Inhibition of RhoA reduces propofol-mediated growth cone collapse, axonal transport impairment, loss of synaptic connectivity, and behavioural deficits.

BACKGROUND: Exposure of the developing brain to propofol results in cognitive deficits. Recent data suggest that inhibition of neuronal apoptosis does not prevent cognitive defects, suggesting mechanisms other than neuronal apoptosis play a role in anaesthetic neurotoxicity. Proper neuronal growth during development is dependent upon growth cone morphology and axonal transport. Propofol modulates actin dynamics in developing neurones, causes RhoA-dependent depolymerisation of actin, and reduces dendritic spines and synapses. We hypothesised that RhoA inhibition prevents synaptic loss and subsequent cognitive deficits. The present study tested whether RhoA inhibition with the botulinum toxin C3 (TAT-C3) prevents propofol-induced synapse and neurite loss, and preserves cognitive function. METHODS: RhoA activation, growth cone morphology, and axonal transport were measured in neonatal rat neurones (5-7 days in vitro) exposed to propofol. Synapse counts (electron microscopy), dendritic arborisation (Golgi-Cox), and network connectivity were measured in mice (age 28 days) previously exposed to propofol at postnatal day 5-7. Memory was assessed in adult mice (age 3 months) previously exposed to propofol at postnatal day 5-7. RESULTS: Propofol increased RhoA activation, collapsed growth cones, and impaired retrograde axonal transport of quantum dot-labelled brain-derived neurotrophic factor, all of which were prevented with TAT-C3. Adult mice previously treated with propofol had decreased numbers of total hippocampal synapses and presynaptic vesicles, reduced hippocampal dendritic arborisation, and infrapyramidal mossy fibres. These mice also exhibited decreased hippocampal-dependent contextual fear memory recall. All anatomical and behavioural changes were prevented with TAT-C3 pre-treatment. CONCLUSION: Inhibition of RhoA prevents propofol-mediated hippocampal neurotoxicity and associated cognitive deficits.

Increased cortical synaptic activation of TrkB and downstream signaling markers in a mouse model of Down Syndrome.

Down Syndrome (DS), trisomy 21, is characterized by synaptic abnormalities and cognitive deficits throughout the lifespan and with development of Alzheimer's disease (AD) neuropathology and progressive cognitive decline in adults. Synaptic abnormalities are also present in the Ts65Dn mouse model of DS, but which synapses are affected and the mechanisms underlying synaptic dysfunction are unknown. Here we show marked increases in the levels and activation status of TrkB and associated signaling proteins in cortical synapses in Ts65Dn mice. Proteomic analysis at the single synapse level of resolution using array tomography (AT) uncovered increased colocalization of activated TrkB with signaling endosome related proteins, and demonstrated increased TrkB signaling. The extent of increases in TrkB signaling differed in each of the cortical layers examined and with respect to the type of synapse, with the most marked increases seen in inhibitory synapses. These findings are evidence of markedly abnormal TrkB-mediated signaling in synapses. They raise the possibility that dysregulated TrkB signaling contributes to synaptic dysfunction and cognitive deficits in DS.

Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system.

Multiple sclerosis is a demyelinating disorder of the central nervous system (CNS), in which an immune attack directed against myelin constituents causes myelin destruction and death of oligodendrocytes, the myelin-producing cells. Here, the efficacy of nerve growth factor (NGF), a growth factor for neurons and oligodendrocytes, in promoting myelin repair was evaluated using the demyelinating model of experimental allergic encephalomyelitis (EAE) in the common marmoset. Surprisingly, we found that NGF delayed the onset of clinical EAE and, pathologically, prevented the full development of EAE lesions. We demonstrate by immunocytochemistry that NGF exerts its antiinflammatory effect by downregulating the production of interferon gamma by T cells infiltrating the CNS, and upregulating the production of interleukin 10 by glial cells in both inflammatory lesions of EAE and normal-appearing CNS white matter. Thus, NGF, currently under investigation in human clinical trials as a neuronal trophic factor, may be an attractive candidate for therapy of autoimmune demyelinating disorders.

Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor.

Some neurodegenerative disorders may be caused by abnormal synthesis or utilization of trophic molecules required to support neuronal survival. A test of this hypothesis requires that trophic agents specific for the affected neurons be identified. Cholinergic neurons in the corpus striatum of neonatal rats were found to respond to intracerebroventricular administration of nerve growth factor with prominent, dose-dependent, selective increases in choline acetyltransferase activity. Cholinergic neurons in the basal forebrain also respond to nerve growth factor in this way. These actions of nerve growth factor may indicate its involvement in the normal function of forebrain cholinergic neurons as well as in neurodegenerative disorders involving such cells.

