Copper distribution in the normal human brain

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.     

Irreversible aggregation of protein synthesis machinery after focal brain ischemia

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2alpha, 4E and 3eta, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia.     

Effect of neonatal handling on brain enkephalin-degrading peptidase activities.

Neonatal handling decreases neutral endopeptidase 24.11 activity in the amygdala. However, this procedure does not affect aminopeptidase activities in any of the brain areas studied. Neonatal handling has been one of the most commonly used strategies to study the plasticity of the nervous system. The crucial role of the opioids in the control of different aspects of behaviour and development has been well documented. Regarding this subject, the endogenous opioid system might mediate some of the effects induced by neonatal handling. In this work, we have studied the effects of neonatal handling on several enkephalin-degrading peptidases, including soluble and membrane-bound aminopeptidases (puromycin-sensitive and -insensitive) and neutral endopeptidase 24.11 in different rat brain areas. Tyrosine aminopeptidase activities were measured fluorimetrically using tyrosine-beta-naphthylamide as substrate and puromycin as selective inhibitor of one of the membrane-enzymes. Dansyl-D-Ala-Gly-Phe(pNO2)-Gly was the fluorogenic substrate for neutral endopeptidase. The reduced neutral endopeptidase 24.11 activity in the amygdala of neonatal handled rats could reduce enkephalin catabolism in this area and it could be responsible for some of the effects induced by neonatal handling.     

Copper in the brain and Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of neurodegenerative disease. The brain is particularly vulnerable to oxidative damage induced by unregulated redox-active metals such as copper and iron, and the brains of AD patients display evidence of metal dyshomeostasis and increased oxidative stress. The colocalisation of copper and amyloid β (Aβ) in the glutamatergic synapse during NMDA-receptor-mediated neurotransmission provides a microenvironment favouring the abnormal interaction of redox-potent Aβ with copper under conditions of copper dysregulation thought to prevail in the AD brain, resulting in the formation of neurotoxic soluble Aβ oligomers. Interactions between Aβ oligomers and copper can further promote the aggregation of Aβ, which is the core component of extracellular amyloid plaques, a central pathological hallmark of AD. Copper dysregulation is also implicated in the hyperphosphorylation and aggregation of tau, the main component of neurofibrillary tangles, which is also a defining pathological hallmark of AD. Therefore, tight regulation of neuronal copper homeostasis is essential to the integrity of normal brain functions. Therapeutic strategies targeting interactions between Aβ, tau and metals to restore copper and metal homeostasis are discussed.     

Effect of Tanakan on postischemic activity of protein synthesis machinery in the rat brain

A growing body of evidence supports the role of free radicals in triggering the functional and metabolic disturbances following transient cerebral ischemia. This study was designed to evaluate whether the extent of reperfusion-induced inhibition of protein synthesis initiation as well as tissue injury can be reduced by Tanakan (Ginkgo biloba extract, EGb 761) (Beaufour-Ipsen Industrie). Rats received Tanakan in the dose of 40 mg/kg/day for 7 days before surgical intervention. Transient forebrain ischemia was induced by 4-vessel occlusion. Rats were subjected to 20 min of ischemia followed by 30 min, 4 h or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. The protective effect was observed, concerning both the protein synthesis and the number of surviving neurons, in the Tanakan-treated groups. Tanakan significantly reduced the ischemia/reperfusion-induced inhibition of translation in the neocortex as well as in the highly sensitive hippocampus. Our results indicate that free radicals play an important role in the development of reperfusion-induced injury, and the treatment of ischemic and reperfused brain with free radical scavengers may reduce the severity of reperfusion damage.     

Dynamics of the translational machinery

The recent growth in single molecule studies of translation has provided an insight into the molecular mechanism of ribosomal function. Single molecule fluorescence approaches allowed direct observation of the structural rearrangements occurring during translation and revealed dynamic motions of the ribosome and its ligands. These studies demonstrated how ligand binding affects dynamics of the ribosome, and the role of the conformational sampling in large-scale rearrangements intrinsic to translation elongation. The application of time-resolved cryo-electron microscopy revealed new conformational intermediates during back-translocation providing an insight into ribosomal dynamics from an alternative perspective. Recent developments permitted examination of conformational and compositional dynamics of the ribosome in real-time through multiple cycles of elongation at the single molecule level. The zero-mode waveguide approach allowed direct observation of the compositional dynamics of tRNA occupancy on the elongating ribosome. The emergence of single molecule in vivo techniques provided insights into the mechanism and regulation of translation at the organismal level.     

