Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

The Hebrew University of Jerusalem

Alanine-to-threonine (A to T) substitutions caused by single nucleotide polymorphisms (SNPs) occur in diverse proteins, and in certain cases these substitutions induce self-aggregation into amyloid fibrils or aggregation in other amyloidogenic proteins. This is compatible with the inverse preferences of alanine to form helices and of threonine to support beta-sheet structures, which are crucial for amyloid fibrils formation. Our interest in these mutations was initiated by studying the potential effects of the A539T substitution in the butyrylcholinesterase BChE-K variant on amyloid fibrils formation in Alzheimer's disease. Other examples are, Parkinson's disease (PD), where A53T alpha-synuclein occurs in Lewy bodies and familial amyloid polyneuropathy (FAP), where an A25T substitution appears in transthyretin (TTR). In peripheral organs, an A34T substitution is found in the light chain immunoglobulin genes of patients with systemic amyloidosis and in familial hypercholesterolemia, an A370T substitution occurs in the LDLR regulator of cholesterol homeostasis. That such substitutions appear in proteins with important cellular functions suggests that they confer antagonistic pleiotropy, providing added value at an earlier age but causing damages and inducing amyloid diseases later on. This, in turn, may explain the evolutionary selection and preservation of these substitutions. The structural effect of residue substitutions and in particular A to T substitutions in amyloidogenic diseases thus merits further attention.

Alanine-to-threonine substitutions and amyloid diseases: butyrylcholinesterase as a case study.

Des substitutions d&#39;alanine-&agrave;-thr&eacute;onine (A &agrave; T) caus&eacute;es par des polymorphismes de nucl&eacute;otides simples (SNP) se produisent dans diverses prot&eacute;ines, et dans certains cas, ces substitutions induisent auto-agr&eacute;gation en fibrilles amylo&iuml;des ou agr&eacute;gation dans d&#39;autres prot&eacute;ines amylo&iuml;dog&egrave;ne. Cela est compatible avec les pr&eacute;f&eacute;rences inverses d&#39;alanine &agrave; h&eacute;lices de forme et de la thr&eacute;onine pour appuyer les structures de b&ecirc;ta-feuille, qui sont cruciales pour la formation de fibrilles amylo&iuml;des. Notre int&eacute;r&ecirc;t pour ces mutations a &eacute;t&eacute; initi&eacute; en &eacute;tudiant les effets potentiels de la substitution de A539T dans la butyrylcholinest&eacute;rase BChE-K variante sur la formation de fibrilles amylo&iuml;des dans la maladie d&#39;Alzheimer. Sont d&#39;autres exemples, la maladie de Parkinson (PD), o&ugrave; A53T alpha-synucl&eacute;ine se produit dans le corps de Lewy et neuropathies amyloides (FAP), o&ugrave; une substitution de A25T appara&icirc;t dans la transthyr&eacute;tine (TTR). Dans les organes p&eacute;riph&eacute;riques, une substitution de A34T se trouve dans les g&egrave;nes d&#39;immunoglobuline de cha&icirc;ne l&eacute;g&egrave;re des patients souffrant de l&#39;amylose syst&eacute;mique et dans l&#39;hypercholest&eacute;rol&eacute;mie, une substitution de A370T se produit dans l&#39;organisme de r&eacute;glementation LDLR de l&#39;hom&eacute;ostasie du cholest&eacute;rol. Que telles substitutions apparaissent en prot&eacute;ines avec des fonctions cellulaires importantes sugg&egrave;re qu&#39;ils conf&egrave;rent pl&eacute;iotropie antagoniste, apportant une valeur ajout&eacute;e &agrave; un &acirc;ge plus pr&eacute;coce, mais causant des dommages-int&eacute;r&ecirc;ts et induire des maladies amylo&iuml;des plus tard. Ceci, &agrave; son tour, peut expliquer la s&eacute;lection &eacute;volutive et la pr&eacute;servation de ces substitutions. L&#39;effet structurel de substitutions de r&eacute;sidus et notamment A &agrave; des substitutions de T dans les maladies amylo&iuml;dog&egrave;ne m&eacute;rite donc plus attention.

