Aggregates In Neuronal Cells Research Articles

Cytoskeleton/Endoplasmic Reticulum Collapse Induced by Prostaglandin J2 Parallels Centrosomal Deposition of Ubiquitinated Protein Aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.

Polyglutamine-Mediated Aggregation and Cell Death

The expansion of CAG repeats is the genetic defect underlying eight neurodegenerative diseases. A common feature of these disorders is the presence of intracellular aggregates in neuronal cells. It is still unclear the significance of these cellular inclusions in the neurodegenerative process, since cell death without aggregate formation has been reported. We have constructed a synthetic fusion protein containing 17 or 43 CAG repeats and the green fluorescent protein that recapitulates the features of CAG-expanded alleles. Expression of 43, but not 17 CAG repeats results in formation of nuclear aggregates in human neuroblastoma cells. Moreover, the normal allele (17 CAG) is sequestered in the inclusion bodies. The presence of nuclear inclusions tightly correlates with apoptosis in cells expressing the protein encoding 43 CAG repeats. Cells harboring nuclear aggregates stop proliferation and undergo apoptosis. Moreover, the inhibition of protein degradation pathway increases intracellular aggregates and cell death. These data indicate that intranuclear aggregates induce apoptosis and suggest that the degradation of unfolded proteins improves cell survival.

The Length of Polyglutamine Tract, Its Level of Expression, the Rate of Degradation, and the Transglutaminase Activity Influence the Formation of Intracellular Aggregates

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.

Cell proliferation, migration and CAM-dependent neurite outgrowth as developmental in vitro endpoints for screening teratogenic potential: Application to valproic acid and related analogues of varying potency

The in vivo teratogenic potential of valproic acid (VPA) and related teratogenic and non-teratogenic analogues has been correlated with their effects on specific in vitro endpoints of cell proliferation, migration and CAM-dependent neurite outgrowth, as these events are common to crucial epochs of development. The (±)-2- n-propyl-4-pentynoic acid [(±)-4-yn-VPA] and S-2- n-propyl-4-pentynoic acid [ S(-)-4-yn-VPA] analogues increased the incidence of neural tube defects in mouse embryos exposed to a single dose, whereas the E-2- n-propyl-2-pentenoic acid ( E-2-en-VPA) analogue and R-2- n-propyl-4-pentynoic acid [ R( + )-4-yn-VPA] enantiomer were without effect. VPA and related analogues tested exerted comparable G1 phase antiproliferative effects in C6 glioma and limb bud cells in a dose range of 0–3 mM; however, their relative potency did not correlate with in vivo teratogenicity. In contrast, VPA and all teratogenic analogues, at 3 mM, inhibited neuronal cell aggregation and limb bud chondrocyte differentiation in a manner that exhibited a reasonable correlation with their in vivo teratogenicity. The teratogenic S(−)-4-yn-VPA and non-teratogenic R( + )-4-yn-VPA enantiomers exhibited a differential inhibition of primary neurone outgrowth of neuntes stimulated by cell adhesion molecules [L1 and N-cadherin (NCAD)]. Half-maximal inhibition was observed at approximately 150 μM for the teratogenic S(−)-4-yn-VPA enantiomer, but not the non-teratogenic R( + )-4-yn-VPA form. These results suggest that in vitro perturbations of differentiation are likely to provide the greatest discriminatory power for in vivo teratogenicity.

Soluble material from Reissner's fiber displays anti-aggregative activity in primary cultures of chick cortical neurons

The subcommissural organ (SCO), which belongs to the circumventricular organs, is a specialized ependymal structure of the brain that secretes glycoproteins into the cerebrospinal fluid (CSF) which condense to form a thread-like structure, Reissner's fiber (RF). The effects of soluble material released by RF were examined in primary cultures of dissociated cortical cells from embryonic (day 8) chick brain. Under serum-free conditions, the presence in the cultures of soluble RF material markedly impaired neuronal cell aggregation. This effect was completely blocked by addition into the culture medium of specific antibodies raised against bovine RF. The anti-aggregative effect of soluble RF material is observed on poly- l -lysine as well as on diffrrent extracellular matrix proteins including collagen and laminin, but was less effective on fibronectin. The continuous exposure of the cultures to soluble RF material for 7 days significantly decreased choline acetyltransferase activity. On the other hand, soluble RF material did not appear to have mitogenic activity on neuronal cultures. Modulation of cell-cell interactions by SCO/RF glycoproteins sterngthens the hypothesis of the involvement of RF in developmental events of the central nervous system.

