Sarah Knippenberg

Significance of behavioural tests in a transgenic mouse model of amyotrophic lateral sclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult-onset motor neuron disorder with marginal therapeutic options. The disease is characterized by progressive degeneration of motor neurons in spinal cord and motor cortex. Transgenic mice carrying the G93A mutation of the superoxide dismutase 1 (SOD1) gene develop a neurodegenerative disease closely mimicking human ALS. Several methods are currently used to record disease onset and progression of the animals in preclinical studies. For the interpretation of these preclinical trials, it is important to assess neurological function as sensitively as possible. In the present study, five different parameters (rotarod performance, weight, footprint analysis for both step length and runtime and the general condition of the mice scored from 1 to 5) were compared with respect to their significance to detect symptom onset and to monitor disease progression in transgenic G93A ALS mice. The rotarod and footprint analyses were performed weekly while the weight was recorded up to three times a week at later time points. General condition was assessed daily. First deficits were detected by rotarod testing and step length analyses. General condition score and weight showed first changes two weeks later. For preclinical testing of novel drug treatments rotarod and footprint analysis for step length therefore seem to be the most effective methods to detect symptom onset and potential treatment induced improvements.

Intracerebroventricular Injection of Encapsulated Human Mesenchymal Cells Producing Glucagon-Like Peptide 1 Prolongs Survival in a Mouse Model of ALS

Background As pharmacological therapies have largely failed so far, stem cell therapy has recently come into the focus of ALS research. Neuroprotective potential was shown for several types of stem and progenitor cells, mainly due to release of trophic factors. In the present study, we assessed the effects of intracerebroventricular injection of glucagon-like peptide 1 (GLP-1) releasing mesenchymal stromal cells (MSC) in mutant SOD1 (G93A) transgenic mice. Methodology/Principal Findings To improve the neuroprotective effects of native MSC, they had been transfected with a plasmid vector encoding a GLP-1 fusion gene prior to the injection, as GLP-1 was shown to exhibit neuroprotective properties before. Cells were encapsulated and therefore protected against rejection. After intracerebroventricular injection of these GLP-1 MSC capsules in presymptomatic SOD1 (G93A) mice, we assessed possible protective effects by survival analysis, measurement of body weight, daily monitoring and evaluation of motor performance by rotarod and footprint analyses. Motor neuron numbers in the spinal cord as well as the amount of astrocytosis, microglial activation, heat shock response and neuronal nitric oxide synthase (nNOS) expression were analyzed by immunohistological methods. Treatment with GLP-1 producing MSC capsules significantly prolonged survival by 13 days, delayed symptom onset by 15 days and weight loss by 14 days and led to significant improvements in motor performance tests compared to vehicle treated controls. Histological data are mainly in favour of anti-inflammatory effects of GLP-1 producing MSC capsules with reduced detection of inflammatory markers and a significant heat shock protein increase. Conclusion/Significance Intracerebroventricular injection of GLP-1 MSC capsules shows neuroprotective potential in the SOD1 (G93A) mouse model.

Decreased mRNA expression of PGC-1α and PGC-1α-regulated factors in the SOD1G93A ALS mouse model and in human sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss. Although the cause of ALS is unknown, oxidative stress, inflammation, and mitochondrial dysfunction have been identified as important components of its pathogenesis. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) plays a central role in the regulation of mitochondrial metabolism and biogenesis via activation of transcription factors, such as nuclear respiratory factors 1 and 2 and mitochondrial transcription factor A (Tfam). Alterations in PGC-1α expression and function have previously been described in models of Huntington and Alzheimer diseases. Moreover, the protective effects of PGC-1α have been shown in animal models of ALS. Levels of PGC-1α correlate with the number of acetylcholine receptor clusters in muscle. This is of particular interest because neurodegeneration in ALS may be a dying-back process. We investigated mRNA and protein expressions of PGC-1α and PGC-1α-regulated factors in the spinal cord and muscle tissues of SOD1 ALS mice and in ALS patients. We detected significant alterations in mRNA expression of PGC-1α and downstream factors with their earliest occurrence in muscle tissue. Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction both in the ALS mouse model and in human sporadic ALS that is probably most relevant in the skeletal muscle.

