Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.

Barley strigolactone signalling mutant hvd14.d reveals the role of strigolactones in abscisic acid-dependent response to drought.

Strigolactones (SLs) are a group of plant hormones involved in many aspects of plant development and stress adaptation. Here, we investigated the drought response of a barley (Hordeum vulgare L.) mutant carrying a missense mutation in the gene encoding the SL-specific receptor HvD14. Our results clearly showed that hvd14.d mutant is hyper-sensitive to drought stress. This was illustrated by a lower leaf relative water content (RWC), impaired photosynthesis, disorganization of chloroplast structure, altered stomatal density and slower closure of stomata in response to drought in the mutant compared to the wild type parent cultivar Sebastian. Although the content of abscisic acid (ABA) and its derivatives remained unchanged in the mutant, significant differences in expression of genes related to ABA biosynthesis were observed. Moreover, hvd14.d was insensitive to ABA during seed germination. Analysis of Arabidopsis thaliana mutant atd14-1 also demonstrated that mutation in the SL receptor resulted in increased sensitivity to drought. Our results indicate that the drought-sensitive phenotype of barley SL mutant might be caused by a disturbed ABA metabolism and/or signalling pathways. These results together uncovered a link between SL signalling and ABA-dependent drought stress response in barley.

Genetic and Physiological Dissection of Photosynthesis in Barley Exposed to Drought Stress.

Balanced photosynthesis under drought is essential for better survival and for agricultural benefits in terms of biomass and yield. Given the current attempts to improve the photosynthetic efficiency for greater crop yield, the explanation of the genetic basis of that process, together with the phenotypic analysis, is significant in terms of both basic studies and potential agricultural application. Therefore, the main objective of this study was to uncover the molecular basis of the photosynthesis process under drought stress in barley. To address that goal, we conducted transcriptomic examination together with detailed photosynthesis analysis using the JIP-test. Using this approach, we indicated that photosynthesis is a process that is very early affected in barley seedlings treated with severe drought stress. Rather than focusing on individual genes, our strategy was pointed to the identification of groups of genes with similar expression patterns. As such, we identified and annotated almost 150 barley genes as crucial core-components of photosystems, electron transport components, and Calvin cycle enzymes. Moreover, we designated 17 possible regulatory interactions between photosynthesis-related genes and transcription factors in barley. Summarizing, our results provide a list of candidate genes for future genetic research and improvement of barley drought tolerance by targeting photosynthesis.

Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a mesenchymal neoplasm displaying variable morphologic and clinical features. To identify pathogenetically important genetic rearrangements, 44 SFTs were analyzed using a variety of techniques. Chromosome banding and fluorescence in situ hybridization (FISH) showed recurrent breakpoints in 12q13, clustering near the NAB2 and STAT6 genes, and single nucleotide polymorphism array analysis disclosed frequent deletions affecting STAT6. Quantitative real-time PCR revealed high expression levels of the 5'-end of NAB2 and the 3'-end of STAT6, which at deep sequencing of enriched DNA corresponded to NAB2/STAT6 fusions. Subsequent reverse-transcriptase PCR (RT-PCR) analysis identified a NAB2/STAT6 fusion in 37/41 cases, confirming that this fusion gene underlies the pathogenesis of SFT. The hypothesis that the NAB2/STAT6 fusions will result in altered properties of the transcriptional co-repressor NAB2--a key regulator of the early growth response 1 (EGR1) transcription factor - was corroborated by global gene expression analysis; SFTs showed deregulated expression of EGR1 target genes, as well as of other, developmentally important genes. We also identified several nonrandom secondary changes, notably loss of material from 13q and 14q. As neither chromosome banding nor FISH analysis identify more than a minor fraction of the fusion-positive cases, and because multiple primer combinations are required to identify all possible fusion transcripts by RT-PCR, alternative diagnostic markers might instead be found among deregulated genes identified at global gene expression analysis. Indeed, using immunohistochemistry on tissue microarrays, the top up-regulated gene, GRIA2, was found to be differentially expressed also at the protein level.

Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders.

The European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or break-apart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.

Updates on the Role of ABSCISIC ACID INSENSITIVE 5 (ABI5) and ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTORs (ABFs) in ABA Signaling in Different Developmental Stages in Plants.

