A new methodological approach for in vitro determination of the role of DNA methylation on transcription factor binding using AlphaScreen® analysis: Focus on CREB1 binding at hBDNF promoter IV.

BACKGROUND: DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding. METHOD: An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation. RESULTS: For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA. COMPARISON WITH EXISTING METHODS: This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin. CONCLUSIONS: The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.

Oxidative Stress in Cerebral Small Vessel Disease Dizziness Patients, Basally and After Polyphenol Compound Supplementation.

BACKGROUND: Leukoaraiosis (LA) is a common radiological finding in elderly, frequently associated with several clinical disorders, including unexplained dizziness. The pathogenesis of LA is multifactorial, with a dysfunction of cerebral microcirculation resulting in chronic hypoperfusion and tissue loss, with oxidative stress involved in this cascade. OBJECTIVE: The aim of this study was to analyse some oxidative stress biomarkers in a cohort of LA patients. METHOD: Fifty-five consecutive patients (33 males, median age 75 years) with LA were recruited. In a subgroup of 33 patients with LA and unexplained dizziness, we have then performed an open study to evaluate if 60-day supplementation with a polyphenol compound may modify these biomarkers and influence quality of life, analysed with the Dizziness Handicap Inventory (DHI) scale. RESULTS: At baseline, blood oxidative stress parameters values were outside normal ranges and compared to matched healthy controls. After the two months supplementation, we observed a significant decrement of advanced oxidation protein products values and a significant improvement of DHI. CONCLUSION: Oxidative stress biomarkers may be useful to detect redox imbalance in LA and to provide non-invasive tools to monitor disease status and response to therapy.

Endothelium and Oxidative Stress: The Pandora's Box of Cerebral (and Non-Only) Small Vessel Disease?

Common cerebral small vessel disease (cSVD) abnormalities are a common neuroradiological finding, especially in the elderly. They are associated with a wide clinical spectrum that leads to an increasing disability, impaired global function outcome and a reduced quality of life. A strong association is demonstrated with age and hypertension and other common vascular risk factors, including diabetes mellitus, dyslipoproteinemia, smoking, low vitamin B12 level, and hyperomocysteinemia. Although these epidemiological associations suggest a systemic involvement, etiopathogenetic mechanisms remain unclear. This review focuses on the potential role of endothelial dysfunction and oxidative stress in the pathogenic cascade leading to cSVD. We stressed on the central role of those pathways, and suggest the importance of quantifying the cerebral (and non-only) "endotheliopathic and oxidative load" and its clinical presentation that could lead to a better determination of vascular risk degree. In addition, understanding underlying pathogenic mechanisms could allow us to slow down the progression of vascular damage and, therefore, prevent the disability due to reiterated microvascular damage.

Epidemiology and cerebrovascular events related to cervical and intracranial arteries dissection: the experience of the city of Pisa.

Spontaneous dissection of cervical arteries (sCAD) is a major cause of ischemic stroke in young patients, with an incidence varying from 1.7 to 3/100,000/year for extracranial internal carotid artery (ICAD) and 1 to 1.9/100,000/year for extracranial vertebral artery (VAD). Reliable epidemiological data on stroke incidence related to sCAD are scarce in Italy. This study aims to evaluate the incidence, clinical features, and outcome of cerebrovascular events related to sCAD and spontaneous intracranial arteries dissections (sIAD) in the city of Pisa (Italy). We retrospectively analyzed consecutive patients admitted between December 1997 and June 2015 with a diagnosis of stroke, TIA, or Bernard-Horner syndrome due to acute cervical or intracranial artery dissection. Considering that our hospital collects presumptively all patients hospitalized with sCAD coming from the referral geographical area, data may provide a good approximation to real incidence of sCAD in our population. Clinical and radiological features, acute treatment and outcome were collected. Seventy-seven cases were included (mean age 48.1±10.4 years, range 23-77,72.7% males), 66 residents in the district of Pisa. Crude incidence rate of cerebrovascular events due to intra or extracranial dissection was 1.88/100,000/year. The incidence of ICAD was 0.80/100,000/year and 0.43/100,000/year for VAD. Stroke occurred in 76.6% of patients. VAD was more prone to cause ischemic stroke and present with cervical pain or focal signs (p < 0.01) than ICAD group, which had older age at onset. sIAD were more frequent in the posterior circle (p = 0.01) and more associated with ischemic lesions. A good outcome (mRS 0-2) was observed in 79% of patients. This is the first epidemiological attempt to investigate impact of sCAD and sIAD in Italy.

