Neuronal cell death

Pyramidal neurons in hippocampal subregions are selectively vulnerable in certain disease states. To investigate, we tested the hypothesis that selective vulnerability in human hippocampus is related to regional differences in neuronal cell death and cell receptor gene expression in CA1 vs. CA3 subregions. We used laser capture microdissection to remove approximately 600 CA1 and 600 CA3 pyramidal neurons each from five fresh-frozen normal post-mortem brains, extracted total RNA and double-amplified mRNA. This was reverse transcribed and labeled for hybridization onto human cDNA array chips containing probes to 10,174 genes and unknown ESTs. RNA from additional microdissections was pooled for replicate hybridizations and quantitative RT-PCR validation. Gene expression differences were few ( < 1%). We found 43 enriched genes in CA1 neuronal samples that included peripheral benzodiazipine receptor-associated protein, nicotinic cholinergic receptor, two chemokine receptors (CCR1 and CCR5) and several transcriptional factors. We found 17 enriched genes in the CA3 neuronal samples that included fibroblast growth factor receptor and prostaglandin-endoperoxide synthase 1. We found no differential gene expression for 23 calcium channel proteins; nine transporter proteins; 55 cell death and apoptotic regulator proteins; and an additional 497 cell receptors, including 24 glutamate receptors. Quantitative RT-PCR of four differentially expressed genes confirmed the microarray data. The results confirm the ability to examine gene expression profiles in microdissected neurons from human autopsy brain. They show only minor gene expression differences between two distinct neuronal populations in the hippocampus and suggest that selective hippocampal vulnerability is due to factors other than intrinsic differential expression in glutamate receptors and cell death genes. D

One of the theories involved in the etiology of Alzheimer's disease (AD) is the oxidative stress hypothesis. The amyloid bpeptide (Ab), a hallmark in the pathogenesis of AD and the main component of senile plaques, generates free radicals in a metal-catalyzed reaction inducing neuronal cell death by a reactive oxygen species mediated process which damage neuronal membrane lipids, proteins and nucleic acids. Therefore, the interest in the protective role of dierent antioxidants in AD such as vitamin E, melatonin and estrogens is growing up. In this review we summarize data that support the involvement of oxidative stress as an active factor in Ab-mediated neuropathology, by triggering or facilitating neurodegeneration, through a wide range of molecular events that disturb neuronal cell homeostasis. 7

Hypoxia-ischemia (HI) brain injury is a major cause of neuronal cell death especially apoptosis in the perinatal period. This study was designated to examine the effect of hydrogen therapy on apoptosis in an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 • C). Immediately after HI insult, pups were placed into a chamber filled with 2% H 2 for 30 min, 60 min, or 120 min, respectively. 24 h after 2% H 2 therapy, the pups were decapitated and brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, and TUNEL staining, as well as caspase-3, caspase-12 activities in the cortex and hippocampus. H 2 treatment in a duration-dependent manner significantly reduced the number of positive TUNEL cells and suppressed caspase-3 and -12 activities. These results indicated H 2 administration after HI appeared to provide brain protection via inhibition of neuronal apoptosis.

Motor neurons degenerate in amyotrophic lateral sclerosis (ALS). The mechanisms for this neuronal cell death are not known, although apoptosis has been implicated. Oxidative damage to DNA and activation of p53 has been identified directly in motor neurons in cases of ALS. We evaluated whether motor neuron degeneration in ALS is associated with changes in the levels and function of the multifunctional protein apurinic/apyrimidinic enodnuclease (APE/Ref-1). APE/Ref-1 functions as an enzyme in the DNA base-excision repair pathway and as a redox-regulation protein for transcription factors. The protein level and localization of APE/Ref-1 are changed in ALS. Immunoblotting showed that APE/Ref-1 protein levels are increased in selectively vulnerable central nervous system (CNS) regions in individuals with ALS compared to age-matched controls. Plasmid DNA repair assay demonstrated that APE from individuals with ALS is competent in repairing apurinic (AP) sites. DNA repair function in nuclear fractions is increased significantly in ALS motor cortex and spinal cord. Immunocytochemistry and single-cell densitometry revealed that APE/Ref-1 is expressed at lower levels in control motor neurons than in ALS motor neurons, which are decreased in number by 42% in motor cortex. APE/Ref-1 is increased in the nucleus of remaining upper motor neurons in ALS, which show a 38% loss of nuclear area. APE-Ref-1 is also upregulated in astrocytes in spinal cord white matter pathways in familial ALS. We conclude that mechanisms for DNA repair are activated in ALS, supporting the possibility that DNA damage is an upstream mechanism for motor neuron degeneration in this disease.