Soluble material from Reissner's fiber displays anti-aggregative activity in primary cultures of chick cortical neurons

The subcommissural organ (SCO), which belongs to the circumventricular organs, is a specialized ependymal structure of the brain that secretes glycoproteins into the cerebrospinal fluid (CSF) which condense to form a thread-like structure, Reissner's fiber (RF). The effects of soluble material released by RF were examined in primary cultures of dissociated cortical cells from embryonic (day 8) chick brain. Under serum-free conditions, the presence in the cultures of soluble RF material markedly impaired neuronal cell aggregation. This effect was completely blocked by addition into the culture medium of specific antibodies raised against bovine RF. The anti-aggregative effect of soluble RF material is observed on poly- l-lysine as well as on diffrrent extracellular matrix proteins including collagen and laminin, but was less effective on fibronectin. The continuous exposure of the cultures to soluble RF material for 7 days significantly decreased choline acetyltransferase activity. On the other hand, soluble RF material did not appear to have mitogenic activity on neuronal cultures. Modulation of cell-cell interactions by SCO/RF glycoproteins sterngthens the hypothesis of the involvement of RF in developmental events of the central nervous system.

Homophilic adhesion mediated by the neural cell adhesion molecule involves multiple immunoglobulin domains.

The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig I-V) and the two fibronectin type III repeats (FnIII 1-2) in the extracellular region of N-CAM are involved in this process have led to ambiguous results. We have generated soluble recombinant proteins corresponding to each of the individual immunoglobulin domains and the combined FnIII 1-2 and prepared polyclonal antibodies specific for each. The purified proteins and antibodies were used in aggregation experiments with fluorescent microspheres and chicken embryo brain cells to determine possible contributions of each domain to homophilic adhesion. The recombinant domains were tested for their ability to bind to purified native N-CAM, to bind to each other, and to inhibit the aggregation of N-CAM on microspheres and the aggregation of neuronal cells. Each of the immunoglobulin domains bound to N-CAM, and in solution all of the immunoglobulin domains inhibited the aggregation of N-CAM-coated microspheres. Soluble Ig II, Ig III, and Ig IV inhibited neuronal aggregation; antibodies against whole N-CAM, the Ig III domain, and the Ig I domain all strongly inhibited neuronal aggregation, as well as the aggregation of N-CAM-coated microspheres. Of all the domains, the third immunoglobulin domain alone demonstrated the ability to self-aggregate, whereas Ig I bound to Ig V and Ig II bound to Ig IV. The combined FnIII 1-2 exhibited a slight ability to self-aggregate but did not bind to any of the immunoglobulin-like domains. These results suggest that N-CAM-N-CAM binding involves all five immunoglobulin domains and prompt the hypothesis that in homophilic cell-cell binding mediated by N-CAM these domains may interact pairwise in an antiparallel orientation.

Changes of endogenous ganglioside composition in mouse cerebrum primary cultures following long-term exposure to phorbol ester

Changes in endogenous gangliosides caused by phorbol 12-myristate 13-acetate (PMA, 162 nM) were examined using mouse cerebrum primary cultures. The total ganglioside content was significantly decreased by 25% and 40% in PMA-treated cultures compared to control cultures on days 1 and 8, respectively. In addition, changes in the pattern of ganglioside composition were also observed in which the percentage of GM1 and GD3 in total gangliosides was significantly increased and, in contrast, the percentage of GD1a and GT1b was reduced. Treatment of neurons with PMA induced the change of morphology. These results suggest that the decrease in the total ganglioside content and changes in ganglioside composition produced by long-term exposure to PMA are related to the appearance of neuronal cell aggregation and neurite fasciculation.

