Franc Llorens

A myriad of miRNA variants in control and Huntington’s disease brain regions detected by massively parallel sequencing

Huntington disease (HD) is a neurodegenerative disorder that predominantly affects neurons of the forebrain. We have applied the Illumina massively parallel sequencing to deeply analyze the small RNA populations of two different forebrain areas, the frontal cortex (FC) and the striatum (ST) of healthy individuals and individuals with HD. More than 80% of the small-RNAs were annotated as microRNAs (miRNAs) in all samples. Deep sequencing revealed length and sequence heterogeneity (IsomiRs) for the vast majority of miRNAs. Around 80–90% of the miRNAs presented modifications in the 3′-terminus mainly in the form of trimming and/or as nucleotide addition variants, while the 5′-terminus of the miRNAs was specially protected from changes. Expression profiling showed strong miRNA and isomiR expression deregulation in HD, most being common to both FC and ST. The analysis of the upstream regulatory regions in co-regulated miRNAs suggests a role for RE1-Silencing Transcription Factor (REST) and P53 in miRNAs downregulation in HD. The putative targets of deregulated miRNAs and seed-region IsomiRs strongly suggest that their altered expression contributes to the aberrant gene expression in HD. Our results show that miRNA variability is a ubiquitous phenomenon in the adult human brain, which may influence gene expression in physiological and pathological conditions.

Neuroinflammatory signals in alzheimer disease and APP/PS1 transgenic mice: Correlations with plaques, tangles, and oligomeric species

Abstract To understand neuroinflammation-related gene regulation during normal aging and in sporadic Alzheimer disease (sAD), we performed functional genomics analysis and analyzed messenger RNA (mRNA) expression by quantitative reverse transcription–polymerase chain reaction of 22 genes involved in neuroinflammation-like responses in the cerebral cortex of wild-type and APP/PS1 transgenic mice. For direct comparisons, mRNA expression of 18 of the same genes was then analyzed in the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 of middle-aged human subjects lacking Alzheimer disease–related pathology and in older subjects with sAD pathology covering Stages I–II/0(A), III–IV/A–B, and V–VI/C of Braak and Braak classification. Modifications of cytokine and immune mediator mRNA expression were found with normal aging in wild-type mice and in middle-aged individuals and patients with early stages of sAD-related pathology; these were accompanied by increased protein expression of certain mediators in ramified microglia. In APP/PS1 mice, inflammatory changes coincided with &bgr;-amyloid (A&bgr;) deposition; increased levels of soluble oligomers paralleled the modified mRNA expression of cytokines and mediators in wild-type mice. In patients with sAD, regulation was stage- and region-dependent and not merely acceleration and exacerbation of mRNA regulation with aging. Gene regulation at first stages of AD was not related to hyperphosphorylated tau deposition in neurofibrillary tangles, A&bgr; plaque burden, concentration of A&bgr;1–40 (A&bgr;40) and A&bgr;1–42 (A&bgr;42), or fibrillar A&bgr; linked to membranes but rather to increased levels of soluble oligomers. Thus, species differences and region- and stage-dependent inflammatory responses in sAD, particularly at the initial stages, indicate the need to identify new anti-inflammatory compounds with specific molecular therapeutic targets.

Emerging functions of myelin-associated proteins during development, neuronal plasticity, and neurodegeneration

Adult mammalian central nervous system (CNS) axons have a limited regrowth capacity following injury. Myelin‐associated inhibitors (MAIs) limit axonal outgrowth, and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo‐A, MAG, and OMgp, share two common neuronal receptors: NgR1, together with its coreceptors [p75(NTR), TROY, and Lingo‐1];and the recently described paired immunoglobulin‐like receptor B (PirB). These proteins impair neuronal regeneration by limiting axonal sprouting. Some of the elements involved in the myelin inhibitory pathways may still be unknown, but the discovery that blocking both PirB and NgR1 activities leads to near‐complete release from myelin inhibition, sheds light on one of the most competitive and intense fields of neuroregeneration study in recent decades. In parallel with the identification and characterization of the roles and functions of these inhibitory molecules in axonal regeneration, data gathered in the field strongly suggest that most of these proteins have roles other than axonal growth inhibition. The discovery of a new group of interacting partners for myelin‐associated receptors and ligands, as well as functional studies within or outside the CNS environment, highlights the potential new physiological roles for these proteins in processes, such as development, neuronal homeostasis, plasticity, and neurodegeneration.—Llorens, F., Gil, V., del Río, J. A. Emerging functions of myelin‐associated proteins during development, neuronal plasticity, and neurodegeneration. FASEB J. 25, 463–475 (2011). www.fasebj.org

A highly expressed miR-101 isomiR is a functional silencing small RNA

BackgroundMicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5’ or 3’ of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5’-trimming variant (5’-isomiR-101).ResultsThe analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5’-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5’-isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5’-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5’-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5’-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5’-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5’-isomiR-101.ConclusionsThese results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.

