The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

Subclinical sinus node dysfunction in patients with atrial fibrillation-Insight from ultrahigh-resolution mapping of human sinoatrial exits.

BACKGROUND: Even a short duration of paroxysmal episodes of atrial fibrillation (AF) is associated with sinus node (SN) remodeling and a reduced SN reserve or dysfunction. The number of earliest atrial activation sites (EASs) during sinus rhythm decreases according to the decrease in the SN reserve. OBJECTIVE: We sought to evaluate the EASs during sinus rhythm using an ultrahigh-density mapping system. METHODS: This study included 35 patients (supraventricular tachycardia [SVT]/paroxysmal atrial fibrillation [PAF]/persistent atrial fibrillation [PsAF] = 5/21/9) who underwent ultrahigh-resolution endocardial mapping of the SN area at rest and during ß-stimulation. The number of EASs was determined by the Lumipoint™ algorithm. RESULTS: The number of EASs was greatest in SVT patients both at rest (SVT/PAF/PsAF = 1.4 ± 0.8/1.0 ± 0/1.0 ± 0, p = .04) and during ß-stimulation (SVT/PAF/PsAF = 2.6 ± 1.0/1.3 ± 0.6/1.0 ± 0, p < .01). The number significantly increased with ß-stimulation as compared to baseline in the PAF patients (p = .02), but not in the PsAF patients. The brain natriuretic peptide (BNP) level was significantly higher in AF than SVT patients (SVT/PAF/PsAF = 12.3 [10.1-14.5]/25.7 [14.8-36.0]/73.4 [57.6-140] pg/ml, p < .01). In the PAF patients, the BNP level was significantly higher in those with unicentric EASs than multicentric EASs during ß-stimulation (28.1 [19.1-46.5] vs. 13.1 [9.4-26.9] pg/ml, p = .03), and the optimal cutoff point for the BNP level predicting unicentric EASs was 21.8 pg/ml (sensitivity 82.6%; specificity 85.7%). CONCLUSIONS: AF patients have a smaller number of EASs and poorer response to ß-stimulation than non-AF patients. An elevated BNP level might predict subclinical SN dysfunction in patients with PAF.

Focal post-Maze atrial tachycardia mimicking macroreentrant tachycardia around the Maze lesion diagnosed using ultra-high-resolution mapping.

Focal post-Maze atrial tachycardia mimicked macroreentrant tachycardia around the Maze lesion.

Pseudo superior vena cava entrance block during sinus rhythm uncovered by continuous atrial pacing.

Usually, superior vena cava (SVC) entrance block is confirmed when SVC potentials disappear during sinus rhythm. We present a case of pseudo SVC entrance block during sinus rhythm, which was uncovered by continuous atrial pacing.

N-Heterocyclic carbene-based C-centered Au(I)-Ag(I) clusters with intense phosphorescence and organelle-selective translocation in cells.

Photoluminescent gold clusters are functionally variable chemical modules by ligand design. Chemical modification of protective ligands and introduction of different metals into the gold clusters lead to discover unique chemical and physical properties based on their significantly perturbed electronic structures. Here we report the synthesis of carbon-centered Au(I)-Ag(I) clusters with high phosphorescence quantum yields using N-heterocyclic carbene ligands. Specifically, a heterometallic cluster [(C)(AuI-L)6AgI2]4+, where L denotes benzimidazolylidene-based carbene ligands featuring N-pyridyl substituents, shows a significantly high phosphorescence quantum yield (Φ = 0.88). Theoretical calculations suggest that the carbene ligands accelerate the radiative decay by affecting the spin-orbit coupling, and the benzimidazolylidene ligands further suppress the non-radiative pathway. Furthermore, these clusters with carbene ligands are taken up into cells, emit phosphorescence and translocate to a particular organelle. Such well-defined, highly phosphorescent C-centered Au(I)-Ag(I) clusters will enable ligand-specific, organelle-selective phosphorescence imaging and dynamic analysis of molecular distribution and translocation pathways in cells.

