Laura Korhonen

Estrogen-Receptor-Dependent Regulation of Neural Stem Cell Proliferation and Differentiation

Estrogen has profound effects on function and plasticity of the nervous system. Receptors for estrogen (ERs) are expressed by neurons in several areas of the brain. Here we demonstrate that embryonic and adult rat neural stem cells (NSC) express ERalpha and ERbeta, 17beta-Estradiol treatment decreased the proliferation of NSC stimulated by epidermal growth factor (EGF), which was due to the upregulation of the cyclin-dependent kinase (CDK) inhibitor, p21(Cip1). The modulatory effect of 17beta-estradiol on EGF was more pronounced in adult NSC. However, 17beta-estradiol alone increased the proliferation of embryonic, but not adult, NSC. The effect of 17beta-estradiol was inhibited by the ER antagonist, ICI-182780, showing an involvement of ERs. 17beta-Estradiol also increased the ratio of neurons to glia cells in embryonic NSC, but not in adult NSC, suggesting an influence on neurogenesis during embryonic development. The data show that estrogen, via ER, affects the proliferation and differentiation of NSC cells, probably acting in conjunction with other factors governing NSC development.

NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and -independent pathways

Inhibitor‐of‐apoptosis proteins (IAPs), including neuronal apoptosis inhibitory protein (NAIP), inhibit cell death. Other IAPs inhibit key caspase proteases which effect cell death, but the mechanism by which NAIP acts is unknown. Here we report that NAIP, through its third baculovirus inhibitory repeat domain (BIR3), binds the neuron‐restricted calcium‐binding protein, hippocalcin, in an interaction promoted by calcium. In neuronal cell lines NSC‐34 and Neuro‐2a, over‐expression of the BIR domains of NAIP (NAIP‐BIR1–3) counteracted the calcium‐induced cell death induced by ionomycin and thapsigargin. This protective capacity was significantly enhanced when NAIP‐BIR1–3 was co‐expressed with hippocalcin. Over‐expression of the BIR3 domain or hippocalcin alone did not substantially enhance cell survival, but co‐expression greatly increased their protective effects. These data suggest synergy between NAIP and hippocalcin in facilitating neuronal survival against calcium‐induced death stimuli mediated through the BIR3 domain. Analysis of caspase activity after thapsigargin treatment revealed that caspase‐3 is activated in NSC‐34, but not Neuro‐2a, cells. Thus NAIP, in conjunction with hippocalcin, can protect neurons against calcium‐induced cell death in caspase‐3‐activated and non‐activated pathways.

19-Nortestosterone influences neural stem cell proliferation and neurogenesis in the rat brain.

Abuse of androgenic anabolic steroids can affect brain function leading to behavioural changes. In this study, the effects of the testosterone analogue, 19‐nortestosterone, on rat neural stem cells was examined. The androgen receptor is expressed by cultured embryonic and adult neural stem cells, and is also present in the ventricular epithelium during development and in the adult brain in, among others, dentate gyrus. In neural stem cells stimulated with epidermal growth factor, nandrolone reduced cell proliferation, especially in adult ones. The decrease was abolished by flutamide, a receptor antagonist. Nandrolone also decreased the BrdU labelling of neural stem cells in the dentate gyrus, demonstrating an effect of the hormone on cell proliferation in vivo. The effect of nandrolone was observed with both female and male rats but it was more pronounced in pregnant rats, indicating an involvement of oestrogen in nandrolone action. Nandrolone also decreased the number of newly born neuronal cells in the dentate gyrus of male rats. The results show that nandrolone has important effects on the proliferation and differentiation of neural stem cells expressing the cognate androgen receptor. The data show that the use of nandrolone may severely affect the formation of neural stem cells and could therefore have long‐term negative consequences in the brain.

Regulation of X-chromosome-linked inhibitor of apoptosis protein in kainic acid-induced neuronal death in the rat hippocampus

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an antiapoptotic protein which inhibits the activity of caspases and suppresses cell death. However, little is known about the presence and function of XIAP in the nervous system. Here we report that XIAP mRNA is expressed in developing and adult rat brain. Using a specific antibody, we observed XIAP-immunoreactive cells in different brain regions, among others, in the hippocampus and cerebral cortex. Kainic acid, which induces delayed cell death of specific neurons, increased the levels of XIAP in the CA3 region of hippocampus. XIAP was, however, largely absent in cells undergoing cell death, as shown by TUNEL labeling and staining for active caspase-3. In cultured hippocampal neurons, XIAP was initially upregulated by kainic acid and then degraded in a process blocked by the caspase-3 inhibitor DEVD. Similarly, recombinant XIAP is cleaved by active caspase-3 in vitro. The results show that there is biphasic regulation of XIAP in the hippocampus following kainic acid and that XIAP becomes a target for caspase-3 activated during cell death in the hippocampus. The degradation of XIAP by kainic acid contributes to neuronal cell death observed in vulnerable neurons of the hippocampus after caspase activation.

