Richard M. Kostrzewa

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson’s disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate‐induced oxytosis. In this study, we explore the protective effects of necrostatin‐1 (Nec‐1), an inhibitor of necroptosis, on glutamate‐induced oxytosis. We show that Nec‐1 inhibits glutamate‐induced oxytosis in HT‐22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec‐1 had no protective effect on free radical‐induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec‐1 has no antioxidant effects. Interestingly, the protective effect of Nec‐1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec‐1 significantly blocks the nuclear translocation of apoptosis‐inducing factor (a marker of caspase‐independent programmed cell death) and inhibits the integration of Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3 (a pro‐death member of the Bcl‐2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec‐1 prevents glutamate‐induced oxytosis in HT‐22 cells through GSH related as well as apoptosis‐inducing factor and Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3‐related pathways.

Neurotoxins and neurotoxic species implicated in neurodegeneration

Neurotoxins, in the general sense, represent novel chemical structures which when administered in vivo orin vitro, are capable of producing neuronal damage or neurodegeneration—with some degree of specificity relating to neuronal phenotype or populations of neurons with specific characteristics (i.e., receptor type, ion channel type, astrocyte-dependence, etc.). The broader term ‘neurotoxin’ includes this categorization but extends the term to include intra- or extracellular mediators involved in the neurodegenerative event, including necrotic and apoptotic factors. Moreover, as it is recognized that astrocytes are essential supportive satellite cells for neurons, and because damage to these cells ultimately affects neuronal function, the term ‘neurotoxin’ might reasonably be extended to include those chemical species which also adversely affect astrocytes. This review is intended to highlight developments that have occurred in the field of ‘neurotoxins’ during the past 5 years, including MPTP/MPP+, 6-hydroxydopamine (6-OHDA), meth-amphetamine; salsolinol; leukoaminochrome-o-semi-quinone; rotenone; iron; paraquat; HPP+; veratridine; soman; glutamate; kainate; 3-nitropropionic acid; peroxynitrite anion; and metals (copper, manganese, lead, mercury). Neurotoxins represent tools to help elucidate intra- and extra-cellular processes involved in neuronal necrosis and apoptosis, so that drugs can be developed towards targets that interrupt the processes leading towards neuronal death.

Supersensitized D1 receptors mediate enhanced oral activity after neonatal 6-OHDA

Enhanced oral responses have been observed in rats that are treated shortly after birth with 6-hydroxydopamine (6-OHDA). A series of studies was conducted to characterize this effect. A dose-response curve demonstrated that the dopamine D1 receptor agonist, SKF 38393, produced a maximal response in 6-OHDA-treated rats at a dose of 0.10 mg/kg (IP). With the D2 receptor antagonist, spiperone, a bell-shaped dose-response curve was seen, with a maximal effect in the 6-OHDA group occurring at 80 micrograms/kg. There were only slight increases in oral activity with different SKF 38393 or spiperone doses in the saline group, indicating that there was an overt supersensitization of D1 receptors in the 6-OHDA-treated rats. The D1 antagonist SCH 23390 (0.30 mg/kg, IP) attenuated the response to both SKF 38393 and spiperone. The oral response to the D2 agonist, quinpirole (0.10 mg/kg, IP) was not preferentially increased in the 6-OHDA group of rats. These findings indicate that the enhanced oral response in neonatal 6-OHDA-treated rats is mediated by supersensitive dopamine D1 receptors. The persistence of the enhanced oral response in 6-OHDA-treated rats at 8 months demonstrates that this sensitization of D1 receptors is a long-lived phenomenon.

Serotonin Neural Adaptations to Ontogenetic Loss of Dopamine Neurons in Rat Brain

Abstract: In rat, the neonatal destruction of nigrostriatal dopamine (DA) neurons by intracerebral administration of 6‐hydroxydopamine entails dramatic changes in serotonin (5‐hydroxytryptamine, 5‐HT) as well as DA function. Most striking is the 5‐HT hyperinnervation of the adult neostriatum, associated with increases in density of various 5‐HT receptor subtypes and enhanced neuronal responsiveness to the iontophoretic application of 5‐HT and its 5‐HT1B/2C and 5‐HT2A/2C receptor agonists, m‐chlorophenylpiperazine and iododimethoxyphenylaminopropane. The topographical distribution of these changes is consistent with up‐regulation and/or increased production and transport of 5‐HT1B and 5‐HT2A receptors by the neostriatal projection neurons, as confirmed for the 5‐HT2A receptor in a recent in situ hybridization study. It is interesting that this study has also shown that increases in both 5‐HT2A binding and mRNA level were abolished by chronic pretreatment with the DA agonists, apomorphine and SKF 38393, suggesting a regulatory influence of DA in the expression of this 5‐HT receptor. D1 receptor binding is known to be slightly reduced in the rostral neostriatum of these rats, a down‐regulation apparently imputable to a reduced rate of synthesis of the receptor. In contrast, D2 receptor binding is increased throughout the DA‐denervated and 5‐HT‐hyperinnervated neostriatum, perhaps due to some post‐transcriptional modifications. Stereotyped and motor behaviors induced by systemic treatment with D1 and D2 agonists are markedly enhanced in these rats (behavioral supersensitivity), although priming is commonly required to unmask a latent D1 supersensitivity. In the case of oral activity, however, overt behavioral supersensitivity is induced by D1 as well as D2 agonists. Moreover, there is overt supersensitivity of oral activity in response to the 5‐HT receptor agonist m‐chlorophenylpiperazine, which is presumably imputable to 5‐HT2C receptors and may be demonstrated even in the absence of supersensitivity to D1 receptor agonist. 5‐HT adaptations, therefore, seem to play a role not only in the abnormal spontaneous behavior, but also in the behavioral supersensitivity to 5‐HT as well as DA receptor agonists in these rats.

