Frontotemporal Dementia Nonsense Mutation of Progranulin Rescued by Aminoglycosides

Frontotemporal dementia (FTD) is an early onset dementia and is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5-26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its function under physiological and pathological conditions remains to be defined. Many FTD-causing nonsense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy. Aminoglycosides are a class of antibiotics that possess a less known function to induce eukaryotic ribosomal readthrough of PTCs to produce a full-length protein. The aminoglycoside-induced readthrough strategy has been utilized to treat multiple human diseases caused by PTCs. In this study, we tested the only clinically approved readthrough small molecule PTC124 and eleven aminoglycosides in a cell culture system on four PTCs responsible for FTD or a related neurodegenerative disease amyotrophic lateral sclerosis. We found that the aminoglycosides G418 and gentamicin B1 rescued the expression of the progranulin R493X mutation. G418 was more effective than gentamicin B1 (~50% rescue vs \u3c 10%), and the effect was dose and time-dependent. The proganulin readthrough protein displayed similar subcellular localization as the wild-type proganulin protein. These data provide an exciting proof-of-concept that aminoglycosides or other readthrough-promoting compounds are a therapeutic avenue for familial FTD caused by proganulin PTC mutations

ALS Mutations of FUS Suppress Protein Translation and Disrupt the Regulation of Nonsense-Mediated Decay

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by preferential motor neuron death. Approximately 15% of ALS cases are familial, and mutations in the fused in sarcoma (FUS) gene contribute to a subset of familial ALS cases. FUS is a multifunctional protein participating in many RNA metabolism pathways. ALS-linked mutations cause a liquid–liquid phase separation of FUS protein in vitro, inducing the formation of cytoplasmic granules and inclusions. However, it remains elusive what other proteins are sequestered into the inclusions and how such a process leads to neuronal dysfunction and degeneration. In this study, we developed a protocol to isolate the dynamic mutant FUS-positive cytoplasmic granules. Proteomic identification of the protein composition and subsequent pathway analysis led us to hypothesize that mutant FUS can interfere with protein translation. We demonstrated that the ALS mutations in FUS indeed suppressed protein translation in N2a cells expressing mutant FUS and fibroblast cells derived from FUS ALS cases. In addition, the nonsense-mediated decay (NMD) pathway, which is closely related to protein translation, was altered by mutant FUS. Specifically, NMD-promoting factors UPF1 and UPF3b increased, whereas a negative NMD regulator, UPF3a, decreased, leading to the disruption of NMD autoregulation and the hyperactivation of NMD. Alterations in NMD factors and elevated activity were also observed in the fibroblast cells of FUS ALS cases. We conclude that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation, both of which likely contribute to motor neuron death

Clinical and Experimental Studies of a Novel P525R FUS Mutation in Amyotrophic Lateral Sclerosis

Objective: To describe the clinical features of a novel fused in sarcoma (FUS) mutation in a young adult female amyotrophic lateral sclerosis (ALS) patient with rapid progression of weakness and to experimentally validate the consequences of the P525R mutation in cellular neuronal models. Methods: We conducted sequencing of genomic DNA from the index patient and her family members. Immunocytochemistry was performed in various cellular models to determine whether the newly identified P525R mutant FUS protein accumulated in cytoplasmic inclusions. Clinical features of the index patient were compared with 19 other patients with ALS carrying the P525L mutation in the same amino acid position. Results: A novel mutation c.1574C\u3eG (p.525P\u3eR) in the in the FUS gene was identified in the index patient. The clinical symptoms are similar to those in familial ALS patients with the P525L mutation at the same position. The P525R mutant FUS protein showed cytoplasmic localization and formed large stress granule–like cytoplasmic inclusions in multiple cellular models. Conclusions: The clinical features of the patient and the cytoplasmic inclusions of the P525R mutant FUS protein strengthen the notion that mutations at position 525 of the FUS protein result in a coherent phenotype characterized by juvenile or young adult onset, rapid progression, variable positive family history, and female preponderance

Frontotemporal dementia nonsense mutation of progranulin rescued by aminoglycosides