Substitutions de l&#39;alanine-&agrave;-thr&eacute;onine et maladies amylo&iuml;des : butyrylcholinest&eacute;rase comme &eacute;tude de cas.

Le sostituzioni di alanina-treonina (A T) causate da polimorfismi a singolo nucleotide (SNPs) si verificano in diverse proteine, e in alcuni casi queste sostituzioni inducono aggregazione in fibrille amiloidi o aggregazione di altre proteine amiloidogeniche. Questo &egrave; compatibile con le preferenze di inverse di alanina per eliche di forma e di treonina a sostenere le strutture beta-sheet, che sono cruciali per la formazione di fibrille amiloidi. Il nostro interesse per queste mutazioni &egrave; stato avviato da studiare i potenziali effetti di sostituzione A539T nella variante Butiril-colinesterasi BChE-K su formazione di fibrille amiloidi nella malattia di Alzheimer. Altri esempi sono, morbo di Parkinson (PD), dove A53T alfa-sinucleina si verifica nei corpi di Lewy e polineuropatia amiloide familiare (FAP), dove una sostituzione A25T appare in transtiretina (TTR). Negli organi periferici, una sostituzione di A34T si trova nei geni per l&#39;immunoglobulina catena leggera dei pazienti con amiloidosi sistemica e nella ipercolesterolemia familiare, una sostituzione di A370T si verifica nel regolatore LDLR di omeostasi del colesterolo. Che tali sostituzioni vengono visualizzati in proteine con importanti funzioni cellulari suggerisce che essi conferiscono pleiotropia antagonista, fornendo valore aggiunto ad un&#39;et&agrave; precedente ma causando danni e indurre malattie amiloide pi&ugrave; tardi. Questo, a sua volta, pu&ograve; spiegare la selezione evolutiva e la conservazione di queste sostituzioni. L&#39;effetto strutturale delle sostituzioni dei residui e in particolare A per sostituzioni di T nelle malattie amiloidogeniche cos&igrave; meriti ulteriore attenzione.

Alanina-treonina sostituzioni e malattie amiloide: Butiril-colinesterasi come un caso di studio.

MicroRNAs (miRs) have emerged as important gene silencers affecting many target mRNAs. Here, we report the identification of 244 miRs that target the 3'-untranslated regions of different cholinesterase transcripts: 116 for butyrylcholinesterase (BChE), 47 for the synaptic acetylcholinesterase (AChE-S) splice variant, and 81 for the normally rare splice variant AChE-R. Of these, 11 and 6 miRs target both AChE-S and AChE-R, and AChE-R and BChE transcripts, respectively. BChE and AChE-S showed no overlapping miRs, attesting to their distinct modes of miR regulation. Generally, miRs can suppress a number of targets; thereby controlling an entire battery of functions. To evaluate the importance of the cholinesterase-targeted miRs in other specific biological processes we searched for their other experimentally validated target transcripts and analyzed the gene ontology enriched biological processes these transcripts are involved in. Interestingly, a number of the resulting categories are also related to cholinesterases. They include, for BChE, response to glucocorticoid stimulus, and for AChE, response to wounding and two child terms of neuron development: regulation of axonogenesis and regulation of dendrite morphogenesis. Importantly, all of the AChE-targeting miRs found to be related to these selected processes were directed against the normally rare AChE-R splice variant, with three of them, including the neurogenesis regulator miR-132, also directed against AChE-S. Our findings point at the AChE-R splice variant as particularly susceptible to miR regulation, highlight those biological functions of cholinesterases that are likely to be subject to miR post-transcriptional control, demonstrate the selectivity of miRs in regulating specific biological processes, and open new venues for targeted interference with these specific processes.

