Differential activation of C1 complement components in rat spinal cord after sciatic nerve injury

A number of new synthetic nociceptin ligands were studied in receptor binding and functional tests in rat brain membranes and in cloned systems. Ligand binding experiments were performed with three different radioprobes developed in our lab. The nociceptin derivatives exhibited high affinity in competition experiments. Receptor-mediated G-protein activation was determined in [³⁵S]GTPgS binding assays. Among the new structures examined, Ac-RYYRIK-ol was found to be only a weak stimulator by itself, whereas this compound inhibited receptor-mediated G-protein activation. These data suggest that Ac-RYYRIK-ol is a high affinity peptide antagonist for the nociceptin receptor.
Acknowledgements:  Supported by the Hungarian Scientific Research Fund OTKA T-035211, T-033078, T-030841, and the Ministry of Education, NKFP 1/027 Hungary.     

A ciliary neuronotrophic factor from peripheral nerve and smooth muscle which is not retrogradely transported

We have found that a CNTF-like molecule which supports ciliary and sympathetic neurons is not retrogradely transported in either sympathetic or parasympathetic nerves. The factor has an apparent M_r of 21 kDa, a pI of 4.9, and is present in peripheral nerves and smooth muscle of the chick. Our experiments indicate that CNTF-like activity does not accumulate on the distal side of ligated chickexpansor nerves. In contrast, there is a clear accumulation of NGF. The activity further differs from NGF in that it is not removed from a smooth muscle of the chick wing by innervating sympathetic fibers. Transection of these fibers does not lead to an accumulation of ciliary activity in theexpansor secundariorum muscle, suggesting that neurons do not actively deplete the muscle of factor by retrograde transport. Finally, recombinant CNTF or semi-purified preparations of CNTF-like activity labelled with¹²⁵I were not transported to the ciliary ganglion of chicks following injection of biologically active material into the eye. Our results suggest either that endogenous CNTF does not act as a survival factorin vivo, or that retrograde transport is not a property inherent to all neuronotrophic molecules.Special issue dedicated to Dr. Lawrence Austin     

Altered spectrum of retrogradely transported axonal proteins in p-bromophenylacetylurea neuropathy

The composition of retrogradely transported axonal proteins was examined by acrylamide gel electrophoresis and gel autoradiography in the experimental neuropathy induced in rats by p-bromophenylacetylurea (BPAU). Protein composition was normal during the early phase of retrograde transport but showed significant abnormalities during a later phase. The early phase consisted of proteins collected distal to a mid-thigh ligature of sciatic nerve between 15 and 24 hours after injection of [³⁵S] methionine into lumbar ventral horn of the spinal cord. In terms of their relative labeling and electrophoretic mobility, these proteins were almost identical in experimental and control rats. Most of the labeled protein bands were also identical in the later phase, collected between 24 and 48 hours, but there were some consistent omissions and additions. Present in controls but missing in BPAU treated rats were three bands at 42, 41, and 25 KDa. In contrast, 4 bands (63, 56, 50, 26 KDa) were more prominent in the experimental rats than in controls. We suspect abnormal post-translational modification or proteolysis of rapidly transported proteins in the terminal or preterminal portion of the neurons exposed to BPAU. This abnormality, in addition to a previously reported premature processing of transported organelles, may underlie the development of peripheral neuropathy.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

Differential proteomics in dequeened honeybee colonies reveals lower viral load in hemolymph of fertile worker bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers.     

The movement of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles

To identify the structures to be rapidly transported through the axons, we developed a new method to permit local cooling of mouse saphenous nerves in situ without exposing them. By this method, both anterograde and retrograde transport were successfully interrupted, while the structural integrity of the nerves was well preserved. Using radioactive tracers, anterogradely transported proteins were shown to accumulate just proximal to the cooled site, and retrogradely transported proteins just distal to the cooled site. Where the anterogradely transported proteins accumulated, the vesiculotubular membranous structures increased in amount inside both myelinated and unmyelinated axons. Such accumulated membranous structures showed a relatively uniform diameter of 50--80 nm, and some of them seemed to be continuous with the axonal smooth endoplasmic reticulum (SER). Thick sections of nerves selectively stained for the axonal membranous structures revealed that the network of the axonal SER was also packed inside axons proximal to the cooled site. In contrast, large membranous bodies of varying sizes accumulated inside axons just distal to the cooled site, where the retrogradely transported proteins accumulated. These bodies were composed mainly of multivesicular bodies and lamellated membranous structures. When horseradish peroxidase was administered in the distal end of the nerve, membranous bodies showing this activity accumulated, together with unstained membranous bodies. Hence, we are led to propose that, besides mitochondria, the membranous components in the axon can be classified into two systems from the viewpoint of axonal transport: "axonal SER and vesiculotubular structures" in the anterograde direction and "large membranous bodies" in the retrograde direction.     

Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo

Brain dynein is a microtubule-activated ATPase considered to be a candidate to function as a molecular motor to transport membranous organelles retrogradely in the axon. To determine whether brain dynein really binds to retrogradely transported organelles in vivo and how it is transported to the nerve terminals, we studied the localization of brain dynein in axons after the ligation of peripheral nerves by light and electron microscopic immunocytochemistry using affinity-purified anti-brain dynein antibodies. Different classes of organelles preferentially accumulated at the regions proximal and distal to the ligated part. Interestingly, brain dynein accumulated both at the regions proximal and distal to the ligation sites and localized not only on retrogradely transported membranous organelles but also on anterogradely transported ones. This is the first evidence to show that brain dynein associates with retrogradely transported organelles in vivo and that brain dynein is transported to the nerve terminal by fast flow. This also suggests that there may be some mechanism that activates brain dynein only for retrograde transport.     

Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways

In plants, animals, and fungi, members of the Dicer family of RNase III-related enzymes process double-stranded RNA (dsRNA) to initiate small-RNA-mediated gene-silencing mechanisms. To learn how C. elegans Dicer, DCR-1, functions in multiple distinct silencing mechanisms, we used a mass-spectrometry-based proteomics approach to identify DCR-1-interacting proteins. We then generated and characterized deletion alleles for the corresponding genes. The interactors are required for production of three species of small RNA, including (1) small interfering RNAs (siRNAs), derived from exogenous dsRNA triggers (exo-siRNAs); (2) siRNAs derived from endogenous triggers (endo-siRNAs); and (3) developmental regulatory microRNAs (miRNAs). One interactor, the conserved RNA-phosphatase homolog PIR-1, is required for the processing of a putative amplified DCR-1 substrate. Interactors required for endo-siRNA production include ERI-1 and RRF-3, whose loss of function enhances RNAi. Our findings provide a first glimpse at the complex biochemical niche of Dicer and suggest that competition exists between DCR-1-mediated small-RNA pathways.     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.     

Prolonged alteration in composition of fast transported protein in axons prevented from regenerating after injury

A previous study revealed a characteristic alteration in the ratio of labeling of two fast axonally transported polypeptides identified as S1 (22,000 daltons) and S2 (18,000 daltons) and their ratio (S2/S1) in rat motoneuron axons following axonal injury and subsequent regeneration. In this study the S2/S1 ratio was determined for axons which were resected and ligated proximally and which did not regenerate to reinnervate muscle. While the initial increase in the S2/S1 ratio following section was the same as that following a crush, the S2/S1 ratio did not return towards normal values after 42 days, but remained elevated for at least 98 days after injury. It is concluded that the return of S2/S1 ratio to normal values, like some other manifestations of the cell body reaction, is delayed if the injured axons do not regenerate.     

How proteomics reveals potential biomarkers in brain diseases

The brain is complex, and so are the proteomics studies of brain tissue and its diseases, including Alzheimer's Disease, Parkinson's Disease and schizophrenia. In this review, general considerations and strategies of proteomics technologies, the advantages and challenges as well as the special needs for brain tissue are described and summarized. In addition, the results of the first studies are presented including a quality evaluation of the candidate proteins for these diseases. A paragraph is dedicated to the efforts of standardization in this field.     

How proteomics reveals potential biomarkers in brain diseases

The brain is complex, and so are the proteomics studies of brain tissue and its diseases, including Alzheimer’s Disease, Parkinson’s Disease and schizophrenia. In this review, general considerations and strategies of proteomics technologies, the advantages and challenges as well as the special needs for brain tissue are described and summarized. In addition, the results of the first studies are presented including a quality evaluation of the candidate proteins for these diseases. A paragraph is dedicated to the efforts of standardization in this field.     

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.