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Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in ‘regulation of RNA splicing’ and ‘mRNA catabolic process’. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouseAS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)—containing the invSINEB2 element—able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.

Alternative splicing (AS) appears to be altered in Huntington’s disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected.Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a strong correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, expanded Htt CAG repeats reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors.Though the mechanisms of this dysregulation remain uncertain, our study unveils network of transcriptionally altered micro-RNAs and RNA-binding proteins (CELF, hnRNPS, PTBP, SRSF) which, in turn, might influence the AS machinery, primarily in neural cells.We suggest that this unbalanced expression of linear and circular RNAs might result in altered neural fitness, contributing to HD striatal vulnerability.

Background With the development of new therapies entering clinical trials for HD, there is an increasing need to develop and validate biomarkers in accessible biofluids, such as blood, to follow disease progression and predict treatment outcomes. Some biomarkers do exist; however, reliable and readily available additional ones will be crucial in assessing the pathogenic process and pharmacologic responses to therapeutic interventions. Aims For the first time in HD, we detect and characterize circRNAs in peripheral blood of a cohort of gender and age-matched controls and HD patients at various stages of the disease. We studied their potential as biomarkers by evaluating their correlation with disease progression and the size of the CAG repeat. Methods 50 blood samples were collected from both HD patients and a healthy cohort of individuals. Total RNA was used for RNA sequencing, and the obtained data were aligned with STAR. DCC program was used to quantify circRNAs and CircTest (R package) to identify differentially expressed circRNA between different disease stages and controls. CAG repeat size correlation was also tested. To validate our findings, candidate circRNAs were quantified via RT-qPCR in our samples. Results Our study led to the discovery of 7, stringently defined circRNAs that possess 3 characteristics: differentially expressed in HD patients (all up-regulated, p-value 0.3 and p-value Conclusions We identified for the first time circRNAs whose expression increases in the blood of HD patients. Currently, we aim to validate these circular molecules in independent blood cohorts as well as in cerebrospinal fluid and plasma samples to fully elucidate whether these highly stable RNA molecules could be used as disease biomarkers.

Background Circular RNAs (circRNAs) are a special group of non-coding RNAs formed by back-splicing. Mostly cytosolic in eukaryotic cells, circRNAs are particularly enriched and conserved in neurons and originate from protein-coding genes to function as global regulators of gene expression. Recent studies showed that circRNAs partake in brain physiology and pathology, contributing to neurological disorders, such as myotonic dystrophy type 1, Parkinson’s and Alzheimer’s Diseases. Here, we identified the first ever known brain enriched RNA circle originating from the Huntington’s Disease gene HTT (CircHTT: 484 nt, Ex 2-6, chr4:3088665-3109150) which is conserved in mouse and minipig. Aims and Methods To functionally characterize circHTT and its implication for HD pathogenesis, we first studied its expression pattern in human and mouse body districts and in iPS-derived neuronal cell lines with different CAG repeats. Then, overexpression and down-regulation of the circle were used to determine the effects on transcription of the HD gene and translation of wild-type and mutant protein. Results CircHTT expression increases significantly with increasing CAG repeats in terminally differentiated cortical neurons. Furthermore, circHtt is significantly more expressed in brain districts of Q111 and zQ175 knock-in mice. These findings indicate that the expression of circHTT/Htt occurs in a CAG repeat dependent manner in neuronal cells, typical hallmark of HD pathology. Strikingly, overexpression of the circular RNA in human HEK293T, PC3, mouse STHdh Q7/7, Q7/111 and Q111/111 cell lines consistently increase wild-type huntingtin, while decreasing mutant huntingtin protein, with no alteration of the HTT/Htt transcript level. Conclusions In conclusion, we identified a brain enriched RNA circle originating from the HD gene locus that is sensitive to the HD mutation and may modulate huntingtin expression. Our observations might pave the way to new trials of therapeutic intervention.

