Department of Neurology, Hannover Medical School Centre for Systems Neurosciences
University of Veterinary Medicine Hannover
Transcriptional regulators and neurotrophic factors in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS)
Histopathological and biochemical studies
in the G93A ALS mouse model and in ALS post mortem tissue
THESIS
submitted in partial fulfillment of the requirements for the degree
- Doctor rerum naturalium -
(Dr. rer. nat.)
by Nadine Thau Hannover, Germany
Supervisor: Prof. Dr. Susanne Petri
Supervision Group: Prof. Dr. Susanne Petri Prof. Dr. Claudia Grothe
Prof. Dr. Wolfgang Baumgärtner
1st Evaluation: Prof. Dr. Susanne Petri Department of Neurology,
Hannover Medical School, Germany Prof. Dr. Claudia Grothe
Department of Neuroanatomy, Hannover Medical School, Germany Prof. Dr. Wolfgang Baumgärtner Department of Pathology,
University of Veterinary Medicine Hannover, Germany
2nd Evaluation: PD Dr. Sibylle Jablonka
Department of Clinical Neurobiology University of Würzburg, Germany
Date of final exam: 5th of October, 2012
Parts of the thesis have been published or submitted previously in:
Thau N, Jungnickel J, Knippenberg S, Ratzka A, Dengler R, Petri S, Grothe C. Prolonged survival and milder impairment of motor function in the SOD1 ALS mouse model devoid of Fibroblast growth factor 2. Neurobiol. Dis. (2012), 47:248-257.PMID: 22542539
Thau N, Knippenberg S, Körner S, Rath KJ, Dengler R, Petri S. Decreased mRNA expression of PGC-1α and PGC-1α regulated factors in the SOD1G93A ALS mouse model and in human sporadic ALS. J Neuropathol Exp Neurol. (2012), 71. PMID: 23147503
Results of the thesis have been presented in form of posters or presentations at following congresses:
25th Arbeitstagung der Anatomischen Gesellschaft (Würzburg, 2008):
Impact of the neurotrophic factor FGF-2 on the neurodegenerative disorder ALS: evidence from mutant mice
20th International Symposium on ALS/MND (Berlin, 2009):
Prolonged survival and enhanced motor performance in SOD-1G93A mice deficient for the neurotrophic factor FGF-2
8th European ALS Consortium Young Investigators Meeting (London, 2010):
Fibroblast growth factor 2 (FGF-2) deficiency prolongs survival and improves motor function in G93A mutant SOD1 mice
9th Göttingen Meeting of the German Neuroscience Society (Göttingen, 2011):
Transcriptional regulators in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) histopathological and biochemical studies in the G93A ALS mouse model and human ALS tissue
9th Meeting of the European Network for the Cure of ALS (Hannover, 2011):
Transcriptional regulators in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS)
histopathological and biochemical studies in the G93A ALS mouse model and human ALS tissue
10th Meeting of the European Network for the Cure of ALS (Dublin, 2012):
Sponsorship:
One part of the thesis was supported by a grant from Deutsche Gesellschaft für Muskelkranke e. V. (DGM) and by a grant of Deutsche Forschungsgemeinschaft (Pe 924-2/2) to SP. The other part was supported by a grant of the German Research Foundation (DFG Pe 924-2/2) to SP.
Content
Content
Introduction... 1
Manuscript I ... 7
Abstract... 8
Manuscript II ... 9
Abstract... 10
Discussion ... 11
Summary ... 17
Zusammenfassung ... 19
References ... 21
Acknowledgements ... 33
Declaration ... 34
Introduction
Introduction
Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motoneuron disease, with a yearly incidence of 1-2 cases in 100 000 and a prevalence of approximately 4-9 in 100 000 individuals. First described in 1869 by the French neurologist Jean-Martin Charcot (1825-1893), ALS leads to progressive loss of upper motoneurons in the motor cortex and lower motoneuron in the brainstem and spinal cord. This results in spasticity, hyperreflexia (upper motoneuron sings), generalized weakness, muscle atrophy, and paralysis (lower motoneuron signs) (MULDER et al.
1986). Failure of the respiratory muscles mostly leads to death within 3-5 years (CLEVELAND u. ROTHSTEIN 2001; ROWLAND u. SHNEIDER 2001). Onset typically occurs at an age between 50 and 60 years. Men have a slightly higher risk to develop ALS (3:2) (MITCHELL u. BORASIO 2007).
Most cases (90%) are classified as sporadic ALS (sALS), as they are not associated with a documented family history. 10% of patients have a hereditary form of ALS (familial ALS; fALS) (MULDER et al. 1986; PRZEDBORSKI et al. 2003). Sporadic and familial forms are clinically and pathologically similar, suggesting a common pathogenesis (BRUIJN et al. 2004). Approximately 20% of the familial cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene (ROSEN et al.
