Apoferritin improves motor deficits in MPTP-treated mice by regulating brain iron metabolism and ferroptosis.

Iron deposition is one of the key factors in the etiology of Parkinson's disease (PD). Iron-free-apoferritin has the ability to store iron by combining with a ferric hydroxide-phosphate compound to form ferritin. In this study, we investigated the role of apoferritin in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice models and elucidated the possible underlying mechanisms. Results showed that apoferritin remarkably improved MPTP-induced motor deficits by rescuing dopaminergic neurodegeneration in the substantia nigra. Apoferritin inhibited MPTP-induced iron aggregation by down-regulating iron importer divalent metal transporter 1 (DMT1). Meanwhile, we also showed that apoferritin prevented MPTP-induced ferroptosis effectively by inhibiting the up-regulation of long-chain acyl-CoA synthetase 4 (ACSL4) and the down-regulation of ferroptosis suppressor protein 1 (FSP1). These results indicate that apoferritin exerts a neuroprotective effect against MPTP by inhibiting iron aggregation and modulating ferroptosis. This provides a promising therapeutic target for the treatment of PD.

Association of genetic and immuno-characteristics with clinical outcomes in patients with RET-rearranged non-small cell lung cancer: a retrospective multicenter study.

BACKGROUND: Rearranged during transfection (RET) has been proven to be a tumorigenic target in non-small cell lung cancers (NSCLCs). In RET-rearranged NSCLCs, molecular features and their impact on prognosis were not well illustrated, and the activity of mainstay therapeutics has not currently been well compared. METHODS: Patients diagnosed with NSCLCs with RET rearrangements were analyzed for concomitant mutations, tumor mutation burden (TMB), PD-L1 expression, T cell receptor repertoire and clinical outcomes with chemotherapy, immune checkpoint inhibitors (ICIs), and multikinase inhibitors (MKIs). RESULTS: Among 129 patients with RET-rearranged NSCLC who were analyzed, 41.1% (53/129) had co-occurring genetic alterations by next-generation sequencing, and concomitant TP53 mutation appeared most frequently (20/53, 37.7%). Patients with concurrent TP53 mutation (n = 15) had shorter overall survival than those without (n = 30; median, 18.4 months [95% CI, 8.6-39.1] vs 24.8 months [95% CI, 11.7-52.8]; P < 0.05). Patients with lower peripheral blood TCR diversity (n = 5) had superior overall survival compared with those with higher diversity (n = 6; median, 18.4 months [95% CI, 16.9-19.9] vs 4.8 months [95% CI, 4.5-5.3]; P = 0.035). An association with overall survival was not observed for PD-L1 expression nor for tumor mutation burden level. Median progression-free survival was not significantly different across chemotherapy, ICIs, and MKIs (median, 3.5 vs 2.5 vs 3.8 months). For patients treated with ICIs, the disease control rate was 60% (6/10) and the objective response rate was 20% (2/10). CONCLUSIONS: RET-rearranged lung cancers can be heterogeneous in terms of concomitant genetic alterations. Patients with concurrent TP53 mutation or high peripheral blood TCR repertoire diversity have relatively inferior overall survival in this series. Outcomes with traditional systemic therapies in general are suboptimal.

[Glial cells are involved in iron accumulation and degeneration of dopamine neurons in Parkinson's disease].

A growing body of evidence suggests that glial cells play an important role in neural development, neural survival, nerve repair and regeneration, synaptic transmission and immune inflammation. As the highest number of cells in the central nervous system, the role of glial cells in Parkinson's disease (PD) has attracted more and more attention. It has been confirmed that nigral iron accumulation contributes to the death of dopamine (DA) neurons in PD. Until now, most researches on nigral iron deposition in PD are focusing on DA neurons, but in fact glial cells in the central nervous system also play an important role in the regulation of iron homeostasis. Therefore, this review describes the role of iron metabolism of glial cells in death of DA neurons in PD, which could provide evidence to reveal the mechanisms underlying nigral iron accumulation of DA neurons in PD and provide the basis for discovering new potential therapeutic targets for PD.

[The Role of Iron and Alpha-synuclein Interacting in Parkinson's Disease].

Both iron and alpha-synuclein accumulation are one of hallmarks of Parkinson's disease (PD). Alpha-synuclein aggregation is often accompanied by abnormal accumulation of iron, indicating that there is a certain link between iron and alpha-synuclein aggregation. Iron promotes alpha-synuclein aggregation by increasing its synthesis and decreasing its degradation. Also, alpha-synuclein regulates iron metabolism through its ferrireductase activity. In this review, we will describe the roles of iron and alpha-synuclein in PD pathogenesis, and the mechanisms of iron and alpha-synuclein interaction.

Lentivirus-mediated Persephin over-expression in Parkinson's disease rats.

Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson's disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson's disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson's disease through protecting dopaminergic neurons.

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes.

It is well known that disrupted brain iron homeostasis was involved in Parkinson's disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 µM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.

Curcumin protects nigral dopaminergic neurons by iron-chelation in the 6-hydroxydopamine rat model of Parkinson's disease.

