Full-length apolipoprotein E protects against the neurotoxicity of an apoE-related peptide.

Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141-149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer's disease where apoE4 confers a significantly greater risk of pathology.

Cathepsin D-mediated proteolysis of apolipoprotein E: possible role in Alzheimer's disease.

Proteolysis of apolipoprotein E (apoE) may be involved in the pathogenesis of Alzheimer's disease (AD). We previously identified aspartic protease(s) as possibly contributing to the proteolysis of apoE in human brain homogenates. The current study used biochemical and immunohistochemical methods to examine whether cathepsin D (catD) and cathepsin E (catE), candidate aspartic proteases, may be involved in apoE proteolysis. CatD was found to proteolyze both lipid-free recombinant full-length human apoE and lipidated human plasma full-length apoE (apoE4/dipalmitoylphosphatidylcholine-reconstituted discs). CatE was found to proteolyze lipid-free recombinant human apoE to a much greater extent than lipidated apoE. This proteolysis, as well as proteolysis of human apoE added to brain homogenates from apoE-deficient mice, was inhibited by pepstatin A (an aspartic protease inhibitor), but not by phenylmethanesulfonyl fluoride (a serine protease inhibitor). The major apoE fragment obtained with catD included the receptor-binding domain and had an apparent molecular weight similar to that found in human brain homogenates. There was little immunoreactivity for catE in AD brain tissue sections. In contrast, qualitative and quantitative analyses of immunostained sections of the frontal cortex revealed that catD and apoE are colocalized in a subset of predominantly dense-core neuritic plaques and in some neurofibrillary tangles. A positive correlation was observed between estimated duration of illness and the percentage of apoE-positive plaques that were also catD-positive. These results suggest that aspartic proteases, catD in particular, may be involved in proteolysis of apoE and perhaps contribute to the generation of apoE fragments previously implicated in AD pathology.

ApoE isoforms affect neuronal N-methyl-D-aspartate calcium responses and toxicity via receptor-mediated processes.

Apolipoprotein E (apoE) alters the pathophysiology of Alzheimer's disease, but its mechanism is not fully understood. We examined the effects of recombinant human apoE3 and apoE4 on the neuronal calcium response to N-methyl-D-aspartate (NMDA), and compared them to their toxicity. ApoE4 (100 nM) significantly increased the resting calcium (by 70%) and the calcium response to NMDA (by 185%), whereas similar changes were not obtained in apoE3-treated neurons. ApoE4, but not apoE3, also significantly increased neurotoxicity, as evidenced by enhanced lactate dehydrogenase release (by 53%) and reduced 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide levels (by 32%). ApoE4-induced changes in the calcium response to NMDA and associated neurotoxicity were blocked by coincubation with MK-801. Both the receptor-associated protein, which inhibits interaction of apoE with members of the LDL receptor family, including the low-density lipoprotein receptor-related protein (LRP), and activated alpha2-macroglobulin, another LRP ligand, prevented apoE4-induced enhancement of the calcium response to NMDA, resting calcium levels, and neurotoxicity. A tandem apoE peptide (100 nM) containing only the receptor binding region residues also eliminated the enhanced calcium signaling and neurotoxicity by apoE4. Taken together, our data demonstrate that differential effects of apoE3 and apoE4 on the calcium signaling in neurons correlate with their effect on neurotoxicity, which are secondary to receptor binding.

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat.

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.

Evidence for reduced accumulation of exogenous neurotrophin by aged sympathetic neurons.

The present study investigated the potential for neurotrophin uptake by cerebrovascular axons and subsequent accumulation in the aged superior cervical ganglion (SCG) following a two week intracerebroventricular infusion of nerve growth factor (NGF). In the SCG from aged rats, NGF protein levels declined significantly compared with the SCG from young adult rats. Following NGF infusion, perivascular axons from both young adult and aged rats showed intense NGF immunostaining. In addition, significant increases in NGF protein were shown using enzyme-linked immunosorbent assay (ELISA) and in counts of NGF immunopositive cell bodies in the SCG when compared with age-matched controls. NGF accumulation in ganglia from aged rats, however, was significantly less when compared with ganglia from young adult rats. The results of the present study suggest that NGF protein is significantly reduced in aged ganglia with the neurons retaining some capacity to take up and transport exogenous neurotrophin. Even so, the potential for NGF accumulation is dramatically reduced in aged rats when compared with that of young adult rats. While previous results have shown robust NGF-induced neurotransmitter responses by sympathetic neurons from the aged animal, the present finding of reduced accumulation of NGF in aged sympathetic neurons suggests an age-related difference in the utilization or transport of NGF.