Parkinson’s disease (PD) is a common neurodegenerative disorder. Neuronal cell death in PD is still poorly understood, despite a wealth of potential pathogenic mechanisms and pathways. Defects in several cellular systems have been implicated as early triggers that start cells down the road toward neuronal death. These include abnormal protein accumulation, particularly of alpha-synuclein; altered protein degradation via multiple pathways; mitochondrial dysfunction; oxidative stress; neuroinflammation; and dysregulated kinase signaling. As dysfunction in these systems mounts, pathways that are more explicitly involved in cell death become recruited. These include JNK signaling, p53 activation, cell cycle re-activation, and signaling through bcl-2 family proteins. Eventually, neurons become overwhelmed and degenerate; however, even the mechanism of final cell death in PD is still unsettled. In this review, we will discuss cell death triggers and effectors that are relevant to PD, highlighting important unresolved issues and implications for the development of neuroprotective therapies.

We examined the neuroprotective role of nicergoline in neuron -microglia or neuron -astrocytes co-cultures. Nicergoline, an ergoline derivative, significantly suppressed the neuronal cell death induced by co-culture with activated microglia or astrocytes stimulated with lipopolysaccharide (LPS) and interferon (IFN)-g. To elucidate the mechanism by which nicergoline exerts a neuroprotective effect, we examined the production of inflammatory mediators and neurotrophic factors in activated microglia and astrocytes following nicergoline treatment. In microglia stimulated with LPS and IFN-g, nicergoline suppressed the production of superoxide anions, interleukin (IL)-1h, IL-6, and tumor necrosis factor (TNF)-a in a dose-dependent manner. In astrocytes, nicergoline also suppressed the production of proinflammatory cytokines and enhanced brain-derived neurotrophic factor (BDNF). Thus, nicergoline-mediated neuroprotection resulted primarily from the inhibition of inflammatory mediators and the upregulation of neurotrophic factors by glial cells.

Pesticides are widely used in agricultural and other settings, resulting in continued human exposure. Pesticide toxicity has been clearly demonstrated to alter a variety of neurological functions. Particularly, there is strong evidence suggesting that pesticide exposure predisposes to neurodegenerative diseases. Epidemiological data has suggested a relationship between pesticide exposure and brain neurodegeneration. However, an increasing debate has aroused regarding this issue. Paraquat is a highly toxic quaternary nitrogen herbicide which has been largely studied as a model for Parkinson's disease providing valuable insight into the possible mechanisms involved in the toxic effects of pesticides and their role in the progression of neurodegenerative diseases. In this work, we review the molecular mechanisms involved in the neurotoxic actions of pesticides, with a particular emphasis on the mechanisms associated with the induction neuronal cell death by paraquat as a model for Parkinsonian neurodegeneration.

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PNQX (9-methyl-amino-6-nitro-hexahydro-benzo(F)quinoxalinedione) is a selective AMPA antagonist with demonstrated neuroprotective effects in focal ischemia in rats. Here we report corresponding effects in mouse hippocampal slice cultures subjected to oxygen and glucose deprivation (OGD) and in transient global cerebral ischemia in gerbils. For in vitro studies, hippocampal slice cultures derived from 7-day-old mice and grown for 14 days, were submersed in oxygen-glucose deprived medium for 30 min and exposed to PNQX for 24 h, starting together with OGD, immediately after OGD, or 2 h after OGD. For comparison, other cultures were exposed to the NMDA antagonist MK-801 using the same protocol. Both PNQX and MK-801 displayed significant neuroprotective effects in all hippocampal subfields when present during and after OGD. When added just after OGD, only PNQX retained some neuroprotective effect. When added 2 h after OGD neither PNQX nor MK-801 had an effect.

Neuronal cell death is a key feature of both normal nervous system development and neuropathological conditions. The Bcl-2 family, via its regulation of both caspase-dependent and caspase-independent cell death pathways, is uniquely positioned to critically control neuronal cell survival. Targeted gene disruptions of specific bcl-2 family members and the generation of transgenic mice overexpressing anti-or proapoptotic Bcl-2 family members have confirmed the importance of the Bcl-2 family in the nervous system. Data from studies of human brain tissue and experimental animal models of neuropathological conditions support the hypothesis that the Bcl-2 family regulates cell death in the mature nervous system and suggest that pharmacological manipulation of Bcl-2 family action could prove beneficial in the treatment of human neurological conditions such as stroke and neurodegenerative diseases. D

This review summarizes recent developments that have contributed to understand how adenosine receptors, particularly A2A receptors, modulate brain injury in various animal models of neurological disorders, including Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD), stroke, Huntington&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (HD), multiple sclerosis, Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) and HIV-associated dementia. It is clear that extracellular adenosine acting at adenosine receptors influences the functional outcome in a broad spectrum of brain injuries, indicating that A2A Rs may modulate some general cellular processes to affect neuronal cells death. Pharmacological, neurochemical and molecular/genetic approaches to the complex actions of A2A receptors in different cellular elements suggest that A2A receptor activation can be detrimental or protective after brain insults, depending on the nature of brain injury and associated pathological conditions. An interesting concept that emerges from these studies is A2A R&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s ability to fine tune neuronal and glial functions to produce neuroprotective effects. While the data presented here clearly highlight the complexity of using adenosinergic agents therapeutically in PD and other neurodegenerative disorders and point out many areas for further inquiry, they also confirm that adenosine receptor ligands, particularly A2A receptor ligands, have many promising characteristics that encourage the pursuit of their therapeutic potential.

Work on acute models of cortical injury has revealed a population of small GABAergic interneurons that are induced to increase their low constitutive expression of neuronal nitric oxide (NO) synthase (nNOS). In some cases, this activation may play a role in NO-mediated degeneration of pyramidal neurons. In this report, we explore the anatomy of various classes of cortical nNOS (+) (nitrergic) neurons, with emphasis on small interneurons, in the medial temporal lobe of subjects with Alzheimer's disease (AD) from two well-characterized cohorts, the Baltimore Longitudinal Study on Aging (BLSA) and the Religious Order Study (ROS). We Wnd that small calbindin (+) cortical interneurons are induced to high levels of NADPHd/nNOS reactivity early in AD and abound in areas with emerging neuroWbrillary pathology, that is, in entorhinal cortex in the beginning of the limbic stage of Braak, in hippocampal CA1 in the mature limbic stage and in temporal neocortex in the late limbic stage. This pattern was robust and signiWcant in the younger of the two AD cohorts studied (BLSA), but persisted as a trend in the older cohort (ROS). In optimally prepared material, we Wnd a signiWcant correlation between numbers of these interneurons and markers of neuronal cell death, for example, caspase-3 activation. Our results show that small cortical inhibitory interneurons represent an extensive signaling system that is induced to higher levels of NADPHd/nNOS expression early in the paralimbic-limbic-neocortical sequence of AD progression. We propose that nNOS/NO signaling initiated in these interneurons can serve as a marker of early cortical injury in AD. The speciWc role played by inhibitory interneurons and NO in the elaboration of spe-ciWc neuropathologies associated with AD, that is, A and neuroWbrillary deposits and cell death deserves further exploration in experimental animal models.

We used seven groups of 8-week-old male ICR mice, with 6 mice in each group, to test if aqueous leaf extract of the Thai medicinal plant Thunbergia laurifolia Linn. (TL) protects against lead poisoning. We found that co-treatment with aqueous TL leaf extract did not affect levels of lead in blood and brain of mice given lead in drinking water at 1 g/L for 8 weeks. However, co-treatment with aqueous TL leaf extract at 100 mg/kg or 200 mg/kg body weight was found to alleviate adverse effects of lead on learning deficit and memory loss, evaluated with water maze swimming test. Further, increased activity of the celldeath marker enzyme caspase-3 was observed in the brain of mice treated with lead, thereby suggesting that the memory loss could be caused by lead-induced loss of neurons in the brain. Co-treatment with aqueous TL leaf extract at 100 mg/kg or 200 mg/kg body weight was found to restore the levels of caspase-3 activity and maintain total anti-oxidant capacity and anti-oxidant enzymes in the brain. TL leaf extract thus reduced neuronal cell death and memory loss caused by lead uptake in mice, and the anti-oxidant activities of the TL leaf extract might account for these effects.

New functions of well-known genes have been revealed frequently. A new example is described in this report. Earlier we have detected an up-regulation of expression of the mitochondrial 16S rRNA gene in non-Hodgkin&#39;s lymphomas. Here we demonstrate that the human mitochondrial 16S rRNA gene encodes a potential oncopeptide, Humanin described recently. This peptide suppresses neuronal cell death induced by mutant genes responsible for familial Alzheimer&#39;s disease (AD). Analysis of the gene coding site structure showed that Humanin mRNA is translated most likely in the cytosol, but not in the mitochondrion in vivo. This led us to suppose that AD could be caused by a block of Humanin mRNA transport from mitochondria into the cytosol. Moreover, our data and reports by others an mitochondrial 16S rRNA transcription and characteristic of transcript structure suggests that Humanin is a potential oncopeptide. Thus, the use of Humanin for the treatment of AD may increase the risk for th...

