Effects of sphingosine, staurosporine, and phorbol ester on neurites of rat sympathetic neurons growing in compartmented cultures

Local application of sphingosine (1-10 microM), an inhibitor of protein kinase C, to NGF-supplied, distal neurites of rat sympathetic neurons in compartmented cultures caused their retraction and/or degeneration within 24 hr. This effect was specific for distal neurites because sphingosine (even at 100 microM) applied to cell bodies and/or proximal neurites did not destroy these regions of the cells, and their distal neurites continued to elongate. However, effects of other agents suggest that the retraction/degeneration observed in distal neurites directly exposed to sphingosine is not mediated by inhibition of protein kinase C: application of staurosporine, another inhibitor of protein kinase C, to distal neurites did not cause retraction or degeneration; treatment of neurons for 24 or more hours with 2 microM phorbol 12-myristate 13-acetate (PMA), used to downregulate protein kinase C activity, slowed neurite extension about 50%, but did not cause degeneration; and neurons pretreated with PMA still displayed retraction/degeneration of neurites when they were subsequently exposed to sphingosine. Also, replacement of NGF supplied to distal neurites with anti-NGF IgG did not cause retraction/degeneration of neurites within 1 d, suggesting that the effect of sphingosine did not arise by interference with the action of NGF. The specificity of the sphingosine-induced retraction/degeneration for distal neurites suggests that this effect operates via specific mechanisms in distal neurites that can trigger their retraction/degeneration. Such mechanisms could play important roles in nerve growth inhibition, nerve fiber retraction, and degeneration that occur normally in the nervous system and in response to injury and disease. Also, the ability of neurites to grow in the presence of PMA suggests that neurite growth is not dependent upon the activity of protein kinase C. However, the reduced rate of neurite extension in the presence of PMA suggests that chronic PMA treatment may affect mechanism(s) that can modulate neurite growth.     

Neurochemical diversity of dystrophic neurites in the early and late stages of Alzheimer's disease

We examined the neurochemical and morphological diversity of abnormal neurites associated with beta-amyloid plaque formation in the early and late stages of Alzheimer's disease. Preclinical Alzheimer's disease was characterised by the presence of abnormal neurites containing either neurofilament or chromogranin A immunoreactivity. All clustered dystrophic neurites in these cases were associated with beta-amyloid plaques. Neurofilament immunoreactive dystrophic neurites in preclinical Alzheimer's disease could be further subclassified into bulb- and ring-like structures, and these abnormal neurites contained both phosphorylated and dephosphorylated neurofilament epitopes. Dystrophic neurites in Alzheimer's disease could be subdivided into predominantly neurofilament, tau, or chromogranin A immunolabeled forms. Some neurofilament immunoreactive neurites had a core region labeled for tau. The neurofilaments of the dystrophic neurites in Alzheimer's disease had the same complement of phosphorylation- and dephosphorylation-dependent epitopes as observed in preclinical cases. Therefore, an abnormal accumulation of variably phosphorylated neurofilaments represent the earliest cytoskeletal alteration associated with dystrophic neurite formation. Furthermore, these data indicate that dystrophic neurites may "mature" through neurofilament-abundant forms to the neurites containing the profoundly altered filaments labeled for tau. The precise morphological and neurochemical changes associated with dystrophic neurite formation suggests that beta-amyloid plaques are causing physical damage to surrounding axons. The resultant axonal sprouting and profound cytoskeletal alterations would follow the chronic stimulation of the stereotypical reaction to such physical trauma.     

Activation of c-Jun N-terminal kinase is required for neurite outgrowth of dopaminergic neuronal cells

Recent studies indicate that activation of stress-activated protein kinases may be implicated in a broad range of biological activities including differentiation. To directly examine whether stress-activated protein kinases are involved in neuronal differentiation, we utilized retinoic acid-induced and spontaneous models of neurite outgrowth in dopaminergic neurons. Here, we show that retinoic acid-induced neurite outgrowth in MN9D dopaminergic neuronal cells was accompanied by activation of c-Jun N-terminal kinase but not p38. Consequently, cotreatment with a specific inhibitor of c-Jun N-terminal kinase or overexpression of c-Jun N-terminal kinase-binding domain of c-Jun N-terminal kinase-interacting protein-1 blocked retinoic acid-induced neurite outgrowth. In primary cultures of dopaminergic neurons, the extent of neurite outgrowth increased spontaneously in a time-dependent manner. When these cultures were treated with a specific inhibitor of c-Jun N-terminal kinase, the total extent of neurites, the primary neurite length and the number of neurites per cell were suppressed significantly. Thus, our data indicate that the c-Jun N-terminal kinase signal seems to play an important role during morphological differentiation in cultured dopaminergic neurons.     

Neurotrophin-3 increases neurite outgrowth and apoptosis in explants of the chicken neural plate

This study examined the effect of neurotrophin-3 (NT-3) on neurite outgrowth and apoptosis of chicken neural plate explants prior to neural tube formation. In situ hybridization revealed that mRNA for the full-length (catalytic) NT-3 receptor, TrkC, was present in, and limited to, the neural plate (including the neural folds) coincident with its formation. Neural plate explants were maintained in vitro on a collagen gel under serum-free conditions in the presence or absence of exogenous NT-3 and/or an antibody to NT-3. In the absence of exogenous NT-3, explants exhibited neurite outgrowth after several days in vitro; apoptotic cells were also seen after 2 days in vitro. This does not appear to be due to endogenous NT-3, since the total number of neurites or apoptotic cells was unchanged if explants were exposed to an NT-3 antibody for the entire culture period. In the presence of exogenous NT-3, neural plate explants exhibited a dose-dependent statistically significant increase in the total number of neurites as compared to explants maintained under control conditions, as well as a statistically significant increase in apoptosis. These NT-3 effects were blocked by an NT-3 antibody. In contrast, NT-3 had no effect on the length of neurites. These findings suggest that NT-3 may play a role during early neural development in vivo.     

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.     

N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) suppresses neuritic degeneration caused by different experimental paradigms including in vitro Wallerian degeneration

Accumulating evidence indicates that neurite degeneration occurs via a distinct mechanism from somal death programs. We have previously shown that neuritic ATP level in sympathetic neurons decreases, whereas somal ATP level remains unaltered during degeneration caused by the microtubule-disrupting agent, vinblastine. Moreover, caspase activation occurs only in cell soma, supporting the view of somal apoptosis and neuritic necrosis. Therefore, the ATP level of neurites is crucial for their degeneration; it appears to correlate with membrane blebbing or beading which precedes late whole fragmentation of neurites under these conditions. Based on these metabolic and morphological criteria, we have tested the effects of various protease inhibitors on vinblastine-induced neurite degeneration in superior cervical ganglia from neonatal mice. Among agents tested, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), the trypsin-like serine protease inhibitor, but not N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the chymotrypsin-like serine protease inhibitor, protected sympathetic neurites from beading formation, neuritic fragmentation and a decrease in their ATP level. The commitment time for the saving effect of TLCK occurred around 7 h following treatment with vinblastine, at a time point after microtubule degradation (2 h) and before massive beading formation (later than 12 h). Moreover, TLCK was also capable of suppressing Wallerian degeneration in culture and neuritic degeneration following withdrawal of NGF in a dose-dependent manner. These results strongly suggest that TLCK intervenes in a common step in the cascade of neuritic degeneration caused by these different experimental paradigms and provides a helpful clue for identifying such a molecular step.     

Newly synthesized catalytic and regulatory components of adenylate cyclase are expressed in neurites of cultured sympathetic neurons

Forskolin- and guanine nucleotide-stimulated adenylate cyclase activities were measured in microdissected sections of neurites from small explants and in dispersed cell cultures of sympathetic ganglion neurons to determine whether a competent system for regulated formation of cAMP, consisting of both catalytic units of adenylate cyclase and regulatory GTP binding proteins, is synthesized during neurite outgrowth and where it is distributed in the neuron. An increase in both guanine nucleotide- and forskolin-dependent activity of adenylate cyclase occurred concomitantly with neurite outgrowth and was directly proportional to neurite length. Separate analysis of adenylate cyclase activity in explant cell bodies or neurites showed that the increased activity was localized entirely in the neurites, while activity in the cell bodies remained virtually constant during growth. Concentric sections of neurites of approximately 500 microns width, which contained similar volumes of neurites as determined with the indicator BCECF (Rink et al., 1982), produced similar levels of cAMP, indicating an even distribution of adenylate cyclase in the neurites. Cell bodies, when stimulated by GTP gamma S, produced 236 +/- 46 attomol cAMP/min (30 degrees C)/cell body and an additional 52.6 +/- 20 attomol cAMP/min (30 degrees C)/neuron were produced with each day of neurite growth (approximately 400 microns). Assuming a turnover number of 2000 min-1, cell bodies and neurites were calculated to contain similar densities of catalytic unit molecules on their surface (9-28 molecules/micron 2). An abundant GTP binding protein, detected by ADP-ribosylation with pertussis toxin, was also widely distributed in the neuron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of patterned electrical activity on neurite outgrowth from mouse sensory neurons

