Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.     

Ontogenic cell death in the nigrostriatal system

Like most neural systems, dopamine neurons of the substantia nigra undergo apoptotic natural cell death during development. In rodents, this occurs largely postnatally and is biphasic with an initial major peak just after birth and a second minor peak on postnatal day 14. As envisioned by classic neurotrophic theory, this event is regulated by interactions with the target of these neurons, the striatum, because a developmental target lesion results in an augmented natural cell death event with fewer nigral dopamine neurons surviving into adulthood. Until recently, the striatal target-derived neurotrophic factors providing developmental support of dopamine neurons were unknown, but there is now growing evidence that glial-cell-line-derived neurotrophic factor (GDNF) serves as a physiologic limiting neurotrophic factor for these neurons during the first phase of natural cell death. During this phase, intrastriatal injection of GDNF diminishes the natural cell death event and neutralizing antibodies augment it. Sustained overexpression of GDNF in the striatum throughout development in a unique double transgenic mouse model results in an increased number of dopamine neurons surviving the first phase of natural cell death. However, this increase does not persist into adulthood. Therefore, other factors or mechanisms must play important roles in the determination of the mature number of nigral dopamine neurons. Further elucidation of these mechanisms will be important in the development of neuroprotective and cell replacement therapies for Parkinsons disease.This work was supported by NS26836, NS38370, DAMD17-03-1-0492, and the Parkinsons Disease Foundation     

Expression of cyclin-dependent kinase 5 and its activator p35 in models of induced apoptotic death in neurons of the substantia nigra in vivo

Cyclin-dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6-hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co-labeling with a neuron-specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.     

Caspase-3 mediated neuronal death after traumatic brain injury in rats

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.     

CEP11004, a novel inhibitor of the mixed lineage kinases, suppresses apoptotic death in dopamine neurons of the substantia nigra induced by 6-hydroxydopamine

There is much evidence that the kinase cascade which leads to the phosphorylation of c-jun plays an important signaling role in the mediation of programmed cell death. We have previously shown that c-jun is phosphorylated in a model of induced apoptotic death in dopamine neurons of the substantia nigra in vivo. To determine the generality and functional significance of this response, we have examined c-jun phosphorylation and the effect on cell death of a novel mixed lineage kinase inhibitor, CEP11004, in the 6-hydroxydopamine model of induced apoptotic death in dopamine neurons. We found that expression of total c-jun and Ser73-phosphorylated c-jun is increased in this model and both colocalize with apoptotic morphology. CEP11004 suppresses apoptotic death to levels of 44 and 58% of control values at doses of 1.0 and 3.0 mg/kg, respectively. It also suppresses, to approximately equal levels, the number of profiles positive for the activated form of capase 9. CEP11004 markedly suppresses striatal dopaminergic fiber loss in these models, to only 22% of control levels. We conclude that c-jun phosphorylation is a general feature of apoptosis in living dopamine neurons and that the mixed lineage kinases play a functional role as up-stream mediators of cell death in these neurons.     

Gene disruption of caspase-3 prevents MPTP-induced Parkinson's disease in mice

The development of Parkinson’s disease is accompanied by concurrent activation of caspase-3 and apoptosis of dopaminergic neurons of human patients and rodent models. The role of caspase-3, a final executioner of apoptosis, in the pathogenesis of Parkinson’s disease, however, remains to be determined. Here, we show that gene disruption of caspase-3 protects mice from 1-methyle-4-phenyl-1,2,3,6-tetrahmydropyridine (MPTP)-induced Parkinsonian syndrome, as reflected by reversal of MPTP-induced bradykinesia and decreased tyrosine hydroxylase expression in the nigra-striatum. MPTP treatment resulted in increased caspase-3 activation and apoptosis in the substantia nigra of wild-type mice at 24 h after the inception of MPTP treatment, as compared with vehicle-treated control animals. Gene disruption of caspase-3 prevented MPTP-induced apoptosis in the substantia nigra. At 7 days after MPTP treatment, tyrosine hydroxylase expression was suppressed and infiltration of activated microglia and astrocytes was markedly increased in the nigra-striatum of wild-type mice. All of these alterations following MPTP treatment were blocked by disruption of caspase-3 in mice. These results clearly indicate that caspase-3 activation is required for the development of MPTP-induced Parkinson’s disease in mice. These findings suggest that activation of caspase-3-mediated apoptosis of dopaminergic neurons in the early stage may play an important role in the pathogenesis of Parkinson’s disease.     