Cholinesterase-Targeting microRNAs Identified in silico Affect Specific Biological Processes.

MicroARN (miRs) est devenus silencieux de g&egrave;ne important affectant plusieurs ARNm cible. Nous rapportons ici, l&#39;identification des 244 miRs qui ciblent les r&eacute;gions non traduites de 3&#39; des transcriptions diff&eacute;rentes cholinest&eacute;rase : 116 pour butyrylcholinest&eacute;rase (BChE), 47 pour le synaptique ac&eacute;tylcholinest&eacute;rase (AChE-S) &eacute;pisser variante et 81 pour l&#39;&eacute;pissure normalement rare variante AChE-R. Parmi ceux-ci, miRs 11 et 6 ciblent AChE-S et AChE-R et transcriptions d&#39;AChE-R et BChE, respectivement. BChE et AChE-S a montr&eacute; aucun chevauchements miRs, attestant de leurs modes distincts de r&eacute;glementation de miR. G&eacute;n&eacute;ralement, les miRs peuvent supprimer un certain nombre de cibles ; contr&ocirc;lant ainsi toute une batterie de fonctions. Pour &eacute;valuer l&#39;importance des miRs cholinest&eacute;rase cibl&eacute;es dans autre biologique sp&eacute;cifique processus nous avons cherch&eacute; &agrave; leurs autres transcriptions de cible valid&eacute;e exp&eacute;rimentalement et analys&eacute; l&#39;ontology de g&egrave;ne enrichi ces transcriptions sont impliqu&eacute;es dans des processus biologiques. Fait int&eacute;ressant, un certain nombre de cat&eacute;gories qui en r&eacute;sultent est &eacute;galement associ&eacute; &agrave; cholinest&eacute;rases. Ils comprennent, pour BChE, r&eacute;ponse au stimulus de glucocortico&iuml;des et AChE, r&eacute;ponse enfant deux termes du d&eacute;veloppement du neurone et la blessant : r&egrave;glement d&#39;axonog&eacute;n&egrave;se et de la r&eacute;glementation de la morphogen&egrave;se dendritique. Ce qui est important, tous les miRs AChE-ciblage est reli&eacute;es &agrave; ces s&eacute;lectionn&eacute; processus &eacute;taient dirig&eacute;es contre l&#39;AChE-R normalement rare &eacute;pissure variante, avec trois d&#39;entre eux, y compris la neurogen&egrave;se r&eacute;gulateur miR-132, qui &eacute;galement dirig&eacute;es contre AChE-S. Notre point de conclusions &agrave; l&#39;AChE-R &eacute;pisser variante comme particuli&egrave;rement sensibles aux r&egrave;glement de miR, mettre en &eacute;vidence les fonctions biologiques des cholinest&eacute;rases qui risquent d&#39;&ecirc;tre soumis &agrave; un contr&ocirc;le post-transcriptionnel miR, d&eacute;montrer la s&eacute;lectivit&eacute; des miRs dans la r&eacute;gulation des processus biologiques sp&eacute;cifiques et ouvrir de nouveaux lieux pour cibl&eacute;e atteinte &agrave; ces proc&eacute;d&eacute;s sp&eacute;cifiques.

Ciblage de cholinest&eacute;rase microARN Identified in silico affectent des processus biologiques sp&eacute;cifiques.