The core abscisic acid (ABA) signaling pathway consists of receptors, phosphatases, kinases and transcription factors, among them ABA INSENSITIVE 5 (ABI5) and ABRE BINDING FACTORs/ABRE-BINDING PROTEINs (ABFs/AREBs), which belong to the BASIC LEUCINE ZIPPER (bZIP) family and control expression of stress-responsive genes. ABI5 is mostly active in seeds and prevents germination and post-germinative growth under unfavorable conditions. The activity of ABI5 is controlled at transcriptional and protein levels, depending on numerous regulators, including components of other phytohormonal pathways. ABFs/AREBs act redundantly in regulating genes that control physiological processes in response to stress during vegetative growth. In this review, we focus on recent reports regarding ABI5 and ABFs/AREBs functions during abiotic stress responses, which seem to be partially overlapping and not restricted to one developmental stage in Arabidopsis and other species. Moreover, we point out that ABI5 and ABFs/AREBs play a crucial role in the core ABA pathway's feedback regulation. In this review, we also discuss increased stress tolerance of transgenic plants overexpressing genes encoding ABA-dependent bZIPs. Taken together, we show that ABI5 and ABFs/AREBs are crucial ABA-dependent transcription factors regulating processes essential for plant adaptation to stress at different developmental stages.

TNF-α and α-synuclein fibrils differently regulate human astrocyte immune reactivity and impair mitochondrial respiration.

Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.

Barley ABI5 (Abscisic Acid INSENSITIVE 5) Is Involved in Abscisic Acid-Dependent Drought Response.

ABA INSENSITIVE 5 (ABI5) is a basic leucine zipper (bZIP) transcription factor which acts in the abscisic acid (ABA) network and is activated in response to abiotic stresses. However, the precise role of barley (Hordeum vulgare) ABI5 in ABA signaling and its function under stress remains elusive. Here, we show that HvABI5 is involved in ABA-dependent regulation of barley response to drought stress. We identified barley TILLING mutants carrying different alleles in the HvABI5 gene and we studied in detail the physiological and molecular response to drought and ABA for one of them. The hvabi5.d mutant, carrying G1751A transition, was insensitive to ABA during seed germination, yet it showed the ability to store more water than its parent cv. "Sebastian" (WT) in response to drought stress. The drought-tolerant phenotype of hvabi5.d was associated with better membrane protection, higher flavonoid content, and faster stomatal closure in the mutant under stress compared to the WT. The microarray transcriptome analysis revealed up-regulation of genes associated with cell protection mechanisms in the mutant. Furthermore, HvABI5 target genes: HVA1 and HVA22 showed higher activity after drought, which may imply better adaptation of hvabi5.d to stress. On the other hand, chlorophyll content in hvabi5.d was lower than in WT, which was associated with decreased photosynthesis efficiency observed in the mutant after drought treatment. To verify that HvABI5 acts in the ABA-dependent manner we analyzed expression of selected genes related to ABA pathway in hvabi5.d and its WT parent after drought and ABA treatments. The expression of key genes involved in ABA metabolism and signaling differed in the mutant and the WT under stress. Drought-induced increase of expression of HvNCED1, HvBG8, HvSnRK2.1, and HvPP2C4 genes was 2-20 times higher in hvabi5.d compared to "Sebastian". We also observed a faster stomatal closure in hvabi5.d and much higher induction of HvNCED1 and HvSnRK2.1 genes after ABA treatment. Together, these findings demonstrate that HvABI5 plays a role in regulation of drought response in barley and suggest that HvABI5 might be engaged in the fine tuning of ABA signaling by a feedback regulation between biosynthetic and signaling events. In addition, they point to different mechanisms of HvABI5 action in regulating drought response and seed germination in barley.

Generation of an induced pluripotent stem cell line (CSC-32) from a patient with Parkinson's disease carrying a heterozygous variation p.A53T in the SNCA gene.

Here, we describe the generation of an induced pluripotent stem cell (iPSC) line, from a male patient diagnosed with Parkinson's disease (PD). The patient carries a heterozygous variation p.A53T in the SNCA gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated iPSC line (CSC-32) preserved the mutation, displayed expression of common pluripotency markers, differentiated into derivatives of the three germ layers, and exhibited a normal karyotype. The clone CSC-32B is presented thereafter; it can be used to study the mechanisms underlying PD pathogenesis.

Human iPSC-Derived Hippocampal Spheroids: An Innovative Tool for Stratifying Alzheimer Disease Patient-Specific Cellular Phenotypes and Developing Therapies.

The hippocampus is important for memory formation and is severely affected in the brain with Alzheimer disease (AD). Our understanding of early pathogenic processes occurring in hippocampi in AD is limited due to tissue unavailability. Here, we report a chemical approach to rapidly generate free-floating hippocampal spheroids (HSs), from human induced pluripotent stem cells. When used to model AD, both APP and atypical PS1 variant HSs displayed increased Aß42/Aß40 peptide ratios and decreased synaptic protein levels, which are common features of AD. However, the two variants differed in tau hyperphosphorylation, protein aggregation, and protein network alterations. NeuroD1-mediated gene therapy in HSs-derived progenitors resulted in modulation of expression of numerous genes, including those involved in synaptic transmission. Thus, HSs can be harnessed to unravel the mechanisms underlying early pathogenic changes in the hippocampi of AD patients, and provide a robust platform for the development of therapeutic strategies targeting early stage AD.

