Distinct role of long 3' UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal neurons.

The brain produces two brain-derived neurotrophic factor (BDNF) transcripts, with either short or long 3' untranslated regions (3' UTRs). The physiological significance of the two forms of mRNAs encoding the same protein is unknown. Here, we show that the short and long 3' UTR BDNF mRNAs are involved in different cellular functions. The short 3' UTR mRNAs are restricted to somata, whereas the long 3' UTR mRNAs are also localized in dendrites. In a mouse mutant where the long 3' UTR is truncated, dendritic targeting of BDNF mRNAs is impaired. There is little BDNF in hippocampal dendrites despite normal levels of total BDNF protein. This mutant exhibits deficits in pruning and enlargement of dendritic spines, as well as selective impairment in long-term potentiation in dendrites, but not somata, of hippocampal neurons. These results provide insights into local and dendritic actions of BDNF and reveal a mechanism for differential regulation of subcellular functions of proteins.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Expression by midbrain dopamine neurons of Sema3A and 3F receptors is associated with chemorepulsion in vitro but a mild in vivo phenotype.

Here we explore the role of semaphorin 3A and 3F (Sema3A, Sema3F) in the formation of the mesotelencephalic pathway. We show that Sema3A and 3F are expressed in the ventral mesencephalon (VM) of E13.5 rat embryos; the receptors Neuropilin 1 and Neuropilin 2, and co-receptors L1CAM, NrCAM, and Plexins A1 and A3 but not A4 are expressed by VM dopaminergic neurons; these neurons bind Sema3A and 3F in vitro which induces collapse of their growth cones and elicits, with different potencies, a repulsive response; and this response is absent in axons from Nrp1 and Nrp2 null embryos. Despite these in vitro effects, only very mild anatomical defects were detected in the organization of the mesotelencephalic pathway in embryonic and adult Nrp1 or Nrp2 null mice. However, the dopaminergic meso-habenular pathway and catecholaminergic neurons in the parafascicular and paraventricular nuclei of the thalamus were significantly affected in Nrp2 null mice. These data are consistent with a model whereby Sema3A and 3F, in combination with other guidance molecules, contributes to the navigation of DA axons to their final synaptic targets.

Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function.

BACKGROUND & AIMS: The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2K(b)-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon gamma-inducible H-2K(b) promoter element. METHODS: Enteric neuronal precursors were isolated from the intestines of E13-mouse fetuses and second day postnatal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33 degrees C, and interferon-gamma for 24 or 48 hours, and then transferred to 39 degrees C in the presence of glial cell line-derived neurotrophic factor for 7 days for further differentiation. Neuronal markers were assessed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Neuronal function was assessed by transplanting these cells into the colons of Piebald or nNOS(-/-) mice. RESULTS: Expression analysis of cells showed the presence of neuronal markers peripherin, PGP9.5, HuD, tau, synaptic marker synaptophysin, characteristic receptors of enteric neurons, Ret, and 5-hydroxytryptamine-receptor subtypes at 33 degrees C and 39 degrees C. Nestin, S-100beta, and alpha-smooth muscle actin were expressed minimally at 39 degrees C. Glial cell line-derived neurotrophic factor resulted in increased phosphorylation of Akt in these cells, similar to primary enteric neurons. Transplantation of cells into the piebald or nNOS(-/-) mice colon improved colonic motility. CONCLUSIONS: We have developed novel enteric neuronal cell lines that have neuronal characteristics similar to primary enteric neurons. These cells can help us in understanding newer therapeutic options for Hirschsprung's disease.

Stigmoid bodies contain type I receptor proteins SorLA/LR11 and sortilin: new perspectives on their function.

Stigmoid bodies (SBs) are structures in the cytoplasm of neurons. SBs are mostly found in the hypothalamic region of the rat and contain a protein called huntingtin-associated protein 1 (HAP1). In a recent publication, large cytoplasmic structures were shown to be immunoreactive for a type I receptor called SorLA/LR11. By light microscopic analysis, these structures appeared similar to SBs in size and in brain regional and subcellular localization. To determine whether these large puncta correspond to HAP1-containing SBs, we used antibodies specific to various domains of the apolipoprotein receptor LR11 to perform immunocytochemistry in rat and mouse brain tissue. Transfection studies using HeLa cells were conducted to demonstrate the specificity of the antibodies. We found that, in both species, antibodies to the domain II (or VSP10 for vacuolar sorting protein 10 domain) of LR11 immunoreact with large cytoplasmic structures. Co-localization immunolabeling experiments in rat brain tissue sections and in neuron cultures showed that these LR11-immunoreactive structures correspond to HAP1-positive SBs. Electron microscopy was performed in rat hypothalamus and further demonstrated the presence of LR11 in SBs and its co-localization with HAP1. LR11-containing SBs were most abundant in the hypothalamus but were also found in many brainstem nuclei, thalamus, and hippocampus. Our data also show that sortilin, another transmembrane protein containing a VPS10 domain, localizes to large cytoplasmic puncta and is found in LR11-positive and Hap1-positive SBs in hypothalamic neuron cultures.

A protocol for isolation and biochemical characterization of stigmoid bodies from rat brain.

Stigmoid bodies (SBs) are structures present in the cytoplasm of neurons. Many brain regions including hypothalamus, thalamus, amygdala, septum, hippocampus, colliculi, and brainstem contain neurons with at least one SB. Despite this widespread distribution their function remains unknown. SBs contain a brain protein called huntingtin-associated protein 1 (HAP1) and have more recently been found to contain the apolipoprotein E receptor LR11 (Lipoprotein Receptor containing 11 LDL binding domains, also called SorLA for sorting protein-related receptor containing LDLR class A repeats) and sortilin. To provide a first step towards further identification of their components and perhaps shed some light on their neurobiological role, we have developed a method for isolating SBs from rat brain. The protocol relies on a combination of centrifugational forces, sucrose gradient, and immunoisolation. Samples enriched in SBs were incubated with antibodies to HAP1B or to LR11 followed by incubation with FITC conjugated secondary antibodies. Anti-FITC coated beads were incubated with samples and SB-bead complexes formed were separated by magnetic sorting without pelleting the complexes during the isolation procedure. Immunopurified SBs, visualized by light and electron microscopy, show similar ultrastructure to those present in neurons.

Gene expression profiles in a transgenic animal model of fragile X syndrome.

Fragile X syndrome is the most common inherited form of mental retardation. Although this syndrome originates from the absence of the RNA-binding protein FMRP, the molecular mechanisms underlying the cognitive deficits are unknown. The expression pattern of 6789 genes was studied in the brains of wild-type and FMR1 knockout mice, a fragile X syndrome animal model that has been associated with cognitive deficits. Differential expression of more than two-fold was observed for the brain mRNA levels of 73 genes. Differential expression of nine of these genes was confirmed by real-time quantitative reverse transcription-polymerase chain reaction and by in situ hybridization. In addition to corroborating the microarray data, the in situ hybridization analysis showed distinct spatial distribution patterns of microtubule-associated protein 2 and amyloid beta precursor protein. A number of differentially expressed genes associated with the fragile X syndrome phenotype have been previously involved in other memory or cognitive disorders.