Dan Lindholm

Regulation of Nerve Growth Factor (NGF) Synthesis in the Rat Central Nervous System: Comparison between the Effects of Interleukin-1 and Various Growth Factors in Astrocyte Cultures and in vivo

In order to obtain information on the physiological regulation of NGF‐synthesis in the central nervous system (CNS) we investigated the effects of a series of growth factors (known to be present in the CNS) in cultures of purified rat astrocytes and compared these effects with those observed after intraventricular injection of the same molecules. After preliminary experiments had shown that 10% fetal calf serum (FCS) produced a marked increase in NGF‐mRNA levels in astrocytes (but neither in microglia nor oligodendrocytes) as demonstrated by Northern blot analysis and in situ hybridization the experiments were performed at low (0.5%) FCS concentrations. Supramaximal concentrations of IL‐1 and various growth factors caused a 5‐ to 7‐fold increase in NGF‐mRNA after 6 h. By 24 h the NGF‐mRNA levels approached control values again, most probably due to inactivation of the added factors since after readdition after 24 h the response was about the same as the initial one. Norepinephrine and 8‐bromo‐cAMP did not change NGF‐mRNA levels. The growth factor‐mediated changes in NGF‐mRNA levels in astrocyte cultures were not consistently reflected by the changes observed after intraventricular injection. IL‐1 produced by far the largest increase in hippocampal NGF‐mRNA after intraventricular injection. This large response to IL‐1 could result from a positive feedback mechanism, since IL‐1β injection not only increases NGF‐mRNA but also IL‐1β‐mRNA in the hippocampus. The understanding of the physiological regulation of NGF synthesis in the CNS is the basis for a rational approach to its pharmacological modification. This, in turn, is an attractive alternative to the (long‐term) infusion of NGF or the transplantation of NGF‐secreting cells with the goal of providing trophic support to the cholinergic neurons of the basal forebrain nuclei. These neurons are consistently affected in the early stages of Alzheimers disease, their impaired function being essentially responsible for the cognitive deficits.

BRAIN-DERIVED NEUROTROPHIC FACTOR PROTECTS AGAINST ISCHEMIC CELL DAMAGE IN RAT HIPPOCAMPUS

The neuroprotective action of brain-derived neurotrophic factor (BDNF) was evaluated in a rat model of transient forebrain ischemia. A continuous intraventricular infusion of BDNF for 7 days starting immediately before the onset of ischemia significantly increased the number of pyramidal cells in the vulnerable CA1 sector of the hippocampus. In situ hybridization experiments suggest the neuroprotection to be mediated via trkB – receptors in the hippocampus. The data indicate a therapeutic potential for the treatment of cerebral ischemia.

The induction of LTP increases BDNF and NGF mRNA but decreases NT-3 mRNA in the dentate gyrus.

We have investigated the expression of the mRNAs for brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in the hippocampus before and after induction of long term potentiation (LTP) of synaptic transmission in the dentate gyrus through stimulation of the perforant path (PP). A unilateral PP stimulation produced a bilateral increase in the mRNA for both BDNF and NGF in granular neurones of the dentate gyrus but not in other neurones in the hippocampus. The mRNA for neurotrophin-3 (NT-3) was bilaterally decreased by LTP but that of NT-4 remained at the basal level. These results suggest that individual neurotrophic factors may play different roles in neuronal plasticity.

Positive feedback between acetylcholine and the neurotrophins nerve growth factor and brain-derived neurotrophic factor in the rat hippocampus.

In the rat hippocampus, nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are synthesized by neurons in an activity‐dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up‐regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high‐affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.

