Annika Kylberg

Distinct cellular patterns of upregulated chemokine expression supporting a prominent inflammatory role in traumatic brain injury

Cerebral gene expressions change in response to traumatic brain injury (TBI), and future trauma treatment may improve with increased knowledge about these regulations. We subjected C57BL/6J mice to injury by controlled cortical impact (CCI). At various time points post-injury, mRNA from neocortex and hippocampus was isolated, and transcriptional alterations studied using quantitative real-time polymerase chain reaction (PCR) and gene array analysis. Spatial distribution of enhanced expression was characterized by in situ hybridization. Products of the upregulated transcripts serve functions in a range of cellular mechanisms, including stress, inflammation and immune responses, and tissue remodeling. We also identified increased transcript levels characterizing reactive astrocytes, oligodendrocytes, and microglia, and furthermore, we demonstrated a novel pattern of scattered cell clusters expressing the chemokine Cxcl10. Notably, a sustained increase in integrin alpha X (Itgax), characterizing antigen-presenting dendritic cells, was found with the transcript located to similar cell clusters. In contrast, T-cell receptor alpha transcript showed only a modest increase. The induced P-selectin (Selp) expression level in endothelial cells, and chemokines from microglia, may guide perivascular accumulation of extravasating inflammatory monocytes differentiating into dendritic cells. In conclusion, our study shows that following TBI, secondary injury chiefly involves inflammatory processes and chemokine signaling, which comprise putative targets for pharmaceutical neuroprotection.

POTENTIATING INTERACTIONS BETWEEN MORPHOGENETIC PROTEIN AND NEUROTROPHIC FACTORS IN DEVELOPING NEURONS

mRNA for bone morphogenetic protein receptor type II (BMPR‐II) was mapped to different neurons in peripheral ganglia and spinal cord of the chicken embryo. The expression of this serine/threonine kinase receptor partially overlaps with that of tyrosine kinase receptors Trk and Ret. Biological activities of osteogenic protein‐1 (OP‐1), a documented ligand for BMPR‐II, were tested in explanted embryonic chicken ganglia and dissociated ganglionic neurons. OP‐1 had only a limited stimulatory effect on neuronal survival. However, OP‐1 combined with either neurotrophin‐3 (NT‐3, a relative of nerve growth factor) or glial cell line–derived neurotrophic factor (GDNF) potentiated neuronal survival three‐ to fourfold. We also show that OP‐1 strongly potentiates nerve fiber outgrowth from ganglia stimulated with NT‐3 or GDNF. Signaling by BMPR‐II in neurons may potentiate the tyrosine kinase pathway activated by NT‐3 and GDNF. The data suggest that morphogenetic proteins may modulate neurotrophic activities during neuronal development and plasticity. J. Neurosci. Res. 53:559–568, 1998.

Blocked map kinase activity selectively enhances neurotrophic growth responses

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.

Appearance of Cxcl10-expressing cell clusters is common for traumatic brain injury and neurodegenerative disorders

Traumatic brain injury (TBI) in the mouse results in the rapid appearance of scattered clusters of cells expressing the chemokine Cxcl10 in cortical and subcortical areas. To extend the observation of this unique pattern, we used neuropathological mouse models using quantitative reverse transcriptase‐polymerase chain reaction, gene array analysis, in‐situ hybridization and flow cytometry. As for TBI, cell clusters of 150–200 μm expressing Cxcl10 characterize the cerebral cortex of mice carrying a transgene encoding the Swedish mutation of amyloid precursor protein, a model of amyloid Alzheimer pathology. The same pattern was found in experimental autoimmune encephalomyelitis in mice modelling multiple sclerosis. In contrast, mice carrying a SOD1G93A mutant mimicking amyotrophic lateral sclerosis pathology lacked such cell clusters in the cerebral cortex, whereas clusters appeared in the brainstem and spinal cord. Mice homozygous for a null mutation of the Cxcl10 gene did not show detectable levels of Cxcl10 transcript after TBI, confirming the quantitative reverse transcriptase‐polymerase chain reaction and in‐situ hybridization signals. Moreover, unbiased microarray expression analysis showed that Cxcl10 was among 112 transcripts in the neocortex upregulated at least threefold in both TBI and ageing TgSwe mice, many of them involved in inflammation. The identity of the Cxcl10+ cells remains unclear but flow cytometry showed increased numbers of activated microglia/macrophages as well as myeloid dendritic cells in the TBI and experimental autoimmune encephalomyelitis models. It is concluded that the Cxcl10+ cells appear in the inflamed central nervous system and may represent a novel population of cells that it may be possible to target pharmacologically in a broad range of neurodegenerative conditions.

