Brain-derived neurotrophic factor reduces cortical cell death by ischemia after middle cerebral artery occlusion in the rat.

Stroke is the major cause of adult brain dysfunction. In an experimental approach to evaluate the possible beneficial effects of administration of neurotrophic factors in stroke, we have used a model of distal middle cerebral artery (MCA) occlusion in adult rats. In this model, we found: (1) a permanent reduction of brain-derived neurotrophic factor (BDNF) and its full-length receptor, TrkB, in the infarcted core; (2) a transient increase in BDNF immunoreactivity in the internal region of the border of the infarct (penumbra area) at 12 h after MCA occlusion; (3) increased truncated TrkB immunoreactivity in astrocytes surrounding the area of the infarction; and (4) increased full-length TrkB immunoreactivity in scattered neurons, distant from the infarct, in ipsilateral and contralateral cortices at 24 and 48 h after MCA occlusion. We next studied the regulation of TrkB expression by BDNF, after ischemia, and its neuroprotective effects in vivo. In control non-ischemic rats, grafting of mock- or BDNF-transfected fibroblasts (F3A-MT or F3N-BDNF cell lines, respectively) in the medial part of the somatosensory cortex increased truncated TrkB immunoreactivity in neighboring astrocytes. Grafting alone also increased full-length TrkB in the vicinity of the mock graft (at 24 and 48 h) and the BDNF-grafted graft (at 4 days). Interestingly, ischemic animals grafted with the mock-transfected cell line did not show any further regulation of TrkB receptors. However, ischemic animals grafted with the BDNF cell line showed an up-regulation of full-length TrkB expression in neurons located in the internal border of the infarct. Analysis of nuclear DNA fragmentation in situ, combined with microtubule-associated protein 2 immunohistochemistry, revealed that most cells dying in the borders of the infarct (penumbra area) at 48 h following MCA occlusion were neurons. No differences in the infarct size were found between MCA occluded, mock-transfected MCA-occluded, and BDNF-transfected MCA-occluded rats. Moreover, cell death was similar in nongrafted and mock-grafted rats subjected to MCA occlusion. However, the number of cells with nuclear DNA breaks was significantly reduced in the penumbra area close to the BDNF graft in ischemic rats. Thus, our results show that BDNF specifically up-regulates its full-length TrkB receptor in cortical neurons of the penumbra area and prevents their death in an in vivo model of focal ischemia.

Phosphorylated map kinase (ERK1, ERK2) expression is associated with early tau deposition in neurones and glial cells, but not with increased nuclear DNA vulnerability and cell death, in Alzheimer disease, Pick's disease, progressive supranuclear palsy and corticobasal degeneration.

Abnormal tau phosphorylation and deposition in neurones and glial cells is one of the major features in taupathies. The present study examines the involvement of the Ras/MEK/ERK pathway of tau phosphorylation in Alzheimer disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), by Western blotting, single and double-labelling immunohistochemistry, and p21Ras activation assay. Since this pathway is also activated in several paradigms of cell death and cell survival, activated ERK expression is also analysed with double-labelling immunohistochemistry and in situ end-labelling of nuclear DNA fragmentation to visualise activated ERK in cells with increased nuclear DNA vulnerability. The MEK1 antibody recognises one band of 45 kD that identifies phosphorylation-independent MEK1, whose expression levels are not modified in diseased brains. The ERK antibody recognises one band of 42 kD corresponding to the molecular weight of phosphorylation-independent ERK2; the expression levels, as well as the immunoreactivity of ERK in individual cells, is not changed in AD, PiD, PSP and CBD. The antibody MAPK-P distinguishes two bands of 44 kD and 42 kD that detect phosphorylated ERK1 and ERK2. MAPK-P expression levels, as seen with Western blotting, are markedly increased in AD, PiD, PSP and CBD. Moreover, immunohistochemistry discloses granular precipitates in the cytoplasm of neurones in AD, mainly in a subpopulation of neurones exhibiting early tau deposition, whereas neurones with developed neurofibrillary tangles are less commonly immunostained. MAPK-P also decorates neurones with Pick bodies in PiD, early tau deposition in neurones in PSP and CBD, and cortical achromatic neurones in CBD. In addition, strong MAPK-P immunoreactivity is found in large numbers of tau-positive glial cells in PSP and CBD, as seen with double-labelling immunohistochemistry. Yet no co-localisation of enhanced phosphorylated ERK immunoreactivity and nuclear DNA fragmentation is found in AD, PiD, PSP and CBD. Finally, activated Ras expression levels are increased in AD cases when compared with controls. These results demonstrate increased phosphorylated (active) ERK expression in association with early tau deposition in neurones and glial cells in taupathies, and suggest activated Ras as the upstream activator of the MEK/ERK pathway of tau phosphorylation in AD.