Thrombin indirectly affects cholinergic cell expression in primary septal cell cultures in a manner distinct from nerve growth factor

The effects of thrombin were examined in primary cultures of dissociated medial septal cells from fetal (embryonic day 17) rat brains. Seven days of continuous exposure of these cultures to thrombin produced a dose-dependent increase in the activity of the enzyme choline acetyltransferase (EC 2.3.1.6) and no change in the number of acetylcholinesterase (EC 3.1.1.7)-positive cells. Maximal induction of choline acetyltransferase activity occurred around 1–2 nM thrombin and was first detected after five days of treatment. In addition, thrombin promoted neuronal cell aggregation, proliferation of the astroglia, and changes in astroglial cell morphology. Neuronal aggregation was first noted after 24 h of treatment, while the proliferative response of the astroglia was first apparent after four days of treatment, slightly prior to the increase in choline acetyltransferase enzymatic activity. In order to see if the induction of the enzyme choline acetyltransferase was dependent upon the astroglial cell response, we included the anti-mitotic agent 5-fluoro-2'-deoxyuridine, to find that astrocyte proliferation, as well as thrombin-induced increase in choline acetyltransferase, were both abolished. In contrast, the aggregation of neurons was not affected. Finally, thrombin-induced changes in choline acetyltransferase could not be antagonized by immunoneutralizing anti-nerve growth factor antibodies and when thrombin was added simultaneously with 100 ng/ml 2.5-S nerve growth factor, the increase in choline acetyltransferase activity was additive. In conclusion, it appears that thrombin affects cholinergic septal neurons indirectly via the responsive astrocytes in a manner distinct from nerve growth factor.

Effects of chlorine dioxide on the developing rat brain

Male and female Long-Evans rat pups, exposed to an oral dose of 14 mg chlorine dioxide (CIO2)/kg/d (postnatal d 1-20), were examined for effects on brain development and for changes in thyroid activity. Body weight reductions were observed on postnatal (pn) d 11, 21, and 35. Forebrain weight and protein content were decreased on pnd 21 and 35, as were the DNA content on d 35 and the number of dendritic spines on cerebral cortical pyramidal cells, a marker for synapse formation. Otherwise, cell proliferation in the forebrain, cerebellum, and olfactory bulbs was normal, as were migration and aggregation of neuronal cells in three areas of the cerebral cortex. Histopathology of the forebrain, cerebellum, and brainstem showed no gross lesions, loss of myelin, or change in the cells staining positive for Nissl substance. Serum T3 and T4 levels, as well as hepatic mitochondrial alpha-glycerophosphate dehydrogenase activity, were unchanged by CIO2 treatment. The results indicated that CIO2 may have central neurotoxic potential. No underlying antithyroid activity was evident.

Polypeptide components and binding functions of neuron-glia cell adhesion molecules.

Neuron-glia cell adhesion molecule (Ng-CAM) has previously been shown to be present exclusively on neurons and to mediate adhesion between neuronal membranes and glial cells. In the present study, its chain structure, binding functions, and relation to N-CAM (the other known CAM on neurons) were investigated further. Three polypeptide components of chicken Ng-CAM (Mr 200,000, 135,000, and 80,000) have been isolated. By using specific antisera against each component, the Mr 135,000 and Mr 80,000 components were found to cross-react antigenically with the Mr 200,000 component but not with each other. The conclusion that the Mr 135,000 and 80,000 components are structurally related to different regions of the Mr 200,000 component was further supported by the finding that 32P could be incorporated in vitro into the Mr 200,000 and 80,000 components but not into the Mr 135,000 component. Ng-CAM appears to be involved in both neuron-glia adhesion and neuron-neuron adhesion by distinguishable mechanisms that appear to involve different sites or conformations of the molecule. Polyclonal antibodies and a monoclonal antibody against Ng-CAM both inhibited adhesion between glia and neurons derived from brain, cerebellum, and retina. In contrast, antibodies against N-CAM (which inhibit neuron-neuron adhesion) did not inhibit neuron-glia adhesion. These findings confirm the proposed function of Ng-CAM in neuron-glia adhesion. In addition, however, Ng-CAM was found to be involved directly or indirectly in neuron-neuron adhesion. Non-cross-reactive polyclonal anti-Ng-CAM and anti-N-CAM antibodies each inhibited the aggregation of neurons from whole brain and cerebellum and the inhibition was greater when both antibodies were present together. In contrast, monoclonal anti-Ng-CAM antibodies were found that inhibited neuron-glia adhesion but did not inhibit neuronal cell aggregation. The amount of Ng-CAM expressed on neurons was not directly predictive of the effect of anti-Ng-CAM antibodies on their homotypic aggregation. Although Ng-CAM and N-CAM can be expressed simultaneously on individual neurons, the ratio of N-CAM to Ng-CAM ranged from 1.5 for cerebellar cells to 10.0 for retinal cells. While, as expected, retinal cell aggregation was inhibitable only by anti-N-CAM, cerebellar cells, which expressed at least as much Ng-CAM as brain cells, showed significantly less inhibition by anti-Ng-CAM antibodies. These findings raise the possibility that Ng-CAM may actually interact with N-CAM to yield non-linear effects. That Ng-CAM and N-CAM may function differently in vivo was suggested by their distribution in sections of brain regions. Within the cerebellum, for example, immunofluorescent anti-N-CAM staining was relatively uniform in all layers; in contrast anti-Ng-CAM staining was absent on dividing external granule cells and was present in greatest abundance on processes of post-mitotic migratory cells in the molecular layer. These observations are consistent with the hypothesis that Ng-CAM mediates neuron-glia adhesion and is thereby also involved in neuronal migration along radial glial cells.

