P. Hughes

Neuroprotective strategies for basal ganglia degeneration: Parkinson’s and Huntington’s diseases

There are three main mechanisms of neuronal cell death which may act separately or cooperatively to cause neurodegeneration. This lethal triplet of metabolic compromise, excitotoxicity, and oxidative stress causes neuronal cell death that is both necrotic and apoptotic in nature. Aspects of each of these three mechanisms are believed to play a role in the neurodegeneration that occurs in both Parkinsons and Huntingtons diseases. Strategies to rescue or protect injured neurons usually involve promoting neuronal growth and function or interfering with neurotoxic processes. Considerable research has been done on testing a large array of neuroprotective agents using animal models which mimic these disorders. Some of these approaches have progressed to the clinical arena. Here, we review neuroprotective strategies which have been found to successfully ameliorate the neurodegeneration associated with Parkinsons and Huntingtons diseases. First, we will give an overview of the mechanisms of cell death and the background of Parkinsons and Huntingtons diseases. Then we will elaborate on a range of neuroprotective strategies, including neurotrophic factors, anti-excitotoxins, antioxidants, bioenergetic supplements, anti-apoptotics, immunosuppressants, and cell transplantation techniques. Most of these approaches hold promise as potential therapies in the treatment of these disorders.

Activity and injury-dependent expression of inducible transcription factors, growth factors and apoptosis-related genes within the central nervous system.

This review primarily discusses work that has been performed in our laboratories and that of our direct collaborators and therefore does not represent an exhaustive review of the current literature. Our aim is to further discuss the role that gene expression plays in neuronal plasticity and pathology. In the first part of this review we examine activity-dependent changes in the expression of inducible transcription factors (ITFs) and neurotrophins with long-term potentiation (LTP) and kindling. This work has identified particular ITFs (Krox-20 and Krox-24) and neurotrophin systems (particularly the brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase-B, Trk-B system) that may be involved in stabilizing long-lasting LTP (i.e. LTP3). We also show that changes in the expression of other ITFs (Fos, Jun-D and Krox-20) and the BDNF/trkB neurotrophin system may play a central role in the development of hippocampal kindling, an animal model of human temporal lobe epilepsy. In the next part of this review we examine changes in gene expression after neuronal injuries (ischemia, prolonged seizure activity and focal brain injury) and after nerve transection (axotomy). We identify apoptosis-related genes (p53, c-Jun, Bax) whose delayed expression selectively increases in degenerating neurons, further suggesting that some forms of neuronal death may involve apoptosis. Moreover, since overexpression of the tumour-suppressor gene p53 induces apoptosis in a wide variety of dividing cell types we speculate that it may perform the same function in post-mitotic neurons following brain injuries. Additionally, we show that neuronal injury is associated with rapid, transient, activity-dependent expression of neurotrophins (BDNF and activinA) in neurons, contrasting with a delayed and more persistent injury-induced expression of certain growth factors (IGF-1 and TGFbeta) in glia. In this section we also describe results linking ITFs and neurotrophic factor expression. Firstly, we show that while BDNF and trkB are induced as immediate-early genes following injury, the injury-induced expression of activinA and trkC may be regulated by ITFs. We also discuss whether loss of retrograde transport of neurotrophic factors such as nerve growth factor following nerve transection triggers the selective and prolonged expression of c-Jun in axotomized neurons and whether c-Jun is responsible for regeneration or degeneration of these axotomized neurons. In the last section we further examine the role that gene expression may play in memory formation, epileptogenesis and neuronal degeneration, lastly speculating whether the expression of various growth factors after brain injury represents an endogenous neuroprotective response of the brain to injury. Here we discuss our results which show that pharmacological enhancement of this response with exogenous application of IGF-1 or TGF-beta reduces neuronal loss after brain injury.

Is c-Jun involved in nerve cell death following status epilepticus and hypoxic-ischaemic brain injury?

Neurons undergoing delayed neuronal death produced by hypoxia-ischaemia (HI) or status epilepticus (SE) showed a massive expression of c-Jun in their nuclei 24 h after the insult. With SE there was also a weaker induction of c-Fos and Jun B in dying neurons. SE induced in the presence of the NMDA antagonist MK-801 produced no delayed c-Jun expression in the hippocampus and nerve cell death did not occur in this region, although there was a delayed c-jun expression in the amygdala/piriform region, and cell death occurred in this area. Activation of central muscarinic receptors with pilocarpine, or block of D2 dopamine receptors with haloperidol, treatments which do not cause neuronal damage, strongly induced Fos and Jun B in hippocampal and striatal neurons, but only induced c-Jun very weakly. Thus, c-Jun may participate in the genetic cascade of events that produce programmed cell death in neurons.

