379 research outputs found

    Age- and stress-associated C. elegans granulins impair lysosomal function and induce a compensatory HLH-30/TFEB transcriptional response.

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    The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases are key contributors to the maintenance of protein homeostasis with age. We previously reported that expression of granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, enhance the accumulation and toxicity of TAR DNA binding protein 43 (TDP-43) in Caenorhabditis elegans (C. elegans). In this study we show that C. elegans granulins are produced in an age- and stress-dependent manner. Granulins localize to the endolysosomal compartment where they impair lysosomal protease expression and activity. Consequently, protein homeostasis is disrupted, promoting the nuclear translocation of the lysosomal transcription factor HLH-30/TFEB, and prompting cells to activate a compensatory transcriptional program. The three C. elegans granulin peptides exhibited distinct but overlapping functional effects in our assays, which may be due to amino acid composition that results in distinct electrostatic and hydrophobicity profiles. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease

    A Molecular Mechanism for Abnormal Prion Protein Accumulation

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    A fundamental event in the pathogenesis of prion disease is the conversion of cellular prion protein into an abnormally folded isoform (PrPSc), which is the infectious causative agent of disease. With progression of disease, PrPSc is replicated and excessively accumulated in most cases. However, the molecular mechanism for excessive accumulation of PrPSc is not well understood. Recently, Sortilin, a member of the VPS10P domain receptor family, has been identified as a sorting receptor that directs prion protein (PrP) to the lysosomal degradation pathway. Moreover, it has been shown that prion infection impairs Sortilin function, resulting in delayed PrPSc degradation. In this chapter, we explain the mechanisms for PrP trafficking into the lysosomal degradation pathway mediated by Sortilin and overaccumulation of PrPSc caused by Sortilin dysfunction

    Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia

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    Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease

    Disorder in Cysteine-Rich Granulin-3 and Its Implication in Alzheimer Disease

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    Granulins (GRNs) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (PGRN) during inflammation. All seven GRNs (1 – 7 or A – G) contain twelve conserved cysteines that form six intramolecular disulfide bonds, rendering this family of proteins unique. GRNs play multiple roles and are involved in a myriad of physiological as well as pathological processes. They are known to a play role in growth and embryonic development, wound healing, and signaling cascades as well as in tumorigenesis. They are also implicated in neurodegenerative diseases like frontotemporal dementia (FTD), Alzheimer disease (AD), and amyotrophic lateral sclerosis (ALS). Despite their manifold functions, there is a paucity in the information about the structure-function relationship of these proteins, especially, with the role of the twelve conserved cysteines and the disulfide linkages in determining their structure and the functions. In this study, the role of disulfide bonds is probed by comparing the structures of the fully reduced GRN-3 (rGRN-3) and native GRN-3. We report that monomeric rGRN-3 is an intrinsically disordered protein (IDP) at low concentrations and undergoes dimerization at higher concentrations to form a fuzzy complex. Interestingly, we show that rGRN-3 is also able to activate NF-kappaB in human neuroblastoma cells in a concentration-dependent manner. We also show that both E. coli and mammalian HEK cells are inefficient in forming correct disulfide linkages and are incapable of generating monomeric native GRN-3 (GRN-3) exclusively, thus, predominately generating multimeric GRN-3 (mGRN-3) with scrambled inter-molecular bonds. We establish that GRN-3 has a more ordered structure as compared to that of mGRN-3 or rGRN-3, stabilized exclusively by the disulfide bonds which form a fulcrum imparting order to an otherwise disordered protein. We determined the potential involvement of GRN-3 in AD pathogenesis by showing that GRN-3 augments A-beta, the protein implicated in AD, aggregation in a concentration- and time-dependent manner and it interacts both with A-beta monomers and oligomers thereby providing a proof of concept for neuroinflammation triggered neurodegeneration

    Bridging the gap between lipid dyshomeostasis and brain disorders : role of prosaposin and progranulin