Brain-derived Neurotrophic Factor is a Survival Factor for Cultured Rat Cerebellar Granule Neurons and Protects them Against Glutamate-induced Neurotoxicity

We have studied the effects of different neurotrophins on the survival and proliferation of rat cerebellar granule cells in culture. These neurons express trkB and trkC, the putative neuronal receptors for brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) respectively. Binding studies using iodinated BDNF and NT‐3 demonstrated that both BDNF and NT‐3 bind to the cerebellar granule neurons with a similar affinity of ˜ 2x10‐9 M. The number of receptors per granule cell was surprisingly high, ∼30x10‐4 and 2x 105 for BDNF and NT‐3, respectively. Both NT‐3 and BDNF elevated c‐fos mRNA in the granule neurons, but only BDNF up‐regulated the mRNA encoding the low‐affinity neurotrophin receptor (p75). In contrast to NT‐3, BDNF acted as a survival factor for the granule neurons. BDNF also induced sprouting of the granule neurons and significantly protected them against neurotoxicity induced by high (1 mM) glutamate concentrations. Cultured granule neurons also expressed low levels of BDNF mRNA which were increased by kainic acid, a glutamate receptor agonist. Thus, BDNF, but not NT‐3, is a survival factor for cultured cerebellar granule neurons and activation of glutamate receptor(s) up‐regulates BDNF expression in these cells.

Endoplasmic Reticulum Stress Inhibition Protects against Excitotoxic Neuronal Injury in the Rat Brain

Elevated brain glutamate with activation of neuronal glutamate receptors accompanies neurological disorders, such as epilepsy and brain trauma. However, the mechanisms by which excitotoxicity triggers neuronal injury are not fully understood. We have studied the glutamate receptor agonist kainic acid (KA) inducing seizures and excitotoxic cell death. KA caused the disintegration of the endoplasmic reticulum (ER) membrane in hippocampal neurons and ER stress with the activation of the ER proteins Bip, Chop, and caspase-12. Salubrinal, inhibiting eIF2α (eukaryotic translation initiation factor 2 subunit α) dephosphorylation, significantly reduced KA-induced ER stress and neuronal death in vivo and in vitro. KA-induced rise in intracellular calcium was not affected by Salubrinal. The results show that ER responses are essential parts of excitotoxicity mediated by glutamate receptor activation and that Salubrinal decreases neuronal death in vivo. Inhibition of ER stress by small molecular compounds may be beneficial for treatment of various neuronal injuries and brain disorders.

Interleukin-6 and Transforming Growth Factor-β1 mRNAs are Induced in Rat Facial Nucleus Following Motoneuron Axotomy

Transection of the rat facial nerve leads to a rapid activation of both astrocytes and microglia around axotomized motoneurons. The factors involved in glial activation in vivo are poorly defined but cytokines have been implicated as major regulators of glial activity in vitro. In the present study we have investigated the expression of cytokine mRNAs in the axotomized facial nucleus that might be involved in glial activation. Eight hours after axotomy unilateral transection of the facial nerve had already induced a rapid accumulation of interleukin (IL)‐6‐mRNA, with a peak at 24 hours. No IL‐6 mRNA was detected on the unoperated control side. Transforming growth factor (TGF)‐β1 mRNA was detected at low levels in the normal facial nucleus, increasing to three times the normal level 2 days after axotomy. After day 7 TGF‐β1 mRNA levels gradually declined, with a second minor peak 21 days after axotomy. In situ hybridization experiments, 4 and 21 days after axotomy, localized TGF‐β1 mRNA to activated microglial cells around regenerating motoneurons, as well as probably some astrocytes. Motoneurons did not express TGF‐β1 mRNA. TGF‐β3 was found to be normally expressed in the facial nucleus but was not regulated by axotomy. No mRNA for IL‐1, tumour necrosis factor‐α or interferon‐γ was found in the regenerating facial nucleus at any point in time. Our data indicate that IL‐6 might act as an early activating signal for glial cells in response to motoneuron axotomy, and that TGF‐β1 expressed by activated glial cells might provide a long‐lasting negative feedback signal to control glial activation.