Frontotemporal dementia (FTD) is an early onset dementia characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5-26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its functions under physiological and pathological conditions remains to be defined. Many FTD-causing non-sense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy. Aminoglycosides are a class of antibiotics that possess a less-known function to induce eukaryotic ribosomal readthrough of PTCs to produce a full-length protein. The aminoglycoside-induced readthrough strategy has been utilized to treat multiple human diseases caused by PTCs. In this study, we tested the only clinically approved readthrough small molecule PTC124 and 11 aminoglycosides in a cell culture system on four PTCs responsible for FTD or a related neurodegenerative disease amyotrophic lateral sclerosis. We found that the aminoglycosides G418 and gentamicin rescued the expression of the progranulin R493X mutation. G418 was more effective than gentamicin (~50% rescue versus <10%), and the effect was dose- and time-dependent. The progranulin readthrough protein displayed similar subcellular localization as the wild-type progranulin protein. These data provide an exciting proof-of-concept that aminoglycosides or other readthrough-promoting compounds are a therapeutic avenue for familial FTD caused by progranulin PTC mutations

H2S suppresses indoleamine 2, 3-dioxygenase 1 and exhibits immunotherapeutic efficacy in murine hepatocellular carcinoma

Abstract Background Over-expression and over-activation of immunosuppressive enzyme indoleamine 2, 3 -dioxygenase 1 (IDO1) is a key mechanism of cancer immune escape. However, the regulation of IDO1 has not been fully studied. The relation between hydrogen sulfide (H2S) and IDO1 is unclear. Methods The influences of endogenous and exogenous H2S on the expression of IDO1, iNOS and NF-κB and STAT3 signaling proteins were investigated using qPCR or western blot, and the production of nitric oxide (NO) was analyzed by nitrate/nitrite assay in Cse −/− mice and MCF-7 and SGC-7901 cells. The effect of H2S on IDO1 activity was investigated by HPLC and in-vitro enzymatic assay. The effect of H2S on tryptophan metabolism was tested by luciferase reporter assay in MCF-7 and SGC-7901 cells. The correlation between H2S-generating enzyme CSE and IDO1 was investigated by immunostaining and heatmaps analysis in clinical specimens and tissue arrays of hepatocellular carcinoma (HCC) patients. The immunotherapeutic effects of H2S on H22 HCC-bearing mice were investigated. Results Using Cse −/− mice, we found that H2S deficiency increased IDO1 expression and activity, stimulated NF-κB and STAT3 pathways and decreased the expression of NO-generating enzyme Inos. Using IDO1-expressing MCF-7 and SGC-7901 cells, we found that exogenous H2S inhibited IDO1 expression by blocking STAT3 and NF-κB pathways, and decreased IDO1 activity via H2S/NO crosstalk, and combinedly decreased the tryptophan metabolism. The negative correlation between H2S-generating enzyme CSE and IDO1 was further validated in clinical specimens and tissue arrays of HCC patients. Additionally, H2S donors effectively restricted the tumor development in H22 HCC-bearing mice via downregulating IDO1 expression, inducing T-effector cells and inhibiting MDSCs. Conclusions Thus, H2S, as a novel negative regulator of IDO1, shows encouraging antitumor immunotherapeutic effects and represents a novel therapeutic target in cancer therapy

Banana MKK1 modulates fruit ripening via the MKK1-MPK6-3/11-4-bZIP21 module

Summary: The mitogen-activated protein kinase (MAPK) cascade consisting of MKKK, MKK, and MPK plays an indispensable role in various plant physiological processes. Previously, we showed that phosphorylation of MabZIP21 by MaMPK6-3 is involved in banana fruit ripening, but the regulatory mechanism by which MKK controls banana fruit ripening remains unclear. Here, ripening-induced MaMKK1 from banana fruit is characterized, and transiently overexpressing and silencing of MaMKK1 in banana fruit accelerates and inhibits fruit ripening, respectively, possibly by influencing phosphorylation and activity of MPK. MaMKK1 interacts with and phosphorylates MaMPK6-3 and MaMPK11-4 mainly at the pTEpY residues, resulting in MPK activation. MaMPK11-4 phosphorylates MabZIP21 to elevate its transcriptional activation ability. Transgenic tomato fruit expressing MabZIP21 ripen quickly with a concomitant increase in MabZIP21 phosphorylation. Additionally, MabZIP21 activates MaMPK11-4 and MaMKK1 transcription to form a regulatory feedback loop. Collectively, here we report a regulatory pathway of the MaMPK6-3/11-4-MabZIP21 module in controlling banana fruit ripening

ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS