TGF-beta induces cell death in the oligodendroglial cell line OLI-neu

We have shown that TGF-beta plays an important role during the period of developmental cell death in the nervous system. Immunoneutralization of TGF-beta prevents ontogenetic neuron death in vivo. Like neurons, oligodendrocytes are generated in excess and eliminated by apoptosis. It has been shown that oligodendrocyte progenitors and newly formed oligodendrocytes are especially susceptible to apoptosis. We choose the oligodendrocyte precursor cell line OLI-neu to address the question if TGF-beta could play a role for the control of oligodendrocyte proliferation and cell death. Flow cytometric analysis revealed that OLI-neu cells arrested in the G1 phase of the cell cycle underwent apoptosis in response to TGF-beta. TUNEL assays, apoptosis ELISA, and caspase assays substantiated the finding that OLI-neu cells died after TGF-beta treatment. Cell death could be inhibited by application of pan-caspase or caspase 8 and 9 inhibitors, whereas the inhibition of calpain was unaffected. Furthermore, we found a reduction of bcl-X(L) at the protein as well as at the mRNA level, while p27 was upregulated. The Smad cascade was activated while TGF-beta reduced the activity of the p42/p44 MAP kinase pathway. Together, these data show that TGF-beta induced apoptotic cell death in cells of oligodendroglial origin, whereby the signaling cascade involved the downregulation of antiapoptotic signaling such as bcl-X(L) leading to the activation of caspases.     

Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse

Research into the molecular mechanisms of epileptic brain injury is hampered by the resistance of key mouse strains to seizure-induced neuronal death evoked by systemically administered excitotoxins such as kainic acid. Because C57BL/6 mice are extensively employed as the genetic background for transgenic/knockout modeling in cell death research but are seizure resistant, we sought to develop a seizure model in this strain characterized by injury to the hippocampal CA subfields. Adult male C57BL/6 mice underwent focally evoked seizures induced by intraamygdala microinjection of kainic acid. Kainic acid (KA) effectively elicited ipsilateral CA3 pyramidal neuronal death within a narrow dose range of 0.1-0.3 microg, with mortality < 10%. With employment of the most consistent (0.3 microg) dose, seizures were terminated 15, 30, 60, or 90 min after KA by diazepam. Damage was largely restricted to the ipsilateral CA3 subfield of the hippocampus, but injury was also consistent within CA1, suggesting that this mouse model better reflects the hippocampal neuropathology of human temporal lobe epilepsy than does the rat, in which CA1 is typically spared. Confirming this CA1 injury as seizure specific and not a consequence of ischemia, we used laser-Doppler flowmetry to determine that cerebral perfusion did not significantly change (97% to 118%) over control. Degenerating cells were > 95% neuronal as determined by neuron-specific nuclear protein (NeuN) counterstaining of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeled (TUNEL) brain sections. Furthermore, TUNEL-positive cells often exhibited the morphological features of apoptosis, and small numbers were positive for cleaved caspase-3. These data establish a mouse model of focally evoked seizures in the C57BL/6 strain associated with a restricted pattern of apoptotic neurodegeneration within the hippocampal subfields that may be applied to research into the molecular basis of neuronal death after seizures.     

