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1.
Neurobiol Dis ; 159: 105507, 2021 11.
Article in English | MEDLINE | ID: mdl-34509608

ABSTRACT

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.


Subject(s)
Benzoxazoles/pharmacology , Dopaminergic Neurons/drug effects , Glucosylceramidase/genetics , Glucosyltransferases/antagonists & inhibitors , Glycosphingolipids/metabolism , Lysosomes/drug effects , Synucleinopathies/metabolism , alpha-Synuclein/drug effects , Animals , Dopaminergic Neurons/metabolism , In Vitro Techniques , Lysosomes/metabolism , Mice , Neurons/drug effects , Neurons/metabolism , Parkinson Disease/genetics , Parkinson Disease/metabolism , Primary Cell Culture , Protein Aggregates , Rats , Synucleinopathies/genetics , alpha-Synuclein/metabolism
2.
Hum Mol Genet ; 28(19): 3244-3254, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31261387

ABSTRACT

Multiple genome-wide association studies (GWAS) in Parkinson disease (PD) have identified a signal at chromosome 4p16.3; however, the causal variant has not been established for this locus. Deep investigation of the region resulted in one identified variant, the rs34311866 missense SNP (p.M393T) in TMEM175, which is 20 orders of magnitude more significant than any other SNP in the region. Because TMEM175 is a lysosomal gene that has been shown to influence α-synuclein phosphorylation and autophagy, the p.M393T variant is an attractive candidate, and we have examined its effect on TMEM175 protein and PD-related biology. After knocking down each of the genes located under the GWAS peak via multiple shRNAs, only TMEM175 was found to consistently influence accumulation of phosphorylated α-synuclein (p-α-syn). Examination of the p.M393T variant showed effects on TMEM175 function that were intermediate between the wild-type (WT) and knockout phenotypes, with reduced regulation of lysosomal pH in response to starvation and minor changes in clearance of autophagy substrates, reduced lysosomal localization, and increased accumulation of p-α-syn. Finally, overexpression of WT TMEM175 protein reduced p-α-syn, while overexpression of the p.M393T variant resulted in no change in α-synuclein phosphorylation. These results suggest that the main signal in the chromosome 4p16.3 PD risk locus is driven by the TMEM175 p.M393T variant. Modulation of TMEM175 may impact α-synuclein biology and therefore may be a rational therapeutic strategy for PD.


Subject(s)
Parkinson Disease/genetics , Polymorphism, Single Nucleotide , Potassium Channels/genetics , alpha-Synuclein/metabolism , Cell Line , Chromosomes, Human, Pair 4/genetics , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Lysosomes/metabolism , Parkinson Disease/metabolism , Phosphorylation , Potassium Channels/metabolism
3.
Proc Natl Acad Sci U S A ; 114(9): 2389-2394, 2017 02 28.
Article in English | MEDLINE | ID: mdl-28193887

ABSTRACT

Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD.


Subject(s)
Autophagosomes/metabolism , Dopaminergic Neurons/metabolism , Lysosomes/metabolism , Mitochondria/metabolism , Potassium Channels/genetics , alpha-Synuclein/chemistry , Animals , Autophagosomes/drug effects , Autophagosomes/pathology , Autophagy/drug effects , Cell Line, Tumor , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/pathology , Gene Expression Regulation , Glucosylceramidase/genetics , Glucosylceramidase/metabolism , Humans , Hydrogen-Ion Concentration , Lysosomes/drug effects , Lysosomes/pathology , Mitochondria/drug effects , Mitochondria/pathology , Models, Biological , Parkinson Disease/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Potassium Channels/deficiency , Primary Cell Culture , Protein Aggregates/drug effects , Rats , alpha-Synuclein/pharmacology
4.
Biochemistry ; 46(12): 3862-71, 2007 Mar 27.
Article in English | MEDLINE | ID: mdl-17323925

ABSTRACT

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.


Subject(s)
Calcium/metabolism , Calmodulin/metabolism , Intermediate Filament Proteins/metabolism , Membrane Glycoproteins/metabolism , Models, Molecular , Nerve Tissue Proteins/metabolism , Animals , Brain Chemistry , Calcium/chemistry , Calmodulin/chemistry , Calmodulin/genetics , Cattle , Humans , Intermediate Filament Proteins/chemistry , Membrane Glycoproteins/chemistry , Mice , Nerve Tissue Proteins/chemistry , Peptides/chemistry , Peptides/metabolism , Peripherins , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinal Rod Photoreceptor Cells/chemistry , Retinal Rod Photoreceptor Cells/metabolism
5.
Biochemistry ; 46(5): 1256-72, 2007 Feb 06.
Article in English | MEDLINE | ID: mdl-17260955

ABSTRACT

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.


Subject(s)
Carrier Proteins/metabolism , Intermediate Filament Proteins/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Adaptor Proteins, Vesicular Transport , Animals , Binding Sites , Carrier Proteins/physiology , Cattle , Cell Line , Cell Membrane , Humans , Intermediate Filament Proteins/physiology , Intracellular Signaling Peptides and Proteins , Membrane Fusion , Membrane Glycoproteins/physiology , Membrane Proteins/metabolism , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/physiology , Optic Disk/ultrastructure , Peripherins , Photoreceptor Cells/ultrastructure , Rats , Retina/chemistry , Retina/cytology
6.
J Biol Chem ; 280(10): 9217-24, 2005 Mar 11.
Article in English | MEDLINE | ID: mdl-15591062

ABSTRACT

Peripherin-2 (also known as peripherin/rds), a photoreceptor specific tetraspanin protein, is required to maintain normal cell structure through its role in renewal processes requiring membrane fusion. It is the first tetraspanin fusogen and has been shown to directly mediate fusion between disk membranes and opposing membranes to maintain the highly ordered structure of rod outer segments. Localized to the C terminus of human, bovine, and murine peripherin-2 is an amphiphilic fusion peptide domain (residues 312-326) and a highly conserved region upstream of this domain that we hypothesize is essential for fusogenic function. Our previous studies indicated that substitution of a threonine for a proline at position 296 within this highly conserved region enhanced fusion activity. In this study we wanted to determine whether this proline is essential with the introduction of three additional substitutions of proline with alanine, leucine, and glutamic acid. Wild type, P296T, P296A, P296L, and P296E mutants of peripherin-2 were expressed as His6-tagged full-length proteins in Madin-Darby canine kidney (MDCK) cells. All of the proteins were localized to intracellular membranes and detected as 42-kDa monomers by Western blot analysis. The wild type, P296A, and P296L assembled into core tetramers; in contrast the P296T and P296E formed higher order oligomers. Fusogenic activity of full-length protein expressed in MDCK membranes and purified protein reconstituted in model membrane liposomes was determined using fluorescence quenching techniques. Fusion activity was decreased in the P296L, P296A, and P296E mutants both in endogenous MDCK membranes and in model liposomes. Collectively, these results suggest that the proline at position 296 is necessary for optimal function.


Subject(s)
Intermediate Filament Proteins/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Cattle , Cell Line , Cell Membrane/metabolism , Conserved Sequence , Dogs , Humans , Intermediate Filament Proteins/chemistry , Intermediate Filament Proteins/genetics , Kinetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Peripherins , Proline , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Rod Cell Outer Segment/enzymology , Transfection
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