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Picking the best isoform: PDE4D isoforms as therapeutic targets in Alzheimer's disease
No full textDe doelstellingen van deze thesis waren 1) het identificeren welke PDE4Disovormen betrokken zijn bij de processen van neuroplasticiteit die door ADgerelateerde pathologie aangedaan worden, en 2) het onderzoeken van het
therapeutische potentieel van gecombineerde behandelingen gericht op het
verlagen van de therapeutische dosis van PDE4(D)-inhibitoren. In het kader van deze
doelstellingen worden in deze thesis een uitgebreide literatuurreview uiteengezet,
alsmede vier experimentele studies waarin gebruik is gemaakt van in silico, in vitro
en in vivo-methoden.
Om te kunnen identificeren welke PDE4D-isovormen betrokken zijn bij de
processen van neuroplasticiteit die aangetast worden door AD-gerelateerde
pathologie, wordt in Hoofdstuk 2 een uitgebreid overzicht gepresenteerd die de
moleculaire biologie van PDE4-enzymen uiteenzet en het potentieel van deze
enzymen als effectief farmacologisch doelwit. In dit hoofdstuk wordt beschreven dat
PDE4-enzymen door vier verschillende genen worden gecodeerd (PDE4A-D) die elk,
door middel van alternatieve promoters en alternatieve splicing, meerdere
isovormen tot expressie kunnen brengen. Bij het refereren naar deze isovormen kan
er onduidelijkheid ontstaan doordat er verschillen bestaan in de gebruikte
nomenclatuur binnen online databases, binnen diersoorten en of er wordt
gerefereerd naar het mRNA of het eiwit van de isovorm. Derhalve is een overzicht
van alle tot dusver bekende isovormen samengesteld (pagina 41) samen met een
vergelijking van humane isovormen en isovormen bij knaagdieren (pagina 126) ter
verduidelijking van de nomenclatuur en classificatie voor het wetenschappelijke veld
en om makkelijker te kunnen refereren naar de veelvuldige isovormen in de volgende
hoofdstukken. In dit hoofdstuk wordt ook samengevat hoe verschillen in
eiwitsequentie tussen PDE4-subtypen en -isovormen leiden tot verschillende
eigenschappen met betrekking tot regulatie van enzymactiviteit, eiwiteiwitinteracties en het binden van inhibitoren door veranderingen in de conformatie
van het enzym. Welke aminozuren betrokken zijn bij alle in de literatuur beschreven post-translationele modificaties en interacties met andere eiwitten is weergegeven
in de figuur op pagina 51. Eveneens wordt het effect van deze modificaties en
interacties op enzymatische activiteit van PDE4-subtypen of -isovormen en PDE4-
inhibitoraffiniteit beschreven. Dit overzicht heeft de aanleiding gevormd om een
grondige analyse uit te voeren naar de wijze waarop de affiniteit van een PDE4-
inhibitor wordt bepaald. Daartoe is een uitgebreid overzicht van PDE4-inhibitoren
samengesteld waarbij affiniteit wordt weergegeven per PDE4-subtype (pagina 69),
isovorm (pagina 75) en conformatie (pagina 79). Tenslotte worden de potentiële
onderliggende mechanismen beschreven die geassocieerd worden met de negatieve
bijwerkingen door PDE4-inhibitie en hoe deze zich verhouden tot therapeutische
doses van verscheidene PDE4-inhibitoren.
Vervolgens, gebruikmakend van de opgedane inzichten omtrent humane
PDE4D isovorm sequenties, werden promoter DNA (hydroxy)methylatie en mRNA
expressie van PDE4D isovormen gemeten in post-mortem hersenmateriaal van de
middelste slaapwending van AD patiënten en gezonde controles (Hoofdstuk 3).
