Stephanie Weber

TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity

Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear—a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function—but not ALS/FTLD-linked mutation, mislocalization, or truncation—for TDP-43-mediated neurotoxicity in vivo.

TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR.

TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5′-GA-3’ and 5′-UG-3′ repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.

Parkinson’s disease-associated DJ-1 modulates innate immunity signaling in Caenorhabditis elegans

DJ-1 is a neuroprotective gene mutated in recessive Parkinson’s disease (PD). In addition to direct protective functions in neurons, DJ-1 regulates neuroinflammatory signaling in primary mouse brain astrocytes. To assess the influence of DJ-1 on innate immunity signaling in vivo, we have generated djr-1 knockout Caenorhabditis elegans. When grown on pathogenic gram-negative bacteria, djr-1−/− worms showed stronger phosphorylation of p38 mitogen-activated protein kinase (PMK-1) and hyper-induction of PMK-1 target genes. Thus, PD-associated DJ-1 contributes to regulation of innate immunity.

Correlation between retinal vessel density profile and circumpapillary RNFL thickness measured with Fourier-domain optical coherence tomography.

Aim To assess circumpapillary retinal vessel density (RVD) profiles and correlate them with retinal nerve fibre layer (RNFL) thickness measured by Fourier domain optical coherence tomography (FD-OCT). Methods RNFL thickness of 106 healthy volunteers was measured using Cirrus FD-OCT. A proprietary software was developed in MATLAB to assess the thickness and position of circumpapillary retinal vessels using the scanning laser ophthalmoscopy fundus image, centred on the optic disc. The individual retinal vessel positions and thickness values were integrated in a 256-sector RVD profile, and intrasubject and intersubject correlations were calculated. Results The mean value±SD for intrasubject correlation between RVD and RNFL was 0.5349±0.1639, with 101 of 106 subjects presenting significant correlation (p<0.05). 181 (out of 256) sectors presented a significant correlation between RVD and RNFL, with a mean value±SD of 0.2600±0.1140 (p<0.05). Conclusions Using our model of the circumpapillary retinal vessel distribution, 70% of the RNFL thickness is influenced by RVD. On average, 7% of the interindividual variance of the RNFL thickness may be explained by RVD. A normative database that takes into account the circumpapillary blood vessels might slightly improve the diagnostic power of RNFL measurement.

Influence of disc-fovea angle and retinal blood vessels on interindividual variability of circumpapillary retinal nerve fibre layer.

Background To assess whether intersubject variability of circumpapillary retinal nerve fibre layer (RNFL) thickness in healthy subjects acquired with spectral domain optical coherence tomography (SD-OCT) can be reduced by considering the disc-fovea angle (DFA), either alone or together with a compensation based on retinal blood vessel distribution (RVD). Methods 106 healthy volunteers underwent SD-OCT examination centred on the optic disc (OD) and on the macula. OD contours and foveal positions were automatically calculated. RVD at 3.4 mm diameter circle was manually assessed. We made two approaches to reduce interindividual variability in RNFL values using compensation processes; RVD compensation: RNFL thickness values were compensated according to RVD variation (RNFLRVD) and DFA compensation: we shifted the RNFL thickness measurements according to the DFA (RNFLDFA). Coefficient of variance (CoV) was calculated in 12 clock hour sectors for original RNFL (RNFLo), RNFLDFA, RNFLRVD and RNFL with both compensation methods (RNFLDFA-RVD). Results Compared with the mean CoV of RNFLO, mean CoV of RNFLDFA, RNFLRVD and RNFLDFA-RVD was changed by −0.71% (p>0.05), −9.51% (p<0.001) and −7.55% (p=0.001), respectively. When compared with RNFLDFA, RNFL DFA-RVD significantly reduced the mean CoV by −6.69% (p=0.001), while compared with RNFLRVD, RNFL DFA-RVD did not significantly increase the mean CoV (+2.20%), (p>0.05). Conclusions Although reaching an improvement in some sectors, rotation of RNFL measurements according to the DFA on average does not reduce intersubject variability of RNFL. However, adjusting for RVD reduced the variance significantly. The results reinforce our work in assessing RVD as an important anatomical factor responsible for intersubject variability in RNFL measurements.

Compensation for retinal vessel density reduces the variation of circumpapillary RNFL in healthy subjects.

