Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease

Abnormal folding of alpha-synuclein (alpha-syn) is thought to lead to neurodegeneration and the characteristic symptoms of Lewy body disease (LBD). Since previous studies suggest that immunization might be a potential therapy for Alzheimer's disease, we hypothesized that immunization with human (h)alpha-syn might have therapeutic effects in LBD. For this purpose, halpha-syn transgenic (tg) mice were vaccinated with halpha-syn. In mice that produced high relative affinity antibodies, there was decreased accumulation of aggregated halpha-syn in neuronal cell bodies and synapses that was associated with reduced neurodegeneration. Furthermore, antibodies produced by immunized mice recognized abnormal halpha-syn associated with the neuronal membrane and promoted the degradation of halpha-syn aggregates, probably via lysosomal pathways. Similar effects were observed with an exogenously applied FITC-tagged halpha-syn antibody. These results suggest that vaccination is effective in reducing neuronal accumulation of halpha-syn aggregates and that further development of this approach might have a potential role in the treatment of LBD.     

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.     

Molecular and Cellular Mechanisms of Lamina-specific Axon Targeting

The specificity of synaptic connections is directly related to the functional integrity of neural circuits. Long-range axon guidance and topographic mapping mechanisms bring axons into spatial proximity of target cells and thus limit the number of potential synaptic partners. Synaptic specificity is then achieved by extracellular short-range guidance cues and cell-surface recognition cues. Neural activity may enhance the precision and strength of specific circuit connections. Here, we focus on one of the final steps of synaptic matchmaking: the targeting of synaptic layers and the mutual recognition of axons and dendrites within these layers.Perception and behavior are critically dependent on synaptic communication between specific neurons. Understanding how neurons achieve such “synaptic specificity” is therefore one of the most fundamental issues in developmental neuroscience. Langley’s notion of “chemical relations” between synaptically connected neurons (Langley 1892) and Sperry’s “chemoaffinity” hypothesis (Sperry 1963) provided a conceptual framework for the development of precise synaptic connections in the central nervous system. Sperry postulated that molecular interactions between neurons and their extracellular environment (including between and amongst axons and dendrites) ensure that connections form only between “appropriate” synaptic partners (Sperry 1963). This hypothesis has been confirmed by experimental work over the last four decades, most importantly by the identification of molecular cues that provide synaptic specificity (see Sanes and Yamagata 2009 for a recent comprehensive review). However, within this broad framework, a number of alternate mechanisms have been shown or proposed to play roles in specific aspects of such targeting processes. Here, we focus on mechanisms that underlie the formation of synaptic layers, a prominent anatomical feature of the visual system as well as many other areas of the CNS.As reviewed previously (O''Leary 2010), the chemoaffinity principle underlies the developmental process of topographic mapping. Indeed, the precision with which neurons preserve the spatial relationships between the visual world and its representation in the brain is remarkable: Across animals ranging from flies to vertebrates, axons that bear signals from adjacent points in visual space invariably choose adjacent targets in the brain (Braitenberg 1967; Lemke and Reber 2005; Sperry 1963). Thus, position-dependent guidance of axons ensures that a visuotopic map develops. However, position in space is just one attribute of a visual stimulus; others include color, brightness, edge detection, and movement. If position in visual space is encoded by localized activation within a two-dimensional field of neurons, then these other features are encoded by local circuits that act both in series and in parallel and are reiterated many times across the field (Fig. 1). These local circuit modules are often envisioned as “columns” that lie orthogonal to the topographic map, with each column corresponding to a pixel in visual space and each level of the column representing a different, specific visual feature within that pixel, such as brightness, color, etc. (Fig. 1). How these columns acquire their laminated structure represents a developmental challenge of extraordinary scale. Although long-range axon guidance and topographic mapping no doubt contribute to restricting the astronomical number of potential synaptic partners, these mechanisms are clearly not sufficient; additional mechanisms must (and do) exist that act on a local scale to provide an additional level of positional information and cell-type-specific “chemoaffinity.”Open in a separate windowFigure 1.Laminae are a fundamental organizing unit of neural circuits. Each column corresponds to a single topographic position (e.g., location on the retina). Within each column, different cell types (shown type A: blue, and type B: red) respond to different features in the visual world, such as motion or luminance. These pixels are repeated many times over and thus cover all of visual space. A simple rule of “Cell type A connects to Layer A, etc.” ensures that functional segregation is maintained in the connections from the retina to the target (parallel processing). Each pixel P1, P2, and P3 connects to a single column (C1, C2, and C3), establishing serial processing. Within each column, there are local circuits that, too, are layer-specific. Thus, laminae ensure functional specificity of both afferent-target connections and local circuit connections.A prominent principle, which guides the formation of connections between specific cell types and is a characteristic feature of CNS architecture, is the concentration of synapses in small areas. These synapse clusters can take the form of planar layers or spherical glomeruli. Although glomeruli are a specialization that appears most prominent in the olfactory system, layers, or laminae, are an almost ubiquitous feature of central nervous system architecture. Indeed, even crude histological stains reveal that axons and dendrites often accumulate in neuropil (cell-body-free areas). Cell-type-specific or single-cell labeling has shown that, within individual neuropil layers, neurites and synapses are not distributed randomly. Rather, synaptic connections arising between neurons with the same or similar functional properties are localized to particular sublaminae that distinguish synapses with different properties (Fig. 1). The structural underpinnings of this functional principle are provided by mechanisms that ensure the lamina-specific branching of the corresponding neurites. How this enormous precision is achieved is the subject of intense investigations in the Drosophila, zebrafish, chick, and mouse visual systems. We will begin by describing three anatomical regions in these model organisms. Then, we will discuss three broad principles of layer-specific targeting in the visual system, namely cell–cell recognition, guidance by matrix cues, and activity-dependent sorting of axon terminals.     

