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Abstract Prion diseases are fatal, infectious, and incurable neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform (PrPSc). In humans, there are sporadic, genetic and infectious etiologies, with sporadic Creutzfeldt-Jakob disease (sCJD) being the most common form. Currently, no treatment is available for prion diseases. Cellular cholesterol is known to impact prion conversion, which in turn results in an accumulation of cholesterol in prion-infected neurons. The major elimination of brain cholesterol is achieved by the brain specific enzyme, cholesterol 24-hydroxylase (CYP46A1). Cyp46A1 converts cholesterol into 24(S)-hydroxycholesterol, a membrane-permeable molecule that exits the brain. We have demonstrated for the first time that Cyp46A1 levels are reduced in the brains of prion-infected mice at advanced disease stage, in prion-infected neuronal cells and in post-mortem brains of sCJD patients. We have employed the Cyp46A1 activator efavirenz (EFV) for treatment of prion-infected neuronal cells and mice. EFV is an FDA approved anti-HIV medication effectively crossing the blood brain barrier and has been used for decades to chronically treat HIV patients. EFV significantly mitigated PrPSc propagation in prion-infected cells while preserving physiological PrPC and lipid raft integrity. Notably, oral administration of EFV treatment chronically at very low dosage starting weeks to months after intracerebral prion inoculation of mice significantly prolonged the lifespan of animals. In summary, our results suggest that Cyp46A1 as a novel therapeutic target and that its activation through repurposing the anti-retroviral medication EFV might be valuable treatment approach for prion diseases.

Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD): whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD.

Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport) per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2), a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2.

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.

IntroductionThe development of organic manure from livestock excreta is a useful source for sustainable crop production in environment-friendly agriculture. Organic manure increases soil microbial activity and organic matter (OM) supply. The excessive use of chemical fertilizers (CFs) leads to air and water pollution caused by toxic chemicals and gases, and soil quality degradation via nutrient imbalance due to supplying specific chemical components. Thus, the use of organic manure will serve as a long-term supply of various nutrients in soil via OM decomposition reaction as well as the maintenance of environment.MethodsIn this study, we aimed to analyze the diverse effects of Hanwoo manure (HM) on plant growth, feed quality, and soil bacterial communities in comparison with CFs, commercial poultry manure (CM), and the combined use of chemical fertilizer and Hanwoo manure (HM+CF). We analyzed the contents of crude matter (protein, fat, fiber, and ash), P, acid detergent fiber (ADF), and neutral detergent fiber (NDF) through feed quality analysis, and the contents or activities of total phenol, total flavonoid, ABTS, nitrite scavenging, and reducing power via the antioxidant assay. Furthermore, the soil microbial communities were determined using 16S rRNA sequencing. We compared the soil bacteria among different soil samples by using amplicon sequence variant (ASV) analysis.Results and discussionWe observed increased OM in the soil of the HM group compared to that of the CF and non-treated groups over a period of two years. Moreover, HM+CF treatment enormously improved plant growth. Organic manure, especially HM, caused an increase in the content of crude ash and phosphorus in plants. There were no significant differences in total polyphenol, total flavonoid, ABTS, nitrite scavenging, and reducing power in plants between HM and CF groups. Finally, we detected 13 soil bacteria (Acidibacter, Algisphaera, Cystobacter, Microvirga, Ohtaekwangia, Panacagrimonas, Pseudarthrobacter, Reryanella, Rhodoligotrophos, Solirubrobacter, Stenotrophobacter, Tellurimicrobium, and Thermomarinilinea) that were considerably correlated with OM and available phosphorus, and three considerably correlated bacteria were specifically distributed in CF or organic manure. The results suggest that HM is a valuable source of organic manure that can replace CF for sustainable crop production.

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr72) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr72, we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr72 does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr72 regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5. [ABSTRACT FROM AUTHOR]

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of lowdensity lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels. [ABSTRACT FROM AUTHOR]

Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport) per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2), a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2. [ABSTRACT FROM AUTHOR]

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration. [ABSTRACT FROM AUTHOR]

There is an increasing body of literature pointing to cytoskeletal proteins as spatial organizers and interactors of organelles. In this study, we identified protein 600 (p600) as a novel microtubule-associated protein (MAP) developmentally regulated in neurons. p600 exhibits the unique feature to interact with the endoplasmic reticulum (ER). Silencing of p600 by RNA interference (RNAi) destabilizes neuronal processes in young primary neurons undergoing neurite extension and containing scarce staining of the ER marker Bip. Furthermore, in utero electroporation of p600 RNAi alters neuronal migration, a process that depends on synergistic actions of microtubule dynamics and ER functions. p600-depleted migrating neurons display thin, crooked, and "zigzag" leading process with very few ER membranes. Thus, p600 constitutes the only known MAP to associate with the ER in neurons, and this interaction may impact on multiple cellular processes ranging from neuronal development to neuronal maturation and plasticity. [ABSTRACT FROM AUTHOR]

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2.Wenowreport that TPX2 plays a role in the cellular response toDNAdouble strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G0 and G1 phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates atDNAdouble strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused byTPX2depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage [ABSTRACT FROM AUTHOR]