Diagnostic utility and sensitivities of matrix Gla protein (MGP), TRPS1 and GATA3 in breast cancer: focusing on metastatic breast cancer, invasive breast carcinoma with special features, and salivary gland-type tumours.

Matrix Gla protein (MGP) and trichorhinophalangeal syndrome type 1 (TRPS1) have recently emerged as novel breast-specific immunohistochemical (IHC) markers, particularly for triple-negative breast cancer (TNBC) and metaplastic carcinoma. The present study aimed to validate and compare the expression of MGP, TRPS1 and GATA binding protein 3 (GATA3) in metastatic breast carcinoma (MBC), invasive breast carcinoma (IBC) with special features, including special types of invasive breast carcinoma (IBC-STs) and invasive breast carcinoma of no special type with unique features, and mammary and non-mammary salivary gland-type tumours (SGTs). Among all enrolled cases, MGP, TRPS1 and GATA3 had comparable high positivity for ER/PR-positive (p=0.148) and HER2-positive (p=0.310) breast carcinoma (BC), while GATA3 positivity was significantly lower in TNBC (p<0.001). Similarly, the positive rates of MGP and TRPS1 in MBCs (99.4%), were higher than in GATA3 (90.9%, p<0.001). Among the IBC-STs, 98.4% of invasive lobular carcinomas (ILCs) were positive for all three markers. Among neuroendocrine tumours (NTs), all cases were positive for TRPS1 and GATA3, while MGP positivity was relatively low (81.8%, p=0.313). In the neuroendocrine carcinoma (NC) subgroup, all cases were positive for GATA3 and MGP, while one case was negative for TRPS1. All carcinomas with apocrine differentiation (APOs) were positive for GATA3 and MGP, while only 60% of the cases demonstrated moderate staining for TRPS1. Among mammary SGTs, MGP demonstrated the highest positivity (100%), followed by TRPS1 (96.0%) and GATA3 (72.0%). Positive staining for these markers was also frequently observed in non-mammary SGTs. Our findings further validate the high sensitivity of MGP and TRPS1 in MBCs, IBC-STs, and breast SGTs. However, none of these markers are capable of distinguishing between mammary and non-mammary SGTs.

Tetramethylpyrazine Nitrone Promotes the Clearance of Alpha-Synuclein via Nrf2-Mediated Ubiquitin-Proteasome System Activation.

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson's disease (PD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent enhancement of the expression of the 20S proteasome core particles (20S CPs) and regulatory particles (RPs) increases proteasome activity, which can promote α-syn clearance in PD. Activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) may reduce oxidative stress by strongly inducing Nrf2 gene expression. In the present study, tetramethylpyrazine nitrone (TBN), a potent-free radical scavenger, promoted α-syn clearance by the ubiquitin-proteasome system (UPS) in cell models overexpressing the human A53T mutant α-syn. In the α-syn transgenic mice model, TBN improved motor impairment, decreased the products of oxidative damage, and down-regulated the α-syn level in the serum. TBN consistently up-regulated PGC-1α and Nrf2 expression in tested models of PD. Additionally, TBN similarly enhanced the proteasome 20S subunit beta 8 (Psmb8) expression, which is linked to chymotrypsin-like proteasome activity. Furthermore, TBN increased the mRNA levels of both the 11S RPs subunits Pa28αß and a proteasome chaperone, known as the proteasome maturation protein (Pomp). Interestingly, specific siRNA targeting of Nrf2 blocked TBN's effects on Psmb8, Pa28αß, Pomp expression, and α-syn clearance. In conclusion, TBN promotes the clearance of α-syn via Nrf2-mediated UPS activation, and it may serve as a potentially disease-modifying therapeutic agent for PD.

ApoE4 dysregulation incites depressive symptoms and mitochondrial impairments in mice.