Expression levels of HMGA2 in adipocytic tumors correlate with morphologic and cytogenetic subgroups.

BACKGROUND: The HMGA2 gene encodes a protein that alters chromatin structure. Deregulation, typically through chromosomal rearrangements, of HMGA2 has an important role in the development of several mesenchymal neoplasms. These rearrangements result in the expression of a truncated protein lacking the acidic C-terminus, a fusion protein consisting of the AT-hook domains encoded by exons 1-3 and parts from another gene, or a full-length protein; loss of binding sites for regulatory microRNA molecules from the 3' untranslated region (UTR) of HMGA2 has been suggested to be a common denominator. METHODS: Seventy adipocytic tumors, representing different morphologic and cytogenetic subgroups, were analyzed by qRT-PCR to study the expression status of HMGA2; 18 of these tumors were further examined by PCR to search for mutations or deletions in the 3'UTR. RESULTS: Type (full-length or truncated) and level of expression varied with morphology and karyotype, with the highest levels in atypical lipomatous tumors and lipomas with rearrangements of 12q13-15 and the lowest in lipomas with 6p- or 13q-rearrangements, hibernomas, spindle cell lipomas and myxoid liposarcomas. All 18 examined tumors showed reduced or absent expression of the entire, or parts of, the 3'UTR, which was not due to mutations at the DNA level. CONCLUSION: In adipocytic tumors with deregulated HMGA2 expression, the 3'UTR is consistently lost, either due to physical disruption of HMGA2 or a shift to production of shorter 3'UTR.

Clinical variability of Waardenburg-Shah syndrome in patients with proximal 13q deletion syndrome including the endothelin-B receptor locus.

Waardenburg-Shah syndrome (Waardenburg syndrome type IV-WS4) is an auditory-pigmentary disorder that combines clinical features of pigmentary abnormalities of the skin, hair and irides, sensorineural hearing loss, and Hirschsprung disease (HSCR). Mutations in the endothelin-B receptor (EDNRB) gene on 13q22 have been found to cause this syndrome. Mutations in both alleles cause the full phenotype, while heterozygous mutations cause isolated HSCR or HSCR with minor pigmentary anomalies and/or sensorineural deafness. We investigated the status of the EDNRB gene, by FISH analysis, in three patients with de novo proximal 13q deletions detected at cytogenetic analysis and examined the clinical variability of WS4 among these patients. Chromosome 13q was screened with locus specific FISH probes and breakpoints were determined at 13q22.1q31.3 in Patients 1 and 3, and at 13q21.1q31.3 in Patient 2. An EDNRB specific FISH probe was deleted in all three patients. All patients had common facial features seen in proximal 13q deletion syndrome and mild mental retardation. However, findings related to WS4 were variable; Patient 1 had hypopigmentation of the irides and HSCR, Patient 2 had prominent bicolored irides and mild bilateral hearing loss, and Patient 3 had only mild unilateral hearing loss. These data contribute new insights into the pathogenesis of WS4.

Cellular alterations identified in pluripotent stem cell-derived midbrain spheroids generated from a female patient with progressive external ophthalmoplegia and parkinsonism who carries a novel variation (p.Q811R) in the POLG1 gene.

Variations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson's disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.

Generation of an induced pluripotent stem cell line (CSC-46) from a patient with Parkinson's disease carrying a novel p.R301C mutation in the GBA gene.

Mutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line was generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-based technology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation, displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further used for studying PD pathogenesis.

Array based characterization of a terminal deletion involving chromosome subband 15q26.2: an emerging syndrome associated with growth retardation, cardiac defects and developmental delay.

BACKGROUND: Subtelomeric regions are gene rich and deletions in these chromosomal segments have been demonstrated to account for approximately 2.5% of patients displaying mental retardation with or without association of dysmorphic features. However, cases that report de novo terminal deletions on chromosome arm 15q are rare. METHODS: In this study we present the first example of a detailed molecular genetic mapping of a de novo deletion in involving 15q26.2-qter, caused by the formation of a dicentric chromosome 15, using metaphase FISH and tiling resolution (32 k) genome-wide array-based comparative genomic hybridization (CGH). RESULTS: After an initial characterization of the dicentric chromosome by metaphase FISH, array CGH analysis mapped the terminal deletion to encompass a 6.48 megabase (Mb) region, ranging from 93.86-100.34 Mb on chromosome 15. CONCLUSION: In conclusion, we present an additional case to the growing family of reported cases with 15q26-deletion, thoroughly characterized at the molecular cytogenetic level. In the deleted regions, four candidate genes responsible for the phenotype of the patient could be delineated: IGFR1, MEF2A, CHSY1, and TM2D3. Further characterization of additional patients harboring similar 15q-aberrations might hopefully in the future lead to the description of a clear cut clinically recognizable syndrome.