2-arachidonoylglycerol levels are increased in leukocytospermia and correlate with seminal macrophages.

Evidence has been produced that macrophages can actively generate endocannabinoids (eCBs) in response to inflammatory stimuli. As eCBs are involved in the control of several physiological processes, including reproduction, here, we explored whether seminal levels of the eCBs, N-arachidonoylethanolamine (AEA), and 2-arachidonoylglycerol (2-AG), were higher in the presence of leukocytospermia, and were correlated with semen concentration of macrophages. The content of AEA and 2-AG was measured by high-performance liquid chromatography/mass spectrometry in seminal plasma of ejaculates from 18 leukocytospermic patients (>1 × 106 leukocytes/mL) and 21 normozoospermic controls. In the same ejaculates, round cells were phenotyped by flow-cytometry as leukocytes (CD45+), macrophages (CD14+), and activated macrophages (CD14+, HLA-DR+). The levels of 2-AG, but not of AEA, were significantly higher in ejaculates from leukocytospermic patients than in controls and exhibited a significant correlation with semen concentration of macrophages and activated macrophages. Significant associations of 2-AG with macrophages and activated macrophages persisted after adjustment for semen volume and sperm concentration. In conclusion, here we provide evidence that seminal plasma levels of 2-AG are higher in the presence of leukocytospermia, as a marker of macrophages activation. Further studies are warranted to elucidate possible clinical implications.

Epigenetic modifications of Dexras 1 along the nNOS pathway in an animal model of multiple sclerosis.

The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1ß (IL-1ß) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.

Promoter-Specific Hypomethylation Correlates with IL-1ß Overexpression in Tuberous Sclerosis Complex (TSC).

In tuberous sclerosis complex (TSC), overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1ß (IL-1ß) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Recent evidence indicates that IL-1ß gene expression can be regulated by DNA methylation of its promoter. In the present study, we hypothesized that hypomethylation in the promoter region of the IL-1ß gene may underlie its overexpression observed in TSC brain tissue. Bisulfite sequencing was used to study the methylation status of the promoter region of the IL-1ß gene in TSC and control samples. We identified hypomethylation in the promoter region of the IL-1ß gene in TSC samples. IL-1ß is overexpressed in tubers, and gene expression is correlated with promoter hypomethylation at CpG and non-CpG sites. Our results provide the first evidence of epigenetic modulation of the IL-1ß signaling in TSC. Thus, strategies that target epigenetic alterations could offer new therapeutic avenues to control the persistent activation of interleukin-1ß-mediated inflammatory signaling in TSC brain.

Dopamine-dependent CB1 receptor dysfunction at corticostriatal synapses in homozygous PINK1 knockout mice.

Recessive mutations in the PTEN-induced putative kinase 1 (PINK1) gene cause early-onset Parkinson's disease (PD). We investigated the interaction between endocannabinoid (eCB) and dopaminergic transmission at corticostriatal synapses in PINK1 deficient mice. Whole-cell patch-clamp and conventional recordings of striatal medium spiny neurons (MSNs) were made from slices of PINK1(-/-), heterozygous PINK1(+/-) mice and wild-type littermates (PINK1(+/+)). In PINK1(+/+) mice, CB1 receptor (CB1R) activation reduced spontaneous excitatory postsynaptic currents (sEPSCs). Likewise, CB1R agonists (ACEA, WIN55,212-3 and HU210) induced a dose-dependent reduction of cortically-evoked excitatory postsynaptic potential (eEPSP) amplitude. While CB1R agonists retained their inhibitory effect in heterozygous PINK1(+/-) mice, conversely, in PINK1(-/-) mice they failed to modulate sEPSC amplitude. Similarly, CB1R activation failed to reduce eEPSP amplitude in PINK1(-/-) mice. Parallel biochemical measurements revealed no significant difference in the levels of the two main eCBs, 2-arachidonoylglycerol (2-AG) and anandamide (AEA) in PINK1(-/-) striata. Similarly, no change was observed in the enzymatic activity of both fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), responsible for eCB hydrolysis. Instead, a significant reduction of binding ability of CB1R agonists was found in PINK1(-/-) mice. Notably, the CB1R-dependent inhibition of synaptic activity was restored either by amphetamine or after chronic treatment with the D2 dopamine receptor agonist quinpirole. Additionally, CB1R binding activity returned to control levels after chronic pretreatment with quinpirole. Consistent with the hypothesis of a close interplay with dopaminergic neurotransmission, our findings show a CB1R dysfunction at corticostriatal synapses in PINK1(-/-), but not in PINK1(+/-) mice, and provide a mechanistic link to the distinct plasticity deficits observed in both genotypes.

