Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

[This corrects the article DOI: 10.1371/journal.pbio.3001480.]

Exposure to N-Ethyl-N-Nitrosourea in Adult Mice Alters Structural and Functional Integrity of Neurogenic Sites

BACKGROUND: Previous studies have shown that prenatal exposure to the mutagen N-ethyl-N-nitrosourea (ENU), a N-nitroso compound (NOC) found in the environment, disrupts developmental neurogenesis and alters memory formation. Previously, we showed that postnatal ENU treatment induced lasting deficits in proliferation of neural progenitors in the subventricular zone (SVZ), the main neurogenic region in the adult mouse brain. The present study is aimed to examine, in mice exposed to ENU, both the structural features of adult neurogenic sites, incorporating the dentate gyrus (DG), and the behavioral performance in tasks sensitive to manipulations of adult neurogenesis. METHODOLOGY/PRINCIPAL FINDINGS: 2-month old mice received 5 doses of ENU and were sacrificed 45 days after treatment. Then, an ultrastructural analysis of the SVZ and DG was performed to determine cellular composition in these regions, confirming a significant alteration. After bromodeoxyuridine injections, an S-phase exogenous marker, the immunohistochemical analysis revealed a deficit in proliferation and a decreased recruitment of newly generated cells in neurogenic areas of ENU-treated animals. Behavioral effects were also detected after ENU-exposure, observing impairment in odor discrimination task (habituation-dishabituation test) and a deficit in spatial memory (Barnes maze performance), two functions primarily related to the SVZ and the DG regions, respectively. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that postnatal exposure to ENU produces severe disruption of adult neurogenesis in the SVZ and DG, as well as strong behavioral impairments. These findings highlight the potential risk of environmental NOC-exposure for the development of neural and behavioral deficits

Data analysis of proliferation, migration and early differentiation assays.

<p>(A–B) Cells in S-phase 1 hour after BrdU injection. Bar graph depicting the BrdU+ cells/mm shows a significant decrease in the proliferative rate of the SVZ (A) and DG (B) in ENU animals. (C–D) SVZ or SGZ-derived cells 30 days after BrdU injection protocol. Bar graph depicting significant decrease in the numbers of BrdU+ cells in the OB (C) and GCL of the DG (D) in ENU-exposed animals. (E–F) Early differentiation of neurons in OB. Bar graph depicting the area fraction (E) and integrated density (F) of doublecortin (Dcx) positive cells in 4 different regions of OB, showing a reduction in all of them after ENU-treatment. *<i>p</i><0.05, **<i>p</i><0.01.</p

Evaluation of GAPS to Dcx expression in the DG of control and ENU animals.

<p>*<i>p</i><0.01.</p

Ultrastructural characterization of the SVZ from ENU-treated animals.

<p>The SVZ of treated animals was altered after ENU-exposure. (A) The type A cells located in the dorsal horn of the SVZ in ENU animals were drastically reduced, and substituted by astrocytic expansions. (B) High magnification of the SVZ dorsal horn with expansions rich in intermediate filaments (asterisks) in ENU animals. (C) Ependymal cell and neuroblasts frequently presented direct contact (arrow heads) in SVZ of treated animals. (D) Synaptic contacts located next to ependymal cell in animals exposed to ENU. (E) Large portions of basal membranes were observed between ependymal cells (arrows). (F) Myelinated and unmyelynated axons (arrows) were located between type A cells that composed chains, in ENU animals. Lv: lateral ventricle. Scale bar: A,F 10 µm, B 500 nm, C–E 2 µm.</p

Ultrastructural characterization of the DG.

<p>(A–B) Ultrastructural images of the SGZ from DG. (A) The control SGZ showed niches formed by precursor cells (arrow heads). (B) The ENU SGZ did not present the typical niches. (C) Cell quantification of the cell population in SGZ under electron microscopy, measured as cells/mm, resulted in a significantly decrease of total cells, due to a reduction in the number of type D cells. Scale bar: 10 µm *<i>p</i><0.01.</p

Ultrastructural characterization of the RMS in ENU animals.

<p>Detail of a typical neuroblasts chain in the RMS of ENU treated animal, surrounded by astrocytic cells. Scale bar: 5 µm.</p

ENU treatment impairs olfactory discrimination.

<p>During the habituation-dishabituation test a cotton swab was repeatedly presented to the mice above a target area and changed every minute. Exploration of the target area was examined. After 5 presentations without odorant, the swab was impregnated with an odorant (Odor A), and presented 6 times. Then, another swab was impregnated with a different odorant, (Odor B), which was also presented 6 times. The dotted lines represent the 1 min bins of exploration time of the target area. Notice that both groups similarly detected Odor A (last No Odor presentation Vs first Odor A presentation; * <i>p</i><0.01 for ENU-treated group; # <i>p</i><0.01 for control group), but when Odor B was presented only control animals discriminated the odor difference, responding to the new stimulus (last Odor A presentation Vs first Odor B presentation; # <i>p</i><0.01).</p