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. 2017 Nov 22;37(47):11366-11376.
doi: 10.1523/JNEUROSCI.0964-17.2017. Epub 2017 Oct 20.

Parkinson's Disease-Associated LRRK2 Hyperactive Kinase Mutant Disrupts Synaptic Vesicle Trafficking in Ventral Midbrain Neurons

Ping-Yue Pan  1   2 , Xianting Li  1   2 , Jing Wang  1   2 , James Powell  1   2 , Qian Wang  1   2 , Yuanxi Zhang  1   2 , Zhaoyu Chen  1   2 , Bridget Wicinski  3   2 , Patrick Hof  3   2 , Timothy A Ryan  4 , Zhenyu Yue  5   2
Affiliations

Parkinson's Disease-Associated LRRK2 Hyperactive Kinase Mutant Disrupts Synaptic Vesicle Trafficking in Ventral Midbrain Neurons

Ping-Yue Pan et al. J Neurosci. .

Abstract

Parkinson's disease (PD) is characterized pathologically by the selective loss of substantia nigra (SN) dopaminergic (DAergic) neurons. Recent evidence has suggested a role of LRRK2, linked to the most frequent familial PD, in regulating synaptic vesicle (SV) trafficking. However, the mechanism whereby LRRK2 mutants contribute to nigral vulnerability remains unclear. Here we show that the most common PD mutation LRRK2 G2019S impairs SV endocytosis in ventral midbrain (MB) neurons, including DA neurons, and the slowed endocytosis can be rescued by inhibition of LRRK2 kinase activity. A similar endocytic defect, however, was not observed in LRRK2 mutant neurons from the neocortex (hereafter, cortical neurons) or the hippocampus, suggesting a brain region-specific vulnerability to the G2019S mutation. Additionally, we found MB-specific impairment of SV endocytosis in neurons carrying heterozygous deletion of SYNJ1 (PARK20), a gene that is associated with recessive Parkinsonism. Combining SYNJ1+/- and LRRK2 G2019S does not exacerbate SV endocytosis but impairs sustained exocytosis in MB neurons and alters specific motor functions of 1-year-old male mice. Interestingly, we show that LRRK2 directly phosphorylates synaptojanin1 in vitro, resulting in the disruption of endophilin-synaptojanin1 interaction required for SV endocytosis. Our work suggests a merge of LRRK2 and SYNJ1 pathogenic pathways in deregulating SV trafficking in MB neurons as an underlying molecular mechanism of early PD pathogenesis.SIGNIFICANCE STATEMENT Understanding midbrain dopaminergic (DAergic) neuron-selective vulnerability in PD is essential for the development of targeted therapeutics. We report, for the first time, a nerve terminal impairment in SV trafficking selectively in MB neurons but not cortical neurons caused by two PARK genes: LRRK2 (PARK8) and SYNJ1 (PARK20). We demonstrate that the enhanced kinase activity resulting from the most frequent G2019S mutation in LRRK2 is the key to this impairment. We provide evidence suggesting that LRRK2 G2019S and SYNJ1 loss of function share a similar pathogenic pathway in deregulating DAergic neuron SV endocytosis and that they play additive roles in facilitating each other's pathogenic functions in PD.

Keywords: LRRK2 G2019S; Parkinson's disease; dopamine neuron; genetic interaction; synaptic vesicle; synaptojanin1/synj1.