Huntington’s disease (HD) is a devastating neurodegenerative disorder caused by an aberrant expansion of the CAG tract within the exon 1 of the HD gene, HTT. HD progressively impairs motor and cognitive capabilities, leading to a total loss of autonomy and ultimate death. Currently, no cure or effective treatment is available to halt the disease. Although the HTT gene is ubiquitously expressed, the striatum appears to be the most susceptible district to the HD mutation with Medium-sized Spiny Neurons (MSNs) (D1R and D2R) representing 95% of the striatal neuronal population. Why are striatal MSNs so vulnerable to the HD mutation? Particularly, why do D1R- and D2R-MSNs display different susceptibility to HD? Here, we highlight significant differences between D1R- and D2R-MSNs subpopulations, such as morphology, electrophysiology, transcriptomic, functionality, and localization in the striatum. We discuss possible reasons for their selective degeneration in the context of HD. Our review suggests that a better understanding of cell type-specific gene expression dysregulation within the striatum might reveal new paths to therapeutic intervention or prevention to ameliorate HD patients’ life expectancy.

Disruptive mutations in the chromodomain helicase DNA binding protein 8 (CHD8) have been recurrently associated with Autism Spectrum Disorders (ASD). In normal cellular physiology, CHD8 co-purifies with MLL1 and MOF transcriptional activation complex, with elongating RNAPII and directly binds to DNA promoters and enhancers regions, thus a regulatory role in transcriptional initiation and elongation could be postulated.Here we investigated how chromatin landscape reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. We interrogated transcriptionally active and repressed regions, as well as active and poised enhancers.CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes. Histone H3K36me3 reduction associated to CHD8-suppression does not functionally impact on global transcriptional levels, but correlated with altered alternative splicing patterns of ∼ 2000 protein coding genes implicated in “RNA splicing”, “mitotic cell cycle phase transition” and “mRNA processing”, especially affecting alternative first exon and exon skipping events.In summary, our results point toward broad molecular consequences of CHD8 suppression, implicating altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

Background Huntington’s Disease (HD), is a hereditary, fatal neurodegenerative disorder due to CAG trinucleotide expansion in exon 1 of the HD gene (HTT). Recent findings revealed that the process of alternative splicing (AS) might be compromised in HD. AS regulation is crucial not only to the establishment of a repertoire of protein coding isoforms extremely relevant for the proper physiological characteristics of the nervous system, but also to the biogenesis of circular RNAs (circRNAs), unusually stable, highly expressed non-coding RNAs produced by the circularization of exons which, seems to have important roles during neuronal development and functioning. Aims Therefore, the overarching goal of our research was to take advantage of genetically engineered Htt knock-in (KI) mouse cellular and in vivo model systems bearing normal or pathological Htt CAG repeat lengths to discover alterations in linear and back-splicing events that might reflect on the levels of expression as well as the composition of a repertoire of proteins, thus contributing to HD striatal vulnerability and pathogenesis. Methods/techniques To discover alterations in linear splicing, we resourced to a publicly available dataset (Langfelder P. et al., 2016, Nature Neuroscience) analyzing full transcriptomic profiling dataset for striatum, cortex and liver at different developmental time points, identifying and quantifying the total numbers of differential AS events for each genotype/sample type and time point. Results/outcome Our analysis demonstrated that specifically for the striatum – most vulnerable to HD degeneration – the total number of detected, differential AS events increased significantly at 6 and 10 month with increasing CAG expansion. Interestingly, not all the AS events increased with CAG-expansion and age, but only a specific subtype of AS event – EXON SKIPPING – revealed to be strongly and specifically affected. On the other hand, we identified and characterized the first circular RNA originating locally from the HTT locus, expressed in whole body, but predominantly in the brain and spinal cord and presenting augmented expression level with increasing HTT CAG size. Moreover, at genome-wide level, data analysis of circRNA-seq showed a decreased circRNAs production when mutant huntingtin is expressed. Specifically, 12 circRNAs, identified by stringent cut-off criteria, showed continuous decreased expression following CAG expansion in neuronal progenitor cells. Since, most of the CAG-sensitive circRNAs have annotated human orthologues, their expression can be further characterized and functionally studied in human cell lines. Conclusions In conclusion, our results support the idea that AS machinery is responding to HD mutational process altering both linear and back-splicing events locally at the HTT locus, but also at genome-wide level. This knowledge will pave the way to new trials of therapeutic intervention aimed to possibly target spliceosomal-circRNAs alterations through specific drugs or genetic manipulation.

Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense-antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

Huntington's disease (HD) is a genetic, fatal autosomal dominant neurodegenerative disorder typically occurring in midlife with symptoms ranging from chorea, to dementia, to personality disturbances (Philos Trans R Soc Lond Ser B Biol Sci 354:957-961, 1999). HD is inherited in a dominant fashion, and the underlying mutation in all cases is a CAG trinucleotide repeat expansion within exon 1 of the HD gene (Cell 72:971-983, 1993). The expanded CAG repeat, translated into a lengthened glutamine tract at the amino terminus of the huntingtin protein, affects its structural properties and functional activities. The effects are pleiotropic, as huntingtin is broadly expressed in different cellular compartments (i.e., cytosol, nucleus, mitochondria) as well as in all cell types of the body at all developmental stages, such that HD pathogenesis likely starts at conception and is a lifelong process (Front Neurosci 9:509, 2015). The rate-limiting mechanism(s) of neurodegeneration in HD still remains elusive: many different processes are commonly disrupted in HD cell lines and animal models, as well as in HD patient cells (Eur J Neurosci 27:2803-2820, 2008); however, epigenetic-chromatin deregulation, as determined by the analysis of DNA methylation, histone modifications, and noncoding RNAs, has now become a prevailing feature. Thus, the overarching goal of this chapter is to discuss the current status of the literature, reviewing how an aberrant epigenetic landscape can contribute to altered gene expression and neuronal dysfunction in HD.

Huntington's disease (HD) is a genetic, fatal autosomal dominant neurodegenerative disorder typically occurring in midlife with symptoms ranging from chorea, to dementia, to personality disturbances (Philos Trans R Soc Lond Ser B Biol Sci 354:957-961, 1999). HD is inherited in a dominant fashion, and the underlying mutation in all cases is a CAG trinucleotide repeat expansion within exon 1 of the HD gene (Cell 72:971-983, 1993). The expanded CAG repeat, translated into a lengthened glutamine tract at the amino terminus of the huntingtin protein, affects its structural properties and functional activities. The effects are pleiotropic, as huntingtin is broadly expressed in different cellular compartments (i.e., cytosol, nucleus, mitochondria) as well as in all cell types of the body at all developmental stages, such that HD pathogenesis likely starts at conception and is a lifelong process (Front Neurosci 9:509, 2015). The rate-limiting mechanism(s) of neurodegeneration in HD still remains elusive: many different processes are commonly disrupted in HD cell lines and animal models, as well as in HD patient cells (Eur J Neurosci 27:2803-2820, 2008); however, epigenetic-chromatin deregulation, as determined by the analysis of DNA methylation, histone modifications, and noncoding RNAs, has now become a prevailing feature. Thus, the overarching goal of this chapter is to discuss the current status of the literature, reviewing how an aberrant epigenetic landscape can contribute to altered gene expression and neuronal dysfunction in HD.

