Search Results (16)

The cerebellum, a key target of ethanol’s toxic effects, is associated with ataxia following alcohol consumption. However, the impact of ethanol on Purkinje cell (PC) mitochondria remains unclear. To investigate how ethanol administration affects mitochondrial dynamics in cerebellar Purkinje cells, we employed a transgenic mouse model expressing mitochondria-targeted yellow fluorescent protein in Purkinje cells (PC-mito-eYFP). Both male and female PC-mito-eYFP mice received an intraperitoneal injection of ethanol or vehicle. One hour after ethanol administration, the animals were perfusion fixed or their cerebellum tissue or isolated mitochondria were collected. Cerebellum sections were analyzed using confocal microscopy to assess changes in mitochondrial length distribution. In vivo superoxide levels were measured using dihydroethidium (DHE), and mitochondrial NAD levels were determined by high-performance liquid chromatography (HPLC). Our findings revealed a sex-dependent response to ethanol administration in mitochondrial size distribution. While male Purkinje cell mitochondria exhibited no significant changes in size, female mitochondria became more fragmented after one hour of ethanol administration. This coincided with elevated phosphorylation of the fission protein Drp1 and increased superoxide production, as measured by DHE fluorescence intensity. Similarly, mitochondrial NAD levels were significantly reduced in female mice, but no changes were observed in males. Our results demonstrate that ethanol induced mitochondrial fragmentation through increased free radical levels, due to reduced NAD and increased p-Drp1, in PC cells of the female cerebellum. Full article

The metastasis-associated protein 1/protein kinase B (MTA1/AKT) signaling pathway has been shown to cooperate in promoting prostate tumor growth. Targeted interception strategies by plant-based polyphenols, specifically stilbenes, have shown great promise against MTA1-mediated prostate cancer progression. In this study, we employed a prostate-specific transgenic mouse model with MTA1 overexpression on the background of phosphatase and tensin homolog (Pten) null (R26^MTA1; Pten^f/f) and PC3M prostate cancer cells which recapitulate altered molecular pathways in advanced prostate cancer. Mechanistically, the MTA1 knockdown or pharmacological inhibition of MTA1 by gnetin C (dimer resveratrol) in cultured PC3M cells resulted in the marked inactivation of mammalian target of rapamycin (mTOR) signaling. In vivo, mice tolerated a daily intraperitoneal treatment of gnetin C (7 mg/kg bw) for 12 weeks without any sign of toxicity. Treatment with gnetin C markedly reduced cell proliferation and angiogenesis and promoted apoptosis in mice with advanced prostate cancer. Further, in addition to decreasing MTA1 levels in prostate epithelial cells, gnetin C significantly reduced mTOR signaling activity in prostate tissues, including the activity of mTOR-target proteins: p70 ribosomal protein S6 kinase (S6K) and eukaryotic translational initiation factor 4E (elF4E)-binding protein 1 (4EBP1). Collectively, these findings established gnetin C as a new natural compound with anticancer properties against MTA1/AKT/mTOR-activated prostate cancer, with potential as monotherapy and as a possible adjunct to clinically approved mTOR pathway inhibitors in the future. Full article

Recent studies have demonstrated the association of APP and Aβ with cancer, suggesting that BACE1 may play an important role in carcinogenesis. In the present study, we assessed BACE1’s usefulness as a therapeutic target in prostate cancer (PCa). BACE1 expression was observed in human PCa tissue samples, patient-derived xenografts (PDX), human PCa xenograft tissue in nude mice, and transgenic adenocarcinoma of the mouse prostate (TRAMP) tissues by immunohistochemistry (IHC) analysis. Additionally, the downstream product of BACE1 activity, i.e., Aβ1-42 expression, was also observed in these PCa tissues by IHC as well as by PET imaging in TRAMP mice. Furthermore, BACE1 gene expression and activity was confirmed in several established PCa cell lines (LNCaP, C4-2B-enzalutamide sensitive [S], C4-2B-enzalutamide resistant [R], 22Rv1-S, 22Rv1-R, PC3, DU145, and TRAMP-C1) by real-time PCR and fluorometric assay, respectively. Treatment with a pharmacological inhibitor of BACE1 (MK-8931) strongly reduced the proliferation of PCa cells in in vitro and in vivo models, analyzed by multiple assays (MTT, clonogenic, and trypan blue exclusion assays and IHC). Cell cycle analyses revealed an increase in the sub-G1 population and a significant modulation in other cell cycle stages (G1/S/G2/M) following MK-8931 treatment. Most importantly, in vivo administration of MK-8931 intraperitoneal (30 mg/kg) strongly inhibited TRAMP-C1 allograft growth in immunocompetent C57BL/6 mice (approximately 81% decrease, p = 0.019). Furthermore, analysis of tumor tissue using the prostate cancer-specific pathway array revealed the alteration of several genes involved in PCa growth and progression including Forkhead O1 (FOXO1). All together, these findings suggest BACE1 as a novel therapeutic target in advanced PCa. Full article