Identification and kainic acid-induced up-regulation of low-affinity p75 neurotrophin receptor (p75NTR) in the nigral dopamine neurons of adult rats

Parkinson's disease is a common and severe debilitating neurological disease that results from massive and progressive degenerative death of dopamine neurons in the substantia nigra, but the mechanisms of neuronal degeneration and disease progression remains largely obscure. We are interested in possible implications of low-affinity p75 neurotrophin receptor (p75NTR), which may mediate neuronal apoptosis in the central nervous system, in triggering cell death of the nigral dopamine neurons. The RT-PCR and immunohistochemistry were carried out to detect if p75NTR is expressed in these nigral neurons and up-regulated by kainic acid (KA) insult in adult rats. It revealed p75NTR-positive immunoreactivity in the substantia nigra, and co-localization of p75NTR and tyrosine hydroxylase (TH) was found in a large number of substantia nigra neurons beside confirmation of p75NTR in the choline acetyltransferase (ChAT)-positive forebrain neurons. Cell count data further indicated that about 47-100% of TH-positive nigral neurons and 98-100% of ChAT-positive forebrain neurons express p75NTR. More interestingly, significant increasing in both p75NTR mRNA and p75NTR-positive neurons occurred rapidly following KA insult in the substantia nigra of animal model. The present study has provided first evidence on p75NTR expression and KA-inducing p75NTR up-regulation in substantia nigra neurons in rodent animals. Taken together with previous data on p75NTR functions in neuronal apoptosis, this study also suggests that p75NTR may play important roles in neuronal cell survival or excitotoxic degeneration of dopamine neurons in the substantia nigra in pathogenesis of Parkinson's disease in human beings.     

A novel in vivo post-translational modification of p53 by PARP-1 in MPTP-induced parkinsonism

Sporadic Parkinson's disease (PD) affects primarily dopaminergic neurons of the substantia nigra pars compacta. There is evidence of necrotic and apoptotic neuronal death in PD, but the mechanisms behind selected dopaminergic neuronal death remain unknown. The tumor suppressor protein p53 functions to selectively destroy stressed or abnormal cells during life and development by means of necrosis and apoptosis. Activation of p53 leads to death in a variety of cells including neurons. p53 is a target of the nuclear enzyme Poly(ADP-ribose)polymerase (PARP), and PARP is activated following DNA damage that occurs following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP is the favored in vivo model of PD, and reproduces the pathophysiology, anatomy and biochemistry of PD. p53 protein normally exhibits a fleeting half-life, and regulation of p53 stability and activation is achieved mainly by post-translational modification. We find that p53 is heavily poly(ADP-ribosyl)ated by PARP-1 following MPTP intoxication. This post-translational modification serves to stabilize p53 and alters its transactivation of downstream genes. These influences of PARP-1 on p53 may underlie the mechanisms of MPTP-induced parkinsonism and other models of neuronal death.     

Cellular and molecular mechanisms of Parkinson's disease: neurotoxins, causative genes, and inflammatory cytokines

1. Parkinson's disease (PD) is considered to be an aging-related neurodegeneration of catecholamine (CA) systems [typically A9 dopamine (DA) neurons in the substantia nigra and A6 noradrenaline (NA) neurons in the locus coeruleus]. The main symptom is movement disorder caused by a DA deficiency at the nerve terminals of fibers that project from the substantia nigra to the striatum. Most PD is sporadic (sPD) without any hereditary history. sPD is speculated to be caused by some exogenous or endogenous substances that are neurotoxic toward CA neurons, which toxicity leads to mitochondrial dysfunction and subsequent oxidative stress resulting in the programmed cell death (apoptosis or autophagy) of DA neurons. 2. Recent studies on the causative genes of rare familial PD (fPD) cases, such as alpha-synuclein and parkin, suggest that dysfunction of the ubiquitin-proteasome system (UPS) and the resultant accumulation of misfolded proteins and endoplasmic reticulum stress may cause the death of DA neurons. 3. Activated microglia, which accompany an inflammatory process, are present in the nigro-striatum of the PD brain; and they produce protective or toxic substances, such as cytokines, neurotrophins, and reactive oxygen or nitrogen species. These activated microglia may be neuroprotective at first in the initial stage, and later may become neurotoxic owing to toxic change to promote the progression toward the death of CA neurons.4. All of these accumulating evidences on sPD and fPD points to a hypothesis that multiple primary causes of PD may be ultimately linked to a final common signal-transduction pathway leading to programmed cell death, i.e., apoptosis or autophagy, of the CA neurons.     

Increases in Bcl-2 and cleavage of caspase-1 and caspase-3 in human brain after head injury.