Apolipoprotein E4 (ApoE4) is involved in the stress-response processes and is hypothesized to be a risk factor for depression by means of mitochondrial dysfunction. However, their exact roles and underlying mechanisms are largely unknown. ApoE4 transgenic mice (B6. Cg-ApoEtm1Unc Cdh18Tg( GFAP-APOE i4)1Hol /J) were subjected to stress (lipopolysaccharides, LPS) to elucidate the aetiology of ApoE4-induced depression. LPS treatment significantly aggravated depression-like behaviours, concurrent with neuroinflammation and impaired mitochondrial changes, and melatonin/Urolithin A (UA) + 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) reversed these effects in ApoE4 mice. Concurrently, ApoE4 mice exhibited mitophagy deficits, which could be further exacerbated by LPS stimulation, as demonstrated by reduced Atg5, Beclin-1 and Parkin levels, while PINK1 levels were increased. However, these changes were reversed by melatonin treatment. Additionally, proteomic profiling suggested mitochondria-related signalling and network changes in ApoE4 mice, which may underlie the exaggerated response to LPS. Furthermore, HEK 293T cells transfected with ApoE4 showed mitochondria-associated protein and mitophagy defects, including PGC-1α, TFAM, p-AMPKα, PINK1 and LC3B impairments. Additionally, it aggravates mitochondrial impairment (particularly mitophagy), which can be attenuated by triggering autophagy. Collectively, ApoE4 dysregulation enhanced depressive behaviour upon LPS stimulation.

eIF4E phosphorylation mediated LPS induced depressive-like behaviors via ameliorated neuroinflammation and dendritic loss.

The translational defect has emerged as a common feature of neurological disorders. Studies have suggested that alterations between opposing and balanced synaptic protein synthesis and turnover processes could lead to synaptic abnormalities, followed by depressive symptoms. Further studies link this phenomenon with eIF4E and TrkB/BDNF signaling. However, the interplay between the eIF4E and TrkB/BDNF signaling in the presence of neuroinflammation is yet to be explored. To illuminate the role of eIF4E activities within LPS-induced neuroinflammation and depression symptomology, we applied animal behavioral, biochemical, and pharmacological approaches. In addition, we sought to determine whether eIF4E dysregulated activities correlate with synaptic protein loss via the TrkB/BDNF pathway. Our results showed that LPS administration induced depressive-like behaviors, accompanied by neuroinflammation, reduced spine numbers, and synaptic protein dysregulation. Concurrently, LPS treatment enhanced eIF4E phosphorylation and TrkB/BDNF signaling defects. However, eFT508 treatment rescued the LPS-elicited neuroinflammation and depressive behaviors, as well as altered eIF4E phosphorylation, synaptic protein expression, and TrkB/BDNF signaling. The causal relation of eIF4E with BDNF signaling was further explored with TrkB antagonist K252a, which could reverse the effects of eFT508, validating the interplay between the eIF4E and TrkB/BDNF signaling in regulating depressive behaviors associated with neuroinflammation via synaptic protein translational regulation. In conclusion, our results support the involvement of eIF4E-associated translational dysregulation in synaptic protein loss via TrkB/BDNF signaling, eventually leading to depressiven-like behaviors upon inflammation-linked stress.

Tetramethylpyrazine nitrone exerts neuroprotection via activation of PGC-1α/Nrf2 pathway in Parkinson's disease models.

