Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Mutations in RNA binding proteins (RBPs) have been linked to the motor neuron disease amyotrophic lateral sclerosis (ALS). Extensive auto-regulation, cross-regulation, cooperation and competition mechanisms among RBPs are in place to ensure proper expression levels of common targets, often including other RBPs and their own transcripts. Moreover, several RBPs play a crucial role in the nervous system by localizing target RNAs in specific neuronal compartments. These include the RBPs FUS, FMRP, and HuD. ALS mutations in a given RBP are predicted to produce a broad impact on such delicate equilibrium. Here we studied the effects of the severe FUS-P525L mutation on common FUS and FMRP targets. Expression profiling by digital color-coded molecular barcoding in cell bodies and neurites of human iPSC-derived motor neurons revealed altered levels of transcripts involved in the cytoskeleton, neural projection and synapses. One of the common targets is HuD, which is upregulated because of the loss of FMRP binding to its 3'UTR due to mutant FUS competition. Notably, many genes are commonly altered upon FUS mutation or HuD overexpression, suggesting that a substantial part of the effects of mutant FUS on the motor neuron transcriptome could be due to HuD gain-of-function. Among altered transcripts, we also identified other common FUS and FMRP targets, namely MAP1B, PTEN, and AP2B1, that are upregulated upon loss of FMRP binding on their 3'UTR in FUS-P525L motor neurons. This work demonstrates that the impairment of FMRP function by mutant FUS might alter the expression of several genes, including new possible biomarkers and therapeutic targets for ALS

EZH2, JMJD3 and UTX epigenetically regulate hepatic plasticity inducing retro-differentiation and proliferation of liver cells

Modification of histones by lysine methylation plays a role in many biological processes, and it is dynamically regulated by several histone methyltransferases and demethylases. The polycomb repressive complex contains the H3K27 methyltransferase EZH2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3), which trigger gene suppression. JMJD3 and UTX have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3, which in turns lead to gene transcriptional activation. EZH2, JMJD3 and UTX have been extensively studied for their involvement in development, immune system, neurodegenerative disease, and cancer. However, their role in molecular mechanisms underlying the differentiation process of hepatic cells is yet to be elucidated. Here, we show that EZH2 methyltransferase and JMJD3/UTX demethylases were deregulated during hepatic differentiation of human HepaRG cells resulting in a strong reduction of H3K27 methylation levels. Inhibition of JMJD3 and UTX H3K27 demethylase activity by GSK-J4 epi-drug reverted phenotype of HepaRG DMSO-differentiated cells and human primary hepatocytes, drastically decreasing expression of hepatic markers and inducing cell proliferation. In parallel, inhibition of EZH2 H3K27me3 activity by GSK-126 epi-drug induced upregulation of hepatic markers and downregulated the expression of cell cycle inhibitor genes. To conclude, we demonstrated that modulation of H3K27 methylation by inhibiting methyl-transferase and dimethyl-transferase activity influences the differentiation status of hepatic cells, identifying a possible new role of EZH2, JMJD3 and UTX epi-drugs to modulate hepatic cell plasticity

3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches

Microglia control glutamatergic synapses in the adult mouse hippocampus

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1−/− mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses

Identification of Molecular Signatures in Neural Differentiation and Neurological Diseases Using Digital Color-Coded Molecular Barcoding

Human pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, represent powerful tools for disease modeling and for therapeutic applications. PSCs are particularly useful for the study of development and diseases of the nervous system. However, generating in vitro models that recapitulate the architecture and the full variety of subtypes of cells that make the complexity of our brain remains a challenge. In order to fully exploit the potential of PSCs, advanced methods that facilitate the identification of molecular signatures in neural differentiation and neurological diseases are highly demanded. Here, we review the literature on the development and application of digital color-coded molecular barcoding as a potential tool for standardizing PSC research and applications in neuroscience. We will also describe relevant examples of the use of this technique for the characterization of the heterogeneous composition of the brain tumor glioblastoma multiforme

Sir2 is involved in the transcriptional modulation of NHP6A in Saccharomyces cerevisiae

The Sir proteins, namely Sir2, 3 and 4, have roles related to heterochromatin, but genome-wide studies have revealed their presence at many euchromatic loci, although the functional meaning of this is still not clear. Nhp6a is an abundant HMG-like protein in yeast, which has a role in transcription by modulating chromatin structure and nucleosome number. Although much is known about its structure and function, information regarding its regulation is scarce. NHP6A, among other genes, emerges in ChIP-on chip studies of global Sir proteins binding, suggesting it could be regulated by SIR. We have investigated NHP6A expression in sir deletion mutants as well as in SIR2 overexpressing conditions. In addition, we have asked if the Sir2 deacetylation activity is involved by using conditions that either inhibit (treatment with nicotinamide) or enhance (calorie restriction conditions) Sir2 activity. We have found that, consistent with previous microarray studies, NHP6A expression undergoes a slight increase in sir mutant strains, but is strongly repressed when SIR2 is overexpressed. In a sir3 mutant strain the gene continues to be transcribed, even in SIR2 overexpressing conditions. In addition, treating the cells with nicotinamide counteracts the SIR2 overexpressing effect. Finally, conditions that are known to potentiate Sir2 deacetylation activity seem to mimic the effect of SIR2 overexpression on NHP6A. Our results suggest that Sir2 is involved in the regulation of NHP6A promoter, acting more as a specific repressor, rather than a long-range silencer. This effect is specific, and the Sir2 deacetylase activity is required for the Sir2 mediated repression of NHP6A. Moreover, the presence of the SIR complex seems required for Sir2 to silence NHP6A

Congenital anomalies of the tubular gastrointestinal tract

Congenital anomalies of the tubular gastrointestinal tract are an important cause of morbidity not only in infants, but also in children and adults. The gastrointestinal (GI) tract, composed of all three primitive germ layers, develops early during embryogenesis. Two major steps in its development are the formation of the gut tube (giving rise to the foregut, the midgut and the hindgut), and the formation of individual organs with specialized cell types. Formation of an intact and functioning GI tract is under strict control from various molecular pathways. Disruption of any of these crucial mechanisms involved in the cell-fate decision along the dorsoventral, anteroposterior, left-right and radial axes, can lead to numerous congenital anomalies, most of which occur and present in infancy. However, they may run undetected during childhood. Therapy is surgical, which in some cases must be performed urgently, and prognosis depends on early diagnosis and suitable treatment. A precise pathologic macroscopic or microscopic diagnosis is important, not only for the immediate treatment and management of affected individuals, but also for future counsel-ling of the affected individual and their family. This is even more true in cases of multiple anomalies or syndromic patterns. We discuss some of the more frequent or clinically important congenital anomalies of the tubular GI, including atresia's, duplications, intestinal malrotation, Meckel's diverticulum and Hirschsprung's Disease

Anthracycline-free neoadjuvant chemotherapy ensures higher rates of pathologic complete response in breast cancer

Neoadjuvant chemotherapy (NCT) is a standard of care for locally advanced and initially inoperable breast cancer. NCT can test chemotherapy efficacy and can be followed by breast-conserving surgery. Considering taxanes as one of the most effective agents, we analyzed the efficacy of a neoadjuvant schedule without anthracyclines and based only on taxanes and carboplatin, trying to avoid cardiotoxicity, which is the most serious side effect correlated with anthracyclines