1993). Currently, more than 100 different mutations distributed throughout the 153- amino acid SOD1 polypeptide have been linked to ALS (INCE et al. 1998; SHAW et al. 1998). The best known physiological function of the SOD1 enzyme is to protect cells from toxic superoxide. SOD1 catalyses the conversion of toxic superoxide
Introduction
SOD1 leads to motoneuron degeneration. As SOD1 knockout mice do not develop ALS-like symptoms, motoneuron loss apparently occurs due to gain of function rather than loss of function of the mutant enzyme (REAUME et al. 1996; BRUIJN et al.
1998).
Based on this knowledge a transgenic mouse model of ALS with a mutation of SOD1 at amino acid position 93 where glycine is replaced by alanine (G93A) was developed (GURNEY et al. 1994). In these animals, weakness typically starts in the hind limbs and progresses towards complete paralysis, associated with increased astrogliosis, activation of microglia, and loss of spinal cord motoneurons as well as muscle atrophy. Symptom onset is characterized by tremor, followed by asymmetric or symmetric paresis of the hind limbs. Histopathological changes can be detected before the disease is phenotypically obvious and pathology closely mimics many aspects of human disease (BRUIJN et al. 2004). Because of the similarity of the sporadic and familial form, progress in elucidating the mechanisms underlying familial ALS may provide insight into both sALS and fALS (BRUIJN et al. 2004).
The exact pathogenesis of ALS still has not been elucidated but appears to be multifactorial, including genetic and environmental factors (ALMER 2003).
Hypotheses include oxidative damage (BEAL 2002a, b; BARBER u. SHAW 2010), mitochondrial dysfunction (BORTHWICK et al. 1999; BEAL 2000; MENZIES et al.
2002; MANFREDI u. XU 2005; DUFFY et al. 2011), growth factor deficiency (EKESTERN 2004), defects in axonal transport (DE VOS et al. 2008; IKENAKA et al.
2012), and glutamate excitotoxicity (HEATH u. SHAW 2002; BRUIJN et al. 2004).
The only known treatment up to now is riluzole, a glutamate antagonist with a complex mechanism, but it only marginally prolongs life expectancy for up to three
Introduction
months (BENSIMON et al. 1994; DOBLE 1996; LACOMBLEZ et al. 1996a;
LACOMBLEZ et al. 1996b; RIVIERE et al. 1998; MILLER et al. 2002; PAILLISSE et al. 2005).
Lack of neurotrophic factors is one of several pathomechanisms which are discussed in ALS (WONG et al. 1998; CLEVELAND 1999; ROWLAND u. SHNEIDER 2001).
Impaired neurotrophic support to motoneurons could occur due to deficiencies in retrograde transport from muscle tissue, anterograde transport from other neurons, paracrine support from neighbouring cells, endocrine support from ependymal cells in the periphery or autocrine support from the neuron itself (EKESTERN 2004). Several in vivo studies have shown protective effects of neurotrophic factors such as IGF-1 (Insulin-like Growth Factor 1) (NAGANO et al. 2005), CNTF (Cilliary Neurotrophic Factor) (GIESS et al. 2002), GDNF (Glial cell line-Derived Neutrophic Factor) (ACSADI et al. 2002; W. LI et al. 2007) and VEGF (Vascular Endothelial Growth Factor) (WANG et al. 2007) in the ALS mouse model (EKESTERN 2004). Some of these neurotrophic factors have already been evaluated in clinical studies in ALS patients. Up to date, none of the tested factors has had significant effects on disease outcome (EKESTERN 2004).
A role of basic fibroblast growth factor (FGF-2) in ALS has been controversially discussed so far: No disease specific alterations in the expression pattern of FGF-2 were found in post mortem spinal cord of ALS patients (PETRI et al. 2008), but increased FGF-2 levels were measured in serum and cerebrospinal fluid of ALS patients (JOHANSSON et al. 2003). Subcutaneous administration of FGF-2 had no
Introduction
Nevertheless it is known that FGF-2 plays a prominent role in the motor system. It could be demonstrated that FGF-2 is expressed in spinal motoneurons and that its expression is lesion-induced regulated (GROTHE et al. 1991; KOSHINAGA et al.