Disruption of spinal cord white matter and sciatic nerve geometry inhibits axonal growth in vitro in the absence of glial scarring.

BACKGROUND: Axons within the mature mammalian central nervous system fail to regenerate following injury, usually resulting in long-lasting motor and sensory deficits. Studies involving transplantation of adult neurons into white matter implicate glial scar-associated factors in regeneration failure. However, these studies cannot distinguish between the effects of these factors and disruption of the spatial organization of cells and molecular factors (disrupted geometry). Since white matter can support or inhibit neurite growth depending on the geometry of the fiber tract, the present study sought to determine whether disrupted geometry is sufficient to inhibit neurite growth. RESULTS: Embryonic chick sympathetic neurons were cultured on unfixed longitudinal cryostat sections of mature rat spinal cord or sciatic nerve that had been crushed with forceps ex vivo then immediately frozen to prevent glial scarring. Neurite growth on uncrushed portions of spinal cord white matter or sciatic nerve was extensive and highly parallel with the longitudinal axis of the fiber tract but did not extend onto crushed portions. Moreover, neurite growth from neurons attached directly to crushed white matter or nerve tissue was shorter and less parallel compared with neurite growth on uncrushed tissue. In contrast, neurite growth appeared to be unaffected by crushed spinal cord gray matter. CONCLUSIONS: These observations suggest that glial scar-associated factors are not necessary to block axonal growth at sites of injury. Disruption of fiber tract geometry, perhaps involving myelin-associated neurite-growth inhibitors, may be sufficient to pose a barrier to regenerating axons in spinal cord white matter and peripheral nerves.

Myelin contributes to the parallel orientation of axonal growth on white matter in vitro.

BACKGROUND: Brain and spinal cord white matter can support extensive axonal growth. This growth is generally constrained to an orientation that is parallel to the longitudinal axis of the fiber tract. This constraint is presumably due to permissive and non-permissive substrates that are interleaved with each other and oriented in parallel within the tract. RESULTS: Embryonic chick sympathetic neurons were cultured on cryostat sections of rat brain and the orientation of neurite growth on white matter was assessed. To determine if haptotaxis is sufficient to guide parallel neurite growth, neurons were cultured under conditions designed to interfere with interactions between growing neurites and factors that act as biochemical contact guidance cues but not interactions with haptotactic cues. Under these conditions, neurites extending on white matter were not exclusively oriented in parallel to the fiber tract, suggesting that biochemical cues are involved. To assess the role of myelin in guiding parallel neurite growth, neurons were cultured on myelin-deficient corpus callosum. These neurons also extended neurites that were not constrained to a parallel orientation. Moreover, preincubation with NGF and treatment with cAMP analogs, manipulations that attenuate overall myelin-mediated inhibition of neurite growth, also led to a reduced parallel orientation of neurite growth. CONCLUSIONS: The present studies suggest that some of the relevant factors that constrain axonal growth on white matter are not haptotactic in nature and appear to be partly mediated by factors that are associated with myelin and may involve myelin-associated "inhibitors".

Absence of p75(NTR) expression reduces nerve growth factor immunolocalization in cholinergic septal neurons.

Septal axons provide a cholinergic innervation to the nerve growth factor (NGF)-producing neurons of the mammalian hippocampus. These cholinergic septal afferents are capable of responding to target-derived NGF because they possess trkA and p75(NTR), the two transmembrane receptors that bind NGF and activate ligand-mediated intracellular signaling. To assess the relative importance of p75(NTR) expression for the responsiveness of cholinergic septal neurons to hippocampally derived NGF, we used three lines of mutant and/or transgenic mice: p75(-/-) mice (having two mutated alleles of the p75(NTR) gene), NGF/p75(+/+) mice (transgenic animals overexpressing NGF within central glial cells and having two normal alleles of the p75(NTR) gene), and NGF/p75(-/-) mice (NGF transgenic animals having two mutated alleles of the p75(NTR) gene). BALB/c and C57B1/6 mice (background strains for the mutant and transgenic lines of mice) were used as controls. Both lines of NGF transgenic mice possess elevated levels of NGF protein in the hippocampus and septal region, irrespective of p75(NTR) expression. BALB/c and C57Bl/6 mice display comparably lower levels of NGF protein in both tissues. Despite differing levels of NGF protein, the ratios of hippocampal to septal NGF levels are similar among BALB/c, C57B1/6, and NGF/p75(+/+) mice. Both p75(-/-) and NGF/p75(-/-) mice, on the other hand, have markedly elevated ratios of NGF protein between these two tissues. The lack of p75(NTR) expression also results in a pronounced absence of NGF immunoreactivity in cholinergic septal neurons of p75(-/-) and NGF/p75(-/-) mice. BALB/c, C57B1/6, and NGF/p75(+/+) mice, on the other hand, display NGF immunoreactivity that appears as discrete granules scattered through the cytoplasm of cholinergic septal neurons. Elevated levels of NGF in the hippocampus and septal region coincide with hypertrophy of cholinergic septal neurons of NGF/p75(+/+) mice but not of NGF/p75(-/-) mice. Levels of choline acetyltransferase (ChAT) enzyme activity are, however, elevated in the septal region and hippocampus of both NGF/p75(+/+) and NGF/p75(-/-) mice, compared with control mice. These data indicate that an absence of functional p75(NTR) expression disrupts the normal cellular immunolocalization of NGF by cholinergic septal neurons but does not affect the ability of these neurons to respond to elevated levels of NGF, as determined by ChAT activity.

Sympathetic neurite growth on central nervous system sections is region-specific and unaltered by aging.

Several lines of evidence suggest that the brain exhibits reduced plasticity with aging. However, a variety of soluble neurite outgrowth-promoting factors, such as neurotrophins, are not decreased in the aged brain, and aged neurons do not possess dramatically reduced growth potential. The possibility that aging results in reduced baseline substrate-bound neurite outgrowth-promoting activity in the central nervous system (CNS) was evaluated using tissue section culture. There were clear differences between brain regions in the extent of neurite outgrowth on both young and aged brain sections. However, no differences in the extent of neurite outgrowth were observed as a function of age. These results suggest that aging of the rat CNS is not accompanied by major alterations in the baseline neurite outgrowth-promoting substrate properties of the tissue.

Remodeling of adult sensory axons in the superior cervical ganglion in response to exogenous nerve growth factor.

In previous studies, we found that a 2-week in vivo intracerebroventricular infusion of nerve growth factor (NGF) elicited a sprouting response by sympathetic perivascular axons associated with the intradural segment of the internal carotid artery. We hypothesized that NGF infused into the ventricular system would be internalized by responsive sympathetic cerebrovascular axons, retrogradely transported to parent cell bodies in the superior cervical ganglion (SCG), and subsequently released into the local ganglionic environment. Because fibers exhibiting immunoreactivity for calcitonin gene related peptide (CGRP) have been localized in the SCG, we used immunohistochemical methods to investigate whether a response by CGRP-immunoreactive axons in the SCG occurred following the proposed transport to and release of exogenous NGF in the ganglion. In consecutive tissue sections of the SCG stained for either CGRP or NGF, we found CGRP pericellular 'baskets' surrounding identified NGF-immunoreactive cell bodies. Nerve growth factor infusion resulted in a significant increase both in the number of CGRP pericellular baskets and in NGF-immunoreactive cell bodies. A significant positive correlation (r=0.95, P<0.05) between the pericellular baskets and NGF-immunoreactive cell bodies was observed, suggesting that intracranial projection neurons in the SCG released infused NGF (or possibly a converted signal) into the local ganglionic environment to elicit remodeling of CGRP fibers to form pericellular baskets. These findings were confirmed in sections double labeled for NGF and CGRP immunoreactivity. This remodeling suggests that exogenous NGF may mediate retrograde transneuronal plasticity, allowing for future in vivo examinations of the mechanisms involved in neurotrophin transport and release.