Specifieke isovormen (PDE4D1, -D3, -D5 en -D8) werden geïdentificeerd die een
verhoogde expressie in AD lieten zien, welke geassocieerd was met veranderingen
in het DNA methylatieprofiel van de corresponderende promoter-regio’s. Bovendien
was verhoogde PDE4D1 en PDE4D3 expressie geassocieerd met verhoogde niveaus
van amyloïde plaques en neurofibrillaire tangles en een lager cognitief vermogen.
Deze studie toonde aan dat, hoewel isovormen voortkomen uit hetzelfde gen,
expressieregulatie van PDE4D in AD wordt beïnvloed op een isovorm-specifieke
wijze.
Om voort te borduren op de gedane bevindingen in humaan materiaal en
deze bevindingen aan te vullen werden er in Hoofdstuk 4 experimenten verricht
gebruikmakend van diermodellen en cellijnen. In overeenstemming met de
observaties in human weefsel werd er een expressieverhoging van dezelfde PDE4D
isovormen gevonden in hersenmateriaal van transgene AD muizen die β-amyloïd pathologie vertonen alsmede in gekweekte hippocampale neuronen blootgesteld
aan β-amyloïd. Het idee dat farmacologische inhibitie van PDE4D neuronale
plasticiteit kan bevorderen werd gevalideerd in dezelfde muriene hippocampale
neuroncellijn. Gebruikmakend van CRISPR-Cas9 gemedieerde genbewerking werd in
dezelfde cellijn bepaald dat specifieke knock-down van één van de lange PDE4D
isovormen (i.e. PDE4D3, PDE4D5, PDE4D7 en PDE4D9) voldoende is om neuronale
plasticiteitsprocessen te verbeteren. Bovendien kon knock-down van één van deze
isovormen bescherming verschaffen tegen het plasticiteit-aantastende effect van βamyloïd. Ten slotte gaf dit hoofdstuk, door het beschrijven van isovorm-specifieke
expressie in de muizenmaag, aan welke van de lange PDE4D isovormen wellicht niet
betrokken zijn bij het induceren van maag-geassocieerde bijwerkingen van PDE4(D)
inhibitie.
Aangezien het werd gevonden dat knock-down van lange PDE4D isovormen
neuronale plasiticteit bevordert, kon het inzicht in de moleculaire biologie van de
verschillende PDE4D isovormen zoals beschreven in Hoofdstuk 2 gebruikt worden
om deze bevindingen in een mechanistisch perspectief te plaatsen. De
enzymactiviteit van PDE4D isovormen wordt differentieel beïnvloed door
fosforylering door ‘protein kinase A’ (PKA) en/of ‘extracellular signal-regulated
kinase’ (ERK) afhankelijk van de isovorm-categorie (i.e. lang, kort en superkort). Om
te begrijpen waarom genetische knock-down van specifiek lange isovormen
neuronale plasticiteit bevorderde, werd er een in silico computationeel model
gecreëerd om te simuleren hoe de verschillende isovorm-categorieën cyclisch
adenosine monofosfaat (cAMP) niveaus reguleren en hoe deze de vervolgsignalering
beïnvloeden gedurende de tijd (Hoofdstuk 5). In dit hoofdstuk werd beschreven dat
specifiek lange PDE4D isovormen het grootste effect uitoefenen op cAMP
signalering en de signalering die daarop volgt. De experimentele data uit het vorige
hoofdstuk wordt hierdoor ondersteund en daardoor kan gesuggereerd worden dat inhibitie van lange PDE4D isovormen de meest effectieve aanpak is om de cAMP
signalering die betrokken is in neuronale plasticiteitsprocessen te bevorderen.
Betreffende de eerste doelstelling van deze thesis kan inhibitie van specifieke
PDE4D isovormen betrokken bij (AD-geassocieerde) geheugenprocessen een
effectievere en/of veiligere behandelstrategie verschaffen. Naast deze aanpak kan de
veiligheid van PDE4(D) inhibitoren ook verbeterd worden door het verlagen van de
therapeutische dosis om het risico op nadelige bijwerkingen te reduceren. In lijn met
de tweede doelstelling van deze thesis, laat Hoofdstuk 6 het potentieel zien van het
combineren van PDE2 en PDE4 inhibitoren om neuroplasticiteit in vitro en
geheugenvorming in vivo te stimuleren. Aangezien werd bevonden dat PDE2 en
PDE4 synergistische acties vertonen, zouden therapeutische doses van iedere
inhibitor verlaagd kunnen worden wat er vervolgens voor zou kunnen zorgen dat het
risico op PDE4-gemedieerde nadelige bijwerkingen geminimaliseerd wordt.
Concluderend, het werk in deze thesis heeft de potentie van PDE4D isovormspecifieke inhibitie en synergistische PDE4/PDE2 inhibitie vastgesteld als nieuwe
strategieën om behandelingen gericht op PDE4 doeltreffender en/of veiliger te
maken ten bate van de behandeling van geheugenproblemen in AD.The goals of this thesis were 1) to identify which PDE4D isoforms are involved
in neuroplasticity processes that are impaired by AD-associated pathology, and 2) to
investigate the therapeutic potential of using combined treatments to reduce the
therapeutic dose of PDE4(D) inhibitors. In light of these goals, this thesis presents a
comprehensive literature review and four experimental studies using in silico, in vitro,
and in vivo approaches.
In order to identify which PDE4D isoforms are involved in neuroplasticity
processes that are impaired by AD-associated pathology, an extensive overview was
established on the molecular biology of PDE4 enzymes and their utility as
pharmacological targets in Chapter 2. It was outlined that PDE4 enzymes are
encoded by four different genes (PDE4A-D) that each give rise to multiple isoforms
owing to the use of alternative promoters and alternative splicing. Confusion can
arise when referring to these isoforms as their nomenclature may differ across online
databases, species and depending on whether one refers to the isoform’s mRNA or
protein. Hence, an overview figure of all known isoforms (page 41) and a comparison
of human and rodent isoforms (page 126) were established to clarify the
nomenclature and classification to the scientific field and to easily refer to the
multitude of isoforms in upcoming chapters. This chapter also summarized how
differences in protein sequence across PDE4 subtypes and isoforms result in different
properties in view of enzymatic activity regulation, protein-protein interactions and
inhibitor binding by changing the enzyme’s conformation. The amino acids involved
in all reported post-translational modifications and interactions with other proteins
were illustrated in an overview figure (page 51). Moreover, the effect of these
modifications and interactions on enzymatic activity of PDE4 subtypes or isoforms
and their affinity to PDE4 inhibitors was reported. This overview provided the
rationale to establish a thorough analysis of the approaches to determine affinities
of PDE4 inhibitors. Therefore, a comprehensive list of PDE4 inhibitors was compiled
indicating affinity per PDE4 subtype (page 69), isoform (page 75) or conformation (page 79). Lastly, potential mechanisms underlying the adverse side effects
associated with PDE4 inhibition were described in relation to the therapeutic dose
ranges of several PDE4 inhibitors.
Next, using the gained knowledge on human PDE4D isoform sequences,
promoter DNA (hydroxy)methylation and mRNA expression of PDE4D isoforms were
measured in post-mortem material of the middle temporal lobe of AD patients and
healthy controls (Chapter 3). Specific isoforms (PDE4D1, -D3, -D5, and -D8)
displaying increased expression in AD were identified, concomitant with changes in
the DNA methylation signatures of associated promoter regions. Moreover,
increased PDE4D1 and PDE4D3 expression was associated with higher levels of
plaque and tangles and lower cognitive performance. This study indicated that,
although stemming from the same gene, expression regulation of PDE4D is impacted
in an isoform-specific manner in AD.
To follow up on and complement these findings using human material,
studies using animal models and cell lines were conducted in Chapter 4. In
correspondence to the observations in human tissue, an upregulation of the same
PDE4D isoforms was found in brain material of transgenic AD mice that exhibit
amyloid-β pathology as well as in cultured hippocampal neurons exposed to
amyloid-β. The notion that pharmacological PDE4D inhibition can promote neuronal
plasticity was validated in the same mouse hippocampal neuronal cell line. Making
use of CRISPR-Cas9 mediated gene-editing, it was determined in the same cell line
that specific knockdown of one of the long PDE4D isoforms (i.e. PDE4D3, PDE4D5,
PDE4D7, and PDE4D9) is sufficient to enhance neuronal plasticity processes.