Influence of biologically active substances isolated from Galleria mellonella on neurons of Lymnaea stagnalis in culture

1. A procedure for isolating biologically active substances from Galleria mellonella using a culture of isolated giant neurons of mollusc Lymnaea stagnalis as a test-system is described. 2. Fractions capable of activating neurites and inhibiting aggregation of neuronal cells within a range of concentrations from 1 to 30 μg/ml were isolated. 3. The fractions obtained have in their chemical composition about 10.5% N, also contain P and S. They have a carbohydrate component.

Two antigenically related neuronal cell adhesion molecules of different specificities mediate neuron-neuron and neuron-glia adhesion.

Previous studies in this laboratory have led to the identification of the neural cell adhesion molecule, N-CAM, a homophilic ligand that mediates adhesion between neurons as well as between neurons and striated muscle precursors. By means of a similar immunological approach but with different assays, we have now identified a cell adhesion molecule on neurons (Ng-CAM) that mediates the heterotypic adhesion between neuronal membranes and glial cells. In this paper, we compare certain aspects of the structure and function of Ng-CAM and embryonic N-CAM from the chicken. Ng-CAM was localized by specific antibodies on neurons but not on glia, and double-staining methods showed that individual neurons contained both Ng-CAM and N-CAM. Embryonic Ng-CAM migrates primarily as a single component of Mr 135,000; its apparent Mr shifted to 127,000 after neuraminidase treatment. In contrast, the embryonic form of N-CAM migrates on NaDodSO4/polyacrylamide gels in the apparent Mr range of 200,000-250,000; after neuraminidase treatment, N-CAM migrates as two components of Mr 170,000 and Mr 140,000. Although both Ng-CAM and N-CAM have calcium-independent binding mechanisms, immunologically based cell adhesion assays suggested that they have different specificities in mediating cell adhesion. Whereas 0.25 micrograms of Ng-CAM partially neutralized the ability of 0.5 mg of polyspecific antineural Fab' fragments to inhibit the heterotypic binding of neuronal membrane vesicles to glial cells and larger amounts of Ng-CAM completely neutralized this inhibition, 20 micrograms of N-CAM had no neutralization activity in this assay. Reciprocally, 0.25 micrograms of N-CAM partially neutralized the ability of 0.5 mg of the same Fab' fragments to inhibit the direct homotypic aggregation of neuronal cells, but 20 micrograms of Ng-CAM had no detectable activity. Although peptide maps of the two cell adhesion molecules differed considerably and despite the differences in binding specificity of these molecules, two independently derived monoclonal antibodies were found to crossreact with both Ng-CAM and N-CAM. Therefore, these different neuronal cell adhesion molecules with distinct binding specificities share at least one antigenic determinant, raising the possibility that they arose from a common evolutionary precursor.