A noninvasive method of electric stimulation was used in cell culture preparations to determine the effects of patterned electrical activity on the morphology and motility of mammalian central nervous system growth cones. Neurites from dorsal root ganglion (DRG) neurons of fetal mice were allowed to grow under the barrier of an insert placed in culture dishes. The insert confined the cell bodies within separate experimental and control compartments, and provided a means of exciting action potentials in the growing neurites by extracellular current pulses delivered across the barrier. A phasic pattern of stimulation caused immediate retraction of the filopodia and lamellipodium. Further outgrowth was halted and in many cases retraction of the neurite ensued. No changes in morphology or growth cone motility were evoked by electric stimulation when action potentials were blocked with 1 microM tetrodotoxin (TTX). These effects depended on the rate, pattern, and duration of stimulation. Phasic stimulation was more effective than stimulation with the same number of impulses delivered at a constant frequency. An important new observation was that cultures exposed to phasic stimulation for several hours contained actively growing neurites with normal growth cones which were insensitive to the stimulus. This apparent accommodation in neurites exposed to chronic stimulation may involve processes that regulate calcium conductance or buffering. Cessation of neurite outgrowth by action potentials could represent one mechanism linking morphological and functional characteristics in the developing CNS of mammals, by stabilizing the outgrowth of neurites forming appropriate synaptic contacts and leading to the retraction of growth cones from collaterals that have not formed appropriate contacts at the time the neuron enters into a functionally active circuit.     

Serum amyloid P component induces neuronal apoptosis and beta-amyloid immunoreactivity

Previously we have reported serum amyloid P component (SAP) induced cell death in cerebro-cortical cultures of rat brain. In this paper we studied the types of target cells and the molecular mechanism of SAP-induced cell death. Immuno-electron and light microscopy revealed that SAP penetrates the plasma membrane and translocates selectively into the nuclei of neurons. Neuronal cells with SAP immunoreactivity exhibit the morphological hallmarks of apoptosis in vitro. The apoptotic mechanism of cell death is also supported by the increased Bax/Bcl-2 ratio. In addition to neurotoxic effects, we detected elevated beta-amyloid (Abeta) immunoreactivity following SAP treatment. This study supports the thesis that SAP plays an important role in the pathomechanism of neurodegenerative diseases, including Alzheimer's disease by inducing neuronal apoptosis.     

Proteasome inhibition arrests neurite outgrowth and causes "dying-back" degeneration in primary culture

Proteasome inhibitors such as lactacystin were first isolated when assaying their ability to stimulate neurite outgrowth in neuronal-like cell lines; however, their effect on neurites in primary culture has been largely neglected. We report here that lactacystin causes immediate arrest of nerve growth factor (NGF)-stimulated neurite outgrowth in sympathetic and sensory explant cultures. This is followed by neurite degeneration that in sympathetic cultures has a distinctive "dying-back" morphology. Remarkably, this occurs even at concentrations below that required to induce neurite outgrowth in PC12 cells. Thus, lactacystin opposes rather than potentiates the effect of NGF on sympathetic neurite outgrowth and the role of the ubiquitin proteasome pathway in growth and long-term maintenance of axons and dendrites differs from that in neuritogenesis in neuronal-like cell lines. Retrograde degeneration caused by blocking of the ubiquitin proteasome pathway may mimic some aspects of gracile axonal dystrophy, a dying-back axonopathy in mice caused by ubiquitin hydrolase (Uch-l1) deficiency, and may be relevant to human neurodegenerative diseases involving ubiquitination or proteasome abnormalities.     

Calpain-mediated cleavage of collapsin response mediator protein(CRMP)-2 during neurite degeneration in mice

Axon or dendrite degeneration involves activation of the ubiquitin-proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two-dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)-2 and CMRP-4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP-2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP-2 or -4 occurred in NGF-deprived neurites from Wld(s) (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1-specific siRNA, we have demonstrated that CRMP-2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down-regulation of caplain 1 expression suppressed CRMP-2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP-2 and -4 play a pivotal role in axonal growth and transport, and the C-terminus region of CRMP-2 is essential for its binding to kinesin-1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.     