1993; GROTHE u. WEWETZER 1996). It has been shown in vitro and in vivo that FGF-2 promotes survival of motoneurons (GROTHE u. UNSICKER 1992; TENG et al. 1999). In the peripheral system FGF-2 prevented motoneurons from decline after nerve lesion (OTTO et al. 1987; L. LI et al. 1994). FGF-2 deficient mice and mice without high-affinity tyrosine kinase FGF receptor 3 (FGFR3) show no lesion-induced cell death (JUNGNICKEL et al. 2004; JUNGNICKEL et al. 2005). In hippocampal progenitor cell lines and primary hippocampal cells it was demonstrated that FGF-2 induces a TNFα-mediated death pathway. Furthermore FGF-2 can induce a switch in death receptor pathways from FasL-mediated to TNFα-mediated (EVES et al. 2001).
However, it depends on activation of different receptors whether FGF-2 promotes cell survival or death. It has been suggested that FGF-2 mediates neuronal cell death after peripheral nerve lesion via FGFR3 signaling (GROTHE et al. 2006).
In order to evaluate the physiological role of FGF-2 in the ALS scenario, double mouse mutants transgenic for the human SOD1G93A mutation and lacking the endogenous FGF-2 gene were established. Mice with only a knock-out for FGF-2 are viable and fertile, show inconspicuous development and behaviour, and have normal life expectancy. The only nervous-system related abnormality was a reduction of differentiated large neurons in the cervical spinal cord (DONO et al. 1998; ORTEGA et al. 1998; ZHOU et al. 1998).
To assess the effect of FGF-2 deficiency, motor performance was determined by specific behaviour tests in both, heterozygous and homozygous FGF-2 deficient
Introduction
mutant SOD1G93A mice. Onset of disease and the survival time was recorded. In addition, we studied motoneuron loss, inflammation and growth factor expression levels.
Another factor that could play a role on different levels in the pathology of ALS is the transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α). Since its discovery (PUIGSERVER et al. 1998) it has been implicated in adaptive thermogenesis, β-oxidation of fatty acids, glucose metabolism, and energy homeostasis (PUIGSERVER u. SPIEGELMAN 2003).
Mitochondrial abnormalities can be found in ALS patients in spinal cord and skeletal muscle (WIEDEMANN et al. 1998; VIELHABER et al. 2000; WIEDEMANN et al.
2002) and also in ALS mouse models (KONG u. XU 1998; MATTIAZZI et al. 2002).
Mitochondria therefore seem to be key players in the pathogenesis of ALS (BEAL 2000; BRUIJN et al. 2004; MANFREDI u. XU 2005). Several studies suggest that alterations in mitochondrial morphology and function occur long before disease onset (M. T. LIN u. BEAL 2006). It has been suggested that mitochondria may be primary targets for SOD1-mediated damage also in non mutant SOD1-related ALS patients (BRUIJN et al. 2004).
PGC-1α regulates mitochondrial metabolism and biogenesis via activation of transcription factors such as nuclear respiratory factor-1 and -2 (NRF-1/-2). NRF-1 and NRF-2 are targets of PGC-1α and stimulate the expression of mitochondrial transcription factor A (Tfam), a mitochondrial matrix protein essential for the replication, maintenance, and transcription of mitochondrial DNA (J. LIN et al. 2005;
Introduction
SCARPULLA 2006). PGC-1α is involved in the regulation of mitochondrial density in neurons (WARESKI et al. 2009).
It is also known that PGC-1α levels correlate with the number of acetylcholine receptor (AChR) clusters in the skeletal muscle (HANDSCHIN et al. 2007). This is of particular interest as there is evidence that neurodegeneration in ALS could be a dying back process (KENNEL et al. 1996; FREY et al. 2000; FISCHER et al. 2004).
In adult human muscle utrophin (Utrn) is preferentially concentrated in acetylcholine receptor (ACHR)-rich crests at the neuromuscular junction (NMJ), where it binds to components of the DAPC and myotendinous junctions (MTJ) (PERKINS u. DAVIES 2002).
Alterations in PGC-1α expression and function have previously been described in models of Huntington’s and Alzheimer’s disease (CUI et al. 2006; WEYDT et al.
2006; SHI u. GIBSON 2007; CHATURVEDI et al. 2009; QIN et al. 2009). Recent studies have shown that PGC-1α and peroxisome proliferator-activated receptor gamma agonists protect motoneurons and neuromuscular junctions, alter disease progression and extend lifespan in animal model of ALS (KIAEI et al. 2005; LIANG et al. 2011; ZHAO et al. 2011; DA CRUZ et al. 2012). The second part of my PhD project therefore consisted in the analysis of mRNA and protein expression of PGC- 1α and downstream factors (NRF-1, NRF-2, Tfam, mnSOD) in spinal cord and muscle tissue of SOD1G93A ALS mice and non-transgenic controls in pre symptomatic, early symptomatic and late symptomatic disease stages as well as in human post mortem brain and spinal cord tissue and in tissue of muscle biopsies of sporadic ALS patients and controls.
