Importin-�� Is a GDP-to-GTP Exchange Factor of Ran: IMPLICATIONS FOR THE MECHANISM OF NUCLEAR IMPORT*

Ran-GTP interacts strongly with importin-β, and this interaction promotes the release of the importin-α-nuclear localization signal cargo from importin-β. Ran-GDP also interacts with importin-β, but this interaction is 4 orders of magnitude weaker than the Ran-GTP·importin-β interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-β promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-β complex stabilizes the complex, which cannot be dissociated by importin-α. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-β has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-β. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-β. Importin-α inhibits the GDP exchange reaction by sequestering importin-β, whereas RanBP1 restores the GDP nucleotide exchange by importin-β by forming a tetrameric complex with importin-β, Ran, and importin-α. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.Ran (Gsp1p in yeast) is a Ras-like GTPase that regulates diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly (1, 2). Like other GTPases, Ran can bind GTP and GDP. Ran-GTP is generated in the nucleus by the guanine exchange factor RCC1 (regulator of chromosome condensation 1), which is associated with the chromatin (3). Ran-GDP is produced in the cytoplasm by the activation of the intrinsic GTPase activity of Ran by RanGAP1 (GTPase-activating protein) (4) and RanBP1 (Ran-binding protein-1, Yrb1p in yeast). The compartmentalization of RanGAP1 (cytoplasm) and RCC1 (nucleus) gives rise to the asymmetric distribution of Ran-GDP (cytoplasm) and Ran-GTP (nucleus) across the nuclear envelope. This asymmetric distribution of Ran-GDP and Ran-GTP plays a central role in nucleocytoplasmic transport by mediating assembly and disassembly of import and export complexes through interaction with the nuclear import machinery (for reviews, see Refs. 5–9).The passage of molecules into the nucleus occurs through the nuclear pore complexes (NPCs)⁶ (10). Nucleocytoplasmic transport is driven by a series of protein-protein interactions and involves several soluble carriers named β-karyopherins. Import carriers are called importins and export carriers are called exportins. The classical nuclear import pathway involves importin-β (Kap95p in yeast) and the adaptor protein importin-α (Kap60p in yeast). In the cytoplasm importin-β binds to importin-α. Their interaction triggers a conformational change of importin-α that increases its affinity for cargo proteins containing a nuclear localization signal (NLS) (11, 12). The translocation of the resulting complex (importin-β·importin-α·NLS) involves interactions with the NPC proteins (nucleoporins), particularly the FXFG-repeat domains (11). The protein cargo is released in the nucleus by the action of Ran-GTP, which induces the dissociation of importin-α from importin-β by forming a stable complex with importin-β. The importins are then recycled to the cytoplasm. Importin-β transfers to the cytoplasm associated with Ran-GTP, and importin-α is exported by CAS (exportin2; Cse1p in yeast) in the form of an importin-α·CAS·Ran-GTP complex (13). Importin-β and importin-α are released from their complexes in the cytoplasm by the combined action of RanBP1 and RanGAP1. Importin-β and importin-α are then able to function in a new cycle of transport, whereas Ran-GDP is transported into the nucleus by NTF2 (nuclear-transport factor-2, Ntf2p in yeast) (14). In the nucleus Ran-GDP is transformed to Ran-GTP by the action of RCC1 (3).The complexity of the nuclear import mechanism is highlighted by the fact that it involves the active participation of soluble factors other than Ran-GTP, importin-β, and importin-α. Indeed, Ran-GDP, RanBP1, and NTF2 have been shown to be involved in the docking and translocation events of nuclear import. Chi et al. (15) have demonstrated that Ran-GDP forms a stable complex with RanBP1 and importin-β; they suggested a role for Ran-GDP in the association of the importin-β·importin-α·NLS complex with the nuclear pore and speculated that the importin-β·importin-α·NLS·Ran-GDP·RanBP1 pentameric complex was the actual translocation complex that moved through the pore. This model has also been adopted by others (16–18) who have proposed that a stable Ran-GDP-containing complex was created on nucleoporin Nup358 (also called RanBP2) and that upon displacement of the importin-β·importin-α·Ran-GDP complex from the RBH (domain homologous to RanBP1) domains of Nup358 by RanBP1, binding of NTF2 triggered translocation to the nucleus. The role of NTF2 as the factor responsible for the translocation of the transport complex through the nuclear envelope has also been proposed by Paschal et al. (19). The role of Ran-GDP and RanBP1 in nuclear import has been demonstrated by a single mutation of a cysteine residue of importin-β; the mutation was required for binding Ran-GDP·RanBP1, but not Ran-GTP·RanBP1, and inhibited the nuclear import in permeabilized cells (20). The active role of RanBP1 in nuclear import has been further demonstrated by Künzler et al. (21), who showed that mutations in the Yrb1 gene encoding the yeast ortholog of RanBP1 impair nucleocytoplasmic transport.Despite considerable evidence for the involvement of Ran-GDP, RanBP1, and NTF2 in nuclear protein import, the precise mechanism by which these molecules regulate this process has been unknown. Here we characterize the interaction between Kap95p and Gsp1p-GDP. We show that this interaction results in GDP-to-GTP exchange on Gsp1p. Furthermore, we demonstrate that Gsp1p, Kap60p, Kap95p, Yrb1p, and Ntf2p interact to regulate the GDP-to-GTP exchange on Gsp1p. We suggest a mechanism of nuclear import additional to the RCC1-dependent pathway that incorporates our observations.     