MicroRNA (MIR) &egrave; emerse come importante gene silencers che interessano molti mRNA bersaglio. Riportiamo l&#39;identificazione di 244 Mir che colpiscono le regioni non tradotta 3&#39; delle trascrizioni differenti colinesterasi: 116 per butirrilcolinesterasi (BChE), 47 per il synaptic acetilcolinesterasi (AChE-S) splice variante e 81 per la variante di splice normalmente rara AChE-R. Di questi, 11 e 6 miRs destinazione AChE-S e mal-R sia trascrizioni AChE-R e BChE, rispettivamente. BChE e mal-S ha mostrato alcuna sovrapposizione miRs, che attesta il loro distinte modalit&agrave; di regolamento di miR. In genere, Mir pu&ograve; sopprimere un certo numero di obiettivi; quindi controllare un&#39;intera batteria di funzioni. Per valutare l&#39;importanza di Mir colinesterasi mirati in altri biologici specifici processi abbiamo cercato per loro altre trascrizioni di destinazione sperimentalmente validati e analizzato il ontology del gene arricchito i processi biologici, in che queste trascrizioni sono coinvolti. &Egrave; interessante notare che, un certo numero di categorie risultanti &egrave; anche legato alla cholinesterases. Essi comprendono, per BChE, risposta a stimolo di glucocorticoidi e AChE, risposta al bambino due termini dello sviluppo del neurone e ferendone: regolamento di assonogenesi e regolamento della morfogenesi dendrite. Importante, tutti di Mir mal-targeting trovato per essere collegato a questi selezionati processi erano diretti contro la mal-R normalmente rara splice variante, con tre di loro, tra cui la neurogenesi regolatore miR-132, diretto anche contro il mal di-S. Il nostro punto di scoperte presso il mal-R splice variante come particolarmente sensibili al regolamento miR, evidenziare quelle funzioni biologiche di cholinesterases che sono suscettibili di essere sottoposti al controllo posttrascrizionale miR, dimostrare la selettivit&agrave; di Mir nel regolare i processi biologici specifici e aprire nuove sedi per interferenze mirate con questi processi specifici.

Colinesterasi-Targeting microRNA identificati in silico possono influenzare i processi biologici specifici.

The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogren's syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9(-/-) mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-ÎºB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogren's syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-ÎºB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9(-/-) mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogren's disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-ÎºB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-ÎºB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogren's syndrome and may be applicable to additional autoimmune syndromes.

Activation of the alternative NFÎºB pathway improves disease symptoms in a model of Sjogren's syndrome.

Le but de notre &eacute;tude &eacute;tait de comprendre si les Toll-like receptor 9 (TLR9) activation pourrait contribuer au contr&ocirc;le de l&#39;inflammation dans le syndrome de Sj&ouml;gren. &Agrave; cette fin, nous avons manipul&eacute; TLR9 signaling in non ob&egrave;ses diab&eacute;tiques (EDL) et des souris TLR9(-/-) agonistiques CpG oligonucl&eacute;otide aptam&egrave;res TLR9 inhibiteurs et l&#39;oligonucl&eacute;otide interne BL-7040. Ensuite, nous avons mesur&eacute; la salivation, marqueurs de la r&eacute;ponse inflammatoire et l&#39;expression des prot&eacute;ines en aval aux voies d&#39;activation de NF-&kappa;B. Enfin, nous avons &eacute;tiquet&eacute; prot&eacute;ines d&#39;int&eacute;r&ecirc;t dans les biopsies des glandes salivaires de patients de Gougerot-Sj&ouml;gren, compar&eacute;es aux sujets t&eacute;moins Sicca syndrome. Nous montrons que, chez les souris NOD BL-7040 active TLR9 pour induire un autre mode d&#39;activation de NF-&kappa;B r&eacute;sultant en une augmentation de la salivation, une r&eacute;ponse anti-inflammatoire &eacute;lev&eacute;e dans les glandes salivaires et r&eacute;duit l&#39;activit&eacute; AChE p&eacute;riph&eacute;rique. Ces effets sont plus importants et aussi suppressible par TLR9 inhibiteurs chez la souris NOD, mais TLR9(-/-) souris &eacute;taient r&eacute;sistantes aux effets des oligonucl&eacute;otides CpG favorisant la salivation et BL-7040. Enfin, les glandes salivaires de patients atteints de la maladie de Sjogren a montr&eacute; augment&eacute; inflammatoires et une diminution des biomarqueurs anti-inflammatoires, outre une baisse des taux de prot&eacute;ines de voie alternatives NF-&kappa;B. En r&eacute;sum&eacute;, nous avons d&eacute;montr&eacute; qu&#39;activation du TLR9 par BL-7040 l&#39;conduit &agrave; non-canoniques activation de NF-&kappa;B, promouvoir le fonctionnement salivaire et r&eacute;gulateur d&#39;en bas l&#39;inflammation. Nous proposons que BL-7040 pourrait &ecirc;tre b&eacute;n&eacute;fique dans le traitement du syndrome de Gougerot-Sj&ouml;gren et peut-&ecirc;tre s&#39;appliquer &agrave; des syndromes auto-immuns suppl&eacute;mentaires.

