LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD

High LRRK2 Levels Fail to Induce or Exacerbate Neuronal Alpha-Synucleinopathy in Mouse Brain

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson’s disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy

Assessment of a Targeted Gene Panel for Identification of Genes Associated With Movement Disorders

International audienceImportance: Movement disorders are characterized by a marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis. Objective: To develop and evaluate a targeted sequencing approach using a customized panel of genes involved in movement disorders. Design, Setting and Participants: We selected 127 genes associated with movement disorders to create a customized enrichment in solution capture array. Targeted high-coverage sequencing was applied to DNA samples taken from 378 eligible patients at 1 Luxembourgian, 1 Algerian, and 25 French tertiary movement disorder centers between September 2014 and July 2016. Patients were suspected of having inherited movement disorders because of early onset, family history, and/or complex phenotypes. They were divided in 5 main movement disorder groups: parkinsonism, dystonia, chorea, paroxysmal movement disorder, and myoclonus. To compare approaches, 23 additional patients suspected of having inherited cerebellar ataxia were included, on whom whole-exome sequencing (WES) was done. Data analysis occurred from November 2015 to October 2016. Main Outcomes and Measures: Percentages of individuals with positive diagnosis, variants of unknown significance, and negative cases; mutational frequencies and clinical phenotyping of genes associated with movement disorders. Results: Of the 378 patients (of whom 208 were male [55.0%]), and with a median (range) age at disease onset of 31 (0-84) years, probable pathogenic variants were identified in 83 cases (22.0%): 46 patients with parkinsonism (55% of 83 patients), 21 patients (25.3%) with dystonia, 7 patients (8.4%) with chorea, 7 patients (8.4%) with paroxysmal movement disorders, and 2 patients (2.4%) with myoclonus as the predominant phenotype. Some genes were mutated in several cases in the cohort. Patients with pathogenic variants were significantly younger (median age, 27 years; interquartile range [IQR], 5-36 years]) than the patients without diagnosis (median age, 35 years; IQR, 15-46 years; P = .04). Diagnostic yield was significantly lower in patients with dystonia (21 of 135; 15.6%; P = .03) than in the overall cohort. Unexpected genotype-phenotype correlations in patients with pathogenic variants deviating from the classic phenotype were highlighted, and 49 novel probable pathogenic variants were identified. The WES analysis of the cohort of 23 patients with cerebellar ataxia led to an overall diagnostic yield of 26%, similar to panel analysis but at a cost 6 to 7 times greater. Conclusions and Relevance: High-coverage sequencing panel for the delineation of genes associated with movement disorders was efficient and provided a cost-effective diagnostic alternative to whole-exome and whole-genome sequencing

Microgliosis in end-stage haSN(A53T) transgenic mouse brain is unaltered by high LRRK2 levels.

<p>DAB-immunohistochemistry for Iba1 shows activated microglia on a representative sagittal brain section of a haSN(A53T) mouse (A and 20×higher magnification from brainstem in B) and a haSN(A53T)/hLRRK2(G2019S) double transgenic mouse (C and 20×higher magnification from brainstem in D). (E) Quantification of the brainstem results. Values represent % of the area in the brainstem that is covered by Iba1-positive microglia. p-value (p = 0.179) was determined by two-tailed, unequal variances Student’s t-test. Dots represent quantifications of single individuals. Control images obtained from a separate experiment but from littermate hLRRK2(G2019S) single transgenic (F and 20×higher magnification from brainstem in G) and from non-transgenic wildtype littermate control (Ntg) (H and 20×higher magnification from brainstem in I) mouse.</p

High LRRK2 transgene levels do not exacerbate α-synuclein-driven phenotypes.

<p>(A) Schematic representation of the four different transgenic lines used to generate double transgenics. (B) 3-Step accelerated rotarod performance of females and males comparing single and double transgenics. The different genotypes and the number of mice per genotype are indicated. p-values were determined by repeated measures ANOVA (group effects for the respective panels: 1: F(1,22) = 0.483, p = 0.494; 2: F(1,26) = 0.000, p = 0.983; 3: F(1,11) = 0.738, p = 0.409; 4: F(1,22) = 2.048, p = 0.166; 5: F(1,16) = 1.255, p = 0.279; 6: F(1,27) = 5.171, p = 0.031). (C) Kaplan-Meier curves showing the time-of-sacrifice when mice had to be killed because of too severe motor deficits (1 = 100% and 0 = 0% of mice alive). The different genotypes, gender, number of mice per genotype and the p-values (nonparametric Kaplan-Meier) are indicated.</p

aSN and phospho-S129-aSN protein levels in spinal cord and forebrain of end-stage disease single and double transgenic mice.