Mitochondrial dysfunction has been described in many neurodegenerative disorders; however, there is less information regarding mitochondrial deficits in Machado–Joseph disease (MJD), a polyglutamine (polyQ) disorder caused by CAG repeat expansion in the ATXN3 gene. In the present study, we characterized the changes in mitochondrial function and biogenesis markers in two MJD models, CMVMJD135 (MJD135) transgenic mice at a fully established phenotype stage and tetracycline-regulated PC6-3 Q108 cell line expressing mutant ataxin-3 (mATXN3). We detected mATXN3 in the mitochondrial fractions of PC6-3 Q108 cells, suggesting the interaction of expanded ATXN3 with the organelle. Interestingly, in both the cerebella of the MJD135 mouse model and in PC6-3 Q108 cells, we found decreased mitochondrial respiration, ATP production and mitochondrial membrane potential, strongly suggesting mitochondrial dysfunction in MJD. Also, in PC6-3 Q108 cells, an additional enhanced glycolytic flux was observed. Supporting the functional deficits observed in MJD mitochondria, MJD135 mouse cerebellum and PC6-3 Q108 cells showed reduced cytochrome c mRNA and protein levels. Overall, our findings show compromised mitochondrial function associated with decreased cytochrome c levels in both cell and animal models of MJD. Full article

The apolipoprotein E4 (APOE4) genotype is predictive of Alzheimer’s disease (AD). The brain is highly enriched with the omega-3 polyunsaturated fatty acid (n3-PUFA), docosahexaenoic acid (DHA). DHA’s metabolism is defective in APOE4 carriers. Flavanol intake can play a role in modulating DHA levels. However, the impact of flavanol co-supplementation with fish oil on brain DHA uptake, status and partitioning, and according to APOE genotype is currently unknown. Here, using a humanised APOE3 and APOE4 targeted replacement transgenic mouse model, the interactive influence of cocoa flavanols (FLAV) and APOE genotype on the blood and subcortical brain PUFA status following the supplementation of a high fat (HF) enriched with DHA from fish oil (FO) was investigated. DHA levels increased in the blood (p < 0.001) and brain (p = 0.001) following supplementation. Compared to APOE3, a higher red blood cell (RBC) DHA (p < 0.001) was evident in APOE4 mice following FO and FLAV supplementation. Although FO did not increase the percentage of brain DHA in APOE4, a 17.1% (p < 0.05) and 20.0% (p < 0.001) higher DHA level in the phosphatidylcholine (PC) fraction in the HF FO and HF FO FLAV groups, and a 14.5% (p < 0.05) higher DHA level in the phosphatidylethanolamine (PE) fraction in the HF FO FLAV group was evident in these animals relative to the HF controls. The addition of FLAV (+/− FO) did not significantly increase the percentage of brain DHA in the group as a whole. However, a higher brain: RBC DHA ratio was evident in APOE3 only (p < 0.05) for HF FLAV versus HF. In conclusion, our data shows only modest effects of FLAV on the brain DHA status, which is limited to APOE3. Full article

