Cortical regulation of dopamine depletion-induced dendritic spine loss in striatal medium spiny neurons.

The proximate cause of Parkinson's disease is striatal dopamine depletion. Although no overt toxicity to striatal neurons has been reported in Parkinson's disease, one of the consequences of striatal dopamine loss is a decrease in the number of dendritic spines on striatal medium spiny neurons (MSNs). Dendrites of these neurons receive cortical glutamatergic inputs onto the dendritic spine head and dopaminergic inputs from the substantia nigra onto the spine neck. This synaptic arrangement suggests that dopamine gates corticostriatal glutamatergic drive onto spines. Using triple organotypic slice cultures composed of ventral mesencephalon, striatum, and cortex of the neonatal rat, we examined the role of the cortex in dopamine depletion-induced dendritic spine loss in MSNs. The striatal dopamine innervation was lesioned by treatment of the cultures with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) or by removing the mesencephalon. Both MPP+ and mesencephalic ablation decreased MSN dendritic spine density. Analysis of spine morphology revealed that thin spines were preferentially lost after dopamine depletion. Removal of the cortex completely prevented dopamine depletion-induced spine loss. These data indicate that the dendritic remodeling of MSNs seen in parkinsonism occurs secondary to increases in corticostriatal glutamatergic drive, and suggest that modulation of cortical activity may be a useful therapeutic strategy in Parkinson's disease.

CSF lipoproteins and Alzheimer's disease.

Alterations in lipid homeostasis have been suggested to play a role in the pathogenesis of Alzheimer's disease (AD). This hypothesis is supported by the observed changes in lipid content and composition of AD brains when compared to age-matched control brains. The association between the e4 allele of the apolipoprotein E gene and increased risk of AD implicates lipoproteins in the pathogenesis. Lipoproteins are macromolecular particles responsible for lipid trafficking and metabolism. CNS lipoproteins are different from their plasma counter parts. We review the current understanding of the structure and functions of CNS lipoproteins. In addition to mediating lipid trafficking and metabolism, there is increasing evidence that apoE-containing lipoproteins are also involved in dendritic remodeling and synaptogensis and maintenance of the synapto-dendritic complexity during aging. Interestingly, these functions have been shown to be apoE-isoform specific with apoE4 lacking the activities of apoE3 and apoE2. In addition to the association between apoE4 and an increased risk of AD, oxidative stress is believed to play a role in the pathogenesis of this disease. Indeed evidence of lipid peroxidation in cerebral spinal fluid (CSF) lipoproteins from AD patients has been observed. Oxidation of lipoproteins not only eliminates their supportive roles in neurite outgrowth and synaptogenesis, but actually transforms them into neurotoxic agents. The elucidation of the pathways and mechanisms by which apoE-isoform and oxidation affect lipoprotein function in the CNS remains a challenge for scientist in the field of neurodegenerative disease.

Human, but not bovine, oxidized cerebral spinal fluid lipoproteins disrupt neuronal microtubules.

Cerebral spinal fluid (CSF) lipoproteins have become a focus of research since the observation that inheritance of particular alleles of the apolipoprotein E gene affects the risk of Alzheimer's disease (AD). There is evidence of increased lipid peroxidation in CSF lipoproteins from patients with AD, but the biological significance of this observation is not known. A characteristic of the AD brain is a disturbance of the neuronal microtubule organization. We have shown previously that 4-hydroxy-2(E)-nonenal, a major product of lipid peroxidation, causes disruption of neuronal microtubules and therefore tested whether oxidized CSF lipoproteins had the same effect. We exposed Neuro 2A cells to human CSF lipoproteins and analyzed the microtubule organization by immunofluorescence. In vitro oxidized human CSF lipoproteins caused disruption of the microtubule network, while their native (nonoxidized) counterparts did not. Microtubule disruption was observed after short exposures (1 h) and lipoprotein concentrations were present in CSF (20 microg/mL), conditions that did not result in loss of cell viability. Importantly, adult bovine CSF lipoproteins, oxidized under identical conditions, had no effect on the microtubule organization of Neuro 2A cells. Comparison of human and bovine CSF lipoproteins revealed similar oxidation-induced modifications of apolipoproteins E and A-I as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Fatty acid analysis revealed substantially lower amounts of unsaturated fatty acids in bovine CSF lipoproteins, when compared to their human counterparts. Our data therefore indicate that oxidized human CSF lipoproteins are detrimental to neuronal microtubules. This effect is species-specific, since equally oxidized bovine CSF lipoproteins left the neuronal microtubule organization unchanged.

Congeners of N(alpha)-acetyl-L-cysteine but not aminoguanidine act as neuroprotectants from the lipid peroxidation product 4-hydroxy-2-nonenal.