INTRODUCTION: Parkinson's disease (PD) is common neurodegenerative disease where oxidative stress and mitochondrial dysfunction play important roles in its progression. Tetramethylpyrazine nitrone (TBN), a potent free radical scavenger, has shown protective effects in various neurological conditions. However, the neuroprotective mechanisms of TBN in PD models remain unclear. OBJECTIVES: We aimed to investigate TBN's neuroprotective effects and mechanisms in PD models. METHODS: TBN's neuroprotection was initially measured in MPP+/MPTP-induced PD models. Subsequently, a luciferase reporter assay was used to detect peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) promoter activity. Effects of TBN on antioxidant damage and the PGC-1α/Nuclear factor erythroid-2-related factor 2 (Nrf2) pathway were thoroughly investigated. RESULTS: In MPP+-induced cell model, TBN (30-300 µM) increased cell survival by 9.95 % (P < 0.05), 16.63 % (P < 0.001), and 24.09 % (P < 0.001), respectively. TBN enhanced oxidative phosphorylation (P < 0.05) and restored PGC-1α transcriptional activity suppressed by MPP+ (84.30 % vs 59.03 %, P < 0.01). In MPTP-treated mice, TBN (30 mg/kg) ameliorated motor impairment, increased striatal dopamine levels (16.75 %, P < 0.001), dopaminergic neurons survival (27.12 %, P < 0.001), and tyrosine hydroxylase expression (28.07 %, P < 0.01). Selegiline, a positive control, increased dopamine levels (15.35 %, P < 0.001) and dopaminergic neurons survival (25.34 %, P < 0.001). Additionally, TBN reduced oxidative products and activated the PGC-1α/Nrf2 pathway. PGC-1α knockdown diminished TBN's neuroprotective effects, decreasing cell viability from 73.65 % to 56.87 % (P < 0.001). CONCLUSION: TBN has demonstrated consistent effectiveness in MPP+-induced midbrain neurons and MPTP-induced mice. Notably, the therapeutic effect of TBN in mitigating motor deficits and neurodegeneration is superior to selegiline. The neuroprotective mechanisms of TBN are associated with activation of the PGC-1α/Nrf2 pathway, thereby reducing oxidative stress and maintaining mitochondrial function. These findings suggest that TBN may be a promising therapeutic candidate for PD, warranting further development and investigation.

Low-dose Cu exposure enhanced α-synuclein accumulation associates with mitochondrial impairments in mice model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that mainly affects the elder population, and its etiology is enigmatic. Both environmental risks and genetics may influence the development of PD. Excess copper causes neurotoxicity and accelerates the progression of neurodegenerative diseases. However, the underlying mechanisms of copper-induced neurotoxicity remain controversial. In this study, A53T transgenic α-synuclein (A53T) mice and their matching wild-type (WT) mice were treated with a low dose of copper (0.13 ppm copper chlorinated drinking water, equivalent to the copper exposure of human daily copper intake dose) for 4 months, and copper poisoning was performed on human A53T mutant SHSY5Y cells overexpressed with α-synuclein (dose of 1/4 IC50), to test the effects of copper exposure on the body. The results of the open field test showed that the moto function of Cu-treated mice was impaired. Proteomics revealed changes in neurodevelopment, transport function, and mitochondrial membrane-related function in Cu-treated WT mice, which were associated with reduced expression of mitochondrial complex (NDUFA10, ATP5A), dopamine neurons (TH), and dopamine transporter (DAT). Mitochondrial function, nervous system development, synaptic function, and immune response were altered in Cu-treated A53T mice. These changes were associated with increased mitochondrial splitting protein (Drp1), decreased mitochondrial fusion protein (OPA1, Mfn1), abnormalities in mitochondrial autophagy protein (LC3BII/I, P62), decreased dopamine neuron (TH) expression, increased α-synuclein expression, inflammatory factors (IL-6, IL-1ß, and TNF-α) release and microglia (Iba1) activation. In addition, we found that Cu2+ (30 µM) induced excessive ROS production and reduced mitochondrial ATP production in human A53T mutant α-synuclein overexpressing SHSY5Y cells by in vitro experiments. In conclusion, low-dose copper treatment altered critical proteins involved in mitochondrial, neurodevelopmental, and inflammatory responses and affected mitochondria's ROS and ATP production levels.

Ibrutinib Delays ALS Installation and Increases Survival of SOD1G93A Mice by Modulating PI3K/mTOR/Akt Signaling.

Amyotrophic lateral sclerosis (ALS) is a fatal multisystem degenerative disorder with minimal available therapeutic. However, some recent studies showed promising results of immunological-based treatment. Here, we aimed to evaluate the efficacy of ibrutinib against ALS-associated abnormalities by targeting inflammation and muscular atrophy. Ibrutinib was administrated orally to SOD1 G93A mice from 6 to 19 weeks for prophylactic administration and 13 to 19 weeks for therapeutic administration. Our results demonstrated that ibrutinib treatment significantly delayed ALS-like symptom onset in the SOD1 G93A mice, as shown by improved survival time and reduced behavioral impairments. Ibrutinib treatment significantly reduced muscular atrophy by increasing muscle/body weight and decreasing muscular necrosis. The ibrutinib treatment also considerably reduced pro-inflammatory cytokine production, IBA-1, and GFAP expression, possibly mediated by mTOR/Akt/Pi3k signaling in the medulla, motor cortex and spinal cord of the ALS mice. In conclusion, our study demonstrated that ibrutinib could delay ALS onset, increase survival time, and reduce ALS progression by targeting inflammation and muscular atrophy via mTOR/Akt/PI3K modulation.