Intraspinal Injection of Human Umbilical Cord Blood-Derived Cells Is Neuroprotective in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons in the spinal cord, brain stem and motor cortex and has only marginal therapeutic options. Adult stem cells have recently come into the focus of neurological research. While replacement of motor neurons by stem cells currently appears not feasible, there is evidence that non-neuronal cells can be neuroprotective. Objective: Therefore, we evaluated the effects of direct intraspinal administration of human umbilical cord blood cells in a G93A transgenic mouse model of ALS before (day 40) and after symptom onset (day 90). Methods: Treatment effects were assessed by survival analysis, behavioral tests, histological and biochemical analyses. Results: Treatment at early stages increased survival, led to significant improvements in motor performance and significantly reduced motor neuron loss and astrogliosis in the spinal cord. Interestingly females tended to respond better to treatment than males. Conclusion: This study confirms the neuroprotective potential of human umbilical cord blood cells and encourages further investigations.

Neural Progenitors Derived From Human Induced Pluripotent Stem Cells Survive and Differentiate Upon Transplantation Into a Rat Model of Amyotrophic Lateral Sclerosis

Human induced pluripotent stem cells (iPSCs) offer hope for personalized regenerative cell therapy in amyotrophic lateral sclerosis (ALS). We analyzed the fate of human iPSC‐derived neural progenitors transplanted into the spinal cord of wild‐type and transgenic rats carrying a human mutated SOD1(G93A) gene. The aim was to follow survival and differentiation of human neural progenitors until day 60 post‐transplantation in two different in vivo environments, one being ALS‐like. iPSC‐derived neural progenitors efficiently engrafted in the adult spinal cord and survived at high numbers. Different neural progenitor, astroglial, and neuronal markers indicated that, over time, the transplanted nestin‐positive cells differentiated into cells displaying a neuronal phenotype in both wild‐type and transgenic SOD1 rats. Although a transient microglial phenotype was detected at day 15, astroglial staining was negative in engrafted cells from day 1 to day 60. At day 30, differentiation toward a neuronal phenotype was identified, which was further established at day 60 by the expression of the neuronal marker MAP2. A specification process into motoneuron‐like structures was evidenced in the ventral horns in both wild‐type and SOD1 rats. Our results demonstrate proof‐of‐principle of survival and differentiation of human iPSC‐derived neural progenitors in in vivo ALS environment, offering perspectives for the use of iPSC‐based therapy in ALS.

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin

Rationale Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly. Objectives To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice. Methods Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis. Measurements and main results We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-β1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFβ1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFβ1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFβ1-exposed mice. Conclusions Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice.

Macrophage-Inducible C-Type Lectin Mincle-Expressing Dendritic Cells Contribute to Control of Splenic Mycobacterium bovis BCG Infection in Mice

ABSTRACT The macrophage-inducible C-type lectin Mincle has recently been identified to be a pattern recognition receptor sensing mycobacterial infection via recognition of the mycobacterial cell wall component trehalose-6′,6-dimycolate (TDM). However, its role in systemic mycobacterial infections has not been examined so far. Mincle-knockout (KO) mice were infected intravenously with Mycobacterium bovis BCG to mimic the systemic spread of mycobacteria under defined experimental conditions. After intravenous infection with M. bovis BCG, Mincle-KO mice responded with significantly higher numbers of mycobacterial CFU in spleen and liver, while reduced granuloma formation was observed only in the spleen. At the same time, reduced Th1 cytokine production and decreased numbers of gamma interferon-producing T cells were observed in the spleens of Mincle-KO mice relative to the numbers in the spleens of wild-type (WT) mice. The effect of adoptive transfer of defined WT leukocyte subsets generated from bone marrow cells of zDC+/DTR mice (which bear the human diphtheria toxin receptor [DTR] under the control of the classical dendritic cell-specific zinc finger transcription factor zDC) to specifically deplete Mincle-expressing classical dendritic cells (cDCs) but not macrophages after diphtheria toxin application on the numbers of splenic and hepatic CFU and T cell subsets was then determined. Adoptive transfer experiments revealed that Mincle-expressing splenic cDCs rather than Mincle-expressing macrophages contributed to the reconstitution of attenuated splenic antimycobacterial immune responses in Mincle-KO mice after intravenous challenge with BCG. Collectively, we show that expression of Mincle, particularly by cDCs, contributes to the control of splenic M. bovis BCG infection in mice.

