Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

BACKGROUND: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. METHODS: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. RESULTS: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. CONCLUSION: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

De novo protein synthesis of syntaxin-1 and dynamin-1 in long-term memory formation requires CREB1 gene transcription in Lymnaea stagnalis.

Consolidation of aversive operant conditioning into long-term memory (LTM) requires CREB-dependent de novo protein synthesis. The newly synthesized proteins are distributed to the synapses in neurons that are involved in memory formation and storage. Accumulating evidence indicates that the presynaptic release mechanisms also play a role in long-term synaptic plasticity. Our understanding of whether the presynaptic proteins undergo de novo synthesis during long-term memory formation is limited. In this study, we investigated the involvement of syntaxin-1, a presynaptic exocytotic protein, and dynamin-1, an endocytotic protein, in the formation of long-term memory. We took advantage of a well-established aversive operant conditioning model of aerial respiratory behavior in the fresh water pond snail Lymnaea stagnalis, and demonstrated that the LTM formation is associated with increased expression of syntaxin-1 and dynamin-1, coincident with elevated levels of CREB1. Partial knockdown of CREB1 gene by double stranded RNA inhibition (dsRNAi) prior to operant conditioning prevented snails from memory consolidation, and reduced the expression of syntaxin-1 and dynamin-1 at both mRNA and protein levels. These findings suggest that CREB1-mediated gene expression is required for the LTM-induced up-regulation of synaptic proteins, syntaxin-1 and dynamin-1, in L. stagnalis. Our study thus offers new insights into the molecular mechanisms that mediate CREB1-dependent long-term memory formation.

Intraocular pressure elevation induces mitochondrial fission and triggers OPA1 release in glaucomatous optic nerve.

PURPOSE: To determine whether elevation of intraocular pressure (IOP) triggers mitochondrial fission and ultrastructural changes and alters optic atrophy type 1 (OPA1) expression and distribution in the optic nerve (ON) of glaucomatous DBA/2J mice. METHODS: IOP in the eyes of DBA/2J mice was measured, and mitochondrial structural changes were assessed by conventional electron microscopy (EM) and EM tomography. Cytochrome c oxidase IV subunit 1 (COX), OPA1, and Dnm1, a rat homologue of dynamin-related protein-1, mRNA were measured by quantitative (q)PCR. COX and OPA1 protein distribution was assessed by immunocytochemistry and Western blot. RESULTS: Excavation of the optic nerve head (ONH), axon loss, and COX reduction were evident in 10-month-old glaucomatous ONHs of eyes with >20 mm Hg IOP elevation. EM analysis showed mitochondrial fission, matrix swelling, substantially reduced cristae volume, and abnormal cristae depletion in 10-month-old glaucomatous ONH axons. The mean length of mitochondrial cross section in these axons decreased from 858.2 +/- 515.3 nm in 3-month-old mice to 583.3 +/- 298.6 nm in 10-month-old glaucomatous mice (P < 0.001). Moderate reductions of COX mRNA were observed in the 10-month-old DBA/2J mice's ONHs. Larger reductions of OPA1 immunoreactivity and gene expression were coupled with larger increases of Dnm1 gene expression in 10-month-old glaucomatous ONH. Subcellular fractionation analysis indicates increased release of both OPA1 and cytochrome c from mitochondria in 10-month-old glaucomatous ONs. CONCLUSIONS: IOP elevation may directly damage mitochondria in the ONH axons by promoting reduction of COX, mitochondrial fission and cristae depletion, alterations of OPA1 and Dnm1 expression, and induction of OPA1 release. Thus, interventions to preserve mitochondria may be useful for protecting against ON degeneration in glaucoma.

Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease.

Synaptic dysfunction is one of the earliest events in the pathogenesis of Alzheimer disease (AD). However, the molecular mechanisms underlying synaptic defects in AD are largely unknown. We report here that beta-amyloid (Abeta), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that is essential for synaptic vesicle recycling and, hence, for memory formation and information processing. The Abeta-induced dynamin 1 decrease occurred in the absence of overt synaptic loss and was also observed in the Tg2576 mouse model of AD. In addition, our results provided evidence that the Abeta-induced decrease in dynamin 1 was likely the result of a calpain-mediated cleavage of dynamin 1 protein and possibly the down-regulation of dynamin 1 gene expression. These data suggest a mechanism to explain the early cognitive loss without a major decline in synapse number observed in AD and propose a novel therapeutic target for AD intervention.

Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells.

The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic beta-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane.