Implication de deux nouveaux partenaires d'interaction de la Caspase-6, DAXX et STK3, dans le vieillissement et la maladie de Huntington

La neurodégénérescence fait partie intégrante de la maladie de Huntington (MH) dont les premiers symptômes moteurs et cognitifs apparaissent vers l’âge de 30 à 40 ans. Cette maladie incurable est causée par une mutation dans le gène codant pour la protéine huntingtin (htt). L’activation de la caspase-6 (casp6) est observée au stade présymptomatique chez l’humain et les modèles murins MH faisant de la casp6 un joueur majeur dans la neurodégénérescence précoce associé à la MH. De plus, le clivage de htt mutant par la casp6 produit un fragment N-terminal neurotoxique essentielle au développement de la MH. Des résultats préliminaires ont permis de révéler l’interaction et le clivage des protéines proapoptotiques Serine/Threonine Kinase 3 (STK3), Death-Domain Associated Protein (DAXX) par la casp6. Des effets proapoptotiques sont associés à leurs fragments et leur production par les caspases pourrait influencer la neurodégénérescence observée dans diverses maladies neurodégénératives et dans le vieillissement normal. Nos résultats dans les souris C57Bl/6 démontrent que l’expression de DAXX varie fortement avec l’âge selon l’organe analysé. Ses divers fragments ne suivent pas la même tendance d’un organe à l’autre suggérant des fonctions différentielles à travers l’organisme et une importante régulation de ses fonctions par des modifications post-traductionnelles. Chez les modèles murins de la MH, les souris YAC128, nous avons constaté une augmentation des fragments à 65 et 70 kDa dans le cortex et une diminution de DAXX entier et du fragment à 70 kDa dans le cervelet soulignant la possibilité de fonctions spécifiques selon les régions cérébrales. Nous avons aussi démontré pour la première fois le clivage de STK3 par la caspase-7 et la production différentielle de fragments par les caspase-3, 6 et 7. L’expression protéique de STK3 augmente globalement à travers l’organisme avec l’âge et dans le cervelet des souris YAC128. Par contre, une diminution de l’expression de STK3 est observée dans le cortex des individus atteints de la MH et des souris YAC128. Finalement, par l’induction de différents stress cellulaires, nous avons constaté la présence d’un mécanisme adaptatif des neurones modèles de la MH impliquant STK3. En conclusion, l’expression de DAXX et STK3 varie avec l’âge à travers l’organisme et est altérée dans la maladie de Huntington. Plus particulièrement, STK3semble être impliqué dans un mécanisme protégeant les neurones de la mort cellulaire dans la MH

Olfactory dysfunction associated with cognitive decline in an elderly population

Abstract: In many neurological disorders, including Alzheimer disease, early olfactory dysfunction is observed. In order to determine if deficits in olfactory memory are present in the elderly and if olfactory dysfunction correlates with cognitive impairment in the aging population, olfactory testing has been done on seniors from the NuAge cohort accepting to participate in the Olfactory Response Cognition and Aging (ORCA) secondary sub-study. The t-Mini Mental Statement Examination and the Telephone Interview for Cognitive Status tests were done to assess cognition levels. Overall, 94% of the ORCA cohort displayed olfactory dysfunction. Deficits in olfactory memory were also present. A correlation was observed between olfactory function and cognitive test scores. Moreover, in women who smoked, there was an association between olfactory memory and cognitive scores. Our results suggest that olfactory dysfunction may predict impending cognitive decline and highlights the need for olfactory training in seniors to improve olfaction and overall well-being

Implication de deux nouveaux partenaires d'interaction de la Caspase-6, DAXX et STK3, dans le vieillissement et la maladie de Huntington

La neurodégénérescence fait partie intégrante de la maladie de Huntington (MH) dont les premiers symptômes moteurs et cognitifs apparaissent vers l’âge de 30 à 40 ans. Cette maladie incurable est causée par une mutation dans le gène codant pour la protéine huntingtin (htt). L’activation de la caspase-6 (casp6) est observée au stade présymptomatique chez l’humain et les modèles murins MH faisant de la casp6 un joueur majeur dans la neurodégénérescence précoce associé à la MH. De plus, le clivage de htt mutant par la casp6 produit un fragment N-terminal neurotoxique essentielle au développement de la MH. Des résultats préliminaires ont permis de révéler l’interaction et le clivage des protéines proapoptotiques Serine/Threonine Kinase 3 (STK3), Death-Domain Associated Protein (DAXX) par la casp6. Des effets proapoptotiques sont associés à leurs fragments et leur production par les caspases pourrait influencer la neurodégénérescence observée dans diverses maladies neurodégénératives et dans le vieillissement normal. Nos résultats dans les souris C57Bl/6 démontrent que l’expression de DAXX varie fortement avec l’âge selon l’organe analysé. Ses divers fragments ne suivent pas la même tendance d’un organe à l’autre suggérant des fonctions différentielles à travers l’organisme et une importante régulation de ses fonctions par des modifications post-traductionnelles. Chez les modèles murins de la MH, les souris YAC128, nous avons constaté une augmentation des fragments à 65 et 70 kDa dans le cortex et une diminution de DAXX entier et du fragment à 70 kDa dans le cervelet soulignant la possibilité de fonctions spécifiques selon les régions cérébrales. Nous avons aussi démontré pour la première fois le clivage de STK3 par la caspase-7 et la production différentielle de fragments par les caspase-3, 6 et 7. L’expression protéique de STK3 augmente globalement à travers l’organisme avec l’âge et dans le cervelet des souris YAC128. Par contre, une diminution de l’expression de STK3 est observée dans le cortex des individus atteints de la MH et des souris YAC128. Finalement, par l’induction de différents stress cellulaires, nous avons constaté la présence d’un mécanisme adaptatif des neurones modèles de la MH impliquant STK3. En conclusion, l’expression de DAXX et STK3 varie avec l’âge à travers l’organisme et est altérée dans la maladie de Huntington. Plus particulièrement, STK3semble être impliqué dans un mécanisme protégeant les neurones de la mort cellulaire dans la MH

LDLR-related protein 10 (LRP10) regulates amyloid precursor protein (APP) trafficking and processing: evidence for a role in Alzheimer’s disease

<p>Abstract</p> <p>Background</p> <p>The Aβ peptide that accumulates in Alzheimer’s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by β- and γ-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent Aβ production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and Aβ production.</p> <p>Results</p> <p>In this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into Aβ, while knockdown of LRP10 expression increases Aβ production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP β-cleavage into Aβ. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.</p> <p>Conclusions</p> <p>The present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer’s disease.</p

Pharmacist-led transitions of care for older adults at risk of drug-related problems : a feasibility study

Background: Transitions of care (TOC) is one of three key action areas identified in the World Health Organization (WHO)’s third Global Patient Safety Challenge, Medication Without Harm, released in 2017. Systematic reviews have shown that TOC interventions can improve health outcomes, although few studies have evaluated the role of the community pharmacist. Objective: To evaluate the feasibility of a pharmacist-led TOC intervention for older adults at risk of drug-related problems. Methods: Pragmatic feasibility study conducted in hospital and community pharmacies in a health region of Quebec, Canada. The interventions consisted of a pharmaceutical care plan developed by the hospital pharmacist and transferred at hospital discharge to the patients’ community pharmacist, who completed patient consultations in the week following discharge and monthly for six months thereafter. Feasibility evaluations included recruitment, retention, time required, types of interventions, and modified classes of medications, based on clinical data entered in an electronic health record accessible to clinicians in all settings. Results: Of the 90 recruited patients, 76 were discharged with a pharmaceutical care plan. The mean age of these 76 subjects was 79.5 years, and 52.6% were female. The most frequent inclusion criteria were 15 or more medications (57.9%), two or more emergency department visits (past three months), or one or more hospitalization (past twelve months) (42.1%). The hospital pharmacist interventions took a mean time of 222 min. The community pharmacist interventions took a mean time of 52 min and 32 min for the first and subsequent visits, respectively. Therapeutic goals were documented for 60.5% of patients. Conclusions: This study shows the feasibility of implementing a pharmacist-led TOC intervention in the Canadian context. Development of the TOC model in three health regions is currently being pursued along with the inclusion of primary care clinics who recently added pharmacists to their interdisciplinary teams

Frameshift indels introduced by genome editing can lead to in-frame exon skipping

<div><p>The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of “multiple of three nucleotides” exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a “multiple of three nucleotides” exon of <i>LGALS8</i> that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.</p></div

Exon skipping in <i>LGALS8</i> in human erythroblasts.

<p>(<b>A</b>) In <i>in vitro</i> differentiated human erythroblasts, three <i>LGALS8</i> mRNA isoforms are expressed. Isoform 1 includes the “multiple of three nucleotides” exon 9 (in red, 126-bp), whereas isoforms 2 and 3 do not. The nonsense variant rs2273865 (p.Leu212Ter) is located in exon 9. At this variant, the minor A-allele has a frequency of 3.5% in populations of European ancestry (ExAC). (<b>B</b>) Eight erythroblast samples are heterozygous at rs2273865 and show strong allelic imbalance (binomial <i>P</i><0.05 for all samples). Numbers in the bars indicate the numbers of reads carrying the T (green) or A (blue) allele. Differential expression of total <i>LGALS8</i> (<b>C</b>), isoform 1 (<b>D</b>), isoform 2 (<b>E</b>), and isoform 3 (<b>F</b>) between erythroblast samples homozygous TT (n = 16) and heterozygous AT (n = 8) at rs2273865. No samples homozygous for the minor allele (AA) were available. (<b>G</b>) The ratio of <i>LGALS8</i> transcripts without exon 9 over transcripts with exon 9 is higher in heterozygous AT than in homozygous TT erythroblast samples. <i>P</i>-values are calculated by linear regression correcting for cell developmental stage.</p

Frameshift indels cause in-frame exon skipping in <i>PHACTR1</i>.

<p>(<b>A</b>) <i>PHACTR1</i> expression levels measured by real-time qPCR in the parental teloHAEC cell line, an unedited clone (sg-E8N23), and clones with CRISPR-Cas9-generated frameshift indels in <i>PHACTR1</i> exon 8 (sg-E8N2 and sg-E8N16), exon 9 (sg-E9N1), and exon 10 (sg-E10N8). Data show mean and standard error of the mean from two biological replicates, done in triplicates. <i>PHACTR1</i> expression levels in sg-E8N2 is 6.2 fold greater than in the parental teloHAEC cell line (Student’s <i>t</i>-test <i>P</i> = 0.0033). (<b>B</b>) Agarose gel electrophoresis profile of the main <i>PHACTR1</i> isoforms detected in cDNA from teloHAEC cells, unedited clones, or clones with a frameshift indel. We assigned a transcript number to each of the <i>PHACTR1</i> isoform that we could Sanger sequence and align to the reference sequence. Unlabeled bands could not be assigned to <i>PHACTR1</i>. Bands in the molecular ladder correspond to 400, 500 and 700-bp. This gel is representative of three independent experiments. (<b>C</b>) Schematic diagram of all the <i>PHACTR1</i> isoforms that we identified in the different teloHAEC cell lines. Transcript numbers correspond to the bands (white numbers) in <b>B</b>. The PCR primers in exon 6 and 11 are depicted. For the isoforms expressed in edited clones, we added the corresponding nucleotide changes introduced by the frameshift indels. (<b>D</b>) Western blot of PHACTR1 in the parental, unedited and edited teloHAEC cells. The arrowhead indicates PHACTR1, lower bands are non-specific proteins recognized by the antibody. PHACTR1 is smaller in sg-E8N2, consistent with skipping of exon 8 or usage of an alternative in-frame start codon downstream of the frameshift indel. For sg-E9N1, the smaller PHACTR1 protein is consistent with skipping of exon 9. We could not detect PHACTR1 proteins in sg-E8N16 and sg-E10N8. We used GAPDH as loading control. This Western blot is representative of three independent experiments.</p

Frameshift indel can lead to exon skipping in the zebrafish <i>adgrl4</i> gene.

<p>(<b>A</b>) <i>adgrl4</i> genomic locus, TALEN target site in exon 2 of the <i>adgrl4</i> gene and a stable mutant line (Δ5) that was analyzed. (<b>B</b>) PCR analysis of <i>adgrl4</i> mRNA transcripts in 10 to 15 pooled embryo samples from control (ctrl), <i>adgrl4</i> Δ5^+/-, and <i>adgrl4</i> Δ5^-/- fishes. (<b>C</b>) Schematic representation of the different transcripts recovered from the bands (white numbers) in <b>B</b>.</p