Sabeluzole accelerates neurite outgrowth in different neuronal cell lines

The outgrowth of neurites in neuronal cell cultures reflects the intrinsic capacity for neurite regeneration and morphological rearrangements after axotomy and in plasticity. The role of fast axonal transport in these neurite outgrowth responses has not been investigated. We have recently shown that sabeluzole (R58735), a new neuro-active compound increases fast axonal transport in cultures of hippocampal neurons. In rat hippocampal neurons, N4 neuroblastoma cells and adult rat dorsal root ganglion cultures, incubation with sabeluzole at an optimal concentration of between 0.1 μM and 0.5 μM enhances neurite outgrowth between 10 and 30%. The relative number of cells with neurite length greater than twice the cell body, is also increased dose-dependently. Time-dependent studies further indicate that the rate of neurite elongation is markedly enhanced during the first 24-48 h. This neurite enhancing effect of sabeluzole is discussed in relation to the enhancement of fast axonal transport.     

Factors regulating the expression of acetylcholinesterase-containing neurons in striatal cultures: effects of chemical depolarization

The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 microM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20 microM) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCl levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2+ channels--verapamil (1 microM) and nifedipine (1 microM)--did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 microM) had a partial effect. When cultures treated with 5 microM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed.     

Induction of autophagy in neurite degeneration of mouse superior cervical ganglion neurons

Emerging lines of evidence show that the mechanisms of neurite degeneration are convergent, with poor neuritic transport, mitochondrial dysfunction and an increase in intra-axonal calcium being the principal convergence points. Nevertheless, the details are unclear. Here, we revealed the induction of autophagy in degenerating neurites of sympathetic neuron initiated by three different experimental paradigms. Autophagosomes were colocalized with collapsed cytoskeletal proteins in neuritic beadings during degeneration. Accumulation of microtubule-associated protein light chain 3-II, which is the most reliable marker for autophagy, was observed in the early stage of neurite degeneration. The autophagy inhibitor 3-methyladenine efficiently suppressed neurite degeneration by protecting neurites from the loss of viability and mitochondrial function. Furthermore, knocking down the key autophagy-related genes Atg7 and Beclin1 significantly delayed axonal and dendritic degeneration after nerve growth factor deprivation. Reduced expression of Atg7 also suppressed neurite fragmentation after transection. Therefore, our present data suggest the critical role of autophagy in neurite degeneration and may provide a valuable clue in understanding the mechanism of axonal and dendritic degeneration.     

Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer's disease activates expression of proapoptotic markers in primary cortical neurons

Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.     

Latency to onset of status epilepticus determines molecular mechanisms of seizure-induced cell death

The molecular mechanisms mediating degeneration in response to neuronal insults, including damage evoked by prolonged seizure activity, show substantial variability across laboratories and injury models. Here we investigate the extent to which the proportion of cell death occurring by apoptotic vs. necrotic mechanisms may be shifted by changing the temporal parameters of the insult. In initial studies with continuous seizures (status epilepticus, SE), signs of apoptotic degeneration were most clearly observed when SE occurred following a long latency (>86 min) after injection of kainic acid as compared with a short-latency SE (<76 min). Therefore, in this study we directly compared short- with long-latency SE for the expression of molecular markers for apoptosis and necrosis in an especially vulnerable brain region (rhinal cortex). Molecular markers of apoptosis (DNA fragmentation, cleavage of ICAD, an inhibitor of "caspase-activated DNase" (CAD), and prevalence of a caspase-generated fragment of alpha-spectrin) were detected following long-latency SE. Short-latency SE resulted in expression of predominantly necrotic features of cell death, such as "non-ladder" pattern of genomic DNA degradation, prevalence of a calpain-generated alpha-spectrin fragment, and absence of ICAD cleavage. Silver staining revealed no significant difference in the extent and spatial distribution of degeneration between long- or short-latency SE. These data indicate that the latency to onset of SE determines the extent to which apoptotic or necrotic mechanisms contribute to the degeneration following SE. The presence of a long latency period, during which multiple brief seizure episodes may occur, favors the occurrence of apoptotic cell death. It is possible that the absence of such "preconditioning" period in short-latency SE favors predominantly necrotic profile.     

Senile plaques in cerebral amyloid angiopathy show accumulation of amyloid precursor protein without cytoskeletal abnormalities.