Proposed animal model of attention deficit hyperactivity disorder

Dopamine (DA) neurons are implicated in the hyperlocomotion of neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of attention deficit hyperactivity disorder (ADHD). Because serotonin (5-HT) neurons mediate some DA agonist effects, we investigated the possible role of 5-HT neurons on locomotor activity. Rats were treated at 3 days after birth with vehicle or 6-OHDA (134 micrograms ICV; desipramine pretreatment, 20 mg/kg IP, 1 h), and at 10 weeks with vehicle or 5,7-dihydroxytryptamine (5,7-DHT; 75 micrograms ICV; pretreatment with desipramine and pargyline, 75 mg/kg IP, 30 min), to destroy DA and/or 5-HT fibers. Intense spontaneous hyperlocomotor activity was produced in rats lesioned with both 6-OHDA and 5,7-DHT. Locomotor time in this group was 550 +/- 17 s in a 600 s session, vs. 127 +/- 13 s in the 6-OHDA group and < 75 s in 5,7-DHT and intact control groups (p < 0.001). Oral activity dose-effect curves established that 5,7-DHT attenuated DA D1 receptor supersensitivity and further sensitized 5-HT2c receptors. Acute treatment with dextroamphetamine (0.25 mg/kg SC) reduced locomotor time in 6-OHDA + 5,7-DHT-lesioned rats to 76 +/- 37 s (p < 0.001). Striatal DA was reduced by 99% and 5-HT was reduced by 30% (vs. 6-OHDA group). Because combined 6-OHDA (to neonates) and 5,7-DHT (to adults) lesions produce intense hyperlocomotion that is attenuated by amphetamine, we propose this as a new animal model of ADHD. The findings suggest that hyperactivity in ADHD may be due to injury or impairment of both DA and 5-HT neurons.

Physical Exercise Alleviates ADHD Symptoms: Regional Deficits and Development Trajectory

The heterogeneous, chronic, and proliferating aspect of attention deficit hyperactivity disorder (ADHD) and comorbidities covers heritability, cognitive, emotional, motor, and everyday behavioral domains that place individuals presenting the condition at some considerable disadvantage. Disruption of “typical developmental trajectories” in the manifestation of gene-environment interactive predispositions implies that ADHD children and adolescents may continue to perform at defective levels as adults with regard to academic achievement, occupational enterprises, and interpersonal relationships, despite the promise of pharmacotherapeutic treatments. Physical exercise provides a plethora of beneficial effects against stress, anxiety, depression, negative affect and behavior, poor impulse control, and compulsive behavior concomitant with improved executive functioning, working memory and positive affect, as well as improved conditions for relatives and care-givers. Brain-derived neurotrophic factor, an essential element in normal brain development that promotes health-associated behaviors and quality-of-life, though reduced in ADHD, is increased markedly by the intervention of regular physical exercise. Functional, regional, and biomarker deficits, as well as hypothalamic–pituitary–adrenal disruptions, have been improved through regular and carefully applied exercise programs. In view of the complications involving ADHD with co-morbidities, such as obesity, the influence of regular physical exercise has not been found negligible. Physical exercise bestows a propensity for eventual manifestation of “redifferentiated” developmental trajectories that may equip ADHD adults with a prognosis that is more adaptive functionally, independent of the applications of other therapeutic agents and treatments.

Novel mechanisms and approaches in the study of neurodegeneration and neuroprotection. A review

Cellular mechanisms involved in neurodegeneration and neuroprotection are continuing to be explored, and this paper focuses on some novel discoveries that give further insight into these processes. Oligodendrocytes and activated astroglia are likely generators of the pro-inflammatory cytokines, such as the tumor necrosis factor family and interleukin family, and these glial support cells express adhesion receptors (e.g., VCAM) and release intercellular adhesion molecules (ICAM) that have a major role in neuronal apoptosis. Even brief exposure to some substances, in ontogeny and sometimes in adulthood, can have lasting effects on behaviors because of their prominent toxicity (e.g., NMDA receptor antagonists) or because they sensitize receptors (e.g., dopamine D2 agonists), possibly permanently, and thereby alter behavior for the lifespan. Cell cycle genes which may be derived from microglia, are the most-recent entry into the neuroprotection schema. Neuroprotection afforded by some common substances (e.g., melatonin) and uncommon substances [e.g., nicotine, green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG), trolox], ordinarily thought to be simple radical scavengers, now are thought to invoke previously unsuspected cellular mechanisms in the process of neuroprotection. Although Alzheimers disease (AD) has features of a continuous spectrum of neural and functional decline,in vivo PET imaging and and functional magnetic resonance imaging, indicate that AD can be staged into an early phase treatable by inhibitors of β and γ secretase; and a late phase which may be more amenable to treatment by drugs that prevent or reverse tau phosphorylation. Neural transplantation, thought to be the last hope for neurally injured patients (e.g., Parkinsonians), may be displaced by non-neural tissue transplants (e.g., human umbilical cord blood; Sertoli cells) which seem to provide similar neurotrophic support and improved behavior-without posing the major ethical dilemma of removing tissue from aborted fetuses. The objective of this paper is to invite added research into the newly discovered (or postulated) novel mechanisms; and to stimulate discovery of additional mechanisms attending neurodegeneration and neuroprotection.