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P 2 residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-L-proline along with four additional scaffold variants were selected as P 2 elements for their predicted ability to clash sterically with a residue of the caspase-3 S 2 pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20-to 60-fold increased selectivity for caspase-2 and $200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.

Huntington’s disease (HD) is a progressive neurodegenerative disorder associated with selective neuronal cell death. Abnormal aggregation of huntingtin protein with polyQ expansion has been shown to be causally linked to HD. Grape seed polyphenolic extract (GSPE) is a natural compound that has previously been shown to interfere with aggregations of proteins involved in neurological disorders, such as amyloid beta peptides (Aβ) and Tau protein. In this study we found that GSPE treatment significantly inhibits polyQ aggregation in phaeochromocytoma (PC)-12 cell line containing an ecdysone-inducible protein comprising the first 17 amino acid of huntingtin plus 103 glutamines fused with enhanced GFP. In vivo feasibility studies using the Q93httexon1 drosophila model of HD, we extended our in vitro evidence and found that flies fed with GSPE had a significantly improved lifespan compared to the control flies. Using the R6/2 rodent model of HD, we found that oral administration of 100 mg/kg/day GSPE (equivalent to 500mg per day in human) significantly attenuated the motor skill decay as well as extended the lifespan in the R6/2 mice relative to vehicle-control mice. Collectively, our studies strongly suggest that GSPE might be able to modulate the onset and/or progression of HD.

In this immunohistochemical study, the age-and stage-dependent accumulation of advanced glycation end-products (AGEs) in Alzheimer's disease (AD) and their relation to the formation of neurofibrillary tangles and neuronal cell death was investigated. For this purpose, the distribution of AGEs in neurons and glia was analyzed in the auditory association area of superior temporal gyrus (Brodmann area 22) of young and old non-demented controls and compared with early-and late-stage AD. A possible co-localization of AGEs with typical hallmarks of AD, such as hyperphosphorylated tau (as a marker for disturbed kinase/phosphatase activity), nNOS (as a marker for nitroxidative stress) and caspase-3 (as a marker of apoptotic cell death), was also investigated. Our results show that the percentage of AGE-positive neurons (and astroglia) increase both with age and, in AD patients, with the progression of the disease (Braak stages). Interestingly, nearly all if those neurons which show diffuse cytosolic AGE immunoreactivity also contain hyperphosphoryated tau, suggesting a link between AGE accumulation and the formation of early neurofibrillary tangles. Many, but not all, neurons show a co-localization of AGEs with other markers of neurodegeneration, such as nNOS and caspase-3.

Huntington's disease (HD) is a genetically transmitted neurodegenerative disorder. The neuropathology in HD is a selective neuronal cell death in several brain regions including cortex. Although changes in synaptic plasticity were shown within the hippocampus and striatum of HD transgenic mice, there are no studies considering neocortical synaptic plasticity abnormalities in HD. We examined the impact of the HD transgene upon learning-dependent plasticity of cortical representational maps. The effect of associative learning, in which stimulation of a row of vibrissae was paired with appetitive stimulus, upon functional representations of vibrissae in the barrel cortex, was investigated with 2-deoxyglucose brain mapping in presymptomatic R6/1 HD mice. In wild-type mice, cortical representation of the row of vibrissae involved in the training was expanded, while in HD mice the representation of this row was not expanded. The results suggest that presymptomatic R6/1 HD transgenic mice show deficits in plasticity of primary somatosensory cortex. D 2004 Elsevier Inc. All rights reserved.

Null mutations affecting members of the transforming growth factor-beta and neurotrophin families result in overlapping patterns of neuronal cell death. This is particularly striking in the cranial sensory nodose-petrosal ganglion complex (NPG), in which loss of either glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4) results in a 30-50% reduction in neuronal survival. It is unknown, however, whether GDNF and any single neurotrophin support survival of the same cells, and if so, whether they are required simultaneously or sequentially during development. To approach these issues we defined survival requirements of nodose and petrosal neurons for GDNF in vitro and in bdnf, gdnf, and bdnf/gdnf null mutant mice, as well as the distribution of GDNF in NPG target tissues. Our analyses focused on the total population of ganglion cells as well as the subset of NPG neurons that are dopaminergic. Neur...

