated as [(wet weight–dry weight)/wet weight × 100%] .
For section preparation, all mice were anesthetized with 4% chloral hydrate, and perfused with saline for 3 min followed by 4% PFA for 5 min. Brain tissues and Dclns were fixed in 4% PFA overnight after perfusion and dehydrated with 20% sucrose solution dissolved in 0.01 M PBS for 3 days. 30 μm slices were cut coronally throughout the midbrain on a cryostat (Leica, Wetzlar, Germany) and mounted onto gelatin-coated slides. The lymph nodes were also cryostat sliced at 30 μm. For CSF tracer experiments, PFA post-fixed midbrain tissues were sliced on a vibratome (Leica) at 100 μm and mounted onto gelatin-coated slides in sequence.
Cerebral dura mater stripping
The skull was separated into the upper half and lower half utilizing microsurgical scissors. A complete skull obtained after brain removed was placed in 20% sucrose for 3 days and then transferred into sterilized water for 2 days. Under a stereomicroscope, the dura mater was carefully dissected from the skull using a pair of ophthalmic forceps, then unfolded on glass slide covered with poly-l-lysine staining.
Hematoxylin-eosin (H&E) staining
Lymph nodes were stained with hematoxylin solution for 5 min and washed with distilled water for 1 h, then placed in 70 and 90% alcohol for 10 min, respectively. The sections were stained with Eosin solution for 2–3 min, dehydrated with pure alcohol, transparented with xylene, and sealed by neutral gum.
Immunohistochemical staining was performed as previously described . The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MAB360) and rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, Wako, Cat # 019–19,741). After rinsing with PBS, the sections were incubated with biotinylated goat anti-mouse or rabbit IgG (11000) for 1 h at room temperature and thereafter incubated with streptavidin-biotin-peroxidase complex (Elite ABC Kit, Vector Laboratories, Cat # PK-7200) and visualized with 2,2′-diaminodiphenyl.
Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody.
Cell counting and image analysis
Immunohistochemically stained sections were observed by an Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA) with an optical fractionator (Stereo Investigator 7, MBF bioscience, Williston, VT, USA). The numbers of TH-, GFAP- and Iba-1-positive cells in SN were assessed as described previously . Briefly, regions of each side of SN were outlined at low magnification (40×). The counting frame size was 50 μm × 50 μm and the sampling grid size was 100 μm × 100 μm. The sampling scheme was designed to have coefficient of error (CE) less than 10% in order to get reliable results. The number of immunoreactive cells mentioned above was counted on 13 serial sections per mouse respectively. The data were presented as the mean of 4 mice in each group. Percentage of GFAP or Iba-1P positive area was also counted by NIH Image J.
The midbrain sections with immunofluorescence and CSF fluorescence tracer were photographed by a digital microscope (Leica Microsystems, Wetzlar, Germany). The image was analyzed using Image J and fluorescence signal of AQP4, α-syn and TR-d3 was measured respectively, by using the interest grayscale threshold analysis . Percentage of fluorescence signal positive area in midbrain was calculated. The AQP4 polarization in SN was obtained by the ratio between the AQP4 fluorescence intensity of the perivascular domains and the adjacent neutrophil domains [27, 28]. Each index was averaged from 6 to 8 midbrain sections per mouse in each group of 4 mice. The images were acquired and analyzed by a double-blind investigator.
Brain homogenate preparation
Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 μl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min at 16000 rpm . To analysis insoluble α-syn oligomeric protein, 5 volumes of Triton X-100 insoluble lysate were added to the precipitated protein obtained from centrifugation, sonication, boiling at 95 °C for 20 min, and centrifuged again for additional 10 min at room temperature . Protein concentration of the samples was determined by a bicinchoninic acid protein assay kit (Kevgen Biotech, Cat # ab6672). Bovine). Bovine serum albumin (1 mg/ml) was used as standard protein.
The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h . Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit anti-phosphorylated Tau Ser396/Ser404 (PHF-1, 1:1000, Abcam, ab184951) then followed by overnight incubation at 4 °C. The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, and signals were detected by chemiluminescence (ECL, Pierce, USA). Images were acquired with Image Quant LAS 4000 mini (GE Healthcare, USA) and analyzed using Image J. Analyses were completed in duplicate and the mean value was calculated.
Soluble and insoluble α-syn from ventral midbrain samples were quantified using a commercial ELISA kit (Abcam, Cat # ab210973). The assay procedure was carried out according to the manufacturer’s instructions. Briefly, samples or standards were added to the 96-well plate and mixed with the antibody. After incubation, the wells were washed to remove unbound material. 3,3′5,5′-tetramethylbenzidine (TMB) substrate was added and incubation was catalyzed by HRP to generate blue colorimetric reaction. The reaction was stopped with 100 μl 1 M HCl and absorbance was read at 450 nm using a 96-well microplate reader (GE Healthcare, USA). All measurements were performed in duplicate and the mean value was calculated.