Supersensitized oral responses to a serotonin agonist in neonatal 6-OHDA-treated rats

Neonatal 6-hydroxydopamine (6-OHDA) treatment of rats is associated with supersensitization of the dopamine D1 agonist induction of oral activity. The present study was conducted to determine whether induced oral responses to serotonin (5-HT) agonists would be similarly altered in this rat model. At 3 days after birth, rats received desipramine HCl (20 mg/kg, IP) 1 h before 6-OHDA HBr (100 micrograms in each lateral ventricle) or saline-ascorbic acid (0.1%) vehicle. At approximately 9 mo, rats were challenged with the mixed 5-HT1C and 5-HT2 receptor agonist, m-chlorophenylpiperazine diHCl (m-CPP 2HCl; 0.30-6.0 mg/kg, IP) and were then observed for 1 min every 10 min over a 60-min period. m-CPP induced oral activity in both the vehicle and 6-OHDA groups, with the responses of the 6-OHDA group being much greater. An m-CPP dose of 3.0 mg/kg produced a maximal response of 63.6 +/- 3.2 oral movements in the 6-OHDA group. A bell-shaped response curve was obtained, with lower and higher doses of m-CPP producing less of an effect. Attenuation of the m-CPP-induced response by the 5-HT receptor antagonist, mianserin HCl (1.0 mg/kg, IP, 30 min before m-CPP), indicates that the m-CPP effect is receptor mediated. These findings demonstrate that neonatal 6-OHDA treatment produces ontogenic long-lived supersensitization of a 5-HT receptor system in rats.

Influence of physical exercise on neuroimmunological functioning and health: aging and stress.

Chronic and acute stress, with associated pathophysiology, are implicated in a variety of disease states, with neuroimmunological dysregulation and inflammation as major hazards to health and functional sufficiency. Psychosocial stress and negative affect are linked to elevations in several inflammatory biomarkers. Immunosenescence, the deterioration of immune competence observed in the aged aspect of the life span, linked to a dramatic rise in morbidity and susceptibility to diseases with fatal outcomes, alters neuroimmunological function and is particularly marked in the neurodegenerative disorders, e.g., Parkinson’s disease and diabetes. Physical exercise diminishes inflammation and elevates agents and factors involved in immunomodulatory function. Both the alleviatory effects of life-long physical activity upon multiple cancer forms and the palliative effects of physical activity for individuals afflicted by cancer offer advantages in health intervention. Chronic conditions of stress and affective dysregulation are associated with neuroimmunological insufficiency and inflammation, contributing to health risk and mortality. Physical exercise regimes have induced manifest anti-inflammatory benefits, mediated possibly by brain-derived neurotrophic factor. The epidemic proportions of metabolic disorders, obesity, and diabetes demand attention; several variants of exercise regimes have been found repeatedly to induce both prevention and improvement under both laboratory and clinical conditions. Physical exercise offers a unique non-pharmacologic intervention incorporating multiple activity regimes, e.g., endurance versus resistance exercise that may be adapted to conform to the particular demands of diagnosis, intervention and prognosis inherent to the staging of autoimmune disorders and related conditions.

Low-dose quinpirole ontogenically sensitizes to quinpirole-induced yawning in rats

It is known that dopamine (DA) receptors can be sensitized by repeated treatments with quinpirole during postnatal development. This study was undertaken to determine whether low-dose quinpirole treatments might sensitize receptors to quinpirole-induced yawning behavior. Rats were treated with quinpirole HCl (50 micrograms/kg per day) or saline at four different periods of ontogeny: a) the 10th day of gestation to day of birth; b) 1st-11th days after birth; c) 12th-22nd days from birth; or d) 23rd-33rd days from birth. The numbers of yawns occurring in 1 h after a challenge dose of quinpirole HCl (50 micrograms/kg, IP) was determined at 6 weeks. Rats exposed prenatally to quinpirole demonstrated increased numbers of yawns following the third dose of quinpirole (2-day interval between doses). In rats exposed postnatally to quinpirole, there was a 70-300% increase in the yawning response, with the greatest response occurring in the group treated with quinpirole from birth to 11 days from birth. The findings demonstrate that quinpirole receptors are sensitized by a low dose of quinpirole, 60-fold lower than previously shown. It is suggested that sensitized receptors are of the DA D3 subclass.