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Known and novel members of the endolysosomal transportome/channelome as candidates to rescue lysosomal storage diseases (LSDs)
Known and novel members of the endolysosomal transportome/channelome as candidates to rescue lysosomal storage diseases (LSDs)
The project presented herein addresses our limited understanding of organellar pharmacology. Specifically, the work was conceived to elucidate the biological relevance of the endolysosomal cation channels (mucolipins/TRPMLs and two-pore channels/TPCs) using novel, selective pharmacological modulators. On one hand, we developed a first-in-field selective TRPML2 agonist, ML2-SA1, which activates the TRPML2 ion channel on early endosomes, recycling endosomes, and lysosomes. We demonstrate how TRPML2 accelerates endosomal traffic, enhancing chemokine secretion and macrophage chemoattraction. TRPML2 activity is particularly important in the rapidly recycling pathway, where it mediates cargo transit directly from sorting endosomes to the plasma membrane. This function is largely conferred by its unqiue activation by membrane stretching, a feature we have shown to rely on a single amino acid in the TRPML2 phosphoinositide binding-pocket (L314). Mutation of L314 into its TRPML1/TRPML3 counterpart (L314R) abrogates TRPML2 osmosensitivity, and impedes the rapidly recycling pathway. These findings provide biological and structural information about TRPML2 function, laying the foundation for future endeavors modulating immune cell response and inflammation through the immune cell-restricted, druggable ion channel. Our primary motivation for investigating the endolysosomal ion channels is development of new treatments for diseases currently lacking therapies. The lysosomal storage diseases (LSDs) represent one such family of diseases, where endolysosomal protein defects result in lysosomal dysfunction and (often) neurodegeneration. Mucolipidosis type IV (MLIV) is caused by dysfunction of the lysosomal TRPML1 ion channel, causing blindness and early-onset neurodegeneration. Aiming to treat LSDs such as MLIV, we investigated the related lysosomal ion channel TPC2. We characterized various TPC2 polymorphisms that increase its activity, and developed agonists for TPC2 that either facilitate high Ca2+ fluxes arresting endosomal motility or Na+ fluxes facilitating lysosomal exocytosis and enhancing autophagy. We used CRISPR/Cas9 to develop new induced pluripotent stem cell (iPSC) models for Neuronal Ceroid Lipofuscinosis (colloquially termed “childhood dementia“) and MLIV, differentiating these into cortical neurons. We used the diseased human neurons to investigate treatments for LSDs, finding the autophagic enhancer tamoxifen and the two-pore channel 2 agonist TPC2-A1-P to counteract LSD phenotypes. TPC2-A1-P restored excessive lysosomal proteolysis, storage defects, and trafficking abnormalities in human MLIV neurons and patient fibroblasts. Similarly, TPC2-A1-P ameliorated LSD phenotypes in Niemann-Pick Disease type C1 fibroblasts (NPC1, also known as childhood Alzheimer’s Disease), another LSD marked by impaired activity of lysosomal cation channels. We finally performed a proof-of-concept in vivo investigation, treating MLIV mice with TPC2-A1-P. While DMSO-treated MLIV mice exhibited gliosis of the cerebellum and hippocampus, TPC2-A1-P-injected mouse brains featured much fewer glial cells, akin to the wild-type controls. These findings demonstrate that pharmacological modulation of the endolysosomal system can restore physiology in a variety of lysosomal storage diseases in vitro and in vivo.
Lysosomal storage diseases, mucolipidosis type IV, TRPML, TRPML1, TRPML2, TPC, TPC2, genetics, endosomes, lysosomes, ion channels, pharmacology, neurodegeneration, iPSCs, CRISPR/Cas9
Krogsaeter, Einar Kleinhans
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Krogsaeter, Einar Kleinhans (2022): Known and novel members of the endolysosomal transportome/channelome as candidates to rescue lysosomal storage diseases (LSDs). Dissertation, LMU München: Faculty of Biology
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Abstract

The project presented herein addresses our limited understanding of organellar pharmacology. Specifically, the work was conceived to elucidate the biological relevance of the endolysosomal cation channels (mucolipins/TRPMLs and two-pore channels/TPCs) using novel, selective pharmacological modulators. On one hand, we developed a first-in-field selective TRPML2 agonist, ML2-SA1, which activates the TRPML2 ion channel on early endosomes, recycling endosomes, and lysosomes. We demonstrate how TRPML2 accelerates endosomal traffic, enhancing chemokine secretion and macrophage chemoattraction. TRPML2 activity is particularly important in the rapidly recycling pathway, where it mediates cargo transit directly from sorting endosomes to the plasma membrane. This function is largely conferred by its unqiue activation by membrane stretching, a feature we have shown to rely on a single amino acid in the TRPML2 phosphoinositide binding-pocket (L314). Mutation of L314 into its TRPML1/TRPML3 counterpart (L314R) abrogates TRPML2 osmosensitivity, and impedes the rapidly recycling pathway. These findings provide biological and structural information about TRPML2 function, laying the foundation for future endeavors modulating immune cell response and inflammation through the immune cell-restricted, druggable ion channel. Our primary motivation for investigating the endolysosomal ion channels is development of new treatments for diseases currently lacking therapies. The lysosomal storage diseases (LSDs) represent one such family of diseases, where endolysosomal protein defects result in lysosomal dysfunction and (often) neurodegeneration. Mucolipidosis type IV (MLIV) is caused by dysfunction of the lysosomal TRPML1 ion channel, causing blindness and early-onset neurodegeneration. Aiming to treat LSDs such as MLIV, we investigated the related lysosomal ion channel TPC2. We characterized various TPC2 polymorphisms that increase its activity, and developed agonists for TPC2 that either facilitate high Ca2+ fluxes arresting endosomal motility or Na+ fluxes facilitating lysosomal exocytosis and enhancing autophagy. We used CRISPR/Cas9 to develop new induced pluripotent stem cell (iPSC) models for Neuronal Ceroid Lipofuscinosis (colloquially termed “childhood dementia“) and MLIV, differentiating these into cortical neurons. We used the diseased human neurons to investigate treatments for LSDs, finding the autophagic enhancer tamoxifen and the two-pore channel 2 agonist TPC2-A1-P to counteract LSD phenotypes. TPC2-A1-P restored excessive lysosomal proteolysis, storage defects, and trafficking abnormalities in human MLIV neurons and patient fibroblasts. Similarly, TPC2-A1-P ameliorated LSD phenotypes in Niemann-Pick Disease type C1 fibroblasts (NPC1, also known as childhood Alzheimer’s Disease), another LSD marked by impaired activity of lysosomal cation channels. We finally performed a proof-of-concept in vivo investigation, treating MLIV mice with TPC2-A1-P. While DMSO-treated MLIV mice exhibited gliosis of the cerebellum and hippocampus, TPC2-A1-P-injected mouse brains featured much fewer glial cells, akin to the wild-type controls. These findings demonstrate that pharmacological modulation of the endolysosomal system can restore physiology in a variety of lysosomal storage diseases in vitro and in vivo.