Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons.

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson's disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2. METHODS: Taking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2G2019S and LRRK2R1441G mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNFα-induced changes in proteins of the NF-κB pathway and optimized western blot analysis to capture α-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls. RESULTS: LRRK2 silencing decreased α-synuclein protein levels in mutated neurons and modified NF-κB transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-κB and α-synuclein pathways converged and found that TNFα modulated α-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-κB dysregulation in mutated neurons, as shown by a protracted recovery of IκBα and a clear impairment in p65 nuclear translocation in the LRRK2 mutants. CONCLUSIONS: Altogether, our results show that LRRK2 mutations affect α-synuclein regulation and impair NF-κB canonical signaling in iPSC-derived neurons. TNFα modulated α-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PD.

Incidence of ultrasonographic signs of pneumothorax in asymptomatic neonates.

INTRODUCTION: Pneumothorax in neonates can be life-threatening. In neonates without respiratory distress, spontaneous pneumothorax can also develop under certain conditions. OBJECTIVES: To determine the incidence of ultrasonographic signs of pneumothorax in asymptomatic neonates using lung ultrasound as the diagnostic tool. METHODS: This was an observational prospective study conducted at the Basurto University Hospital (Bilbao, Spain) between January 2018 and December 2020. Lung ultrasound was performed during routine examination of asymptomatic neonates admitted to the maternity ward. RESULTS: Of a total of 204 asymptomatic neonates included in the study, 21 (10.3%) presented ultrasound signs of pneumothorax (Group A), and 183 (89.7%) had a normal lung ultrasound (Group B). Lung ultrasound was performed after a mean of 19 h of life (range 9-34). The presence of A-lines behind the sternum in the anterior transverse plane, at the intermammillary level, was observed in 100% of patients in Group A compared to no cases in Group B (p < 0.0001). The neonates of Group A presented the lung point located in the midclavicular line, indicative of a mild air leak. CONCLUSIONS: Lung ultrasound allows precise detection of suspected small-sized air leaks that can be detected in asymptomatic neonates. The true incidence of pneumothorax in asymptomatic neonates may be substantially higher than previously described in the literature.

LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1.

Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson's disease. Fibroblasts from Parkinson's disease patients with compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type PINK1, which induced a significant downregulation of LRRK2 (PARK8). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant PINK1 fibroblasts. To examine the interaction between the two PARK genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type PINK1 efficiently downregulated LRRK2 levels. Additionally, we confirmed a dysregulation of LRRK2 expression in fibroblasts from patients with a different homozygous mutation in PINK1 exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson's disease fibroblasts and neurons, suggest a convergent pathway for these PARK genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson's disease.

Leucine-rich repeat kinase 2 modulates cyclooxygenase 2 and the inflammatory response in idiopathic and genetic Parkinson's disease.

Inflammatory mechanisms are activated in aging and late-onset neurodegenerative diseases, such as Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both idiopathic and familial forms of PD. Here, we investigated the involvement of LRRK2 in inflammatory pathways using primary dermal fibroblasts from patients with 2 common mutations in LRRK2 (G2019S and R1441G), idiopathic PD and age-matched healthy individuals. Basal cyclooxygenase (COX)-2 RNA levels were very high in the fibroblasts of all patients. Remarkably, LRRK2 silencing experiments significantly reduced basal COX-2 levels and COX-2 induction after a pro-inflammatory stimulus. Additionally, in samples from patients with the R1441G mutation and with idiopathic PD, we found a prominent cytoplasmic re-distribution of human antigen R, a protein that, among others, stabilizes COX-2 RNA. Furthermore, the response to lipopolysaccharide was defective in these 2 groups, which showed weak induction of pro-inflammatory cytokines and reduced NFκB transcriptional activation. In summary, we describe multiple defects in inflammatory pathways in which LRRK2 appears to be critically involved. Further studies are required to establish the therapeutic implications of inflammatory dysregulation in the pathophysiology of Parkinson's disease.

Buspirone anti-dyskinetic effect is correlated with temporal normalization of dysregulated striatal DRD1 signalling in L-DOPA-treated rats.