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    Brain tissue is the second-highest lipid-containing tissue after adipose in the human body. Lipid homeostasis maintenance requires the coordination of multiple cellular organelles. Pathogenesis of brain disorders, especially Parkinson’s disease (PD), usually involves different dysfunctional pathways that intimately link to lipid homeostasis. Indeed, lipid dyshomeostasis, especially disruption in sphingolipid metabolism, is a critical factor of several common brain disorders, including PD and schizophrenia, and rarer, Gaucher’s disease and Niemann-Picks disease, and probably underlies the dysfunctional pathways contributing to these diseases. Up till now, explanations of various lipid alterations in different disease conditions are lacking, let alone a factor that unifies the principles underlying lipid dyshomeostasis across different diseases. However, recently two lysosomal proteins, prosaposin (PSAP) and progranulin (PGRN), displayed the potential to lend mechanisms to widespread lipid changes across different diseases, which may be a good example to bridge the gap between lipid dyshomeostasis and brain disorders. Both PSAP and PGRN are pleiotropic proteins that show lipid metabolism modulatory functions and neuroprotective effects in the brain and link to different brain disorders. Therefore, the current thesis aims to decipher the role of PSAP and PGRN in three brain conditions, PD, L-DOPA-induced dyskinesia (LID), and schizophrenia. Regarding PD, in Paper I, we report that PSAP levels correlate mainly with core motor symptoms, while PGRN correlates with non-motor symptoms. In mice, PSAP deficiency in DA neurons (cPSAPDAT) causes behavior defects, DA neurotransmission impairment, and synaptic plasticity disruption. By contrast, mice with PSAP-deficiency in serotonin neurons (cPSAPSERT) show normal behaviors and intact serotonin neurotransmission. Spatial lipidomics unraveled systemic lipid changes in the whole brain, with relation to mitochondrial and peroxisomal functions, in cPSAPDAT mice. On the contrary, cPSAPSERT mice only displayed contained lipid accumulation in the dorsal raphe nucleus (DRN). Metabolic analyses demonstrate that the difference in de novo synthesis of NAD+ between DA and serotonin neurons caused divergent lipid changes in these two mouse lines. cPSAPDAT mice are more vulnerable to α-syn toxicity due to exacerbated aggregation of p-Ser129 α-synuclein that can be reversed by PSAP overexpression, compared to control mice. PSAP overexpression protected wild-type mice against both α-syn- and 6-OHDA-induced toxicity. Consistently, PSAP delivered by encapsulated-cell biodelivery (ECB) devices in the striatum protected rats against α-syn-toxicity. In all, PSAP critically modifies the pathogenesis of PD, especially lipid dyshomeostasis, and serves as a potential therapeutic target for PD. The unfolded protein response (UPR) has long been associated with PD and serves as an important regulator of lipid homeostasis. In Paper II, the unfolded protein response (UPR) related proteins and their transcription levels have been quantitatively confirmed to be changed in PD brains. Meanwhile, UPR is not affected in the periphery of PD patients, which indicates that peripheral UPR is independent of its central counterpart. Regarding LID and schizophrenia, sphingolipid changes have been found in both diseases, and PSAP and PGRN have been genetically linked to schizophrenia. In Paper III and IV, consistent with these facts, PSAP levels are found to be elevated in LID animals. PSAP differentially modulates lipid metabolism in striatal and non-striatal DRD1 neurons and affects basic physiological functions more of the latter. Meanwhile, PSAP deficiency decreases the susceptibility of striatal DRD1 neurons to L-DOPA-induced malfunction that presents as LID. In contrast with PSAP in LID, PSAP and PGRN are decreased in postmortem cingulate tissue from schizophrenia patients. Moreover, PSAP and PGRN downregulation in the cingulate induces widespread brain immediate early gene (IEG) changes and schizophrenic behaviors in mice, which provide evidence for a causative role of PSAP and PGRN in schizophrenia. Therefore, PSAP, together with PGRN, may take part in the pathogenesis of both LID and schizophrenia, though in opposite ways. All in all, this thesis sheds light on the role of PSAP and PGRN in multiple brain disorders and proposes that PSAP and PGRN may serve as a bridge between lipid dyshomeostasis and brain disorders

    Progranulin is expressed within motor neurons and promotes neuronal cell survival

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    <p>Abstract</p> <p>Background</p> <p>Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U) associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival.</p> <p>Results</p> <p><it>In situ </it>hybridisation and immunohistochemical techniques revealed that the <it>progranulin </it>gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human <it>progranulin </it>gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months). This is mediated at least in part through an anti-apoptotic mechanism. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. Knockdown of progranulin expression using shRNA technology further reduced cell survival.</p> <p>Conclusion</p> <p>Neurons are among the most long-lived cells in the body and are subject to low levels of toxic challenges throughout life. We have demonstrated that progranulin is abundantly expressed in motor neurons and is cytoprotective over prolonged periods when over-expressed in a neuronal cell line. This work highlights the importance of progranulin as neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including motor neuron disease.</p