SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.

A human minisatellite hosts an alternative transcription start site for NPRL3 driving its expression in a repeat number-dependent manner.

Minisatellites, also called variable number of tandem repeats (VNTRs), are a class of repetitive elements that may affect gene expression at multiple levels and have been correlated to disease. Their identification and role as expression quantitative trait loci (eQTL) have been limited by their absence in comparative genomic hybridization and single nucleotide polymorphisms arrays. By taking advantage of cap analysis of gene expression (CAGE), we describe a new example of a minisatellite hosting a transcription start site (TSS) which expression is dependent on the repeat number. It is located in the third intron of the gene nitrogen permease regulator like protein 3 (NPRL3). NPRL3 is a component of the GAP activity toward rags 1 protein complex that inhibits mammalian target of rapamycin complex 1 (mTORC1) activity and it is found mutated in familial focal cortical dysplasia and familial focal epilepsy. CAGE tags represent an alternative TSS identifying TAGNPRL3 messenger RNAs (mRNAs). TAGNPRL3 is expressed in red blood cells both at mRNA and protein levels, it interacts with its protein partner NPRL2 and its overexpression inhibits cell proliferation. This study provides an example of a minisatellite that is both a TSS and an eQTL as well as identifies a new VNTR that may modify mTORC1 activity.

The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.

Transposable elements (TEs) compose about half of the mammalian genome and, as embedded sequences, up to 40% of long noncoding RNA (lncRNA) transcripts. Embedded TEs may represent functional domains within lncRNAs, providing a structured RNA platform for protein interaction. Here we show the interactome profile of the mouse inverted short interspersed nuclear element (SINE) of subfamily B2 (invSINEB2) alone and embedded in antisense (AS) ubiquitin C-terminal hydrolase L1 (Uchl1), an lncRNA that is AS to Uchl1 gene. AS Uchl1 is the representative member of a functional class of AS lncRNAs, named SINEUPs, in which the invSINEB2 acts as effector domain (ED)-enhancing translation of sense protein-coding mRNAs. By using RNA-interacting domainome technology, we identify the IL enhancer-binding factor 3 (ILF3) as a protein partner of AS Uchl1 RNA. We determine that this interaction is mediated by the RNA-binding motif 2 of ILF3 and the invSINEB2. Furthermore, we show that ILF3 is able to bind a free right Arthrobacter luteus (Alu) monomer sequence, the embedded TE acting as ED in human SINEUPs. Bioinformatic analysis of Encyclopedia of DNA Elements-enhanced cross-linking immunoprecipitation data reveals that ILF3 binds transcribed human SINE sequences at transcriptome-wide levels. We then demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3. This work unveils the existence of a specific interaction between embedded TEs and an RNA-binding protein, strengthening the model of TEs as functional modules in lncRNAs.-Fasolo, F., Patrucco, L., Volpe, M., Bon, C., Peano, C., Mignone, F., Carninci, P., Persichetti, F., Santoro, C., Zucchelli, S., Sblattero, D., Sanges, R., Cotella, D., Gustincich, S. The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.

Genome-wide analysis of mammalian promoter architecture and evolution.

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules.

BACKGROUND: 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). Selective degeneration of A9 neurons occurs in Parkinson's disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown. RESULTS: By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains. CONCLUSIONS: Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

An Air-Well sparging minifermenter system for high-throughput protein production.

BACKGROUND: Over the last few years High-Throughput Protein Production (HTPP) has played a crucial role for functional proteomics. High-quality, high yield and fast recombinant protein production are critical for new HTPP technologies. Escherichia coli is usually the expression system of choice in protein production thanks to its fast growth, ease of handling and high yields of protein produced. Even though shake-flask cultures are widely used, there is an increasing need for easy to handle, lab scale, high throughput systems. RESULTS: In this article we described a novel minifermenter system suitable for HTPP. The Air-Well minifermenter system is made by a homogeneous air sparging device that includes an air diffusion system, and a stainless steel 96 needle plate integrated with a 96 deep well plate where cultures take place. This system provides aeration to achieve higher optical density growth compared to classical shaking growth without the decrease in pH value and bacterial viability. Moreover the yield of recombinant protein is up to 3-fold higher with a considerable improvement in the amount of full length proteins. CONCLUSIONS: High throughput production of hundreds of proteins in parallel can be obtained sparging air in a continuous and controlled manner. The system used is modular and can be easily modified and scaled up to meet the demands for HTPP.