Studies on neuronal death in the mouse model of Niemann-Pick C disease

A mouse model of Niemann-Pick disease type C (NPC) carries a genetic defect that causes biochemical changes in lipid levels and a progressive neuropathology that parallels the effects of NPC disease in humans. It is a moot point whether or not the loss of Purkinje and other neuronal cells proceeds by apoptotic death. Therefore, we have introduced into these mice a transgene expressing human Bcl-2 protein which has previously been demonstrated to prevent developmental neuronal death and death induced by a variety of stimuli. The human Bcl-2 transgene was driven by the neuron-specific enolase promoter and was abundantly expressed in Purkinje and other neuronal cells. npc1(-/-)/bcl-2 transgenic mice did not show a significant delay in the onset of neurological disorders. Neuropathological examination of the npc1(-/-)/bcl-2 transgenic mice did not disclose significant differences in numbers of surviving Purkinje cells between the npc1(-/-), tg(+) and npc1(-/-), tg(-) mice. When the npc1(-/-) mice were treated with minocycline, a drug which was shown to inhibit apparent apoptotic death in other mouse models of neurological disease, no delay in onset of neurological disorders were observed in either npc1(-/-), or npc1(-/-) /mdrla(-/-) mice (mdr1a deficiency was used to enhance brain availability of minocycline). Caspase-1 levels were not altered in npc1(-/-) mice, with or without minocycline treatment. These results suggest that Purkinje cell loss in npc1(-/-) mice does not proceed by an apoptotic pathway that can be inhibited by Bcl-2 or minocycline.     

Potent depressant action of baclofen on hippocampal epileptiform activity in vitro: possible use in the treatment of epilepsy

Effects of baclofen on the cortical electrical paroxysm were examined using slices of the guinea pig hippocampus. A very low concentration of baclofen (10(-8) M) readily suppressed spontaneous epileptiform discharges of CA3 pyramidal cells, whereas synaptic transmission between mossy fibers and CA3 pyramidal cells were not affected even by 10(-4) M of this drug. Such a strikingly selective depressant action of baclofen on the epileptiform activity raises a possibility that this drug may be effective in the treatment of epilepsy.     

Catecholamine-induced oligodendrocyte cell death in culture is developmentally regulated and involves free radical generation and differential activation of caspase-3

Oligodendrocyte cultures were used to study the toxic effects of catecholamines. Our results showed that catecholamine-induced toxicity was dependent on the dose of dopamine or norepinephrine used and on the developmental stage of the cultures, with oligodendrocyte progenitors being more vulnerable. A role for oxidative stress and apoptosis on the mechanism of action of catecholamines on oligodendrocyte cell death was next assessed. Catecholamines caused a reduction in intracellular glutathione levels, an accumulation in reactive oxygen species and in heme oxygenase-1, the 32 kDa stress-induced protein. All these changes were prevented by N-acetyl-L-cysteine, a thiocompound with antioxidant activity and a precursor of glutathione, and were more pronounced in progenitors than mature cells, which could contribute to their higher susceptibility. Apoptotic cell death, as assessed by activation of caspase-9 and -3 and cleavage of poly(ADP-ribose) polymerase (a substrate of caspase-3), was only observed in oligodendrocyte progenitors. Pretreatment with zVAD, a general caspase inhibitor, prevented activation of caspase-9 and -3, DNA fragmentation, and decreased progenitors cell death. Furthermore, the expression levels of procaspase-3 and the ratio of the proapoptotic protein bax to antiapoptotic protein bcl-xl were several folds higher in immature than mature oligodendrocytes. Taken together, these results strongly suggest that the catecholamine-induced cytotoxicity in oligodendrocytes is developmentally regulated, mediated by oxidative stress, and have characteristics of apoptosis in progenitor cells.     

Impaired glutamate homeostasis and programmed cell death in a chronic MPTP mouse model of Parkinson's disease

The pathogenesis of Parkinson's disease is not fully understood, but there is evidence that excitotoxic mechanisms contribute to the pathology. However, data supporting a role for excitotoxicity in the pathophysiology of the disease are controversial and sparse. The goal of this study was to determine whether changes in glutamate signaling and uptake contribute to the demise of dopaminergic neurons in the substantia nigra. Mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid or vehicle (probenecid or saline alone). Extracellular levels of glutamate in the substantia nigra were substantially increased, and there was an increase in the affinity, but no change in the velocity, of glutamate transport after MPTP/probenecid treatment compared to vehicle controls. In addition, the substantia nigra showed two types of programmed death, apoptosis (type I) and autophagic (type II) cell death. These data suggest that increased glutamate signaling could be an important mechanism for the death of dopaminergic neurons and trigger the induction of programmed cell death in the chronic MPTP/probenecid model.     

