PFN1 phosphorylation marks protein aggregation and white matter pathology in ALS

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is the most common form of motor neuron disease. In familial ALS, Multiple mutations of, PFN1 gene a well-known actin-binding protein have been linked to ALS disease recently. Phosphorylation in many degenerative conditions plays an important role in disease mechanism but its potential role in ALS remains not fully understood. We sought to look further into not previously studied phosphorylation of PFN1 as a potential contributor to aggregation and toxicity in ALS. Using different histochemistry and cytochemistry and molecular biology approaches, we observed that phosphorylation on Profilin shows a very distinctive pattern in PFN1C71G andSOD1G93A disease models. This modification is abundantly found in both astrocytes and white matter which latter indeed marks a staining pattern that is indistinguishable between two ALS mice model compared to controls. Interestingly, pPFN1 reactive areas colocalized with Myelin in the spinal cord are frequently found in the proximity of CD68 positive macrophages. Moreover, biochemical fractionation using ultracentrifugation detects endogenous pPFN1 in the highly insoluble fraction of protein lysate from both PFN1C71G andSOD1G93A model. Finally, a similar staining pattern to the ALS mice model was also observed in human sporadic ALS cases. Overall, our results suggest for the first time a role for phosphorylation of PFN1 in protein aggregation and white matter pathology in ALS that will shed more light on the mechanism of disease and developing potential therapeutics in near future

Experimental Verification of a Predicted Intronic MicroRNA in Human NGFR Gene with a Potential Pro-Apoptotic Function

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells

miRNAs as therapeutic agents in neurodegeneration : a pilot study

L’échec des différents essais cliniques souligne la nécessité de développer des nouvelles thérapies pour la maladie d'Alzheimer (MA), la cause la plus commune de démence. Les microARNs (miARNs) sont les ARNs non-codants les plus étudiés et ils jouent un rôle important dans la modulation de l'expression des gènes et de multiples voies de signalisation. Des études antérieures, dont celles de mon laboratoire d’accueil, ont permis de développer l’hypothèse que certains membres de la famille miR-15/107 (c.-à-d. miR-15ab, miR-16, miR-195, miR-424, and miR-497) pourraient être utilisés comme agents thérapeutiques dans MA. En effet, cette famille avait le potentiel de réguler de multiples gènes associés à MA, tels que la protéine précurseur de l'amyloïde (APP), la β-secrétase (BACE1), et la protéine Tau. Tel que démontré dans ce projet de thèse, j’ai choisi miR-16 comme cible thérapeutique potentielle pour MA parmi tous les membres de la famille. L’essai luciférase dans ce projet confirme que miR-16 peut réguler simultanément APP et BACE1, directement par une interaction avec la région non-codante en 3’ de l’ARNm). Notamment, nous observons aussi une réduction de la production des peptides amyloïdes et de la phosphorylation de Tau après une augmentation de miR-16 en cellule. J’ai ensuite validé mes résultats in vivo dans la souris en utilisant une méthode de livraison de miR-16 via une pompe osmotique implanté dans le cerveau. Dans ce cas, l'expression des protéines d’intérêts (APP, BACE1, Tau) a été mesurée par immunobuvardage et PCR à temps réel. Après validation, ces résultats ont été complémentés par une étude protéomique (iTRAQ) du tronc cérébral et de l'hippocampe, deux régions associées à la maladie. Ces données m’ont permis d’identifier d'autres protéines régulées par miR-16 in vivo, incluant α-Synucléine, Transferrine receptor1, et SRm300. Une autre observation intéressante : les voies régulées par miR-16 in vivo sont directement en lien avec le stress oxydatif et la neurodégénération. En résumé, ce travail démontre l’efficacité et la faisabilité d’utiliser un miARN comme outil thérapeutique pour la maladie d’Alzheimer. Ces résultats rentrent dans un cadre plus vaste de découvrir de nouvelles cibles pour MA, et en particulier la forme sporadique de la maladie qui représente plus de 95% de tous les cas. Évidemment, la découverte d’une molécule pouvant cibler simultanément les deux pathologies de la maladie (plaques amyloïdes et hyper phosphorylation de tau) est nouvelle et intéressante, et ce domaine de recherche ouvre la porte aux autres petits ARNs non-codants dans MA et les maladies neurodégénératives connexes.Failure at different clinical trials emphasizes the need for developing new therapeutics for Alzheimer disease (AD) as the most common cause of dementia. MicroRNAs (miRNA) are the most studied groups of non-coding RNAs and have a critical role in modulating multiple signaling pathways and fine-tuning gene expression. Supporting evidence from other studies, including host lab, suggest that multiple members of the miR-15/107 family (miR-15ab, miR-16, miR-195, miR-424, and miR-497) could be used as therapeutic agents in AD. The potential ability of this miRNA family to modify disease pathway by multiple targeting of AD-associated genes such as Amyloid precursor protein (APP), β-site amyloid-β precursor protein cleaving enzyme (BACE1) and microtubule-associated protein Tau is of attention. Based on documented results in this study I chose miR-16 as candidate therapeutic miRNA in AD. This choice is based on data obtain from cells and in vitro luciferase assay indicating the role of this miRNA in the simultaneous regulation of APP, BACE1 (directly by targeting 3’UTR of these genes). Decrease in Tau phosphorylation and amyloid beta peptides were further observed following increased miR-16 levels. Furthermore, I validated these results in vivo by delivering miR-16 oligos using Osmotic pumps implanted subcutaneously to deliver oligos to lateral ventricles of mouse brain also providing a wide distribution of these oligos. Expression of desired protein targets was measured by western blot and qPCR in different brain regions. Results demonstrated a context-dependent action of delivered miR-16 increase on the potential AD involved targets in mouse brain. These results were complemented by proteomics study of Brainstem and Hippocampus regions. Data indicated the potential regulation of other proteins by miR-16 in vivo such as α-Synuclein in Brainstem and Transferrin receptor1 and SRm300 in Hippocampus. The increase in miR-16 levels in vivo and in vitro was sufficient to downregulate the protein product of these genes confirmed by western blot. Enrichment study predicted oxidative stress and neurodegeneration as top terms in close connection with miR-16. This work provided a proof-of-principle for possibility and efficiency of miRNA replacement based therapeutics delivered to CNS using miR-16 a member of the miR-15/107 family. Understanding the molecular mechanisms involved in the regulation of AD-related genes could have important implications for sporadic AD, which accounts for more than 95% of all cases with no effective therapy available. Multi-target therapy by non-coding RNA in AD is an emerging concept that would have the potential to change the way that therapeutics is developed for AD and other neurodegenerative diseases with complex nature and no effective therapy available

