Sarcoma Protein Research Articles

Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration.

Aggregation of fused in sarcoma (FUS) protein, and mutations in FUS gene, are causative to a range of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. To gain insights into the molecular mechanism whereby FUS causes neurodegeneration, we generated transgenic Drosophila melanogaster overexpressing human FUS in the photoreceptor neurons, which exhibited mild retinal degeneration. Expression of familial ALS-mutant FUS aggravated the degeneration, which was associated with an increase in cytoplasmic localization of FUS. A carboxy-terminally truncated R495X mutant FUS also was localized in cytoplasm, whereas the degenerative phenotype was diminished. Double expression of R495X and wild-type FUS dramatically exacerbated degeneration, sequestrating wild-type FUS into cytoplasmic aggregates. Notably, replacement of all tyrosine residues within the low-complexity domain, which abolished self-assembly of FUS, completely eliminated the degenerative phenotypes. Taken together, we propose that self-assembly of FUS through its low-complexity domain contributes to FUS-induced neurodegeneration.

Abstract A27: BET bromodomain proteins in Ewing sarcoma regulate a specific transcriptional program, tumorigenicity and apoptosis by interacting with EWS-FLI1 and the positive transcription elongation factor

Abstract Introduction: Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors that are genetically defined by balanced chromosomal EWS/ETS translocations. Previously, we reported the use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) and the associated strong down-regulation of the predominant EWS-ETS protein EWS-FLI1. Here we analyzed the mechanistic effects of this treatment by EWS-FLI1 interaction studies and the evaluation of possible down-stream players in order to shed further light into this intricate network and to develop refined treatment regimens for ES patients. Experimental procedures: Function of BRDs was analyzed by application of specific inhibitors (JQ1, I-BET151), RNA interference (RNAi) with the generation of stable and inducible knockdowns or knockouts by the generation of BRD4 CRISPR/Cas9 cell lines. To analyses the resulting changes Co-IP, ChIP-qPCR, RT-PCR, western blotting, cell cycle analysis, proliferation and invasion assays, whole transcriptome analysis via microarrays as well as xenograft mouse models were utilized. Summary: By use of JQ1 we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner, which was confirmed by treatment with I-BET151. Microarray analysis revealed JQ1 treatment to block the typical ES associated expression program. The effect on this expression program was partially mimicked by RNAi with BRD3 or BRD4 expression but not by BRD2 blockade. Furthermore, knockout studies of BRD4 by CRISPR/Cas9 as well as knockdowns of individual BRD2, 3 or 4 did not recapitulate proliferation restrictions as observed for JQ1, hinting towards an interdependency for all 3 proteins. Subsequent functional studies on the protein level demonstrated an interaction of EWS-FLI1 with BRD4 as well as phosphorylated CDK9 as an essential partner of the positive transcription elongation factor (p-TEFb). Additional analysis revealed a particular regulation of the X-linked inhibitor of apoptosis protein (XIAP) and CASP8 and FADD-like regulator (CFLAR), as a result of CDK9 activity. Treatment of ES cells with a specific CDK9 inhibitor further demonstrated a rapid down regulation of EWS-FLI1 expression and block of contact dependent growth. Furthermore, CDK9i induced apoptosis in ES as depicted by cleavage of Caspase 8 or PARP, which we similarly observed after JQ1 treatment. Conclusion: Here we demonstrate a possible interdependency of BET proteins regulating the ES specific expression profile and a possible substitution effect observed after knockdown or knockout of individual BRD proteins. We further demonstrate that ES are also susceptible to treatment with inhibitors targeting the p-TEFb complex that resemble features observed after JQ1 treatment. Most interestingly, we observed EWS-FLI1 to interact with BRD4 and CDK9 to presumably regulate an ES specific expression profile and suppress activation of apoptotic mechanisms. Hence treatment of ES with BRD inhibitors in combination with a CDK9i seems a new treatment option that could significantly improve the particular need to specifically block the pathognomonic EWS-ETS transcriptional program and malignant phenotype of ES. Citation Format: Tim Hensel, Chiara Giorgi, Fiona Becker-Dettling, Julia Calzada-Wack, Frauke Neff, Oxana Schmidt, Shudong Wang, Beat W. Schäfer, Stefan Burdach, Günther HS Richter. BET bromodomain proteins in Ewing sarcoma regulate a specific transcriptional program, tumorigenicity and apoptosis by interacting with EWS-FLI1 and the positive transcription elongation factor [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A27.