Identification of a Sphingolipid α-Glucuronosyltransferase That Is Essential for Pollen Function in Arabidopsis

Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars; however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants.     

Nuclear Recruitment of Neuronal Nitric-oxide Synthase by α-Syntrophin Is Crucial for the Induction of Mitochondrial Biogenesis

Neuronal nitric-oxide synthase (nNOS) has various splicing variants and different subcellular localizations. nNOS can be found also in the nucleus; however, its exact role in this compartment is still not completely defined. In this report, we demonstrate that the PDZ domain allows the recruitment of nNOS to nuclei, thus favoring local NO production, nuclear protein S-nitrosylation, and induction of mitochondrial biogenesis. In particular, overexpression of PDZ-containing nNOS (nNOSα) increases S-nitrosylated CREB with consequent augmented binding on cAMP response element consensus sequence on peroxisome proliferator-activated receptor γ co-activator (PGC)-1α promoter. The resulting PGC-1α induction is accompanied by the expression of mitochondrial genes (e.g., TFAM, MtCO1) and increased mitochondrial mass. Importantly, full active nNOS lacking PDZ domain (nNOSβ) does not localize in nuclei and fails in inducing the expression of PGC-1α. Moreover, we substantiate that the mitochondrial biogenesis normally accompanying myogenesis is associated with nuclear translocation of nNOS. We demonstrate that α-Syntrophin, which resides in nuclei of myocytes, functions as the upstream mediator of nuclear nNOS translocation and nNOS-dependent mitochondrial biogenesis. Overall, our results indicate that altered nNOS splicing and nuclear localization could be contributing factors in human muscular diseases associated with mitochondrial impairment.     

A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity,but Helicase Activity Is Dispensable

Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase α-primase (pol-prim), constituting the viral primosome. Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro.     

Diosgenin from Dioscorea bulbifera: Novel Hit for Treatment of Type II Diabetes Mellitus with Inhibitory Activity against α-Amylase and α-Glucosidase

Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, ¹H NMR and ¹³C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus.     

Ectopic Localization of Mitochondrial ATP Synthase: A Target for Anti-Angiogenesis Intervention?

A receptor for angiostatin was identified on the surface of endothelial cells as F₁–F₀ ATP synthase (Moser et al., 1999). Proc. Natl. Acad. Sci. U.S.A. 96, 2811–2816. This ectopic ATP synthase catalyzes ATP synthesis and is inhibited by angiostatin over a wide pH range. Endothelial cells grown at normal pH suffer no ill effects from this angiostatin-mediated inhibition of ATP synthase, whereas endothelial cells grown at low, tumor-like extracellular pH cannot maintain a normal intracellular pH and die. Angiostatin inhibits both ATP synthesis and ATP hydrolysis (Moser et al., 2001) and interferes with intracellular pH regulation (Wahl and Grant, 2002; Wahl et al., 2002). Although angiostatin administered intravenously is cleared from the circulation in a matter of minutes, angiostatin-mimetics that are more stable have potential for clinical application. An angiostatin-mimetic activity has recently been observed using a polyclonal antibody against the β catalytic subunit of ATP synthase. In order to explore the mechanism of action of angiostatin and its mimetics, further work needs to be done to evaluate clinical applicability, specificity, and contraindications for this class of therapeutics.     