<p>Tris-soluble and -insoluble fractions of spinal cord and forebrain lysates were immunoblotted and stained with antibodies detecting total α-synuclein (aSN) or specifically phosphorylated S129-aSN (paSN). β-actin (βAc) levels were measured as loading control and for normalization. For reference, LRRK2 levels detected via immunoblot are shown comparing single and double-transgenics. Different α-synuclein protein species/forms are marked as follows: mo, monomer; ol, oligomer; tr, truncated. For reference, in the upper panels the performance and specificity of the antibodies are illustrated in the two right lanes comparing WT and KO (aSN knock-out) brain samples and were added to indicate unspecific cross-reactive proteins (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone.0036581.s007" target="_blank">Figure S7</a>). Graphs represent quantifications of monomeric aSN and paSN/aSN, all normalized to βAc. Circles represent individual mice, the means are indicated as horizontal bars and % are normalized to the levels in haSN(A53T) single transgenics. p-values were determined by two-tailed, unequal variances Student’s t-test. Genotypes: aSN = haSN(A53T), aSN/LRRK2 = haSN(A53T)/hLRRK2(G2019S), Ntg = non-transgenic wildtype littermate control and KO = aSN knock-out mice.</p

Motor assessment and aSN/Tau protein characterization in hLRRK2(G2019S) mice.

<p>(A) Motor skill learning of 4-month-old male and 6-month-old female hLRRK2(G2019S) and Ntg controls in the 3-step accelerated rotarod task over four consecutive days. The number of mice per genotype is indicated. Three batches of animals were included in this graph (single transgenic and Ntg animals from experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone-0036581-g003" target="_blank">Figure 3B</a> as well as a separate batch). p-values were determined by repeated measure ANOVA (group effect males: F(1,119) = 9.42, p = 0.003, group effect females: F(1,52) = 3.74, p = 0.059). (B) Novelty-induced horizontal locomotor activity of 7.3- and 28.2-month-old hLRRK2(G2019S) and Ntg mice. Bar graphs show the sum of the distance travelled from 5–30 min and from 35–60 min. The number of mice per genotype is indicated. p-values were determined either by repeated measure ANOVA (group effect males 7.3 M: F(1,16) = 4.044, p = 0.061; group effect males 28.2 M: F(1,16) = 0.093, p = 0.764) or by two-tailed, unequal variances Student’s t-test. (C) Western blotting of forebrain homogenates from 15-month-old hLRRK2(G2019S) (TG) and Ntg male mice. Lower panel: Shown are levels of mouse α-synuclein (aSN) and phospho-α-synuclein Ser129 (paSN) as well as mouse microtubule-associated protein Tau and phospho-Tau Ser202/Thr205 (pTau). β-actin (βAc) was used as loading control and for normalization. Upper panel shows the results of the immunoblot quantifications. Circles represent individual mice, the means (% normalized to Ntg) are indicated as horizontal bars. p-values were determined by two-tailed, unequal variances Student’s t-test. Ntg: non-transgenic wildtype littermate control.</p

NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies

Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped young patient cohort

International audienceThe etiopathogenesis of critical COVID-19 remains unknown. Indeed given major confounding factors (age and comorbidities), true drivers of this condition have remained elusive. Here, we employ an unprecedented multi-omics analysis, combined with artificial intelligence, in a young patient cohort where major comorbidities have been excluded at the onset. Here, we established a three-tier cohort of individuals younger than 50 years without major comorbidities. These included 47 “critical” (in the ICU under mechanical ventilation) and 25 “non-critical” (in a non-critical care ward) COVID-19 patients as well as 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cells proteomics, cytokine profiling and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing and structural causal modeling led to key findings. Critical patients were characterized by exacerbated inflammation, perturbed lymphoid/myeloid compartments, coagulation and viral cell biology. Within a unique gene signature that differentiated critical from non-critical patients, several driver genes promoted critical COVID-19 among which the upregulated metalloprotease ADAM9 was key. This gene signature was supported in a second independent cohort of 81 critical and 73 recovered COVID-19 patients, as were ADAM9 transcripts, soluble form and proteolytic activity. Ex vivo ADAM9 inhibition affected SARS-CoV-2 uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, COVID-19 cohort, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. The key driver, ADAM9, interfered with SARS-CoV-2 biology. A repositioning strategy for anti-ADAM9 therapeutic is feasible