. Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the gene coding for a lysosomal ion channel Mucolipin 1 (TRPML1). MLIV causes severe developmental delays, psychomotor retardation, cognitive disabilities, corneal cloudiness and degeneration of the retina. At the cellular level, accumulation of phospholipids and gangliosides is detected, and virtually all patients'cells contain enfolded membranous structures of unknown origin. Since TRPML1 localizes in lysosomes and its downregulation causes severe lysosomal storage phenotype, TRPML1 was suggested to regulate lysosomal function. At present, there is no complete model that explains TRPML1 function in the lysosomes. TRPML1 was suggested to directly regulate intracellular membrane traffic by modulating fusion and/or fission of the organelles within the lower portions of the endocytic pathway. An alternative model suggests that TRPML1 affects lysosomal hydrolysis by regulating lysosomal acidification. The present project aims to directly answer the two main question in MLIV pathogenesis: whether TRPML1 directly regulates membrane traffic in the endocytic pathway and which aspect of TRPML1 permeability makes it indispensable for the proper function of the endocytic pathway. This will be accomplished by a combination of molecular, biochemical and physiological techniques. Specifically, an siRNA driven TRPML1 downregulation system will be used to establish the direct and immediate effects of TRPML1 downregulation on membrane traffic and lysosomal lipid hydrolysis. A structure function analysis of ion permeation through TRPML1 and its close relative TRPML3 will be performed to identify the structural determinants of the unique aspects of TRPML1 ion permeation. The ion permeation through TRPML1 will be specifically modulated in order to establish the aspects of TRPML1 permeability that make TRPML1 indispensable for the proper function of the endocytic pathway. The experiments planned in this proposal will establish the direction for the search for MLIV treatments, identify the new roles for intracellular ion channels in regulation of membrane traffic, lysosomal metabolism and organellar ion homeostasis and provide novel structural information regarding the basic mechanisms of ion channel permeation, rectification and block.

Public Health Relevance

. Finding whether MLIV is a result of membrane traffic delays or inefficient lipid hydrolysis in lysosomes is key to formulating strategies for MLIV treatment as it defines whether the enzyme replacement therapies will be effective as an MLIV treatment. If TRPML1 loss affects lysosomal ion homeostasis and causes inactivation of lysosomal enzymes, then the future MLIV therapies may focus on enzyme replacement using enzymes modified to work in MLIV specific lysosomal ionic environment. Furthermore, pharmacological strategies can be formulated to specifically change lysosomal ionic environment to compensate the TRPML1 loss.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD058577-04
Application #
8337338
Study Section
Developmental Brain Disorders Study Section (DBD)
Program Officer
Oster-Granite, Mary Lou
Project Start
2011-09-26
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
4
Fiscal Year
2012
Total Cost
$270,400
Indirect Cost
$89,109
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Kukic, Ira; Kelleher, Shannon L; Kiselyov, Kirill (2014) Zn2+ efflux through lysosomal exocytosis prevents Zn2+-induced toxicity. J Cell Sci 127:3094-103
Coblentz, Jessica; St Croix, Claudette; Kiselyov, Kirill (2014) Loss of TRPML1 promotes production of reactive oxygen species: is oxidative damage a factor in mucolipidosis type IV? Biochem J 457:361-8
Kukic, Ira; Lee, Jeffrey K; Coblentz, Jessica et al. (2013) Zinc-dependent lysosomal enlargement in TRPML1-deficient cells involves MTF-1 transcription factor and ZnT4 (Slc30a4) transporter. Biochem J 451:155-63
Colletti, Grace A; Miedel, Mark T; Quinn, James et al. (2012) Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis. J Biol Chem 287:8082-91
Rybalchenko, Volodymyr; Ahuja, Malini; Coblentz, Jessica et al. (2012) Membrane potential regulates nicotinic acid adenine dinucleotide phosphate (NAADP) dependence of the pH- and Ca2+-sensitive organellar two-pore channel TPC1. J Biol Chem 287:20407-16
Colletti, Grace A; Kiselyov, Kirill (2011) TRPML1. Adv Exp Med Biol 704:209-19
Kiselyov, Kirill; Colletti, Grace A; Terwilliger, Austen et al. (2011) TRPML: transporters of metals in lysosomes essential for cell survival? Cell Calcium 50:288-94
Kiselyov, Kirill; van Rossum, Damian B; Patterson, Randen L (2010) TRPC channels in pheromone sensing. Vitam Horm 83:197-213
Kiselyov, Kirill; Yamaguchi, Soichiro; Lyons, Christopher W et al. (2010) Aberrant Ca2+ handling in lysosomal storage disorders. Cell Calcium 47:103-11