Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions.

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).

OPA1 haploinsufficiency induces a BNIP3-dependent decrease in mitophagy in neurons: relevance to Dominant Optic Atrophy.

Dominant optic atrophy (DOA) is because of mutations in the mitochondrial protein OPA1. The disease principally affects retinal ganglion cells, whose axons degenerate leading to vision impairments, and sometimes other neuronal phenotypes. The exact mechanisms underlying DOA pathogenesis are not known. We previously demonstrated that the main role of OPA1, as a mitochondrial fusogenic and anti-apoptotic protein, are inhibited by interaction with the stress inducible pro-apoptotic BNIP3 protein. Because BNIP3 was recently reported to participate in autophagy and mitophagy, we tested the involvement of these processes in DOA pathogenesis. Using an in vitro neuronal model of DOA, we identified a BNIP3 down-regulation that reduced autophagy and mitophagy. Restoring BNIP3 had a biphasic effect, first rescuing autophagy and mitophagy levels but later leading to cell death. Similarly, in an in vivo mouse model of DOA, we showed that BNIP3 levels are decreased in young adult mice and increase to normal levels upon aging, paralleling disease progression. Altogether, our results indicate that the relationship between OPA1 and BNIP3 may have important bearings on DOA pathogenesis.

Amplifying mitochondrial function rescues adult neurogenesis in a mouse model of Alzheimer's disease.

Adult hippocampal neurogenesis is strongly impaired in Alzheimer's disease (AD). In several mouse models of AD, it was shown that adult-born neurons exhibit reduced survival and altered synaptic integration due to a severe lack of dendritic spines. In the present work, using the APPxPS1 mouse model of AD, we reveal that this reduced number of spines is concomitant of a marked deficit in their neuronal mitochondrial content. Remarkably, we show that targeting the overexpression of the pro-neural transcription factor Neurod1 into APPxPS1 adult-born neurons restores not only their dendritic spine density, but also their mitochondrial content and the proportion of spines associated with mitochondria. Using primary neurons, a bona fide model of neuronal maturation, we identified that increases of mitochondrial respiration accompany the stimulating effect of Neurod1 overexpression on dendritic growth and spine formation. Reciprocally, pharmacologically impairing mitochondria prevented Neurod1-dependent trophic effects. Thus, since overexpression of Neurod1 into new neurons of APPxPS1 mice rescues spatial memory, our present data suggest that manipulating the mitochondrial system of adult-born hippocampal neurons provides neuronal plasticity to the AD brain. These findings open new avenues for far-reaching therapeutic implications towards neurodegenerative diseases associated with cognitive impairment.

Identification and implication of tissue-enriched ligands in epithelial-endothelial crosstalk during pancreas development.

Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. BMP7 and WNT7B, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and SEMA6D, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced BMP7 limited development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of BMP7 in the intercellular communications contributing to vessel development and organization during pancreas organogenesis.

The Multifunctional Sorting Protein PACS-2 Controls Mitophagosome Formation in Human Vascular Smooth Muscle Cells through Mitochondria-ER Contact Sites.

Mitochondria-associated ER membranes (MAMs) are crucial for lipid transport and synthesis, calcium exchange, and mitochondrial functions, and they also act as signaling platforms. These contact sites also play a critical role in the decision between autophagy and apoptosis with far reaching implications for cell fate. Vascular smooth muscle cell (VSMC) apoptosis accelerates atherogenesis and the progression of advanced lesions, leading to atherosclerotic plaque vulnerability and medial degeneration. Though the successful autophagy of damaged mitochondria promotes VSMC survival against pro-apoptotic atherogenic stressors, it is unknown whether MAMs are involved in VSMC mitophagy processes. Here, we investigated the role of the multifunctional MAM protein phosphofurin acidic cluster sorting protein 2 (PACS-2) in regulating VSMC survival following a challenge by atherogenic lipids. Using high-resolution confocal microscopy and proximity ligation assays, we found an increase in MAM contacts as in PACS-2-associated MAMs upon stimulation with atherogenic lipids. Correspondingly, the disruption of MAM contacts by PACS-2 knockdown impaired mitophagosome formation and mitophagy, thus potentiating VSMC apoptosis. In conclusion, our data shed new light on the significance of the MAM modulatory protein PACS-2 in vascular cell physiopathology and suggest MAMs may be a new target to modulate VSMC fate and favor atherosclerotic plaque stability.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype.

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE-/-) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

Autophagy in Metabolic Age-Related Human Diseases.

Autophagy is a highly conserved homeostatic cellular mechanism that mediates the degradation of damaged organelles, protein aggregates, and invading pathogens through a lysosome-dependent pathway. Over the last few years, specific functions of autophagy have been discovered in many tissues and organs; however, abnormal upregulation or downregulation of autophagy has been depicted as an attribute of a variety of pathologic conditions. In this review, we will describe the current knowledge on the role of autophagy, from its regulation to its physiological influence, in metabolic age-related disorders. Finally, we propose to discuss the therapeutic potential of pharmacological and nutritional modulators of autophagy to treat metabolic diseases.

Vascular smooth muscle cell death, autophagy and senescence in atherosclerosis.

In the present review, we describe the causes and consequences of loss of vascular smooth muscle cells (VSMCs) or their function in advanced atherosclerotic plaques and discuss possible mechanisms such as cell death or senescence, and induction of autophagy to promote cell survival. We also highlight the potential use of pharmacological modulators of these processes to limit plaque progression and/or improve plaque stability. VSMCs play a pivotal role in atherogenesis. Loss of VSMCs via initiation of cell death leads to fibrous cap thinning and promotes necrotic core formation and calcification. VSMC apoptosis is induced by pro-inflammatory cytokines, oxidized low density lipoprotein, high levels of nitric oxide and mechanical injury. Apoptotic VSMCs are characterized by a thickened basal lamina surrounding the cytoplasmic remnants of the VSMC. Inefficient clearance of apoptotic VSMCs results in secondary necrosis and subsequent inflammation. A critical determinant in the VSMC stress response and phenotypic switching is autophagy, which is activated by various stimuli, including reactive oxygen and lipid species, cytokines, growth factors and metabolic stress. Successful autophagy stimulates VSMC survival, whereas reduced autophagy promotes age-related changes in the vasculature. Recently, an interesting link between autophagy and VSMC senescence has been uncovered. Defective VSMC autophagy accelerates not only the development of stress-induced premature senescence but also atherogenesis, albeit without worsening plaque stability. VSMC senescence in atherosclerosis is likely a result of replicative senescence and/or stress-induced premature senescence in response to DNA damaging and/or oxidative stress-inducing stimuli. The finding that VSMC senescence can promote atherosclerosis further illustrates that normal, adequate VSMC function is crucial in protecting the vessel wall against atherosclerosis.

Methods for Measuring Autophagy in Mice.

Autophagy is a dynamic intracellular process that mediates the degradation of damaged cytoplasmic components by the lysosome. This process plays important roles in maintaining normal cellular homeostasis and energy balance. Measuring autophagy activity is critical and although the determination of autophagic flux in isolated cells is well documented, there is a need to have reliable and quantitative assays to evaluate autophagy in whole organisms. Because mouse models have been precious in establishing the functional significance of autophagy under physiological or pathological conditions, we present in this chapter a compendium of the current available methods to measure autophagy in mice, and discuss their advantages and limitations.