Genetically modified bone morphogenetic protein signalling alters traumatic brain injury-induced gene expression responses in the adult mouse

Three genetic mouse models were examined to define effects of bone morphogenetic protein (BMP) signalling on gene expression in normal and injured adult brain. CaMKII‐Cre eliminated the BMP receptor Acvr1 (Alk2) and the common TGFβ superfamily signal mediator Smad4 or activated a constitutively active Acvr1 in postnatal forebrain neurons. All mutants followed mendelian ratios, with no overt phenotypic changes. In situ hybridization demonstrated normal patterns of the dendritic marker MAP2 (Mtap2) throughout cortex despite neuron‐specific losses of Acvr1 or Smad4. However, strong up‐regulation of Mtap2 transcript in these mice was found by quantitative RT‐PCR (qRT‐PCR), indicating that Mtap2 is normally suppressed by BMP. Traumatic brain injury (TBI) resulted in increases of histone‐associated DNA fragments in both control and Smad4‐deficient cortex. Several cell‐type‐specific transcripts known to be involved in injury‐related responses were measured by qRT‐PCR. Gfap mRNA was strongly up‐regulated in controls as well as in the loss‐of‐BMP‐signalling mutants. Notably, activated Acvr1 signalling gave significantly lower TBI‐induced up‐regulations of Gfap and Phox2a mRNA levels, indicating reductions in astroglial and neuronal reactions to injury. Strong impairment in injury‐induced Timp1 transcript up‐regulation was also seen in these mice. In contrast, osteopontin (Spp1) transcript levels in activated microglia were not reduced by Acvr1 signalling. Altogether, the data suggest that BMP signalling is dispensable in adult cortical neurons but that augmented BMP signalling affects molecular changes associated with neuronal lesions.

Interacting chemokine signals regulate dendritic cells in acute brain injury.

Brain trauma is known to activate inflammatory cells via various chemokine signals although their interactions remain to be characterized. Mice deficient in Ccl3, Ccr2 or Cxcl10 were compared with wildtype mice after controlled cortical impact injury. Expression of Ccl3 in wildtypes was rapidly upregulated in resident, regularly spaced reactive microglia. Ccl3-deficiency enhanced endothelial expression of platelet selectin and invasion of peripheral inflammatory cells. Appearance of Ccr2 transcripts, encoding the Ccl2 receptor, reflected invasion of lysozyme 2-expressing phagocytes and classical antigen-presenting dendritic cells expressing major histocompatibility complex class II. Ccr2 also directed clustered plasmacytoid dendritic cells positive for the T-cell attracting chemokine Cxcl10. A reduction in Ccr2 and dendritic cells was found in injured wildtype cortex after cyclophosphamide treatment resembling effects of Ccr2-deficiency. The findings demonstrate the feasibility to control inflammation in the injured brain by regulating chemokine-dependent pathways.

Molecular cloning of the chicken trkA and its expression in early peripheral ganglia