Declines in serum uric acid level after catheter ablation of atrial fibrillation.

BACKGROUND: Few studies have examined whether catheter ablation for AF patients improves biomarkers other than serum levels of brain natriuretic peptide (BNP) and renal function. This study was to explore whether catheter ablation for atrial fibrillation (AF) patients affects uric acid (UA), glucose and lipid metabolism. METHODS AND RESULTS: A total of 206 patients (66.6 ± 10.4 years; 132 men) who underwent initial AF ablation without changes to oral medications were included. Baseline BNP and UA levels significantly decreased at 1 year after ablation (p < 0.05 each). Changes in UA level correlated significantly with pre-procedural UA level (r = 0.57). In multivariable logistic regression modeling, pre-procedural UA level, persistent AF, and hemoglobin A1c (p < 0.05 each) were independent predictors of post-procedural UA level decline. Significant improvements in both persistent and paroxysmal AF patients were identified, and the magnitude of post-procedural serum UA level decline after ablation (ΔUA) was significantly greater in patients with persistent AF (0.8 ± 1.0 mg/dl) than in those with paroxysmal AF (0.2 ± 0.8 mg/dl, p < 0.001). Of the 48 patients with high UA level before procedure, 28 patients showed improvement in UA level to normal range. CONCLUSIONS: Catheter ablation for AF patients significantly improved serum UA levels without obvious influences of heart failure, renal function, or inflammation, suggesting that AF ablation may be effective for AF patients with hyperuricemia. Trial registration The study was approved by the Research Ethics Committee of University of Fukui (no. 20210132) and clinical trial registration (UMIN000044669).

Evaluation of cryoballoon pulmonary vein isolation lesions during the acute and chronic phases using a high-resolution mapping system.

BACKGROUND: An acute cryothermal ablation lesion contains both reversible and irreversible elements. However, differences in lesions created with cryoballoon pulmonary vein isolation (PVI) between the acute and chronic phases have not been fully elucidated. METHODS: We retrospectively analyzed 23 consecutive patients with atrial fibrillation who underwent cryoballoon PVI during the initial procedure followed by a second ablation procedure. In all patients, cryoballoon PVI lesions were evaluated with high-resolution voltage mapping just after PVI (acute phase) and during the second session (chronic phase). We compared the area and width of the non-isolated left atrial posterior wall (NI-LAPW) with voltage ≥ 0.5 mV during both sessions. RESULTS: PVI was successfully achieved in all patients. Cryoballoon PVI lesions were re-evaluated at 11 [2-17] months post-procedure. During the chronic phase, NI-LAPW width became significantly larger at the level of the roof (change, 5.8 ± 5.5 mm; p < 0.001) and at the level of the carina (change, 3.3 ± 7.0 mm; p < 0.05), and NI-LAPW area became significantly larger (change, 1.5 ± 1.9 cm2; p < 0.001) compared with the acute phase. Eight patients without any PV reconnections also had larger NI-LAPW areas (change, 1.3 ± 1.2 cm2; p < 0.05) during the chronic phase. Conduction resumption confined to the right carina was observed in 1 (4.3%) patient who presented with circumferential PVI that included the carina during the first session. CONCLUSION: Acute cryoballoon PVI lesions significantly regressed during the chronic phase. PV reconnections and the isolation area should be carefully re-evaluated during the second procedure.

Evaluation of interatrial conduction pattern after pulmonary vein isolation using an ultrahigh-resolution electroanatomical mapping system.