In vivo models of hypoxic-ischemic brain injury have shown altered expression of a number of genes that are important in regulating neuronal survival. However, it is not clear as to whether hypoxia alone can alter the expression of genes regulating neuronal survival. We Ž . Ž . hypothesized that 1 hypoxia alone alters the expression of bcl-2 in neurons, 2 the severity and duration of hypoxia influence bcl-2 Ž . expression, and 3 the alteration of bcl-2 expression has an important role in regulating neuronal survival during hypoxia. Embryonic rat neocortical neurons cultured for 7-10 days were exposed to 0.1, 1, or 3% oxygen for various durations and were removed for analyses at 24-h intervals. Under all hypoxic conditions, neurons exhibited morphologic changes, as assessed by electron microscopy and Annexin V staining, consistent with apoptosis. Immunoblot and immunofluorescence analyses revealed an increase in neuronal bcl-2 protein during hypoxic exposure. Quantitative immunofluorescence analyses of bcl-2 immunostained neurons indicated that expression of bcl-2 was altered by the duration and severity of hypoxia. Attenuation of bcl-2 expression by antisense oligonucleotides decreased the proportion of surviving neurons by ; 50% after 48 h of exposure to 0.1% oxygen. We conclude that observed increase in bcl-2, in part, plays an important role in neuronal survival during exposure to hypoxia. q

During ischemic brain injury, glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca 2+ overload and neuronal cell death. Here we show that glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells. During and shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. In these cells, mitochondrial membrane potential collapsed, nuclei swelled, and intracellular debris were scattered in the incubation medium. Neurons surviving the early necrotic phase recovered mitochondrial potential and energy levels. Later, they underwent apoptosis, as shown by the formation of apoptotic nuclei and by chromatin degradation into high and low molecular weight fragments. These results suggest that mitochondrial function is a critical factor that determines the mode of neuronal death in excitotoxicity.

Thyrotropin-releasing hormone (TRH) and TRH analogues have been reported to be neuroprotective in experimental models of spinal cord injury and head injury. We have previously shown that a diketopiperazine structurally related to the TRH metabolite cyclo-his-pro reduces neuronal cell death in vitro and in vivo. Here we report the neuroprotective activity of other cyclic dipeptides in multiple in vitro models of neuronal injury and after controlled cortical impact (CCI) in mice. Using primary neuronal cultures, three novel dipeptides were compared to the previously reported diketopiperazine as well as to vehicle controls; each of the compounds reduced cell death after direct physical trauma or trophic withdrawal. Two of these peptides also protected against glutamate toxicity and b-amyloid-induced injury; the latter also strongly inhibited glutamate-induced increases in intracellular calcium. Treatment with each of the test compounds resulted in highly significant improvement of motor and cognitive recovery after CCI, as well as markedly reducing lesion volumes as shown by high field magnetic resonance imaging. DNA microarray studies following fluid percussion induced traumatic brain injury (TBI) in rats showed that treatment with one of these dipeptides after injury significantly down-regulated expression of mRNAs for cell cycle proteins, aquaporins, cathepsins and calpain in ipsilateral cortex and/or hippocampus, while up-regulating expression of brain-derived neurotrophic factor, hypoxia-inducible factor and several heat-shock proteins. Many of these mRNA expression changes were paralleled at the protein level. The fact that these small peptides modulate multiple mechanisms favoring neuronal cell survival, as well as their ability to improve functional outcome and reduce posttraumatic lesion size, suggests that they may have potential utility in clinical head injury.

Neuronal cell death is in many cases regulated by competitive interactions between pro-and antiapoptotic proteins of the Bcl-2 family. In this study we have identified two splice variants of the rat proapoptotic molecule Bad, which differ in their carboxy-terminal regions. Both splice variants of Bad interacted with the antiapoptotic molecule Bcl-w as shown by yeast two-hybrid assay and by coimmunoprecipitation experiments from transfected cells. mRNA expression for the two variants of bad were detected in all neonatal and adult rat tissues tested. Overexpression of either of the two isoforms of Bad in nerve growth factor (NGF)-maintained sympathetic neurons by microinjection induced the cell death of these neurons, which was neutralized by co-expression of Bcl-w. Overexpression of Bcl-w in sympathetic neurons also counteracted death induced by NGF deprivation, which was not reduced by co-expression of either of the two Bad variants. The results suggest that Bcl-w, Bad-␣, and Bad-␤ may participate in the regulation of apoptosis in the sympathetic nervous system.