Cannabinoid CB2 receptor (CB2R) stimulation delays rubrospinal mitochondrial-dependent degeneration and improves functional recovery after spinal cord hemisection by ERK1/2 inactivation.

Spinal cord injury (SCI) is a devastating condition of CNS that often results in severe functional impairments for which there are no restorative therapies. As in other CNS injuries, in addition to the effects that are related to the primary site of damage, these impairments are caused by degeneration of distal regions that are connected functionally to the primary lesion site. Modulation of the endocannabinoid system (ECS) counteracts this neurodegeneration, and pharmacological modulation of type-2 cannabinoid receptor (CB2R) is a promising therapeutic target for several CNS pathologies, including SCI. This study examined the effects of CB2R modulation on the fate of axotomized rubrospinal neurons (RSNs) and functional recovery in a model of spinal cord dorsal hemisection (SCH) at the cervical level in rats. SCH induced CB2R expression, severe atrophy, and cell death in contralateral RSNs. Furthermore, SCH affected molecular changes in the apoptotic cascade in RSNs - increased cytochrome c release, apoptosome formation, and caspase-3 activity. CB2R stimulation by its selective agonist JWH-015 significantly increased the bcl-2/bax ratio, reduced cytochrome c release, delayed atrophy and degeneration, and improved spontaneous functional recovery through ERK1/2 inactivation. These findings implicate the ECS, particularly CB2R, as part of the endogenous neuroprotective response that is triggered after SCI. Thus, CB2R modulation might represent a promising therapeutic target that lacks psychotropic effects and can be used to exploit ECS-based approaches to counteract neuronal degeneration.

Endocannabinoid signaling and food addiction.

Overeating, frequently linked to an increasing incidence of overweight and obesity, has become epidemic and one of the leading global health problems. To explain the development of this eating behavior, new hypotheses involve the concept that many people might be addicted to food by losing control over their ability to regulate food intake. Among the different neurotransmitter networks that partake in the reward circuitry within the brain, a large body of evidence supports the involvement of the endocannabinoid system. Indeed, its dysfunctions might contribute to food addiction, by regulating appetite and food preference through central and peripheral mechanisms. Here, we review and discuss the role of endocannabinoid signaling in the reward circuitry, and the possible therapeutic exploitation of strategies based on its fine regulation.

Activity of anandamide (AEA) metabolic enzymes in rat placental bed.

Endocannabinoids are endogenous lipid mediators, with anandamide (AEA) being the first member identified. It is now widely accepted that AEA influences early pregnancy events and its levels, which primarily depend on its synthesis by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degradation by a fatty acid amide hydrolase (FAAH), must be tightly regulated. Previous studies demonstrated that AEA levels require in situ regulation of these respective metabolic enzymes, and thus, any disturbance in AEA levels may impact maternal remodeling processes occurring during placental development. In this study, the activities of the AEA-metabolic enzymes that result in the establishment of proper local AEA levels during rat gestation were examined. Here, we demonstrate that during placentation NAPE-PLD and FAAH activities change in a temporal manner. Our findings suggest that NAPE-PLD and FAAH create the appropriate AEA levels required for tissue remodeling in the placental bed, a process essential to pregnancy maintenance.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

Involvement of cannabinoid receptor-1 activation in mitochondrial depolarizing effect of lipopolysaccharide in human spermatozoa.

