Mutations in the SPG3A gene encoding the GTPase atlastin interfere with vesicle trafficking in the ER/Golgi interface and Golgi morphogenesis.

Mutations in SPG3A causing autosomal dominant pure spastic paraplegia led to identification of atlastin, a new dynamin-like large GTPase. Atlastin is localized in the endoplasmic reticulum, the Golgi, neurites and growth cones and has been implicated in neurite outgrowth. To investigate whether it exerts its activity in the early secretory system, we expressed normal and mutant atlastin in cell culture. Pathogenic mutations in the GTPase domain interfered with the maturation of Golgi complexes by preventing the budding of vesicles from the endoplasmic reticulum, whereas mutations in other regions of the protein disrupted fission of endoplasmic reticulum-derived vesicles or their migration to their Golgi target. Atlastin, therefore, plays a role in vesicle trafficking in the ER/Golgi interface. Furthermore, atlastin partially co-localized with proteins of the p24/emp/gp25L family that regulate vesicle budding and trafficking in the early secretory pathway, and co-immunoprecipitated p24, suggesting a functional relationship that should be further explored.

Protective effect of the alpha2-adrenoceptor antagonist, dexefaroxan, against spatial memory deficit induced by cortical devascularization in the adult rat.

The alpha2-adrenoceptor antagonist, dexefaroxan, has been shown in the rat to have neuroprotective and plastic effects against degenerative structural changes in elements of the basalocortical cholinergic system that result from cortical devascularization [Neuroscience 115 (2002) 41]. The present study, using the same experimental protocol, examined the functional consequences of cortical devascularization and dexefaroxan treatment in the Morris water maze memory test. Rats were first trained to find the hidden platform in the test, and then subjected to the devascularization procedure. Thirty-one days later, lesioned rats exhibited a significant deficit in recalling the platform location, compared with sham control animals. A 28-day subcutaneous infusion with dexefaroxan (0.63, 2.5, and 10 mg rat(-1) day(-1)), starting from the moment of the devascularization, protected against this spatial memory deficit.

Protective effects of the alpha 2-adrenoceptor antagonist, dexefaroxan, against degeneration of the basalocortical cholinergic system induced by cortical devascularization in the adult rat.

It has been hypothesized [Colpaert, F.C., 1994. In: Briley, M., Marien, M. (Eds.), Noradrenergic Mechanisms in Parkinson's Disease. CRC Press, Boca Raton, FL, pp. 225-254] that a deficiency in the noradrenergic system originating from the locus coeruleus is a decisive factor in the progression of central neurodegenerative disorders including Alzheimer's disease, and that treatments which boost noradrenergic transmission (e.g. via blockade of alpha(2)-adrenoceptors) could provide both symptomatic and trophic benefits against the disease. Studies in the rat in vivo demonstrating that the selective alpha(2)-adrenoceptor antagonist dexefaroxan increases acetylcholine release in the cortex, improves measures of cognitive performance and protects against excitotoxin lesions, support this concept. As a further test of the hypothesis, we investigated the effect of dexefaroxan in a rat model of unilateral cortical devascularization that induces a loss of the cortical cholinergic terminal network and a retrograde degeneration of the cholinergic projections that originate in the nucleus basalis magnocellularis. Lesioned and sham-operated rats received a 28-day subcutaneous infusion of dexefaroxan (0.63 mg/rat/day) or vehicle, delivered by osmotic minipumps implanted on the day of the cortical devascularization procedure. In lesioned rats, the dexefaroxan treatment was associated with a significantly higher number and size of vesicular acetylcholine transporter-immunoreactive boutons in comparison to the vehicle treatment; this effect was most marked within cortical layer V. Dexefaroxan also significantly reduced the atrophy of cholinergic neurons within the nucleus basalis magnocellularis. Dexefaroxan had no observable effect on any of these parameters in sham-operated cohorts. These results show that systemically administered dexefaroxan mitigates cholinergic neuronal degeneration in vivo, and provide further evidence for a therapeutic potential of the drug in neurodegenerative diseases such as Alzheimer's disease, where central cholinergic function is progressively compromised.

Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action.