Organization of the ENaC-regulatory machinery

The control of fluid and electrolyte homeostasis in vertebrates requires the integration of a diverse set of signaling inputs, which control epithelial Na(+) transport, the principal ionic component of extracellular fluid. The key site of regulation is a segment of the kidney tubules, frequently termed the aldosterone-sensitive distal nephron, wherein the epithelial Na(+) channel (or ENaC) mediates apical ion entry. Na(+) transport in this segment is strongly regulated by the salt-retaining hormone, aldosterone, which acts through the mineralocorticoid receptor (MR) to influence the expression of a selected set of target genes, most notably the serine-threonine kinase SGK1, which phosphorylates and inhibits the E3 ubiquitin ligase Nedd4-2. It has long been known that ENaC activity is tightly regulated in vertebrate epithelia. Recent evidence suggests that SGK1 and Nedd4-2, along with other ENaC-regulatory proteins, physically associate with each other and with ENaC in a multi-protein complex. The various components of the complex are regulated by diverse signaling networks, including steroid receptor-, PI3-kinase-, mTOR-, and Raf-MEK-ERK-dependent pathways. In this review, we focus on the organization of the targets of these pathways by multi-domain scaffold proteins and lipid platforms into a unified complex, thereby providing a molecular basis for signal integration in the control of ENaC.     

Copper deficiency in the developing rat brain: evidence for abnormal mitochondria.

Abstract— Copper deficiency was produced in developing rats by feeding a low copper diet to rats during gestation and lactation and providing the offspring the same diet. The progeny developed similar to those of an earlier model based on preconception depletion followed by marginal supplementation during gestation. Copper levels were greatly reduced in the brain, iron levels were slightly depressed, and no differences in zinc content were found. Electron microscopic examination of brain tissue revealed the presence of enlarge mitochondria from copper-deficient animals. Isolated mitochondria from copper-deficient rats showed a 30% reduction in the rate of both succinate and glutamate oxidation, and for glutamate, the respiratory control ratio (RCR) was decreased by 60%. Difference spectra displayed a four-fold reduction in cytochrome a+a₃ and slight increases in cytochrome b, c₁ and c. Enzyme analysis of isolated mitochondria revealed a five-fold decrease in cytochrome oxidase, slight increases in succinic dehydrogenase and fumarase, and small decreases in hexokinase and monoamine oxidase. No difference in peroxidation of brain lipids was evident. Determination of metabolites from fast frozen tissue suggested that the copper-deficient brain was in a more reduced state based on a doubling of both the lactate/pyruvate and α-glycerol-P/dihydroxyacetone-P ratios. Creatine-P, ATP, and ADP levels were not different.     

Copper brain homeostasis: role of amyloid precursor protein and prion protein

The main proteins associated with Alzheimer's and prion diseases (amyloid precursor protein (APP) and prion protein (PrP(C)), respectively, have binding sites for copper and it has therefore been suggested that they play a role in copper metabolism. Here, we review evidence indicating that the copper binding domains (CuBD) of APP and PrP(C) are able to modulate the oxidation state of copper, and prevent neurotoxic effects and memory impairments induced by copper. Results with transgenic and other animal models have established the relation between these pathogenic proteins and copper. In particular, APP transgenic models, suggest a beneficial effect for copper in AD.     

Postmortem stability of somatostatin-like immunoreactivity in mouse brain under conditions simulating handling of human autopsy material

The postmortern stability of somatostatin-like immunoreactivity (SLI) has been investigated in mice cooled after death at a rate which approximates the rate of cooling of the human brain in situ under normal mortuary conditions. Significant changes in SLI levels were observed within the first few hours after death, but concentrations measured 6–24 hr after death seem to be similar to those existing at the time of death.     

Structural biology of the macroautophagy machinery

Macroautophagy is a conserved degradative process mediated through formation of a unique double- membrane structure, the autophagosome. The discovery of autophagy-related （Atg） genes required for autophagosome formation has led to the characterization of approximately 20 genes mediating this process. Recent structural studies of the Atg proteins have provided the molecular basis for their function. Here we summarize the recent progress in elucidating the structural basis for autophagosome formation.     

Evolution of the chloroplast division machinery

Chloroplasts are photosynthetic organelles derived from endosymbiotic cyanobacteria during evolution. Dramatic changes occurred during the process of the formation and evolution of chloroplasts, including the large-scale gene transfer from chloroplast to nucleus. However, there are still many essential characters remaining. For the chloroplast division machinery, FtsZ proteins, Ftn2, SulA and part of the division site positioning system—MinD and MinE are still conserved. New or at least partially new proteins, such as FtsZ family proteins FtsZ1 and ARC3, ARC6H, ARC5, PDV1, PDV2 and MCD1, were introduced for the division of chloroplasts during evolution. Some bacterial cell division proteins, such as FtsA, MreB, Ftn6, FtsW and FtsI, probably lost their function or were gradually lost. Thus, the chloroplast division machinery is a dynamically evolving structure with both conservation and innovation.     

