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. 2024 Jan 31;10(1):31.
doi: 10.1038/s41531-024-00643-z.

Phosphorylation of AQP4 by LRRK2 R1441G impairs glymphatic clearance of IFNγ and aggravates dopaminergic neurodegeneration

Heng Huang #  1 , Lishan Lin #  1 , Tengteng Wu  2 , Cheng Wu  3 , Leping Zhou  4 , Ge Li  5 , Fengjuan Su  1 , Fengyin Liang  1 , Wenyuan Guo  2 , Weineng Chen  1 , Qiuhong Jiang  1 , Yalun Guan  5 , Xuejiao Li  5 , Pingyi Xu  2 , Yu Zhang  5 , Wanli Smith  6 , Zhong Pei  7
Affiliations

Phosphorylation of AQP4 by LRRK2 R1441G impairs glymphatic clearance of IFNγ and aggravates dopaminergic neurodegeneration

Heng Huang et al. NPJ Parkinsons Dis. .

Abstract

Aquaporin-4 (AQP4) is essential for normal functioning of the brain's glymphatic system. Impaired glymphatic function is associated with neuroinflammation. Recent clinical evidence suggests the involvement of glymphatic dysfunction in LRRK2-associated Parkinson's disease (PD); however, the precise mechanism remains unclear. The pro-inflammatory cytokine interferon (IFN) γ interacts with LRRK2 to induce neuroinflammation. Therefore, we examined the AQP4-dependent glymphatic system's role in IFNγ-mediated neuroinflammation in LRRK2-associated PD. We found that LRRK2 interacts with and phosphorylates AQP4 in vitro and in vivo. AQP4 phosphorylation by LRRK2 R1441G induced AQP4 depolarization and disrupted glymphatic IFNγ clearance. Exogeneous IFNγ significantly increased astrocyte expression of IFNγ receptor, amplified AQP4 depolarization, and exacerbated neuroinflammation in R1441G transgenic mice. Conversely, inhibiting LRRK2 restored AQP4 polarity, improved glymphatic function, and reduced IFNγ-mediated neuroinflammation and dopaminergic neurodegeneration. Our findings establish a link between LRRK2-mediated AQP4 phosphorylation and IFNγ-mediated neuroinflammation in LRRK2-associated PD, guiding the development of LRRK2 targeting therapy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. LRRK2 R1441G impairs glymphatic system clearance.
a Schema showing the intracisternal injection of fluorescein isothiocyanate (FITC)-dextran for in vivo two-photon imaging. b Representative image of FITC-dextran along the perivascular space (PVS) penetrating the brain parenchyma of LRRK2 WT and R1441G mice, 100 μm below the cortical surface (scale bar = 200 μm). c Statistic analysis of the fluorescence intensity of FITC-dextran in the PVS shown in (b). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis. d Representative immunostaining of AQP4 and GFAP in the LRRK2 WT and R1441G mice (scale bar = 20 μm). e Schematic diagram illustrating the calculation process for AQP4 polarity (scale bar = 100 μm). f Quantification of (d). LRRK2 R1441G mutant led to a decrease in perivascular AQP4 polarity compared to LRRK2 WT group. Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Independent-sample t-tests were used for analysis.
Fig. 2
Fig. 2. LRRK2 interacts with AQP4 and phosphorylates AQP4.
a Immunoblotting of proteins from brain lysates of LRRK2 WT and R1441G transgenic mice subjected to co-immunoprecipitation (co-IP) by an antibody against LRRK2. b Quantification of (a). Datasets are expressed as means ± standard error of mean. n = 6 per group. One-way ANOVA and Tukey’s post hoc test were used for analysis. c Immunoblot of proteins from HeLa cell lysates transfected with FLAG-LRRK2 different domains and HA-AQP4, subjected to co-IP by antibody to HA. d Immunoblotting of proteins from brain lysates of the LRRK2 WT, LRRK2 R1441G, LRRK2 R1441G+GTPase inhibitor and LRRK2 R1441G+Mli-2 groups. e Quantification of (d). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. f Immunoblotting of phosphorylated serine (p-Ser) in AQP4 pulled down by LRRK2 from brain lysates of the LRRK2 WT, LRRK2 R1441G, LRRK2 R1441G+GTPase inhibitor and LRRK2 R1441G+Mli-2 groups. g Quantification of (f). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. h Immunoblot of proteins from HeLa cell lysates transfected with FLAG-LRRK2 WT/G2019S and HA-AQP4, subjected to co-IP by antibody to LRRK2. i Quantification of (h). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis.
Fig. 3
Fig. 3. LRRK2 regulates AQP4 polarity.
a HeLa cells (red) are seeded on the top and primary culture of astrocytes (purple) is seeded on the bottom of the filter. b Representative images of the membrane compartmentalization of AQP4 at the endfeet processes of primary astrocytes from the LRRK2 WT and R1441G transgenic mice treated with or without a GTPase inhibitor/Mli-2 groups (scale bar = 20 μm). c Representative images of AQP4 polarity in the LRRK2 WT and R1441G transgenic mice treated with or without a GTPase inhibitor/Mli-2 groups (scale bar = 20 μm). d Quantification of (c). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. e Immunoblotting of membrane proteins from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without a GTPase inhibitor/Mli-2. f Quantification of (e). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis.
Fig. 4
Fig. 4. IFNγ is involved in LPS-induced neuroinflammation in mutant LRRK2 mice.
a Representative images of tyrosine hydroxylase (TH) in the control and experimental groups (scale bar = 100 μm). b Immunoblotting of TH from midbrain lysates of LRRK2 WT and R1441G transgenic mice treated with or without lipopolysaccharide (LPS). c Quantification of (b). Datasets are expressed as means ± standard error of mean. n = 6 per group. **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. d Immunoblotting of TH from midbrain lysates of LRRK2 WT and R1441G mice treated with or without a GTPase inhibitor/LPS. e Quantification of (d). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. f Representative images of Iba-1 and GFAP in the control and experimental groups (scale bar = 100 μm). g Quantification of Iba-1 in (f). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. h Quantification of GFAP in (f). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. i Immunoblotting of IFNγ from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without LPS. j Quantification of (i). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; ***p < 0.001. Two-way ANOVA and Tukey’s post hoc test were used for analysis. k Immunoblotting of IFNγ from brain lysates of LRRK2 WT and R1441G mice treated with or without a GTPase inhibitor/LPS. l Quantification of (k). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis.
Fig. 5
Fig. 5. LRRK2 R1441G impairs the clearance of IFNγ through the glymphatic system.
a Schematic of Intrastriatum injection of fluorescein isothiocyanate (FITC)-IFNγ (anteroposterior, +1.5 mm; mediolateral, −1.5 mm; and dorsoventral, +2.5 mm from the bregma). b Representative images of brain sections showing the coverage of FITC-IFNγ in the parenchyma of FVB mice treated with or without an AQP4 inhibitor(scale bar = 2 mm). c Quantification of (b). Datasets are expressed as means ± standard error of mean. n = 6 per group. **p < 0.01. Independent-sample t-tests were used for analysis. d Representative images of deep cervical lymph nodes (dCLNs) showing the fluorescence intensity of the FITC-IFNγ(scale bar = 250 μm). e Quantification of (d). Datasets are expressed as means ± standard error of mean. n = 6 per group. **p < 0.01. Independent-sample t-tests were used for analysis. f Representative images of brain sections showing FITC-IFNγ coverage in the brain parenchyma of LRRK2 WT and R1441G mice treated with or without a GTPase inhibitor(scale bar = 2 mm). g Quantification of (f). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. h Representative images showing the FITC-IFNγ drainage into the dCLNs(scale bar = 250 μm). i Quantification of (h). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis.
Fig. 6
Fig. 6. IFNγ upregulates IFNGR-2 expression in astrocytes, increases LRRK2 expression, and reduces AQP4 polarity in LRRK2 R1441G mice.
a Immunoblotting of LRRK2 and IFNγR from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without IFNγ. b Quantification of LRRK2 in (a). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. c Quantification of IFNγR in (a). Datasets are expressed as means ± standard error of mean. n = 6 per group. **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. d Immunoblotting of LRRK2 and IFNγR from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without IFNγ and a GTPase inhibitor. e Quantification of LRRK2 in (d). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. f Quantification of IFNγR in (d). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis. g Representative images of IFNγR and GFAP in the control and experimental groups (scale bar = 50 μm). h Representative images of IFNγR and Iba-1 in the control and experimental groups (scale bar = 50 μm). i Representative images of AQP4 polarity in the control and experimental groups (scale bar = 20 μm). j Quantification of (i). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05; **p < 0.01. Two-way ANOVA and Tukey’s post hoc test were used for analysis. k Immunoblotting of membrane AQP4 from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without IFNγ. l Quantification of (k). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis. m Immunoblotting of membrane AQP4 from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without IFNγ and a GTPase inhibitor. n Quantification of (m). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis. o Immunoblotting of TH from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without IFNγ. p Quantification of (o). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis. q Immunoblotting of TH from brain lysates of LRRK2 WT and R1441G transgenic mice treated with or without IFNγ and a GTPase inhibitor. r Quantification of (q). Datasets are expressed as means ± standard error of mean. n = 6 per group. *p < 0.05. Two-way ANOVA and Tukey’s post hoc test were used for analysis.
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