Developing small molecule agonistic ligands for tyrosine kinase receptors has been difficult, and it is generally thought that such ligands require bivalency. Moreover, multisubunit receptors are difficult to target, because each subunit contributes to ligand affinity, and each subunit may have distinct and sometimes opposing functions. Here, the nerve growth factor receptor subunits p75 and the tyrosine kinase TrkA were studied using artificial ligands that bind specifically to their extracellular domain. Bivalent TrkA ligands afford robust signals. However, genuine monomeric and monovalent TrkA ligands afford partial agonism, activate the tyrosine kinase activity, cause receptor internalization, and induce survival and differentiation in cell lines and primary neurons. Monomeric and monovalent TrkA ligands can synergize with ligands that bind the p75 subunit. However, the p75 ligands used in this study must be bivalent, and monovalent p75 ligands have no effect. These findings will be useful in designing and developing screens of small molecules selective for tyrosine kinase receptors and indicate that strategies for designing agonists of multisubunit receptors require consideration of the role of each subunit. Last, the strategy of using anti-receptor mAbs and small molecule hormone mimics as receptor ligands could be applied to the study of many other heteromeric cell surface receptors.

A designed peptidomimetic agonistic ligand of TrkA nerve growth factor receptors.

A proteolytically stable small molecule beta-turn peptidomimetic, termed D3, was identified as an agonist of the TrkA neurotrophin receptor. D3 binds the Ig-like C2 region of the extracellular domain of TrkA, competes the binding of another TrkA agonist, affords selective trophic protection to TrkA-expressing cell lines and neuronal primary cultures, and induces the differentiation of primary neuronal cultures. These results indicate that a small beta-turn peptidomimetic can activate a tyrosine kinase neurotrophin receptor that normally binds a relatively large protein ligand. Agents such as D3 that bind the extracellular domain of Trk receptors will be useful pharmacological agents to address disorders where Trk receptors play a role, by targeting populations selectively.

Reorganization of cholinergic terminals in the cerebral cortex and hippocampus in transgenic mice carrying mutated presenilin-1 and amyloid precursor protein transgenes.

Cholinergic deficits are one of the most consistent neuropathological landmarks in Alzheimer's disease (AD). We have examined transgenic mouse models (PS1M146L, APPK670N,M671L) and a doubly transgenic line (APPK670N,M671L + PS1M146L) that overexpress mutated AD-related genes [presenilin-1 (PS1) and the amyloid precursor protein (APP)] to investigate the effect of AD-related gene overexpression and/or amyloidosis on cholinergic parameters. The size of the basal forebrain cholinergic neurons and the pattern of cholinergic synapses in the hippocampus and cerebral cortex were revealed by immunohistochemical staining for choline acetyltransferase and the vesicular acetylcholine transporter, respectively. At the time point studied (8 months), no apparent changes in either the size or density of cholinergic synapses were found in the PS1M146L mutant relative to the nontransgenic controls. However, the APPK670N,M671L mutant showed a significant elevation in the density of cholinergic synapses in the frontal and parietal cortices. Most importantly, the double mutant (APPK670N,M671L + PS1M146L), which had extensive amyloidosis, demonstrated a prominent diminution in the density of cholinergic synapses in the frontal cortex and a reduction in the size of these synapses in the frontal cortex and hippocampus. Nonetheless, no significant changes in the size of basal forebrain cholinergic neurons were observed in these three mutants. This study shows a novel role of APP and a synergistic effect of APP and PS1 that correlates with amyloid load on the reorganization of the cholinergic network in the cerebral cortex and hippocampus at the time point studied.

A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat.

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter-IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

A TrkA-selective, fast internalizing nerve growth factor-antibody complex induces trophic but not neuritogenic signals.

Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to TrkA and/or p75 receptors. We report a comparative study of the binding, internalization, and biological activity of NGF versus that of NGF in association with an anti-NGF monoclonal antibody (mAb NGF30), directed against the C termini of NGF. NGF.mAb complexes do not bind p75 effectively but bind TrkA with high affinity. After binding, NGF. mAb complexes stimulate internalization faster and to a larger degree than NGF. NGF.mAb-induced activation of TrkA, Shc, and MAPK is transient compared with NGF-induced activation; yet NGF and NGF. mAb afford identical trophic responses. In contrast, NGF induces Suc-1-associated neurotrophic activating protein phosphorylation and neuritogenic differentiation, but NGF.mAb does not. Thus, an absolute separation of trophic and neuritogenic function is seen for NGF.mAb, suggesting that biological response modifiers of neurotrophins can afford ligands with selected activities.

TrkA antagonists decrease NGF-induced ChAT activity in vitro and modulate cholinergic synaptic number in vivo.