The RNA binding protein EWS is broadly involved in the regulation of pri-miRNA processing in mammalian cells.

The Ewing Sarcoma protein (EWS) is a multifaceted RNA binding protein (RBP) with established roles in transcription, pre-mRNA processing and DNA damage response. By generating high quality EWS–RNA interactome, we uncovered its specific and prevalent interaction with a large subset of primary microRNAs (pri-miRNAs) in mammalian cells. Knockdown of EWS reduced, whereas overexpression enhanced, the expression of its target miRNAs. Biochemical analysis revealed that multiple elements in target pri-miRNAs, including the sequences flanking the stem–loop region, contributed to high affinity EWS binding and sequence swap experiments between target and non-target demonstrated that the flanking sequences provided the specificity for enhanced pri-miRNA processing by the Microprocessor Drosha/DGCR8. Interestingly, while repressing Drosha expression, as reported earlier, we found that EWS was able to enhance the recruitment of Drosha to chromatin. Together, these findings suggest that EWS may positively and negatively regulate miRNA biogenesis via distinct mechanisms, thus providing a new foundation to understand the function of EWS in development and disease.

Chapter 21 - Concepts and classification of neurodegenerative diseases

Neurodegenerative diseases are disorders characterized by progressive loss of neurons associated with deposition of proteins showing altered physicochemical properties in the brain and in peripheral organs. Molecular classification of neurodegenerative disease is protein-based. This emphasizes the role of protein-processing systems in the pathogenesis. The most frequent proteins involved in the pathogenesis of neurodegenerative diseases are amyloid-β, prion protein, tau, α-synuclein, TAR-DNA-binding protein 43kDa, and fused-in sarcoma protein. There are further proteins associated mostly with hereditary disorders such as proteins encoded by genes linked to trinucleotide repeat disorders, neuroserpin, ferritin, and familial cerebral amyloidoses. The clinical presentations are defined by the distinct involvement of functional systems and do not necessarily indicate the molecular pathologic background. Seeding of pathologic proteins and hierarchic involvement of anatomic regions is commonly seen in neurodegenerative diseases. Overlap of neurodegenerative diseases and combinations of different disorders is frequent. Translation of neuropathologic categories of neurodegenerative diseases into in vivo detectable biomarkers is only partly achieved but intensive research is performed to reach this goal.

Diagnosing, monitoring and managing behavioural variant frontotemporal dementia.

Behavioural variant frontotemporal dementia is characterised by insidious changes in personality and interpersonal conduct that reflect progressive disintegration of the neural circuits involved in social cognition, emotion regulation, motivation and decision making. The underlying pathology is heterogeneous and classified according to the presence of intraneuronal inclusions of tau, TDP-43 or, occasionally, fused in sarcoma proteins. Biomarkers to detect these histopathological changes in life are increasingly important with the development of disease-modifying drugs. A number of gene abnormalities have been identified, the most common being an expansion in the C9orf72 gene, which together account for most familial cases. The 2011 international consensus criteria propose three levels of diagnostic certainty: possible, probable and definite. Detailed history taking from family members to elicit behavioural features underpins the diagnostic process, with support from neuropsychological testing designed to detect impairment in decision making, emotion processing and social cognition. Brain imaging is important for increasing the level of diagnosis certainty over time. Carer education and support remain of paramount importance.

Comprehensive profiling of H-Ras signalling in angiosarcoma endothelium.

The MS1/SVR system, in which MS1 represents immortalized endothelial cells and SVR represents MS1 cells transformed with oncogenic human-rat sarcoma protein (H-Ras), has been used for around 20 years as a valuable tool to study angiogenesis and carcinogenesis. Despite the use of these cells in numerous studies, a comprehensive profile of the signalling differences due to oncogenic H-Ras transformation has not been performed previously. In this study, we profiled the well-known MS1 and SVR cell lines using a combination of both Western blot and gene chip assays.

Diverse Supramolecular Nanofiber Networks Assembled by Functional Low-Complexity Domains.

