Excessive Autophagy Contributes to Neuron Death in Cerebral Ischemia

Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo. Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.     

A brain aggregate model gives new insights into the pathobiology and treatment of prion diseases

Brain aggregates (BrnAggs) derived from fetal mouse brains contain mature neurons and glial cells. We determined that BrnAggs are consistently infected with Rocky Mountain Laboratory scrapie strain prions and produce increasing levels of the pathogenic form of the prion protein (PrP). Their abundant dendrites undergo degeneration shortly after prion infection. Treatment of prion-infected BrnAggs with drugs, such as a γ-secretase inhibitors and quinacrine (Qa), which stop PrP formation and dendritic degeneration, mirrors the results from rodent studies. Because PrP is trafficked into lysosomes by endocytosis and autophagosomes by phagocytosis in neurons of prion strain-infected BrnAggs, we studied the effects of drugs that modulate subcellular trafficking. Rapamycin (Rap), which activates autophagy, markedly increased light-chain 3-II (LC3-II)-positive autophagosomes and cathepsin D-positive lysosomes in BrnAggs but could not eliminate the intracellular PrP within them. Adding Qa to Rap markedly reduced the number of LC3-II-positive autolysosomes. Rap + Qa created a competition between Rap increasing and Qa decreasing LC3-II. Rapamycin + Qa decreased total PrP by 56% compared with that of Qa alone, which reduced PrP by 37% relative to Rap alone. We conclude that the decrease was dominated by the ability of Qa to decrease the formation of PrP. Therefore, BrnAggs provide an efficient in vitro tool for screening drug therapies and studying the complex biology of prions.     

Normal inhibitory avoidance learning and anxiety, but increased locomotor activity in mice devoid of PrP(C).

Prions are the causative agents of transmissible spongiform encephalopathies. The transmissible agent (PrP(Sc)) is an abnormal form of PrP(C), a normal neuronal protein. The physiological role of PrP(C) remains unclear. In the present report, we evaluated behavioral parameters in Prnp(0/0) mice devoid of PrP(C). Prnp(0/0) mice showed normal short- and long-term retention of a step-down inhibitory avoidance task and normal behavior in an elevated plus maze test of anxiety. During a 5-min exploration of an open field, Prnp(0/0) mice showed normal number of rearings, defecation, and latency to initiate locomotion, but a significant increase in the number of crossings. The results suggest that Prnp(0/0) mice show normal fear-motivated memory, anxiety and exploratory behavior, and a slight increase in locomotor activity during exploration of a novel environment.     

HIF-1α/Beclin1-Mediated Autophagy Is Involved in Neuroprotection Induced by Hypoxic Preconditioning

Hypoxic preconditioning (HPC) exerts a protective effect against hypoxic/ischemic brain injury, and one mechanism explaining this effect may involve the upregulation of hypoxia-inducible factor-1 (HIF-1). Autophagy, an endogenous protective mechanism against hypoxic/ischemic injury, is correlated with the activation of the HIF-1α/Beclin1 signaling pathway. Based on previous studies, we hypothesize that the protective role of HPC may involve autophagy occurring via activation of the HIF-1α/Beclin1 signaling pathway. To test this hypothesis, we evaluated the effects of HPC on oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptosis and autophagy in SH-SY5Y cells. HPC significantly attenuated OGD/R-induced apoptosis, and this effect was suppressed by the autophagy inhibitor 3-methyladenine and mimicked by the autophagy agonist rapamycin. In control SH-SY5Y cells, HPC upregulated the expression of HIF-1α and downstream molecules such as BNIP3 and Beclin1. Additionally, HPC increased the LC3-II/LC3-I ratio and decreased p62 levels. The increase in the LC3-II/LC3-I ratio was inhibited by the HIF-1α inhibitor YC-1 or by Beclin1-short hairpin RNA (shRNA). In OGD/R-treated SH-SY5Y cells, HPC also upregulated the expression levels of HIF-1α, BNIP3, and Beclin1, as well as the LC3-II/LC3-I ratio. Furthermore, YC-1 or Beclin1-shRNA attenuated the HPC-mediated cell viability in OGD/R-treated cells. Taken together, our results demonstrate that HPC protects SH-SY5Y cells against OGD/R via HIF-1α/Beclin1-regulated autophagy.     

