Cyclic-NDGA Effectively Inhibits Human γ-Synuclein Fibrillation, Forms Nontoxic Off-Pathway Species, and Disintegrates Preformed Mature Fibrils.

Parkinson's disease arises from protein misfolding, aggregation, and fibrillation and is characterized by LB (Lewy body) deposits, which contain the protein α-synuclein (α-syn) as their major component. Another synuclein, γ-synuclein (γ-syn), coexists with α-syn in Lewy bodies and is also implicated in various types of cancers, especially breast cancer. It is known to seed α-syn fibrillation after its oxidation at methionine residue, thereby contributing in synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule inhibitors and modulators of γ-syn fibrillation remains largely unexplored. This work reveals the modulatory properties of cyclic-nordihydroguaiaretic acid (cNDGA), a natural polyphenol, on the structural and aggregational properties of human γ-syn employing various biophysical and structural tools, namely, thioflavin T (ThT) fluorescence, Rayleigh light scattering, 8-anilinonaphthalene-1-sulfonic acid binding, far-UV circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) spectroscopy, atomic force microscopy, ITC, molecular docking, and MTT-toxicity assay. cNDGA was observed to modulate the fibrillation of γ-syn to form off-pathway amorphous species that are nontoxic in nature at as low as 75 µM concentration. The modulation is dependent on oxidizing conditions, with cNDGA weakly interacting (Kd ∼10-5 M) with the residues at the N-terminal of γ-syn protein as investigated by isothermal titration calorimetry and molecular docking, respectively. Increasing cNDGA concentration results in an increased recovery of monomeric γ-syn as shown by sodium dodecyl sulfate and native-polyacrylamide gel electrophoresis. The retention of native structural properties of γ-syn in the presence of cNDGA was further confirmed by far-UV CD and FTIR. In addition, cNDGA is most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and is also capable of disintegrating the preformed mature fibrils. These findings could, therefore, pave the ways for further exploring cNDGA as a potential therapeutic against γ-synucleinopathies.

Special Issue "Neurobiology of Protein Synuclein".

Synucleins are a family of proteins consisting of α, ß, and γ synuclein (syn) [...].

The Herbal Combination Shu Gan Jie Yu Regulates the SNCG/ER-a/AKT-ERK Pathway in DMBA-Induced Breast Cancer and Breast Cancer Cell Lines Based on RNA-Seq and IPA Analysis.

BACKGROUND: Soothing the liver (called Shu Gan Jie Yu in Chinese, SGJY) is a significant therapeutic method for breast cancer in TCM. In this study, 3 liver-soothing herbs, including Cyperus rotundus L., Citrus medica L. var. sarcodactylis Swingle and Rosa rugosa Thunb. were selected and combined to form a SGJY herbal combinatory. THE AIM OF THE STUDY: To investigate the inhibiting effect of SGJY on breast cancer in vivo and vitro, and to explore the potential mechanisms. MATERIALS AND METHODS: SGJY herbal combination was extracted using water. A breast cancer rat model was developed by chemical DMBA by gavage, then treated with SGJY for 11 weeks. The tumor tissue was preserved for RNA sequencing and analyzed by IPA software. The inhibition effects of SGJY on MCF-7 and T47D breast cancer cells were investigated by SRB assay and cell apoptosis analysis, and the protein expression levels of SNCG, ER-α, p-AKT and p-ERK were measured by western blotting. RESULTS: SGJY significantly reduced the tumor weight and volume, and the level of estradiol in serum. The results of IPA analysis reveal SGJY upregulated 7 canonical pathways and downregulated 16 canonical pathways. Estrogen receptor signaling was the key canonical pathway with 9 genes downregulated. The results of upstream regulator analysis reveal beta-estradiol was the central target; the upstream regulator network scheme showed that 86 genes could affect the expression of the beta-estradiol, including SNCG, CCL21 and MB. Additionally, SGJY was verified to significantly alter the expression of SNCG mRNA, CCL21 mRNA and MB mRNA which was consistent with the data of RNA-Seq. The inhibition effects of SGJY exhibited a dose-dependent response. The apoptosis rates of MCF7 and T47D cells were upregulated. The protein expression of SNCG, ER-α, p-AKT and p-ERK were all significantly decreased by SGJY on MCF-7 and T47D cells. CONCLUSION: The results demonstrate that SGJY may inhibit the growth of breast cancer. The mechanism might involve downregulating the level of serum estradiol, and suppressing the protein expression in the SNCG/ER-α/AKT-ERK pathway.