Self-assembling supramolecular nanofibers, common in the natural world, are of fundamental interest and technical importance to both nanotechnology and materials science. Despite important advances, synthetic nanofibers still lack the structural and functional diversity of biological molecules, and the controlled assembly of one type of molecule into a variety of fibrous structures with wide-ranging functional attributes remains challenging. Here, we harness the low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an essential cellular nuclear protein with slow kinetics of amyloid fiber assembly, to construct random copolymer-like, multiblock, and self-sorted supramolecular fibrous networks with distinct structural features and fluorescent functionalities. We demonstrate the utilities of these networks in the templated, spatially controlled assembly of ligand-decorated gold nanoparticles, quantum dots, nanorods, DNA origami, and hybrid structures. Owing to the distinguishable nanoarchitectures of these nanofibers, this assembly is structure-dependent. By coupling a modular genetic strategy with kinetically controlled complex supramolecular self-assembly, we demonstrate that a single type of protein molecule can be used to engineer diverse one-dimensional supramolecular nanostructures with distinct functionalities.

Fused in Sarcoma Neuropathology in Neurodegenerative Disease.

Abnormal intracellular accumulation of the fused in sarcoma (FUS) protein is the characteristic pathological feature of cases of familial amyotrophic lateral sclerosis (ALS) caused by FUS mutations (ALS-FUS) and several uncommon disorders that may present with sporadic frontotemporal dementia (FTLD-FUS). Although these findings provide further support for the concept that ALS and FTD are closely related clinical syndromes with an overlapping molecular basis, important differences in the pathological features and results from experimental models indicate that ALS-FUS and FTLD-FUS have distinct pathogenic mechanisms.

The glycosylation stoichiometry of EWS species in neuronal cells.

Although Ewing sarcoma protein (EWS) is known to be glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc), the dynamics and stoichiometry of its glycosylation remain obscure. Here, we report a dynamic change in the glycosylation stoichiometry of EWS species during neuronal differentiation of embryonic carcinoma P19 cells. Our findings suggest that O-GlcNAc glycosylation participates in the regulation of EWS functions in neuronal cells.

PI3K/Akt Activated by GPR30 and Src Regulates 17β-Estradiol-Induced Cultured Immature Boar Sertoli Cells Proliferation.

Sertoli cell (SC) is a key element in the process of spermatogenesis. Accumulating research show that estrogen plays an important role in regulating boar SC proliferation. However, it is unclear whether phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) is involved in this process. In the present study, the role of PI3K/Akt on the 17β-estradiol-induced piglet SC proliferation was explored. In addition, we also explained the roles of G-protein-coupled estrogen receptor (GPR30) and Sarcoma protein (Src) in this process. Our study demonstrated that, 17β-estradiol induced activation of PI3K in a time-dependent manner. Both G-15 (an antagonist of GPR30, 0.1 μmol/L) and PP2 (an inhibitor of Src, 2.0 μmol/L) inhibited 17β-estradiol-induced activation of PI3K, reduced SC proliferation, and decreased messenger RNA (mRNA) and protein expression of S-phase kinase-associated protein 2 (Skp2). We also found that 17β-estradiol induced activation of Akt in a time-dependent manner. Both LY294002 (an inhibitor of PI3K) and 10-DEBC (an inhibitor of Akt) significantly reduced 17β-estradiol-induced SC proliferation and reduced mRNA and protein expression of Skp2. In addition, LY294002 inhibited 17β-estradiol-induced activation of Akt. The results indicated that 17β-estradiol regulates SC proliferation by activating PI3K/Akt. Both GPR30 and Src are involved in 17β-estradiol-induced phosphorylation of PI3K/Akt. Activation of PI3K/Akt enhances the expression of Skp2, which promotes SC proliferation.

High EZH2 expression is correlated to metastatic disease in pediatric soft tissue sarcomas.

BackgroundEnhancer of Zeste Drosophila Homologue 2 (EZH2) is a key regulator of transcription as a member of polycomb repressive complex 2 (PRC2) which exerts repression of downstream genes and is correlated to invasiveness and progression of different tumours. Therefore, we evaluated the expression of PRC2 proteins in pediatric soft tissue sarcoma (rhabdomyosarcoma, RMS and extraosseous Ewing sarcoma, EES) correlating them to the clinical outcome of the patients.MethodsWe analyzed PRC2 protein expression by quantitative real time PCR, western blotting and immunohistochemistry in 17 soft tissue sarcomas (11 RMS and 6 EES) enrolled at Paediatric Oncology Units of the Second University of Naples. Expression analysis was performed for EZH2, SUZ12 and EED.ResultsEnhancer of Zeste Drosophila Homologue 2 was expressed with a different degree in 60 % of samples. Interestingly, the magnitude of EZH2 up regulation was significantly higher in patients presenting lymph node and/or distant metastases at the diagnosis. Moreover, patients overexpressing EZH2 had a lower probability of survival compared to patients negative or with low EZH2 expression.ConclusionsOur study suggests that high EZH2 expression is associated to increased aggressiveness of the disease. Therefore, drugs that control its activity could be potentially used in the epigenetic target treatment of tumors with these alterations.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches.

ObjectiveKirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted.MethodsIn the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein.ResultsInterestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values.ConclusionThus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein.

청색광에 의한 마우스 망막손상에서 선택적 광수용세포의 사멸

목적: 본 연구는 망막색소상피층에 색소가 존재하는 mouse에서 청색광으로 인해 광수용세포 손상이 일어날 수 있는지 확인하고, 광수용세포 중 특이적 세포에서 세포사멸이 유도되는지 조사하여 청색광에 의해 야기될 수 있는 연령관련 황반변성의 기전 규명과 치료제 개발에 도움이 되고자 진행되었다. 방법: C57black mice를 24시간 암순응 시켜 463 nm의 청색광을 <TEX>$2800{\pm}10lux$</TEX>로 조사한 후 1일, 3일, 7일째에 안구를 적출하였다. 청색광의 자극은 GFAP(Glial fibrillary acidic protein)단백질의 발현을 이용하여 확인하였고, 광수용세포의 세포사멸은 TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling)을 사용하여 분석하였다. Western blotting으로 ERK(Extracellular signal-regulated kinases), c-JUN, SRC(Sarcoma) 단백질 발현을 확인하였고, 막대세포와 원뿔세포의 손상 정도를 비교하기 위해 면역염색으로 분석하였다. 결과: 청색광을 조사한 후 1, 3, 7일이 지난 망막은 대조군 보다 전체적으로 두께가 감소하였고, 각 얼기층보다 핵층에서 두께 감소를 확인할 수 있었다. 또한 청색광을 조사한 후 1일 지난 Muller glia에서 GFAP 단백질이 증가하는 것을 확인하였다. TUNEL 염색에서는 청색광을 조사한 후 1일 지난 망막의 광수용세포에서 가장 많은 발현을 보였다. 세포사멸 기전 과정 중 하나임을 확인하기 위해 ERK, c-JUN, SRC 단백질 활성을 확인한 결과 청색광을 조사한 망막에서 phosphorylated ERK는 증가하였고 phosphorylated SRC는 조사 후 1일에서만 증가를 나타내었으며, 반대로 phosphorylated c-JUN은 조사 후 1일에서만 감소하였다. 청색광을 조사한 망막에서 막대세포 발색단인 로돕신과 원뿔세포의 발색단인 옵신이 감소하였으며, 옵신의 감소량은 로돕신의 감소량보다 큰 것을 확인하였다. 결론: 본 연구는 청색광이 망막에 손상자극을 주고, ERK와 SRC 신호전달과 관련하여 광수용세포의 세포사멸을 일으킬 수 있으며 청색광이 광수용세포 중 원뿔세포의 세포사멸을 직접적으로 유도하여 망막 손상을 야기할 수 있다는 가능성을 제시하였다. Purpose: The study was conducted to determine that photoreceptors of mouse having pigment in RPE(retinal pigment epithelium) can be damaged by blue-light and apoptosis of specific cells among photoreceptors are induced by blue-light, and to assist the investigation of AMD(Age-related macular degeneration) mechanisms and development of AMD drugs. Methods: C57Black mice were injured by irradiating <TEX>$2800{\pm}10lux$</TEX> of 463 nm LED for 6 hours after 24 hours dark adaptation and eyes were enucleated 1, 3, 7 days. Damage of retina induced by blue-light was determined by western blotting GFAP(Glial fibrillary acidic protein) expression. In the light-injured retina, cell death of photoreceptors was determined by TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. ERK(Extracellular signal-regulated kinases), JNK, and SRC(sarcoma) expression were assessed by western blotting to determine regulated pathway. Blue light-injured retina were immunostained with antibodies against Opsin and Rhodopsin as markers of photoreceptors to compared the damage cone cells with rod cells. Results: After 1, 3 and 7 days from exposure to blue-light, thickness of retina was more decreased than control, and more decreased at nuclear layer than at outer plexiform layer and GFAP expression was increased day 1 after blue-light injured. While phosphorylated ERK and SRC protein expressions at day 1 were increased after blue-light injured, phosphorylated c-JUN was decreased. Fluorescence intensity analysis showed that markers of cone and rod cells were decreased after blue-light injured and Opsin was more decreased than Rhodopsin. Conclusions: The study suggests possibilities that the blue-light promotes retinal damage and causes apoptotic cell death via ERK and SRC pathway in mouse retina, and blue-light retinal damage is more induced cone cells apoptosis than rod cells directly.