Evolution of the chloroplast division machinery

Chloroplasts are photosynthetic organelles derived from endosymbiotic cyanobacteria during evolution.Dramatic changes occurred during the process of the formation and evolution of chloroplasts,including the large-scale gene transfer from chloroplast to nucleus.However,there are still many essential characters remaining.For the chloroplast division machinery,FtsZ proteins,Ftn2,SulA and part of the division site positioning system- MinD and MinE are still conserved.New or at least partially new proteins,such as FtsZ family proteins FtsZl and ARC3,ARC6H,ARC5,PDV1,PDV2 and MCD1,were introduced for the division of chloroplasts during evolution.Some bacterial cell division proteins,such as FtsA,MreB,Ftn6,FtsW and Ftsl,probably lost their function or were gradually lost.Thus,the chloroplast division machinery is a dynamically evolving structure with both conservation and innovation.     

A glimpse of the machinery.

Asymmetric mRNA localization within cells plays an important part in both development and physiology. Recent studies have provided a glimpse of the conserved molecular machinery that directs the localization of specific mRNAs.     

Taking apart the endocytic machinery

The formation of clathrin-coated endocytic vesicles is driven by a complex and highly dynamic molecular machinery. In this issue, Idrissi et al. (Idrissi, F.-Z., H. Grötsch, I.M. Fernández-Golbano, C. Presciatto-Baschong, H. Riezman, and M.-I. Geli. 2008. J. Cell Biol. 180:1219–1232) reveal some of the secrets of this machinery by analyzing the localizations of nine endocytic proteins during vesicle budding in yeast using quantitative immunoelectron microscopy.More than 50 different proteins are thought to have roles in the formation of clathrin-coated endocytic vesicles. These proteins assemble together at the plasma membrane, forming the molecular machinery that drives budding of endocytic vesicles. Although clathrin-mediated endocytosis has been studied already for more than four decades, an understanding of the molecular mechanisms of the process is still quite limited. The difficulty of unraveling the molecular mechanisms is not only a result of the large number of involved proteins but is also a result of the dynamic nature of the endocytic machinery. Endocytic proteins are recruited to the site of vesicle formation in a sequential manner, each protein having its specific times of arrival and departure. The composition of the endocytic machinery can change in a matter of seconds. Many of the recent insights into the process of clathrin-mediated endocytosis have come from imaging of fluorescently labeled proteins in living cells using fluorescence microscopy. Light microscopy provides a good temporal resolution of dynamic events, but its spatial resolution is quite limiting when studying endocytic vesicle budding. On the other hand, electron microscopy offers much better spatial resolution but only provides still images.In this issue, one study (see Idrissi et al. on p. 1219) uses immunoelectron microscopy to study the localizations of nine different proteins at sites of endocytosis in yeast cells. Most of the proteins involved in clathrin-mediated endocytosis in yeast are conserved throughout eukaryotes, including mammals, making yeast a good model system for studying the basic mechanisms of endocytosis. However, only a few studies have addressed the organization of the endocytic machinery at the ultrastructural level in yeast (Mulholland et al., 1994; Young et al., 2004; Rodal et al., 2005). Idrissi et al. (2008) start by looking at clathrin, Pan1 (Eps15 homologue), and Sla1 (intersectin-like protein), which, when visualized in living cells by fluorescence microscopy, show similar behaviors. These proteins accumulate at the plasma membrane, forming small fluorescent spots that are initially nonmotile but then move ∼200 nm from the surface toward the interior of the cell at a constant speed for ∼10 s, after which the spots are rapidly disassembled (Kaksonen et al., 2005; Newpher et al., 2005). Idrissi et al. (2008) show by immunoelectron microscopy that clathrin, Pan1, and Sla1 each localize to tips of plasma membrane invaginations, which are ∼50 nm in diameter and have variable lengths up to 180 nm (Fig. 1). This confirms the earlier hypothesis that the movement of these proteins seen in living cells corresponds to the invagination of a clathrin-coated pit, not to the movement of an already budded vesicle. Importantly, these observations show that the length of the endocytic invagination can be used as an indicator for its age.Open in a separate windowFigure 1. Schematic model showing the localization of nine proteins on an endocytic invagination. An invagination of intermediate length (∼100 m) is depicted. The coat proteins, including clathrin, coat the tip of the invagination. Rvs167, Las17, and Bbc1 occupy the neck region below the tip. Myo5 concentrates to the base of the invagination. Actin and actin-binding protein Abp1 form a shell covering the whole invagination. The quantitative