External micromorphology of the frons plate and its adjacent areas in workers of the oriental hornet

The micromorphology of the frons and the adjacent regions in young workers (1–24 hr of age) of the Oriental hornet Vespa orientalis and some adult worker hornets is described. The young workers still lack globular secretions at the bases of the setae. Such secretions do occur at the bases of the setae in the adult workers and are composed mainly of the elements Si, Al, S, Mg, Ca, Cl, and Fe. Contiguous with each of the ocelli is a gland that also secretes minerals. Young workers usually have a relatively large concentration of Ca in the glands behind the paired ocelli. The gland associated with the median anterior ocellus opens by an elongated sutura coronalis in the frons and contains various elements, predominantly Si, but also Ca. The setae (hairs) on the frons are arranged in concentric circles around each of the ocelli; they are long in the upper part of the frons but shorten gradually toward the tip of the frons. The tip lacks hair, but the cuticle bears elevated scales that project as cuticular protuberences. It appears that the ocelli and their associated glands, and the entire frons plate with its hairs and glands at the bases of the hairs in this region, comprise an equilibrium “organ” that senses changes in gravity. © 1992 Wiley-Liss, Inc.     

Differential effects of UCHL1 modulation on alpha-synuclein in PD-like models of alpha-synucleinopathy

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn) within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg) and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy) in control cells, in cells over expressing a-syn UCH-L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/aggregated a-syn species in the PD brain.     

Ambiguities in results obtained with 2D gel replicon mapping techniques.

Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed to map replication origins in eukaryotic plasmids and chromosomal DNA (1-11). The neutral/neutral technique, which requires less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment. We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used, neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order to minimize possible misinterpretations.     

Effects of temperature and sink activity on the transport of 14C-labelled indol-3yl-acetic acid in the intact pea plant (Pisum sativum L.)