BDNF and NT-3 induce intracellular Ca2+ elevation in hippocampal neurones

The effects of neurotrophins on intracellular Ca2+ levels in rat hippocampal neurones were studied in vitro using fura-2 fluorescence microscopy. BDNF and NT-3, but not NGF, rapidly increased cytoplasmic Ca2+ concentrations in these neurones ten-fold to reach 1 microM. Moreover in some of the neurones both BDNF and NT-3 elicited Ca2+ responses, indicative of the presence of functional receptors for these neurotrophins in the same cell. In these cultures approximately 80% of the hippocampal neurones were stained with antibodies against full-length TrkB. The expression of functional TrkB was also confirmed by RNA analysis. These results demonstrate the presence of functional receptors for BDNF and NT-3 in hippocampal neurones.

Autocrine—paracrine Regulation of Hippocampal Neuron Survival by IGF‐1 and the Neurotrophins BDNF, NT‐3 and NT‐4

In contrast to sympathetic and sensory neurons in the peripheral nervous system, the neurotrophic requirements for neurons in the central nervous system (CNS) have not been clearly identified. The inactivation of specific neurotrophic factors and their receptors by gene targeting has shown that there are no major changes in neuron numbers in the CNS. This suggests an overlap between the action of different neurotrophic factors in the brain during development. Here we have studied the survival of hippocampal neurons prepared from embryonic rats, using different culture conditions. Whereas the hippocampal neurons survive well in culture when plated at high density, they die at lower cell densities in the absence of appropriate neurotrophic factors. Under the latter conditions, both insulin‐like growth factor‐1 (IGF‐1) and the neurotrophins—brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and neurotrophin‐4 (NT‐4)—rescued a large proportion of cultured neurons. In addition, hippocampal neurons from BDNF knockout mice exhibited enhanced cell death compared with cells from wild‐type animals. BDNF and IGF‐1 both increased the survival of the hippocampal neurons lacking BDNF, showing complementary action for these factors in supporting survival. Blocking antibodies against NT‐3 and IGF‐1 decreased hippocampal neuron survival at low cell densities, showing autocrine or paracrine action of the factors. At higher cell densities, however, the antibodies had no effect, demonstrating that there is a sufficient amount of endogenous factors supporting survival under these conditions. The present results show that hippocampal neurons depend for survival on local neurotrophic factors such as IGF‐1, BDNF and NT‐3, which act in an autocrine/paracrine manner. The multifactorial support of hippocampal neurons ensures a maximal degree of neuron survival even in the absence of an individual factor.

Transgenic expression and activation of PGC-1α protect dopaminergic neurons in the MPTP mouse model of Parkinson’s disease

Mitochondrial dysfunction and oxidative stress occur in Parkinson’s disease (PD), but little is known about the molecular mechanisms controlling these events. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator that is a master regulator of oxidative stress and mitochondrial metabolism. We show here that transgenic mice overexpressing PGC-1α in dopaminergic neurons are resistant against cell degeneration induced by the neurotoxin MPTP. The increase in neuronal viability was accompanied by elevated levels of mitochondrial antioxidants SOD2 and Trx2 in the substantia nigra of transgenic mice. PGC-1α overexpression also protected against MPTP-induced striatal loss of dopamine, and mitochondria from PGC-1α transgenic mice showed an increased respiratory control ratio compared with wild-type animals. To modulate PGC-1α, we employed the small molecular compound, resveratrol (RSV) that protected dopaminergic neurons against the MPTP-induced cell degeneration almost to the same extent as after PGC-1α overexpression. As studied in vitro, RSV activated PGC-1α in dopaminergic SN4741 cells via the deacetylase SIRT1, and enhanced PGC-1α gene transcription with increases in SOD2 and Trx2. Taken together, the results reveal an important function of PGC-1α in dopaminergic neurons to combat oxidative stress and increase neuronal viability. RSV and other compounds acting via SIRT1/PGC-1α may prove useful as neuroprotective agents in PD and possibly in other neurological disorders.