Interatrial conduction consists of various muscular bundles, including the Bachmann bundle. In this study, we investigated interatrial activation patterns using ultrahigh-resolution left atrial endocardial mapping. This study investigated 58 patients who underwent catheter ablation of atrial arrhythmia via an ultrahigh-resolution mapping system (Rhythmia) at our hospital from May 2020 to January 2021. Left atrial voltage maps and activation maps were acquired after the ablation procedure during right atrial appendage (RAA) pacing. We defined left atrial breakout sites (LABSs) as centrifugal activation patterns shown by the LUMIPOINT Activation Search Tool. The distance between each LABS in the left atrial anterior wall and the superior border of the interatrial septum (DLABS-IAS) was measured on the shell of the electroanatomical map, and anterior LABSs were divided equally into roof- and septal-side groups. Fifty-three (91%) patients underwent cryoballoon pulmonary vein isolation. Ultrahigh-resolution left atrial mapping was successfully performed in all patients (6831 ± 2158 points). A total of 82 LABSs were identified in left atrial anterior wall; 34 patients had single LABS and 24 patients had dual LABSs. The mean DLABS-IAS was 10.3 ± 9.6 mm. Seven patients also exhibited posterior LABS near the interatrial raphe below the right inferior pulmonary vein. Patients with a single roof-side LABS had significantly shorter left atrial activation times than those with a single septal-side LABS (81.6 ± 13.2 ms vs. 93.5 ± 13.7 ms, p < 0.05). Interatrial conduction patterns during RAA pacing varied between patients and affected the left atrial activation time.

Conduction delay across the cavotricuspid isthmus block line caused by the gap near the inferior vena cava: the role of conduction block in the lower lateral right atrium.

The electrophysiological properties of the gap associated with the cavotricuspid isthmus (CTI) block line near the inferior vena cava (IVC) are not fully elucidated. Of 143 patients who underwent CTI block line ablation between September 2020 and April 2021, high-resolution CTI gap mapping was performed for 15 patients. Four patients were identified as having a gap near the IVC (IVC-side gap) despite wide double potentials (DPs) with > 90 ms intervals at the block line. Detailed gap mapping during coronary sinus ostial pacing was performed before and after touch-up ablation. CTI conduction delays caused by an IVC-side gap were classified into 3 patterns: (1) conduction delay at the IVC-side gap without detouring gap conduction, (2) detouring gap conduction due to intrinsic lower lateral right atrium (LLRA)-IVC functional block, and (3) detouring gap conduction due to LLRA-IVC conduction block created by lateral deviation of the CTI ablation line. In Pattern 2, IVC-side gap conduction traveled backward toward the crista terminalis below the LLRA-IVC junction and came back forward again above the border. One patient presented with a head-to-bottom activation pattern of the lateral right atrium (pseudo-CTI block). Pattern 3 was caused by lateral deviation of initial RF deliveries and presented with the same course as intrinsic LLRA-IVC functional block. All patients had wide DP intervals near the tricuspid annulus (mean, 112 ms) and just above the gap site (mean, 109 ms). An IVC-side gap associated with the CTI block line can present with various conduction delay patterns.

Ultra-high resolution mapping of reverse typical atrial flutter: electrophysiological properties of a right atrial posterior wall and interatrial septum activation pattern.

PURPOSE: We aimed to elucidate the right atrial posterior wall (RAPW) and interatrial septum (IAS) conduction pattern during reverse typical atrial flutter (clockwise AFL: CW-AFL). METHODS: This study included 30 patients who underwent catheter ablation of CW-AFL (n = 11) and counter-clockwise AFL (CCW-AFL; n = 19) using an ultra-high resolution mapping system. RAPW transverse conduction block was evaluated by the conduction pattern on propagation maps and double potentials separated by an isoelectric line. The degree of blockade was evaluated by the %blockade, which was calculated by the length of the blocked area divided by the RAPW length. IAS activation patterns were also investigated dependent on the propagation map. RESULTS: The average %blockade of the RAPW was significantly smaller in patients with CW-AFL than those with CCW-AFL (25 [3-74]% vs. 67 [57-75]%, p < 0.05). CW-AFL patients exhibited 3 different RAPW conduction patterns: (1) a complete blockade pattern (3 patients), (2) moderate (> 25% blockade) blockade pattern (2 patients), and (3) little (< 25% blockade) blockade pattern (6 patients). In contrast, the little blockade pattern was not observed in CCW-AFL patients. Of 11 CW-AFL patients, 4, including all patients with an RAPW complete blockade pattern, had an IAS activation from the wavefront from the anterior tricuspid annulus (TA), and 6 had an IAS activation from the wavefronts from both the anterior TA and RAPW. One patient had IAS activation dominantly from the wavefront from the RAPW. CONCLUSIONS: RAPW transverse conduction blockade during CW-AFL was less frequent than during CCW-AFL, which possibly caused various IAS activation patterns.