Abstract IA13: Mechanisms of chromatin regulation by the EWS-FLI1 fusion protein in Ewing sarcoma

Abstract Our laboratory uses genomic approaches to identify and characterize abnormalities in gene regulation in cancer. Large scale sequencing studies have shown that transcriptional regulators, including transcription factors, chromatin modifiers and histones, are frequently altered in cancer and may thus contribute to transformation through widespread changes in gene expression programs. This is particularly evident in pediatric tumors, which have low numbers of recurrent genetic events and where alterations in a transcriptional regulator may be the sole genetic “driver”. In the case of Ewing sarcoma, the second most common pediatric bone cancer, translocations involving EWS and the ETS transcription factor FLI1 generate a fusion protein that has been shown to be essential for the growth and survival of tumor cells. We recently used genome-wide chromatin profiling to study the mechanisms of action of EWS-FLI1 and found that it utilizes two distinct patterns of chromatin remodeling to either activate or repress enhancers. At repeats of the GGAA ETS recognition motif, which are poorly conserved in evolution, EWS-FLI1 multimers induce chromatin opening and create active enhancers de novo. These repeat enhancers can loop to physically interact with target promoters, as demonstrated by chromosome conformation capture assays. In contrast, at conserved enhancers containing canonical ETS motifs, EWS-FLI1 displaces wild-type ETS transcription factors and leads to significant reductions in enhancer activity. These different chromatin remodeling mechanisms are associated with the repression of tumor suppressors and mesenchymal lineage genes and the activation of oncogenes and potential therapeutic targets. Our studies thus illustrate how chromatin profiling can reveal mechanisms of chromatin regulation that are critical in establishing an oncogenic regulatory program in cancer. Citation Format: Miguel N. Rivera. Mechanisms of chromatin regulation by the EWS-FLI1 fusion protein in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA13.

Molecular Pathological Classification of Neurodegenerative Diseases: Turning towards Precision Medicine.

Neurodegenerative diseases (NDDs) are characterized by selective dysfunction and loss of neurons associated with pathologically altered proteins that deposit in the human brain but also in peripheral organs. These proteins and their biochemical modifications can be potentially targeted for therapy or used as biomarkers. Despite a plethora of modifications demonstrated for different neurodegeneration-related proteins, such as amyloid-β, prion protein, tau, α-synuclein, TAR DNA-binding protein 43 (TDP-43), or fused in sarcoma protein (FUS), molecular classification of NDDs relies on detailed morphological evaluation of protein deposits, their distribution in the brain, and their correlation to clinical symptoms together with specific genetic alterations. A further facet of the neuropathology-based classification is the fact that many protein deposits show a hierarchical involvement of brain regions. This has been shown for Alzheimer and Parkinson disease and some forms of tauopathies and TDP-43 proteinopathies. The present paper aims to summarize current molecular classification of NDDs, focusing on the most relevant biochemical and morphological aspects. Since the combination of proteinopathies is frequent, definition of novel clusters of patients with NDDs needs to be considered in the era of precision medicine. Optimally, neuropathological categorizing of NDDs should be translated into in vivo detectable biomarkers to support better prediction of prognosis and stratification of patients for therapy trials.

Endosialin and Associated Protein Expression in Soft Tissue Sarcomas: A Potential Target for Anti-Endosialin Therapeutic Strategies.

Endosialin (CD248, TEM-1) is expressed in pericytes, tumor vasculature, tumor fibroblasts, and some tumor cells, including sarcomas, with limited normal tissue expression, and appears to play a key role in tumor-stromal interactions, including angiogenesis. Monoclonal antibodies targeting endosialin have entered clinical trials, including soft tissue sarcomas. We evaluated a cohort of 94 soft tissue sarcoma samples to assess the correlation between gene expression and protein expression by immunohistochemistry for endosialin and PDGFR-β, a reported interacting protein, across available diagnoses. Correlations between the expression of endosialin and 13 other genes of interest were also examined. Within cohorts of soft tissue diagnoses assembled by tissue type (liposarcoma, leiomyosarcoma, undifferentiated sarcoma, and other), endosialin expression was significantly correlated with a better outcome. Endosialin expression was highest in liposarcomas and lowest in leiomyosarcomas. A robust correlation between protein and gene expression data for both endosialin and PDGFR-β was observed. Endosialin expression positively correlated with PDGFR-β and heparin sulphate proteoglycan 2 and negatively correlated with carbonic anhydrase IX. Endosialin likely interacts with a network of extracellular and hypoxia activated proteins in sarcomas and other tumor types. Since expression does vary across histologic groups, endosialin may represent a selective target in soft tissue sarcomas.