A new allele of α1-antitrypsin: PI * NADELAIDE

The allele PI NADELAIDE (PI NADE) was named in accord with nomenclature guidelines and specifies a new co-dominant variant of alpha 1AT. Discovery was achieved by IEF and the isoelectric point of NADE is between N and NHAM. Familial inheritance of PI NADE was demonstrated and both PI M2NADE and PI M3NADE phenotypes were observed. The mobility of PI NADE is identical to PI M by both starch and agarose electrophoresis. PI NADE apparently confers normal alpha 1AT serum concentrations and is probably unrelated to disease.     

Integrin αXβ2 Is a Leukocyte Receptor for Candida albicans and Is Essential for Protection against Fungal Infections

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)β(2) in the pathogenesis of fungal infection was assessed. Both purified α(X)β(2) and α(X)β(2)-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)β(2). Mice deficient in α(X)β(2) were more prone to systemic infection with the LD(50) fungal inoculum decreasing 3-fold in α(X)β(2)-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 10(4) cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)β(2)-deficient mice within 9 d. Organs taken from α(X)β(2)-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)β(2) is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)β(2) to control fungal invasion.     

Selective Transport of α-Mannosidase by Autophagic Pathways: IDENTIFICATION OF A NOVEL RECEPTOR,Atg34p*

In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy.     

Functional Analysis of the α-1,3-Glucan Synthase Genes agsA and agsB in Aspergillus nidulans: AgsB Is the Major α-1,3-Glucan Synthase in This Fungus

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and ¹³C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.     

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

Background

The Ca_vβ subunits of high voltage-activated Ca²⁺ channels control the trafficking and biophysical properties of the α₁ subunit. The Ca_vβ-α₁ interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that βs regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the α-interaction domain (AID) be a rigid structure.

Methodology/Principal Findings

The present study tests this hypothesis by altering the flexibility and orientation of this region in α₁2.2, then testing for Ca_vβ regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished β2a and β3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of β2a to produce non-inactivating currents. Orientation of Ca_vβ with respect to α₁2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca_vβ subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca_vβ subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms β-sheet. The orientation of β with respect to α was confirmed by the bimolecular fluorescence complementation assay.

Conclusions/Significance

These results show that the orientation of the Ca_vβ subunit relative to the α₁2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca_vβ to regulate channel activity.     

7 The Molecular Biology of Tryptophan Synthase: A Model for Protein–Protein Interaction

Abstract

The use of plastic produced from non-renewable resources constitutes a major environmental problem of the modern society. Polylactide polymers (PLA) have recently gained enormous attention as one possible substitution of petroleum derived polymers. A prerequisite for high quality PLA production is the provision of optically pure lactic acid, which cannot be obtained by chemical synthesis in an economical way. Microbial fermentation is therefore the commercial option to obtain lactic acid as monomer for PLA production. However, one major economic hurdle for commercial lactic acid production as basis for PLA is the costly separation procedure, which is needed to recover and purify the product from the fermentation broth. Yeasts, such as Saccharomyces cerevisiae (bakers yeast) offer themselves as production organisms because they can tolerate low pH and grow on mineral media what eases the purification of the acid. However, naturally yeasts do not produce lactic acid. By metabolic engineering, ethanol was exchanged with lactic acid as end product of fermentation. A vast amount of effort has been invested into the development of yeasts for lactic acid production since the first paper on this topic by Dequin and process insight. If pH stress is used as basis for DNA microarray analyses, in order to improve the host, what exactly is addressed? Growth? Or productivity? They might be connected, but can be negatively correlated. A better growing strain might not be a better producer. So if the question was growth, the answer might not be what was initially intended (productivity).

A major task for the future is to learn to ask the right questions – a lot of studies intended to lead to better productivity, did lead to interesting results, but NOT to better production strains.

Taking together what we learned from lactic acid production with yeasts, we see a bright future for bulk and fine chemical production with these versatile hosts.     

A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317–326 Peptide

TAT-RasGAP_317–326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP_317–326 sequence for the anticancer activities of TAT-RasGAP_317–326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP_317–326.     

Infection Structure–Specific Expression of β-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of β-Glucan–Triggered Immunity in Maize

β-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of β-1,3-glucan is unknown. We functionally characterized the β-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of β-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading β-glucan–triggered immunity.     

A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor: A NOVEL DUAL MECHANISM OF MANNITOL FOR THE TREATMENT OF PARKINSON DISEASE (PD)*

The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.     

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.     

Seeded Aggregation and Toxicity of α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES*

The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.