Metastasis of breast cancer to bones alters the tumor immune microenvironment.

BACKGROUND: Bone is one of the most frequent sites for breast cancer metastasis. Breast cancer bone metastasis (BCBM) leads to skeletal morbidities including pain, fractures, and spinal compression, all of which severely impact quality of life. Immunotherapy is a promising therapy for patients with advanced cancer, but whether it may provide benefit to metastatic bone cancer is currently unknown. Thus, a better understanding of the immune landscape of bone-disseminated breast cancers may reveal new therapeutic strategies. In this study, we use histopathological analysis to investigate changes within the immune microenvironment of primary breast cancer and paired BCBM. METHODS: Sixty-three patients with BCBM, including 31 with paired primary and bone metastatic lesions, were included in our study. The percentage of stroma and stromal tumor-infiltrating lymphocytes (TILs) was evaluated by histopathological analysis. The quantification of stromal TILs (CD4 + and CD8 +), macrophages (CD68 + and HLA-DR +), programmed cell death protein 1 (PD-1), and programmed cell death protein ligand 1 (PD-L1) was evaluated through immunohistochemical (IHC) staining. Statistical analysis was performed with paired t test, Wilcoxon test, spearman correlation test, and univariate and multivariate cox regression. RESULTS: Median survival after BCBM pathological diagnosis was 20.5 months (range: 3-95 months). Of the immune parameters measured, none correlated with survival after bone metastasis was diagnosed. Compared to the primary site, bone metastases exhibited more tumor stroma (mean: 58.5% vs 28.87%, p < 0.001) and less TILs (mean: 8.45% vs 14.03%, p = 0.042), as determined by H&E analysis. The quantification of primary vs metastatic tissue area with CD4 + (23.95/mm2 vs 51.69/mm2, p = 0.027 and with CD8 + (18.15/mm2 vs 58.95/mm2, p = 0.004) TILs similarly followed this trend and was reduced in number for bone metastases. The number of CD68 + and HLA-DR + macrophages showed no significant difference between primary sites and bone metastases. PD-1 expression was present in 68.25% of the bone metastasis, while PD-L1 expression was only present in 7.94% of the bone metastasis. CONCLUSIONS: Our findings suggest that compared to the primary breast cancer site, bone metastases harbor a less active immune microenvironment. Despite this relatively dampened immune landscape, expression of PD-1 and PD-L1 in the bone metastasis indicates a potential benefit from immune checkpoint inhibitors for some BCBM cases.

Matrix Gla protein (MGP), GATA3, and TRPS1: a novel diagnostic panel to determine breast origin.

BACKGROUND: Metastatic breast carcinoma is commonly considered during differential diagnosis when metastatic disease is detected in females. In addition to the tumor morphology and documented clinical history, sensitive and specific immunohistochemical (IHC) markers such as GCDFP-15, mammaglobin, and GATA3 are helpful for determining breast origin. However, these markers are reported to show lower sensitivity in certain subtypes, such as triple-negative breast cancer (TNBC). MATERIALS AND METHODS: Using bioinformatics analyses, we identified a potential diagnostic panel to determine breast origin: matrix Gla protein (MGP), transcriptional repressor GATA binding 1 (TRPS1), and GATA-binding protein 3 (GATA3). We compared MGP, TRPS1, and GATA3 expression in different subtypes of breast carcinoma of (n = 1201) using IHC. As a newly identified marker, MGP expression was also evaluated in solid tumors (n = 2384) and normal tissues (n = 1351) from different organs. RESULTS: MGP and TRPS1 had comparable positive expression in HER2-positive (91.2% vs. 92.0%, p = 0.79) and TNBC subtypes (87.3% vs. 91.2%, p = 0.18). GATA3 expression was lower than MGP (p < 0.001) or TRPS1 (p < 0.001), especially in HER2-positive (77.0%, p < 0.001) and TNBC (43.3%, p < 0.001) subtypes. TRPS1 had the highest positivity rate (97.9%) in metaplastic TNBCs, followed by MGP (88.6%), while only 47.1% of metaplastic TNBCs were positive for GATA3. When using MGP, GATA3, and TRPS1 as a novel IHC panel, 93.0% of breast carcinomas were positive for at least two markers, and only 9 cases were negative for all three markers. MGP was detected in 36 cases (3.0%) that were negative for both GATA3 and TRPS1. MGP showed mild-to-moderate positive expression in normal hepatocytes, renal tubules, as well as 31.1% (99/318) of hepatocellular carcinomas. Rare cases (0.6-5%) had focal MGP expression in renal, ovarian, lung, urothelial, and cholangiocarcinomas. CONCLUSIONS: Our findings suggest that MGP is a newly identified sensitive IHC marker to support breast origin. MGP, TRPS1, and GATA3 could be applied as a reliable diagnostic panel to determine breast origin in clinical practice.