The bcl-2 and caspase families are important regulators of programmed cell death in experimental models of ischemic, excitotoxic, and traumatic brain injury. The Bcl-2 family members Bcl-2 and Bcl-xL suppress programmed cell death, whereas Bax promotes programmed cell death. Activated caspase-1 (interleukin-1beta converting enzyme) and caspase-3 (Yama/Apopain/Cpp32) cleave proteins that are important in maintaining cytoskeletal integrity and DNA repair, and activate deoxyribonucleases, producing cell death with morphological features of apoptosis. To address the question of whether these Bcl-2 and caspase family members participate in the process of delayed neuronal death in humans, we examined brain tissue samples removed from adult patients during surgical decompression for intracranial hypertension in the acute phase after traumatic brain injury (n=8) and compared these samples to brain tissue obtained at autopsy from non-trauma patients (n=6). An increase in Bcl-2 but not Bcl-xL or Bax, cleavage of caspase-1, up-regulation and cleavage of caspase-3, and evidence for DNA fragmentation with both apoptotic and necrotic morphologies were found in tissue from traumatic brain injury patients compared with controls. These findings are the first to demonstrate that programmed cell death occurs in human brain after acute injury, and identify potential pharmacological and molecular targets for the treatment of human head injury.     

Glial Cell Line-Derived Neurotrophic Growth Factor Inhibits Apoptotic Death of Postnatal Substantia Nigra Dopamine Neurons in Primary Culture

Abstract: Glial cell line-derived neurotrophic factor (GDNF) was identified on the basis of its ability to enhance the development of embryonic mesencephalic dopamine neurons. It remains unknown whether GDNF is a physiologically relevant trophic factor for these neurons. We have shown that natural cell death among dopamine neurons of the substantia nigra occurs largely postnatally. To investigate whether GDNF may have the ability to support these neurons during their period of natural cell death, we have used a postnatal primary culture model. We find that GDNF is able to support the viability of postnatal nigral dopamine neurons by inhibiting apoptotic death. This ability of GDNF shows both regional specificity for the nigra and cellular specificity for the dopamine phenotype. Among eight other neurotrophic factors previously reported to support embryonic dopamine neurons, GDNF was unique in this ability. Thus, GDNF meets this criterion for a physiologically relevant trophic factor for dopamine neurons of the substantia nigra.     

The TrkB-Positive Dopaminergic Neurons are Less Sensitive to MPTP Insult in the Substantia Nigra of Adult C57/BL Mice

Tyrosine kinase receptors TrkB and TrkC mediate neuroprotective effects of the brain-derived neurotrophic factor (BDNF) and neurotrophins in the dopaminergic nigro-striatal system, but it is obscure about their responses or expression changes in the injured substantia nigra under Parkinson’s disease. In present study, immunofluorescence, Fluoro-Jade staining and laser scanning confocal microscopy were applied to investigate distribution and changes of TrkB and TrkC in the dopamine neurons of the substantia nigra by comparison of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. It revealed that TrkB and TrkC-immunoreactivities were substantially localized in cytoplasm and cell membrane of the substantia nigra neurons of control adults. While neurons double-labeled with tyrosine hydroxylase (TH)/TrkB, or TH/TrkC were distributed in a large numbers in the substantia nigra of controls, they apparently went down at 36.2–65.7% of normal level, respectively following MPTP insult. In MPTP model, cell apoptosis or degeneration of nigral neurons were confirmed by caspase-3 and Fluoro-Jade staining. More interestingly, TH/TrkB-positive neurons survived more in cell numbers in comparison with that of TH/TrkC-positive ones in the MPTP model. This study has indicated that TrkB-containing dopamine neurons are less sensitive in the substantia nigra of MPTP mouse model, suggesting that specific organization of Trks may be involved in neuronal vulnerability to MPTP insult, and BDNF-TrkB signaling may play more important role in protecting dopamine neurons and exhibit therapeutic potential for Parkinson’s disease.     

Mechanisms of p75-mediated death of hippocampal neurons. Role of caspases

Neurotrophins support neuronal survival and differentiation via Trk receptors, yet can also induce cell death via the p75 receptor. In these studies, we investigated signaling mechanisms governing p75-mediated death of hippocampal neurons, specifically the role of caspases. Although p75 is structurally a member of the Fas/TNFR1 receptor family, caspase-8 was not required for p75-mediated death, unlike other members of this receptor family. In contrast, p75-mediated neuronal death was associated with mitochondrial loss of cytochrome c and required Apaf-1 and caspase-9, -6, and -3. In particular, caspase-6 plays a central role in mediating neurotrophin-induced death, illuminating a novel role for this caspase. Inhibition of DIABLO/Smac, which blocks inhibitor of apoptosis proteins, protected cells from death, whereas simultaneous inhibition of both DIABLO/Smac and MIAP3 allowed trophin-induced death to proceed. In vivo, pilocarpine-induced seizures, previously shown to up-regulate p75 expression and increase neurotrophin production, caused activation of caspase-6 and -3 and cleavage of poly(ADP-ribose) polymerase in p75-expressing hippocampal neurons. In p75(-/-) mice, no activated caspase-3 was detected, and there was a marked reduction in the number of dying neurons after pilocarpine treatment compared with wild type mice. Neurotrophin-induced p75-mediated death is likely to play an important role in mediating neuronal loss consequent to brain injury.     