Upregulation of Parkinson's disease-associated protein alpha-synuclein suppresses tumorigenesis via interaction with mGluR5 and gamma-synuclein in liver cancer.

Numerous epidemiological studies suggest a link between Parkinson's disease (PD) and cancer, indicating that PD-associated proteins may mediate the development of cancer. Here, we investigated a potential role of PD-associated protein α-synuclein in regulating liver cancer progression in vivo and in vitro. We found the negative correlation of α-synuclein with metabotropic glutamate receptor 5 (mGluR5) and γ-synuclein by analyzing the data from The Cancer Genome Atlas database, liver cancer patients and hepatoma cells with overexpressed α-synuclein. Moreover, upregulated α-synuclein suppressed the growth, migration, and invasion. α-synuclein was found to associate with mGluR5 and γ-synuclein, and the truncated N-terminal of α-synuclein was essential for the interaction. Furthermore, overexpressed α-synuclein exerted the inhibitory effect on hepatoma cells through the degradation of mGluR5 and γ-synuclein via α-synuclein-dependent autophagy-lysosomal pathway (ALP). Consistently, in vivo experiments with rotenone-induced rat model of PD also confirmed that, upregulated α-synuclein in liver cancer tissues through targeting on mGluR5/α-synuclein/γ-synuclein complex inhibited tumorigenesis involving in ALP-dependent degradation of mGluR5 and γ-synuclein. These findings give an insight into an important role of PD-associated protein α-synuclein accompanied by the complex of mGluR5/α-synuclein/γ-synuclein in distant communications between PD and liver cancer, and provide a new strategy in therapeutics for the treatment of liver cancer.

Sncg, Mybpc1, and Parm1 Classify subpopulations of VIP-expressing interneurons in layers 2/3 of the somatosensory cortex.

Neocortical vasoactive intestinal polypeptide-expressing (VIP+) interneurons display highly diverse morpho-electrophysiological and molecular properties. To begin to understand the function of VIP+ interneurons in cortical circuits, they must be clearly and comprehensively classified into distinct subpopulations based on specific molecular markers. Here, we utilized patch-clamp RT-PCR (Patch-PCR) to simultaneously obtain the morpho-electric properties and mRNA profiles of 155 VIP+ interneurons in layers 2 and 3 (L2/3) of the mouse somatosensory cortex. Using an unsupervised clustering method, we identified 3 electrophysiological types (E-types) and 2 morphological types (M-types) of VIP+ interneurons. Joint clustering based on the combined electrophysiological and morphological features resulted in 3 morpho-electric types (ME-types). More importantly, we found these 3 ME-types expressed distinct marker genes: ~94% of Sncg+ cells were ME-type 1, 100% of Mybpc1+ cells were ME-type 2, and ~78% of Parm1+ were ME-type 3. By clarifying the properties of subpopulations of cortical L2/3 VIP+ interneurons, this study establishes a basis for future investigations aiming to elucidate their physiological roles.

Effects of the Methylation Levels for the Breast Cancer Associated Genes BCSG1 and BRCA1 on Cellular Proliferation and Migration.

Objective: The aim of this study was to determine whether the methylation patterns of the breast cancer-specific gene 1 (BCSG1) and the breast cancer susceptibility gene 1 (BRCA1) can be used as biomarkers for predicting the occurrence and development of breast cancer. Methods: Methylation-specific polymerase chain reaction (PCR) was used to detect the methylation status of the BCSG1 and BRCA1 genes in ductal infiltrating carcinomas of the breast; carcinoma in situ of the breast; fibroadenoma of the breast and adjacent normal tissues. Quantitative real-time PCR and immunohistochemistry were used to detect the expression levels of BCSG1 and BRCA1. The BCSG1 and BRCA1 genes were knocked down by siRNA to study their effect of BCSG1 and BRCA1 on the behaviour of breast cancer cell lines. Results: The BCSG1 gene was hypomethylated in breast cancer tissues, and its mRNA as well as its protein levels showed elevated expression compared to normal adjacent tissues. In contrast, the BRCA1 gene was hypermethylated in breast cancer tissues and showed correspondingly decreased mRNA and protein expression levels. In vitro experiments demonstrated that BCSG1 could promote the proliferation and migration of breast cancer cells. After inhibiting the methylation, the expression of both the BCSG1 and BRCA1 genes were increased. Conclusion: Abnormal methylation patterns of the BCSG1 and BRCA1 genes are associated with the development of breast cancer. Thus, methylatedion analyses of these genes have biomarker potential for breast cancer prognoses.