Activation de la voie alternative de NF&kappa;B am&eacute;liore les sympt&ocirc;mes de la maladie dans un mod&egrave;le du syndrome de Gougerot-Sj&ouml;gren.

Lo scopo del nostro studio &egrave; stato quello di capire se Toll-like receptor 9 (TLR9) attivazione potrebbe contribuire al controllo dell&#39;infiammazione nella sindrome di Sjogren. A tal fine, abbiamo manipolato TLR9 segnalazione nei non obesi diabetici (NOD) e TLR9(-/-) nei topi utilizzando CpG agonistica del oligonucleotide aptameri, TLR9 inibitori e il oligonucleotide interno BL-7040. Abbiamo quindi misurato salivazione, marcatori di risposta infiammatoria e l&#39;espressione delle proteine a valle di percorsi di attivazione di NF-&kappa;B. Infine, abbiamo etichettato proteine di interesse per la biopsia delle ghiandole salivari da pazienti con la sindrome di Sjogren, rispetto ai controlli sindrome Sicca. Mostriamo che in topi NOD BL-7040 attiva TLR9 per indurre un&#39;alternativa modalit&agrave; di attivazione di NF-&kappa;B con conseguente aumento della salivazione, elevata risposta antinfiammatoria nelle ghiandole salivari e ridotta attivit&agrave; AChE periferico. Questi effetti sono stati pi&ugrave; importanti e anche sopprimibile da TLR9 inibitori nei topi NOD, ma TLR9(-/-) topi sono resistenti agli effetti salivazione-promozione di oligonucleotidi CpG e BL-7040. Ultima, ghiandole salivari di pazienti con malattia di Sjogren hanno mostrati aumentato biomarkers anti-infiammatori infiammatoria e diminuito, oltre alla diminuzione dei livelli di proteine di percorso alternative NF-&kappa;B. In sintesi, abbiamo dimostrato che attivazione di TLR9 da BL-7040 conduce non canonico attivazione di NF-&kappa;B, promuovere il funzionamento salivare e gi&ugrave;-regolazione di infiammazione. Noi proponiamo che BL-7040 potrebbe essere utile nel trattamento della sindrome di Sjogren e possono essere applicabili alle sindromi autoimmuni supplementari.

Attivazione della via alternativa NF&kappa;B migliora i sintomi della malattia in un modello della sindrome di Sjogren.

Genetic studies link inherited errors in RNA metabolism to familial neurodegenerative disease. Here, we report such errors and the underlying mechanism in sporadic Alzheimer's disease (AD). AD entorhinal cortices presented globally impaired exon exclusions and selective loss of the hnRNP A/B splicing factors. Supporting functional relevance, hnRNP A/B knockdown induced alternative splicing impairments and dendrite loss in primary neurons, and memory and electrocorticographic impairments in mice. Transgenic mice with disease-associated mutations in APP or Tau displayed no alterations in hnRNP A/B suggesting that its loss in AD is independent of AÎ² and Tau toxicity. However, cholinergic excitation increased hnRNP A/B levels while in vivo neurotoxin-mediated destruction of cholinergic neurons caused cortical AD-like decrease in hnRNP A/B and recapitulated the alternative splicing pattern of AD patients. Our findings present cholinergic-mediated hnRNP A/B loss and impaired RNA metabolism as important mechanisms involved in AD.