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 Parkinson's and Huntington's 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 Parkinson's and Huntington's diseases. First, we will give an Progress in Neurobiology 60 0301-0082/00/$ -see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 -0 0 8 2 ( 9 9 ) 0 0 0 3 2 -5 overview of the mechanisms of cell death and the background of Parkinson's and Huntington's 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. #

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that overexpression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.

Aging is associated with mitochondrial dysfunctions, which trigger membrane leakage, release of reactive species from oxygen and nitrogen and subsequent induction of peroxidative reactions that result in biomolecules' damaging and releasing of metals with amplification of free radicals discharge. Free radicals induce neuronal cell death increasing tissue loss, which could be associated with memory detriment. These pathological events are involved in cardiovascular, neurodegenerative and carcinogenic processes. Dietary bioactive compounds from different functional foods, herbs and nutraceuticals (ginseng, ginkgo, nuts, grains, tomato, soy phytoestrogens, curcumin, melatonin, polyphenols, antioxidant vitamins, carnitine, carnosine, ubiquinone, etc.) can ameliorate or even prevent diseases. Protection from chronic diseases of aging involves antioxidant activities, mitochondrial stabilizing functions, metal chelating activities, inhibition of apoptosis of vital cells, and induction of cancer cell apoptosis. Functional foods and nutraceuticals constitute a great promise to improve health and prevent aging-related chronic diseases.

Huntington’s disease (HD) is a progressive neurodegenerative disorder associated with selective neuronal cell death. Abnormal aggregation of huntingtin protein with polyQ expansion has been shown to be causally linked to HD. Grape seed polyphenolic extract (GSPE) is a natural compound that has previously been shown to interfere with aggregations of proteins involved in neurological disorders, such as amyloid beta peptides (Aβ) and Tau protein. In this study we found that GSPE treatment significantly inhibits polyQ aggregation in phaeochromocytoma (PC)-12 cell line containing an ecdysone-inducible protein comprising the first 17 amino acid of huntingtin plus 103 glutamines fused with enhanced GFP. In vivo feasibility studies using the Q93httexon1 drosophila model of HD, we extended our in vitro evidence and found that flies fed with GSPE had a significantly improved lifespan compared to the control flies. Using the R6/2 rodent model of HD, we found that oral administration of 100 mg/kg/day GSPE (equivalent to 500mg per day in human) significantly attenuated the motor skill decay as well as extended the lifespan in the R6/2 mice relative to vehicle-control mice. Collectively, our studies strongly suggest that GSPE might be able to modulate the onset and/or progression of HD.

The neuroprotective potency of N-PEP-12, a novel, proprietary compound consisting of biopeptides and amino acids was investigated. Lesion models have been applied in neuronal cultures of embryonic chicken cortex, pre-treated with N-PEP-12 from the first day onwards. On day 8 in vitro neurons were lesioned and cell viability was measured 24 and 48 hours later. To simulate acute brain ischemia, cytotoxic hypoxia was induced by sodium cyanide or by iodoacetate and excitotoxicity by L-glutamate. Ionomycin for up to 48 hours induced calcium overload. The cytoskeleton was disrupted by addition of colchicine. N-PEP-12 shows dose-dependent neuroprotection in all different models. The effect size depends on the recovery time but also on the extent of the lesion. In cases of mild to moderate lesion pronounced dose-dependent effects could be demonstrated. This indicates that chronic exposure to N-PEP-12 is able to prevent neuronal cell death associated to conditions occurring during normal aging and neurological disorders like ischemic stroke, hypoxia, brain trauma, or AD.

The phosphatidylinositol-3-kinase (PI3-K) pathway has been suggested to play a pivotal role in neuronal survival. Although PI3-K has been recently identified as a neuroprotectant, there are no reports regarding the effect of a direct PI3-K activator on A␤-induced neurotoxicity. We investigated whether direct PI3-K activation prevents A␤-induced neurotoxicity. To evaluate the effect of A␤ on neuronal cells, we treated primary cultured cortical neurons with several doses of A␤ for 72 h. To investigate the protective effect that PI3-K activation has on A␤-induced neurotoxicity, cells were simultaneously treated with several doses of a PI3-K activator for 72 h. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, and DAPI staining showed that A␤ decreased neuronal cell viability in a concentration-dependent manner and also that PI3-K activation effectively prevented A␤-induced neuronal cell death. A␤ significantly decreased survival signals, including phosphorylated Akt, glycogen synthase kinase-3␤, and heat shock transcription factor-1. A␤ also increased death signals, such as phosphorylated tau (pThr231) and activated caspase-3. Treatment with a PI3-K activator restored the survival signals and inhibited the death signals. These results suggest that the neurotoxic effect of A␤ can be partially prevented by PI3-K activation.