Dopamine replacement with l-DOPA is the most effective therapy in Parkinson's disease. However, with chronic treatment, half of the patients develop an abnormal motor response including dyskinesias. The specific molecular mechanisms underlying dyskinesias are not fully understood. In this study, we used a well-characterized animal model to first establish the molecular differences between rats that did and did not develop dyskinesias. We then investigated the molecular substrates implicated in the anti-dyskinetic effect of buspirone, a 5HT1A partial agonist. Striatal protein expression profile of dyskinetic animals revealed increased levels of the dopamine receptor (DR)D3, ΔFosB and phospho (p)CREB, as well as an over-activation of the DRD1 signalling pathway, reflected by elevated ratios of phosphorylated DARPP32 and ERK2. Buspirone reduced the abnormal involuntary motor response in dyskinetic rats in a dose-dependent fashion. Buspirone (4 mg/kg) dramatically reduced the presence and severity of dyskinesias (by 83%) and normalized DARPP32 and ERK2 phosphorylation ratios, while the increases in DRD3, ΔFosB and pCREB observed in dyskinetic rats were not modified. Pharmacological experiments combining buspirone with 5HT1A and DRD3 antagonists confirmed that normalization of both pDARPP32 and pERK2 is required, but not sufficient, for blocking dyskinesias. The correlation between pDARPP32 ratio and dyskinesias was significant but not strong, pointing to the involvement of convergent factors and signalling pathways. Our results suggest that in dyskinetic rats DRD3 striatal over-expression could be instrumental in the activation of DRD1-downstream signalling and demonstrate that the anti-dyskinetic effect of buspirone in this model is correlated with DRD1 pathway normalization.

Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells.

Human embryonic and induced pluripotent stem cells are potential cell sources for regenerative approaches in Parkinson disease. Inductive differentiation protocols can generate midbrain dopamine neurons but result in heterogeneous cell mixtures. Therefore, selection strategies are necessary to obtain uniform dopamine cell populations. Here, we developed a selection approach using lentivirus vectors to express green fluorescent protein under the promoter region of FOXA2, a transcription factor that is expressed in the floor plate domain that gives rise to dopamine neurons during embryogenesis. We first validated the specificity of the vectors in human cell lines against a promoterless construct. We then selected FOXA2-positive neural progenitors from several human pluripotent stem cell lines, which demonstrated a gene expression profile typical for the ventral domain of the midbrain and floor plate, but failed to enrich for dopamine neurons. To investigate whether this was due to the selection approach, we overexpressed FOXA2 in neural progenitors derived from human pluripotent stem cell lines. FOXA2 forced expression resulted in an increased expression of floor plate but not mature neuronal markers. Furthermore, selection of the FOXA2 overexpressing fraction also failed to enrich for dopamine neurons. Collectively, our results suggest that FOXA2 is not sufficient to induce a dopaminergic fate in this system. On the other hand, our study demonstrates that a combined approach of promoter activation and lentivirus vector technology can be used as a versatile tool for the selection of a defined cell population from a variety of human pluripotent stem cell lines.

Fast and efficient neural conversion of human hematopoietic cells.

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation.

The existence of endogenous neural progenitors in the nigrostriatal system could represent a powerful tool for restorative therapies in Parkinson's disease. Sox-2 is a transcription factor expressed in pluripotent and adult stem cells, including neural progenitors. In the adult brain Sox-2 is expressed in the neurogenic niches. There is also widespread expression of Sox-2 in other brain regions, although the neurogenic potential outside the niches is uncertain. Here, we analyzed the presence of Sox-2(+) cells in the adult primate (Macaca fascicularis) brain in naïve animals (N = 3) and in animals exposed to systemic administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine to render them parkinsonian (N = 8). Animals received bromodeoxyuridine (100 mg/kg once a day during five consecutive days) to label proliferating cells and their progeny. Using confocal and electron microscopy we analyzed the Sox-2(+) cell population in the nigrostriatal system and investigated changes in the number, proliferation and neurogenic potential of Sox-2(+) cells, in control conditions and at two time points after MPTP administration. We found Sox-2(+) cells with self-renewal capacity in both the striatum and the substantia nigra. Importantly, only in the striatum Sox-2(+) was expressed in some calretinin(+) neurons. MPTP administration led to an increase in the proliferation of striatal Sox-2(+) cells and to an acute, concomitant decrease in the percentage of Sox-2(+)/calretinin(+) neurons, which recovered by 18 months. Given their potential capacity to differentiate into neurons and their responsiveness to dopamine neurotoxic insults, striatal Sox-2(+) cells represent good candidates to harness endogenous repair mechanisms for regenerative approaches in Parkinson's disease.

Correction: Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation.

[This corrects the article on p. e66377 in vol. 8.].