Mice were perfused from the left cardiac ventricle with cold PBS for 10 min. Meninges were dissected with fine forceps and digested in RPMI-1640 medium (Sigma-Aldrich, Cat # R0883) + 1.4 U/ml Collagenase VIII (Sigma-Aldrich, Cat # C2139) + 1 mg/ml DNase1 (Sigma-Aldrich, Cat # 11119915001) for 15 min at 37 °C . Dclns were removed and smashed in dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal calf serum. Digested meninges and smashed Dclns were dissociated using pipetting, and were passed through 70-μm filters. Cell suspension was incubated with antibody dye CD3 (1:400, eBioscience, Cat # 145-2C11), CD4 (1:400, eBioscience Cat # 17–0041-83) or CD8 (1:400, BD Bioscience Cat # 335787) for 30 min at 4 °C. Data were acquired on an LSRII cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using the Flowjo Pro software (FlowJo, LLC, Ashland, OR, USA). The number of cells used for each analysis was 10,000 for the meningeal samples and 50,000 for the Dclns. Mean value of each sample was obtained from three independent experiments.
All data were expressed as means ± SEM. Statistical analyses were performed with Prism 6 (Graph-Pad, La Jolla, CA, USA). Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test or Student-t test as indicated in the figure legends. p < 0.05 was considered to have statistical significance.
LDclns blocks meningeal lymphatic drainage
LDclns decreases glymphatic influx of CSF tracer
We then observed interactive effects of LDclns and α-syn overexpression on glymphatic transport function of mice. After injecting TR-d3 into the cisterna magna for 30 min, fluorescent signal of TR-d3 was clearly observed in the whole SN region of WT-sham mice, but the fluorescence intensity was reduced in the other three groups, especially, in A53T-LDclns mice (Fig. 1e). The percentage of TR-d3 diffusion area into the midbrain of WT-LDclns mice, A53T-sham mice and A53T-LDclns mice was 65, 67 and 42% of WT-sham mice respectively (Fig. 1g).
LDclns exacerbates α-syn aggregation in A53T mouse brain
We also analyzed whether LDclns would affect clearance of other macromolecules from the brain. Results showed that protein levels of total Tau and PHF-1, one type of phosphorylated Tau, were higher in A53T-LDclns mice than those in WT-LDclns mice and A53T mice (all p < 0.05; Additional file 3: Figure S3a-c). This result is consistent with the view that there is a synergistic interaction between α-syn and tau in mediating neurodegeneration in PD, as α-syn may increase tau aggregation [32–34] and treating A53T mice with tau oligomer specific monoclonal antibody can alleviate PD-like pathophysiological phenotypes .
In addition, we examined whether blocking meningeal lymphatics affects clearance of excessive ISF from the brain. Brain water content was not different between WT mice and A53T mice with or without LDclns (Additional file 4: Figure S4). These data are consistent with a pervious study showed that lack of dural lymphatic vessels compromises CNS macromolecule clearance, but does not affect CNS water homeostasis . This might be due to a compensatory role of other water transport mechanisms including transport across the blood-brain-barrier and absorption at the arachnoid granulations [11, 15].
LDclns exacerbates reactive gliosis and inflammatory cytokine production in SN of A53T mice
Ldclns aggravates impairment of AQP4 polarization in SN of A53T mice
AQP4 deletion impairs clearance of interstiatal α-syn from the parenchyma
LDclns causes dopaminergic neuronal loss and motor impairments of A53T mice
A53T mice transgenically expressing mutant human α-syn can simulate the progressive pathological process of PD with appearance of motor impairments from 7 to 8 months old [43–45]. The present study found that 24-week old A53T mice do not show obvious abnormal behavioral performance in the pole test and rotoard test at baseline, despite a slight decline in total distance of movement during the open field testing when compared with WT mice. These A53T mice also do not show a significant loss of midbrain dopaminergic neurons. However, α-syn positive inclusion bodies are observed within neurons including TH-positive cells, suggesting that intracellular aggregation of α-syn is one of the early pathological events in this PD mouse model. Furthermore, the present results have revealed extracellular aggregation of α-syn especially in the perivascular space of SN in A53T mice. Impaired paravascular influx of CSF tracer further suggests that dysfunctions of the glymphatic clearance occur in the early stage of PD-like pathology. The decrease of the glymphatic clearance is mainly due to AQP4 mislocalization that caused by activated astrocytes, to be discussed later.
In addition, studies on human postmortem brains and cellular/animal models of PD have suggested a close interrelation between α-syn and autophagy [46–49]. Autophagy facilitates α-syn degradation and its impairment may favor α-syn inclusion formation . On the other hand, accumulation of oligomeric α-syn is thought to be cytotoxic and damaging autophagy-lysosomal function subsequently, resulting in a pathological cycle leading to neuronal death . The present results show that blocking meningeal lymphatic drainage alters expression levels of autophagy marker LC3II/LC3I in A53T mice but not in WT mice. Likewise, the level of insoluble α-syn only increases in A53T-ligated mice. These data suggest that intercellular autophagy degradation may play a compensatory role in clearance of α-syn in the healthy brain when CNS lymphatic drainage is blocked. However, this compensatory mechanism is insufficient due to a long-term over-production of α-syn combined with meningeal lymphatic dysfunction. Taken together, the present results suggest that decline of glymphatic clearance is involved in the early pathological process of A53T transgenic PD mouse model. This finding supports the view that the glymphatic system has an essential role in maintaining metabolism homeostasis of macromolecules in the brain and its alteration is involved in the pathogenesis of macromolecule aggregation related neurodegeneration [16, 50].
