Jordi Ballabriga

Transforming growth factor-α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal and pathologic brain

Abstract Transforming growth factor α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity is observed in the majority of neurons, and in maturing astrocytes, in the developing and adult brain of humans and different species of animals. TGF-α and EGF-R co-localize in most neurons and maturing astrocytes, suggesting that most TGF-α producing cells are EGF-R-expressing cells. TGF-α and EGF-R immunoreactivity decrease in damaged areas following different insults. However, EGF-R appears in reactive glia, mostly reactive astrocytes, within and surrounding the damaged areas. TGF-α and EGF-R immunoreactivity is found in neurons of patients affected by Alzheimers disease and other forms of dementia, and in neurons of patients suffering from epilepsy owing to different causes, thus pointing to the conclusion that TGF-α does not play a significant role in these pathologies. However, EGF-R immunoreactivity occurs in reactive astrocytes and microglia in subacute but not chronic lesions in human cases. Since TGF-α is a membrane-anchored growth factor, which may be cleaved leading to the formation of soluble forms, and both the membrane-anchored and soluble forms have the capacity to activate the EGF-R, it is feasible that TGF-α in the nervous system may act upon EGF-R-containing neurons through different mechanisms. In addition to distant effects resulting from the release of soluble TGF-α, local effects may be produced by establishing direct cell-to-cell contacts (juxtacrine stimulation), or in cells expressing both TGF-α and EGF-R (autocrine stimulation).

BDNF Up‐Regulates TrkB Protein and Prevents the Death of CA1 Neurons Following Transient Forebrain Ischemia

The neurotrophin family of growth factors, which includes Nerve Growth Factor (NGF), Brain‐Derived Neurotrophic Factor (BDNF), Neurotrophin‐3 (NT3) and Neurotrophin‐4/5 (NT4/5) bind and activate specific tyrosine kinase (Trk) receptors to promote cell survival and growth of different cell populations. For these reasons, growing attention has been paid to the use of neurotrophins as therapeutic agents in neurodegeneration, and to the regulation of the expression of their specific receptors by the ligands. BDNF expression, as revealed by immunohistochemistry, is found in the pre‐subiculum, CA1, CA3, and dentate gyrus of the hippocampus. Strong TrkB immunoreactivity is present in most CA3 neurons but only in scattered neurons of the CA1 area. Weak TrkB immunoreactivity is found in the granule cell layer of the dentate gyrus. Unilateral grafting of BDNF‐transfected fibroblasts into the hippocampus resulted in a marked increase in the intensity of the immunoreaction and in the number of TrkB‐immunoreactive neurons in the granule cell layer of the dentate gyrus, pre‐subiculum and CA1 area in the vicinity of the graft. No similar effects were produced after the injection of control mock‐transfected fibroblasts. Delayed cell death in the CA1 area was produced following 5 min of forebrain ischemia in the gerbil. The majority of living cells in the CA1 area at the fourth day were BDNF/TrkB immunoreactive. Unilateral grafting of control mock‐transfected or BDNF fibroblasts two days before ischemia resulted in a moderate non‐specific protection of TrkB‐negative, but not TrkB‐positive cells, in the CA1 area of the grafted side. This finding is in line with a vascular and glial reaction, as revealed, by immunohistochemistry using astroglial and microglial cell markers. This astroglial response was higher in the grafted side than in the contralateral side in ischemic gerbils, but no differences were seen between BDNF‐producing and non‐BDNF‐producing grafts. However, grafting of BDNF‐producing fibroblasts two days before ischemia significantly and specifically prevented nerve cells from dying in the CA1 area of the ipsilateral hippocampus. Cell survival was associated with increased TrkB immunoreactivity as the majority of living cells were TrkB immunoreactive. Thus, our results show that BDNF is able to up‐regulate the expression of TrkB in control and pathological states, and that BDNF prevention of neuronal death following transient forebrain ischemia is associated with increased expression of its specific receptor.