Preclinical Evaluation of miR-15/107 Family Members as Multifactorial Drug Targets for Alzheimer's Disease

Alzheimer's disease (AD) is a multifactorial, fatal neurodegenerative disorder characterized by the abnormal accumulation of Aβ and Tau deposits in the brain. There is no cure for AD, and failure at different clinical trials emphasizes the need for new treatments. In recent years, significant progress has been made toward the development of miRNA-based therapeutics for human disorders. This study was designed to evaluate the efficiency and potential safety of miRNA replacement therapy in AD, using miR-15/107 paralogues as candidate drug targets. We identified miR-16 as a potent inhibitor of amyloid precursor protein (APP) and BACE1 expression, Aβ peptide production, and Tau phosphorylation in cells. Brain delivery of miR-16 mimics in mice resulted in a reduction of AD-related genes APP, BACE1, and Tau in a region-dependent manner. We further identified Nicastrin, a γ-secretase component involved in Aβ generation, as a target of miR-16. Proteomics analysis identified a number of additional putative miR-16 targets in vivo, including α-Synuclein and Transferrin receptor 1. Top-ranking biological networks associated with miR-16 delivery included AD and oxidative stress. Collectively, our data suggest that miR-16 is a good candidate for future drug development by targeting simultaneously endogenous regulators of AD biomarkers (i.e., Aβ and Tau), inflammation, and oxidative stress

mir-6165 overexpression in U87 cell line induces apoptosis.

<p>A) PI staining of U87 cells 34 hours post transfection was done to investigate the effect of mir-6165 on cell cycle. A dramatic change was observable toward sub-G1 stage in the cells overexpressing mir-6165 compared to negative controls (a′- d′).B) Annexin-PI staining of the U87 cells overexpressing mir-6165 shows, the most of the cells have entered early apoptosis stage compared to negative control and the result is consistent with PI staining in the previous section (a″- d″). The gate setting distinguished between living (bottom left), necrotic (top left), early apoptotic (bottom right), and late apoptotic (top right) cells. Repeated Measures ANOVA analysis shows that the changes observed in flow cytometry of U87 cells is extremely significant (p<0.05) between negative controls and the cells overexpressing mir-6165 (e″).</p

Prediction of pre-mir-6165 within the 4^th intron of human NGFR gene.

<p>A) Position of predicted hairpin structure within the human NGFR gene is shown in the 4^th intorn. This hairpin is predicted to produce Hsa-mir-6165 which is shown as red colored sequence on the stem loop. B) Prediction of Drosha enzyme 5' and 3' cutting sites on the sequence of stem loop by Microprocessor SVM. C) Blat search result shows a strong conservation of Hsa-mir-6165 between human, rhesus, dog and elephant.</p

Additional file 1 of Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Additional file 1: Table S1. List of Primary Antibodies used in the Study. Table S2. List of Secondary Antibodies used in the Study

Detection of Hsa-mir-6165 in the brain derived cell lines and biopsies.

