Gzalpha deficient mice: enzyme levels in the autonomic nervous system, neuronal survival and effect of genetic background

Our laboratory has generated a genetically mutant mouse in which the alpha subunit of the heterotrimeric GTP binding protein, Gz has been made dysfunctional by homologous recombination to determine its in vivo function. These animals show a characteristic failure to thrive phenotype. Gzα is expressed in a variety of nervous system tissues as well as in the adrenal medulla. We therefore examined the autonomic nervous system of the Gzα deficient mouse by measuring the activity of tyrosine hydroxylase and choline acetyltransferase in the superior cervical ganglia, submaxillary gland and the adrenal medulla. Preliminary results using animals of mixed BALB/c and C57BL/6 strains gave inconsistent results. Further experiments demonstrated differences in the activity of tyrosine hydroxylase and choline acetyltransferase between BALB/c and C57BL/6 mouse strains. The analysis of the pure strains showed a reduction in the size and enzyme levels of the adrenal gland and submaxillary glands of the Gzα deficient mouse suggesting a role for adrenal insufficiency and/or nutritional disorders for the failure to thrive phenotype. The survival of sympathetic and sensory neurons was also examined in the Gzα deficient mouse and in the presence of pertussis toxin, sympathetic but not sensory neuronal survival in Gzα deficient mice was significantly attenuated. This suggests that in vivo other pertussis toxin sensitive G proteins may be recruited to compensate for the loss of Gzα

Evidence for phosphatidylinositol 4-kinase and actin involvement in the regulation of 125I-b-nerve growth factor retrograde axonal transport

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled βNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I- βNGF retrograde transport. PAO (20 nM to 200 μM) also inhibited NGF- dependent survival of both sympathetic and sensory neuronal populations. F- actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-βNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-βNGF retrograde axonal transport in vivo

Differential mRNA expression and subcellular locations of P13-kinase isoforms in sympathetic and sensory neurons

Phosphatidylinositol 3-kinase (PI3-kinase) enzymes are key signalling molecules in the PC12 and neuronal cell survival pathway and are also involved in the regulation of retrograde axonal transport of nerve growth factor (NGF), with sympathetic neurons more sensitive to the effects of wortmannin/LY294002 than sensory neurons (Bartlett et al. [1997]; Brain Res. 761:257-262; Reynolds et al. [1998] Brain Res. 798:6774). In this article, we characterized the mRNA expression of PI3-kinase isoforms in mouse sympathetic superior cervical ganglia (SCG) and sensory trigeminal ganglia (TGG) and examined the subcellular locations of immunoreactivity of the PI3-kinase isoforms in mouse cultured SCG and dorsal root ganglion (DRG) neurons. Both the SCG and the TGG express mRNA for the p110α, β, γ, δ, and vps34p PI3- kinase isoforms, but the TGG and not the SCG express mRNA for the p170 PI3- kinase isoform. In cultured SCG and DRG neurons, p110α, β, and immunoreactivity is in the SCG and DRG growth cones, and predominantly in puncta throughout the growth cone varicosity. However, in the cell bodies immunoreactivity varied, p110α is localized predominantly at the plasma membrane, while p110β and γ is localized in the perinuclear region of the cells. In addition, unlike other cell types, wortmannin has little effect on actin filament polymerization in either mouse cultured SCG or DRG neurons

Evidence for phosphatidylinositol 4-kinase and actin involvement in the regulation of 125I-β-nerve growth factor retrograde axonal transport

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled βNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I- βNGF retrograde transport. PAO (20 nM to 200 μM) also inhibited NGF- dependent survival of both sympathetic and sensory neuronal populations. F- actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-βNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-βNGF retrograde axonal transport in vivo.</p

Differential mRNA expression and subcellular locations of PI3-kinase isoforms in sympathetic and sensory neurons

Phosphatidylinositol 3-kinase (PI3-kinase) enzymes are key signalling molecules in the PC12 and neuronal cell survival pathway and are also involved in the regulation of retrograde axonal transport of nerve growth factor (NGF), with sympathetic neurons more sensitive to the effects of wortmannin/LY294002 than sensory neurons (Bartlett et al. [1997]; Brain Res. 761:257-262; Reynolds et al. [1998] Brain Res. 798:6774). In this article, we characterized the mRNA expression of PI3-kinase isoforms in mouse sympathetic superior cervical ganglia (SCG) and sensory trigeminal ganglia (TGG) and examined the subcellular locations of immunoreactivity of the PI3-kinase isoforms in mouse cultured SCG and dorsal root ganglion (DRG) neurons. Both the SCG and the TGG express mRNA for the p110α, β, γ, δ, and vps34p PI3- kinase isoforms, but the TGG and not the SCG express mRNA for the p170 PI3- kinase isoform. In cultured SCG and DRG neurons, p110α, β, and immunoreactivity is in the SCG and DRG growth cones, and predominantly in puncta throughout the growth cone varicosity. However, in the cell bodies immunoreactivity varied, p110α is localized predominantly at the plasma membrane, while p110β and γ is localized in the perinuclear region of the cells. In addition, unlike other cell types, wortmannin has little effect on actin filament polymerization in either mouse cultured SCG or DRG neurons.</p