Regulation of GABAA and Glutamate Receptor Expression, Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

Background Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Methodology/Principal Findings Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice. Conclusions/Significance Present results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABAA and AMPA-Kainate neurotransmission. New PrPc functions have recently been described, which point to PrPc as a target for putative therapies in Alzheimers disease. However, our results indicate that a “gain of function” strategy in Alzheimers disease, or a “loss of function” in prionopathies, may impair PrPc function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Developmental analysis of Lingo‐1/Lern1 protein expression in the mouse brain: Interaction of its intracellular domain with Myt1l

Lingo‐1 (also known as Lern1) is a component of the Nogo receptor complex that mediates intracellular signaling in response to myelin associated inhibitors (MAIs): NogoA, MAG, and Omgp. Signaling through Nogo receptor extends to more than its well known role in preventing axon regeneration after lesion in the CNS, being implicated in neuronal functional maturation. Using Lingo‐1‐deficient mice, it has been demonstrated that Lingo‐1 plays relevant roles in oligodendrocyte differentiation during brain development, and that treatment with Lingo‐1 antagonists can improve axon regeneration after lesion in adult mice by decreasing MAI mediated signaling. However, a detailed description of the pattern of expression of Lingo‐1 protein in correlation with the other partners of Nogo receptor is missing. Here, we show that components of the Nogo receptor complex, Lingo‐1, NgR1, p75, and TROY coexist in mouse brain in a defined time window only at later postnatal stages. We have also determined the Lingo‐1 distribution showing expression in particular subsets of neurons, but not in myelinating mature oligodendrocytes. Surprisingly, Lingo‐1 is expressed at early developmental stages without NgR1, which supports the notion that Lingo‐1 may participate in other activities in developing neurons different from oligodendrocyte maturation or axon extension inhibition in the adult. Finally, we propose that the intracellular domain of Lingo‐1 contributes to signaling and show that it interacts with the postmitotic neuronal specific zinc finger protein Myt1l, suggesting that Lingo‐1 may regulate Myt1l transcription factor activity by affecting its subcellular localization.

Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7–PSD-95 binding

Cellular prion protein neuroprotection against kainate is due to its ability to modulate glutamate receptor 6/7–mediated neurotransmission and JNK3 pathway activation.

Subtype and Regional-Specific Neuroinflammation in Sporadic Creutzfeldt–Jakob Disease

The present study identifies deregulated cytokines and mediators of the immune response in the frontal cortex and cerebellum of sporadic Creutzfeldt–Jakob disease (sCJD) MM1 and VV2 subtypes compared to age-matched controls. Deregulated genes include pro- and anti-inflammatory cytokines, toll-like receptors, colony stimulating factors, cathepsins, members of the complement system, and members of the integrin and CTL/CTLD family with particular regional and sCJD subtype patterns. Analysis of cytokines and mediators at protein level shows expression of selected molecules and receptors in neurons, in astrocytes, and/or in microglia, thus suggesting interactions between neurons and glial cells, mainly microglia, in the neuroinflammatory response in sCJD. Similar inflammatory responses have been shown in the tg340 sCJD MM1 mice, revealing a progressive deregulation of inflammatory mediators with disease progression. Yet, inflammatory molecules involved are subjected to species differences in humans and mice. Moreover, inflammatory-related cell signaling pathways NFκB/IKK and JAK/STAT are activated in sCJD and sCJD MM1 mice. Together, the present observations show a self-sustained complex inflammatory and inflammatory-related responses occurring already at early clinical stages in animal model and dramatically progressing at advanced stages of sCJD. Considering this scenario, measures tailored to modulate (activate or inhibit) specific molecules could be therapeutic options in CJD.

Genetic and Transcriptomic Profiles of Inflammation in Neurodegenerative Diseases: Alzheimer, Parkinson, Creutzfeldt-Jakob and Tauopathies

Polymorphisms in certain inflammatory-related genes have been identified as putative differential risk factors of neurodegenerative diseases with abnormal protein aggregates, such as sporadic Alzheimer’s disease (AD) and sporadic Parkinson’s disease (sPD). Gene expression studies of cytokines and mediators of the immune response have been made in post-mortem human brain samples in AD, sPD, sporadic Creutzfeldt-Jakob disease (sCJD) subtypes MM1 and VV2, Pick’s disease (PiD), progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration linked to mutation P301L in MAPT Frontotemporal lobar degeneration-tau (FTLD-tau). The studies have disclosed variable gene regulation which is: (1) disease-dependent in the frontal cortex area 8 in AD, sPD, sCJD MM1 and VV2, PiD, PSP and FTLD-tau; (2) region-dependent as seen when comparing the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 (FC) in AD; the substantia nigra, putamen, FC, and angular gyrus in PD, as well as the FC and cerebellum in sCJD; (3) genotype-dependent as seen considering sCJD MM1 and VV2; and (4) stage-dependent as seen in AD at different stages of disease progression. These observations show that regulation of inflammation is much more complicated and diverse than currently understood, and that new therapeutic approaches must be designed in order to selectively act on specific targets in particular diseases and at different time points of disease progression.

The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases

The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPSc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays—such as protein misfolding cyclic amplification (PMCA)—which are based on sonication, the RT-QuIC technique is based on prion seed–induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimers and Parkinsons disease.