The role of NGF in pregnancy-induced degeneration and regeneration of sympathetic nerves in the guinea pig uterus.

In the guinea pig, pregnancy is associated with a generalised depletion of noradrenaline in uterine sympathetic nerves and, in the areas of the uterus surrounding the foetus, by a complete degeneration of sympathetic nerve fibres. These pregnancy-induced changes have been interpreted as a selective effect of placental hormones on the system of short sympathetic fibres arising from the paracervical ganglia. An alternative explanation is that pregnancy affects the neurotrophic capacity of the uterus. We measured NGF-protein levels in the guinea pig uterine horn, tubal end and cervix at early pregnancy, late pregnancy and early postpartum, using a two-site enzyme-linked immunosorbent assay. For comparative purposes the distribution and relative density of noradrenaline-containing sympathetic nerve fibres were assessed histochemically, and tissue levels of noradrenaline were measured biochemically, using high-performance liquid chromatography with electrochemical detection. In all the uterine regions analysed, NGF-protein levels showed a decline at term pregnancy, but in no case was this change statistically significant. After delivery, NGF-protein levels showed a marked increase in the cervix as well as in both the fertile and empty horns. These results suggest that alterations in NGF-protein do not account for the impairment of uterine sympathetic innervation during pregnancy, but may contribute to their recovery after delivery.

White matter of the CNS supports or inhibits neurite outgrowth in vitro depending on geometry.

Axonal regeneration is normally limited within myelinated fiber tracts in the CNS of higher vertebrates. Numerous studies suggest that CNS myelin contains inhibitors that may contribute to abortive axonal growth. In contrast to the evidence of myelin-associated neurite inhibitors, embryonic neurons transplanted into the CNS can regenerate extensively within myelinated tracts in vivo. It has been speculated that embryonic neurons do not yet express the appropriate receptors for myelin-associated inhibitors. Recently, however, extensive regeneration from transplanted adult neurons has also been reported within myelinated tracts of the CNS, casting doubt on the role myelin-associated inhibitors play in abortive regeneration. The present study reexamined the potential of white matter to support neurite growth in vitro. By the use of Neurobasal medium, neurons were cultured onto unfixed cryostat sections of mature rat CNS tissue. As documented previously, robust neuronal attachment and neurite outgrowth occurred on gray matter but these neurites were sharply inhibited by white matter. In addition, however, increased rates of neuronal attachment directly to white matter occurred with neurite outgrowth comparable in length with that on gray matter but limited to directions parallel to the fiber tract. Frequently, the same section of white matter was found to inhibit neurite outgrowth from neurons on gray matter while supporting parallel neurite outgrowth from neurons on white matter. These results suggest that whether white matter supports or inhibits axonal growth depends on the geometric relationship between the axon and the fiber tract; more specifically, white matter supports parallel growth but inhibits nonparallel growth.

Truncated apolipoprotein E (ApoE) causes increased intracellular calcium and may mediate ApoE neurotoxicity.

Apolipoprotein E (apoE)-related synthetic peptides, the 22 kDa N-terminal thrombin-cleavage fragment of apoE (truncated apoE), and full-length apoE have all been shown to exhibit neurotoxic activity under certain culture conditions. In the present study, protease inhibitors reduced the neurotoxicity and proteolysis of full-length apoE but did not block the toxicity of truncated apoE or a synthetic apoE peptide, suggesting that fragments of apoE may account for its toxicity. Additional experiments demonstrated that both truncated apoE and the apoE peptide elicit an increase in intracellular calcium levels and subsequent death of embryonic rat hippocampal neurons in culture. Similar effects on calcium were found when the apoE peptide was applied to chick sympathetic neurons. The rise in intracellular calcium and the hippocampal cell death caused by the apoE peptide were significantly reduced by receptor-associated protein, removal of extracellular calcium, or administration of the specific NMDA glutamate receptor antagonist MK-801. These results suggest that apoE may be a source of both neurotoxicity and calcium influx that involves cell surface receptors. Such findings strengthen the hypothesis that apoE plays a direct role in the pathology of Alzheimer's disease.