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated I sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultra-structural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal

In sympathetic but not sensory neurones, phosphoinositide-3 kinase is important for NGF-dependent survival and the retrograde transport of 125I-β NGF

The way in which the same ligands and receptors have different functional effects in different cell types must depend on subtle differences in the second messenger cascades. Sensory and sympathetic neurones both retrogradely transport nerve growth factor (NGF) and depend on NGF for their developmental survival. NGF binding to the high affinity tyrosine kinase (TrkA) receptors initiates second messenger signalling cascades, one of which includes the activation of phosphoinositide-3 kinase (PI3-kinase). We demonstrate that 100-fold higher concentrations of the PI3-kinase inhibitor, Wortmannin, are required to inhibit the survival effects and retrograde axonal transport of NGF in sensory neurones than in sympathetic neurones. Similarly, although less potently than Wortmannin, the PI3-kinase inhibitor LY294002 required a 10-fold higher concentration to inhibit the survival effects of NGF in sensory than in sympathetic neurones. Inhibitors of other second messengers, including staurosporine, pertussis and cholera toxins, failed to have an effect on the transport of the NGF receptor complex in both cell types. Also, Wortmannin did not affect the structural integrity of the sympathetic nerve terminals. As PI3-kinase is present in both neuronal populations, this suggests that the Wortmannin sensitive isoform of PI3- kinase (p110) is essential in sympathetic neurones both for survival and for NGF-TrkA receptor complex trafficking. As sensory neurones also depend on NGF for their developmental survival and endocytose and retrogradely transport the NGF-TrkA receptor complex, this population of neurones may either recruit a different isoform of PI3-kinase or utilize PI3-kinase independent signalling pathways for these cellular functions.</p

Solh, the mouse homologue of the Drosophila melanogaster small optic lobes gene: organization, chromosomal mapping, and localization of gene product to the olfactory bulb.

The Drosophila melanogaster small optic lobes gene (sol) is required for normal development of the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein products of sol and its human homologue SOLH contain zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal domain of unknown function. Long-distance PCR was used to amplify genomic DNA for Solh, the mouse homologue of sol, following the identification of mouse Solh expressed sequence tags. The nucleotide sequence of the Solh coding region (6.0 kb) was determined. The predicted Solh protein of 1095 amino acid residues shows 89% identity (93% similarity) to the human homologue. Solh was localized by in situ hybridization to band A3.3 on mouse Chromosome 17, in a region of maintained homology with human 16p13.3. Antipeptide antibodies were prepared and verified by demonstration of specific reactivity with recombinant human SOLH protein prepared by in vitro transcription/translation and expression in insect cells using the baculovirus system. The antibodies were used to show that the Solh protein localizes to the olfactory bulb in mouse and rat brain, suggesting that it could have an analogous role in development of sensory system neurons in Drosophila and in mammals. (C) 2000 Academic Press

Hypertolerance to morphine in G(zalpha)-deficient mice

Our laboratory has generated a mouse deficient in the alpha (α) subunit of the G protein, G(z), (G(zα)) gene and we have examined the involvement of G(zα) in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by th

Genetic tracing of subpopulation neurons in the prethalamus of mice (Mus musculus)

Genetic labeling based on the Cre/lox reporter system has allowed the creation of fate maps for progenitor cells and their offspring. In the diencephalon, pools of progenitors express the plp transcripts in the zona limitans intrathalamica (ZLI), the basal plate of the diencephalon (bpD), and the posterior part of the hypothalamus. We used plp-Cre transgenics crossed with either Rosa26-lox-lacZ (R26R) or actin-lox gfp (Z/EG) reporter mice to investigate the progeny of plp-expressing ventricular cells in the diencephalon. We describe the subpopulations of prethalamic neurons derived from plp-activated progenitors, their possible migratory routes as development proceeds, and their final positional identity. Neurons derived from plp-expressing progenitors issued from the ZLI contribute to GABAergic cells in the zona incerta, the subgeniculate nucleus, the ventral lateral geniculate, and the intergeniculate leaflet. Plp+ progenitors in the bpD and posterior hypothalamus appear to generate glutamatergic neurons in the subthalamic nucleus and GABAergic neurons in the mammillary and retromammillary tegmentum derivatives. In all these nuclei the contribution of plp+ progenitors is only partial, illustrating the heterogeneity of origin of neurons in prethalamic and caudal hypothalamic nuclei.Grant sponsor: DD was a fellow of the Ministère de l’Enseignement Supérieur et de la Recherche and European Leukodystrophy Association; Grant sponsor: INSERM; Grant sponsor: Association de Recherche sur la Sclérose En Plaques (to J.L.T.); Grant sponsor: National Multiple Sclerosis Society; Grant number: TR-3762-A-1 (to B.Z.); Grant sponsor: DFG (CMPB) and BMBF (to K.A.N.); Grant sponsor: EEC; Grant number: LSHG-CT-2004- 512003; Grant sponsor: EUREXPRESS; Grant numbers: ACOMP06/076, BFU2005-23722-E/BFI, MEC BFU2005-09085; Grant sponsor: ELA Fondation de Recherche, France (to S.M.).Peer Reviewe