This work intends to assess circumpapillary retinal vessel density (RVD) at a 3.46 mm diameter circle and correlate it with circumpapillary retinal nerve fiber layer (RNFL) thickness measured with Fourier-Domain Optical Coherence Tomography. Furthermore, it aims to evaluate the reduction of intersubject variability of RNFL when considering RVD as a source of information for RNFL distribution. For that, 106 healthy subjects underwent circumpapillary RNFL measurement. Using the scanning laser ophthalmoscope fundus image, thickness and position of retinal vessels were assessed and integrated in a 256-sector RVD profile. The relationship between local RVD value and local RNFL thickness was modeled by linear regression. RNFL was then compensated for RVD variation by regression formulas. A strong statistically significant intrasubject correlation was found for all subjects between RVD and RNFL profiles (mean R = 0.769). In the intersubject regression analysis, 247 of 256 RNFL sectors showed a statistically significant positive correlation with RVD (mean R = 0.423). RVD compensation of RNFL resulted in a relative reduction of up to 20% of the intersubject variance. In conclusion, RVD in a 3.46mm circle has a clinically relevant influence on the RNFL distribution. RVD may be used to develop more individualized normative values for RNFL measurement, which might improve early diagnosis of glaucoma.

Regulation of mRNA Translation by MID1: A Common Mechanism of Expanded CAG Repeat RNAs.

Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT) RNA, which is linked to Huntingtons disease (HD), at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analyzed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2), Ataxin3 (ATXN3), and Ataxin7 (ATXN7), that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3, and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3, and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs.

Retinal Blood Vessel Distribution Correlates With the Peripapillary Retinal Nerve Fiber Layer Thickness Profile as Measured With GDx VCC and ECC.

Purpose:Aim of the present study was to evaluate whether there is a correlation between retinal blood vessel density (RVD) and the peripapillary retinal nerve fiber layer (RNFL) thickness profile. Methods:RNFL thickness of 106 healthy subjects was measured using scanning laser polarimetry, GDx variable corneal compensation (VCC), and GDx enhanced corneal compensation (ECC). A proprietary software was developed in MATLAB to measure the peripapillary retinal vessels using scanning laser ophthalmoscopy fundus images, centered on the optic disc measured by Cirrus spectral domain optical coherence tomography. The individual retinal vessel positions and thickness values were integrated in a 64-sector RVD profile and intrasubject and intersubject correlations were calculated. Results:The mean R value±SD for intrasubject correlation between RVD and RNFL thickness measured with GDx VCC and GDx ECC was 0.714±0.157 and 0.629±0.140, with 105 of 106 subjects presenting significant correlations. In the intersubject linear regression analysis for GDx VCC, 33 of 64 (52%) sectors presented a significant Pearson correlation coefficient between RNFL thickness and RVD values, with a mean R value of 0.187±0.135 (P<0.05). Conclusions:Peripapillary RNFL thickness profiles correlate with the RVD over 50% of the sectors and might explain up to 26% of the interindividual variance of the peripapillary RNFL thickness values as measured with GDx VCC. To our opinion, taking into account RVD might reduce interindividual variation in peripapillary RNFL thickness profiles measured with scanning laser polarimetry.

Resveratrol induces dephosphorylation of Tau by interfering with the MID1-PP2A complex

The formation of paired helical filaments (PHF), which are composed of hyperphosphorylated Tau protein dissociating from microtubules, is one of the pathological hallmarks of Alzheimer’s disease (AD) and other tauopathies. The most important phosphatase that is capable of dephosphorylating Tau at AD specific phospho-sites is protein phosphatase 2 A (PP2A). Here we show that resveratrol, a polyphenol, significantly induces PP2A activity and reduces Tau phosphorylation at PP2A-dependent epitopes. The increase in PP2A activity is caused by decreased expression of the MID1 ubiquitin ligase that mediates ubiquitin-specific modification and degradation of the catalytic subunit of PP2A when bound to microtubules. Interestingly, we further show that MID1 expression is elevated in AD tissue. Our data suggest a key role of MID1 in the pathology of AD and related tauopathies. Together with previous studies showing that resveratrol reduces β-amyloid toxicity they also give evidence of a promising role for resveratrol in the prophylaxis and therapy of AD.

MicroRNAs miR-19, miR-340, miR-374 and miR-542 regulate MID1 protein expression

The MID1 ubiquitin ligase activates mTOR signaling and regulates mRNA translation. Misregulation of MID1 expression is associated with various diseases including midline malformation syndromes, cancer and neurodegenerative diseases. While this indicates that MID1 expression must be tightly regulated to prevent disease states specific mechanisms involved have not been identified. We examined miRNAs to determine mechanisms that regulate MID1 expression. MicroRNAs (miRNA) are small non-coding RNAs that recognize specific sequences in their target mRNAs. Upon binding, miRNAs typically downregulate expression of these targets. Here, we identified four miRNAs, miR-19, miR-340, miR-374 and miR-542 that bind to the 3’-UTR of the MID1 mRNA. These miRNAs not only regulate MID1 expression but also mTOR signaling and translation of disease associated mRNAs and could therefore serve as potential drugs for future therapy development.