Brain-derived neurotrophic factor is induced as an immediate early gene following N-methyl-d-aspartate receptor activation

Recent studies show that focal brain injury, cerebral ischaemia, hypoglycaemia and seizures increase the expression of c-fos and brain-derived neurotrophic factor in brain. Here we report that hippocampal focal brain injury transiently induces the immediate early genes c-fos, jun-B, c-jun and krox-24 (zif-268) messenger RNA and protein and brain-derived neurotrophic factor messenger RNA in rat dentate gyrus neurons, an effect that was blocked by the N-methyl-D-aspartate receptor antagonist MK-801. Prior administration of the protein synthesis inhibitor cycloheximide super-induced immediate early gene messenger RNA, abolished immediate early gene protein induction, but had no effect on injury-mediated induction of brain-derived neurotrophic factor messenger RNA. Thus, while N-methyl-D-aspartate receptor activation results in the induction of both immediate early genes and brain-derived neurotrophic factor messenger RNA, de novo synthesis of immediate early gene proteins is not critical for the increased expression of brain-derived neurotrophic factor messenger RNA seen in brain after focal injury. These results suggest that brain-derived neurotrophic factor is induced after injury as an immediate early gene.

Expression of the activin axis and neuronal rescue effects of recombinant activin A following hypoxic-ischemic brain injury in the infant rat

Neurotrophic factors are induced in the brain in response to injury and may restrict the extent of neuronal loss and facilitate recovery. We have previously reported a strong neuronal induction of activin betaA subunit mRNA expression after a hypoxic-ischemic (HI) injury in the rat brain. Here, we further extended our studies to examine a role for the activin inhibitory binding protein, follistatin after injury and also to determine the potential of activin as a neuronal rescue agent. Ribonuclease protection assay (RPA) was used to quantify the time course of the mRNA expression of activin betaA subunit and follistatin, following a 60-min HI brain injury. Activin betaA subunit mRNA level increased in the contralateral hemisphere 5 h after injury and returned to normal at 10 h post injury. In contrast, follistatin mRNA levels decreased in the same hemisphere at 5 and 10 h after injury. The effect of intracerebroventrically (i. c.v.) administered recombinant human activin A or its antagonist, inhibin A, on neuronal death after a 15-min HI brain injury was determined for a number of brain regions. One microgram activin A (n=23) reduced the neuronal loss in the hippocampal CA1/2 region, dorsolateral striatum but not in the parietal cortex. In contrast, 1 microg of inhibin A (n=18) did not have a significant effect on the extent of neuronal loss in any of the affected regions. This pattern of neuroprotection was consistent with the distribution of immunoreactivity for the activin receptor type II subunit. These results demonstrate that activin A, but not its functional antagonist inhibin A, can enhance the survival of injured hippocampal and striatal neurons. Since follistatin is thought to exert a neutralising effect on activin A activity, the down-regulation of follistatin expression post injury may be allowing activin A to become more accessible to neurons after injury. Overall, these results suggest a role of the activin axis in modulating the survival of specific populations of injured neurons.

Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus

The basal expression of the protein products of the inducible immediate early genes (IEGs), Fos, Jun, and Krox 24, was investigated in rat hippocampus using immunocytochemical visualization methods with antisera specific for Fos only, Fos and the Fos-related antigens (FRAs), the Jun family, and Krox 24 (previously described as TIS 8, egr-1, NGF-IA or zif 268). In the normal adult rat brain basal levels of Jun, Krox 24 and Fos-related antigens but not Fos were seen within the hippocampus. More specifically very high basal levels of Jun were seen in the dentate granule cells with high basal Krox 24 levels seen in the CA1-subiculum region of the rat hippocampus. Basal FRAs but not Fos-positive cells were seen at low levels in the dentate granule cells. The implications of these results to the functioning of IEG proteins in hippocampal neurons is discussed.

Administration of recombinant human Activin-A has powerful neurotrophic effects on select striatal phenotypes in the quinolinic acid lesion model of Huntington's disease.