Kainic acid—induced excitotoxicity is associated with a complex c‐Fos and c‐Jun response which does not preclude either cell death or survival

c-fos and c-jun mRNA induction and c-Fos and c-Jun protein expression were examined in the brains of adult rats subjected to systemic kainic acid (KA) injection at convulsant doses. Induction of c-fos and c-jun mRNA, as seen with in situ hybridization, occurred in the piriform and entorhinal cortices, neocortex, amygdala, hippocampus, dentate gyrus, and discrete thalamic nuclei. This was followed by c-Fos protein expression, as revealed with immunohistochemistry, in the same regions. However, the distribution of c-Jun protein expression differed depending on the antibody used. The distribution of cells immunostained with the antibody c-Jun (AB-1) was similar to that of c-jun mRNA, but the distribution of cells immunostained with the antibody c-Jun/AP1 (N) was restricted to a few neurons in the pyramidal cell layer of CA1 and CA3, layer II of the piriform and entorhinal cortices, basal amygdala, and discrete thalamic nuclei. Although the regional distribution of c-Fos- and c-Jun-immunoreactive cells in the hippocampus, layer II of the entorhinal and piriform cortices, basal amygdala, and discrete thalamic nuclei matched the distribution of cells committed to dying, c-Fos- and c-Jun-immunoreactive cells in the neocortex and dentate gyrus survived. Therefore, the present data show that c-fos and c-jun are not predictors of either cell death or survival, but rather, markers of cells sensitive to KA excitotoxicity. Western blots to c-Fos showed a double band at p62 in samples containing the hippocampus and entorhinal and piriform cortices (hip samples) and in samples containing the neocortex (cortex samples). The upper band was abolished following preincubation of the samples with alkaline phosphatase, thus suggesting c-Fos phosphorylation. Western blots to c-Jun (AB-1) showed a single band at about p39 in hip and cortex. However, Western blots to c-Jun/AP1 (N) identified two bands. One band at about p39 was seen in control rats and the cortex of KA-treated rats. Another band at p26 was observed only in hip samples of KA-treated rats. In addition, decreased c-Jun N-terminal kinase 1 (JNK-1) expression, as revealed on Western blots, was coincidental with the appearance of the p26 c-Jun-immunoreactive band in KA-treated rats. These results show that c-Fos and different Jun-related antigens are expressed following KA excitotoxicity, and that posttranslational modifications involving phosphorylation of c-Fos and Jun(s) may occur following KA injection. These results also stress the necessity of examining the composition of Fos and Jun-related antigens and the metabolic state of Fos and Jun(s) in different experimental models of nervous system injury.

bFGF and FGFR-3 immunoreactivity in the rat brain following systemic kainic acid administration at convulsant doses: localization of bFGF and FGFR-3 in reactive astrocytes, and FGFR-3 in reactive microglia

Strong bFGF immunoreactivity was observed in reactive astrocytes, as shown by double-labeling immunohistochemistry of bFGF and GFAP, from days 7 up to 30 (last time point examined) following kainic acid (KA) injection at convulsant doses in the adult rat. bFGF was not found in OX-42-positive reactive microglia. A few reactive glia co-localized FGFR-3 and GFAP, whereas the majority of cells expressing FGFR-3 were OX-42-immunoreactive. This was further supported by the observation that only approximately 10% of reactive glia co-localized bFGF and FGFR-3. These results show that reactive astrocytes are a major source of bFGF during the subacute stages of tissue damage following KA injection and that reactive astrocytes and, most particularly, reactive microglia are putative targets of bFGF through FGFR-3.

Bcl-2, Bax, and Bcl-x expression in the CA1 area of the hippocampus following transient forebrain ischemia in the adult gerbil

Abstract Delayed neuronal death was produced in the CA1 area of the hippocampus following 5 min of forebrain ischemia in adult gerbils. Immunohistochemistry and Western blotting to Bcl-2, Bax, and Bcl-x was examined in control (age-matched, non-operated and sham-operated) and ischemic gerbils. Bcl-2 immunoreactivity was low in CA1 neurons, but Bax was highly expressed in CA1 neurons of control gerbils. Moderate Bcl-x immunoreactivity was observed in control CA1 neurons. Strong Bcl-2 and Bcl-x immunoreactivity was found in CA1 neurons following ischemia. Bcl-2, Bax, and Bcl-x were localized in dying cells, thus suggesting that expression of Bcl-2 was not sufficient to prevent nerve cells from dying. Although the Bcl-x antibody does not discriminate between Bcl-xL and Bcl-xS content in tissue sections, Western blots disclosed a marked increase in the intensity of the band corresponding to Bcl-xS, but not of the band corresponding to Bcl-xL in ischemic hippocampi, thus indicating that the increase in Bcl-xS is associated with delayed cell death following transient forebrain ischemia in the adult gerbil.