Increased Expression of Alpha-, Beta-, and Gamma-Synucleins in Brainstem Regions of a Non-Human Primate Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by cell loss in the substantia nigra and the presence of alpha-synuclein (α-syn)-containing neuronal Lewy bodies. While α-syn has received major interest in the pathogenesis of PD, the function of beta- and gamma-synucleins (ß-syn and γ-syn, respectively) is not really known. Yet, these proteins are members of the same family and also concentrated in neuronal terminals. The current preclinical study investigated the expression levels of α-, ß-, and γ-synucleins in brainstem regions involved in PD physiopathology. We analyzed synuclein expression in the substantia nigra, raphe nuclei, pedunculopontine nucleus, and locus coeruleus from control and parkinsonian (by MPTP) macaques. MPTP-intoxicated monkeys developed a more or less severe parkinsonian score and were sacrificed after a variable post-MPTP period ranging from 1 to 20 months. The expression of the three synucleins was increased in the substantia nigra after MPTP, and this increase correlates positively, although not very strongly, with cell loss and motor score and not with the time elapsed after intoxication. In the dorsal raphe nucleus, the expression of the three synucleins was also increased, but only α- and γ-Syn are linked to the motor score and associated cell loss. Finally, although no change in synuclein expression was demonstrated in the locus coeruleus after MPTP, we found increased expression levels of γ-Syn, which are only correlated with cell loss in the pedunculopontine nucleus. Altogether, our data suggest that these proteins may play a key role in brainstem regions and mesencephalic tegmentum. Given the involvement of these brain regions in non-motor symptoms of PD, these data also strengthen the relevance of the MPTP macaque model of PD, which exhibits pathological changes beyond nigral DA cell loss and α-synucleinopathy.

Multiomic Analysis of Neurons with Divergent Projection Patterns Identifies Novel Regulators of Axon Pathfinding.

Axon pathfinding is a key step in neural circuits formation. However, the transcriptional mechanisms regulating its progression remain poorly understood. The binary decision of crossing or avoiding the midline taken by some neuronal axons during development represents a robust model to investigate the mechanisms that control the selection of axonal trajectories. Here, to identify novel regulators of axon guidance, this work compares the transcriptome and chromatin occupancy profiles of two neuronal subpopulations, ipsilateral (iRGC) and contralateral retinal ganglion cells (cRGC), with similar functions but divergent axon trajectories. These analyses retrieved a number of genes encoding for proteins not previously implicated in axon pathfinding. In vivo functional experiments confirm the implication of some of these candidates in axonal navigation. Among the candidate genes, γ-synuclein is identified as essential for inducing midline crossing. Footprint and luciferase assays demonstrate that this small-sized protein is regulated by the transcription factor (TF) Pou4f1 in cRGCs. It is also shown that Lhx2/9 are specifically expressed in iRGCs and control a program that partially overlaps with that regulated by Zic2, previously described as essential for iRGC specification. Overall, the analyses identify dozens of new molecules potentially involved in axon guidance and reveal the regulatory logic behind the selection of axonal trajectories.

Synuclein Analysis in Adult Xenopus laevis.

The α-, ß- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, ß- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, ß- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, ß- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.

Gamma synuclein promotes cancer metastasis through the MKK3/6-p38MAPK cascade.