快速动眼睡眠剥夺对大鼠学习、记忆能力及海马组织自噬的影响

目的 探讨快速动眼(rapid eye movement,REM)睡眠剥夺后与大鼠认知相关的行为学变化及脑内海马组织中自噬相关蛋白的表达水平。方法 健康成年雄性大鼠经过筛选后分为空白对照组(CC组)、环境对照组(TC组)、睡眠剥夺组(SD组),每组各6只; 采用改良多平台睡眠剥夺法(modified multiple platform method,MMPM)建立睡眠剥夺模型,连续剥夺5 d后利用Morris水迷宫检测大鼠认知功能; 用蛋白质印记法(Western Blot,WB)检测自噬相关微管蛋白(LC3)及SQSTM1/P62的表达水平变化。结果 与CC组和TC组比较,SD组大鼠毛色无光泽、易激惹、体重下降(P<0.05)。SD组与其他2组比较,逃逸潜伏期延长、目标象限时间减少(P<0.05)。WB显示SD组与其他2组比较,大鼠脑内海马组织自噬相关蛋白LC3-II表达水平上升,P62水平下降(P<0.05)。CC组与TC组大鼠比较,体重、学习记忆能力、海马组织自噬蛋白表达水平均无明显差异(P>0.05)。结论 睡眠剥夺后可损害大鼠学习及记忆功能,海马组织中自噬水平上调提示自噬活动可能参与睡眠剥夺介导的认知功能障碍过程。     

Aged PrP null mice show defective processing of neuregulins in the peripheral nervous system

A prion, a protease-resistant conformer of the cellular prion protein (PrP(C)), is the causative agent of transmissible spongiform encephalopathies or prion diseases. While this property is well established for the aberrantly folded protein, the physiological function of PrP(C) remains elusive. Among different putative functions, the non-pathogenic protein isoform PrP(C) is involved in several cellular processes. Here, we show that PrP(C) regulates the cleavage of neuregulin-1 proteins (NRG1). Neuregulins provide key axonal signals that regulate several processes, including glial cells proliferation, survival and myelination. Interestingly, mice devoid of PrP(C) (Prnp?/?) were recently shown to have a late-onset demyelinating disease in the peripheral nervous system (PNS) but not in the central nervous system (CNS). We found that NRG1 processing is developmentally regulated in the PNS and, by comparing wildtype and Prnp?/? mice, that PrP(C) influences NRG1 processing in old, but not in young, animals. In addition, we found that also the processing of neuregulin-3, another neuregulin family member, is altered in the PNS of Prnp?/? mice. These differences in neuregulin proteins processing are not paralleled in the CNS, thus suggesting a different cellular function for PrP(C) between the CNS and the PNS.     

The cellular prion protein (PrPC) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation

Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP(C)). In various reports, it has been postulated that PrP(C) may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP(C) is not known. To investigate whether PrP(C) can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP(C) from PrP knockout (PrP(-/-)) neuronal cells and examined the role of PrP(C) under apoptosis and/or serum-deprived condition. We found that PrP(-/-) cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP(C). The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP(-/-) cells. In addition, Ca(2+) levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP(-/-) cells. These results strongly suggest that PrP(C) may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP(C) and consequent reduction of anti-apoptotic capacity of PrP(C) may be one of the pathogenic mechanisms of prion diseases.     

Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model

Dopamine at 100–500 μM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1–2 h after treatment we found an increase in hypoxia-inducible factor-1α (HIF-1α) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1α and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12 h of 500 μM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6 h of treatment with both 100 and 500 μM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.     

Neuronal stimulation induces autophagy in hippocampal neurons that is involved in AMPA receptor degradation after chemical long-term depression

Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP-lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K-Akt-mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.     

Clioquinol induces autophagy in cultured astrocytes and neurons by acting as a zinc ionophore

Recent studies have demonstrated that clioquinol, an antibiotic with an anti-amyloid effect, acts as a zinc ionophore under physiological conditions. Because increases in labile zinc may induce autophagy, we examined whether clioquinol induces autophagy in cultured astrocytes in a zinc-dependent manner. Within 1h of exposure to 0.1-10 μM clioquinol, the levels of microtubule-associated protein 1 light chain 3 (LC3)-II, a marker of autophagy, began to increase in astrocytes. Confocal live-cell imaging of GFP-LC3-transfected astrocytes showed the formation of LC3(+) autophagic vacuoles (AVs), providing a further indication that clioquinol induced autophagy. Addition of 3-methyladenine or small-interfering RNA against autophagy-related gene 6 (ATG6/Beclin-1) blocked clioquinol-induced increases in LC3-II. FluoZin-3 fluorescence microscopy showed that, like the zinc ionophore pyrithione, clioquinol increased intracellular zinc levels in the cytosol and AVs in an extracellular zinc-dependent manner. Zinc chelation with N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) reduced, and addition of zinc increased the levels of LC3-II and LC3(+) puncta, indicating that zinc influx plays a key role therein. Moreover, astrocytes and SH-SY5Y cells expressing mutant huntingtin (mHttQ74) accumulated less aggregates when treated with clioquinol, and this effect was reversed by TPEN. These results indicate that clioquinol-induced autophagy is likely to be physiologically functional. The present study demonstrates that clioquinol induces autophagy in a zinc-dependent manner and contributes to clearance of aggregated proteins in astrocytes and neurons. Hence, in addition to its metal-chelating effect in and around amyloid beta (Aβ) plaques, clioquinol may contribute to the reduction of Aβ loads by activating autophagy by increasing or normalizing intracellular zinc levels in brain cells.     

In situ hybridization analysis of PrP mRNA in human CNS tissues

Expression of the prion protein gene (Prnp) and production of the PrP protein are essential requirements for acquisition and spread of transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) in humans. Here we have developed an in situ hybridization method for use on human post-mortem central nervous system (CNS) tissues in order to determine those cell which are transcribing the Prnp gene and thus expressing PrP mRNA. Tissues from 11 adult individuals (age range 21-79 years) were analysed. Similar to previous studies in other animal systems, it was shown that PrP production occurs primarily in neuronal populations throughout the human brain. Neurones of the hippocampus, cortex, thalamus, cerebellum and medulla all synthesize PrP mRNA at readily detectable levels. No age-related differences were observed between the cases studied. It was also found that the ependymal cells produced PrP mRNA; these were the only non-neuronal cell type expressing the Prnp gene in the CNS. It is hoped that the information produced here will be helpful in understanding the pathology associated with CJD and other prion diseases in humans.     

DT-diaphorase Protects Against Autophagy Induced by Aminochrome-Dependent Alpha-Synuclein Oligomers

Alpha-synuclein (SNCA) oligomers have been reported to inhibit autophagy. Aminochrome-induced SNCA oligomers are neurotoxic, but the flavoenzyme DT-diaphorase prevents both their formation and their neurotoxicity. However, the possible protective role of DT-diaphorase against autophagy impairment by aminochrome-induced SNCA oligomers remains unclear. To test this idea, we used the cell line RCSN-3NQ7SNCA, with constitutive expression of a siRNA against DT-diaphorase and overexpression SNCA, and RCSN-3 as control cells. A significant increase in LC3-II expression was observed in RCSN-3 cells treated with 20 μM aminochrome and 10 μM rapamycin followed by a decrease in cell death compared to RCSN-3 cells incubated with 20 μM aminochrome alone. The incubation of RCSN-3NQ7SNCA cells with 20 μM aminochrome and 10 μM rapamycin does not change the expression of LC3-II in comparison with RCSN-3NQ7SNCA cells incubated with 20 μM aminochrome alone. The incubation of both cell lines preincubated with 100 nM bafilomycin and 20 μM aminochrome increases the level of LC3-II. Under the same conditions, cell death increases in both cell lines in comparison with cells incubated with 20 μM aminochrome. These results support the protective role of DT-diaphorase against SNCA oligomers-induced autophagy inhibition.     