Deletion of the SCN gene cluster on 2q24.4 is associated with severe epilepsy: an array-based genotype-phenotype correlation and a comprehensive review of previously published cases.

PURPOSE: To characterize a deletion of chromosome 2q at the molecular level in a patient suffering from severe epilepsy resembling severe myoclonic epilepsy of infancy/Dravet's syndrome (SMEI/DS) and to correlate other cases harboring deletions in the same region to morphological and clinical data. METHODS: Array-based comparative genomic hybridization (array CGH) was performed on DNA from the patient. Forty-three previously published cases reporting deletions within region 2q21-q31 were collected and analyzed regarding their cytogenetic and clinical data. RESULTS: A del(2)(q24.3q31.1) was detected in the patient, spanning a 10.4-megabase (Mb) region between 165.18 and 175.58Mb, harboring 47 genes. FISH analysis was performed, confirming this deletion. Twenty-two of the 43 previously published cases were seizure-positive. The most common dysmorphic features were ear abnormalities, microcephaly, micrognathia and brachysyndactyly for all patients as well as for solely the seizure-positive and -negative ones. For the 22 seizure-positive cases chromosome subband 2q24.3 constituted the smallest commonly deleted region among the majority of the cases, where subbands 2q22.1 and 2q33.3 represented the most proximal and distal breakpoint, respectively. CONCLUSIONS: Based on the early age of presentation and the severity of the epilepsy reported for the majority of the seizure-positive cases it was concluded that SMEI/DS could be the epileptic encephalopathy associated with deletions within the 2q22.1-q33.3 region, due to haploinsuffiency of SCN1A and/or complete or partial deletion of other voltage-gated sodium channel genes caused by the aberration. Furthermore, our study supports that array CGH is a competent technique for screening SCN1A mutation-negative patients diagnosed with SMEI/DS-like epilepsies and dysmorphic features, generating rapid and high-resolution data of genomic imbalances present in the patients.

The POU5F1P1 pseudogene encodes a putative protein similar to POU5F1 isoform 1.

POU5F1, which encodes a transcriptional factor, has two alternatively spliced transcripts, 1 and 2, as well as six pseudogenes. Transcript 1 is considered to be a key regulator of cellular pluripotency and self-renewal. The POU5F1 pseudogene, POU5F1P1 on 8q24, encodes a protein with 95% homology with the isoform 1 of POU5F1. It is located 15 kbp downstream of the SNP rs6983267, which is strongly associated with an increased risk of prostate and colon cancer, and within the amplified region in a variety of human malignancies. The previous finding of expressed sequence tags suggests that POU5F1P1 can be expressed. We showed that a putative POU5F1P1 protein is localized in the nucleus, acts as a transcriptional activator and regulates the expression in a similar way to the POU5F1 isoform 1. However, POU5F1P1 was a weaker activator than isoform 1 of POU5F1, possibly due to the amino acid substitutions.

Generation of an induced pluripotent stem cell line (CSC-44) from a Parkinson's disease patient carrying a compound heterozygous mutation (c.823C>T and EX6 del) in the PARK2 gene.

Mutations in the PARK2 gene, which encodes PARKIN, are the most frequent cause of autosomal recessive Parkinson's disease (PD). We report the generation of an induced pluripotent stem cell (iPSC) line from a 78-year-old patient carrying a compound heterozygous mutation (c.823C>T and EX6del) in the PARK2 gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line CSC-44 exhibits expression of common pluripotency markers, in vitro differentiation into the three germ layers and normal karyotype. This iPSC line can be used to explore the association between PARK2 mutations and PD.

Generation of a human induced pluripotent stem cell line (CSC-42) from a patient with sporadic form of Parkinson's disease.

Skin fibroblasts were collected from a 44-year-old patient with sporadic case of Parkinson's disease (PD). The non-integrating Sendai virus vector encoding OCT3/4, SOX2, c-MYC and KLF4 was used to reprogram fibroblasts into induced pluripotent stem cells (iPSCs). Generated iPSCs had normal karyotypes, expressed common stem cell markers, and were capable of differentiating into all three germ layers. Generated line could be used for PD modeling to understand the mechanisms that influence the disorder.

Generation of an integration-free induced pluripotent stem cell line (CSC-43) from a patient with sporadic Parkinson's disease.

An induced pluripotent stem cell (iPSC) line was generated from a 36-year-old patient with sporadic Parkinson's disease (PD). Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line (CSC-43) exhibits expression of common pluripotency markers, in vitro differentiation into three germ layers and normal karyotype. This iPSC line can be used to study the mechanisms underlying the development of PD.