Methylazoximethanol acetate-induced cell death in the granule cell layer of the developing mouse cerebellum is associated with caspase-3 activation, but does not depend on the tissue-type plasminogen activator.

Methylazoximethanol (MAM) acetate-induced cell death in the external granule cell layer of the developing cerebellum affects clusters of cells with morphological features of apoptosis. This is accompanied by selective induction of active caspase-3 expression and increased c-Jun/AP-1 (N) immunoreactivity in dying cells, as revealed with immunohistochemistry. Since the antibody to cJun/AP-1 (N) cross-reacts with epitopes emerging after caspase-mediated proteolysis during apoptosis, these results indicate that MAM-induced cell death is associated with active caspase-3 expression and function in dying cells. In order to investigate the involvement of tissue-type plasminogen activator (tPA), which has been implicated in certain forms of neuronal cell death, MAM-induced cell death has been examined in tPA-/- and tPA+/+ mice. No differences in the number of dying cells, as seen with haematoxylin and eosin staining and in situ end-labelling of fragmented nuclear DNA-processed sections, were seen between tPA-/- and tPA+/+ mice. These results indicate that tPA is not involved in MAM-induced cell death in the developing brain.

Brain-derived neurotrophic factor in Huntington disease.

Trophic factors, administered systemically or delivered via genetically-modified cells grafted into target regions, have been proposed as putative therapeutic agents in human neurodegenerative disorders. In parallel to the study of the beneficial effects in experimental models of particular diseases, a crucial aspect of the study of trophic factors is the gathering of information about the actual trophic factor expression in human diseased states. Brain-derived neurotrophic factor (BDNF) promotes survival and growth of various nerve cell populations during normal development and following various insults in the developing and adult brain. In particular, BDNF prevents cell death of certain striatal populations in excitotoxic models of Huntington disease (HD) following intrastriatal injection of quinolinic acid to the adult rodent brain. The present study examines BDNF expression, by gel electrophoresis and Western blotting, and immunohistochemistry, in the brains of patients who had suffered from HD. Reduced BDNF expression, ranging from 53 to 82%, has been found in the caudate and putamen in HD when compared with age-matched controls. No modifications in BDNF expression levels have been seen in the parietal cortex, temporal cortex and hippocampus. Furthermore, immunohistochemistry has shown reduced BDNF immunoreactivity in caudate neurons, but not in cortical neurons in HD when compared with controls. These data demonstrate selective BDNF decay in regions that are vulnerable to HD, and suggest, in combination with results in experimental models, that a BDNF surplus may have beneficial effects in the treatment of HD.

Expression of synaptic proteins in the developing rat cerebellum following ionizing radiation.

Various proteins regulating neurotransmission release and synaptic vesicle exocytosis have been implicated in axonal elongation and synaptic maturation. In the present study, immunohistochemistry to the presynaptic membrane proteins syntaxin-I and synaptosomal-associated protein of 25 kDa (SNAP-25) synaptic vesicle-associated proteins synaptophysin and synapsin-I and the neuronal maturation and axonal growth-related protein GAP-43, has been carried out in the normal developing cerebellum and following a single dose of ionizing radiation (2 Gy gamma-rays) at postnatal day 1. Our aim has been to learn about the morphological and possible functional modalities that occur during the progression of neuronal connectivity in normal and abnormal development. Expression of all these proteins is associated with the arrival of afferents in the subcortical white matter and with the maturation of the internal granule cell layer and molecular layer during normal development. In addition, SNAP-25 and GAP-43 are strongly expressed in granule cells of the external granule cell layer, thus suggesting that these proteins are involved in cell elongation of granule cells. Apoptosis appears at 3 h and peaks at 6 h following ionizing radiation. Radiation-induced apoptosis in the external granule cell layer produces a transient decrease in the expression of SNAP-25 and GAP-43 in the external granule cell layer. The external granule cell layer recovers at 48 h and external granule cells of proliferating cells also express SNAP-25 and GAP-43, thus indicating that proliferating cells in this layer are equipped with proteins involved in cell elongation. Furthermore, expression of synaptophysin, synapsin-I, syntaxin-I and SNAP-25 is the same in the cerebellum of irradiated and normal rats from this time to adulthood (3 months). These results point to the likelihood that recovery of the cerebellar cortex occurs following a single exposure of ionizing radiation during postnatal development.

BDNF and full-length and truncated TrkB expression in Alzheimer disease. Implications in therapeutic strategies.