Synaptosomal glutamate release and uptake in mice lacking the cellular prion protein

Glutamate plays a central role in the fast excitatory synaptic transmission and is a key neurotransmitter involved in several neurophysiological processes. Glutamate levels on the synaptic cleft are related to neural excitability, neuroplasticity, and neuronal damage associated with excitotoxicity. Mice lacking the cellular prion protein (PrP(c)) gene (Prnp) present a decreased astrocytic glutamate uptake in cultures, higher neuronal excitability in vitro and sensitivity to pro-convulsant drugs in vivo, and age-dependent memory impairment. Here, we investigate if PrP(c) might be involved in neuronal uptake and release of glutamate. For this purpose, we compared synaptosomal preparations from the cerebral cortex, entorhinal cortex, hippocampus, cerebellum, and olfactory bulb of 3- or 9-month-old PrP(c) null mice and with respective wild-type controls. Although we observed differences in synaptosomal glutamate release and uptake regarding the age of mice and the brain structure studied, these differences were similar for PrP(c) null mice and their respective wild-type controls. Therefore, despite a possible correlation between neuronal glutamate transporters, excitability, and neuronal damage, our results suggest that PrP(c) expression is not critical for neuronal glutamate transport.     

Decreased hyperlocomotion induced by MK-801, but not amphetamine and caffeine in mice lacking cellular prion protein (PrP(C))

The cellular prion protein (PrP(C)) has been involved in several neurodegenerative disorders however it has been proposed that it is also be implicated in psychotic disorders. We investigated the effect of three psychotropic drugs in locomotor activity of PrP(C) knockout (Prnp(O/O)) and wild-type mice. The NMDA receptor channel blocker MK-801 (0.25 mg/kg), the indirect dopamine agonist amphetamine (1 mg/kg) and the adenosine receptor antagonist caffeine (10 mg/kg) were administered i.p. after 60 min of habituation and locomotion was monitored for 3 h. Prnp(O/O) mice presented a diminished hyperlocomotor response to MK-801 treatment but normal response to amphetamine and caffeine compared to wild type mice. These results suggest that lack of PrP(C) leads to a functional alteration in the glutamatergic system, whereas the regulation of both dopaminergic and adenosinergic systems are preserved. Finally, lack of PrP(C) seems not to exacerbate the response to these psychotropic drugs, which modulate neurotransmitter systems possibly involved in schizophrenia and psychotic disorders.     

Micro RNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

Micro RNA-9(mi R-9) has been shown to promote the differentiation of bone marrow mesenchymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study confirmed that increased autophagic activity improved the efficiency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that mi RNAs adjust the autophagic pathways. This study used mi R-9-1 lentiviral vector and mi R-9-1 inhibitor to modulate the expression level of mi R-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3(LC3)-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Results showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron specific enolase and microtubule-associated protein 2 increased in the mi R-9+ group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when mi R-9 was overexpressed, demonstrating that mi R-9 can promote neuronal differentiation by increasing autophagic activity.     

Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke

The temporal dynamics of neuronal autophagy and apoptosis in the ischemic penumbra following stroke remains unclear.Therefore,in this study,we investigated the dynamic changes in autophagy and apoptosis in the penumbra to provide insight into potential therapeutic targets for stroke.An adult Sprague-Dawley rat model of permanent ischemic stroke was prepared by middle cerebral artery occlusion.Neuronal autophagy and apoptosis in the penumbra post-ischemia were evaluated by western blot assay and immunofluorescence staining with antibodies against LC3-Ⅱ and cleaved caspase-3,respectively.Levels of both LC3-Ⅱ and cleaved caspase-3 in the penumbra gradually increased within 5 hours post-ischemia.Thereafter,levels of both proteins declined,especially LC3-Ⅱ.The cerebral infarct volume increased slowly 1–4 hours after ischemia,but subsequently increased rapidly until 5 hours after ischemia.The severity of the neurological deficit was positively correlated with infarct volume.LC3-Ⅱ and cleaved caspase-3 levels were high in the penumbra within 5 hours after ischemia,and after that,levels of these proteins decreased at different rates.LC3-Ⅱ levels were reduced to a very low level,but cleaved caspase-3 levels remained high 72 hours after ischemia.These results indicate that there are temporal differences in the activation status of the autophagic and apoptotic pathways.This suggests that therapeutic targeting of these pathways should take into consideration their unique temporal dynamics.     

Necrostatin-1 protection of dopaminergic neurons

Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson's disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range(5–30 μM) elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson's disease.     

Autophagy after experimental intracerebral hemorrhage.

Autophagy contributes to ischemic brain injury, but it is not clear if autophagy occurs after intracerebral hemorrhage (ICH). This study examined whether ICH-induced cell death is partly autophagic. It then examined the role of iron in inducing this form of cell death after ICH. Male, adult Sprague-Dawley rats received an infusion of autologous whole blood or ferrous iron into the right basal ganglia. Control rats (sham) had a needle insertion. The rats were killed at 1, 3, 7, or 28 days later. Some rats were treated with either deferoxamine or vehicle after ICH. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and cathepsin D, a lysosomal biomarker, were measured by Western blot analysis and immunohistochemistry. Immunofluorescent double-labeling was used to identify the cell types expressing cathepsin D. Electron microscopy was performed to examine the cellular ultrastructure changes after ICH. We found that conversion of LC3-I to LC3-II, cathepsin D expression, and vacuole formation are increased in the ipsilateral basal ganglia after ICH. Intracerebral infusion of iron also resulted in enhanced conversion of LC3-I to LC3-II and increased cathepsin D levels. Deferoxamine (an iron chelator) treatment significantly reduced the conversion of LC3-I to LC3-II and cathepsin D levels after ICH. Our results demonstrated that autophagy occurs after ICH, and iron has a key role in ICH-induced autophagy. This also suggests that iron-induced autophagy may play a role in brain injury in other diseases associated with iron overload.     

The Designer Drug 3-Fluoromethcathinone Induces Oxidative Stress and Activates Autophagy in HT22 Neuronal Cells

Synthetic cathinones are psychoactive substances, derivatives of a natural psychostimulant cathinone. Although many synthetic cathinones have lost their legal status in many countries, their abuse still continues worldwide. Recently, they have been reported to exert neurotoxic effects in vitro and in vivo. The molecular mechanisms of their action have not been fully elucidated. Recently, they have been linked to the induction of oxidative stress, autophagy, and apoptosis. The aim of this study was to investigate whether 3-fluoromethcathinone (3-FMC), a synthetic cathinone, is able to induce oxidative stress, autophagy, and apoptosis in HT22 immortalized mouse hippocampal cells. We found that treatment of HT22 cells with this compound results in a concentration-dependent increase in the intracellular production of reactive oxygen species. Moreover, 3-FMC induced concentration-dependent conversion of cytosolic LC3-I to membrane-bound LC3-II and formation of autophagic vacuoles. Additionally, the level of p62/SQSTM1 protein decreased after 3-FMC treatment, suggesting that accumulation of autophagic vacuoles resulted from activation rather than inhibition of autophagy. Our results also showed that 3-FMC at millimolar concentration is able to induce caspase-dependent apoptotic cell death in HT22 cells. Our findings suggest that abuse of 3-FMC may disturb neuronal homeostasis and impair functioning of the central nervous system.