Huntington disease is characterized by the selective loss of striatal neurons, particularly of medium-sized spiny glutamate decarboxylase67 staining/GABAergic projection neurons which co-contain the calcium binding protein calbindin. Lesioning of the adult rat striatum by intrastriatal injection of the N-methyl-D-aspartate receptor agonist quinolinic acid (100 nmol) results in a pattern of striatal neuropathology seven days later that resembles that seen in the Huntington brain. Using this animal model of human Huntingtons disease we investigated the effect of daily intrastriatal infusion of the nerve cell survival molecule ActivinA (single bolus dose of 0.73 microg daily for seven days) on the quinolinic acid-induced degeneration of various striatal neuronal phenotypes. By seven days, unilateral intrastriatal infusion of quinolinic acid produced a partial but significant loss (P < 0.01) in the number of striatal neurons immunoreactive for glutamate decarboxylase (to 51.0+/-5.8% of unlesioned levels), calbindin (to 58.7+/-5.1%), choline acetyltransferase (to 68.6+/-6.1%), NADPH-diaphorase (to 47.4+/-5.4%), parvalbumin (to 58.8+/-4.1%) and calretinin (to 60.6+/-8.6%) in adult rats that were administered intrastriatal phosphate-buffered saline for seven days following quinolinic acid. In contrast, in rats that received intrastriatal recombinant human ActivinA once daily for seven days following quinolinic acid, phenotypic degeneration was significantly attenuated in several populations of striatal neurons. Treatment with ActivinA had the most potent protective effect on the striatal cholinergic interneuron population almost completely preventing the lesion induced decline in choline acetyltransferase expression (to 95.1+/-5.8% of unlesioned levels, P < 0.01). ActivinA also conferred a significant protective effect on parvalbumin (to 87.5+/-7.7%, P < 0.01) and NADPH-diaphorase (to 77.5+/-7.5%, P < 0.01) interneuron populations but failed to prevent the phenotypic degeneration of calretinin neurons (to 56.6+/-5.5%). Glutamate decarboxylase67 and calbindin-staining nerve cells represent largely overlapping populations and both identify striatal GABAergic projection neurons. We found that ActivinA significantly attenuated the loss in the numbers of neurons staining for calbindin (to 79.7+/-6.6%, P < 0.05) but not glutamate decarboxylase67 (to 61.1+/-5.9%) at seven days following quinolinic acid lesioning. Taken together these results suggest that exogenous administration of ActivinA can rescue both striatal interneurons (labelled with choline acetyltransferase, parvalbumin, NADPH-diaphorase) and striatal projection neurons (labelled by calbindin) from excitotoxic lesioning with quinolinic acid. Longer-term studies will be required to determine whether these surviving calbindin-expressing projection neurons recover their ability to express the glutamate decarboxylase67/GABAergic phenotype. These results therefore suggest that treatment with ActivinA may help to prevent the degeneration of vulnerable striatal neuronal populations in Huntingtons disease.

Muscarinic receptor-mediated induction of Fos protein in rat brain

Recent studies have shown that the centrally active muscarinic agonist pilocarpine induces c-fos mRNA in rat cortex. Here we describe the localization of muscarinic receptor-induced FOS protein, within the rat central nervous system (CNS), following administration of pilocarpine (25 mg/kg). High levels of FOS induction were apparent in many forebrain structures including the primary olfactory (piriform) cortex, nucleus accumbens, amygdala, hippocampus, neocortex and supra-optic nucleus of the hypothalamus. Within the neocortex FOS induction followed a laminar distribution being highest in layers 4 and 6 with lower induction seen in layers 2 and 5. Other areas showing FOS induction included the striatum, septum, inferior colliculus, thalamus, hypothalamus and several brainstem nuclei. Both atropine (10 mg/kg) and pirenzepine (100 mg/kg) reduced FOS induction suggesting that a pirenzepine-sensitive muscarinic receptor was involved. The possible significance of muscarinic-mediated FOS induction, to cholinergic kindling and the cholinergic hypothesis of learning and memory, is discussed.

Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge

Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity (mossy fibre sprouting) associated with kindling. Conversely, the role in kindling of those genes whose expression was significantly attenuated by MK801 (Fos, Jun-D, Krox-20, trkB and BDNF) requires further examination.

Activation of pirenzepine-sensitive muscarinic receptors induces a specific pattern of immediate-early gene expression in rat brain neurons

Accumulating evidence suggests that immediate-early gene transcription factors such as c-Fos, form part of an intracellular signalling pathway linking the activation of neuronal receptors by neurotransmitters to changes in neuronal gene expression. Recently it has been demonstrated that the centrally active muscarinic receptor agonist pilocarpine induces both c-fos mRNA and protein in rat brain. In this report using immunocytochemical and in situ hybridization techniques we demonstrate for the first time that in addition to c-fos, pilocarpine administration increases the neuronal expression of jun-B, krox-20 and krox-24 (zif-268) but not related c-jun and jun-D genes in rat cortex and hippocampus. Pretreatment of animals with atropine or pirenzepine significantly reduced induction of c-fos, jun-B, krox-20 and krox-24 genes in both hippocampus and cortex. These results show that activation of pirenzepine-sensitive muscarinic receptors results in a specific pattern of immediate-early gene expression in rat brain neurons. We suggest that the combinatorial complexity of immediate-early gene induction may allow receptor-specific control of gene expression in vivo.