Huntington's disease (HD) is a monogenic autosomal dominant, fatal disorder due to CAG trinucleotide expansion in exon 1 of the HD gene (HTT) (The Huntington's Disease Collaborative Research Group, 1993; Figure ​Figure1A).1A). Nowadays, there is no cure or effective treatment for the disease which presents with motor, cognitive and psychiatric dysfunction. Although typically conceived as a “neurodegenerative disease,” mainly affecting the GABAergic medium-sized spiny neurons (MSN) of the striatum and deep layers of the cortex (Rosas et al., 2003), an increasing body of evidence has surfaced indicating that abnormal neurodevelopment might also have a crucial role in HD (Mehler and Gokhan, 2000, 2001; Humbert, 2010). Neurodegenerative diseases have been classically defined as chronic and progressive disorders of the nervous system affecting neurologic and behavioral functions, which start with specific biochemical changes that ultimately lead to distinct histopathologic and clinical syndromes. On the contrary, neurodevelopmental disorders result from an anomaly of brain maturation, during fetal or early postnatal life, which is postulated to alter the structure and/or function of neuronal and synaptic populations (Harrison, 1995).

TRAF and TNF receptor-associated protein (TTRAP) is a multifunctional protein that can act in the nucleus as a 5'-tyrosyl DNA phosphodiesterase and in the cytoplasm as a regulator of cell signaling. In this paper we show that in response to proteasome inhibition TTRAP accumulates in nucleolar cavities in a promyelocytic leukemia protein-dependent manner. In the nucleolus, TTRAP contributes to control levels of ribosomal RNA precursor and processing intermediates, and this phenotype is independent from its 5'-tyrosyl DNA phosphodiesterase activity. Our findings suggest a previously unidentified function for TTRAP and nucleolar cavities in ribosome biogenesis under stress.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover from the toxin attack. The fact that the majority of the human transcriptome encodes for non-coding RNAs (ncRNAs) raises the question: do host cells deploy non-coding transcripts to rapidly control the most energy-consuming process in cells--i.e., host translation--to counteract the infection? Here, we discuss the intriguing possibility that membrane-damaging toxins induce, in the host, the expression of toxin-specific long non-coding RNAs (lncRNAs), which act as sponges for other molecules, encoding small peptides or binding target mRNAs to depress their translation efficiency. Unravelling the function of host-produced lncRNAs upon bacterial infection or membrane damage requires an improved understanding of host lncRNA expression patterns, their association with polysomes and their function during this stress. This field of investigation holds a unique opportunity to reveal unpredicted scenarios and novel approaches to counteract antibiotic-resistant infections.

The CAG repeat expansion in the Huntington's disease gene HTT extends a polyglutamine tract in mutant huntingtin that enhances its ability to facilitate polycomb repressive complex 2 (PRC2). To gain insight into this dominant gain of function, we mapped histone modifications genome-wide across an isogenic panel of mouse embryonic stem cell (ESC) and neuronal progenitor cell (NPC) lines, comparing the effects of Htt null and different size Htt CAG mutations. We found that Htt is required in ESC for the proper deposition of histone H3K27me3 at a subset of ‘bivalent’ loci but in NPC it is needed at ‘bivalent’ loci for both the proper maintenance and the appropriate removal of this mark. In contrast, Htt CAG size, though changing histone H3K27me3, is prominently associated with altered histone H3K4me3 at ‘active’ loci. The sets of ESC and NPC genes with altered histone marks delineated by the lack of huntingtin or the presence of mutant huntingtin, though distinct, are enriched in similar pathways with apoptosis specifically highlighted for the CAG mutation. Thus, the manner by which huntingtin function facilitates PRC2 may afford mutant huntingtin with multiple opportunities to impinge upon the broader machinery that orchestrates developmentally appropriate chromatin status.