Recent studies have identified meningeal lymphatic vessels in human and rodent brains [51, 52]. They align with dural blood vessels and exit the cranium via the foramina together with venous sinuses, arteries and cranial nerves [53, 54]. Functional studies have revealed that meningeal lymphatic vessels mainly contribute to the delivery of CSF macromolecules within the subarachnoid space into the Dclns [11, 16, 17, 55, 56]. For example, Da Mesquita and colleagues (2018) have demonstrated that disruption of meningeal lymphatic vessels decreases paravascular influx of macromolecules into the brain and efflux of macromolecules from the ISF in young-adult mice . Our results show that intracisternal fluorescent tracer drainage into the Dclns is almost completely abrogated after blocking their afferent lymphatic vessels. Our results also demonstrate that astrocytes with damaged AQP4 polarity are activated and the levels of α-syn and tau proteins are increased in the midbrain of WT-LDclns mice. Furthermore, blocking meningeal lymphatic drainage in WT mice leads to an increase in inflammatory cytokines accompanied with mild dysfunctions of motor and coordination. Similarly, early studies reported that ablation of Dclns results in cognitive impairment, which is associated with disruption of the normal flow of meningeal T-cells into the Dclns . We have demonstrated that numbers of CD3+, CD4+ and CD8+ T cells are significantly increased in the meninges, but decreased in the Dclns after ligation of their afferent lymphatic vessels. These findings together with previous studies highlight the functional communications between the glymphatic system and extracranial lymphatic drainage pathway [15–17, 56, 58].
LDclns significantly aggravates α-syn pathology in A53T mice. As mentioned above, there is no obvious motor abnormality in 24-week old A53T mice compared to WT controls. However, A53T mice that received LDclns show significant dysfunctions of coordination and balance that evidenced by decreases in the total distance of spontaneous movement in the open field and the time of staying on the rotarod, and increases in T-turn and T-total in the pole test. Consistent with behavioral alterations, blocking meningeal drainage aggravates glymphatic clearance dysfunction causing worse deposition of α-syn, glial activation, inflammation, and dopaminergic neuronal loss. Our results strongly suggest that overexpression of α-syn leads to reactive astrogliosis, which impairs AQP4 polarity and in turn hampers glymphatic clearance function. However, a considerable proportion of excessive α-syn can still be cleared from the brain parenchyma due to functional preservation of extracranial lymphatic drainage thereby alleviating the PD-like pathological process in A53T mice.
In summary, we have showed that blocking extracranial lymphatic drainage markedly exacerbates accumulation of α-syn in the brain parenchyma of A53T mice and subsequently leads to extensive reactive astrogliosis and AQP4 polarity destruction, thus exacerbating glymphatic clearance deficits and promoting α-syn-related pathology (Fig. 9b). However, it should be noted that the lymphatic system is rich in collateral circulation, likely after LDclns, compensatory effects of other extracranial lymphatic systems including the cribriform plate route need to be analyzed. The results suggest that both intracellular degradation and extracellular clearance of α-syn are impaired in A53T mice, but which one is an initial event needs to be further explored. In addition, pathogenic point mutations in the α-syn gene, such as A53T and A30P missense mutations, are linked to familial PD. However, most neurodegenerative disorders involving Lewy bodies are associated with abnormal accumulation of wild-type α-syn . Neuronal over-expression of wild-type human α-syn in mice resulted in progressive accumulation of α-syn in neurons, associated with loss of dopaminergic terminals in the basal ganglia and with motor impairment . Whether brain lymphatic clearance dysfunction is also involved in pathology of this PD model and patients with AD warrants further investigation.
Our results demonstrate an impairment of glymphatic clearance of α-syn in the brain of A53T mice, and disruption of meningeal lymphatic drainage further exacerbates α-syn-related pathology. Findings suggest that lymphatic clearance dysfunction is crucial in the onset and development of PD, and might be therapeutically targeted to alleviate age-associated neurodegenerative disorders.
We thank Dr. Weiguo Peng (University of Rochester Medical Center) for modifying the manuscript. Ms. Jianhua Ding for her excellent technical support.
This work was supported by grants from the National Natural Science Foundation of China (81671070 and 81473196).
Availability of data and materials
All data mentioned in this article are available on published article.
MX and GH designed the study. WZ and TP completed pathological analysis. WZ and WF performed Western blotting and ELISA experiments. WZ and YZ performed fluorescence tracing and behavioral experiments. WZ and ML analyzed data. MX wrote the manuscript with WZ, ML and RD. All authors read and approved the final manuscript.
All procedures were approved by Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1601153).
Consent for publication
The authors declare that they have no competing interests.
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