Evidence for cholinergic neurites in senile plaques.

In the neocortices and amygdalae of young and aged macaques, cholinergic axons were identified by means of a monoclonal antibody to bovine choline acetyltransferase. Many fine, linear, immunoreactive profiles were seen in these animals. In the older animals, some cholinergic axons showed multifocal enlargements along their course. In some instances, neurites with choline acetyltransferase immunoreactivity were associated with deposits of amyloid (visualized with thioflavin T fluorescence). The appearance of these amyloid-associated abnormal cholinergic processes was similar to that of neurites in senile plaques, as shown by conventional silver impregnation techniques. Cholinergic systems thus give rise to some of the neurites within senile plaques.

Molecular studies in Alzheimer's disease.

In the past decade, there has been an explosion of information relating to the molecular neurobiology of Alzheimer's disease (AD). Molecular dissection of the neuropathology of AD has provided insight into the pathogenesis of this disease and has defined areas where investigation may prove useful in elucidating the cause of this disorder and suggest new treatments.

c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

NGF signaling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway.

The target-derived neurotrophic factor "nerve growth factor" (NGF) signals through TrkA to promote the survival, differentiation, and maintenance of neurons. How the NGF signal in axon terminals is conveyed to the cell body is unknown. The "signaling endosome hypothesis" envisions that NGF-TrkA complexes are internalized at the axon terminal and retrogradely transported to the cell body. Following NGF treatment, we found that clathrin-coated vesicles contained NGF bound to TrkA together with activated signaling proteins of the Ras-MAP kinase pathway. Evidence that these vesicles could signal was their ability in vitro to activate Elk, a downstream target of Erk1/2. Our results point to the existence of a population of signaling endosomes derived from clathrin-coated membranes in NGF-treated cells.

p140trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression is induced by NGF.

Nerve growth factor (NGF) appears to act as a neurotrophic factor for basal forebrain and caudate-putamen cholinergic neurons. The mechanism by which NGF transduces its signal in these neurons is yet to be defined. Recent data indicate that the product of the trk gene, p140trk, is a critical component of the NGF receptor. Herein, we show that p140trk mRNA is highly restricted in its distribution in the adult rat forebrain, that it is present in cholinergic neurons, and that most if not all cholinergic neurons contain p140trk mRNA. Furthermore, induction of trk expression by NGF suggests that neurotrophin-mediated up-regulation of their receptor tyrosine kinases is an important feature of their actions and that neurotrophins may regulate the activity of responsive neurons through increasing the level of their receptors.

Developmental regulation of nerve growth factor and its receptor in the rat caudate-putamen.

In prior studies, nerve growth factor (NGF) administration induced a robust, selective increase in the neurochemical differentiation of caudate-putamen cholinergic neurons. In this study, expression of NGF and its receptor was examined to determine whether endogenous NGF might serve as a neurotrophic factor for these neurons. The temporal pattern of NGF gene expression and the levels of NGF mRNA and protein were distinct from those found in other brain regions. NGF and high-affinity NGF binding were present during cholinergic neurochemical differentiation and persisted into adult-hood. An increase in NGF binding during the third postnatal week was correlated with increasing choline acetyltransferase activity. The data are consistent with a role for endogenous NGF in the development and, possibly, the maintenance of caudate-putamen cholinergic neurons.

Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector.

To study posttranslational mechanisms for the control of nerve growth factor (NGF), we used a recombinant vaccinia virus vector to independently express the two major NGF transcripts in a variety of mammalian cell lines. The two major transcripts contain NGF (12.5 kilodaltons [kDa]) at the C-terminus and differ by alternative splicing of an N-terminal exon, so that the large precursor (34 kDa) had 67 amino acids upstream of an internal signal peptide and the smaller precursor (27 kDa) had this signal peptide at its N-terminus. In L929 cells, expression of either NGF transcript with the vaccinia virus vector gave rise to an apparently identical intracellular 35-kDa glycosylated precursor formed by cleavage of the primary gene product after the signal peptide. These cells also secreted biologically active NGF. To determine whether NGF processing is restricted by cell type, we infected a variety of mammalian cell lines with both recombinant viruses; all accumulated the same 35-kDa precursor and secreted NGF. Thus, many types of cells have the machinery to process and secrete NGF. However, NGF accumulated intracellularly (presumably in secretory granules) in cells with a regulated pathway of secretion (e.g., AtT-20 and HIT cells). In these cells, a membrane-permeable cyclic AMP analog, 8-bromo-cyclic AMP, stimulated NGF secretion. This suggests a mechanism for the regulation of NGF levels in which specific secretagogues, e.g., neurotransmitters, control NGF secretion.