Etiologies and risk factors for dementia

Dementia is understood as a clinical syndrome characterized by impairment in memory impairment along with cognitive deficits in other domains. Over the years, understanding about the causes of dementias has improved. Broadly, dementias can be classified as irreversible degenerative dementias and reversible dementias. Alzheimer's disease is the prototype of degenerative dementias and is characterized by the accumulation of beta-amyloid protein (called as amyloid plaques) outside the neurons and accumulation of tau protein (called tau tangles) inside the neurons. Vascular dementias are characterized by cerebrovascular insults which lead to pathological brain changes that impair cognition. The pathological hallmark of Lewy body dementia is the presence of α-synuclein neuronal inclusions, also known as Lewy bodies, accompanied by neuronal loss. Frontotemporal dementias are characterized by abnormal deposits of the microtubule-associated protein tau, the trans-activation response TAR DNA-binding protein with molecular weight 43 kDa (TDP-43), and the fused in sarcoma protein. Reversible dementias are characterized by the primary illness and may not present with characteristic brain deposits as seen with many degenerative dementias.

Pur-alpha functionally interacts with FUS carrying ALS-associated mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to motor neuron loss. Fused in sarcoma (FUS) protein carrying ALS-associated mutations localizes to stress granules and causes their coalescence into larger aggregates. Here we show that Pur-alpha physically interacts with mutated FUS in an RNA-dependent manner. Pur-alpha colocalizes with FUS carrying mutations in stress granules of motoneuronal cells differentiated from induced pluripotent stem cells and that are derived from ALS patients. We observe that both Pur-alpha and mutated FUS upregulate phosphorylation of the translation initiation factor eukaryotic translation initiation factor 2 alpha and consistently inhibit global protein synthesis. In vivo expression of Pur-alpha in different Drosophila tissues significatively exacerbates the neurodegeneration caused by mutated FUS. Conversely, the downregulation of Pur-alpha in neurons expressing mutated FUS significatively improves fly climbing activity. All these findings suggest that Pur-alpha, through the control of mRNA translation, might be involved in the pathogenesis of ALS associated with the mutation of FUS, and that an alteration of protein synthesis may be directly implicated in the disease. Finally, in vivo RNAi-mediated ablation of Pur-alpha produced locomotion defects in Drosophila, indicating a pivotal role for this protein in the motoneuronal function.

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

The fused in sarcoma protein forms cytoplasmic aggregates in motor neurons derived from integration-free induced pluripotent stem cells generated from a patient with familial amyotrophic lateral sclerosis carrying the FUS-P525L mutation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons (MNs) and has no effective treatment. Mutations in the fused in sarcoma (FUS) gene and abnormal aggregation of FUS protein have been reported in ALS. However, the mechanisms involved in ALS are poorly understood. Clinical drug trails have failed due to a lack of appropriate disease models, including a lack of access to MNs from ALS patients. Induced pluripotent stem (iPS) cells derived from patients with ALS provide an indispensable resource for in vitro mechanistic studies and for future patient-specific cell-based therapies. Previous reports demonstrated that viral-based ALS-iPS cells generated from fibroblasts harvested from Caucasian populations are ideal for basic research; however, ALS-iPS cells are precluded from cell-based therapeutic applications because of the risks associated with the integration of viral sequences into the genome and inconvenience associated with dermal biopsies. To establish a model for use in clinical applications, using episomal vectors, we generated an integration-free iPS cell line from peripheral blood mononuclear cells (PBMCs) harvested from a familial ALS (FALS) patient carrying the FUS-P525L mutation and a healthy control. Furthermore, we successfully differentiated ALS patient-specific iPS cells into MNs and subsequently detected cytoplasmic mislocalization and formation of FUS protein aggregates in MNs due to the FUS-P525L mutation. Our findings offer a cell-based disease model for use in further elucidating ALS pathogenesis and provide a tool for exploring gene repair coupled with cell replacement therapy.