immunoelectron microscopy analysis is then applied to six other proteins involved in endocytosis: Rvs167, Las17, Bbc1, Myo5, actin, and Abp1 (Fig. 1). Rvs167 is a homologue of mammalian amphiphysin, a protein involved in pinching vesicles off from the tips of clathrin-coated pits (Takei et al., 1999). The other studied proteins are regulators or components of the actin cytoskeleton, which, in yeast, is essential for endocytosis, specifically for the movement of clathrin and other coat-associated proteins away from the cell surface (i.e., for the membrane invagination; Kubler and Riezman, 1993; Kaksonen et al., 2003). This analysis reveals many exciting details about the dynamic organization of the endocytic machinery. The yeast amphiphysin homologue Rvs167 is shown to localize to the tubular area of the membrane invagination, just below its clathrin-coated tip. Las17 (yeast Wiskott-Aldrich syndrome protein), a strong activator of the actin filament nucleator Arp2/3 (Winter et al., 1999), and Bbc1, an inhibitor of Las17 (Rodal et al., 2003), both localize to the same area as Rvs167. Myo5, a type I myosin, which is both an actin-dependent molecular motor and an activator of the Arp2/3 complex (Sun et al., 2006), localizes mostly to the base of the invagination, where the membrane has a negative curvature. Genetic experiments together with live cell imaging have suggested that Las17 and Myo5 are both needed sequentially for actin-driven invagination of the membrane (Sirotkin et al., 2005; Sun et al., 2006; Galletta et al., 2008). Las17 has a role in initiating the actin polymerization at endocytic sites, whereas Myo5 is needed for the subsequent internalization process. Interestingly, these two major activators of the Arp2/3 complex localize slightly differently: Myo5 closer to the base of the invagination and Las17 in the middle. This suggests that actin polymerization may be spatially restricted to different areas during different stages of endocytosis. Analysis of actin and actin filament–binding protein Abp1 reveals that they are localized throughout the invagination. However, when compared with the other proteins, immunogold labeling for actin and Abp1 is significantly further away from the lipid bilayer, suggesting that the actin cytoskeleton forms an outer shell covering the rest of the endocytic protein machinery.Using the invagination length as an indicator for the age of the endocytic site, Idrissi et al. (2008) are able to add the time dimension to their data, revealing some interesting temporal dynamics of protein localizations. The temporal order of protein recruitment derived from the electron microscopy data matches observations made using live cell imaging, but the localizations can now be seen at much higher resolution and in relation to the shape of the membrane. Bbc1 and Rvs167 colocalize with Las17, but they appear only on longer, older invaginations. Similarly, Las17 localization precedes Myo5 accumulation, which is consistent with their postulated order of function. The shortest invaginations (<50 nm) show very little labeling for actin. This may mean that the initial membrane bending is independent of actin and could be caused by clathrin or other coat proteins. In older invaginations, actin shows an intriguing distribution. The initial continuous labeling is split into two. Part of the staining localizes to the base of the invagination, and another part localizes to the tip. Similar behavior is also described for Myo5, which initially is concentrated at the base of the invagination but later also appears at the tip. It is not clear whether this staining pattern reflects two separate structures or whether one structure breaks into two. However, this finding shows that the organization of the actin cytoskeleton associated with the endocytic sites may be more complex than previously thought.One of the key events on the endocytic pathway, vesicle scission, still escapes analysis. Scission and the following disassembly are probably too transient to be caught in fixed cells frequently enough to yield sufficient data for analysis. Other very transient events may also go undetected because they could get smeared as a result of the averaging of data from tens of different invaginations. For these very transient events, live cell imaging is likely to remain the method of choice (Merrifield et al., 2005). However, the superior resolution offered by electron microscopy will clearly continue to provide critical insights. Idrissi et al. (2008) analyzed localizations of nine different proteins. At least 40 yeast proteins involved in endocytic internalization remain to be studied. The rich collections of endocytic mutants will also provide many interesting samples for analysis. What happens to the organization of the endocytic machinery when one of the Arp2/3 activators is mutated? Does the shape or size of the invagination change if one of the coat components is deleted? What would be the effect of inhibiting the motor activity of Myo5? These are just a few examples of exciting questions that can now be addressed.