Nerve growth factor activates persistent Rap1 signaling in endosomes.

We investigated a role for endogenous Rap1, a small monomeric GTP-binding protein of the Ras family, in nerve growth factor (NGF) signaling in PC12 cells. Although both epidermal growth factor (EGF) and NGF caused transient activation of Ras, only NGF induced the activation of Rap1. Moreover, Rap1 activation was sustained for hours, an effect that matched the sustained activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate the molecular basis for Rap1 activation, we examined complexes containing C3G, a guanine nucleotide exchange factor for Rap1, and CrkL, an adapter protein known to influence Rap1 signaling. NGF induced the formation of a long-lived complex containing C3G/CrkL/Shp2/Gab2/TrkA. Linking the complex to Rap1 activation, we coprecipitated activated TrkA and activated MAPK with activated Rap1 in NGF-treated cells. Confocal microscopy and subcellular fractionation showed that activated Rap1 and the other proteins of the signaling complex were present in endosomes. Pretreatment of PC12 cells with brefeldin A (BFA), which disrupts the Golgi and endosomal compartments, had little effect on Ras activation but strongly inhibited NGF-induced Rap1 activation and continuing MAPK activation. We propose that endosomes are a site from which NGF induces the prolonged activation of Rap1 and MAPK.

NGF signals through TrkA to increase clathrin at the plasma membrane and enhance clathrin-mediated membrane trafficking.

Neurotrophin (NT) signals may be moved from axon terminals to neuron cell bodies via signaling endosomes-organelles in which NTs continue to be bound to their activated receptors. Suggesting that clathrin-coated membranes serve as one source of signaling endosomes, in earlier studies we showed that nerve growth factor (NGF) treatment increased clathrin at the plasma membrane and resulted in colocalization of clathrin with TrkA, the receptor tyrosine kinase for NGF. Strikingly, however, we also noted that most clathrin puncta at the surface of NGF-treated cells did not colocalize with TrkA, raising the possibility that NGF induces a general increase in clathrin-coated membrane formation. To explore this possibility further, we examined the distribution of clathrin in NGF- and BDNF-treated cells. NGF signaling in PC12 cells robustly redistributed the adaptor protein AP2 and the clathrin heavy chain (CHC) to surface membranes. Using confocal and epifluorescence microscopy, as well as biochemical assays, we showed the redistribution of clathrin to be attributable to the activation of TrkA. Significantly, NGF signaled through TrkA to induce an increase in clathrin-mediated membrane trafficking, as revealed in the increased endocytosis of transferrin. In that BDNF treatment increased AP2 and clathrin at the surface membranes of hippocampal neurons, these findings may represent a physiologically significant response to NTs. We conclude that NT signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking and speculate that this effect contributes to their trophic actions via the increased internalization of receptors and other proteins that are present in clathrin-coated membranes.

Nerve growth factor in Alzheimer's disease: to treat or not to treat?

Several hypotheses can be proposed to link neurotrophic factors with neurodegenerative diseases. Not surprisingly, different hypotheses suggest completely different approaches to therapy; some would suggest use of neurotrophic factors, while others would propose that the actions of these factors be blocked. It has been suggested that NGF be used to prevent the loss of basal forebrain cholinergic neurons in Alzheimer's disease (AD). At this time it is not possible to conclude whether or not NGF is implicated in the causation or progression of this disorder. Nevertheless, experimental studies in animals have given a strong rationale for its use. Given the lack of an effective treatment for this disorder, the careful approach to NGF trials outlined by an ad hoc committee of the National Institute on Aging should be pursued.

Nerve growth factor reverses neuronal atrophy in a Down syndrome model of age-related neurodegeneration.