Prolonged survival and milder impairment of motor function in the SOD1 ALS mouse model devoid of fibroblast growth factor 2

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss in brain and spinal cord. Mutations in the superoxide dismutase (SOD) 1 gene account for 10-20% of familial ALS patients. The ALS-mouse model over-expressing a mutant human SOD1 (G93A) gene closely mimics human ALS disease. The cause for the selective death of motoneurons is still unclear, but among several pathomechanisms discussed, loss of neurotrophic factors is one possibility. Basic fibroblast growth factor 2 (FGF-2) plays a prominent role in the motor system. In order to evaluate a role of FGF-2 in ALS pathogenesis, double mouse mutants transgenic for the human SOD1 mutation and lacking the endogenous FGF-2 gene were generated. Both heterozygous and homozygous FGF-2 deficient mutant SOD1 mice showed a significant delay in disease onset and less impaired motor performance in comparison to mutant SOD1 mice with normal FGF-2 levels. Survival of the double mouse mutants was significantly prolonged for two weeks. Motoneuron numbers were significantly higher in the double mutants and astrocytosis was diminished at disease endstage. While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1 animals, our results revealed a protective effect of FGF-2 reduction. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors with proven protective effects in the ALS mouse model, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in muscle and spinal cord tissue of double mutant animals.

Therapeutic Potential of N-Acetyl-Glucagon-Like Peptide-1 in Primary Motor Neuron Cultures Derived From Non-Transgenic and SOD1-G93A ALS Mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons (MN) in the motor cortex, brain stem, and spinal cord. In the present study, we established an ALS in vitro model of purified embryonic MNs, derived from non-transgenic and mutant SOD1-G93A transgenic mice, the most commonly used ALS animal model. MNs were cultured together with either non-transgenic or mutant SOD1-G93A astrocyte feeder layers. Cell viability following exposure to kainate as excitotoxic stimulus was assessed by immunocytochemistry and calcium imaging. We then examined the neuroprotective effects of N-acetyl-GLP-1(7-34) amide (N-ac-GLP-1), a long-acting, N-terminally acetylated, C-terminally truncated analog of glucagon-like peptide-1 (GLP-1). GLP-1 has initially been studied as a treatment for type II diabetes based on its function as insulin secretagogue. We detected neuroprotective effects of N-ac-GLP-1 in our in vitro system, which could be attributed to an attenuation of intracellular calcium transients, not only due to these antiexcitotoxic capacities but also with respect to the increasing knowledge about metabolic deficits in ALS which could be positively influenced by N-ac-GLP-1, this compound represents an interesting novel candidate for further in vivo evaluation in ALS.

Altered Expression of DJ-1 and PINK1 in Sporadic ALS and in the SOD1G93A ALS Mouse Model

Mitochondrial dysfunction is an important mechanism in the pathogenesis of neurodegenerative diseases such as Parkinson disease and amyotrophic lateral sclerosis (ALS). DJ-1 and PTEN-induced putative kinase 1 (PINK1) are important proteins for the maintenance of mitochondrial function and protection against cell death. Mutations in the genes coding for these proteins cause familial forms of Parkinson disease. Recent studies have postulated that changes in the expression of both proteins are also involved in pathologic mechanisms in ALS mouse models. Here, we studied the mRNA and protein expression of PINK1 and DJ-1 in postmortem brain and spinal cord tissue and muscle biopsy samples from ALS patients and controls and in brain, spinal cord, and gastrocnemius muscle of SOD1(G93A) ALS mice at different disease stages. We found significant decreases of PINK1 and DJ-1 mRNA levels in muscle tissue of SOD1(G93A) mice. Together with the significant decrease of PINK1 mRNA levels in human ALS muscle tissue, statistically nonsignificant reduction of DJ-1 mRNA levels, and reduced immunostaining for PINK1 in human ALS muscle, the results suggest potential pathophysiologic roles for these proteins in both mutant SOD1 transgenic mice and in sporadic ALS(G93A).