MIR Is a Novel ERM-like Protein That Interacts with Myosin Regulatory Light Chain and Inhibits Neurite Outgrowth

The ERM protein family members ezrin, radixin, and moesin are cytoskeletal effector proteins linking actin to membrane-bound proteins at the cell surface. Here we report on the cloning of myosin regulatory light chain interacting protein (MIR), a protein with an ERM-homology domain and a carboxyl-terminal RING finger, that is expressed, among other tissues, in brain. MIR is distributed in cultured COS cells, in a punctated manner as shown using enhanced green fluorescent protein (EGFP)-tagged MIR and by staining with a specific antibody for MIR. In the yeast two-hybrid system and in transfected COS cells, MIR interacts with myosin regulatory light chain B, which in turn regulates the activity of the actomyosin complex. Overexpression of MIR cDNA in PC12 cells abrogated neurite outgrowth induced by nerve growth factor (NGF) without affecting TrkA signaling. The results show that MIR, a novel ERM-like protein, affects cytoskeleton interactions regulating cell motility, such as neurite outgrowth.

Expression of c-Met in developing rat hippocampus: evidence for HGF as a neurotrophic factor for calbindin D-expressing neurons.

Hepatocyte growth factor‐scatter factor (HGF) is expressed in different parts of the nervous system, and has been shown to exhibit neurotrophic activity. Here we show that c‐Met, the receptor for HGF, is expressed in developing rat hippocampus, with the highest levels during the first postnatal weeks. To study the function of HGF, hippocampal neurons were prepared from embryonic rats and treated with different HGF concentrations. In these cultures, HGF increased the number of neurons expressing the 28‐kDa calcium‐binding protein (calbindin D) in a dose‐dependent manner. The effect of HGF was larger than that observed with either brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 (NT‐3), and cotreatment of the cultures with HGF and the neurotrophins was additive with respect to calbindin D neurons. Besides affecting the number of neurons, HGF significantly increased the degree of sprouting of calbindin D‐positive neurons, suggesting an influence on neuronal maturation. BDNF and NT‐3 stimulated neurite outgrowth of calbindin D neurons to a much smaller degree. In contrast to calbindin D neurons, HGF did not significantly increase the number of neurons immunoreactive with the neurotransmitter γ‐aminobutyric acid (GABA) in the hippocampal cultures. Immunohistochemical studies showed that c‐Met‐, calbindin D‐ and HGF‐immunoreactive cells are all present in the dentate gyrus and partly colocalize within neurons. These results show that HGF acts on calbindin D‐containing hippocampal neurons and increases their neurite outgrowth, suggesting that HGF plays an important role for the maturation and function of these neurons in the hippocampus.

Partial resistance to malonate-induced striatal cell death in transgenic mouse models of Huntington's disease is dependent on age and CAG repeat length

Transgenic Huntingtons disease (HD) mice, expressing exon 1 of the HD gene with an expanded CAG repeat, are totally resistant to striatal lesion induced by excessive NMDA receptor activation. We now show that striatal lesions induced by the mitochondrial toxin malonate are reduced by 70–80% in transgenic HD mice compared with wild‐type littermate controls. This occurred in 6‐ and 12‐week‐old HD mice with 150 CAG repeats (line R6/2) and in 18‐week‐old, but not 6‐week‐old, HD mice with 115 CAG repeats (line R6/1). Therefore, we show for the first time that the resistance to neurotoxin in transgenic HD mice is dependent on both the CAG repeat length and the age of the mice. Importantly, most HD patients develop symptoms in adulthood and exhibit an inverse relationship between CAG repeat length and age of onset. Transgenic mice expressing a normal CAG repeat (18 CAG) were not resistant to malonate. Although endogenous glutamate release has been implicated in malonate‐induced cell death, glutamate release from striatal synaptosomes was not decreased in HD mice. Malonate‐induced striatal cell death was reduced by 50–60% in wild‐type mice when they were treated with either the NMDA receptor antagonist MK‐801 or the caspase inhibitor zVAD‐fmk. These two compounds did not reduce lesion size in transgenic R6/1 mice. This might suggest that NMDA receptor‐ and caspase‐mediated cell death pathways are inhibited and that the limited malonate‐induced cell death still occurring in HD mice is independent of these pathways. There were no changes in striatal levels of the two anti cell death proteins Bcl‐XL and X‐linked inhibitor of apoptosis protein (XIAP), before or after the lesion in transgenic HD mice. We propose that mutant huntingtin causes a sublethal grade of metabolic stress which is CAG repeat length‐dependent and results in up‐regulation over time of cellular defense mechanisms against impaired energy metabolism and excitotoxicity.

