The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic efects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has signifcantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques

Development and characterization of TDP-43 in vivo models of ALS

Die neurodegenerative Erkrankung Amyotrophe Lateralsklerose (ALS) ist vor allem durch den Verlust von Motoneuronen im Kortex sowie im Rückenmark charakterisiert. In nahezu allen ALS Patienten aggregiert das Kernprotein transactive response DNA binding protein of 43 kDa (TDP-43) im Zytoplasma von Neuronen, wo es auch hyper-phosphoryliert, -ubiquitiniert sowie fragmentiert wird. Bislang ist nicht klar, wie es zur Aggregation kommt und diese das Absterben von Motoneuronen induziert. Um diese pathologischen Mechanismen zu verstehen, wurden verschiedene Tiermodelle mit TDP-43 Überexpression oder Gen-Knock-out entwickelt. Allerdings weisen diese Modelle entweder keine oder eine zu starke Pathologie auf, wodurch sie für Grundlagenforschung oder Medikamententestungen nur bedingt verwendet werden können. Aus diesem Grund wurde in dieser Doktorarbeit ein Modell entwickelt, welches einen milden, aber spezifischen ALS Phänotyp zeigt. Dafür wurden Adeno-assoziierte Viruspartikel, die humanes TDP-43 und/oder GFP exprimieren, in den adulten Motorkortex injiziert, welcher als Startpunkt von ALS gilt. Das motorische und kognitive Verhalten der Tiere wurde über einen Zeitraum von 6 Monaten analysiert, wobei motorische Defizite bereits nach einem Monat in TDP-43-exprimierenden Mäusen eintraten und sich im Verlauf der Studie verschlechterten. Zusätzlich zeigten die Tiere ein erhöhtes Angstlevel sowie Beeinträchtigungen im assoziativen Lernen. Nach der 6-monatigen Inkubationszeit wurden Kortex- und Rückenmarksproben auf spezifische TDP-43 Pathologien mittels biochemischer und histologischer Methoden untersucht. Dabei detektierten wir an der Injektionsstelle hyper-ubiquitiniertes TDP-43, verstärkte inflammatorische Prozesse sowie einen Verlust von kortikalem Gewebe und Neuronen. Zusammenfassend kann gesagt werden, dass das TDP-43 Mausmodell typische ALS-Merkmale aufweist und für weitere TDP-43 Studien sowie für Austestung von neuen, potentiellen ALS Medikamenten herangezogen werden kann.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of upper motor neurons in the brain and lower motor neurons in the spinal cord. Malfunctions of the nuclear protein transactive response DNA binding protein of 43 kDa (TDP-43) have been described in most ALS patients. Pathological TDP-43 mislocalizes to the cytoplasm, where it is hyper-phosphorylated, ubiquitinated, truncated and aggregated in inclusion bodies. However, the mechanism causing TDP-43 dysfunction and leading to neuronal death has not been identified yet. Recently, various transgenic TDP-43 rodent models have been generated either lacking or displaying fatal ALS pathology. Hence, it has been challenging to investigate TDP-43 function or to use these models for drug screening studies. Thus, the aim of this project was to develop a novel TDP-43 mouse model showing specific and mild ALS pathology by injecting adeno-associated viruses expressing human TDP-43 into neural regions comprising motor neurons. To this end, we injected TDP-43 and/or GFP into the adult motor cortex, which has been described as one of the “starting points” of ALS in humans. Behavioral impairments were monitored longitudinally for 6 months. Afterwards, cellular malfunctions caused by TDP-43 overexpression in the motor cortex were investigated histologically and biochemically. Behavioral analyses revealed beginning of motor abnormalities 1 month after injection and significant deficits after 3 months. Additionally, increased anxiety levels and cognitive decline were detectable after 6 months. TDP-43 was highly ubiquitinated at the injection site and induced an ALS-like cellular pathology, such as increased inflammation and neuronal loss in the motor cortex. By contrast, late-phase neurodegeneration of spinal motor neurons was not observed. Together, we generated a novel ALS mouse model, which mimics pathologies of early ALS and carries the potential to be used as a tool for drug testing analyses.vorgelegt von Barbara Scherz, M.Sc.Zusammenfassungen in Deutsch und EnglischKarl-Franzens-Universität Graz, Dissertation, 2018OeBB(VLID)294582

mTh1 driven expression of hTDP-43 results in typical ALS/FTLD neuropathological symptoms

<div><p>Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models.</p></div

Quantification of hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.

