BDNF but not NT-4 is required for normal flexion reflex plasticity and function

Neurotrophins can directly modulate the function of diverse types of central nervous system synapses. Brain-derived neurotrophic factor (BDNF) might be released by nociceptors onto spinal neurons and mediate central sensitization associated with chronic pain. We have studied the role of BDNF and neurotrophin-4 (NT-4), both ligands of the trkB tyrosine kinase receptor, in synaptic transmission and reflex plasticity in the mouse spinal cord. We used an in vitro spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents in BDNF- and NT-4-deficient mice. In situ hybridization studies show that both these neurotrophins are synthesized by sensory neurons, and NT-4, but not BDNF, also is expressed by spinal neurons. BDNF null mutants display selective deficits in the ventral root potential (VRP) evoked by stimulating nociceptive primary afferents whereas the non-nociceptive portion of the VRP remained unaltered. In addition, activity-dependent plasticity of the VRP evoked by repetitive (1 Hz) stimulation of nociceptive primary afferents (termed wind-up) was substantially reduced in BDNF-deficient mice. This plasticity also was reduced in a reversible manner by the protein kinase inhibitor K252a. Although the trkB ligand NT-4 is normally present, reflex properties in NT-4 null mutant mice were normal. Pharmacological studies also indicated that spinal N-methyl-d-aspartate receptor function was unaltered in BDNF-deficient mice. Using immunocytochemistry for markers of nociceptive neurons we found no evidence that their number or connectivity was substantially altered in BDNF-deficient mice. Our data therefore are consistent with a direct role for presynaptic BDNF release from sensory neurons in the modulation of pain-related neurotransmission.

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice.

The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor alpha (TNF-alpha), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-alpha in experimental CJD, we investigated the expression of TNF-alpha in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1 alpha and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-alpha, interleukin 1 alpha, and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, beta-actin expression remained unchanged. Progressively increased expression of TNF-alpha in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-alpha. The collective findings of TNF-alpha overexpression during the course of clinical disease suggest that TNF-alpha may mediate the myelin sheath vacuolation observed in experimental CJD.

Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.

Operant conditioning of H-reflex changes synaptic terminals on primate motoneurons.

Operant conditioning of the primate triceps surae H-reflex, the electrical analog of the spinal stretch reflex, creates a memory trace that includes changes in the spinal cord. To define the morphological correlates of this plasticity, we analyzed the synaptic terminal coverage of triceps surae motoneurons from animals in which the triceps surae H-reflex in one leg had been increased (HRup mode) or decreased (HRdown mode) by conditioning and compared them to each other and to motoneurons from unconditioned animals. Motoneurons were labeled by intramuscular injection of cholera toxin-horseradish peroxidase. A total of 5055 terminals on the cell bodies and proximal dendrites of 114 motoneurons from 14 animals were studied by electron microscopy. Significant differences were found between HRup and HRdown animals and between HRup and naive (i.e., unconditioned) animals. F terminals (i.e., putative inhibitory terminals) were smaller and their active zone coverage on the cell body was lower on motoneurons from the conditioned side of HRup animals than on motoneurons from the conditioned side of HRdown animals. C terminals (i.e., terminals associated with postsynaptic cisterns and rough endoplasmic reticulum) were smaller and the number of C terminals in each C complex (i.e., a group of contiguous C terminals) was larger on motoneurons from the conditioned side of HRup animals than on motoneurons either from the conditioned side of HRdown animals or from naive animals. Because the treatment of HRup and HRdown animals differed only in the reward contingency, the results imply that the two contingencies had different effects on motoneuron synaptic terminals. In combination with other recent data, they show that H-reflex conditioning produces a complex pattern of spinal cord plasticity that includes changes in motoneuron physiological properties as well as in synaptic terminals. Further delineation of this pattern should reveal the contribution of the structural changes described here to the learned change in behavior.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.

Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.

Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions.

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 --> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (alpha-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence of vacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.

Embryonic expression patterns of the neural cell adhesion molecule gene are regulated by homeodomain binding sites.

During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.

Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.