Glycogen synthase kinase-3, especially the beta form (GSK-3β), plays key roles in oxidative stress-induced neuronal cell death, an important pathogenic mechanism of various neurodegenerative diseases. Although the neuroprotective effects of GSK-3β inhibitors have been described, the optimal level of GSK-3β inhibition for neuronal cell survival has not yet been determined. We investigated the effect of varying GSK-3β activity on the viability of oxidative stress-injured neuronally differentiated PC12 (nPC12) cells and intracellular signals related with the GSK-3β and caspase-3 pathways. Compared to the nPC12 control cells treated with only 100 μM H 2 O 2 , treatment of 50-200 nM GSK-3β inhibitor II or 25-500 nM GSK-3β inhibitor VIII reduced the increased enzyme activity by about 50% and protected the cells against H 2 O 2-induced oxidative stress. The optimal concentration of GSK-3β inhibitor II enhanced heat shock transcription factor-1 levels, decreased levels of phosphorylated tau (Ser202) and cytosolic cytochrome c, activated caspase-3, and cleaved poly (ADP-ribose) polymerase. In contrast, higher concentrations of GSK-3β inhibitor II (more than 500 nM) induced neuronal cell death and showed opposite effects relative to the above described intracellular signals. These results suggest that optimized inhibitor levels for modulating GSK-3β activity may prevent apoptosis induced by oxidative stress associated with neurodegenerative diseases.

Here we report that the OP nerve agent, VX, induces apoptoticlike cell death in cultured rat cortical neurons. The VX effects on neurons were concentrationdependent, with an IC 50 of approximately 30 M. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 µM D-2-amino-5phosphonovalerate (APV) diminished 30 M VX-induced total cell death, as assessed by ala-marBlue TM assay and Hoechst staining. In contrast, neither antagonists of -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

Histone deacetylase-related protein (HDRP), an alternatively spliced and truncated form of histone deacetylase-9 that lacks a C-terminal catalytic domain, protects neurons from death. In an effort to understand the mechanism by which HDRP mediates its neuroprotective effect, we screened for proteins in the brain that interact with HDRP by using a yeast two-hybrid assay. One of the HDRP-interacting proteins identified in this screen was amino enhancer of split (AES), a 197–amino acid protein belonging to the Groucho family. Interaction between HDRP and AES was verified by in vitro binding assays, coimmunoprecipitation, and colocalization studies. To investigate the significance of the HDRP-AES association to the regulation of neuronal survival, we used cultured cerebellar granule neurons, which undergo apoptosis when treated with low potassium (LK) medium. We found that in contrast to HDRP, whose expression is markedly reduced by LK treatment, AES expression was not appreciably altered. Forced expression of AES in healthy neurons results in cell death, an action that is blocked by the coexpression of HDRP. AES is a truncated version of larger Groucho-related proteins, one of which is transducin-like enhancer of split (TLE)-1. We found that the expression of TLE1 is reduced in LK-treated neurons and the forced expression of TLE1 blocks LK-induced neuronal death as well as death induced by AES. Our results show that AES has apoptotic activity in neurons and suggest that neuroprotection by HDRP is mediated by the inhibition of this activity through direct interaction. © 2008 Wiley-Liss, Inc.

Endocannabinoids are thought to function as retrograde messengers, which modulate neurotransmitter release by activating presynaptic cannabinoid receptors. Anandamide and 2-arachidonoylglycerol (2-AG) are the two best studied endogenous lipids which can act as endocannabinoids. Together with the proteins responsible for their biosynthesis, inactivation and the cannabinoid receptors, these lipids constitute the endocannabinoid system. This system is proposed to be involved in various neurodegenerative diseases such as Parkinson's and Huntington's diseases as well as Multiple Sclerosis. It has been demonstrated that the endocannabinoid system can protect neurons against glutamate excitotoxicity and acute neuronal damage in bothin vitro andin vivo models. In this paper we review the data concerning the involvement of the endocannabinoid system in neurodegenerative diseases in which neuronal cell death may be elicited by excitotoxicity. We focus on the biosynthesis of endocannabinoids and on their modes of action in animal models of these neurodegenerative diseases.

