Mitochondrial localization and function of the lysosomal enzyme glucocerebrosidase

Biallelic mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), cause the lysosomal storage disease Gaucher's disease (GD). These mutations have been recently identified as the strongest risk factor for Parkinson’s disease (PD) and other synucleinopathies. In the lysosome, GCase catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glycosphingolipid (GSL), to ceramide and glucose and of glucosylsphingosine to glucose and sphingosine. Interestingly, a reduction in GCase activity has also been observed in sporadic PD as well as with ageing. The mechanisms leading to neurodegeneration in GBA1 carriers remain unclear. To explore the mechanisms involved in GBA1-linked neurodegeneration, we assessed the interactome of wild-type (WT) and mutant GCase. To this end, we developed an inducible T-Rex HEK cell model overexpressing V5-Flag-tagged WT, p.E326K, or p.L444P mutant GCase. We chose these two mutants as p.L444P is a severe mutation leading to a neuropathic form of GD and as it is one of the most common severe GBA1-PD mutations. p.E326K is another common mutation in GBA1-PD. However, unlike most other GBA1 mutations, p.E326K homozygous mutations on their own are not linked to GD. The overexpression model was validated by the analysis of GCase expression and protein level, as well as co-localization with the lysosomal marker LAMP1. We performed Flag co-immunoprecipitation and analyzed the eluates by quantitative tandem mass tag liquid chromatography mass spectrometry. Interestingly, we found that 13.3% of GCase interactors are mitochondrial proteins. As mitochondrial dysfunction has previously been linked to GBA1, we further explored a potential direct link between GCase and mitochondrial dysfunction. First, we validated the mitochondrial localization of GCase by split-GFP experiments, in which the interaction between mitochondrial matrix targeted GFP1-10 and WT and mutant GBA1-GFP-S11ß lead to green fluorescence. We demonstrated that the import of GCase into the mitochondrial matrix is HSC70, translocase of outer mitochondrial membrane (TOM)-, and translocase of inner mitochondrial membrane (TIM)-complex dependent. Removal of the internal mitochondrial targeting like sequences prevented import of GCase into mitochondria. In addition, we found an increased interaction between HSP60 and LONP1 with mutant GCase, suggesting their potential role in the refolding or degradation of mutant GCase in mitochondria. Furthermore, we found a decreased interaction of mutant GCase with the complex I (CI) assembly factor TIMMDC1 and the CI subunit NDUFA10. To validate these results in a model relevant to GBA1-PD, we generated induced pluripotent stem cells (iPSCs) from p.L444P and p.E326K heterozygous PD patients’ fibroblasts. Next we employed genome editing (zinc finger nucleases and CRISPR-Cas9) to generate corresponding isogenic gene-corrected controls. Gene-correction rescued GCase protein level and activity. Mutant and isogenic controls did not show differences in midbrain neuron differentiation potential. The interaction with HSP60 and LONP1 was validated by co-immunoprecipitation with endogenous GCase in neural precursor cell (NPC) lysates. Preliminary data point towards CI assembly defects in mutant and GBA1 KO NPCs as well as midbrain neurons. These data confirm the potential involvement of GCase in CI assembly. Improving GCase trafficking to mitochondria could be a potential therapeutic target as overexpression of GCase improved mitochondrial dysfunction in T-Rex HEK cells. Our data provide evidence for a novel mitochondrial role of GCase, showing its potential involvement in the maintenance of CI integrity by modulating the stability of the assembly factor TIMMDC1

Neuronopathic Gaucher disease models reveal defects in cell growth promoted by Hippo pathway activation

Gaucher Disease (GD), the most common lysosomal disorder, arises from mutations in the GBA1 gene and is characterized by a wide spectrum of phenotypes, ranging from mild hematological and visceral involvement to severe neurological disease. Neuronopathic patients display dramatic neuronal loss and increased neuroinflammation, whose molecular basis are still unclear. Using a combination of Drosophila dGBA1b loss-of-function models and GD patient-derived iPSCs differentiated towards neuronal precursors and mature neurons we showed that different GD- tissues and neuronal cells display an impairment of growth mechanisms with an increased cell death and reduced proliferation. These phenotypes are coupled with the downregulation of several Hippo transcriptional targets, mainly involved in cells and tissue growth, and YAP exclusion from nuclei. Interestingly, Hippo knock-down in the GBA-KO flies rescues the proliferative defect, suggesting that targeting the Hippo pathway can be a promising therapeutic approach to neuronopathic GD.A combination of Drosophila dGBA1b loss-of-function models and Gaucher Disease (GD) patient-derived iPSCs reveals an impairment in GD neuronal cell growth and that Hippo pathway hyperactivation contributes to the impairment

Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism

Mutations in GBA1, the gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher's disease, are the most frequent genetic risk factor for Parkinson's disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson's Disease.

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases

Claudin-12 is not required for blood-brain barrier tight junction function.

BACKGROUND The blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. METHODS We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. RESULTS Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice. CONCLUSIONS Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions

Critical review of strategic planning research in hospitality and tourism

Strategic planning remains one of the most popular management tools, but theoretical and empirical developments in the academic literature have been a slow burn. This paper addresses this gap and provides an up-to-date review of hospitality and tourism strategic planning research. We review strategic planning research from 1995 to 2013 in seven leading tourism academic journals, and adopt a modern and broad conceptualization of strategic planning. While there is some awareness of effective tourism strategic planning processes, academic research has not kept pace with practice. To stimulate a resurgence of research interest, we provide future research directions. We observe a methodological introspection and present some new research methodologies, which are critically important in researching the turbulent, chaotic and nonlinear tourism environment

Phenomenon-based research in management and organisation science: When is it rigorous and does it matter?

Recently, the editors of Long Range Planning called for more phenomenon-based research. Such research focuses on identifying and reporting on new or recent phenomena of interest and relevance to management and organisation science. In this article, we explore the nature of phenomenon-based research and develop a research strategy that provides guidelines for researchers seeking to make this type of scientific inquiry rigorous and relevant. Phenomenon-based research establishes and describes the empirical facts and constructs that enable scientific inquiry to proceed. An account of the study of open source software development illustrates the research strategy. Rigorous phenomenon-based research tackles problems that are relevant to management practice and fall outside the scope of available theories. Phenomenon-based research also bridges epistemological and disciplinary divides because it unites diverse scholars around their shared interest in the phenomenon and their joint engagement in the research activities: identification, exploration, design, theorising and synthesis