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

LINE-1 regulates cortical development by acting as long non-coding RNAs.

Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides.

SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with huntingtin protein and promotes its atypical ubiquitination to enhance aggregate formation.

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.

The E3 Ubiquitin Ligase TRAF6 Interacts with the Cellular Prion Protein and Modulates Its Solubility and Recruitment to Cytoplasmic p62/SQSTM1-Positive Aggresome-Like Structures.

The cellular prion protein (PrPC) is a ubiquitous glycoprotein highly expressed in the brain where it is involved in neurite outgrowth, copper homeostasis, NMDA receptor regulation, cell adhesion, and cell signaling. Conformational conversion of PrPC into its insoluble and aggregation-prone scrapie form (PrPSc) is the trigger for several rare devastating neurodegenerative disorders, collectively referred to as prion diseases. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To better dissect the role of ubiquitination in PrPC physiology, we focused on the E3 RING ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here, we report that PrPC interacts with TRAF6 both in vitro, in cells, and in vivo, in the mouse brain. Transient overexpression of TRAF6 indirectly modulates PrPC ubiquitination and triggers redistribution of PrPC into the insoluble fraction. Importantly, in the presence of wild-type TRAF6, but not a mutant lacking E3 ligase activity, PrPC accumulates into cytoplasmic aggresome-like inclusions containing TRAF6 and p62/SQSTM1. Our results suggest that TRAF6 ligase activity could exert a role in the regulation of PrPC redistribution in cells under physiological conditions. This novel interaction may uncover possible mechanisms of cell clearance/reorganization in prion diseases.

Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disorder with progressive motor dysfunction and cognitive decline. The disease is caused by a CAG repeat expansion in the IT15 gene, which elongates a polyglutamine stretch of the HD protein, Huntingtin. No therapeutic treatments are available, and new pharmacological targets are needed. Retrotransposons are transposable elements (TEs) that represent 40% and 30% of the human and Drosophila genomes and replicate through an RNA intermediate. Mounting evidence suggests that mammalian TEs are active during neurogenesis and may be involved in diseases of the nervous system. Here we show that TE expression and mobilization are increased in a Drosophila melanogaster HD model. By inhibiting TE mobilization with Reverse Transcriptase inhibitors, polyQ-dependent eye neurodegeneration and genome instability in larval brains are rescued and fly lifespan is increased. These results suggest that TE activation may be involved in polyQ-induced neurotoxicity and a potential pharmacological target.

Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein.

Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and ß-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and ß- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal α-syn fragments.

Specific transcriptional programs differentiate ICOS from CD28 costimulatory signaling in human Naïve CD4+ T cells.

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes.

Analysis of LINE1 Retrotransposons in Huntington's Disease.

Transposable elements (TEs) are mobile genetic elements that made up about half the human genome. Among them, the autonomous non-LTR retrotransposon long interspersed nuclear element-1 (L1) is the only currently active TE in mammals and covers about 17% of the mammalian genome. L1s exert their function as structural elements in the genome, as transcribed RNAs to influence chromatin structure and as retrotransposed elements to shape genomic variation in somatic cells. L1s activity has been shown altered in several diseases of the nervous system. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of a CAG repeat in the HTT gene which leads to a gradual loss of neurons most prominently in the striatum and, to a lesser extent, in cortical brain regions. The length of the expanded CAG tract is related to age at disease onset, with longer repeats leading to earlier onset. Here we carried out bioinformatic analysis of public RNA-seq data of a panel of HD mouse models showing that a decrease of L1 RNA expression recapitulates two hallmarks of the disease: it correlates to CAG repeat length and it occurs in the striatum, the site of neurodegeneration. Results were then experimentally validated in Htt Q111 knock-in mice. The expression of L1-encoded proteins was independent from L1 RNA levels and differentially regulated in time and tissues. The pattern of expression L1 RNAs in human HD post-mortem brains showed similarity to mouse models of the disease. This work suggests the need for further study of L1s in HD and adds support to the current hypothesis that dysregulation of TEs may be involved in neurodegenerative diseases.

SINEUPs: a novel toolbox for RNA therapeutics.

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.

Rrs1 is involved in endoplasmic reticulum stress response in Huntington disease.