Limbic seizure-induced changes in extracellular amino acid levels in the hippocampal formation: a microdialysis study of freely moving rats

Limbic seizure-activity was induced by injecting kainic acid into the amygdala of rats. Extracellular levels of amino acids were monitored by microdialysis in the hippocampus. No changes were detected in the levels of glutamate and aspartate. The level of glycine also remained unchanged, whereas GABA showed an increase of approximately 35%. The level of glutamine decreased by approximately 30%, and that of serine by approximately 20%. The results indicate that increased turnover may exist in the glutamate transmitter pool. In addition, impairment of GABA-release seems not to be a pathogenetic factor in seizure-induced hippocampal neuron loss. It is concluded that even during sustained seizure-activity, the extracellular level of glutamate, is maintained within narrow limits. A proposed index for excitatory neurodegeneration, glutamate x glycine/GABA, was found to be decreased in this seizure model. We therefore suggest that seizure-induced neuron death is not reflected by alterations in the extracellular levels of glutamate and aspartate, thought to act as direct neurotoxins.     

Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury

Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type-1 receptor (TNFR1) and type-2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild-type (WT) animals and in mice lacking TNFR1 (TNFR1 KO) or TNFR2 (TNFR2 KO). Lack of TNFR1, but not of TNFR2, significantly decreased the inflammatory response and tissue damage elicited by the cryolesion at both 3 and 7 days postlesion, with decreased gliosis, lower IL-1beta immunostaining, and a reduction of apoptosis markers. Cryolesion produced a clear induction of the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, and TNF-alpha; this induction was significantly lower in the TNFR1 KO mice. Host response genes (ICAM-1, A20, EB22/5, and GFAP) were also induced by the cryolesion, but to a lesser extent in TNFR1 KO mice. Lack of TNFR1 signaling also affected the expression of apoptosis/cell death-related genes (Fas, Rip, p53), matrix metalloproteinases (MMP3, MMP9, MMP12), and their inhibitors (TIMP1), suggesting a role of TNFR1 in extracellular matrix remodeling after injury. However, GDNF, NGF, and BDNF expression were not affected by TNFR1 deficiency. Overall, these results suggest that TNFR1 is involved in the early establishment of the inflammatory response and that its deficiency causes a decreased inflammatory response and tissue damage following brain injury.     

Steroid-triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria

Neuronal death occurs during normal development and disease and can be regulated by steroid hormones. In the hawkmoth, Manduca sexta, individual accessory planta retractor (APR) motoneurons undergo a segment-specific pattern of programmed cell death (PCD) at pupation that is triggered directly and cell autonomously by the steroid hormone 20-hydroxyecdysone (20E). APRs from abdominal segment six [APR(6)s] die by 48 hours after pupal ecdysis (PE; entry into the pupal stage), whereas APR(4)s survive until adulthood. Cell culture experiments showed previously that 20E acts directly on APRs to trigger PCD, with intrinsic segmental identity determining which APRs die. The APR(6) death pathway includes caspase activation and loss of mitochondrial function. We used transmission electron microscopy to investigate the ultrastructure of APR somata before and during PCD. APR(4)s showed normal ultrastructure at all stages examined, as did APR(6)s until approximately stage PE. During APR(6) death, there was massive accumulation of autophagic bodies and vacuoles, mitochondria became ultracondensed and aggregated into compact clusters, and ribosomes aggregated in large blocks. Nuclear ultrastructure remained normal, without chromatin condensation, until the nuclear envelope fragmented late in the death process. Light microscopic immunocytochemistry showed that dying APR(6)s were TUNEL-positive, which is diagnostic of fragmented DNA. These observations indicate that the steroid-induced, caspase-dependent, cell-autonomous PCD of APR(6)s is autophagic, not apoptotic, and support an early role for mitochondrial alterations during PCD. This system permits the study of neuronal death in response to its bona fide developmental signal, the rise in a steroid hormone.     

Human umbilical cord mesenchymal stem cell-derived exosome suppresses programmed cell death in traumatic brain injury via PINK1/Parkin-mediated mitophagy

Aims

Recently, human umbilical cord mesenchymal stem cell (HucMSC)-derived exosome is a new focus of research in neurological diseases. The present study was aimed to investigate the protective effects of HucMSC-derived exosome in both in vivo and in vitro TBI models.

Methods

We established both mouse and neuron TBI models in our study. After treatment with HucMSC-derived exosome, the neuroprotection of exosome was investigated by the neurologic severity score (NSS), grip test score, neurological score, brain water content, and cortical lesion volume. Moreover, we determined the biochemical and morphological changes associated with apoptosis, pyroptosis, and ferroptosis after TBI.

Results

We revealed that treatment of exosome could improve neurological function, decrease cerebral edema, and attenuate brain lesion after TBI. Furthermore, administration of exosome suppressed TBI-induced cell death, apoptosis, pyroptosis, and ferroptosis. In addition, exosome-activated phosphatase and tensin homolog-induced putative kinase protein 1/Parkinson protein 2 E3 ubiquitin–protein ligase (PINK1/Parkin) pathway-mediated mitophagy after TBI. However, the neuroprotection of exosome was attenuated when mitophagy was inhibited, and PINK1 was knockdown. Importantly, exosome treatment also decreased neuron cell death, suppressed apoptosis, pyroptosis, and ferroptosis and activated the PINK1/Parkin pathway-mediated mitophagy after TBI in vitro.

Conclusion

Our results provided the first evidence that exosome treatment played a key role in neuroprotection after TBI through the PINK1/Parkin pathway-mediated mitophagy.     

Selective death of newborn neurons in hippocampal dentate gyrus following moderate experimental traumatic brain injury

Memory impairment is one of the most significant residual deficits following traumatic brain injury (TBI) and is among the most frequent complaints heard from patients and their relatives. It has been reported that the hippocampus is particularly vulnerable to TBI, which results in hippocampus-dependent cognitive impairment. There are different regions in the hippocampus, and each region is composed of different cell types, which might respond differently to TBI. However, regional and cell type-specific neuronal death following TBI is not well described. Here, we examined the distribution of degenerating neurons in the hippocampus of the mouse brain following controlled cortical impact (CCI) and found that the majority of degenerating neurons observed were in the dentate gyrus after moderate (0.5 mm cortical deformation) CCI-TBI. In contrast, there were only a few degenerating neurons observed in the hilus, and we did not observe any degenerating neurons in the CA3 or CA1 regions. Among those degenerating cells in the dentate gyrus, about 80% of them were found in the inner granular neuron layer. Analysis with cell type-specific markers showed that most of the degenerating neurons in the inner granular neuron layer are newborn immature neurons. Further quantitative analysis shows that the number of newborn immature neurons in the dentate gyrus is dramatically decreased in the ipsilateral hemisphere compared with the contralateral side. Collectively, our data demonstrate the selective death of newborn immature neurons in the hippocampal dentate gyrus following moderate injury with CCI in mice. This selective vulnerability of newborn immature dentate neurons may contribute to the persistent impairment of learning and memory post-TBI and provide an innovative target for neuroprotective treatment strategies.     