Gamma synuclein (SNCG) is a neuronal protein that is also aberrantly overexpressed in various types of human cancer. SNCG overexpression promotes cancer invasion and metastasis. However, the mechanisms that drive cancer metastasis upon SNCG expression remain elusive. Elucidation of the mechanisms underlying the promotion of cancer metastasis by SNCG may help discover therapeutic avenues for SNCG-overexpressed cancer. Here, we show that SNCG promotes transforming growth factor-ß (TGF-ß)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Mechanistically, SNCG promotes p38MAPK phosphorylation by interacting with the MAPK kinase 3/6 (MKK3/6) and prevents their degradation. SNCG knockdown leads to a decrease in TGF-ß-induced phosphorylation of MKK3/6; and abrogates the induction of matrix metalloproteinase (MMP)-9 expression by TGF-ß and its target gene Twist1. Furthermore, p38MAPK inhibition abrogates the promotion of MMP-9 expression and cancer cell invasion by SNCG. Both p38MAPK and MMP inhibitors can suppress the promotion of cancer cell invasion by SNCG. Finally, overexpression of SNCG in liver cancer cells promotes lung metastasis, which can be suppressed by the p38MAPK inhibitor. Together, our data uncover a previously unknown role of SNCG in promoting TGF-ß-MKK3/6-p38MAPK signaling. This study highlights the critical role of p38MAPK in the promotion of cancer metastasis by SNCG, and indicates that p38MAPK inhibitor may serve as a potential therapeutic for SNCG-overexpressed cancer.

Long Noncoding RNA Solute Carrier Family 25 Member 21 Antisense RNA 1 Inhibits Cell Malignant Behaviors and Enhances Radiosensitivity of Gastric Cancer Cells by Upregulating Synuclein Gamma Expression.

Long noncoding RNAs (LncRNAs) were reported to be implicated in the progression of gastric cancer (GC). This study aimed to explore the role of solute carrier family 25 member 21 antisense RNA 1 (SLC25A21-AS1) in radiosensitivity of GC cells. In the present study, reverse transcription quantitative polymerase chain reaction (RT-qPCR) showed that the expression of SLC25A21-AS1 and synuclein gamma (SNCG) was downregulated in GC tissues and cells, while the expression of microRNA-15a-5p (miR-15a-5p) was upregulated in GC tissues and cells. The expression of SLC25A21-AS1 was elevated in GC cells after radiation treatment. SLC25A21-AS1 overexpression enhanced GC cell radiosensitivity, inhibited cell proliferation and promoted apoptosis. SLC25A21-AS1 overexpression also facilitated the DNA damage caused by radiation in GC cells. Mechanically, SLC25A21-AS1 interacted with miR-15a-5p and negatively regulated miR-15a-5p expression in GC cells. SNCG was directly targeted by miR-15a-5p at the 3' untranslated region (3'UTR). In GC tissues, the expression of SNCG was negatively correlated with that of miR-15a-5p, but was positively correlated with that of SLC25A21-AS1. Rescue assays revealed that SNCG silencing rescued the tumor-suppressive effect of overexpressed SLC25A21-AS1 on GC cells. The enhanced radiosensitivity caused by SLC25A21-AS1 overexpression was also reduced by SNCG knockdown. In conclusion, lncRNA SLC25A21-AS1 inhibits cell malignant behaviors and enhances cell radiosensitivity in GC by elevating SNCG expression.

Synaptic vesicle binding of α-synuclein is modulated by ß- and γ-synucleins.

α-synuclein, ß-synuclein, and γ-synuclein are abundantly expressed proteins in the vertebrate nervous system. α-synuclein functions in neurotransmitter release by binding to and clustering synaptic vesicles and chaperoning SNARE-complex assembly. Pathologically, aggregates originating from soluble pools of α-synuclein are deposited into Lewy bodies in Parkinson's disease and related synucleinopathies. The functions of ß-synuclein and γ-synuclein in presynaptic terminals remain poorly studied. Using in vitro liposome binding studies, circular dichroism spectroscopy, immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments on isolated synaptic vesicles in combination with subcellular fractionation of brains from synuclein mouse models, we show that ß-synuclein and γ-synuclein have a reduced affinity toward synaptic vesicles compared with α-synuclein, and that heteromerization of ß-synuclein or γ-synuclein with α-synuclein results in reduced synaptic vesicle binding of α-synuclein in a concentration-dependent manner. Our data suggest that ß-synuclein and γ-synuclein are modulators of synaptic vesicle binding of α-synuclein and thereby reduce α-synuclein's physiological activity at the neuronal synapse.

Up and Down γ-Synuclein Transcription in Dopamine Neurons Translates into Changes in Dopamine Neurotransmission and Behavioral Performance in Mice.