Bcl-2, Bax and Bcl-x expression following hypoxia-ischemia in the infant rat brain

Abstract Severe hypoxic-ischemic cerebral damage was produced in 8-day-old rats following permanent bilateral carotid artery occlusion and 15 min of ischemia. Cellular damage consisted of early necrosis and appearance of cells with apoptotic-like morphology (karyorrhectic cells) and cells with granular chromatin degeneration in the cerebral cortex, hippocampus, thalamus, striatum and amygdala. Expression of Bcl-2, Bax and Bcl-x was examined in control and hypoxic-ischemic rats using immunohistochemistry and Western blotting. Members of the Bcl-2 family were expressed in the vast majority of, if not all, neurons in control pups. Bcl-2, Bax and Bcl-x immunoreactivity decreased in necrotic cells, but about 60% of cells with apoptotic-like morphology and cells with granular chromatin degeneration were stained with antibodies to Bcl-2, Bax or Bcl-x. Although the total number of stained cells decreased with time, recruitment of cells with apoptotic morphology and cells with granular chromatin degeneration was still found up to 48 h. Western blots showed a band at about p28 and p21, respectively for Bcl-2 and Bax in control and hypoxic-ischemic pups at 6, 12 and 24 h. Two bands at about p37 and p30, representing Bcl-xL and Bcl-xS, respectively, were found in samples stained with antibodies to Bcl-x. No gross changes in the intensity of these bands were observed in ischemic pups at 6, 12 and 24 h, except for a slight decrease in Bcl-2 at 24 h, and a slight and inconstant increase of the putative Bcl-xS at 12 h. These results suggest that Bcl-2, Bax, Bcl-xL and Bcl-xS do not play a leading role in the fate of damaged nerve cells following a severe hypoxic-ischemic insult of the developing brain.

MULTIPLE NEUROTROPHIC SIGNALS CONVERGE IN SURVIVING CA1 NEURONS OF THE GERBIL HIPPOCAMPUS FOLLOWING TRANSIENT FOREBRAIN ISCHEMIA

Delayed cell death involving the CA1 area of the hippocampus was produced following 5 minutes of transient forebrain ischemia in gerbils. Cell death mainly affected CA1 pyramidal neurons, whereas parvalbumin‐immunoreactive (parv‐ir) cells were spared. Synaptophysin immunoreactivity was observed in the strata oriens and radiatum of CA1 for months, although immunoreactivity decreased in gerbils surviving 1 year post‐ischemia. Golgi studies disclosed a few pyramidal neurons with dendrites, variably covered with dendritic spines, in the CA1 area of 1‐year surviving gerbils. In the normal gerbil, the majority of CA1 neurons expressed brain‐derived neurotrophic factor (BDNF), tyrosine protein kinase C (TrkC), fibroblast growth factor receptor 1 (Flg), transforming growth factor‐alpha (TGF‐alpha), and epidermal growth factor‐receptor (EGF‐R), but only a minority of cells were tyrosine protein kinase B (TrkB)‐immunoreactive. Marked reduction in the number of BDNF‐, TrkC‐, Flg‐, TGF‐alpha‐, and EGF‐R‐ir cells was observed in CA1 from 24 hours to 1 year after ischemia. In contrast, TrkB‐ir cells survived the ischemic insult. Double‐labeling immunohistochemistry disclosed that about 90% of surviving BDNF‐ir and 85% of TrkB‐ir neurons co‐localized parvalbumin in the CA1 area. In control gerbils, only about 5% of BDNF‐ir cells in CA1 co‐expressed TrkB. However, TrkB co‐localized in about 95% of surviving BDNF‐ir neurons in CA1 in ischemic gerbils. In addition, parvalbumin was co‐expressed in about 90% of TrkC‐, 95% Flg‐, and 85% EGF‐R‐ir surviving neurons in the stratum pyramidale of CA1. Finally, basic fibroblast growth factor (bFGF) was expressed by reactive astrocytes from day 4 onwards. These data show that the subpopulation of TrkB‐/parv‐ir neurons in CA1 survive the ischemic episode and that multiple neurotrophic signals converge in surviving neurons of the gerbil hippocampus following transient forebrain ischemia. J. Comp. Neurol. 394:416–430, 1998.