Spinocerebellar ataxia type 1 (SCA1) is a fatal, dominantly inherited neurodegenerative disease caused by the expansion of CAG repeats in the Ataxin-1 (ATXN1) gene. SCA1 is characterized by the early and prominent pathology of the cerebellar Purkinje cells that results in balance and coordination deficits. We previously demonstrated that cerebellar astrocytes contribute to SCA1 pathogenesis in a biphasic, stage of disease-dependent manner. We found that pro-inflammatory transcriptional regulator nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling in astrocytes has a neuroprotective role during early-stage SCA1. Here, we sought to examine whether further inducing NF-κB activation in astrocytes of SCA1 model mice at an early stage of the disease has therapeutic benefits. To perform this task, we created a novel Slc1a3-CreER^T/IKKβ_CA/ATXN1[82Q] triple transgenic mouse model in which TMX injection at 4 weeks of age results in the expression of constitutively active inhibitor of kB kinase beta (IKKβ_CA), the main activator of NF-κB signaling. As we evaluated SCA1-like phenotypes, we noticed that ATXN1[82Q] mice did not exhibit motor deficits anymore, even at very late stages of the disease. We sequenced the mutant ATXN1 gene and discovered that the CAG repeat number had decreased from 82 to 71. However, despite the loss of motor phenotype, other well-characterized SCA1-changes, including atrophy of Purkinje cell dendrites, hallmarks of cerebellar astrogliosis and microgliosis, and Purkinje cell disease-associated gene expression changes, were still detectable in ATXN1[71Q] mice. We found delayed PC atrophy and calbindin reduction in SCA1 mice expressing IKKβ_CA in astrocytes implicating beneficial effects of increased NF-κB signaling on Purkinje cell pathology. The change in the motor phenotype of SCA1 mice with CAG reduction prevented us from evaluating the neuroprotective potential of IKKβ_CA on motor deficits in these mice. Full article

Background: The N-methyl-D-aspartate receptor (NMDAR) is a target in current treatments for Alzheimer’s disease (AD). The human prion protein (PrPC) has an important role in the pathophysiology of AD. We hypothesized that PrPC modulates NMDA signaling, thus being a process associated with Alzheimer’s disease. Methods: NMDAR signaling was characterized in the absence or presence of PrPC in cAMP level determination, mitogen-activated protein kinase (MAPK) pathway and label-free assays in homologous and heterologous systems. Bioluminescence resonance energy transfer was used to detect the formation of NMDAR-PrPC complexes. AXIS™ Axon Isolation Devices were used to determine axonal transport of Tau and pTau proteins in cortical primary neurons in the absence or presence of PrPC. Finally, proximity ligation assays were used to quantify NMDA-PrPC complex formation in neuronal primary cultures isolated from APP_Sw/Ind transgenic mice, an Alzheimer’s disease model expressing the Indiana and Swedish mutated version of the human amyloid precursor protein (APP). Results: We discovered a direct interaction between the PrPC and the NMDAR and we found a negative modulation of NMDAR-mediated signaling due to the NMDAR-PrPC interaction. In mice primary neurons, we identified NMDA-PrPC complexes where PrPC was capable of blocking NMDAR-mediated effects. In addition, we observed how the presence of PrPC results in increased neurotoxicity and neuronal death. Similarly, in microglial primary cultures, we observed that PrPC caused a blockade of the NMDA receptor link to the MAPK signaling cascade. Interestingly, a significant increase in NMDA-PrPC macromolecular complexes was observed in cortical neurons isolated from the APP_Sw,Ind transgenic model of AD. Conclusions: PrPC can interact with the NMDAR, and the interaction results in the alteration of the receptor functionality. NMDAR-PrPC complexes are overexpressed in neurons of APP_Sw/Ind mouse brain. In addition, PrPC exacerbates axonal transport of Tau and pTau proteins. Full article