The synuclein family consists of α-, ß-, and γ-Synuclein (α-Syn, ß-Syn, and γ-Syn) expressed in the neurons and concentrated in synaptic terminals. While α-Syn is at the center of interest due to its implication in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, limited information exists on the other members. The current study aimed at investigating the biological role of γ-Syn controlling the midbrain dopamine (DA) function. We generated two different mouse models with: (i) γ-Syn overexpression induced by an adeno-associated viral vector and (ii) γ-Syn knockdown induced by a ligand-conjugated antisense oligonucleotide, in order to modify the endogenous γ-Syn transcription levels in midbrain DA neurons. The progressive overexpression of γ-Syn decreased DA neurotransmission in the nigrostriatal and mesocortical pathways. In parallel, mice evoked motor deficits in the rotarod and impaired cognitive performance as assessed by novel object recognition, passive avoidance, and Morris water maze tests. Conversely, acute γ-Syn knockdown selectively in DA neurons facilitated forebrain DA neurotransmission. Importantly, modifications in γ-Syn expression did not induce the loss of DA neurons or changes in α-Syn expression. Collectively, our data strongly suggest that DA release/re-uptake processes in the nigrostriatal and mesocortical pathways are partially dependent on substantia nigra pars compacta /ventral tegmental area (SNc/VTA) γ-Syn transcription levels, and are linked to modulation of DA transporter function, similar to α-Syn.

The amyloid concentric ß-barrel hypothesis: Models of synuclein oligomers, annular protofibrils, lipoproteins, and transmembrane channels.

Amyloid beta (Aß of Alzheimer's disease) and α-synuclein (α-Syn of Parkinson's disease) form large fibrils. Evidence is increasing however that much smaller oligomers are more toxic and that these oligomers can form transmembrane ion channels. We have proposed previously that Aß42 oligomers, annular protofibrils, and ion channels adopt concentric ß-barrel molecular structures. Here we extend that hypothesis to the superfamily of α, ß, and γ-synucleins. Our models of numerous synuclein oligomers, annular protofibrils, tubular protofibrils, lipoproteins, and ion channels were developed to be consistent with sizes, shapes, molecular weights, and secondary structures of assemblies as determined by electron microscopy and other studies. The models have the following features: (1) all subunits have identical structures and interactions; (2) they are consistent with conventional ß-barrel theory; (3) the distance between walls of adjacent ß-barrels is between 0.6 and 1.2 nm; (4) hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains are relatively optimal; and (5) residues that are identical among distantly related homologous proteins cluster in the interior of most oligomers whereas residues that are hypervariable are exposed on protein surfaces. Atomic scale models of some assemblies were developed.

A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG.

The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets γ-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism of fat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.

A Novel Ruthenium-Fluvastatin Complex Downregulates SNCG Expression to Modulate Breast Carcinoma Cell Proliferation and Apoptosis via Activating the PI3K/Akt/mTOR/VEGF/MMP9 Pathway.

Breast cancer is the most common cause of malignancy and cancer-related morbidity and death worldwide that requests effective and safe chemotherapy. Evaluation of metallodrug-based anticancer agents and statins as chemotherapeutics with fewer side effects is a largely unexplored research field. Synthesis and characterization of the ruthenium-fluvastatin complex were achieved using multiple spectroscopic techniques and thus further examined to evaluate its chemotherapeutic prospects in both MDA-MB-231 and MCF-7 cancer lines and eventually in vivo models of DMBA-induced mammary carcinogenesis in rodents. Our studies indicate that the metal and ligand chelation was materialized by the ligand's functional groups of carbonyl (=O) oxygen and hydroxyl (-OH), and the complex has been observed to be crystalline and able to chelate with CT-DNA. The complex was able to reduce cell proliferation and activate apoptotic events in breast carcinoma cell lines MCF-7 and MDA-MB-231. In addition, the complex was able to modify p53 expressions to interfere with apoptosis in the carcinoma of the breast, stimulated by the intrinsic apoptotic path assisted by Bcl2 and Bax in vivo, yet at the same point, controlling the PI3K/Akt/mTOR/VEGF pathway, as obtained from western blotting, correlates with the MMP9-regulated tumor mechanisms. Our research reveals that ruthenium-fluvastatin chemotherapy may disrupt, rescind, or interrupt breast carcinoma progression by modifying intrinsic apoptosis as well as the antiangiogenic cascade, thereby taking the role of a potential candidate in cancer therapy for the immediate future.