17beta-estradiol attenuates programmed cell death in cortical pericontusional zone following traumatic brain injury via upregulation of ERalpha and inhibition of caspase-3 activation

Pericontusional zone (PCZ) of traumatic cerebral contusion is a target of pharmacological intervention. It is well studied that 17beta-estradiol has a protective role in ischemic brain injury, but its role in brain protection of traumatic brain damage deserves further investigation, especially in pericontusional zone. Here we show that 17beta-estradiol enhances the protein expression and mRNA induction of estrogen alpha receptor (ERalpha) and prevents from programmed cell death in cortical pericontusional zone. ERalpha specific antagonist blocks this protective effect of 17beta-estradiol. Caspase-3 activation occurs in cortical pericontusional zone of the oil-treated injured rat brain and its activation is inhibited by 17beta-estradiol treatment. Additionally, ERalpha specific antagonist reverses this inhibition. Pan-caspase inhibitor also protect cortical pericontusional zone from programmed cell death. Our present study indicates 17beta-estradiol protects from programmed cell death in cortical pericontusional zone via enhancement of ERalpha and decrease of caspase-3 activation.     

Cellular and Molecular Mechanisms of Parkinson’s Disease: Neurotoxins, Causative Genes, and Inflammatory Cytokines

1. Parkinson’s disease (PD) is considered to be an aging-related neurodegeneration of catecholamine (CA) systems [typically A9 dopamine (DA) neurons in the substantia nigra and A6 noradrenaline (NA) neurons in the locus coeruleus]. The main symptom is movement disorder caused by a DA deficiency at the nerve terminals of fibers that project from the substantia nigra to the striatum. Most PD is sporadic (sPD) without any hereditary history. sPD is speculated to be caused by some exogenous or endogenous substances that are neurotoxic toward CA neurons, which toxicity leads to mitochondrial dysfunction and subsequent oxidative stress resulting in the programmed cell death (apoptosis or autophagy) of DA neurons.2. Recent studies on the causative genes of rare familial PD (fPD) cases, such as alpha–synuclein and parkin, suggest that dysfunction of the ubiquitin–proteasome system (UPS) and the resultant accumulation of misfolded proteins and endoplasmic reticulum stress may cause the death of DA neurons.3. Activated microglia, which accompany an inflammatory process, are present in the nigro-striatum of the PD brain; and they produce protective or toxic substances, such as cytokines, neurotrophins, and reactive oxygen or nitrogen species. These activated microglia may be neuroprotective at first in the initial stage, and later may become neurotoxic owing to toxic change to promote the progression toward the death of CA neurons.4. All of these accumulating evidences on sPD and fPD points to a hypothesis that multiple primary causes of PD may be ultimately linked to a final common signal-transduction pathway leading to programmed cell death, i.e., apoptosis or autophagy, of the CA neurons.Special Issue dedicated to Dr. Julie Axelrod     

Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.     

Characterization of the execution pathway of developing motoneurons deprived of trophic support

Avian spinal motoneurons have been well characterized with regard to developmental programmed cell death (PCD). Approximately 50% of the neurons originally generated undergo cell death as they innervate their target muscles, and target derived trophic support plays an important role in regulating survival of these neurons. To investigate events mediating motoneuron PCD, we have examined the role of Bcl-2 family proteins, cytochrome C, and caspase-9 in this process. We report that while protein levels of Bcl-2, Bcl-xL, and Bax do not change within motoneurons as they become committed to die, a translocation of Bax from the cytosol to organelle membranes and the nucleus occurs coincident with the time when motoneurons become committed to cell death. In addition, cytochrome C is released from mitochondria to the cytosol in dying cells prior to the activation of caspases. Consequently, an enhanced caspase-9-like activity was detected in dying cells, and this activity was upstream and necessary for the appearance of a caspase-3-like activity. These results allow us to further define some of the critical events that mediate the execution phase of motoneuron death following trophic factor deprivation.     

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra,but are not required for axon degeneration

Activation of c‐jun N‐terminal kinase (JNK) by the mitogen‐activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson’s disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6‐hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.