Increased generation of neurotoxic lipid peroxidation products is proposed to contribute to the pathogenesis of Alzheimer's disease (AD). Current antioxidant therapies are directed at limiting propagation of brain lipid peroxidation. Another approach would be to scavenge the reactive aldehyde products of lipid peroxidation. N(alpha)-acetyl-L-cysteine (NAC) and aminoguanidine (AG) react rapidly and irreversibly with 4-hydroxy-2-nonenal (HNE) in vitro, and both have been proposed as potential scavengers of HNE in biological systems. We have compared NAC, AG, and a series of congeners as scavengers of HNE and as neuroprotectants from HNE. Our results showed that while both NAC and AG had comparable chemical reactivity with HNE, only NAC and its congeners were able to block HNE-protein adduct formation in vitro and in neuronal cultures. Moreover, NAC and its congeners, but not AG, effectively protected brain mitochondrial respiration and neuronal microtubule structure from the toxic effects of HNE. We conclude that NAC and its congeners, but not AG, may act as neuroprotectants from HNE.

Cerebrospinal fluid lipoproteins in Alzheimer's disease.

Interest in cerebrospinal fluid (CSF) lipoproteins has been stimulated by the association of certain alleles of the human apolipoprotein E gene (APOE) with an increased risk of Alzheimer's disease (AD), and because apolipoprotein E (apoE) is one of the major apolipoproteins in CSF. CSF lipoproteins (d < 1.210 g/ml fraction) are distinct from their plasma counterparts, and in AD patients CSF may contain novel particles. The protein concentration of CSF lipoproteins is reduced in AD patients. Moreover, the molecular distribution of apoE- and apoAII-containing apolipoproteins in CSF is dictated by APOE. The lipid composition suggests that CSF lipoproteins from AD patients may have undergone increased free radical-mediated damage; experimental data support the possibility that this may occur both before and after lipoprotein assembly. Finally, human CSF lipoproteins oxidized ex vivo are neurotoxic to neuronal cells in culture and disrupt microtubule structure, an activity not observed with oxidized bovine CSF lipoproteins. CSF lipoproteins may represent a means whereby apoE influences the outcome of free radical-mediated damage to brain.

The lipid peroxidation product 4-hydroxynonenal inhibits neurite outgrowth, disrupts neuronal microtubules, and modifies cellular tubulin.

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.

Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer's disease and are neurotoxic when oxidized ex vivo.

Brain regional oxidative damage is thought to be a central mechanism in the pathogenesis of Alzheimer's disease (AD). Recent studies of cerebrospinal fluid (CSF) have suggested that increased lipid peroxidation of CSF and CSF lipoproteins also may occur in AD patients. In the present study, we determined the susceptibility of human CSF to ex vivo lipid peroxidation and tested the hypothesis that oxidized CSF lipoproteins may be neurotoxic. Whole CSF or a CSF lipoprotein fraction (d < 1.210 g/mL) was oxidized with 2,2'-azobis(2-amidino-propane)dihydrochloride (AAPH), a hydrophilic free-radical generator. Kinetics of CSF lipid peroxidation were followed by a standard fluorescence product accumulation assay. Oxidation of AD CSF yielded significantly shorter fluorescent lag times than controls, indicating reduced antioxidant capacity. Electrophoretic mobilities of CSF apolipoproteins were specifically reduced upon oxidation of CSF with AAPH, suggesting that lipoproteins are primary targets of CSF lipid peroxidation. Cultured neuronal cells were exposed to physiological concentrations of isolated CSF lipoproteins oxidized with increasing concentrations of AAPH; the resulting neurotoxicity showed a significant linear AAPH concentration-response relationship. These results suggest that oxidized CSF lipoproteins may contribute to the pathogenesis of neurodegeneration in AD.

Distribution of reducible 4-hydroxynonenal adduct immunoreactivity in Alzheimer disease is associated with APOE genotype.

Two major risk factors for late-onset familial and sporadic Alzheimer disease (AD), a leading cause of dementia worldwide, are increasing age and inheritance of the epsilon4 allele of the apolipoprotein E gene (APOE4). Several isoform-specific effects of apoE have been proposed; however, the mechanisms by which apoE isoforms influence the pathogenesis of AD are unknown. Also associated with AD is increased lipid peroxidation in the regions of the brain most damaged by disease. 4-hydroxynonenal (HNE), the most potent neurotoxic product of lipid peroxidation, is thought to be deleterious to cells through reactions with protein nucleophiles. We tested the hypothesis that accumulation of the most common forms of HNE-protein adducts, borohydride-reducible adducts, is associated with AD and examined whether there was a relationship to APOE. Our results demonstrated that reducible HNE adducts were increased in the hippocampus, entorhinal cortex, and temporal cortex of patients with AD. Furthermore, our data showed that the pattern of reducible HNE adduct accumulation was related to APOE genotype; AD patients homozygous for APOE4 had pyramidal neuron cytoplasmic accumulation of reducible HNE adducts, while AD APOE3 homozygotes had both pyramidal neuron and astrocyte accumulation of reducible HNE adducts. This is in contrast to our previous observations that a distinct HNE protein adduct, the pyrrole adduct, accumulates on neurofibrillary tangles in AD patients. We conclude that APOE genotype influences the cellular distribution of increased reducible HNE adduct accumulation in AD.