Moreover, knockdown of one of these isoforms could protect against plasticityimpairing effect of amyloid-β. Lastly, this chapter indicated which of the long PDE4D
isoforms may not be involved in stomach-associated side effects of PDE4(D)
inhibition by describing the isoform-specific expression in the mouse stomach. Since knockdown of long-form PDE4D isoforms specifically was found to
promote neuronal plasticity, the understanding of the molecular biology of the
different PDE4D isoforms described in Chapter 2 could be used to put these findings
into a mechanistic perspective. The enzyme activity of PDE4D isoforms is
differentially altered by phosphorylation by protein kinase A (PKA) and/or
extracellular signal-regulated kinase (ERK) depending on the isoform category (i.e.
long, short, and supershort). In order to understand why genetic knockdown of long
isoforms specifically enhanced neuronal plasticity, an in silico computational model
was established to simulate how the different isoform categories regulate cyclic
adenosine monophosphate (cAMP) levels and control downstream signaling over
time (Chapter 5). Here, it was described that long PDE4D isoforms specifically exert
the largest control on cAMP and its downstream signaling. Hence, supporting the
experimental data of the previous chapter, inhibition of long PDE4D isoforms may
provide the most profound approach to increase cAMP signaling involved in
neuronal plasticity processes.
In view of the first goal of this thesis, inhibition of specific PDE4D isoforms
involved in (AD-associated) memory processes could provide a more efficacious
and/or safer therapeutic strategy. Besides this approach, the safety of PDE4(D)
inhibitors can be improved by lowering the required therapeutic dose to reduce the
risk of adverse side effects. Following up on the second goal of this thesis, Chapter
6 demonstrates the potential of combining PDE2 and PDE4 inhibitors to promote
neuroplasticity in vitro and stimulate memory consolidation in vivo. As it was found
that PDE2 and PDE4 inhibitors exert synergistic actions, the therapeutic doses of each
inhibitor could be lowered, which would minimize the risk of PDE4-mediated adverse
side effects.
Concludingly, the work in this thesis has established the potential of PDE4D
isoform-specific inhibition and synergistic PDE4/PDE2 inhibition as new strategies to make PDE4-targeting therapies more efficacious and/or safer for the benefit of
treating memory deficits in AD
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Pharmacological inhibition of phosphodiesterase 7 enhances consolidation processes of spatial memory
No full textAugmentation of cAMP signaling through inhibition of phosphodiesterases (PDE) is known to enhance plasticity and memory. Inhibition of PDE4 enhances consolidation into memory, but less is known about the role of other cAMP specific PDEs. Here, we tested the effects of oral treatment with a selective inhibitor of PDE7 of nanomolar potency on spatial and contextual memory. In an object location task, doses of 0.3-3 mg/kg administered 3 h after training dose-dependently attenuated time-dependent forgetting in rats. Significant enhancement of memory occurred at a dose of 3 mg/kg with corresponding brain levels consistent with PDE7 inhibition. The same dose given prior to training augmented contextual fear conditioning. In mice, daily dosing before training enhanced spatial memory in two different incremental learning paradigms in the Barnes Maze. Drug treated mice made significantly less errors locating the escape in a probe-test 24 h after the end of training, and they exhibited hippocampal-dependent spatial search strategies more frequently than controls, which tended to show serial sampling of escape locations. Acquisition and short-term memory, in contrast, were unaffected. Our data provide evidence for a role of PDE7 in the consolidation of hippocampal-dependent memory. We suggest that targeting PDE7 for memory enhancement may provide an alternative to PDE4 inhibitors, which tend to have undesirable gastrointestinal side-effects
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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