Neurodegenerative Lysosomal Storage Disorders: TPC2 Comes to the Rescue!

Abstract

Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca²⁺ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca²⁺-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo.

Keywords: BK; COPD; TRPA1; TRPM2; TRPML; TRPML3; TRPV2; asthma; cystic fibrosis; emphysema; lung injury.

Figure 1

Effect of TPC2 inhibitors on cholesterol accumulation and lactosylceramide trafficking. (A) Representative confocal images showing filipin staining to visualize choles-terol accumulation and TO-PRO3 as nuclear staining. Experiments were performed in human fibroblasts: control (HF CTR), Niemann-Pick type C1 (HF NPC1) and mucolipidosis type IV (HF MLIV) treated with either DMSO, SG005 (3 µM), SG094 (3 µM) or tetrandrine (3 µM). (B) Bar plot showing the filipin intensity per cell (expressed as cholesterol load). Shown are mean values ± SEM. n > 3 biological replicates for each tested condition. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. Two-way ANOVA, post hoc Tukey’s multiple comparisons test. (C) Representative confocal images of LacCer (green) and LysoTracker (LyTr; red) in HF MLIV treated with DMSO, SG005 (1 µM) or SG094 (1 µM).

Figure 2

Effect of TPC2-A1-P on cholesterol accumulation in selected patient fibroblasts (HF) and electrophysiological characterization of a novel patient mutation (MCOLN1^T121M/T121M). (A) Effect of TPC2-A1-P or the TRPML1 activators ML-SA1 and MK6-83 on cholesterol accumulation in WT, MLIV and selected patient fibroblasts, carrying MLIV-causing point mutations as indicated, visualized by filipin. MCOLN1^T121M/T121M is a severely mislocalized variant (B) found to be homozygously expressed in a patient from a Yazidi family, recently diagnosed with MLIV (Prof. Thorsten Marquardt, University of Münster, Münster, Germany). The patient, an 18-year-old woman, showed a comparably mild clinical phenotype for her age (ability to walk and talk, delayed development with retinal degeneration (risk of blindness), reduced iron (39 µg/dL (60–140)) and ferritin (6 µg/L (16–92)) levels, reduced Hb, HCT, MCV and MCHC (iron deficiency anemia), slightly deranged liver function (ASAT: 56 U/L (<30) and ALAT: 42 U/L (<30))). Electrophysiology revealed TRPML1^T121M/T121M to retain some residual channel activity. (C–F) Shown are representative currents (I-V traces) from vacuolin-enlarged LE/LY, isolated from WT or patient fibroblasts (HF), activated by ML-SA1 or ML1-SA1 (=EVP169 = selective TRPML1 agonist). * p-value < 0.05; *** p-value < 0.001; **** p-value < 0.0001.

Figure 3

Effect of sphingomyelin on TPC2 and TRPML1 activities. (A,B) Representative measurements (I/Cm -V traces) of transiently transfected PM variants of hTPC2 (A) and hTRPML1 (B) in HEK cells. Channels were activated by small molecule agonists (10 µM TPC2-A1P and 10 µM ML-SA1), respectively. Subsequently, bath solution was completely exchanged for a solution containing agonist plus 20 µM sphingomyelin (SM). Bath solution contained: 138 mM NaCl, 6 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 5.5 mM D-glucose (adjusted to pH 7.4 with NaOH) and pipette solution contained 140 mM K-MSA, 5 mM KOH, 4 mM NaCl, 0.39 mM CaCl₂, 1 mM EGTA and 20 mM HEPES (pH was adjusted with KOH to 7.2).

Figure 4

Effect of TRPML1 activation in different LSD and WT iPSC-derived cortical neurons (endolysosomal patch clamp experiments as described previously [12]). (A) Representative measurements (I/Cm -V traces) from apilimod-enlarged LE/LY, isolated from WT, CLN mutants or MLIV knockout iPSC derived cortical neurons activated with ML-SA1 (10 µM). (B) Statistical analysis of TRPML1 activation at −80 mV depicted as mean values ± SEM (WT, CLN D416G, CLN R405R or CLN ko; n > 5); each dot represents a single measurement from distinct neuronal differentiations. An unpaired t-test was applied to quantify statistical significance; * p-value < 0.05, **** p-value < 0.0001.