Enhanced neurotrophin-induced axon growth in myelinated portions of the CNS in mice lacking the p75 neurotrophin receptor.

Axonal growth in the adult mammalian CNS is limited because of inhibitory influences of the glial environment and/or a lack of growth-promoting molecules. Here, we investigate whether supplementation of nerve growth factor (NGF) to the CNS during postnatal development and into adulthood can support the growth of sympathetic axons within myelinated portions of the maturing brain. We have also asked whether p75(NTR) plays a role in this NGF-induced axon growth. To address these questions we used two lines of transgenic mice overexpressing NGF centrally, with or without functional expression of p75(NTR) (NGF/p75(+/+) and NGF/p75(-/-) mice, respectively). Sympathetic axons invade the myelinated portions of the cerebellum, beginning shortly before the second week of postnatal life, in both lines of NGF transgenic mice. Despite the presence of central myelin, these sympathetic axons continue to sprout and increase in density between postnatal days 14 and 100, resulting in a dense plexus of sympathetic fibers within this myelinated environment. Surprisingly, the growth response of sympathetic fibers into the cerebellar white matter of NGF/p75(-/-) mice is enhanced, such that both the density and extent of axon ingrowth are increased, compared with age-matched NGF/p75(+/+) mice. These dissimilar growth responses cannot be attributed to differences in cerebellar levels of NGF protein or sympathetic neuron numbers between NGF/p75(+/+) and NGF/p75(-/-) mice. Our data provide evidence demonstrating that growth factors are capable of overcoming the inhibitory influences of central myelin in the adult CNS and that neutralization of the p75(NTR) may further enhance this growth response.

NGF expression in the aged rat pineal gland does not correlate with loss of sympathetic axonal branches and varicosities.

The factors that determine the ability of some, but not all neurons, to sustain their axonal projections during aging remain largely unknown. Because sympathetic neurons remain responsive to nerve growth factor (NGF) in old age, it has been proposed that the selective decrease observed in the sympathetic innervation to some targets in aged rats may be the result of a deficit in target-derived NGF. In this study we utilized two different techniques to demonstrate decreased target innervation by sympathetic fibers in the aged rat pineal gland, which is an appropriate and relevant model for examining mechanisms of neuron-target interactions in aging. Tyrosine hydroxylase immunoreactive profiles were quantified in pineal glands of young and aged male Sprague-Dawley rats. The density of tyrosine hydroxylase-immunoreactive fibers was 30% lower in aged pineals, although the remaining fibers contained 20% more tyrosine hydroxylase-immunoreactivity. Othograde tracing of the pineal sympathetic innervation using biotinylated dextran revealed that average axon length, varicosity numbers, branch point numbers, and numbers of terminations were all decreased by approximately 50% in aged tissues, indicating possible functional deficits. These findings suggest that whole branches, along with their associated varicosities were lost in old age. A sensitive quantitative ribonuclease protection assay and a two-site ELISA assay were used to examine whether reduced NGF availability might correlate with sympathetic nerve atrophy. No significant differences were detected in either NGF mRNA or NGF protein levels when comparing young and aged pineal glands, suggesting that atrophy in aged sympathetic neurons is not causally related to reduced availability of NGF at the target. Our results indicate that mechanisms other than NGF expression need to be explored in order to explain the age-related axonal regression observed in this target.

Uninjured aged sympathetic neurons sprout in response to exogenous NGF in vivo.