The velocity and intensity of basipetal transport of ¹⁴C-labelled indol-3yl-acetic acid (IAA) applied to the apical bud of the intact pea plant were influenced by the temperature to which the stem was exposed and were not influenced by changes in the temperature of the root system when this was controlled independently between 5°C and 35°C. The velocity of transport increased steadily with temperature to a maximum in excess of 35°C and then fell sharply with further increase in temperature. The Q₁₀ for velocity, determined from Arrhenius plots, was low (ca. 1.3). Transport intensity increased to a maximum at about 25°C (Q₁₀=2.2) and then declined gradually with further increase in temperature. It is suggested that transport velocity and transport intensity are controlled independently.The characteristics of auxin transport through the stem were not affected by removal of the root system, or by the withdrawl of root aeration. Labelled IAA did not pass a region of the stem cooled to about 1.0°C, or through a narrow zone of stem tissue killed by heat treatment. In the latter case the heat treatment was shown not to interfere with the upward transport of water in the xylem. Labelled IAA continued to move into, and to accumulate in, the tissues immediately above a cooled or heat-killed region of the stem. It was concluded that the long-distance basipetal transport of auxin through the stem of the intact plant is driven by the transporting cells themselves and is independent of the activity of sinks for the transported auxin.The fronts of the observed tracer profiles in the stem were closely fitted by error function diffusion analogue curves. However, diffusion of IAA alone could not account for the observed characteristics of the transport and it is suggested that the curvilinear fronts of the profiles resulted from a diffusive mixing of exogenous IAA (or IAA-carrier complexes) with endogenous IAA already in the transport pathway.Abbreviations IAA indol-3yl-acetic acid - IAAsp indol-3yl-acetyl aspartic acid - CFM methyl 2-chloro-9-hydroxyfluorene-9-carboxylate (morphactin) - TIBA 2,3,5-triiodobenzoic acid - ABA abscisic acid     

