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.

Background: TPX2 is as an essential protein for mitosis, but its nuclear function is unknown. Results: TPX2 goes to DNA double strand breaks and regulates ␥-H2AX levels upon ionizing radiation. Conclusion:We discover a novel and the first nuclear function for TPX2. Significance: Our study provides new insights into the physiological and oncological roles of TPX2.

Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1-6). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein ␣COP. Ndel1 promotes the interaction between Lis1, ␣COP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/␣COP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration.The cytoskeleton constitutes a highly organized structure formed by the interconnection of three filamentous networks (microtubules (MTs), 3 intermediate filaments (IFs), and microfilament) and their associated proteins (7-10). The dynamics of these networks regulate essential intracellular functions such as transport and trafficking and, therefore, impact on cell division, morphology, and integrity (7-10). Named according to their intermediate diameter (10 nm), IFs constitute the largest family of cytoskeletal proteins. Up to 60 genes encoding for IFs are expressed in a tissue-specific and spatio-temporal manner in mammals (7,11,12). After synthesis in the cytoplasm, a fraction of soluble IF proteins is rapidly incorporated into the polymeric filamentous network, referred to as the "insoluble" fraction (7, 12). In parallel, individual IF subunits, dimers, or small oligomers that constitute the "soluble" fraction translocate rapidly along MTs via molecular motors in structures termed particles (13,14). This model is particularly relevant for the assembly of the IF vimentin (15, 16) and more controversial in the case of neuronal IFs (NFs) (17-19).The neurofilament proteins (NF-L, NF-M, and NF-H) are the most abundant IFs in mature central nervous system neurons (12). We recently demonstrated that Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, a protein implicated in nuclear distribution during hyphal growth, binds directly to the key subunit NF-L, thereby regulating NF assembly (5). Ndel1 also interacts with the Disrupted-in-Schizophrenia protein 1 (DISC1) to regulate MT dynamics during neurite outgrowth in neuroblastoma PC-12 cells and during neuronal migration in the developing cortex. Interestingly, Ndel1 associates with the molecular motors dynein/dynactin (20 -22), and numerous independent reports documented the dynein tran...

Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrPSc accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases.

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.

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.

Background:The Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a key factor for spindle assembly; its deregulation is associated with numerous cancers. Results: Phosphorylation of TPX2 at Thr 72 regulates its spindle assembly functions via Aurora A and Eg5. Conclusion: Proper regulation of TPX2 phosphorylation at Thr 72 is required for spindle assembly. Significance: Our study provides new mechanistic insights into the spindle and cancers-associated roles of TPX2.

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