RESUMO
Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of ß-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.
Assuntos
Sequência de Aminoácidos , Proteínas PrPSc , Doenças Priônicas , Agregação Patológica de Proteínas , Deleção de Sequência , Animais , Linhagem Celular , Humanos , Proteínas PrPSc/química , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Coelhos , Ovinos , SolubilidadeRESUMO
Tauopathies represent a vast family of neurodegenerative diseases, the most well-known of which is Alzheimer's disease. The symptoms observed in patients include cognitive deficits and locomotor problems and can lead ultimately to dementia. The common point found in all these pathologies is the accumulation in neural and/or glial cells of abnormal forms of Tau protein, leading to its aggregation and neurofibrillary tangles. Zebrafish transgenic models have been generated with different overexpression strategies of human Tau protein. These transgenic lines have made it possible to highlight Tau interacting factors or factors which may limit the neurotoxicity induced by mutations and hyperphosphorylation of the Tau protein in neurons. Several studies have tested neuroprotective pharmacological approaches. On few-days-old larvae, modulation of various signaling or degradation pathways reversed the deleterious effects of Tau mutations, mainly hTauP301L and hTauA152T. Live imaging and live tracking techniques as well as behavioral follow-up enable the analysis of the wide range of Tau-related phenotypes from synaptic loss to cognitive functional consequences.
Assuntos
Modelos Animais de Doenças , Tauopatias , Peixe-Zebra , Proteínas tau/metabolismo , Animais , Animais Geneticamente Modificados , Humanos , Terapia de Alvo Molecular , Proteínas tau/genéticaRESUMO
Brain-derived neurotrophic factor (BDNF) dysregulations contribute to the neurotoxicity in neurodegenerative pathologies and could be efficiently targeted by therapies. In Alzheimer's disease (AD), although the relationship between BDNF and amyloid load has been extensively studied, how Tau pathology affects BDNF signaling remains unclear. Using the TAU-P301L transgenic zebrafish line, we investigated how early Tau-induced neurotoxicity modifies BDNF signaling. Alterations in BDNF expression levels were observed as early as 48 h post fertilization in TAU-P301L zebrafish embryos while TrkB receptor expression was not affected. Decreasing BDNF expression, using a knockdown strategy in wild-type embryos to mimic Tau-associated decrease, did not modify TrkB expression but promoted neurotoxicity as demonstrated by axonal outgrowth shortening and neuronal cell death. Moreover, the TrkB antagonist ANA-12 reduced the length of axonal projections. Rescue experiments with exogenous BDNF partially corrected neuronal alterations in TAU-P301L by counteracting primary axonal growth impairment but without effect on apoptosis. Importantly, the axonal rescue was proved functionally effective in a behavioral test, at a similar level as obtained with the GSK3ß inhibitor LiCl, known to decrease TAU phosphorylation. Finally, treatment with a TrkB agonist, 7,8-dihydroxyflavone, led to comparable results and allowed full rescue of locomotor response. We provided here strong evidence that Tau neurotoxicity provoked alterations in BDNF system and that BDNF pathway might represent an efficient therapeutic target.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Fármacos Neuroprotetores , Tauopatias/tratamento farmacológico , Peixe-Zebra , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Comportamento Animal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/agonistas , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Morte Celular , Humanos , Larva , Cultura Primária de Células , Receptor trkB/biossíntese , Proteínas Recombinantes/farmacologia , Tauopatias/genéticaRESUMO
Retinitis pigmentosa (RP) is one of the most common forms of inherited retinal degeneration with 1/4,000 people being affected. The vision alteration primarily begins with rod photoreceptor degeneration, then the degenerative process continues with cone photoreceptor death. Variants in 71 genes have been linked to RP. One of these genes, PDE6a is responsible for RP43. To date no treatment is available and patients suffer from pronounced visual impairment in early childhood. We used the novel zebrafish pde6aQ70X mutant, generated by N-ethyl-N-nitrosourea at the European Zebrafish Resource Centre, to better understand how PDE6a loss of function leads to photoreceptor alteration. Interestingly, zebrafish pde6aQ70X mutants exhibited impaired visual function at 5 dpf as evidenced by the decrease in their visual motor response (VMR) compared to pde6a WT larvae. This impaired visual function progressed with time and was more severe at 21 dpf. These modifications were associated with an alteration of rod outer segment length at 5 and 21 dpf. In summary, these findings suggest that rod outer segment shrinkage due to Pde6a deficiency begins very early in zebrafish, progresses with time. The zebrafish pde6aQ70X mutant represents an ideal model of RP to screen relevant active small molecules that will block the progression of the disease.
RESUMO
BACKGROUND: Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. However, the access to fermentable cell wall sugars by carbohydrate degrading enzymes is impeded by lignins. These complex polymers are made from reactive oxidized monolignols in the cell wall. Little is known about the laccase-mediated oxidation of monolignols in grasses, and inactivation of the monolignol polymerization mechanism might be a strategy to increase the yield of fermentable sugars. RESULTS: LACCASE5 and LACCASE8 are inactivated in a Brachypodium double mutant. Relative to the wild type, the lignin content of extract-free mature culms is decreased by 20-30% and the saccharification yield is increased by 140%. Release of ferulic acid by mild alkaline hydrolysis is also 2.5-fold higher. Interfascicular fibers are mainly affected while integrity of vascular bundles is not impaired. Interestingly, there is no drastic impact of the double mutation on plant growth. CONCLUSION: This work shows that two Brachypodium laccases with clearly identified orthologs in crops are involved in lignification of this model plant. Lignification in interfascicular fibers and metaxylem cells is partly uncoupled in Brachypodium. Orthologs of these laccases are promising targets for improving grass feedstock for cellulosic biofuel production.
RESUMO
Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a ß-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPC structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPC nor for the formation of a stable prion structure.