Anti-depressive-like behaviors of APN KO mice involve Trkb/BDNF signaling related neuroinflammatory changes.

Major depression disorder is a severe mental illness often linked with metabolic disorders. Adiponectin is an adipocyte-secreted circulatory hormone with antidiabetic and glucose/lipid modulation capacities. Studies have demonstrated the pathophysiological roles of adiponectin involved in various neurological disorders, including depression. However, the underlying mechanisms are poorly understood. Here we showed that adiponectin deprivation enhanced antidepressive-like behaviors in the LPS-induced model of depression. APN KO mice displayed increased cytokines (both pro and anti-inflammatory), accompanied by an impaired expression of adiponectin receptors (mRNA/protein level) and decreasing IBA-1 level in the cortex and primary microglia of LPS treated APN KO mice. Further, LPS-treatment significantly reduced p-NFκB expression in the microglia of APN KO mice. However, the Bay11-7082 treatment recovered p-NFκB expression in the cortex of APN KO mice in the presence of LPS. Interestingly, the antidepressant potentials of APN KO mice were abolished by TrkB antagonist K252a, IKK inhibitor Bay11-7082, and AdipoRon suggesting crosstalk between TrkB/BDNF signaling and NFκB in depression. Furthermore, the effects of Bay11-7082 were abolished by a TrkB/BDNF activator (7,8-DHF), indicating a critical role of TrkB/BDNF signaling. Taken together, these findings showed that dysregulated neuroinflammatory status and BDNF signaling might underlie the antidepressive-like behaviors of APN KO mice. NFκB elicited BDNF changes may be accountable for the pathogenesis of LPS induced depression, where APN might present an alternative therapeutic target for depressive disorders.

Distribution Characteristics and Prognostic Value of Immune Infiltration in Oligometastatic Breast Cancer.

BACKGROUND: To assess the distribution characteristics and the prognostic value of immune infiltration in female oligometastatic breast cancer patients. METHODS: We retrospectively analyzed the clinicopathological data of oligometastatic breast cancer (OMBC) patients diagnosed between June 2000 and January 2020. Immune markers were quantified by immunohistochemistry on FFPE tissues in paired normal breast tissues, primary breast cancers and oligometastatic lesions. Survival analyses were performed using the Kaplan-Meier curves and Cox-proportional hazards model. RESULTS: A total of 95 female OMBC patients visited Sun Yat-sen University Cancer Center between June 2000 and January 2020, and 33 of them had matched normal breast tissues, primary cancers and oligometastatic lesions and were reviewed in immune infiltration analysis. CD8 of primary tumors had a higher expression than that in matched normal tissues. The expressions of CD8 and FOXP3 were higher in the primary sites than that in the oligometastatic lesions. CD3, CD4 and CD8 were significantly lower in the intratumoral regions than that in the peritumoral regions both in primary and oligometastatic lesions. Notably, the high percentage of CD3 in the intratumoral oligometastatic lesions predicted the longer PFS and OS, and higher CD4 in the same lesions also predicted a better OS. There was obviously positive correlation between CD4/CD3 and Ki-67 in primary cancers and negative correlation between CD4/CD3 and ER in oligometastatic sites. CONCLUSION: We explored immune distribution and evolution in time and space in OMBC to provide new understandings for biological behaviors of this disease and further divided patients in different prognosis.