The induction of Rrs1 expression is one of the earliest events detected in a presymptomatic knock-in mouse model of Huntington disease (HD). Rrs1 up-regulation fulfills the HD criteria of dominance, striatal specificity, and polyglutamine dependence. Here we show that mammalian Rrs1 is localized both in the nucleolus as well as in the endoplasmic reticulum (ER) of neurons. This dual localization is shared with its newly identified molecular partner 3D3/lyric. We then show that both genes are induced by ER stress in neurons. Interestingly, we demonstrate that ER stress is an early event in a presymptomatic HD mouse model that persists throughout the life span of the rodent. We further show that ER stress also occurs in postmortem brains of HD patients.

Antisense Transcription in Loci Associated to Hereditary Neurodegenerative Diseases.

Natural antisense transcripts are common features of mammalian genes providing additional regulatory layers of gene expression. A comprehensive description of antisense transcription in loci associated to familial neurodegenerative diseases may identify key players in gene regulation and provide tools for manipulating gene expression. We take advantage of the FANTOM5 sequencing datasets that represent the largest collection to date of genome-wide promoter usage in almost 2000 human samples. Transcription start sites (TSSs) are mapped at high resolution by the use of a modified protocol of cap analysis of gene expression (CAGE) for high-throughput single molecule next-generation sequencing with Helicos (hCAGE). Here we present the analysis of antisense transcription at 17 loci associated to hereditary Alzheimer's disease, Frontotemporal Dementia, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease. We focused our analysis on libraries derived from brain tissues and primary cells. We also screened libraries from total blood and blood cell populations in the quest for peripheral biomarkers of neurodegenerative diseases. We identified 63 robust promoters in antisense orientation to genes associated to familial neurodegeneration. When applying a less stringent cutoff, this number increases to over 400. A subset of these promoters represents alternative TSSs for 24 FANTOM5 annotated long noncoding RNA (lncRNA) genes, in antisense orientation to 13 of the loci analyzed here, while the remaining contribute to the expression of additional transcript variants. Intersection with GWAS studies, sample ontology, and dynamic expression reveals association to specific genetic traits as well as cell and tissue types, not limited to neurodegenerative diseases. Antisense transcription was validated for a subset of genes, including those encoding for Microtubule-Associated Protein Tau, α-synuclein, Parkinsonism-associated deglycase DJ-1, and Leucin-Rich Repeat Kinase 2. This work provides evidence for the existence of additional regulatory mechanisms of the expression of neurodegenerative disease-causing genes by previously not-annotated and/or not-validated antisense long noncoding RNAs.

Structural properties of polyglutamine aggregates investigated via molecular dynamics simulations.

Polyglutamine (polyQ) beta-stranded aggregates constitute the hallmark of Huntington disease. The disease is fully penetrant when Q residues are more than 36-40 ("disease threshold"). Here, based on a molecular dynamics study on polyQ helical structures of different shapes and oligomeric states, we suggest that the stability of the aggregates increases with the number of monomers, while it is rather insensitive to the number of Qs in each monomer. However, the stability of the single monomer does depend on the number of side-chain intramolecular H-bonds, and therefore on the number of Qs. If such number is lower than that of the disease threshold, the beta-stranded monomers are unstable and hence may aggregate with lower probability, consistently with experimental findings. Our results provide a possible interpretation of the apparent polyQ length dependent-toxicity, and they do not support the so-called "structural threshold hypothesis", which supposes a transition from random coil to a beta-sheet structure only above the disease threshold.

Huntingtin polyQ Mutation Impairs the 17ß-Estradiol/Neuroglobin Pathway Devoted to Neuron Survival.

Among several mechanisms underlying the well-known trophic and protective effects of 17ß-estradiol (E2) in the brain, we recently reported that E2 induces the up-regulation of two anti-apoptotic and neuroprotectant proteins: huntingtin (HTT) and neuroglobin (NGB). Here, we investigate the role of this up-regulation. The obtained results indicate that E2 promotes NGB-HTT association, induces the localization of the complex at the mitochondria, and protects SK-N-BE neuroblastoma cells and murine striatal cells, which express wild-type HTT (i.e., polyQ7), against H2O2-induced apoptosis. All E2 effects were completely abolished in HTT-knocked out SK-N-BE cells and in striatal neurons expressing the mutated form of HTT (mHTT; i.e., polyQ111) typical of Huntington's disease (HD). As a whole, these data provide a new function of wild-type HTT which drives E2-induced NGB in mitochondria modulating NGB anti-apoptotic activity. This new function is lost by HTT polyQ pathological expansion. These data evidence the existence of a novel E2/HTT/NGB neuroprotective axis that may play a relevant role in the development of HD therapeutics.