Surfactant secretion in LRRK2 knock-out rats: changes in lamellar body morphology and rate of exocytosis

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca(2+) release upon ATP treatment and significant triggering of LB exocytosis. These findings are in line with the strong Ca(2+)-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca(2+) signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca(2+) signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP.

Figure 1. LBs are significantly enlarged in LRRK2 -/- rats.

(A) LBs (green) in cells that were metabolically labeled with Bodipy phosphatidylcholine (left, asterisks indicate nuclei, scale bar  = 10 µm). LB size was significantly (p<0.006) increased in cells from LRRK2 -/- animals (diameter: 2.26 µm±0.04, n = 4 animals) compared to LBs in cells from wt animals (diameter: 1.95 µm±0.07, n = 4 animals) (middle). Size distribution diagram for LB sizes derived from wt and LRRK2 -/- animals (right, n = 4 animals). (B) LBs (red) in cells that were stained with lipophilic dye Nile red (left, asterisks indicate nuclei, scale bar  = 10 µm). LB size was significantly (p<0.0003) increased in cells from LRRK2 -/- animals (diameter: 2.29 µm±0.03, n = 4 animals) compared to LBs in cells from wt animals (diameter: 1.98 µm±0.03, n = 4 animals) (middle). Size distribution diagram for LB sizes derived from wt and LRRK2 -/- animals (right, n = 4 animals). In all experiments 25 cells were analyzed in each animal corresponding to approx. 350 – 450 LBs per animal. Images were taken from LRRK2 –/- cells.

Figure 2. Expression and localization of LB markers are not affected by LRRK2 -/-.

Immunocytochemical localization of Lamp1, ABCa3, and surfactant proteins SP-B and SP-C in ATII isolated from LRRK2 -/- rats (A) and ATII cells isolated from wt rats (B). Lamp1 and ABCa3 localized to LB limiting membrane, whereas SP-B and SP-C are inside the vesicle together with the lipid component of surfactant. No obvious differences in localization of these markers in cells isolated from KO and wt rats were observed. Arrows indicate individual LBs in type II cells. Scale bar = 10 µM

Figure 3. ATP stimulation results in an increased fusion response in LRRK2 -/- cells.

Response is expressed as the fraction of cells with at least 1 fusion within 10(A), and as a number of fusions/cell in responding cells (B). n denotes number of animals. Equal numbers of experiments were conducted in each animal.

Figure 4. ATP accelerates LB fusion and increases intracellular Ca²⁺-release in LRRK2 -/- cells.

LB fusion response time histograms (bars) and fura-2 ratios (lines) in response to stimulation of ATII cells. (A – D) Fusion delay histograms from wt and LRRK2 -/- cells following stimulation with 100 µM ATP (A), 300 nM PMA (B), 100 µM ATP and 300 nM PMA (C) and 1 µM ionomycin (D). Stimulation with ATP resulted in a significant left shift in the fusion delay histograms in LRRK2 -/- cells. Delay histograms were derived from pooled data from 3 to 6 animals per experimental condition and from 45 to 256 individual fusions. Cells were stimulated at t = 0 s. Fura-2 traces were derived from 7 to 116 cells. Panels represent wt cells (left), LRRK2 -/- cells (middle) and overlay of wt and -/- delay histograms (right). (E) Resting [Ca²⁺]_c is not significantly enhanced in cells from LRRK2 -/- animals (145.2±27.3 nM, n = 4 in LRRK2 -/- cells and 121.2±26.5 nM, n = 4 in wt cells, respectively, p = 0.55, approx. 15 to 20 cells were analysed for each animal). (F) Changes in [Ca²⁺]_c following stimulation with 100 µM ATP (Cells were stimulated at t = 0 s, n = 3 and 2 for LRRK2 -/- and wt cells respectively). (G) Peak [Ca²⁺]_c following stimulation with 100 µM ATP in LRRK2 -/- cells (n = 3) compared to wt cells (n = 2).

Figure 5. DPPC concentration in supernatant as a measure of surfactant secretion.

DPPC concentration was measured in supernatant of unstimulated cells and of cells stimulated with either 100 µM ATP or 300 nM PMA for 30 minutes. DPPC concentration in supernatant was significantly higher in wt cells following stimulation with 100 µM ATP.

Figure 6. Actin coating and compression of fused LBs are not altered in LRRK2 -/- cells.

A) Immunostaining of ATII cells from wt (upper image series) and LRRK2 -/- (lower image series) animals, labeled for myosin IIA, filamentous actin (phalloidin) and LB membrane marker ABCa3. Cells were fixed 3 minutes after stimulation with 100 µM ATP. Myosin IIA and phalloidin staining of ABCa3 positive organelles indicate actin coating of LBs following exocytic fusion with plasma membrane (arrows). Scale bar  = 10 µm. B) Percentage of LBs coated with actin-GFP following fusion after 100 µM ATP stimulation in live-cell experiments and C) fraction of actin coats that contracted within 30 s and 60 s following fusion. Data were derived from 2 wt and 2 LRRK2 -/- animals, and up to 67 fusions and 46 compression events were analysed for each animal. No significant difference was observed between wt and LRRK2 -/- cells.