Herein, we assessed the stage-specific efficacy of inositol hexaphosphate (IP6, phytic acid), a bioactive food component, on prostate cancer (PCa) growth and progression in a transgenic mouse model of prostate cancer (TRAMP). Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water for ~8–15 weeks. Pathological assessments at study endpoint indicated that tumor grade is arrested at earlier stages by IP6 treatment; IP6 also prevented progression to more advanced forms of the disease (~55–70% decrease in moderately and poorly differentiated adenocarcinoma incidence was observed in advanced stage TRAMP cohorts). Next, we determined whether the protective effects of IP6 are mediated via its effect on the expansion of the cancer stem cells (CSCs) pool; results indicated that the anti-PCa effects of IP6 are associated with its potential to eradicate the PCa CSC pool in TRAMP prostate tumors. Furthermore, in vitro assays corroborated the above findings as IP6 decreased the % of floating PC-3 prostaspheres (self-renewal of CSCs) by ~90%. Together, these findings suggest the multifaceted chemopreventive-translational potential of IP6 intervention in suppressing the growth and progression of PCa and controlling this malignancy at an early stage. Full article

Pancreatic cancer (PC) is a deadly disease with a grim prognosis. Pancreatic tumor derived factors (TDF) contribute to the induction of an immunosuppressive tumor microenvironment (TME) that impedes the effectiveness of immunotherapy. PC-induced microRNA-155 (miRNA-155) represses expression of Src homology 2 (SH2) domain-containing Inositol 5′-phosphatase-1 (SHIP-1), a regulator of myeloid cell development and function, thus impacting anti-tumor immunity. We recently reported that the bioflavonoid apigenin (API) increased SHIP-1 expression which correlated with the expansion of tumoricidal macrophages (TAM) and improved anti-tumor immune responses in the TME of mice with PC. We now show that API transcriptionally regulates SHIP-1 expression via the suppression of miRNA-155, impacting anti-tumor immune responses in the bone marrow (BM) and TME of mice with PC. We discovered that API reduced miRNA-155 in the PC milieu, which induced SHIP-1 expression. This promoted the restoration of myelopoiesis and increased anti-tumor immune responses in the TME of heterotopic, orthotopic and transgenic SHIP-1 knockout preclinical mouse models of PC. Our results suggest that manipulating SHIP-1 through miR-155 may assist in augmenting anti-tumor immune responses and aid in the therapeutic intervention of PC. Full article

Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrP^C), PrP^Sc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies. Full article

Conformational conversion of the cellular isoform of prion protein, PrP^C, into the abnormally folded, amyloidogenic isoform, PrP^Sc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrP^C into PrP^Sc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrP^C into PrP^Sc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrP^C into PrP^Sc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrP^C into PrP^Sc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrP^C into PrP^Sc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrP^C to convert into PrP^Sc after prion infection. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in aging populations. Recently, the regulation of neurolipid-mediated signaling and cerebral lipid species was shown in AD patients. The triple transgenic mouse model (3xTg-AD), harboring βAPP_Swe, PS1_M146V, and tau_P301L transgenes, mimics many critical aspects of AD neuropathology and progressively develops neuropathological markers. Thus, in the present study, 3xTg-AD mice have been used to test the involvement of the neurolipid-based signaling by endocannabinoids (eCB), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (S1P) in relation to the lipid deregulation. [³⁵S]GTPγS autoradiography was used in the presence of specific agonists WIN55,212-2, LPA and CYM5442, to measure the activity mediated by CB₁, LPA₁, and S1P₁ G_i/0 coupled receptors, respectively. Consecutive slides were used to analyze the relative intensities of multiple lipid species by MALDI Mass spectrometry imaging (MSI) with microscopic anatomical resolution. The quantitative analysis of the astrocyte population was performed by immunohistochemistry. CB₁ receptor activity was decreased in the amygdala and motor cortex of 3xTg-AD mice, but LPA₁ activity was increased in the corpus callosum, motor cortex, hippocampal CA1 area, and striatum. Conversely, S1P₁ activity was reduced in hippocampal areas. Moreover, the observed modifications on PC, PA, SM, and PI intensities in different brain areas depend on their fatty acid composition, including decrease of polyunsaturated fatty acid (PUFA) phospholipids and increase of species containing saturated fatty acids (SFA). The regulation of some lipid species in specific brain regions together with the modulation of the eCB, LPA, and S1P signaling in 3xTg-AD mice indicate a neuroprotective adaptation to improve neurotransmission, relieve the myelination dysfunction, and to attenuate astrocyte-mediated neuroinflammation. These results could contribute to identify new therapeutic strategies based on the regulation of the lipid signaling in familial AD patients. Full article