Motile areas of leech neurites are rich in microfilaments and two actin-binding proteins: gelsolin and profilin.

Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.

Electrical activity, growth cone motility and the cytoskeleton.

The development of the nervous system takes place in two main steps: first an extensive preliminary network is formed and then it is pruned and trimmed to establish the final form. This refinement is achieved by mechanisms that include cell death, selective growth and loss of neurites and the stabilization and elimination of synapses. The focus of this review is on selective neurite retraction during development, with particular emphasis on the role of electrical activity. In many developing vertebrate and invertebrate neurones, the frequency and duration of ongoing impulse activity determine the final arborizations and the pattern of connections. When impulse traffic is silenced, axons fail to retract branches that had grown to inappropriate destinations in the mammalian visual system, cerebellum and neuromuscular junctions. Similarly, in crustaceans, Drosophila melanogaster and leeches, refinements in axonal morphology during development are influenced by impulse activity. From experiments made in culture, it has been possible to mimic these events and to show a clear link between the density of voltage-activated calcium channels in a neurite and its retraction following stimulation. The distribution of these calcium channels in turn is determined by the substratum with which the neurites are in contact or by the formation of synapses. Several lines of evidence suggest that calcium entry into the growth cone leads to collapse by disruption of actin filaments. One candidate for coupling membrane events to neurite retraction is the microfilament-associated protein gelsolin which, in its calcium-activated state, severs actin filaments. Open questions that remain concern the differential effects of activity on dendrites and axons as well as the mechanisms by which the growth cone integrates information derived from stimuli in the cell and in the extracellular environment.

Disruption of microfilaments in growth cones following depolarization and calcium influx.

Depolarization of leech neurons growing on extracellular matrix extract (ECM) leads to cessation of neurite outgrowth, rounding up of the peripheral regions of the growth cone, loss of filopodia, and neurite retraction. These responses depend on the influx of calcium (Neely, 1993). The aim of the present experiments was to analyze how the cytoskeleton becomes reorganized as growth cones change their morphology. Immunocytochemistry revealed a loss of microfilaments in the tips of neurites growing on ECM after depolarization. Leech neurons cultured on a different substrate, the plant lectin concanavalin A (ConA), continue to grow during and after depolarization (Grumbacher-Reinert and Nicholls, 1992; Neely, 1993). As expected, we did not observe any change in the distribution of microfilaments after depolarization on ConA. Since there is evidence that this lack of response is due to a reduced calcium influx during depolarization of neurons on ConA (Ross et al., 1988), the effect of the calcium ionophore A23187 on the outgrowth of these cells was analyzed. In the absence of depolarization, this ionophore caused cessation of growth cone motility and a loss of microfilaments, while microtubules were not affected. Cytochalasin D, a microfilament-disrupting agent, induced changes in growth cone morphology and neurite retraction similar to those observed after depolarization and calcium influx. Application of phalloidin, a drug that stabilizes microfilaments, inhibited depolarization-induced retraction of neurites on ECM. By contrast, stabilization of microtubules with taxol did not prevent depolarization from inducing changes in growth cone morphology and neurite growth. These experiments show that changes in growth cone morphology and motility of leech neurons induced by depolarization and calcium influx are accompanied by a dramatic change in the organization of microfilaments, but not microtubules.

Substrate-dependent interactions of leech microglial cells and neurons in culture.

The principal aim of the present experiments has been to analyze the properties of microglial cells and their role in nerve regeneration. In the leech, damage to the CNS has been shown to be followed by accumulation of laminin and microglial cells at the site of injury (Masuda-Nakagawa et al., 1990. Proc. R. Soc. Lond. B. 241:201-206; and 1993. Proc. Natl. Acad. Sci. USA 90:4966-4970). Procedures were devised for isolating these small, wandering cells from the CNS of the leech. In culture, they were reliably identified by their sizes, shapes, and phagocytotic activity. Their morphology, motility, and interactions with neurons were influenced by the substrate molecules on which they were plated. On the plant lectin concanavalin A (Con A) microglia had a rounded shape and remained stationary. By contrast on extracts of leech extracellular matrix (ECM) enriched with laminin the cells were mobile and spindle-shaped with long processes. On Con A, neuronal growth cones avoided microglial cells, whereas on ECM extract the presence of a microglial cell did not influence neurite growth. Microglial cells showed immunoreactivity on both substrates when stained with a monoclonal antibody against leech laminin. Together these results suggest that microglial cells are influenced in their properties by molecules in the environment and that they could contribute to neuronal outgrowth at the site of an injury.