Gram-negative bacteria frequently involved in urogenital tract infections release the endotoxin lipopolysaccharide (LPS); its receptor, toll-like receptor-4 (TLR4), has been recently identified in human spermatozoa, and its direct activation has been suggested in mediating adverse effects of LPS on human spermatozoa. However, the underlying signal transduction remains to be clarified. In other cell types, LPS induces the generation of endocannabinoids, which are involved in mediating endotoxin effects. In human spermatozoa, which exhibit a completely functional endocannabinoid system, the activation of cannabinoid receptor-1 (CB1) inhibited sperm mitochondrial membrane potential (ΔΨm). In this study, we tested the hypothesis of a contribution of CB1 activation by sperm-generated endocannabinoids in the adverse effects exerted by LPS on human spermatozoa. The exposure of motile sperm suspensions to E. coli LPS produced a significant decrease in sperm ΔΨm, assessed at flow cytometry with JC-1, similar to that induced by Metanandamide (Met-AEA), a non-hydrolyzable analogue of the endocannabinoid AEA. The LPS-induced inhibition of ΔΨm was prevented by the selective CB1 cannabinoid receptor antagonist, SR141716. However, the inhibition of ΔΨm induced by either LPS or Met-AEA did not affect sperm motility. Consistent with this finding, the CB1-mediated inhibition of ΔΨm was neither associated to mitochondrial generation of reactive oxygen species as evaluated by flow cytometry with MytoSox Red nor to apoptosis pathway activation as evaluated with cytoflorimetric assay for activated caspase-9 and caspase-3. Any oxidative genomic damage was also ruled out with the cytoflorimetric quantification of the oxidized base adduct 8-hydroxy-2'-deoxyguanosine. In conclusion, E. coli LPS inhibited sperm ΔΨm through the activation of CB1, but this effect was not accompanied to the activation of mitochondrial dysfunction-related apoptotic/oxidative mechanisms, which could affect sperm motility and genomic integrity.

GPR55 and its interaction with membrane lipids: comparison with other endocannabinoid-binding receptors.

A number of integral membrane G protein-coupled receptors (GPCRs) share common structural features (including palmytoilated aminoacid residues and consensus sequences specific for interaction with cholesterol) that allow them to interact with lipid rafts, membrane cholesterol-rich microdomains able to regulate GPCR signalling and functions. Among GPCRs, type-1 and type-2 cannabinoid receptors, the molecular targets of endocannabinoids (eCBs), control many physiological and pathological processes through the activation of several signal transduction pathways. Recently, the orphan GPR55 receptor has been proved to be activated by many eCBs, thus leading to the hypothesis that it might be the "type-3" cannabinoid receptor. While the biological activity of eCBs and the influence of membrane lipids on their functions are rather well established, information regarding GPR55 is still scarce and often controversial. Based on this background, here we shall review current data about GPR55 pharmacology and signalling, highlighting its involvement in several pathophysiological conditions. We shall also outline the structural features that allow GPR55 to interact with cholesterol and to associate with lipid rafts; how the latter lipid microdomains impact the biological activity of GPR55 is also addressed, as well as their potential for the discovery of new therapeutics useful for the treatment of those human diseases that might be associated with alterations of GPR55 activity.

Endocannabinoid signaling in Alzheimer’s disease: current knowledge and future directions.

The importance of the endocannabinoid system (ECS) in the modulation functions of the central nervous system has been extensively investigated during the last few years. In particular, accumulated evidence has implicated ECS in the pathophysiology of Alzheimer’s disease (AD), that is a progressive, degenerative, and irreversible disorder characterized by the accumulation in the brain of beta-amyloid fragments forming insoluble plaques, and of intracellular neurofibrillary tangles (NTFs) associated with synaptic and neuronal loss. In all the processes involved in the formation of both plaques and NFTs, the key-role played by the ECS has been documented. Here, we review current knowledge and future directions of ECS modulation both in animal models of AD and in human tissues, underlying the role of endocannabinoid signaling in the development of AD hallmarks. Overall, the available data suggest that next generation therapeutics might target distinct ECS elements, for instance CB2 receptor or fatty acid amide hydrolase, as a promising approach to halt or at least to slow down disease progression.

