Study of cellular processes affected by lysosomal disorders
: importance for mitochondrial morphology and cholesterol homeostasis

  • Guillaume Van Beersel

Student thesis: Doc typesDoctor of Sciences


LSDs (Lysosomal storage diseases) represent diverse sets of conditions resulting from an impaired uptake, sorting or digestion of the material captured by cells during endocytosis or autophagy. These autosomal recessive diseases can be caused by defects in soluble enzymes, in non-enzymatic lysosomal proteins or even in non-lysosomal proteins, resulting, in fine, to the storage of specific macromolecules inside the organelles of the endo-lysosomal system. Numerous and complex biochemical and signaling cascades have been shown to be activated/altered in response to the original lysosomal disorder and are now known or suspected to play an essential role in the cell dysfunction and death. Such “pathogenic mechanisms” include, for example: alterations of cellular calcium homeostasis and impairment of autophagy, the onset of oxidative stress and inflammation (and autoimmune response), an endoplasmic reticulum stress response as well as changes in the lipid homeostasis and mitochondrial dysfunction. However, even if changes in mitochondrial morphology have been already described in more than ten LSDs, the molecular mechanisms underlying these defects in response to the lysosomal storage and more generally the impact of lysosomal dysfunction on mitochondria remain poorly characterized. The first part of this work has been dedicated to the study of the effect of the TPP-1 (TriPeptidyl-Peptidase-I) deficiency (a lysosomal hydrolase deficient in the LINCL (Late Infantile Neuronal Ceroid Lipofuscinosis)) on the mitochondrial population of human skin fibroblasts. We found that a TPP-1 deficiency induces a fragmentation of the mitochondrial network in these cells, probably caused by a reduction in the abundance of both Mitofusin 1 and Mitofusin 2, two proteins that are essential for the mitochondrial fusion process. Surprisingly, this aberrant mitochondrial morphology is not accompanied by changes either in the matrix calcium concentration or in the abundance of ROS (Reactive Oxygen Species) in basal conditions. In addition, changes in matrix calcium concentration and ROS abundance observed in response to an appropriate stimulus are similar in TPP-1 deficient cells than in cells that recovered TPP-1 activity. We also found that BNIP3 (BCL2/adenovirus E1B 19 kDa protein-Interacting Protein 3), a pro-apoptotic BH3-only protein, is over-expressed in TPP-1 deficient cells. By using a RNA interference approach, we showed that BNIP3 is not involved in the fragmentation of the mitochondrial network in LINCL cells in basal conditions but that this protein can sensitize mitochondria of TPP-1 deficient cells to further mitochondrial fragmentation induced by antimycin A, an inhibitor of the complex III of the mitochondrial respiratory chain, well-known to induce ROS production. These results open the way to further investigation on the role of mitochondrial dysfunction in the pathogenesis of the LINCL. The perturbation of the lipid homeostasis is another characteristic shared by several types of lysosomal disorders. Indeed, a sequestration of cholesterol inside the endo-lysosomal system has been observed in several of these diseases and was shown to lead to the activation of SREBPs (Sterol Regulatory Element-Binding Proteins) transcription factors, key actors in the regulation of lipid metabolism. In the second part of this work, we were interested to determine whether or not this phenomenon could be observed in a sucrose-induced lysosomal storage, a model commonly used to study cell response to lysosomal storage. Indeed, while the relevance of this “sucrosome” model in the study of the effects of a lysosomal storage is widely accepted, the question to know whether it could be relevant to study the molecular mechanisms by which the lysosomal storage triggers broader cellular dysfunctions is still unclear. Here, we showed that the non-esterified cholesterol distribution is modified in 143B cells incubated with sucrose and demonstrated that this aberrant free cholesterol distribution is associated with an increase in the activity of SREBPs. Moreover, we found that the activation of these transcription factors is associated with an increase in the expression of HMG-CoA synthase and mevalonate kinase, the products of two of their target genes, accompanied by an increase in total lipid biosynthesis. Finally, using siRNA interference we demonstrate that SREBP-2 but not SREBP-1a is responsible for the up-regulation of genes encoding these two enzymes. Our results therefore confirmed that the sucrosome model is relevant in the study of the molecular mechanisms and signaling pathways activated in response to a cholesterol sequestration in the endo-lysosomal system resulting from a lysosomal storage, a phenomenon observed in several LSDs. This experimental model, contrary to cell lines derived from LSD patients, presents some assets such as its perfect reversibility when cells are pre-incubated with the invertase but also the possibility to study the putative transitory molecular events occurring at the early onset of the lysosomal overload. Finally, in our research of cellular processes that could be impaired in response of a lysosomal dysfunction, we sought to identify putative other transcription factors and signaling pathways activated in the brain of a mouse model knockout for TPP-1. These experiments allowed us to identify several candidates for which DNA-binding activity seems to be increased in the brain of 120 day-old TPP-1 deficient mice. The majority of them have been described to be involved in the differentiation or the activation of cells of the hematopoietic lineage including the microglial cells and therefore suggest that the TPP-1 deficiency triggers directly (or indirectly) the activation of an inflammatory response in the brain of TPP-1 knockout mice. However, the putative role of these transcription factors and the inflammation in the LINCL pathogenesis remains to be determined
Date of Award22 Feb 2013
Original languageEnglish
Awarding Institution
  • University of Namur
SupervisorMichel Jadot (Supervisor), Thierry ARNOULD (Supervisor), Martine Raes (President), Benoit Muylkens (Jury), Olivier De Backer (Jury), P. Lobel (Jury) & J N Octave (Jury)

Cite this