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G₁) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.In response to low levels of the DNA-alkylating agent methyl methanesulfonate (MMS), wild-type yeast cells slow their progression through S phase, while cells lacking the appropriate upstream checkpoint kinase (Mec1 in the budding yeast Saccharomyces cerevisiae; Rad3 in the fission yeast Schizosaccharomyces pombe) or the appropriate downstream checkpoint kinase (Rad53 in budding yeast, Cds1 in fission yeast) fail to do so. Other DNA-damaging agents also cause a checkpoint-dependent slowing of S phase, in vertebrates as well as in yeasts. This slowing of S phase in response to DNA damage is sometimes called the “intra-S-phase” checkpoint (3, 6, 22, 23, 26, 28, 36, 37, 45, 53). Here we shall refer to it as the “S-phase damage” checkpoint.Prior to this report, the downstream portions of the checkpoint pathway(s) that slow S phase in response to DNA damage in fission yeast were unclear. However, the upstream portions of these pathways in fission yeast and other organisms have been partially elucidated, and downstream mechanisms in other organisms have been partially clarified. In all studied systems, upon detection of DNA damage in S phase, checkpoint proteins initiate a phosphorylation cascade that ultimately leads to slowing of replication. Upstream signaling in these systems involves the activation of one or more of the phosphatidylinositol-3-kinase-like protein kinases (PIK kinases; ATR and/or ATM in humans, Mec1 and/or Tel1 in budding yeast, and Rad3 in fission yeast). The activated PIK kinases then phosphorylate several proteins, including certain Ser/Thr kinases (Chk1 and/or Chk2 in humans, Rad53 in budding yeast, and Cds1 in fission yeast). These kinases, in turn, phosphorylate other substrates that, directly or indirectly, mediate the slowing of S phase (reviewed in reference 3).In budding yeast, two different mechanisms were shown to slow S phase upon DNA damage by MMS. Of these, one mechanism, inhibition of late-firing origins, depended on the Mec1-Rad53 checkpoint pathway (45, 53), while the other mechanism, inhibition of replication forks, appeared to be a direct consequence of DNA damage rather than a result of checkpoint activation (53). Tercero and Diffley (53) found that, in MMS-treated cells with mutations in the RAD53 gene, unregulated origin firing compensated for checkpoint-independent replication fork slowing, thus permitting a relatively normal overall rate of DNA synthesis. The mechanism by which the Rad53 protein modulates late origin activity is not yet clear, but one possibility is inhibition (by Rad53-catalyzed phosphorylation) of Dbf4, the regulatory subunit of the Cdc7-Dbf4 kinase, which is essential for initiation of replication (7, 8, 14, 55).In vertebrates, at least three different pathways have been shown to contribute to the slowing of S phase after DNA damage. In some cases checkpoint-mediated phosphorylation of Dbf4 inhibits progression through S phase by downregulating origin firing (7, 14), as may take place in budding yeast. In other cases, checkpoint-mediated phosphorylation leads to inhibition and destruction of the protein phosphatase Cdc25A, which is an activator of Cdk2. Cdk2 is the S-phase-specific cyclin-dependent kinase. Cdk2 activity is crucial for initiation of DNA replication and is modulated by inhibitory phosphorylation at Tyr-15. Cdc25A activates Cdk2 by dephosphorylating Tyr-15. Thus, when Cdc25A is phosphorylated by checkpoint kinases after DNA damage and subsequently destroyed, Cdk2 can no longer promote initiation of DNA replication (9, 27). The third mechanism by which vertebrate cells can slow progression through S phase is inhibition of replication fork movement. In vertebrate cells, slowing of replication forks in response to DNA damage is frequently checkpoint dependent; in contrast, in budding yeast, such slowing appeared to be checkpoint independent. In the tested cases, fork slowing has proved to be dependent on the PIK kinase ATR (homologous to budding yeast Mec1 and fission yeast Rad3) and on the Ser/Thr kinase Chk1 (a functional analogue of budding yeast''s Rad53 and fission yeast''s Cds1). In each of these cases, the checkpoint response to DNA damage led to inhibition of origin firing as well as to inhibition of replication fork movement (42, 44, 54). The precise mechanism leading to slowing of replication fork movement has not been fully worked out, but the mechanism appears to involve interactions between Chk1 and the proteins Tim and Tipin (54), whose yeast homologues (Swi1 and Swi3 in fission yeast, Tof1 and Csm3 in budding yeast) form a “replication fork protection complex” that is associated with replication forks (19, 33).Although it is clear that slowing of S phase in response to MMS-induced DNA damage in fission yeast requires both the Rad3 and Cds1 kinases, the pathways operating downstream of Cds1 have been uncertain. We obtained results indicating that Cdc25, which was already known to be a target of Cds1 in hydroxyurea (HU)-treated cells, is also a target of Cds1 in MMS-treated cells, because both overproduction of Cdc25 and conversion of Tyr-15 on Cdc2 (the major cyclin-dependent kinase of fission yeast; also known as Cdk1) to a nonphosphorylatable residue (Cdc2-Y15F; this mutation rendered Cdc2 constitutively active) were sufficient to prevent MMS-induced slowing of S phase (23). We concluded that, in fission yeast, the Rad3→Cds1⊣Cdc25→Cdc2 pathway forms a checkpoint signaling module very similar to the corresponding one of vertebrates. However, Kommajosyula and Rhind were not able to repeat our observations regarding the roles of Cdc25 and Cdc2 (22), so the relevance of Cdc25 and Cdc2 to checkpoint-induced slowing of S phase in fission yeast has remained uncertain until now. In addition, whether S phase in MMS-treated fission yeast cells is slowed by inhibition of origin firing, by reduction in rate of fork movement, or by a combination of these has been equally unclear.In order to resolve these issues, we initiated the series of experiments reported in this paper. To measure the rate of progression through S phase, we followed S phase by flow cytometry and by two-dimensional (2D) gel electrophoresis in cells released from a G₁ block (achieved by incubating cells bearing a cdc10 temperature-sensitive mutation at the restrictive temperature, then releasing to the permissive temperature [21, 23]). We found that, in MMS-treated, checkpoint-competent cells, the firing of early origins near centromeres was partially delayed but that the firing of late origins near telomeres was unaffected. Furthermore, the lifetimes of replication intermediates (RIs) were prolonged, consistent with slowing of replication forks. These effects were completely abrogated both in cells lacking the Cds1 kinase and in cells overproducing the Cdc25 phosphatase, showing that these effects were checkpoint dependent and that the relevant checkpoint pathway probably involved inhibition of Cdc25.