2,4-Dinitrophenol blocks neurodegeneration and preserves sciatic nerve function after trauma.

Preventing the harm caused by nerve degeneration is a major challenge in neurodegenerative diseases and in various forms of trauma to the nervous system. The aim of the current work was to investigate the effects of systemic administration of 2,4-dinitrophenol (DNP), a compound with newly recognized neuroprotective properties, on sciatic-nerve degeneration following a crush injury. Sciatic-nerve injury was induced by unilateral application of an aneurysm clip. Four groups of mice were used: uninjured, injured treated with vehicle (PBS), injured treated with two intraperitoneal doses of DNP (0.06 mg DNP/kg every 24 h), and injured treated with four doses of DNP (every 12 h). Animals were sacrificed 48 h post injury and both injured and uninjured (contralateral) sciatic nerves were processed for light and electron microscopy. Morphometric, ultrastructural, and immunohistochemical analysis of injured nerves established that DNP prevented axonal degeneration, blocked cytoskeletal disintegration, and preserved the immunoreactivity of amyloid precursor protein (APP) and Neuregulin 1 (Nrg1), proteins implicated in neuronal survival and myelination. Functional tests revealed preservation of limb function following injury in DNP-treated animals. Results indicate that DNP prevents nerve degeneration and suggest that it may be a useful small-molecule adjuvant in the development of novel therapeutic approaches in nerve injury.

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration.

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.

Formation of soluble oligomers and amyloid fibrils with physical properties of the scrapie isoform of the prion protein from the C-terminal domain of recombinant murine prion protein mPrP-(121-231).

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.

Identification of a neurofilament-like protein in the protocerebral tract of the crab Ucides cordatus.

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of approximately 160 kDa, similar to the mammalian NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.

Amyloidogenicity and cytotoxicity of recombinant mature human islet amyloid polypeptide (rhIAPP).

Pancreatic amyloid plaques formed by the pancreatic islet amyloid polypeptide (IAPP) are present in more than 95% of type II diabetes mellitus patients, and their abundance correlates with the severity of the disease. IAPP is currently considered the most amyloidogenic peptide known, but the molecular bases of its aggregation are still incompletely understood. Detailed characterization of the mechanisms of amyloid formation requires large quantities of pure material. Thus, availability of recombinant IAPP in sufficient amounts for such studies constitutes an important step toward elucidation of the mechanisms of amyloidogenicity. Here, we report, for the first time, the successful expression, purification and characterization of the amyloidogenicity and cytotoxicity of recombinant human mature IAPP. This approach is likely to be useful for the production of other amyloidogenic peptides or proteins that are difficult to obtain by chemical synthesis.