Fluoxetine regulates eEF2 activity (phosphorylation) via HDAC1 inhibitory mechanism in an LPS-induced mouse model of depression.

BACKGROUND: Selective serotonin reuptaker inhibitors, including fluoxetine, are widely studied and prescribed antidepressants, while their exact molecular and cellular mechanism are yet to be defined. We investigated the involvement of HDAC1 and eEF2 in the antidepressant mechanisms of fluoxetine using a lipopolysaccharide (LPS)-induced depression-like behavior model. METHODS: For in vivo analysis, mice were treated with LPS (2 mg/kg BW), fluoxetine (20 mg/kg BW), HDAC1 activator (Exifone: 54 mg/kg BW) and NH125 (1 mg/kg BW). Depressive-like behaviors were confirmed via behavior tests including OFT, FST, SPT, and TST. Cytokines were measured by ELISA while Iba-1 and GFAP expression were determined by immunofluorescence. Further, the desired gene expression was measured by immunoblotting. For in vitro analysis, BV2 cell lines were cultured; treated with LPS, exifone, and fluoxetine; collected; and analyzed. RESULTS: Mice treated with LPS displayed depression-like behaviors, pronounced neuroinflammation, increased HDAC1 expression, and reduced eEF2 activity, as accompanied by altered synaptogenic factors including BDNF, SNAP25, and PSD95. Fluoxetine treatment exhibited antidepressant effects and ameliorated the molecular changes induced by LPS. Exifone, a selective HDAC1 activator, reversed the antidepressant and anti-inflammatory effects of fluoxetine both in vivo and in vitro, supporting a causing role of HDAC1 in neuroinflammation allied depression. Further molecular mechanisms underlying HDAC1 were explored with NH125, an eEF2K inhibitor, whose treatment reduced immobility time, altered pro-inflammatory cytokines, and NLRP3 expression. Moreover, NH125 treatment enhanced eEF2 and GSK3ß activities, BDNF, SNAP25, and PSD95 expression, but had no effects on HDAC1. CONCLUSIONS: Our results showed that the antidepressant effects of fluoxetine may involve HDAC1-eEF2 related neuroinflammation and synaptogenesis.

Tetramethylpyrazine nitrone improves motor dysfunction and pathological manifestations by activating the PGC-1α/Nrf2/HO-1 pathway in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons that results in skeletal muscle atrophy, weakness and paralysis. Oxidative stress plays a key role in the pathogenesis of ALS, including familial forms of the disease arising from mutation of the gene coding for superoxide dismutase (SOD1). We have used the SOD1G93A ALS mouse model to investigate the efficacy of 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel tetramethylpyrazine derivative armed with a powerful free-radical scavenging nitrone moiety. TBN was administered to mice by intraperitoneal or intragastric injection after the onset of motor deficits. TBN slowed the progression of motor neuron disease as evidenced by improved motor performance, reduced spinal motor neuron loss and the associated glial response, and decreased skeletal muscle fiber denervation and fibrosis. TBN treatment activated mitochondrial antioxidant activity through the PGC-1α/Nrf2/HO-1 pathway and decreased the expression of human SOD1. These findings suggest that TBN holds promise as a therapeutic agent for ALS.

Melatonin Act as an Antidepressant via Attenuation of Neuroinflammation by Targeting Sirt1/Nrf2/HO-1 Signaling.

Physical or psychological stress can cause an immunologic imbalance that disturbs the central nervous system followed by neuroinflammation. The association between inflammation and depression has been widely studied in recent years, though the molecular mechanism is still largely unknown. Thus, targeting the signaling pathways that link stress to neuroinflammation might be a useful strategy against depression. The current study investigated the protective effect of melatonin against lipopolysaccharide (LPS)-induced neuroinflammation and depression. Our results showed that LPS treatment significantly induced depressive-like behavior in mice. Moreover, LPS-treatment enhanced oxidative stress, pro-inflammatory cytokines including TNFα, IL-6, and IL-1ß, NF-κB phosphorylation, and glial cell activation markers including GFAP and Iba-1 in the brain of mice. Melatonin treatment significantly abolished the effect of LPS, as indicated by improved depressive-like behaviors, reduced cytokines level, reduced oxidative stress, and normalized LPS-altered Sirt1, Nrf2, and HO-1 expression. However, the melatonin protective effects were reduced after luzindole administration. Collectively, it is concluded that melatonin receptor-dependently protects against LPS-induced depressive-like behaviors via counteracting LPS-induced neuroinflammation.