Cholinergic-associated loss of hnRNP-A/B in Alzheimer's disease impairs cortical splicing and cognitive function in mice.

Des &eacute;tudes g&eacute;n&eacute;tiques lien Erreurs h&eacute;rit&eacute;es dans le m&eacute;tabolisme de RNA de maladie neurod&eacute;g&eacute;n&eacute;rative familial. Nous rapportons ici, ces erreurs et le m&eacute;canisme sous-jacent dans la maladie d&#39;Alzheimer sporadique (AD). Cortex entorhinal AD pr&eacute;sent&eacute; globalement alt&eacute;r&eacute;e exon exclusions et perte s&eacute;lective de la hnRNP A / B facteurs d&#39;&eacute;pissage. Soutenir la pertinence fonctionnelle, hnRNP A / knockdown B induite par des d&eacute;ficiences &eacute;pissage alternatifs et la perte de la dendrite dans des neurones primaires et d&eacute;ficiences de m&eacute;moire et electrocorticographic chez la souris. Des souris transg&eacute;niques pr&eacute;sentant des mutations associ&eacute;es &agrave; la maladie dans l&#39;APP ou Tau n&#39;affichent aucune alt&eacute;ration dans hnRNP A / B, sugg&eacute;rant que sa perte en AD est ind&eacute;pendant de toxicit&eacute; A&beta; et Tau. Cependant, excitation cholinergique a augment&eacute; A de hnRNP / B niveaux tandis qu&#39;in vivo neurotoxine induite par la destruction des neurones cholinergiques a provoqu&eacute; corticale AD-comme diminution hnRNP A / B et r&eacute;capitul&eacute; le patron &eacute;pissage alternatif des patients atteints de ma. Nos r&eacute;sultats pr&eacute;sentent hnRNP m&eacute;diation cholinergique A / B perte et m&eacute;tabolisme de RNA douteux comme des m&eacute;canismes importants impliqu&eacute;s dans AD.

Cholinergiques associ&eacute;es &agrave; la perte des hnRNP-A/B dans la maladie d&#39;Alzheimer alt&egrave;re l&#39;&eacute;pissage corticale et la fonction cognitive chez les souris.

Studi genetici link errori ereditati nel metabolismo del RNA malattia neurodegenerativa familiare. Riportiamo tali errori ed il meccanismo sottostante in sporadico della malattia di Alzheimer (AD). Cortecce entorinale annuncio presentato globalmente compromessa esone esclusioni e perdita selettiva di hnRNP A / B fattori di splicing. Supporto di pertinenza funzionale, hnRNP A / atterramento B indotta da menomazioni splicing alternativi e perdita di dendrite in neuroni primari e disturbi della memoria ed electrocorticographic nei topi. Topi transgenici con mutazioni associate a malattie APP o Tau non visualizzato alterazioni in hnRNP A / B, suggerendo che la sua perdita in annuncio &egrave; indipendente di tossicit&agrave; A&beta; e Tau. Tuttavia, eccitazione colinergico aumentato hnRNP A / B livelli mentre in vivo neurotossina-mediata distruzione dei neuroni colinergici causata corticale diminuzione simile AD hnRNP A / B e ricapitolato il pattern di splicing alternativo dei pazienti AD. I nostri risultati presentano hnRNP colinergica mediata A / B perdita e alterato metabolismo del RNA come meccanismi importanti coinvolti nell&#39;annuncio.

Geula Hanin

Department of Biological Chemistry and The Edmond & Lily Safra Center for Brain Sciences

The Hebrew University of Jerusalem

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

Geula Hanin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Chemistry and The Edmond & Lily Safra Center for Brain Sciences

The Hebrew University of Jerusalem

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

Department of Biological Chemistry and The Edmond & Lily Safra Center for Brain Sciences