Excess membrane binding of monomeric alpha-, beta- and gamma-synuclein is invariably associated with inclusion formation and toxicity.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs ß-synuclein (ßS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-ß component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in ßS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Gamma-synuclein is a novel prognostic marker that promotes tumor cell migration in biliary tract carcinoma.

Gamma-synuclein (SNCG) promotes invasive behavior and is reportedly a prognostic factor in a range of cancers. However, its role in biliary tract carcinoma (BTC) remains unknown. Consequently, we investigated the clinicopathological significance and function of SNCG in BTC. Using resected BTC specimens from 147 patients with adenocarcinoma (extrahepatic cholangiocarcinoma [ECC, n = 96]; intrahepatic cholangiocarcinoma [ICC, n = 51]), we immunohistochemically evaluated SNCG expression and investigated its correlation with clinicopathological factors and outcomes. Furthermore, cell lines with high SNCG expression were selected from 16 BTC cell lines and these underwent cell proliferation and migration assays by siRNAs. In the results, SNCG expression was present in 22 of 96 (22.9%) ECC patients and in 10 of 51 (19.6%) ICC patients. SNCG expression was significantly correlated with poorly differentiated tumor in both ECC and ICC (p = 0.01 and 0.03, respectively) and with perineural invasion and lymph node metastases in ECC (p = 0.04 and 0.003, respectively). Multivariate analyses revealed that SNCG expression was an independent poor prognostic factor in both OS and RFS in both ECC and ICC. In vitro analyses showed high SNCG expression in three BTC cell lines (NCC-BD1, NCC-BD3, and NCC-CC6-1). Functional analysis revealed that SNCG silencing could suppress cell migration in NCC-BD1 and NCC-CC6-1 and downregulate cell proliferation in NCC-CC6-1 significantly. In conclusion, SNCG may promote tumor cell activity and is potentially a novel prognostic marker in BTC.

IGFBPs mediate IGF-1's functions in retinal lamination and photoreceptor development during pluripotent stem cell differentiation to retinal organoids.

Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.

γ-Synuclein is Closely Involved in Autophagy that Protects Colon Cancer Cell from Endoplasmic Reticulum Stress.

BACKGROUND: In previous studies, we provided evidence suggesting the involvement of γ-synuclein in growth, invasion, and metastasis of colon cancer cells in vitro and in vivo. Among γ-synuclein downstream genes, the microtubule-associated protein 1 Light Chain 3 (LC3), an autophagy gene, was screened by gene expression profile chip analysis. OBJECTIVE: We planned to investigate the functional effects of γ-synuclein on autophagy induced by ER stress in colon cancer cells. METHODS: We investigated the functional effects of γ-synuclein on autophagy and apoptosis induced by Thapsigargin (TG), ER stress-inducing agent, in colon cancer cell lines using immunofluorescence staining, RT-PCR, western blot, CCK8 test, flow cytometry analysis, and transmission electron microscopy. To further determine how γ-synuclein regulated autophagy and apoptosis, PD98059 (ERK inhibitor), SP600125 (ERK inhibitor), anisomycin (JNK activator), and c-Jun siRNA were used respectively in γ-synuclein siRNA transfected HCT116 cells. Then, autophagy proteins, apoptosis proteins, and pathway proteins were detected by western blot analysis. The expression of autophagy genes was assessed by RT-PCR. RESULTS: Our data showed that ER stress-induced colon cancer cells autophagy mainly in the early stage (0-24h) and apoptosis mainly in the late stage (24-48h). ER stress up-regulated γ-synuclein gene and protein expression in colon cancer cells, accompanied by autophagy. γ-synuclein protected HCT116 cells by enhancing autophagy in the early stage (0-24h) through activation of ERK and JNK pathway and inhibiting apoptosis in the late stage (24-48h) through inhibition of the JNK pathway. γ-synuclein could promote autophagy via the JNK pathway activation of ATG genes, LC3, Beclin 1, and ATG7. γ-synuclein may play a role in the transition between autophagy and apoptosis in our model. CONCLUSION: Overall, we provided the first experimental evidence to show that γ-synuclein may play an important role in autophagy that protects colon cancer cells from ER stress. Therefore, our data suggest a new molecular mechanism for γ-synuclein-mediated CRC progression.