Memantine Improves Cognitive Function and Alters Hippocampal and Cortical Proteome in Triple Transgenic Mouse Model of Alzheimer's Disease.

Memantine is a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist clinically approved for moderate-to-severe Alzheimer's disease (AD) to improve cognitive functions. There is no report about the proteomic alterations induced by memantine in AD mouse model yet. In this study, we investigated the protein profiles in the hippocampus and the cerebral cortex of AD-related transgenic mouse model (3×Tg-AD) treated with memantine. Mice (8-month) were treated with memantine (5 mg/kg/bid) for 4 months followed by behavioral and molecular evaluation. Using step-down passive avoidance (SDA) test, novel object recognition (NOR) test and Morris water maze (MWM) test, it was observed that memantine significantly improved learning and memory retention in 3xTg-AD mice. By using quantitative proteomic analysis, 3301 and 3140 proteins in the hippocampus and the cerebral cortex respectively were identified to be associated with AD abnormalities. In the hippocampus, memantine significantly altered the expression levels of 233 proteins, among which PCNT, ATAXIN2, TNIK, and NOL3 were up-regulated, and FLNA, MARK 2 and BRAF were down-regulated. In the cerebral cortex, memantine significantly altered the expression levels of 342 proteins, among which PCNT, PMPCB, CRK, and MBP were up-regulated, and DNM2, BRAF, TAGLN 2 and FRY1 were down-regulated. Further analysis with bioinformatics showed that memantine modulated biological pathways associated with cytoskeleton and ErbB signaling in the hippocampus, and modulated biological pathways associated with axon guidance, ribosome, cytoskeleton, calcium and MAPK signaling in the cerebral cortex. Our data indicate that memantine induces higher levels of proteomic alterations in the cerebral cortex than in the hippocampus, suggesting memantine affects various brain regions in different manners. Our study provides a novel view on the complexity of protein responses induced by memantine in the brain of AD.

Dysregulation of Myosin Complex and Striated Muscle Contraction Pathway in the Brains of ALS-SOD1 Model Mice.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease characterized by cortical and spinal motor neuron degeneration, some inherited cases of which are caused by mutations in the gene coding for copper-zinc superoxide dismutase-1 (SOD1). The SOD1G93A mutant model mouse, which expresses large amounts of mutant SOD1, develops adult-onset neurodegeneration of spinal motor neurons and progressive motor deficits leading to paralysis. We used the Tandem Mass Tag technique to investigate the proteome profile of hippocampus, cerebral cortex, and medulla oblongata of the SOD1G93A mutant model mice as compared with that of wild-type (WT) mice. Fifteen proteins were significantly increased or decreased (i.e., changed) in all three tissues. Gene ontology analysis revealed that the changed proteins were mainly enriched in negative regulation of reactive oxygen species, myosin complex and copper ion binding. In the Striated Muscle Contraction Pathway, most of the identified proteins were decreased in the SOD1G93A mice compared with the WT mice. Myosin-1 (MYH1), fructose-2,6-bisphosphatase TIGAR (TIGAR), and sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1) were significantly reduced in mutant vs WT mice, as confirmed by Western blot analysis. Since myosins and tropomyosins are specific for synapse function and drive actin dynamics in the maturation of dendritic spines, changes in these proteins may contribute to perturbations of brain neuronal circuitry in addition to spinal motor neuron disease.

Hippocampal Proteomic Alteration in Triple Transgenic Mouse Model of Alzheimer's Disease and Implication of PINK 1 Regulation in Donepezil Treatment.