The abnormal neurites that surround beta-amyloid in senile plaques (SP) in Alzheimer disease contain beta-amyloid precursor protein (beta APP) or abnormal filaments which react with antibodies to tau. Occasionally, beta APP and abnormal filaments are present in the same neurite. Whether both types of abnormal neurites are reactive to the presence of beta-amyloid or they are instead independent from each other is unknown. To begin to clarify this issue, we comparatively studied beta APP and tau-epitopes in SP from cases of classical Alzheimer disease and cases of cerebral amyloid angiopathy, with SP but without neurofibrillary pathology. In subjects with cerebral amyloid angiopathy, about one-third of SP, the same percentage as in Alzheimer disease, were beta APP reactive in the absence of tau-reactivity. beta APP epitopes were ultrastructurally localized in dense bodies of probable lysosomal origin, adjacent to the core of SP. These results demonstrate that beta APP and tau-reactive cytoskeletal alterations occur independently in the neurites of SP. The presence of beta APP in dystrophic neurites of SP and the localization of beta APP in lysosomes suggest that beta APP containing dystrophic neurites may play a role in the extracellular deposition of amyloid.     

Relationship between apolipoprotein E and the amyloid deposits and dystrophic neurites of Alzheimer's disease

T. C. Dickson, H. L. Saunders and J. C. Vickers (1997) Neuropathology and Applied Neurobiology , 23, 483–491
Relationship between apolipoprotein E and the amyloid deposits and dystrophic neurites of Alzheimer's disease
Although the inheritance of certain apolipoprotein E (ApoE) alleles has been recognized as a genetic risk factor for Alzheimer's disease, the role of ApoE in the pathology underlying this disease is unclear. Several reports have emphasized the association of ApoE with either β-amyloid plaque formation or the development of neurofibrillary pathology. Utilization of multiple label immunohistochemical methods enabled us to examine directly the localization of ApoE immunoreactivity relative to β- amyloid plaques, dystrophic neurites and neurofibrillary tangles. In Alzheimer's disease cases, β-amyloid plaques showing high ApoE immunoreactivity were localized to layers II, III and V of the neocortex. In layer I, β-amyloid plaques were unlabelled for ApoE relative to β-amyloid. Dense core plaques labelled for β-amyloid often had only the central portions labelled for ApoE. Conversely, ApoE labelled spherical structures within some plaques were not immunoreactive for β-amyloid or dystrophic neurite markers. Unlike β-amyloid labelled plaques, all ApoE immunoreactive plaques were associated with dystrophic neurites. In preclinical Alzheimer's disease cases, most plaques were double labelled for β-amyloid and ApoE. ApoE did not label dystrophic neurites or the early stages of neurofibrillary tangle formation, indicating that ApoE may not be directly involved in neurofibrillary pathology. The specific presence of ApoE in plaques associated with dystrophic neurites in demented patients suggests that ApoE may contribute toward a higher degree of β- amyloid fibrillogenesis, enhancing the ability of certain plaques to cause damage to surrounding axons.     

Ascorbic acid protects SH-SY5Y neuroblastoma cells from apoptosis and death induced by beta-amyloid

beta-Amyloid causes apoptosis and death in cultured neurons that may be mediated by generation of reactive oxygen species. Since ascorbic acid concentrations are relatively high in brain, we tested whether and how this antioxidant might protect cultured SH-SY5Y neuroblastoma cells from apoptotic cell death. SH-SY5Y cells did not contain ascorbate in culture but readily took it up to achieve intracellular concentrations several-fold those of GSH. Treatment of cells with 2-10 microM beta-amyloid(25-35) decreased both intracellular ascorbate and GSH without affecting rates of ascorbate transport, which suggests that the peptide induces an oxidant stress in the cells. Overnight culture of cells with 10-20 microM beta-amyloid(25-35) induced apoptosis in SH-SY5Y cells when measured as externalization of phosphatidylserine by annexin V binding, as DNA fragmentation in the TUNEL assay, and as caspase-3 activity in cell lysates. Pre-loading cells with ascorbate substantially prevented apoptosis measured by these assays as well as cell death. In addition to preventing apoptosis, ascorbate loading of SH-SY5Y cells also decreased basal rates of generation of endogenous beta-amyloid. Together, these results support the notion that beta-amyloid induces apoptosis and death in neurons due to oxidant stress and suggest that intracellular ascorbate effectively prevents this toxicity.     

beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity.

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.