Role of substrate and calcium in neurite retraction of leech neurons following depolarization.

The aim of these experiments was to analyze how depolarization influences neurite outgrowth in leech neurons and what role the substrate and Ca2+ play in this response. Neurons in culture were exposed to 60 mM extracellular K+ for 30 min, which induced retraction of a subset of neurites growing on extracellular matrix substrate (ECM), a response comparable to that observed after electrical stimulation (Grumbacher-Reinert and Nicholls, 1992). After normal medium had been restored, the neurites continued to retract for about 1 hr to approximately 80% of the total starting neurite length. Retraction was reversible and regrowth began after the cells had been in normal medium for about 3 hr. Similar depolarization-induced neurite retraction was observed in both Retzius and anterior pagoda cells. Retraction was inhibited by raised extracellular Mg2+, suggesting a mechanism dependent on calcium. The effect of high K+ on neurite outgrowth was also influenced by the substrate on which the cells were plated. Cells plated on concanavalin A (ConA) did not retract but continued to extend processes during exposure to high K+. To understand the different behavior of cells grown on ECM and ConA, the morphology of growth cones was analyzed by scanning electron microscopy. The growth cones of cells grown on ECM and exposed to high K+ revealed retraction of lamellipodial and filopodial structures. On ConA, however, no differences were observed between growth cones of cells exposed to high K+ and those of control cells. These results demonstrate the importance of substrate molecules in the responses of growth cones to depolarization and therefore in the differentiation of neurons.

HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits.

The ultrastructure and biochemical characteristics of HMW-2, the Sertoli cell cytoplasmic dynein isolated from rat testes, were analyzed. Electron microscopic studies revealed a two-headed two-stem structure with dimensions very similar to other dyneins. We found that, like other cytoplasmic dyneins, both heads have an approximately spherical shape with a central cavity. Heavy chain analysis suggested the presence of only one type of heavy chain, a finding that was supported by the simple Michaelis-Menten kinetics displayed by the HMW-2-associated ATPase activity. In addition, dissociation of the HMW-2 complex resulted in a single type of dynein subunit sedimenting at 11.8 S. This fraction contained all the polypeptides present in the undissociated HMW-2. Ultrastructurally the HMW-2 subunits were composed of one globular domain with a tail. The simplest interpretation is that HMW-2 is a dimer of nearly identical subunits, each containing one heavy chain, one 90-kDa intermediate chain, and two light chains.

The Sertoli cell cytoskeleton: a target for toxicant-induced germ cell loss.

Numerous studies in recent years have elucidated fundamental properties of axoplasmic structure, biochemistry, and function. The structural role of the cytoskeletal elements, the orientation of MTs within the axon, the phenomenon of MT-dependent transport, and the identity and direction of movement of two MT motors--kinesin and MAP-1C--have been revealed. For many years to come, researchers investigating the structure and function of the Sertoli cell cytoskeleton will be able to adapt techniques gleaned from work on the axonal cytoskeleton. Innovative thinking will be required to apply these techniques to the special circumstances of the male reproductive system; however, the underlying questions are similar. For example, knowledge of several fundamental properties of transport processes in the Sertoli cell would facilitate the toxicologic evaluation of this system. What is the orientation of MTs within the Sertoli cell cytoplasm? Are the fast-growing (+) ends of all MTs in the Sertoli cell cytoplasm directed toward the lumen? This is an important question because the direction of MT-dependent transport involving known MT motors is dependent upon the MT orientation. Which of the Sertoli cell transport pathways are MT-dependent pathways? What are the MT motors involved in these pathways? Ultrastructural examination following exposure to specific cytoskeleton-disrupting agents has highlighted the importance of AFs, IFs, and MTs in the Sertoli cell. Future research will focus on the nature of those molecules which integrate these cytoskeletal components into a dynamic whole, the regulatory systems which control this integration, and the role of an integrated cytoskeleton in Sertoli cell function and testicular homeostasis. Toxicology will be an active participant in this process of scientific discovery. The selective nervous system and testicular toxicants may be useful tools in revealing similarities in the cytoskeletal organization of these apparently disparate organ systems. By searching for common targets in the testis and nervous system, the mechanisms of action of these agents may be more easily, and more confidently, determined.

Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein).

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.