Donepezil is a clinically approved acetylcholinesterase inhibitor (AChEI) for cognitive improvement in Alzheimer's disease (AD). Donepezil has been used as a first-line agent for the symptomatic treatment of AD, but its ability to modify disease pathology and underlying mechanisms is not clear. We investigated the protective effects and underlying mechanisms of donepezil in AD-related triple transgenic (APPSwe/PSEN1M146V/MAPTP301L) mouse model (3×Tg-AD). Mice (8-month old) were treated with donepezil (1.3 mg/kg) for 4 months and evaluated by behavioral tests for assessment of cognitive functions, and the hippocampal tissues were examined by protein analysis and quantitative proteomics. Behavioral tests showed that donepezil significantly improved the cognitive capabilities of 3×Tg-AD mice. The levels of soluble and insoluble amyloid beta proteins (Aß1-40 and Aß1-42) and senile plaques were reduced in the hippocampus. Golgi staining of the hippocampus showed that donepezil prevented dendritic spine loss in hippocampal neurons of 3×Tg-AD mice. Proteomic studies of the hippocampal tissues identified 3131 proteins with altered expression related to AD pathology, of which 262 could be significantly reversed with donepezil treatment. Bioinformatics with functional analysis and protein-protein interaction (PPI) network mapping showed that donepezil significantly elevated the protein levels of PINK 1, NFASC, MYLK2, and NRAS in the hippocampus, and modulated the biological pathways of axon guidance, mitophagy, mTOR, and MAPK signaling. The substantial upregulation of PINK 1 with donepezil was further verified by Western blotting. Donepezil exhibited neuroprotective effects via multiple mechanisms. In particular, PINK 1 is related to mitophagy and cellular protection from mitochondrial dysfunction, which might play important roles in AD pathogenesis and represent a potential therapeutic target.

Substantial protection against MPTP-associated Parkinson's neurotoxicity in vitro and in vivo by anti-cancer agent SU4312 via activation of MEF2D and inhibition of MAO-B.

We have previously demonstrated the unexpected neuroprotection of the anti-cancer agent SU4312 in cellular models associated with Parkinson's disease (PD). However, the precise mechanisms underlying its neuroprotection are still unknown, and the effects of SU4312 on rodent models of PD have not been characterized. In the current study, we found that the protection of SU4312 against 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in PC12 cells was achieved through the activation of transcription factor myocyte enhancer factor 2D (MEF2D), as evidenced by the fact that SU4312 stimulated myocyte enhancer factor 2 (MEF2) transcriptional activity and prevented the inhibition of MEF2D protein expression caused by MPP+, and that short hairpin RNA (ShRNA)-mediated knockdown of MEF2D significantly abolished the neuroprotection of SU4312. Additionally, Western blotting analysis revealed that SU4312 potentiated pro-survival PI3-K/Akt pathway to down-regulate MEF2D inhibitor glycogen synthase kinase-3beta (GSK3ß). Furthermore, using the in vivo PD model of C57BL/6 mice insulted with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that intragastrical administration of SU4312 (0.2 and 1 mg/kg) greatly ameliorated Parkinsonian motor defects, and restored protein levels of MEF2D, phosphorylated-Ser473-Akt and phosphorylated-Ser9-GSK3ß. Meanwhile, SU4312 effectively reversed the decrease in protein expression of tyrosine hydroxylase in substantia nigra pars compacta dopaminergic neurons, inhibited oxidative stress, maintained mitochondrial biogenesis and partially prevented the depletion of dopamine and its metabolites. Very encouragingly, SU4312 was able to selectively inhibit monoamine oxidase-B (MAO-B) activity both in vitro and in vivo, with an IC50 value of 0.2 µM. These findings suggest that SU4312 provides therapeutic benefits in cellular and animal models of PD, possibly through multiple mechanisms including enhancement of MEF2D through the activation of PI3-K/Akt pathway, maintenance of mitochondrial biogenesis and inhibition of MAO-B activity. SU4312 thus may be an effective drug candidate for the prevention or even modification of the pathological processes of PD.

Characterization of the Molecular Mechanisms Underlying the Chronic Phase of Stroke in a Cynomolgus Monkey Model of Induced Cerebral Ischemia.

Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.