Cholinergic neurons are known to respond in vivo to the administration of nerve growth factor (NGF) by a prominent and selective increase of choline acetyl transferase activity and by cholinergic synaptogenesis in the rat brain. By using a synthetic TrkA antagonist we demonstrated that endogenously produced NGF is involved in the continual re-modeling of cholinergic neuronal connections during adulthood, acting through TrkA receptors.

Involvement of protease-activated receptor-1 in the in vitro development of mesencephalic dopaminergic neurons.

In situ hybridization studies have revealed high levels of protease (thrombin)-activated receptor-1 messenger RNA in the mesencephalon of rats, suggesting that dopaminergic neurons are a target for thrombin's actions. We have evaluated the effect of thrombin receptor activation, either by thrombin or by thrombin receptor agonist peptide, a 14 amino acid agonist of protease-activated receptor-1, on tyrosine hydroxylase-positive neurons. Pure cultures of rat mesencephalic neurons or co-cultures of mesencephalic neurons and glial cells were treated with either thrombin or thrombin receptor agonist peptide the day after plating. Tyrosine hydroxylase-positive cell counting, [3H]dopamine uptake and morphometric analysis were performed on day 5. Thrombin and thrombin receptor agonist peptide influenced neurite elongation, branching and the number of primary, secondary and tertiary neurites of tyrosine hydroxylase-positive neurons. In pure cultures, the most significant effects of thrombin and thrombin receptor agonist peptide were to delay branching and to increase the centrifugal growth of neurites without affecting the total neuritic length. Thrombin (up to 10 nM) and thrombin receptor agonist peptide did not affect the number of tyrosine hydroxylase-positive neurons or [3H]dopamine uptake. Neurotrophin-4 also influenced the morphology of tyrosine hydroxylase-positive neurons. The increase of neuritic length initiated by this neurotrophin is complementary to the radial elongation induced by protease-activated receptor-1 activation. When neurons were cultured in the presence of glial cells, the effects of thrombin and thrombin receptor agonist peptide on most of these parameters were larger than those observed with pure cultures. Thus, thrombin is able to initiate a complex remodelling of the architecture of tyrosine hydroxylase-positive neurons through the activation of protease-activated receptor-1. These results provide further support for the involvement of protease-activated receptor-1 activation in the development and differentiation of the central nervous system.

Pharmacological characterization of protease-activated receptor (PAR-1) in rat astrocytes.

The proteolytic action of thrombin on its receptor (protease-activated receptor-1 or PAR-1) results in a conformational change in which the new N-terminal sequence auto-activates the receptor. Peptide analogs of this N-terminal sequence (TRAPs) are able to mimic the effect of thrombin and an extensive search has led to the definition of the structural requirement for the agonist and antagonist activity on thrombin receptors in several peripheral systems. Thrombin plays an important role in central and peripheral nervous system development and PAR-1 is present in neurons and astrocytes. We have now characterized thrombin receptors pharmacologically in cultured rat astrocytes by using [3H]thymidine incorporation and reversal of stellation induced by Bt2cAMP as end-points. Thrombin increased [3H]thymidine incorporation into DNA with an EC50 of 1 nM and induced a complete reversion of cell stellation. The effects of thrombin on [3H]thymidine incorporation were mimicked by TRAP-14 (EC50 = 3 microM) and a peptide containing non-natural amino acids Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (A6Y; EC50 = 0.8 microM). Similarly, these two peptides reversed Bt2cAMP-induced stellation. The effect of thrombin, TRAP-14 and A6Y on [3H]thymidine incorporation into DNA was significantly prevented by L9R, a 9-amino-acid peptide (Leu-Val-Arg-D-Cys-Gly-Lys-His-Ser-Arg; IC50 = 180 microM against thrombin and TRAP-14 and 800 microM against A6Y) previously described as an antagonist in human platelet aggregation. L9R antagonized also thrombin effects on astrocyte morphology. These results demonstrate that rat astrocytes express PAR-1 receptors which are pharmacologically similar to those previously characterized in human platelets.

Transduction mechanisms involved in thrombin receptor-induced nerve growth factor secretion and cell division in primary cultures of astrocytes.

In astrocytes, thrombin and thrombin receptor-activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [3H]-thymidine incorporation into DNA (EC50 = 1 nM and +650% at 100 nM for thrombin; EC50 = 3 microM and +600% at 100 microM for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 nM thrombin or 100 microM TRAP-14). The [3H] thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as Gi or G(o), is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.

Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons.

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (-83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (-80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 microM and -63% at 100 microM) and in co-cultures (+25% at 0.1 microM and -28% at 100 microM). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.