OBJECTIVE: Curcumin is a plant polyphenolic compound and a major component of spice turmeric (Curcuma longa). It has been reported to possess free radical-scavenging, iron-chelating, and anti-inflammatory properties in different tissues. Our previous study showed that curcumin protects MES23.5 dopaminergic cells from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The present study aimed to explore this neuroprotective effect in the 6-OHDA-lesioned rat model of Parkinson's disease in vivo. METHODS: Rats were given intragastric curcumin for 24 days. 6-OHDA lesioning was conducted on day 4 of curcumin treatment. Dopamine content was assessed by high-performance liquid chromatography with electrochemical detection, tyrosine hydroxylase (TH)-containing neurons by immunohistochemistry, and iron-containing cells by Perls' iron staining. RESULTS: The dopamine content in the striatum and the number of TH-immunoreactive neurons decreased after 6-OHDA treatment. Curcumin pretreatment reversed these changes. Further studies demonstrated that 6-OHDA treatment increased the number of iron-staining cells, which was dramatically decreased by curcumin pretreatment. CONCLUSION: The protective effects of curcumin against 6-OHDA may be attributable to the iron-chelating activity of curcumin to suppress the iron-induced degeneration of nigral dopaminergic neurons.

Recombinant adenovirus-mediated expression of GHS-R1a in HEK 293 cells.

OBJECTIVE: To construct the recombinant adenovirus vector carrying human growth hormone secretagogue receptor type 1a (GHS-R1a), for genetic transfection. METHODS: The full-length human GHS-R1a gene was obtained by PCR amplification and then cloned into the shuttle plasmid pAdTrack-CMV. The linearized plasmid pAdTrack-CMV-GHS-R1a was co-transformed into Escherichia coli (E. coli) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy1. The HEK293 cells were then infected with adenoviruses. The expression of GHS-R1a was indicated by green fluorescent protein (GFP), and confirmed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot. RESULTS: Enzymatic digestion of pAdGHS-R1a yielded a large fragment (approximately 30 kb) and a small fragment (4.5 kb), indicating the successful construction of recombinant adenovirus expression vector. Expression of GFP was observed by confocal laser scanning microscopy at 24 h after infection. RT-PCR and Western blot further confirmed that GHS-R1a was efficiently expressed in 293 cells. CONCLUSION: Recombinant adenovirus (AdGHS-R1a) is successfully constructed, and the target gene can be expressed efficiently in 293 cells, which provide a valuable tool for further studying the function of GHS-R1a.

Rosmarinic acid inhibits 6-OHDA-induced neurotoxicity by anti-oxidation in MES23.5 cells.

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. It is found in several herbs in the Lamiaceae family, such as Perilla frutescens. RA has been reported to exert anti-oxidative effects on rat erythrocyte, liver, and kidney cells. However, little is known about the effects of RA on dopaminergic cells. In the present study, we investigated whether RA could protect MES23.5 dopaminergic cells from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. The results showed that RA pretreatment significantly prevented 6-OHDA-induced cell viability reduction. Further experiments demonstrated that 6-OHDA induced intracellular reactive oxygen species generation and decreased the mitochondria membrane potential (DeltaPsim). These effects could be partially reversed by RA pretreatment. However, RA had no direct chemical reaction with 6-OHDA extracellularly in a cell-free system. Taken together, these results suggest that RA could exert its protective effects against 6-OHDA-induced neurotoxicity through its anti-oxidation properties. Thus, we propose that RA should be viewed as a potential chemotherapeutic in Parkinson's disease patients.

Rg1 reduces nigral iron levels of MPTP-treated C57BL6 mice by regulating certain iron transport proteins.

Elevated iron levels in the substantia nigra (SN) participate in neuronal death in Parkinson's disease, in which the misregulation of iron transporters such as divalent metal transporter (DMT1) and ferroportin1 (FP1) are involved. Our previous work observed that nigral iron levels were increased in MPTP-treated mice and Ginsenoside Rg1 which is one of the main components of ginseng, had neuroprotective effects against MPTP toxicity. Whether Rg1 could reduce nigral iron levels to protect the dopaminergic neurons? And whether its neuroprotective effect is achieved by regulating certain iron transporters? The present studies showed that Rg1 pre-treatment increased the dopamine and its metabolites contents in the striatum, as well as increased tyrosine hydroxylase expression in the SN. Further experiments observed that Rg1 pre-treatment substantially attenuated MPTP-elevated iron levels, decreased DMT1 expression and increased FP1 expression in the SN. These results suggest that the neuroprotective effect of Rg1 on dopaminergic neurons against MPTP is due to the ability to reduce nigral iron levels, which is achieved by regulating the expressions of DMT1 and FP1.

Over-expressed human divalent metal transporter 1 is involved in iron accumulation in MES23.5 cells.

Elevated iron accumulation has been reported in brain regions in some neurodegenerative disorders. However, the mechanism for this is largely unknown. Divalent metal transporter 1 (DMT1) is an important divalent cation transporter. The aim of the present study is to construct recombinant adenovirus encoding human DMT1 with iron responsive element (DMT1+IRE) and infect MES23.5 dopaminergic cells in order to investigate the relationship between increased DMT1+IRE expression and iron accumulation. The human DMT1 gene was obtained by RT-PCR from tissues of human duodenum. AdDMT1+IRE was successfully constructed and identified by PCR, restriction endonuclease analyses and DNA sequencing, respectively. It was able to efficiently infect MES23.5 cells, which was confirmed by RT-PCR and Western blots. When incubated with 100 microM ferrous iron for 6h, the intracellular iron levels dramatically increased in AdDMT1+IRE infected MES23.5 cells compared to the solely adenovirus infected cells. Meanwhile, the levels of hydroxyl free radicals and malondialdehyde (MDA) in these cells increased. This led to the activation of caspase-3. The apoptosis in AdDMT1+IRE infected cells was shown with hypercondensed nuclei using Hoechst staining. Analysis of DNA extracted from these cells showed the typical "ladder pattern", indicating the formation of mono- and oligonucleosomes. These results suggested that increased DMT1+IRE expression in MES23.5 cells caused the increased intracellular iron accumulation. This resulted in the increased oxidative stress leading to ultimate cell apoptosis.