Long-term use of HU210 adversely affects spermatogenesis in rats by modulating the endocannabinoid system.

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of Δ9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm's endocannabinoid system. Long-term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users. HU210, a selective agonist for CB1 and CB2 cannabinoid receptors impairs spermatogenesis and sperm motility and deregulates the endocannabinoid system.

5-Lipoxygenase and cyclooxygenase-2 in the lungs of pigs naturally affected by enzootic pneumonia and porcine pleuropneumonia.

Enzootic pneumonia by Mycoplasma hyopneumoniae and pleuropneumonia by Actinobacillus pleuropneumoniae are among the most common and economically relevant pulmonary diseases in swine herds. We herein investigated the activity and expression of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) in healthy and diseased porcine lungs, by means of immunohistochemical, immunochemical and biochemical assays. Diseased lungs showed a significantly higher activity and expression of 5-LOX and COX-2 in a wide range of cell types, thus suggesting the likely involvement of both enzymes in the pathogenesis of bacterial porcine pneumonia. Consistently, increased enzyme activities were paralleled by increased leukotriene B(4) (LTB(4)), a 5-LOX product and prostaglandin E(2) (PGE(2)), a COX-2 product, content in diseased versus healthy lungs.

Pin1 contribution to Alzheimer's disease: transcriptional and epigenetic mechanisms in patients with late-onset Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles and senile plaques are hallmarks of Alzheimer's disease (AD) although the molecular basis of their coexistence remains elusive. The peptidyl-prolyl cis/trans isomerase Pin1 acts on both tau and amyloid precursor protein to regulate their functions by influencing tau phosphorylation and amyloid precursor protein processing. OBJECTIVE: In order to identify potential biomarkers for AD in easily accessible cells and to gain insight into the relationship between the brain and peripheral compartments in AD pathology, we investigated Pin1 expression and activity in the peripheral blood mononuclear cells of subjects with late-onset AD (LOAD) and age-matched controls (CT). METHODS: Gene and protein expression, promoter methylation, Ser(16) phosphorylation and activity of Pin1 were evaluated in 32 samples from subjects with LOAD and in 28 samples from CT. RESULTS: In LOAD subjects, there was a statistically significant reduction in Ser(16) phosphorylation (-30%; p = 0.041) and promoter methylation (-8%; p = 0.001), whereas Pin1 expression was significantly increased (+74%; p = 0.018). CONCLUSION: The modifications of Pin1 found in LOAD subjects support its involvement in the pathogenesis of the disease with an important role being played by epigenetic mechanisms.

Distinct regulation of nNOS and iNOS by CB2 receptor in remote delayed neurodegeneration.

Hemicerebellectomy results in remote delayed degeneration of precerebellar neurons. We have reported that such a lesion induces type 2 cannabinoid receptor (CB(2)) expression in precerebellar neurons and that stimulation of CB(2), but not CB(1), has neuroprotective effects. In this study, we found that in the same model, the CB(2) agonist JWH-015 enhances neuronal nitric oxide synthase (nNOS) expression in axotomized neurons and that CB(2)-mediated neuroprotection is abrogated by pharmacological inhibition of nNOS. JWH-015 prevented the axotomy-induced upregulation of inducible NOS (iNOS) in astrocytes but had no effect on endothelial NOS (eNOS). In addition, we observed that JWH-015 significantly reduces hemicerebellectomy-induced neuroinflammatory responses and oxidative/nitrative stress. With regard to the signaling pathways of CB(2)/nNOS-mediated neuroprotection, we noted nNOS-dependent modulation of the expression of anti-oxidative (Hsp70) and anti-apoptotic (Bcl-2) proteins. These findings shed light on the interactions between the endocannabinoid and nitrergic systems after focal brain injury, implicating distinct functions of nNOS activation and iNOS inhibition in CB(2) signaling, which protect neurons from axotomy-induced cell death.