The effect of oxygenation level on cerebral post-traumatic apoptotsis is modulated by the 18-kDa translocator protein (also known as peripheral-type benzodiazepine receptor) in a rat model of cortical contusion

Aims: Hyperbaric hyperoxia has been shown to reduce apoptosis in brain injury. As the 18-kDa translocator protein (TSPO), also known as peripheral-type benzodiazepine receptor, is closely associated with the mitochondrial transition pore and because of its role in mitochondrial respiration and apoptosis, we hypothesized that reduction of apoptosis by hyperoxia may involve the TSPO. Methods: TSPO and transferase-mediated dUTP nick end labelling (TUNEL) immunopositivity was first assessed in cortical contusion, created by dynamic cortical deformation, by immunohistochemistry in rats exposed to normoxia [(dynamic cortical deformation (DCD)], normobaric hyperoxia or hyperbaric hyperoxia [hyperbaric oxygen therapy (HBO)]. In a second step, transmembrane mitochondrial potential (Δψ_M) and caspase 9 activity were assessed in the injured area in comparison with the noninjured hemisphere. Measurements were performed in DCD and HBO groups. A third group receiving both HBO and the TSPO ligand PK11195 was investigated as well. Results: TSPO correlated quantitatively and regionally with TUNEL immunopositivity in the perilesional area. Hyperoxia reduced both the number of TSPO expressing and TUNEL positive cells in the perilesional area, and this effect proved to be pressure dependent. After contusion, we demonstrated a dissipation of Δψ_M in isolated mitochondria and an elevation of caspase 9 activity in tissue homogenates from the contused area, both of which could be substantially reversed by hyperbaric hyperoxia. This protective effect of hyperoxia was reversed by PK11195. Conclusions: The present findings suggest that the protective effect of hyperoxia may be due to a negative regulation of the proapoptotic function of mitochondrial TSPO, including conservation of the mitochondrial membrane potential.     

Evidence for the spontaneous production but massive programmed cell death of new neurons in the subcallosal zone of the postnatal mouse brain

In the last 10 years, many studies have reported that neural stem/progenitor cells spontaneously produce new neurons in a subset of adult brain regions, including the hippocampus, olfactory bulb (OB), cerebral cortex, substantia nigra, hypothalamus, white matter and amygdala in several mammalian species. Although adult neurogenesis in the hippocampus and OB has been clearly documented, its occurrence in other brain regions is controversial. In the present study, we identified a marked accumulation of new neurons in the subcallosal zone (SCZ) of Bax-knockout mice in which programmed cell death (PCD) of adult-generated hippocampal and OB neurons has been shown to be completely prevented. By contrast, in the SCZ of wild-type (WT) mice, only a few immature (but no mature) newly generated neurons were observed, suggesting that virtually all postnatally generated immature neurons in the SCZ were eliminated by Bax-dependent PCD. Treatment of 2-month-old WT mice with a caspase inhibitor, or with the neurotrophic factor brain-derived neurotrophic factor, promoted the survival of adult-generated neurons, suggesting that it is the absence of sufficient neurotrophic signaling in WT SCZ that triggers the Bax-dependent, apoptotic PCD of newly generated SCZ neurons. Furthermore, following focal traumatic brain injury to the posterior brain, SCZ neurogenesis in WT mice was increased, and a subset of these newly generated neurons migrated toward the injury site. These data indicate that the adult SCZ maintains a neurogenic potential that could contribute to recovery in the brain in response to the injury-induced upregulation of neurotrophic signaling.     

Ectopic expression of cell cycle markers in models of induced programmed cell death in dopamine neurons of the rat substantia nigra pars compacta

There is increasing evidence that proteins normally involved in the cell cycle can regulate neuronal programmed cell death (PCD). However, it remains unknown whether cell cycle markers are expressed in normal, postmitotic, postmigratory neurons undergoing PCD in vivo. We have previously shown that natural cell death occurs postnatally in dopamine neurons of the substantia nigra pars compacta (SNpc). PCD can be induced postnatally in these neurons either by intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA) or by medial forebrain bundle (MFB) axotomy. At the time of induction of death in these models, these neurons are long postmitotic and postmigratory. We have studied three cell cycle markers in these models: 5-bromo-2'-deoxyuridine (BrdU) incorporation (a marker of S phase), cdc2 protein expression (a marker of G2 phase), and expression of MPM2 (a marker of M phase), an epitope phosphorylated by cdc2. We report here that postmitotic dopaminergic neurons undergoing PCD in the SNpc following 6-OHDA and axotomy lesions incorporate BrdU and overexpress cdc2, but do not express MPM2. This is the first in vivo evidence that postmitotic dopamine neurons of the SNpc undergoing apoptosis express markers for S phase and G2 phase. These results raise the possibility that cell cycle regulatory proteins may play a role in the demise of dopaminergic neurons in Parkinson's disease, in which PCD has been postulated to play a role.     

Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease

One promising therapy for the treatment of Parkinson's disease is transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted cells die, many via apoptosis. In this study we attempted to prevent anoikis-induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function. We utilized the extracellular matrix molecule tenascin-C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell-matrix and cell-cell interactions. In vitro, both tenascin- and L1 Ab-treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control. Additionally, cell survival assays determined that tenascin and L1 Ab-treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions. In contrast to the culture results, tenascin- and L1 Ab-treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 microl of 100,000 cells/microl). However, under low-density conditions (3 microl of 3,000 cells/microl), tenascin augmented grafted THir neuron survival. These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo. In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long-term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation.     

Retinal ganglion cell death in a DBA/2J mouse model of glaucoma Microglial activation and intraocular pressure

BACKGROUND: Retinal microglia has been shown to reactivate in a murine model of pigmentary glaucoma. However, the relationship between microglial activation and intraocular pressure (IOP) elevation and retinal ganglion cell (RGC) death is still unclear. OBJECTIVE: To verify that microglial activation and tumor necrosis factor alpha (TNF-α) expression is involved in RGC death with elevated IOP and prolonged time of glaucomatous optic nerve lesion in a DBA/2J mouse model of glaucoma. DESIGN, TIME AND SETTING: This randomized, controlled, animal experiment was performed at the Peking University Third Hospital, Peking University Eye Center, China between December 2006 and May 2008. MATERIALS: DBA/2J mice and C57BL/6J mice (Jackson Laboratory, USA), rat anti-mouse CD11b monoclonal antibody (Serotec, UK), and goat anti-TNF-α polyclonal antibody (Sigma, USA) were used in this study. METHODS: A total of 100 female, DBA/2J mice at 3, 6, 9,12, and 14 months of age (20 mice per age group) were used for the glaucoma model, and 18 C57BLV6J mice at 3, 9,14 months of age (6 mice per age group) were used as normal controls. The anterior segment of the eye was observed using a slit-lamp biomicroscope. IOP was measured using a microneedle system. Morphology and number of retinal microglia were observed using immunohistochemistry. RGCs were quantified using Nissl staining. Co-localization of TNF-a and microglia was observed using double-labeling immunofluorescence. Excavation of the optic nerve head was observed utilizing hematoxylin-eosin staining. MAIN OUTCOME MEASURES: The following parameters were measured: IOP levels, numbers of RGCs and activated microglia, and TNF-α expression. RESULTS: In 6-month-old DBA/2J mice, dispersed pigment was observed, and some mice developed increased IOP. At 9 months of age, IOP levels reached a peak. In 3-month-old DBA/2J mice, microglia were activated. In 6-month-old DBA/2J mice, the number of activated microglia was significantly increased and migrated to the outer retinal layer. In 9-month-old mice, TNF-α expression was co-localized with microglia. Significant RGC loss occurred in mice aged 9 to 14 months, with the presence of optic nerve fiber loss and optical nerve head excavation. IOP returned to normal levels at 12 months of age, but microglia remained activated, which was consistent with RGC loss. CONCLUSION: Retinal microglial activation was partially attributed to increased IOP. Activated microglia might be mainly responsible for RGC loss. TNF-α expression was evident in the inner retinal layer. However, the relationship between TNF-α and RGC loss remains poorly understood.