Brain-derived neurotrophic factor (BDNF), and full-length and truncated tyrosin kinase B receptor (TrkB) protein expression were examined by Western blotting and immunohistochemistry in the frontal cortex and hippocampus of individuals affected by long-lasting severe Alzheimer disease (AD) and age-matched controls. Since preliminary processing studies in the brains of rats have shown loss of immunoreactivity depending on the postmortem delay in tissue processing and on the type, duration, and temperature of the fixative solution, only human samples obtained up to 6 hours (h) after death for biochemical and morphological studies and fixed by immersion in 4% paraformaldehyde for 24 h for morphological studies were included in the present series. Decreased BDNF and full-length TrkB expression accompanied by increased truncated TrkB expression, as revealed by Western blotting, was observed in the frontal cortex of patients with AD. Immunohistochemistry disclosed reduced BDNF and full-length TrkB immunoreactivity in neurons. BDNF decrease was equally observed in tangle-bearing and non-tangle-bearing neurons, as revealed with double-labeling immunohistochemistry to BDNF and phosphorylated tau or phosphorylated neurofilament epitopes. Full-length TrkB immunoreactivity was largely decreased in tangle-bearing neurons, whereas only moderate decreases occurred in neurons with granulovacuolar degeneration. Strong BDNF immunoreactivity was observed in dystrophic neurites surrounding senile plaques, whereas strong TrkB expression occurred in reactive glial cells, including those surrounding senile plaques. Finally, truncated TrkB immunoreactivity was observed in individual neurons and in reactive glial cells in the cerebral cortex and white matter in AD. These results show decay in the expression of BDNF and TrkB in AD neurons, accompanied by altered BDNF, and full-length and truncated TrkB expression in dystrophic neurites and reactive glial cells, respectively, in this disease. The present results demonstrate selective decline of the BDNF/TrkB neurotrophic signaling pathway in the frontal cortex and hippocampus in AD and provide supplemental data that may be relevant in discussing the suitability of the use of BDNF as a therapeutic agent in patients with AD.

Brain-derived neurotrophic factor does not prevent ionizing radiation-induced apoptosis in the developing rat brain.

In an attempt to learn whether brain-derived neurotrophic factor (BDNF) is able to protect neural cells from ionizing radiation-induced apoptosis in the developing brain, BDNF-transfected (F3N-BDNF) and mock-transfected (F3A MT) fibroblasts were grafted into the right germinal periventricular zone of rats at postnatal day 1 (P1). The rats were later irradiated at P3 with a single dose (2 Gy whole body irradiation) of gamma rays. Age-matched non-irradiated and non-grafted irradiated rats at P3 were used as controls. No differences in the number of apoptotic cells were observed between the ipsilateral and contralateral side of either F3N-BDNF- or F3A-MT-grafted irradiated rats Furthermore, counts of apoptotic cells were similar in non-grafted irradiated and grafted irradiated rats. These results show that BDNF has no capacity to prevent ionizing radiation-induced apoptosis in the developing rat brain.

BDNF, and full length and truncated TrkB expression in the hippocampus of the rat following kainic acid excitotoxic damage. Evidence of complex time-dependent and cell-specific responses.

Systemic administration of kainic acid (KA) at convulsant doses results in irreversible cell damage and neuron loss in the hilus of the dentate gyrus and in the CA1 area of the hippocampus. This is followed by reactive astrocytosis in these regions, and sprouting of mossy fibers into the molecular layer of the dentate gyrus. Since trophic factors are probably implicated in the cellular responses to the excitotoxic insult, and early induction of BDNF and TrkB mRNAs has been observed following KA injection, the present study examines BDNF, full-length and truncated TrkB protein expression in the hippocampus, as revealed by immunohistochemistry, up to 30 days following KA administration to adult rats. Reduction in BDNF and full-length TrkB immunoreactivity preceding neuron loss is observed in the damaged areas. However, transient increase in BDNF immunoreactivity is observed in surviving CA1 neurons and in granule cells of the dentate gyrus. In contrast, full-length TrkB immunoreactivity progressively increases in the molecular layer of the dentate gyrus up to day 30 following KA administration. A second peak in BDNF immunoreactivity is observed in reactive astrocytes, as revealed with double-labeling immunohistochemistry to BDNF and GFAP, in the plexiform layers of CA1 and, to a lesser degree, in the molecular layer of the dentate gyrus. In addition, strong truncated TrkB immunoreactivity is found in reactive astrocytes, as revealed with double-labeling immunohistochemistry to truncated TrkB and GFAP, in the same regions. These results, in concert with previous observations in the same model of hippocampal damage, suggest that BDNF participates in the early response to excitotoxic damage, and that expression of full-length TrkB at strategic sites in the molecular layer of the dentate gyrus has a role in the regenerative response linked to mossy fiber sprouting. Interestingly, delayed expression of BDNF and truncated TrkB in reactive astrocytes may act as negative regulators of neurite growth in devastated regions, such as the CA1 area, which are impoverished of putative postsynaptic sites.