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Figures

Figure 1.
Figure 1.
PD-linked LRRK2 G2019S mutation leads to MB neuron-specific impairment in SV endocytosis. A, B, Representative pHluorin traces (in black) in response to 10 Hz, 10 s field stimulations (short black bars) obtained from midbrain (A) and cortical (B) neurons with different genetic backgrounds: from left to right: GS ctrl (N = 18), GStg (N = 21), WT ctrl (N = 11), and WTtg (N = 11). Red traces represent the fitted single exponential decay function. Scale bars: A, 25 s, 0.15% NH4Cl response; B, 25 s, 0.25% NH4Cl response. C, D, Comparison of endocytosis time constants (τ obtained from the single exponential decay fit) (top two panels) and the amount of exocytosis by normalizing to the peak of the NH4Cl response (bottom two panels) for cultured midbrain neurons (C) and cultured cortical neurons (D) derived from different transgenic backgrounds. For LRRK2 immunofluorescence in cultured MB neurons, see Figure 1-1. For comparison of exocytosis and endocytosis kinetics between TH+ and TH− neurons, see Figure 1-2.
Figure 2.
Figure 2.
LRRK2 G2019S overexpression in rat MB neurons, but not in hippocampal neurons, leads to slowed endocytosis. A, Endocytosis time constant after 10 Hz, 10 s and 10 Hz, 30 s stimulations in WT rat MB neurons (N = 14) and those overexpressing either LRRK2 G2019S (N = 23) or LRRK2 WT (N = 10). B, Endocytosis time constant after 10 Hz, 10 s (left) and 10 Hz, 30 s (right) stimulations in WT rat hippocampal (HP) neurons (N = 10) and those overexpressing LRRK2 G2019S (LRRK2 GS OE, N = 10). C, Representative immunofluorescence image for rat MB culture transfected with vMAT2-pHluorin and mouse LRRK2. D, Summary for the relative LRRK2 overexpression level at the cell body in different neurons. LRRK2 GS was overexpressed at a similar 50% level in both the MB and the HP neurons.
Figure 3.
Figure 3.
Impaired SV trafficking is mediated by hyperactivity of LRRK2 PD mutant G2019S. A, Experimental scheme of LRRK2 kinase inhibitor test for each neuron. B, C, Comparison of endocytosis time constant (B) and normalized exocytosis (C) before and after 30 min incubation with G1023 (1 μm) for GS ctrl cortical neurons (N = 10), GStg cortical neurons (N = 10), GS ctrl MB neurons (N = 9), and GStg MB neurons N = 9). p values are from paired t tests.
Figure 4.
Figure 4.
Combination of LRRK2 G2019S and SYNJ1 heterozygosity leads to impairment in both exocytosis and endocytosis. A, Representative pHluorin traces in response to 10 Hz, 10 s field stimulations obtained from midbrain neurons with the indicated genotypes. Red traces overlaying the decay phase of the pHluorin traces represent the fitted single exponential functions for endocytosis time constants. Scale bar, 25 s, 0.2% NH4Cl response. B, Representative pHluorin traces in response to 10 Hz, 2 min continuous stimulation in the presence bafilomycin (1 μm) for comparison of exocytosis kinetics. Overlaying red traces represent the fitted single exponential functions for exocytosis time constants. Scale bar, 25 s. C, Summary of endocytosis time constants for the following midbrain neurons: SYNJ1+/+ (N = 19), SYNJ1+/− (N = 22), and SYNJ1+/− neurons expressing LRRK2 G2019S (SYNJ1+/− OE GS, N = 12). D, Summary of exocytosis time constants for a subset of these neurons in C: SYNJ1+/+, N = 9; SYNJ1+/−, N = 14; SYNJ1+/− OE GS, N = 10).
Figure 5.
Figure 5.
Dtg mice display altered motor functions. A–D, Male mice at 1 year of age with the following genotypes: WT littermates (N = 21, black), SYNJ1+/− (N = 12, cyan), and LRRK2 G2019S transgenic (LRRK2 GS) (N = 10, purple) and LRRK2GS; SYNJ1+/− (Dtg) mice (N = 23, red) are subjected to open field test (A, B), accelerated Rota-rod test (C), and challenging beam traversal test (D). A, Scattered and box plot summarizing total distance and movement time in the open field test. B, Summary of time animal spent in the center versus the periphery of the open field chamber for all littermates. C, Summary of the duration animal spent on the accelerated Rota-rod. D, Number of steps and total number of slips from averaging 5 separate trials in the challenging beam traversal test for all littermates. E, Representative images showing TH immunolabeling (DAB enhanced, brown) and Nissl counterstaining (blue) at the ventral midbrain from a Dtg mouse and a WT littermate. Scale bars, 20 μm. F, Stereology analysis estimating total number of TH and Nissl-positive neurons in Dtg mice (N = 5) and WT littermates (N = 6) as well as the percentage of TH-positive neurons did not reveal any difference.
Figure 6.
Figure 6.
LRRK2 phosphorylates synj1 and phosphomimetic synj1 display reduced endophilin interaction. A, Radioactive kinase assay showing enhanced 32P signal for synj1 after incubation with 15 nm Invitrogen full-length human LRRK2 G2019S protein. B, Mass spectrometry analysis reveals a threonine at 1205 within the proline-rich domain of human synj1 as the phosphorylation substrate of LRRK2 G2019S. This threonine also exhibits conservation in two rodent forms of synj1. C, Purified kinase-dead (D1994A), WT, and kinase gain-of-function mutant (G2019S) of LRRK2 were used to incubate with synj1 immunoprecipitated by anti-synj1 antibody in a kinase assay, followed by immunoblotting with anti-pT1205 synj1 antibody (N = 5). D, Summary of the pT1205 synj1 signal in C from four separate experiments. E, Reduced pT1205 signal in response to increasing amount of LRRK2 kinase inhibitor (G1023) (0, 0.2, 1, and 5 μm). F, Coimmunoprecipitation of synj1 by endophilinA (endoA). WT and mutant forms of synj1, including FLAG-synj1 S1202A, FLAG- synj1 T1205E, and FLAG-synj1 T1205A, were coexpressed with GFP-endoA in HEK293T cells. Coexpression of WT synj1 and GFP was used as a negative control for IP. G, Box plot summarizing the reduced endophilinA binding for synj1 T1205E and synj1 T1205A (N = 6).
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