Pituitary adenylate cyclase‐activating polypeptide promotes the survival of basal forebrain cholinergic neurons in vitro and in vivo: comparison with effects of nerve growth factor

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a member of the vasointestinal polypeptide gene family for which neurotrophic activity has been postulated. PACAP mRNA is expressed in the developing and adult hippocampus, which is the principal target region of septal cholinergic neurons. We therefore studied the effects of PACAP on septal cholinergic neurons. In primary cultures from septum of embryonic and postnatal rats, PACAP increased the number of neurons immunohistochemically stained for the low‐affinity nerve growth factor (NGF) receptor p75 and for the enzyme choline acetyltransferase (ChAT). PACAP also caused a corresponding increase in ChAT activity. In comparison, NGF had a greater effect than PACAP on the number of p75‐ and ChAT‐positive neurons in these cultures. In vivo, following fimbria fornix transection, the number of immunohistochemically stained septal cholinergic neurons fell significantly to 18% in rats given continuous intracerebroventricular infusion of vehicle, whereas in rats given NGF the number of these neurons did not differ significantly from unoperated controls. In PACAP‐treated rats the number was 48% of unoperated values, which represented a significant increase compared with vehicle‐treated rats and a significant decrease compared with NGF‐treated rats or unoperated controls. Double‐staining experiments revealed that most ChAT‐positive neurons in rat medial septum also express PACAP receptor 1. Together the results show that PACAP promotes the survival of septal cholinergic neurons in vitro, and after injury in vivo, suggesting that PACAP acts as a neurotrophic factor influencing the development and maintenance of these neurons.

Hippocalcin protects against caspase-12-induced and age-dependent neuronal degeneration

Hippocalcin is a neuronal calcium binding protein, but its physiological function in brain is unknown. We show here that hippocampal neurons from hippocalcin-deficient mice are more vulnerable to degeneration, particularly using thapsigargin, elevating intracellular calcium. Caspase-12 was activated in neurons lacking hippocalcin, while calpain was unchanged. Neuronal viability was accompanied by endoplasmic reticulum (ER) stress and a change in the relative induction of the ER chaperone, BiP/GRP78. Neuronal apoptosis inhibitor protein (NAIP), known to interact with hippocalcin, was not altered, but hippocampal neurons from gene-deleted mice were more sensitive to excitotoxicity caused by kainic acid. In addition, an age-dependent increase in neurodegeneration occurred in the gene-deleted mice, showing that hippocalcin contributes to neuronal viability during aging.

Increase in Bcl‐2 phosphorylation and reduced levels of BH3‐only Bcl‐2 family proteins in kainic acid‐mediated neuronal death in the rat brain

Kainic acid induces excitotoxicity and nerve cell degeneration in vulnerable regions of rat brain, most markedly in hippocampus and amygdala. Part of the cell death following kainic acid is apoptotic as shown by caspase 3 activation and chromatin condensation. Here we have studied the regulation of pro‐ and anti‐apoptotic proteins belonging to the Bcl‐2 family in rat hippocampus and amygdala by kainic acid in relationship to ensuing neuronal death. The pro‐apoptotic protein Bax was up‐regulated in hippocampus 6 h after kainic acid administration. The increase in Bax was followed by the appearance of TdT‐mediated dUTP nick end labelling‐positive cells which were prominent at 24 h. Immunohistochemistry for active Bax revealed a punctated labelling of neurons in the CA3 and hilar regions of hippocampus as well as in amygdala. Double staining for NeuN, a marker for nerve cells, and TdT‐mediated dUTP nick end labelling showed that mainly neurons undergo degeneration after kainic acid treatment. In contrast to Bax, the pro‐apoptotic BH3‐only Bcl‐2 proteins Bim and Harakiri/DP5 were down‐regulated by kainic acid. This was also observed for the anti‐apoptotic proteins Bcl‐x and Bcl‐w. Immunoreactive Bcl‐2 was up‐regulated in hippocampus after kainic acid together with an increase in the phosphorylation of serine‐87 in Bcl‐2, suggesting a post‐transcriptional modification of the protein. This was confirmed using immunoprecipitation of total Bcl‐2 from hippocampus and amygdala which revealed an increase in serine‐87 phospho‐Bcl‐2 after kainic acid. Inhibition of the c‐jun N‐terminal protein kinase pathway reduced both serine‐87 phosphorylation and cell death after kainic acid. This indicates an important role of Bcl‐2 phosphorylation in controlling neuronal death after kainic acid. In contrast to the situation in trophic factor‐deprived neurons, no up‐regulation of Bim or Harakiri/DP5 proteins occurred after kainic acid, suggesting alternative pathways for regulation of cell death in excitotoxicity. The results indicate that not only the relative levels of Bcl‐2 family proteins but also conformation changes and post‐translational modifications contribute to neuronal death following kainic acid.