The extent to which the loss of plasticity by aged neurons is due to changes in the neuronal environment or to a loss of growth potential of the neurons has not been determined. In previous studies we observed that young adult cerebrovascular axons undergo a sprouting response following a 2-week intracerebroventricular infusion of nerve growth factor (15 microg; NGF). The present study used electron microscopy to examine the innervation of the intradural segment of the internal carotid artery of the aged rat and to determine whether aged sympathetic perivascular axons would respond to in vivo infusion of NGF. Young adult and aged Fischer 344 female rats received a 2-week intracranial infusion of NGF (15 microg) or vehicle (VEH) and were perfused for electron microscopy. Although there was no change in the total number of perivascular axons associated in aged VEH when compared with young adult VEH, a significant reduction was observed in aged VEH when total axons and sympathetic axons were expressed per microm2 vascular wall, reflecting an age-related increase in blood vessel size. Following NGF infusion, aged sympathetic axons were significantly increased by 192% compared with aged VEH cases. These results suggest that there is a proportional reduction in sympathetic cerebrovascular neurons with aging but that they exhibit robust sprouting in response to an exogenous neurotrophin.

Sympathetic neurite outgrowth is greater on plaque-poor vs. plaque-rich regions of Alzheimer's disease cryostat sections.

Senile plaques are a characteristic histopathological feature of Alzheimer's disease (AD) and are associated with altered neuritic morphology. Numerous individual plaque components, most notably beta-amyloid, have been studied for their possible effects on neurite outgrowth in culture. However, the effect of senile plaques on neuronal morphology and function is difficult to assess. In the present study, the effect of senile plaques on neurite outgrowth was studied by culturing embryonic chick sympathetic neuronal explants on Alzheimer's tissue sections. Explants were cultured for 3 days on amygdala tissue sections from AD as well as non-AD patients in serum-free medium. Neurite outgrowth on plaque-rich regions was compared with outgrowth on plaque-poor regions of the same tissue section, and with outgrowth on non-AD tissue, through colocalization of the living explants and the underlying plaques. Explants growing on plaque-rich regions showed significantly less neurite outgrowth compared with those on plaque-poor regions in the same section or on control brain tissue. These results suggest that plaques are poor substrates for neurite outgrowth as compared with non-plaque areas of the same tissue sections, and support the hypothesis that components of the senile plaques may inhibit neurite outgrowth.

Rat microglia exhibit increased density on Alzheimer's plaques in vitro.

The relationship of microglia to senile plaques was investigated by culturing glial cells derived from neonatal rat brain on cryostat sections of Alzheimer's disease (AD) or control brain. Rat microglia were identified by their uptake of DiI-acetylated LDL. Plaques were colocalized using Thioflavin-S staining. Although the number of microglia attached to AD tissue sections did not differ significantly from the number on control brain tissue, the density of microglia on senile plaques was significantly greater than on nonsenile plaque areas of the same sections. These results suggest that microglia may have a higher affinity for senile plaques than for nonsenile plaque regions of AD brain tissue and are consistent with the hypothesis that microglia respond to plaques.

Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated.

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.

Sympathetic axons invade the brains of mice overexpressing nerve growth factor.

Transgenic mice that overexpress nerve growth factor (NGF) in cells producing glial fibrillary acidic protein were used to determine whether sympathetic axons will invade the undamaged, postnatal mammalian brain. By using reverse transcriptase-polymerase chain reaction, NGF mRNA transgene expression was detectable in the hippocampi and cerebella of transgenic mice but not in age-matched, wild type mice. Elevated levels of NGF protein were detected in the hippocampi and cerebella of postnatal and adult transgenic animals as well as in conditioned media from transgenic cerebellar astrocytes in culture. The brains of these transgenic mice were found to contain postganglionic sympathetic fibers, as identified by their immunohistochemical staining for tyrosine hydroxylase and by their disappearance following superior cervical ganglionectomy. In the cerebellum, a robust plexus of sympathetic fibers was evident in the deep white matter and in the inferior cerebellar peduncles. These axons within the cerebellum were observed as early as 14 days after birth and dramatically increased in number with age. Sympathetic axons were also associated with the large blood vessels of the hippocampal fissure and were present within the hilar region of the dentate gyrus. NGF immunoreactivity was present within the sympathetic axons as well as within glial cells in the transgenic cerebellum and hippocampus. Wild type mice, however, lacked similar patterns of immunostaining. These results demonstrate that elevated expression of NGF in the intact mammalian brain results in the growth of sympathetic axons into the central nervous system in the absence of injury.