Clearance of Alzheimer's Abeta peptide: the many roads to perdition.

The amyloid hypothesis of Alzheimer's disease (AD) maintains that the accumulation of the amyloid beta protein (Abeta) is a critical event in disease pathogenesis. A great deal of both academic and commercial research has focused on the mechanisms by which Abeta is generated. However, investigations into the mechanisms underlying Abeta clearance have blossomed over the last several years. This minireview will summarize pathways involved in the removal of cerebral Abeta, including enzymatic degradation and receptor-mediated efflux out of the brain.

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice.

Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.

Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells.

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

Nhp6, an HMG1 protein, functions in SNR6 transcription by RNA polymerase III in S. cerevisiae.

Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S. cerevisiae at elevated temperatures. We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III. Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect. Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1. Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter. Thus, Nhp6 has a direct role in transcription complex assembly at SNR6.

Association of membrane-bound amyloid precursor protein APP with the apolipoprotein E receptor LRP.

In order to identify cell surface proteins that interact with the amyloid precursor protein (APP), we biotinylated H4 human neuroglioma cells in culture with a water soluble biotinylating agent, immunoprecipitated APP with an antibody specific to the intracellular domain, and probed the precipitated proteins with anti-biotin. In human neuroglioma cells overexpressing APP751, we found a high molecular weight protein that immunoprecipitated with APP. This band was identified as the low density lipoprotein receptor-related protein (LRP) by three criteria: first, the band immunolabeled with anti-LRP antibodies; second, the band bound the LRP receptor associated protein, RAP; and third, this band was present in LRP-expressing fibroblasts, but not LRP-deficient fibroblasts. In complementary experiments, we found that APP co-precipitated with LRP, with a preference for an isoform of APP containing the Kunitz protease inhibitor domain. Interaction of APP and LRP on the surface of living cells was demonstrated by crosslinking APP and LRP with the water-soluble cross-linking agent BS(3). APP and LRP were shown by confocal microscopy to colocalize in perinuclear structures, but to primarily remain separate in vesicles and on the cell surface. We propose that full-length APP can transiently interact with the receptor LRP on the cell surface, affecting the processing and intracellular transport of APP.

The structure and function of nuclear lamins: implications for disease.

The nuclear lamins polymerize to form the nuclear lamina, a fibrous structure found on the inner face of the nuclear membrane. The lamins also appear to form structures within the nucleoplasm. These various lamin structures help to establish and maintain the shape and strength of the interphase nucleus, but recent work also suggests that the lamins have a role in nuclear processes such as DNA replication. Furthermore, mutations in the human lamin A/C gene have recently been linked to several diseases, including Emery-Dreifuss muscular dystrophy. This review discusses the nature of these mutations and the possible effects of lamin mutations on nuclear function.

Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.

Neurological disorders develop in most people infected with human immunodeficiency virus type 1 (HIV-1). However, the underlying mechanisms remain largely unknown. Here we report that binding of HIV-1 transactivator (Tat) protein to low-density lipoprotein receptor-related protein (LRP) promoted efficient uptake of Tat into neurons. LRP-mediated uptake of Tat was followed by translocation to the neuronal nucleus. Furthermore, the binding of Tat to LRP resulted in substantial inhibition of neuronal binding, uptake and degradation of physiological ligands for LRP, including alpha2-macroglobulin, apolipoprotein E4, amyloid precursor protein and amyloid beta-protein. In a model of macaques infected with a chimeric strain of simian-human immunodeficiency virus, increased staining of amyloid precursor protein was associated with Tat expression in the brains of simian-human immunodeficiency virus-infected macaques with encephalitis. These results indicate that HIV-1 Tat may mediate HIV-1-induced neuropathology through a pathway involving disruption of the metabolic balance of LRP ligands and direct activation of neuronal genes.

Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells.

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.

Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42.

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu(2+) and Zn(2+) bind amyloid beta (Abeta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu(2+)-binding sites on synthetic Abeta1-40 and Abeta1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Abeta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu(2+)-binding site on Abeta1-42 (log K(app) = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu(2+) contamination (customarily approximately 0.1 microM). In contrast, Abeta1-40 has much lower affinity for Cu(2+) at this site (estimated log K(app) = 10.3), explaining why this peptide is less self-aggregating. The greater Cu(2+)-binding affinity of Abeta1-42 compared with Abeta1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Abeta precipitation was confirmed by the demonstration that Abeta peptide (10 microM) remained soluble for 5 days only in the presence of high-affinity Cu(2+)-selective chelators.

Interactions between the tetratricopeptide repeat-containing transcription factor TFIIIC131 and its ligand, TFIIIB70. Evidence for a conformational change in the complex.

In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC. A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70. To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction. TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70.TFIIIC.DNA complexes. In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580).TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 +/- 23 nm. CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6-8. CD spectra showed that trifluoroethanol induced significant alpha-helical structure in TFIIIC131-(1-580). A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of alpha-helicity.

Copper catalyzed oxidation of Alzheimer Abeta.

Abeta derived from amyloid plaques of Alzheimer's disease-affected brain contain several oxidative posttranslational modifications. In this study we have characterized the amino acid content of human amyloid-derived Abeta and compared it with that of human synthetic Abeta subjected to metal-catalyzed oxidation. Human amyloid derived Abeta has an increased content of arginine (46%) and glutamate/glutamine residues (28%), but a decreased content of histidine residues (-32%) as compared to the expected amino acid content. Incubation of synthetic human Abeta with Cu(II), but not Fe(III), in the presence of H2O2 similarly induced a decrease in histidine residues (-79%), but also a decrease in tyrosine residues (-28%). Our results suggest that histidine and tyrosine are most vulnerable to metal mediated oxidative attack, consistent with our earlier findings that Cu coordinated via histidine residues is redox competent. Our results suggest that the loss of histidine residues in human amyloid-derived Abeta may be a result of Cu oxidation, and that unidentified post-translational mechanisms operate to modify other amino acids of Abeta in vivo.

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication.

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.

Alzheimer's disease, beta-amyloid protein and zinc.

Alzheimer's disease (AD) is characterized by amyloid deposits within the neocortical parenchyma and the cerebrovasculature. The main component of these predominantly extracellular collections, Abeta, which is normally a soluble component of all biological fluids, is cleaved out of a ubiquitously expressed parent protein, the amyloid protein precursor (APP), one of the type 1 integral membrane glycoproteins. Considerable evidence has indicated that there is zinc dyshomeostasis and abnormal cellular zinc mobilization in AD. We have characterized both APP and Abeta as copper/zinc metalloproteins. Zinc, copper and iron have recently been reported to be concentrated to 0.5 to 1 mmol/L in amyloid plaque. In vitro, rapid Abeta aggregation is mediated by Zn(II), promoted by the alpha-helical structure of Abeta, and is reversible with chelation. In addition, Abeta produces hydrogen peroxide in a Cu(II)/Fe(III)-dependent manner, and the hydrogen peroxide formation is quenched by Zn(II). Moreover, zinc preserves the nontoxic properties of Abeta. Although the zinc-binding proteins apolipoprotein E epsilon4 allele and alpha(2)-macroglobulin have been characterized as two genetic risk factors for AD, zinc exposure as a risk factor for AD has not been rigorously studied. Based on our findings, we envisage that zinc may serve twin roles by both initiating amyloid deposition and then being involved in mechanisms attempting to quench oxidative stress and neurotoxicity derived from the amyloid mass. Hence, it remains debatable whether zinc supplementation is beneficial or deleterious for AD until additional studies clarify the issue.

Repression of ribosome and tRNA synthesis in secretion-defective cells is signaled by a novel branch of the cell integrity pathway.

The transcription of ribosomal DNA, ribosomal protein (RP) genes, and 5S and tRNA genes by RNA polymerases (Pols) I, II, and III, respectively, is rapidly and coordinately repressed upon interruption of the secretory pathway in Saccharomyces cerevisiae. We find that repression of ribosome and tRNA synthesis in secretion-defective cells involves activation of the cell integrity pathway. Transcriptional repression requires the upstream components of this pathway, including the Wsc family of putative plasma membrane sensors and protein kinase C (PKC), but not the downstream Bck1-Mkk1/2-Slt2 mitogen-activated protein kinase cascade. These findings reveal a novel PKC effector pathway that controls more than 85% of nuclear transcription. It is proposed that the coordination of ribosome and tRNA synthesis with cell growth may be achieved, in part, by monitoring the turgor pressure of the cell.

Review: the dynamics of the nuclear lamins during the cell cycle-- relationship between structure and function.

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.

Presenilin 2 interacts with sorcin, a modulator of the ryanodine receptor.

Perturbed Ca(2+) homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with sorcin, a penta-EF-hand Ca(2+)-binding protein that serves as a modulator of the ryanodine receptor intracellular Ca(2+) channel. The association of endogenous sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated sorcin and a subset of the functional PS2 complexes were co-localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the sorcin/PS2 interaction was greatly enhanced by treatment with the Ca(2+) ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca(2+) channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca(2+) homeostasis.

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.

The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.

Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 >> A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.