In patients with Huntington's disease (HD), the protein huntingtin (Htt) has an expanded polyglutamine (poly-Q) tract. HD results in early loss of medium spiny neurons in the striatum, which impairs motor and cognitive functions. Identifying the physiological role and molecular functions of Htt may yield insight into HD pathogenesis. We found that Htt promotes signaling by mTORC1 [mechanistic target of rapamycin (mTOR) complex 1] and that this signaling is potentiated by poly-Q-expanded Htt. Knocking out Htt in mouse embryonic stem cells or human embryonic kidney cells attenuated amino acid-induced mTORC1 activity, whereas overexpressing wild-type or poly-Q-expanded Htt in striatal neuronal cells increased basal mTOR activity. Striatal cells expressing endogenous poly-Q-expanded Htt showed an increase in the number and size of mTOR puncta on the perinuclear regions compared to cells expressing wild-type Htt. Pull-down experiments indicated that amino acids stimulated the interaction of Htt and the guanosine triphosphatase (GTPase) Rheb (a protein that stimulates mTOR activity), and that Htt forms a ternary complex with Rheb and mTOR. Pharmacologically inhibiting PI3K (phosphatidylinositol 3-kinase) or knocking down Rheb abrogated mTORC1 activity induced by expression of a poly-Q-expanded amino-terminal Htt fragment. Moreover, striatum-specific deletion of TSC1, encoding tuberous sclerosis 1, a negative regulator of mTORC1, accelerated the onset of motor coordination abnormalities and caused premature death in an HD mouse model. Together, our findings demonstrate that mutant Htt contributes to the pathogenesis of HD by enhancing mTORC1 activity.

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10−8) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10−10). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC50-value of 0.7 μM, while human A549 adenocarcinoma cells were relatively resistant with an IC50-value of 164 μM CdCl2. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl2 and was concentration-dependent between 1–100 μM CdCl2, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 μM CdCl2. Furthermore, cadmium (1 μM, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd2+, other toxic heavy metal ions (Hg2+, Pb2+, Ni2+, Fe2+, CrO4 2−, Cu2+ or Co2+) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn2+ which caused apotosis at high concentrations (>150 μM) whereas it protected against cadmium-induced apoptosis at low concentrations (10–50 μM).

We investigated the induction of apoptosis by cadmium in NIH 3T3 murine fibroblasts. Apoptosis was triggered effectively by 10 microM CdCl2 within 24 h, under which conditions cell viability was reduced by 50%. Cadmium-induced apoptosis was demonstrated by both morphological and biochemical analysis. We have shown that cadmium concentrations of 5-20 microM caused nuclear fragmentation. Moreover, internucleosomal DNA fragmentation was evoked by 10-25 microM CdCl2 within 24 h, as detected by the formation of ladder patterns in DNA electrophoresis. Since the induction of programmed cell death occurs together with modifications in the cell cycle, we examined the ability of cadmium to block cell divisions by using a 5-bromo2-deoxy-uridine incorporation assay. Our results indicate that about 40% of treated cells are blocked in G0-G1 phase when exposed to 10 microM cadmium for 27 h. Finally, we addressed the question of whether the effect of cadmium could be prevented by suppressing apoptosis. Over-expression of the anti-apoptotic protein Bcl-2 in NIH 3T3 cells protects against cadmium toxicity, thus suggesting a role for Bcl-2 in the regulation of cadmium-induced apoptosis.