Atrophy and dysfunction of certain neurons, including cholinergic neurons in the basal forebrain, are key features of the neuropathology of Alzheimer's disease (AD). Since all individuals with Down syndrome (DS) develop AD neuropathology by the 4th decade, we reasoned that a genetic model of DS, the trisomy 16 (Ts 16) mouse, may provide an animal model to study the neurodegeneration in AD. Ts 16 mice fail to survive birth; to evaluate neurons for long periods in vivo required transplantation of fetal tissue. We previously demonstrated that Ts 16 basal forebrain cholinergic neurons (BFCNs) undergo age-related atrophy similar to DS and AD, and now show that a specific neurotrophic factor, nerve growth factor (NGF), acts to reverse Ts 16-induced atrophy of BFCNs and stimulates hypertrophy of these cells. As NGF levels were not decreased in the host, abnormalities intrinsic to Ts 16 BFCNs presumably caused the atrophy. Our results suggest that NGF may be useful in reversing cholinergic neurodegeneration in DS and AD.

Acceleration of scrapie in neonatal Syrian hamsters.

Prions cause Creutzfeldt-Jakob disease, Gerstmann-Sträussler syndrome, and kuru of humans as well as scrapie of animals. Prolonged incubation periods, from months to decades, precede clinical disease. In studies on the biochemical characteristics of prions, weanling Syrian hamsters have been used extensively because they have relatively short incubation periods. In studies reported here, inoculation of neonatal hamsters significantly shortened the scrapie incubation period even further. Our results show that the scrapie incubation period in hamsters is a function of age. The interval between inoculation and death from scrapie plotted as a function of age (0 to 30 days) gave a correlation coefficient (r) of 0.86. The duration of clinical disease was also shortened in the hamsters inoculated as neonates compared with weanlings. Intraventricular injection of nerve growth factor prior to inoculation of neonates with scrapie significantly diminished the acceleration observed with scrapie alone in neonates. Histopathologic studies of brain from scrapie-inoculated neonates showed more extensive neuronal loss in the hippocampus and neocortex as well as a more profound gliosis in the caudate compared with animals inoculated as weanlings. Our results demonstrate an age-dependent acceleration of scrapie in neonatal hamsters and may provide a new experimental system for defining factors that modify the pathogenesis of prion diseases.

Changes in the localization of brain prion proteins during scrapie infection.

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures.

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Catecholaminergic neurites in senile plaques in prefrontal cortex of aged nonhuman primates.

Immunocytochemical studies, using a polyclonal antibody directed against tyrosine hydroxylase, identified catecholaminergic axons in prefrontal cortex of young and aged nonhuman primates. Aged monkeys, who showed cortical senile plaques in silver stains, had swollen tyrosine hydroxylase-immunoreactive axons in neocortex. Some of these abnormal processes were associated with deposits of amyloid (visualized by thioflavin-T fluorescence) and were similar in appearance to neurites demonstrated by silver impregnation methods. This study provides evidence for structural abnormalities in catecholaminergic axons/nerve terminals in the neocortices of aged primates.

A comprehensive study of the spatiotemporal pattern of beta-amyloid precursor protein mRNA and protein in the rat brain: lack of modulation by exogenously applied nerve growth factor.

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons, a population that degenerates and dies in Alzheimer's disease (AD). It has been suggested that NGF be used to treat AD patients. However, in vivo administration of NGF to the developing hamster brain was shown to induce the expression of the beta-amyloid precursor protein (beta APP) gene. The association of alterations in beta APP gene expression with AD-like neuropathological changes and cognitive impairment in animals, and with AD-like neurodegeneration in Down syndrome patients suggests that NGF-mediated increases in beta APP expression could negate or attenuate NGF's neurotrophic activity in AD treatment trials. The present study was undertaken to explore further the influence of NGF on beta APP expression, and to determine which, if any, of the beta APP mRNAs is altered in response to NGF treatment. We first examined the spatiotemporal pattern of beta APP-695 and Kunitz protease inhibitor (KPI)-containing beta APP mRNA expression in the rat brain. Specific oligonucleotide probes were used to show that these mRNAs are present during embryonic development. In addition, we evaluated postnatal expression in nine brain regions and showed that beta APP mRNAs were readily detected in all regions at postnatal day 2. In human brain, the relative levels of beta APP-695 and beta APP-KPI mRNA and their protein are discordant, in that the level of beta APP-695 mRNA is slightly higher than that of beta APP-KPI, but beta APP-KPI protein predominates. In contrast, the several-fold excess of beta APP-695 mRNA relative to beta APP-KPI mRNA in the rat brain was also reflected at the protein level. Surprisingly, administration of exogenous NGF failed to affect rat beta APP mRNA levels either in vitro or during postnatal development in vivo.