Current evidence suggests that amyloid ␤ peptides (A␤) may play a major role in the pathogenesis of Alzheimer's disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by A␤ 1-40 and A␤ 1-42 peptides as well as the relationships between apoptosis and oxidative stress. A␤ peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by ␣-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38 MAPK , involved early elevation of p53 protein levels. Pretreatment of neurons with ␣-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of A␤ induced apoptosis. These results suggest that oxidative stress -mediated neuronal apoptosis induced by amyloid ␤ operates by eliciting a SAPK-dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2.

p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

Parkinson's disease (PD) is a movement disorder characterized by a progressive loss of nigrostriatal dopaminergic neurons. Microglia activation and neuroinflammation have been associated with the pathogenesis of PD. Indeed, cytokines have been proposed as candidates that mediate the apoptotic cell death of dopaminergic neurons seen in PD. In this study, we investigated the effect of two natural polyphenols, resveratrol and quercetin, on neuroinflammation. For glial cells, we observed that lipopolysaccharide (LPS)-induced mRNA levels of two proinflammatory genes, interleukin 1-a and tumor necrosis factor-a, are strongly decreased by treatments with resveratrol or quercetin. We also undertook microglial-neuronal coculture to examine the influence of resveratrol and quercetin on dopaminergic neuronal cell death evoked by LPS-activated microglia. Cytotoxicity assays were performed to evaluate the percentage of cell death, with apoptotic cells identified by both the TdT-mediated dUTP nick end labeling technique and the detection of cleaved caspase-3. We report that treatment of N9 microglial cells with resveratrol or quercetin successfully reduced the inflammation-mediated apoptotic death of neuronal cells in our coculture system. Altogether our results demonstrate that resveratrol and quercetin diminished apoptotic neuronal cell death induced by microglial activation and suggest that these two phytoestrogens may be potent antiinflammatory compounds. V V C 2007 Wiley-Liss, Inc.

Abbreviations: Olfactory mucosa (OM), olfactory epithelium (OE), olfactory receptors neurons (ORNs), sarafotoxin (S6C) Endothelin as a neuroprotective factor for ORNs ABSTRACT In mammals, the olfactory receptors neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory receptor neurons express preferentially ET B receptors, while ET A receptors are rather present in non-neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect using a primary culture of olfactory receptor neurons. While a serum deprivation leads to a massive decrease of the density of olfactory receptor neurons in the primary cultures, ET-1 rescued part of the neuronal population through both ET A and ET B receptors. This effect was mainly anti-apoptotic as it reduces cleaved caspase-3 signal and nuclear fragmentation. Furthermore, the olfactory epithelium of ET B -deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory receptor neurons directly through ET B and indirectly by limiting non neuronal cells death through ET A .

The zebrafish is an excellent model organism in which to study the retina&#39;s response to photoreceptor degeneration and/or acute injury. While much has been learned about the retinal stem and progenitor cells that mediate the damage response, several questions remain that cannot be addressed by acute models of injury. The development of genetic models, such as the XOPS-mCFP transgenic line, should further efforts to understand the nature of the signals that promote rod progenitor proliferation and differentiation following photoreceptor loss. This in turn may help to refine future approaches in higher vertebrates aimed at enhancing retinal progenitor cell activity for therapeutic purposes.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved. Cells and viruses. The cervical HeLa-UVM cancer cell line (5) (here simply termed HeLa) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. CVB3, an H3 Woodrupt strain (donated by E. Jeon, Samsung Medical Center, Seoul, Korea), was grown and titered with HeLa cells. Viral stocks were prepared by infecting 90% confluent cultures of HeLa cells at a multiplicity of infectivity (MOI) of 10. After infection for 16 h at 37°C, the suspension was freeze-thawed three times. The cell lysates were centrifuged and the supernatants were harvested and stored at Ϫ75°C. The amount of virus was measured by plaque assay. The titer of CVB3 H3 was 10 9 PFU/ml.

Purpose: To determine whether repeated seizures contribute to hippocampal sclerosis, we investigated whether cell loss in the (para) hippocampal region was related to the severity of chronic seizure activity in a rat model for temporal lobe epilepsy (TLE).

Apoptosis is a programmed cell death that plays a critical role during the development of the nervous system and in many chronic neurodegenerative diseases, including Alzheimer's disease (AD). This pathology, characterized by a progressive degeneration of cholinergic function resulting in a remarkable cognitive decline, is the most common form of dementia with high social and economic impact. Current therapies of AD are only symptomatic, therefore the need to elucidate the mechanisms underlying the onset and progression of the disease is surely needed in order to develop effective pharmacological therapies. Because of its pivotal role in neuronal cell death, apoptosis has been considered one of the most appealing therapeutic targets, however, due to the complexity of the molecular mechanisms involving the various triggering events and the many signaling cascades leading to cell death, a comprehensive understanding of this process is still lacking.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimer's Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of β-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3β. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.