Safety and durability of cavo-tricuspid isthmus linear ablation in the current era: Single-center 9-year experience from 1078 procedures.

BACKGROUND: Cavo-tricuspid isthmus (CTI) linear ablation is performed not only for atrial flutter (AFL) but empirically during atrial fibrillation (AF) ablation in real-world practice.  PURPOSE: We sought to evaluate the safety and durability of the CTI ablation.  METHODS: This retrospective study included 1078 consecutive patients who underwent a CTI ablation. AFL was documented before or during the procedure in 249 (23.1%) patients, and an empirical CTI and AF ablation were performed in 829 (76.9%) patients.  RESULTS: CTI block was successfully created in 1051 (97.5%) patients with a 10.3 ± 6.6 min total radiofrequency time. Repeat procedures were performed for recurrent arrhythmias in 187 (17.3%) patients at a median of 11.0 (5.0-30.0) months postprocedure, and conduction resumption was identified in 68/174 (39.1%). Among those undergoing a CTI ablation with an AF ablation, the durability was significantly higher in those with than without documented AFL (78.1% vs. 58.2%, p = .031).  The total radiofrequency time was significantly shorter (9.0 ± 5.3 vs. 10.0 ± 6.4 [mins], p = .024) and durability significantly higher (78.1 vs. 58.7[%], p = .043) in the large-tip than irrigated-tip catheter group. Iatrogenic AFL was observed after the empiric CTI ablation in 11 (1.3%) patients. Procedure-related complications occurred in 15 (1.4%) patients. Eight patients experienced coronary artery spasms, including one with ventricular fibrillation following ST elevation on the ward. The other six patients experienced transient atrioventricular block and one experienced cardiac tamponade requiring drainage.  CONCLUSIONS: Despite a high acute CTI ablation success, the conduction block durability was relatively low after the empiric ablation. An empiric CTI ablation at the time of the AF ablation is not recommended.

The impact of electrical connections between left ipsilateral pulmonary veins on the time-to-isolation values in cryoballoon ablation.

BACKGROUND: The time-to-isolation (TTI) may be a physiological predictor of durable isolations, and TTI-guided dosing strategies are widely performed in cryoballoon ablation. We sought to investigate the impact of the order of targeting the pulmonary veins (PVs) on the TTI values of left ipsilateral PVs. METHODS: This study included 144 atrial fibrillation patients who underwent PV isolations using 28-mm fourth-generation cryoballoons. In 101 patients, the left superior PV (LSPV) was targeted and followed by the left inferior PV (LIPV) (group 1), and the LIPV was targeted and followed by the LSPV in the remaining 43 (group 2). RESULTS: The total LSPV and LIPV freeze durations were 193 ± 60 and 171 ± 40 s, respectively. Real-time PV isolation monitoring was capable in 137 (95.1%) LSPVs and 119 (82.6%) LIPVs and in 112 (77.8%) patients (78 in group 1 and 34 in group 2) in both LSPVs and LIPVs. Among them, the LSPV TTI was significantly longer in group 1 than that in group 2 (54.8 ± 32.1 vs. 34.1 ± 17.3 s, p < 0.0001), while the LIPV TTI was significantly shorter in group 1 than that in group 2 (23.7 ± 11.8 vs. 39.2 ± 19.4 s, p < 0.0001). The ΔTTI ((TTI in LSPV)-(TTI in LIPV)) was significantly greater in group 1 than that in group 2 (31.1 ± 31.4 vs. - 5.0 ± 25.9 s, p < 0.0001). In 5 patients (3 in group 1 and 2 in group 2), initially targeted left PVs were not isolated despite complete vein occlusions, while they were by subsequent applications at the other ipsilateral PVs. CONCLUSIONS: In CB ablation, the order of targeting PVs highly influenced the TTI of the left PVs owing to the presence of electrical connections between left ipsilateral PVs.

