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Insights into Neuronal Ceroid Lipofuscinosis (CLN1) point to the involvement of cilia pathology in the disease

ABSTRACT: Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1 result in early onset neurodegeneration, therefore, we hypothesized that the levels of proteins extracted from purified brain membranes of newborn mice will differ. One of the main effects of palmitoylation is changing the hydrophobic index of proteins and thus affecting these proteins interactions with different membranal domains. Assuming that PPT1 substrates are over palmitoylated in Ppt1-/- brains it is possible that its localization to specific membranal domain is different than in the wild-type brains. We isolated Triton X-100 soluble membranes from three wild-type and three Ppt1-/- brains and identified the proteins by PalmPISC. Many of the identified proteins were found to be palmitoylated. A second feature, which was noted is that many of the differential proteins (62 out of 88, 70%) can be detected in the cilia proteome (http://v3.ciliaproteome.org/cgi-bin/index.php). This observation led to test whether ciliary proteins differ between wild-type and Ppt1-/-. Cilia were prepared from newborn dissociated brain cell cultures and analyzed by MS

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Peter James

LAB HEAD: Peter James

PROVIDER: PXD001893 | Pride | 2021-12-14

REPOSITORIES: Pride

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			Action	DRS
	121019_KH_G1_1_B1TSplusHA_01.RAW	Raw
	121019_KH_G1_2_B2TSplusHA_01.RAW	Raw
	121019_KH_G1_7_B1TSminusHA_01.RAW	Raw
	121019_KH_G1_8_B2TSminusHA_01.RAW	Raw
	121022_KH_G1_11_B9TSminusHA_01.RAW	Raw

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Project description:Brown adipose tissue (BAT) is a thermogenic organ that dissipates stored energy as heat to maintain body temperature in infants and small mammals. This process may also provide protection from development of diet-induced obesity. We found that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, while potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibited reduced expression of BAT-related genes in peripheral white adipose tissue and accumulated significantly more fat than wild-type controls when fed a high fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and suggest that a decrease in peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice. Primary BAT cell cultures were established as previously reported with some minor modifications (Cannon B., 2001; NÃ©chad M., 1983). Four- to 6-week-old mice were euthanized under aseptic conditions and interscapular BAT (IBAT) dissected, finely minced in digestion buffer containing 123 mM NaCl, 5mM HCl, 1.3 mM CaCl2, 5mM glucose, 1.5% (w/v) crude bovine serum albumin fraction V, 100 mM HEPES, 0.2% collagenase type II (Sigma-Aldrich Corp., St. Louis, MO), pH 7.4, and digested in 10 ml of the same buffer for 30 min at 37Â°C. The supernatant obtained was then filtered using a 250 Âµm nylon mesh and kept in ice for 15 minutes to allow fat droplets and mature cells to float. The resulting infranatant was filtered through a 30 Âµm nylon mesh to enrich the fraction with precursor cells and centrifuged at 700g for 10 min. The pellet obtained was washed once in warm DMEM and resuspended in DMEM medium containing 10% newborn calf serum, 10 mM HEPES, 4mM glutamine, 25 Âµg/ml sodium ascorbate (Sigma-Aldrich Corp., St. Louis, MO)., 100U/ml penicillin and 100mg/ml streptomycin. Cells were seeded onto 35mm dish plates. Insulin treatment was started on day 1 at 50 nM and the concentration increased to 100 nM at day 5 and 200 nM at day 7. Differentiated/confluent cells were collected for biochemical analysis (~day 9). To identify genes regulated by ATX and LPA signaling, 9 DIV primary BAT cultures were treated with the vehicle (DMSO), specific ATX inhibitor HA155 (2.5ÂµM), or LPA (1-Oleoly-LPA, 5ÂµM) (Avanti Polar Lipids, AL, USA) during differentiation. Treatment of the BAT cultures did not affect confluence or markers of proliferation. RNA was extracted from three cultures per condition using TRIzol reagent (Invitrogen, Carlsbad, CA) as described previously (Blalock EM., 2003).

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Insights into Neuronal Ceroid Lipofuscinosis (CLN1) point to the involvement of cilia pathology in the disease

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