Previous studies have demonstrated the presence of hemoglobin α-chain and β-chain in neurons of the rodent and human brain thus indicating that hemoglobin is a normal component of nerve cells and that hemoglobin may play a role in intraneuronal oxygen homeostasis. Progressing with these studies, hemoglobin expression has been examined in selected cell population in the brains of Alzheimer's disease (AD), argyrophilic grain disease (AGD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Double labeling immunofluorescence and confocal microscopy revealed reduced hemoglobin α-chain and β-chain in practically all neurons with small amounts of granular or punctuate hyperphosphorylated tau deposits and in neurons with tangles in the hippocampus and frontal cortex in AD and in the hippocampus in AGD; in ballooned neurons containing αB-crystallin in the amygdala in AD and AGD; and in about 80% of neurons with punctuate α-synuclein deposits and in neurons with Lewy bodies in the substantia nigra pars compacta and in vulnerable neurons of the medulla oblongata in PD and DLB; and in neurons with Lewy bodies in the frontal cortex in DLB. Hemoglobin immunoreactivity was also observed in the core of neuritic plaques and in diffuse plaques, but not in dystrophic neurites. Loss of hemoglobin was specific as neuroglobin was present equally in neurons with and without abnormal protein inclusions, and erythropoietin receptor was expressed equally in neurons without and in neurons with abnormal protein aggregates in AD, AGD, PD, and DLB.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the Substantia Nigra and the formation of ubiquitin- and alpha-synuclein (aSYN)-positive cytoplasmic inclusions called Lewy bodies (LBs). Although most PD cases are sporadic, families with genetic mutations have been found. Mutations in PARK7/DJ-1 have been associated with autosomal recessive early-onset PD, while missense mutations or duplications of aSYN (PARK1, PARK4) have been linked to dominant forms of the disease. In this study, we identify the E3 ubiquitin ligase tumor necrosis factor-receptor associated factor 6 (TRAF6) as a common player in genetic and sporadic cases. TRAF6 binds misfolded mutant DJ-1 and aSYN. Both proteins are substrates of TRAF6 ligase activity in vivo. Interestingly, rather than conventional K63 assembly, TRAF6 promotes atypical ubiquitin linkage formation to both PD targets that share K6-, K27- and K29- mediated ubiquitination. Importantly, TRAF6 stimulates the accumulation of insoluble and polyubiquitinated mutant DJ-1 into cytoplasmic aggregates. In human post-mortem brains of PD patients, TRAF6 protein colocalizes with aSYN in LBs. These results reveal a novel role for TRAF6 and for atypical ubiquitination in PD pathogenesis.

Mutations in PARK7 DJ-1 have been associated with autosomal-recessive early-onset Parkinson's disease (PD). This gene encodes for an atypical peroxiredoxin-like peroxidase that may act as a regulator of transcription and a redox-dependent chaperone. Although large gene deletions have been associated with a loss-of-function phenotype, the pathogenic mechanism of several missense mutations is less clear. By performing a yeast two-hybrid screening from a human fetal brain library, we identified TRAF and TNF receptor-associated protein (TTRAP), an ubiquitin-binding domain-containing protein, as a novel DJ-1 interactor, which was able to bind the PD-associated mutations M26I and L166P more strongly than wild type. TTRAP protected neuroblastoma cells from apoptosis induced by proteasome impairment. In these conditions, endogenous TTRAP relocalized to a detergent-insoluble fraction and formed cytoplasmic aggresome-like structures. Interestingly, both DJ-1 mutants blocked the TTRAP protective activity unmasking a c-jun N-terminal kinase (JNK)- and p38-MAPK (mitogen-activated protein kinase)-mediated apoptosis. These results suggest an active role of DJ-1 missense mutants in the control of cell death and position TTRAP as a new player in the arena of neurodegeneration.

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for α- and β-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing α- and β-globin chains, changes in genes involved in O 2 homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.

Cadmium, a toxic environmental contaminant, exerts adverse effects on different cellular pathways such as cell proliferation, DNA damage and apoptosis. In particular, the modulation of Ca(2+) homeostasis seems to have an important role during Cd(2+) injury, but the precise assessment of Ca(2+) signalling still remains poorly understood. We used aequorin-based probes specifically directed to intracellular organelles to study Ca(2+) changes during cadmium injury. We observed that cadmium decreased agonist-evoked endoplasmic reticulum (ER) Ca(2+) signals and caused a 40% inhibition of sarcoplasmic-ER calcium ATPases activity. Moreover, time course experiments correlate morphological alterations, processing of xbp-1 mRNA and caspase-12 activation during cadmium administration. Finally, the time response of ER to cadmium injury was compared with that of mitochondria. In conclusion, we highlighted a novel pathway of cadmium-induced cell death triggered by ER stress and involving caspase-12. Mitochondria and ER pathways seemed to share common time courses and a parallel activation of caspase-12 and caspase-9 seemed likely to be involved in acute cadmium toxicity.