High-Resolution Mapping and Ablation of Atrial Tachycardias Involving the Lateral Left Atrium.

Background The lateral left atrium (LA) is often associated with atrial tachycardia (AT) because of its complex anatomy. We sought to characterize ATs associated with the lateral LA, including the posterolateral mitral isthmus (MI) and left atrial ridge. Methods and Results Twenty-eight lateral LA-associated ATs were mapped with high-resolution mapping systems and entrainment pacing. The vein of Marshall was mapped with a 1.8-Fr mapping catheter when possible. ATs were associated with the posterolateral MI in 18 ATs (14 perimitral, 3 small reentry, and 1 focal AT). All patients had undergone MI area ablation, and all ATs were successfully eliminated. During 27.0 (interquartile range, 10.5-40.0) months of follow-up, all were free from any atrial tachyarrhythmias, with 3 patients on antiarrhythmics. Of 10 ATs involving the ridge or Marshall bundle, 3 were ridge related, 3 were Marshall bundle related based on vein of Marshall mapping, and 1 was a persistent left superior vena cava related AT. All 7 patients had undergone MI linear ablation. The critical isthmus was in the LA-ridge junction or the LA-Marshall bundle junction. Bidirectional conduction block between the LA and ridge or Marshall bundle was created. Two patients had the critical isthmus in the other area. The remaining patient had micro-reentry in the ridge. All 10 ATs were terminated during ablation at the critical isthmus. During 12.0 (5.2-31.7) months of follow-up, all were free from any atrial tachyarrhythmias, with 7 patients on antiarrhythmics. Conclusions Most ATs occurred after MI area ablation. An high resolution mapping-guided approach is highly effective for identifying the mechanism.

A new insight into the lens: cytosolic PLAAT phospholipases degrade organelles to make the lens transparent.

All membrane-bound organelles are degraded during the terminal differentiation of lens fiber cells. How these organelles are degraded has been a long-standing question in biology. We recently revealed that PLAAT (phospholipase A and acyltransferase)-family phospholipases degrade organelles in the lens independently of macroautophagy. Here, we discuss the mechanism and physiological relevance of this new mode of intracellular degradation.

Premature ventricular contraction originating from the distal left anterior fascicle: The usefulness of a multipolar catheter with small electrodes in mapping presystolic Purkinje potential and pace mapping.

Mapping and localizing presystolic Purkinje potentials are crucial for determining the optimal ablation site for fascicular premature ventricular contractions (PVCs). Here we present a case of PVCs originating from the distal left anterior fascicle (LAF). Activation mapping using a multipolar catheter with small electrodes demonstrated early presystolic Purkinje potentials during the PVCs. A moderately good pace-map match was also obtained near the successful ablation site. This case demonstrates the activation pattern of PVCs originating from the distal LAF and the usefulness of multipolar catheters with small electrodes for the mapping of fascicular PVCs.

Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

Cardiac rehabilitation after catheter ablation of atrial fibrillation in patients with left ventricular dysfunction.