<p>A) NGFR and mir-6165expression profile in some glioma cell lines is compared to non-glioma NT2 cell line Daoy, 1321N1, U87 (glioma cell lines) and NT2 (non glioma cell line) were used for detection of Hsa-mir-6165 expression. U48 small neucleolar RNA was used as internal control for the amplifications. In glioma cell lines Hsa-mir-6165 expression level was higher than NT2 cell line. B) Relative, Hsa-mir-6165 and its precursor expression levels in various human glioma tissue samples. The expression level of Hsa-mir-6165 in the tumor samples were compared to the lowest grade of tumors. U48 small nucleolar RNA gene (SNORD48) was used for normalizing the expression levels. Error bars indicate standard deviation (SD) of duplicate experiments. Pearson’s test confirmed a positive correlation between NGFR and its intronic miRNA (p = 0.0065). In all of the high grades (HG) tissue samples, the level of NGFR and mir-6165 were higher than the low grad (LG) samples.</p

Additional file 2 of Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Additional file 2: Figure S1. Citrullinated proteins accumulate as foci in reactive astroglia (dotted circles) in early disease stages and become widespread in late disease stages in ALS mouse models. (A, B) Double immunofluorescence staining for GFAP and citrullinated protein in ventral horn spinal cords of SOD1G93A and PFN1C71G mice, respectively. Figure S2. PC is not increased in microglia in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for IBA1 and citrullinated proteins in the ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for IBA1 and citrullinated proteins in the ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. Figure S3. PCs are not increased in oligodendrocytes in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for Olig2 and citrullinated proteins in ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for Olig2 and citrullinated proteins in the ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. Figure S4. PAD2 expression is increased progressively in astrocytes in the spinal cord white matter of ALS mouse models. (A) Double immunofluorescence staining for GFAP and PAD2 in spinal cord white matter in SOD1G93A mice. (B, C) Quantification of fluorescent intensity of GFAP and PAD2, respectively, in (A). (D) Double immunofluorescence staining for GFAP and PAD2 in spinal cord white matter in PFN1C71G mice. (E, F) Quantification of fluorescent intensity of GFAP and PAD2, respectively, in (D). The ages of nTg mice, n, and statistics are as described in Fig. 2. Figure S5. PAD2 and citrullinated proteins are colocalized in astrocytes but not in aggregates in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for PAD2 and citrullinated proteins in the ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for PAD2 and citrullinated proteins in the ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. Arrowheads point to PAD2-negative but citrulline-positive protein aggregates. The ages of nTg mice are as described in Fig. 2. Figure S6. PAD2 expression is not increased in microglia in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for IBA1 and PAD2 in ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for IBA1 and PAD2 in ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. The blue color in the merge panel represents the nucleus as stained by DAPI. Figure S7. PAD2 expression is not increased in oligodendrocytes in the spinal cord of ALS mouse models. (A, B) Double immunofluorescence staining for Olig2 and PAD2 in ventral horn gray matter of SOD1G93A and PFN1C71G mice, respectively. (C, D) Double immunofluorescence staining for Olig2 and PAD2 in ventral lateral white matter of SOD1G93A and PFN1C71G mice, respectively. The ages of nTg mice are as described in Fig. 2. The blue color in the merge panel represents the nucleus as stained by DAPI. Figure S8. Disease-specific protein aggregates are observed in spinal cords from ALS mouse models. (A) Filter trap assay and quantification of mutant SOD1 protein aggregation. (B) Filter trap assay and quantification of mutant PFN1 protein aggregation. n = 4 in each group. Statistics: unpaired t-test for comparing transgenic mice with their age matched controls. *p < 0.05. ****p < 0.0001. Figure S9. Microscopy and Image processing workflow for quantitative analyses of immunofluorescence-stained spinal cord sections. (A) An example for quantifying PAD2 fluorescent intensity in astrocytes in ventral horn gray matter of a paralyzed PFN1C71G mouse. GFAP was used as a marker to identify astrocytes. The GFAP (red) image was thresholded using ImageJ, and GFAP-positive areas were identified as regions of interest (ROI). The ROI was overlayed on the corresponding immunostained PAD2 (green) image, and PAD2 fluorescent intensity was quantified exclusively within the defined ROI. (B) As in (A) but showing white matter. (C) An example of quantifying citrullinated protein fluorescent intensity in axons of nTg spinal cord white matter. NF-L was used as a marker to identify axons. The NF-L (Green) image was thresholded and NF-L-positive areas were identified as ROI. These ROIs were overlayed on the immunostained citrullinated proteins (red) image, and citrullinated protein fluorescent intensity was quantified exclusively within the defined ROI. (D) Same as (C) but in white matter of a paralyzed PFN1C71G mouse. Figure S10. Procedure for colocalization analysis in the spinal cord of ALS mouse models. Levels of colocalization between citrullinated protein aggregates and other proteins were analyzed using JACoP plugin in ImageJ. (A) PLP- and citrullinated protein-stained images (top) were thresholded to remove background and other weakly stained signals (bottom). (B) The Costes’ mask was generated showing colocalization (white), background (black), PLP (red), and citrullinated proteins (green). The result (colocalization) was expressed as Manders’ overlap coefficient (M2)

mir-6165 overexpression in Hela cell line.

<p>A) PI staining of Hela cells overexpressing Hsa-mir-6165 did not show any significant change in the stages of cell cycle after 34 hours post transfection (a′- c′). B) Annexin-PI staining of the Hela cells shown in figures a″- d″. Repeated Measures ANOVA analysis shows that the changes observed in annexin test of Hela cells were not significant between negative controls (scramble) and the test group (e″).</p