The ability of cadmium to disrupt calcium homeostasis has been known since a long time, but the precise cellular targets of its toxic action are still debated. A great problem in the interpretation of data has been associated with the ability of cadmium to strongly bind traditional calcium probes. Aequorin, the well-characterized calcium-sensitive photoprotein, was used as intracellular calcium indicator during cadmium injury in NIH 3T3 murine fibroblasts. NIH 3T3 cells were transfected with a cDNA construct containing aequorin fused to a truncated glutamate receptor, which directs the probe to the outer surface of intracellular membranes. At first, we tested if different cadmium concentrations were able to modify the rate of light emission by aequorin showing that cadmium concentrations15 microM were ineffective on aequorin luminescence. Hence, aequorin chimeras revealed as a useful tool in the analyses of Cd2+/Ca2+ interference. To directly investigate the role of Cd2+ in Ca2+ homeostasis, we have started to selectively measure the free Ca2+ concentration in different cell compartments. Here, we report that cadmium reduces the transient free calcium signal after stimulation of cells with bradykinin. Further studies are in progress to clarify the role of mitochondria and endoplasmic reticulum in cadmium-induced alterations of Ca2+ homeostasis in order to link signal transduction modifications with the onset of apoptosis induced by cadmium exposure.

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC50-value of 0.7 microM, while human A549 adenocarcinoma cells were relatively resistant with an IC50-value of 164 microM CdCl2. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl2 and was concentration-dependent between 1-100 microM CdCl2, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 microM CdCl2. Furthermore, cadmium (1 microM, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd2+, other toxic heavy metal ions (Hg2+, Pb2+, Ni2+, Fe2+, CrO4(2-), Cu2+ or Co2+) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn2+ which caused apotosis at high concentrations (150 microM) whereas it protected against cadmium-induced apoptosis at low concentrations (10-50 microM).

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

Rare individuals with inactivating mutations in the Huntington’s disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development., Author summary The HTT gene mutated in Huntington’s Disease (HD) has essential roles during normal development. However, still not fully understood are the functional consequences of its partial inactivation. Our genetic study provides a comprehensive description of the effects of progressively more severe decreases in expression of Htt, the murine HTT counterpart. The most severe Htt decrease leads to lethality of early embryos, while intermediate but still severely reduced Htt dosages yield a variety of recessively inherited developmental abnormalities affecting body size, skin, skeletal and ear formation, and hematopoiesis. Comparative molecular analysis of differentiating wild-type cells and cells lacking Htt function further elucidate genes networks dysregulated during organ development. These nominate chromatin regulators and short non-coding RNAs as key molecular mediators. Together these findings demonstrate that Htt is required from conception to support normal embryonic and fetal development.

In various mammalian cells, two group IIb metals, cadmium and zinc, induce several morphological and biochemical effects that are salient features of programmed cell death. In C6 rat glioma cells, cadmium caused externalization of phosphatidylserine, breakdown of the mitochondrial membrane potential, activation of caspase-9, internucleosomal DNA fragmentation, chromatin condensation, and nuclear fragmentation. In NIH3T3 murine fibroblasts, cadmium-induced apoptosis was inhibited by overexpression of the antiapoptotic protein Bcl-2. Cadmium-induced DNA fragmentation in C6 cells was independent of inhibition of protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase, Ca-calmodulin-dependent protein kinase, and protein kinase G. Zinc at moderate concentrations (10-50 microM) protected against programmed cell death induced by cadmium, whereas deprivation of zinc by the membrane-permeable chelator N,N,N',N-terakis-(2-pyridylmethyl)ethylenediamine (TPEN) caused cell death with features characteristic of apoptosis. On the other hand, at elevated extracellular levels (150-200 microM), zinc alone caused programmed cell death in C6 cells. Zinc-induced apoptosis was independent of inhibition of PKA, PKC, guanylate cyclase and MAPK, but it was suppressed in the presence of 100 microM lanthanum chloride.