TrkA immunoreactivity in reactive astrocytes in human neurodegenerative diseases and colchicine-treated rats

Abstract It has been shown that nerve growth factor (NGF) administration is capable of curbing tissue damage in several neurodegenerative disorders. As a first step to learning about the possible functional role of NGF in the astroglial response during neurodegeneration, we have analyzed the expression of the functional receptor for NGF, TrkA, in human neurodegenerative diseases which are accompanied by reactive astrocytosis, as well as in human astrocytomas. We have compared these results with those observed in reactive astrocytes following colchicine-induced cellular damage to adult rats. In the human brain, strong TrkA immunoreactivity is observed in reactive astrocytes in a number of unrelated diseases, including Alzheimer’s disease, Huntington’s disease, progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, multifocal leukoencephalopathy and residual hypoxic encephalopathy. Neoplastic astrocytes in grade II and III astrocytomas display strong TrkA immunoreactivity. In the rat brain, reactive astrocytes following mechanical needle injury and colchicine administration show strong TrkA immunoreactivity. The presence of TrkA receptors in reactive astrocytes from different human neurodegenerative diseases and experimentally induced models in rats, and in neoplastic astrocytes suggests that NGF may participate in the astroglial response to different types of injury and neoplastic proliferation. Since astroglial cells are capable of producing NGF, it is plausible that this neurotrophin may function as an autocrine or paracrine factor in TrkA-expressing reactive and neoplastic glial cells.

Transient forebrain ischemia in the adult gerbil is associated with a complex c-Jun response

C-JUN expression in the hippocampus of gerbils subjected to 5 min of transient forebrain ischemia was examined with immunohistochemistry and western blotting using two c-Jun antibodies raised against two different amino acid sequences. Both c-Jun antibodies showed increased immunoreactivity at 6 and 12 h postischemia in the stratum pyramidale of CA3 and granule cell layer of the dentate gyrus. No immunostaining was detected in CA1 up to the 7th day. Western blots showed increased c-Jun immunoreactivity at 6 and 12 h. However, the antibody c-Jun (AB-1) detected a single band at about p39 in normal and post-ischemic states, whereas the antibody c-Jun/AP-1 (N) recognized a band at about p39 in normal and post-ischemic gerbils, and a p62 phosphorylated double-band at 6 and 12 h following ischemia. In addition, increased c-Jun N-terminal kinase-1 (JNK-1) expression was observed on western blots at 6 and 12 h postischemia. These results suggest that different c-Jun-related responses, some of which probably indicate post-translational changes of the c-Jun protein, occur in the hippocampus of the gerbil following transient forebrain ischemia.

BDNF and TrkB co-localize in CA1 neurons resistant to transient forebrain ischemia in the adult gerbil.

Delayed cell death of projection cells in the CA1 area of the hippocampus is produced in the adult gerbil following 5 minutes (min) of transient forebrain ischemia. Parvalbumin-immunoreactive local-circuit neurons are resistant to the ischemic insult. Brain-Derived Neurotrophic Factor (BDNF) immunoreactivity is localized in all neurons of the CA1 area in control gerbils. However, TrkB immunoreactivity is observed in a minority of BDNF-immunoreactive neurons in the CA1 area. The number of BDNF-immunoreactive cells in CA1 is dramatically reduced in ischemic gerbils as early as 24 h after ischemia, but the number of TrkB-immunoreactive cells in the CA1 area is maintained following ischemia. Moreover, about 90% of BDNF-immunoreactive cells and about 85% of TrkB-immunoreactive cells in ischemic gerbils co-localize the calcium-binding protein parvalbumin. Finally, BDNF and TrkB are coexpressed in about 95% of CA1 neurons surviving the ischemic insult. These results indicate that a subpopulation of CA1 hippocampal neurons coexpressing TrkB, parvalbumin and BDNF is resistant to transient forebrain ischemia in the gerbil. These results also suggest that a subpopulation of CA1 hippocampal neurons in the gerbil hippocampus is endowed with a putative BDNF/TrkB autocrine regulatory loop that may be involved in both cell survival and synaptic remodeling of the damaged gerbil hippocampus following transient forebrain ischemia.