IQGAP1 expression was analyzed in: (1) primary prostate cancer, (2) xenografts produced from LNCaP, DU145, and PC3 cells, (3) tumor of PTEN^−/− and TRAMP mice, and (4) castration resistant PC (CRPC) produced by LNCaP xenografts and PTEN^−/− mice. IQGAP1 downregulations occurred in CRPC and advanced PCs. The downregulations were associated with rapid PC recurrence in the TCGA PanCancer (n = 492, p = 0.01) and MSKCC (n = 140, p = 4 × 10⁻⁶) cohorts. Differentially expressed genes (n = 598) relative to IQGAP1 downregulation were identified with enrichment in chemotaxis, cytokine signaling, and others along with reductions in immune responses. A novel 27-gene signature (Sig27gene) was constructed from these DEGs through random division of the TCGA cohort into a Training and Testing population. The panel was validated using an independent MSKCC cohort. Sig27gene robustly predicts PC recurrence at (hazard ratio) HR 2.72 and p < 2 × 10⁻¹⁶ in two independent PC cohorts. The prediction remains significant after adjusting for multiple clinical features. The novel and robust nature of Sig27gene underlie its great translational potential as a prognostic biomarker to predict PC relapse risk in patients with primary PC. Full article

The human cellular prion protein (PrP^C) is a glycosylphosphatidylinositol (GPI) anchored membrane glycoprotein with two N-glycosylation sites at residues 181 and 197. This protein migrates in several bands by Western blot analysis (WB). Interestingly, PNGase F treatment of human brain homogenates prior to the WB, which is known to remove the N-glycosylations, unexpectedly gives rise to two dominant bands, which are now known as C-terminal (C1) and N-terminal (N1) fragments. This resembles the β-amyloid precursor protein (APP) in Alzheimer disease (AD), which can be physiologically processed by α-, β-, and γ-secretases. The processing of APP has been extensively studied, while the identity of the cellular proteases involved in the proteolysis of PrP^C and their possible role in prion biology has remained limited and controversial. Nevertheless, there is a strong correlation between the neurotoxicity caused by prion proteins and the blockade of their normal proteolysis. For example, expression of non-cleavable PrP^C mutants in transgenic mice generates neurotoxicity, even in the absence of infectious prions, suggesting that PrP^C proteolysis is physiologically and pathologically important. As many mouse models of prion diseases have recently been developed and the knowledge about the proteases responsible for the PrP^C proteolysis is accumulating, we examine the historical experimental evidence and highlight recent studies that shed new light on this issue. Full article

There are two central phenomena in prion disease: prion replication and prion neurotoxicity. Underlying them both is the conversion of a host-encoded ubiquitously expressed protein, prion protein (PrP^C), into a partially-protease resistant isoform, PrP^Sc, which accumulates in the brain. PrP^Sc is associated with both pathology and infectivity. In the absence of PrP^C, PrP^Sc cannot be generated and PrP-null mice do not propagate infectivity or develop pathology on infection with scrapie. However, while PrP^C expression is fundamental to both prion infectivity and neurodegeneration, the uncoupling of these processes is a growing concept in the field. This dissociation is evident in subclinical states of prion infection, where PrP^Sc levels are high in the absence of disease, and in transgenic mice experiments, where, despite extra-neuronal PrP^Sc accumulation, even in very high amounts, there is no neurotoxicity. Both these models have further implications. Thus depleting PrP^C from neurons (but not glia) of prion-infected mice prevents clinical disease, and detaching it from the surface of cells by removing its anchor does the same. The uncoupling toxicity and infectivity has implications for the nature of the neurotoxic species; these mouse models suggest that the site for the generation of this species is intra-neuronal. This review considers the role of neuronal surface-expressed PrP^C in mediating toxicity in prion infection, and the dissociation of this from the deposition of PrP^Sc. Full article