Few studies have examined the efficacy and safety of cardiac rehabilitation in patients with atrial fibrillation (AF) who underwent AF ablation. We explored the feasibility of additional cardiac rehabilitation after AF ablation in patients with a reduced left ventricular ejection fraction (LVEF). Fifty-four patients with heart failure (HF) and a reduced LVEF (HFrEF) (LVEF < 50%; 67.1 ± 11.6 years; 43 men) who underwent initial AF ablation procedures were included. Fourteen (25.9%) patients underwent cardiac rehabilitation (rehabilitation-group) and the remaining 40 (74.1%) did not (non-rehabilitation-group) after the procedure. The rehabilitation-group patients were relatively older, more likely female (p = 0.024), and had more likely a history of an HF hospitalization (p < 0.01) and cardiac device implantation (p = 0.041). The baseline LVEF was significantly lower (p = 0.043) and brain natriuretic peptide (BNP) (p < 0.01) and C-reactive protein (CRP) (p < 0.01) values were significantly higher in the rehabilitation-group. The 6-min walk distance significantly improved after 21.4 ± 11.5 days of cardiac rehabilitation during hospitalization (226.1 ± 155.9 vs. 398.1 ± 77.5 m, p = 0.016) without any adverse events. During an 18.9 ± 6.3 month follow-up period, the freedom from AF recurrence (p = 0.52) and re-hospitalizations due to HF (p = 0.63) were similar between the 2 groups. No death or strokes were observed. During the follow-up period, the LVEF significantly improved similarly in both groups, and the change in the BNP and CRP values significantly decreased in the rehabilitation-group. Despite the rehabilitation-group patients having a more severe HF status, the clinical outcomes and AF freedom were similar between the 2 groups, suggesting the favorable impact of cardiac rehabilitation after AF ablation in HFrEF patients.

Ultrahigh resolution electroanatomical mapping of the transverse conduction of the right atrial posterior wall in cases with and without typical atrial flutter.

INTRODUCTION: The right atrial posterior wall (RAPW) is known to form a conduction barrier during typical atrial flutter (AFL). We evaluated the transverse conduction properties of RAPW in patients with and without typical AFL using an ultrahigh resolution electroanatomical mapping system. METHODS AND RESULTS: This study included 41 patients who underwent catheter ablation of AF, typical or atypical AFL, in whom we performed RAPW mapping with an ultrahigh resolution mapping system during typical AFL and coronary sinus ostial pacing with three different pacing cycle lengths (PCLs) (1) PCL1: PCL within 40 ms of the AFL cycle length in patients with typical AFL or 250-300 ms for those without, (2) PCL2: 400 ms, (3) PCL3: PCL just faster than the sinus rate. Local RAPW conduction block was evaluated by propagation mapping and local double potentials separated by an isoelectric line. The functional block was defined as areas blocked during shorter PCLs but conductive during longer PCLs. The degree of blockade was calculated by dividing the blocked length by RAPW length (%blockade). Only two patients demonstrated a fixed complete RAPW block (100%, %blockade). Thirty-one patients demonstrated a partial block of RAPW, and the %blockade during PCL1-3 was 49.4 ± 19.8%, 39.5 ± 19.2%, and 35.0 ± 22.9% in this group, respectively. Functional block areas were frequently observed above the fixed block area adjacent to the RA-inferior vena cava junction. Transverse conduction block was more frequently observed in patients with typical AFL at any longitudinal level of RAPW. CONCLUSION: RAPW transverse conduction block is lower-side dominant and greater in patients with typical AFL than those without.

A case of outflow tract premature ventricular contractions with very distant exit sites suspected to have a single origin.

Outflow tract premature ventricular contractions sometimes demonstrate multiple exit sites in the right and left outflow tracts with preferential pathways. Here we present a case of outflow tract premature ventricular contractions, which were eliminated by ablation from the right ventricular outflow tract accompanied by additional ablation from the very distant endocardial left ventricular outflow tract. The findings during the ablation indicated there was a single origin with multiple exit sites rather than multiple origins for each QRS morphology. This case illustrates that the preferential pathways can demonstrate very distant multiple exit sites.

Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation.

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.