The neurotrophin tyrosine kinase receptors trkA, trkB, and trkC have been isolated and sequenced from several mammalian species. Their cognate ligands nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐4 (NT‐4), and neurotrophin‐3 (NT‐3) act as survival and trophic factors for neurons in the peripheral nervous system (PNS). In this study we have focused on the isolation and expression of the chicken trkA homologue. In addition to a near full‐length cDNA sequence described, including an extracellular six amino‐acid motif earlier found in neuronal TrkA in human and rat, a novel insert of 150 base pairs (bp) between subdomains IX and X in the otherwise well‐conserved intracellular kinase domain is reported. Phylogenetic analysis showed the relationship between chicken trkA and the mammalian trkA receptors. Comparisons of the extracellular domains showed some amino‐acid motifs of putative NGF binding function to be well conserved in chicken TrkA. The early expression of trkA mRNA, including the alternatively spliced insert form, was localized by in situ hybridization. As early as embryonal day 3 (E3), trkA mRNA is expressed in the condensing dorsal root ganglia, and at E4 distinct trkA mRNA expression appears in the primary sympathetic chain ganglia. Finally, using a reverse transcriptase‐polymerase chain reaction (RT‐PCR) approach, we found that among several tested growth factors only fibroblast growth factor‐2 (FGF‐2) upregulated trkA mRNA expression in E9 sympathetic ganglion explants. This upregulation of trkA was corroborated by subsequent NGF‐stimulated fiber outgrowth.

Specificity of neurotrophin-3 determined by loss-of-function mutagenesis

Neurotrophin‐3 (NT‐3) is a member of the family of neurotrophic factors, which also includes nerve growth factor (NGF) and which have specific activities on different subsets of vertebrate neurons. The aim of this study was to determine which residues in NT‐3 direct its specificity to the cognate TrkC receptor. It was possible to replace 80% of the residues in NT‐3 with NGF residues without loss of specific activity. Residues D72, Y85, R87, W101, S107, and A111, together with either the residues F12, V18, V20, M37, V42, F54, and K57 or the variable regions IV and V, accounted for the specificity of NT‐3. It is concluded that NGF and NT‐3 use overlapping as well as separated regions for determination of specificities for their cognate receptors TrkA and TrkC, respectively. J. Neurosci. Res. 50:496–503, 1997.

Engineering cells to secrete growth factors

The neutrotrophins stimulate survival and differentiation of a range of target neurons. A wealth of evidence suggests that central cholinergic neurons depend on nerve growth factor (NGF) for trophic support. Grafts of NGF-producing cells rescue axotomized basal forebrain cholinergic neurons and reduce cholinergic cell death in the medial septum. Skeletal muscle cells, immortalized from embryonic day 15 (E15) rat embryos for transplantation purposes, were transfected with a human NGF construct and individual clones tested for NGF production by a biological assay using embryonic sympathetic ganglia. Clone RM22 showed a consistent ability to produce human recombinant NGF in high concentration; RM22 cells were grafted to the rat brain, following fimbria-fornix lesions, in order to examine the influence of these cells on basal forebrain cholinergic neurons. The results suggest that implantation of genetically modified cells, engineered by the introduction of expression plasmids or viral constructs to produce NGF or other neurotrophins may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinsons disease. Moreover, the use of genetically engineered cells may be used to study the effects of administering tailor-made neurotrophins with novel activity profiles.

Normal nigrostriatal innervation but dopamine dysfunction in mice carrying hypomorphic tyrosine hydroxylase alleles

We investigated the use of the mouse tyrosine hydroxylase (TH) gene to drive knock‐in constructs in catecholaminergic neurons. Two targeting constructs representing truncated forms of either of the BMP receptors ALK‐2 or BMPR‐II preceded by an internal ribosome entry site (IRES) were introduced into the 3′ untranslated region of TH. An frt‐flanked neomycin‐resistance (neor) cassette was placed in the 3′ end of the targeting constructs. Mice homozygous for the knock‐in alleles showed various degrees of hypokinetic behavior, depending mainly on whether the neor cassette was removed. In situ hybridization and immunohistochemistry showed that TH mRNA and protein were variously down‐regulated in these mouse strains. Reduced levels of dopamine and noradrenalin were found in several brain areas. However, number and morphology of neurons in substantia nigra and their projections to striatum appeared normal in the neor‐positive TH hypomorphic mice as examined by markers for L‐aromatic amino acid decarboxylase and the dopamine transporter. Elimination of the neor cassette from the knock‐in alleles partially restored TH and dopamine levels. The present neor‐positive TH hypomorphic mice show that nigrostriatal innervation develops independently of TH and should find use as a model for conditions of reduced catecholamine synthesis, as seen in, for example, L‐dihydroxyphenylalanine‐responsive dystonia/infantile parkinsonism.