<p>Quantitative hTDP-43 expression in hypothalamus (A), medulla oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS samples. (D, E) Representative overview image and magnifications of hTDP-43 labeling of a brain section (olfactory bulb (1), cortex (2), hippocampus (3), thalamus (4), hypothalamus (5) and medulla oblongata (6)) of 3 months old TAR6/6 (D) and ntg (E) mice. Scale bars: Overview images = 1000 μm; magnification images = 20 μm. (A, B) 1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5; TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (C) n as in A, B exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between age groups. *p<0.05, **p<0.01, ***p<0.001.</p

Quantification of neuron loss in the spinal cord of 3 months old TAR6/6 mice.

<p>(A, C) Number of ChAT⁺ neurons in the ventral horn of the cervical (A) and lumbar (C) spinal cord grey matter. (B, D) Representative images of ChAT labeling in the cervical (B) and lumbar (D) spinal cord of 3 months old TAR6/6 mice compared to ntg animals. Nuclei of cells were stained by DAPI. (A) ntg: n = 4, TAR6/6: n = 3. (C) ntg: n = 3, TAR6/6: n = 3. Unpaired Student’s t-test followed by Welch’s correction. Mean+SEM. *p<0.05.</p

Posttranslational changes in TAR6/6 mice.

<p>(A-D) Localization of tTDP-43, CTF-35 and hTDP-43 after cellular fractionation of 3 months old TAR6/6 mice. (A) Cell fractions of midbrain and cortex samples of 3 months old ntg, TAR6 and TAR6/6 mice were analyzed by Western blotting and probed with the indicated antibodies (GAPDH as cytoplasmic and HDAC3 as nuclear marker; long exp. = long time ECL exposure for 30 min). One representative example of 4 is shown (C: cytoplasm, N: nucleus). (B- D) Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 levels in cell fractions (cyt: cytoplasm, nuc: nucleus). Nuclear tTDP-43 and CTF-35 levels of ntg and hTDP-43 of TAR6/6 were set as 100%. (E-H) Soluble and insoluble TDP-43 protein levels in brains of 3 months old TAR6/6 mice. Brain samples were separated into RIPA soluble (R) and RIPA insoluble / UREA fractions (U). Three representative examples of 5 are shown. Fractions were analyzed by Western blotting and probed with the indicated antibodies (long exp. = long time ECL exposure for 30 min). Coomassie Blue staining was performed as loading control for UREA fraction. (F-H) Densitometric analysis of tTDP-43, CTFs and hTDP-43 levels in RIPA and UREA fractions. For tTDP-43 and CTF evaluation, RIPA fraction of ntg was set as 100%, whereas for hTDP-43 analysis, RIPA sample of TAR6/6 was set as 100%. (B-D; F-H) Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between fractions. *p<0.05, **p<0.01, ***p<0.001.</p

Immunofluorescent double-labeling of human and total TDP-43.

<p>Cortical and hippocampal tissue of three-months old ntg (A) and TAR6/6 (B) mice was labeled with antibodies against human TDP-43 (green) and total TDP-43 (red). Tissue was additionally labeled with NeuN antibody (white) and DAPI (blue) to visualize neuronal somata and nuclei, respectively. The merged images in the right column show human TDP-43, total TDP-43 and DAPI labeling. (C) The preferred nuclear localization of both human TDP-43 and total TDP-43 is obvious when the outlines of NeuN-positive somata are projected on the TDP-43 channels. Scale bars: Overview images = 500 μm; magnification images = 20 μm; (C) = 20 μm.</p

Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.

<p>(A) Brain homogenates, (E) hippocampal and (I) spinal cord homogenates from ntg, TAR6 and TAR6/6 mice at the age of 1.5, 3 and 6 months were analyzed by Western blotting and probed with the indicated antibodies (long exp. = long time ECL exposure for 30 min). One representative example of 3 is shown. Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 levels normalized to β-tubulin levels of brain (B- D), hippocampal (F-H) and spinal cord (J-L) homogenates. Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between age groups. *p<0.05, **p<0.01, ***p<0.001.</p