Manuscript I
Manuscript I
Published in Neurobiology of Disease 2012
Prolonged survival and milder impairment of motor function in the SOD1 ALS mouse model devoid of Fibroblast growth factor 2
Nadine Thau1*§, Julia Jungnickel2, Sarah Knippenberg1, Andreas Ratzka2, Reinhard Dengler1*, Susanne Petri1*#, Claudia Grothe2*#
1 Hannover Medical School, Department of Neurology, Hannover, Germany
2 Hannover Medical School, Institute of Neuroanatomy, Hannover, Germany
* Centre for Systems Neuroscience (ZSN), Hannover, Germany
# contributed equally
§ corresponding author
Preface - about this manuscript
The cause for the selective death of motoneurons in ALS is still unclear, but among several pathomechanisms discussed, loss of neurotrophic factors is one possibility.
While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1G93A animals, our results revealed a protective effect of FGF-2 reduction: double mutants showed a delay in disease onset, less impaired motor performance, prolonged survival, higher motoneuron numbers and astrocytosis was diminished at disease endstage. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors with proven protective effects in the ALS mouse model, CNTF and GDNF, in muscle and spinal cord tissue.
The manuscript has been published in Neurobiology of Disease, Volume 47, Pages 248–257. PMID: 22542539
Manuscript I
Abstract
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.
Manuscript II
Manuscript II
Published in Journal of Neuropathology and Experimental Neurology 2012
Decreased mRNA expression of PGC-1α and PGC-1α regulated factors in the SOD1G93A ALS mouse model and in human sporadic ALS
Nadine Thau*§, Sarah Knippenberg, Sonja Körner, Klaus Jan Rath, Reinhard Dengler*, Susanne Petri*
Hannover Medical School, Department of Neurology, Hannover, Germany
* Centre for Systems Neuroscience (ZSN), Hannover, Germany
§ corresponding author
Preface - about this manuscript
Transcriptional coactivator PGC-1α plays a central role in the regulation of mitochondrial metabolism and biogenesis via activation of downstream factors such as NRF-1/-2 and Tfam. Alterations in expression and function have previously been described in models of Huntington’s and Alzheimer’s disease. Protective effects of PGC-1α were found in animal models of ALS. In the present study, we investigated the mRNA and protein expression of PGC-1α and PGC-1α-regulated factors in SOD1G93A ALS mice and non-transgenic controls (spinal cord & muscle) as well as in tissue of ALS and control patients (brain, spinal cord & muscle). Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction and impaired antioxidant defense both in the SOD1G93A ALS mouse model and in human sporadic ALS.
The manuscript has been published in Journal of Neuropathology and Experimental Neurology, Volume 71. PMID: 23147503
Manuscript II
Abstract
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss. While the exact cause of ALS still remains elusive, oxidative stress, inflammation and mitochondrial dysfunction have been identified as important contributors. PGC-1α plays a central role in the regulation of mitochondrial metabolism and biogenesis via activation of transcription factors such as NRF-1, NRF-2 and Tfam. Alterations in its expression and function have previously been described in models of Huntington’s and Alzheimer’s disease.
Moreover, protective effects could be shown in animal models of ALS. It is also known that PGC-1α levels correlate with the number of acetylcholine receptor clusters in muscle. This is of particular interest as there is evidence that neurodegeneration in ALS is a dying-back process.
In the present study, we investigated the mRNA and protein expression of PGC-1α and PGC-1α-regulated factors in SOD1G93A ALS mice as well as in ALS patients. We could detect significant alterations in mRNA expression of PGC-1α and downstream factors with 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, probably most relevant in the skeletal muscle.
Discussion
Discussion
The precise mechanisms leading to motoneuron death in ALS remain unknown up to know. The only treatment with marginal protective efficacy so far is riluzole, a glutamate antagonist, but it just prolongs life expectancy for several months (BENSIMON et al. 1994; DOBLE 1996; LACOMBLEZ et al. 1996a; LACOMBLEZ et al. 1996b; RIVIERE et al. 1998; MILLER et al. 2002; PAILLISSE et al. 2005). ALS etiology appears to be multifactorial, including genetic and environmental causes (ALMER 2003). Pathomechanisms include amongst others oxidative damage, mitochondrial dysfunction, growth factor deficiency, defects in axonal transport, abnormal protein aggregation and glutamate excitotoxicity. So diverse the hypotheses of the mechanisms are, so multitudinous are the approaches of therapeutic strategies.
The aim of the first study within this PhD project was to evaluate the role of the growth factor FGF-2 in ALS pathogenesis. Therefore we generated double mouse mutants transgenic for the human SOD1G93A mutation and either heterozygous or homozygous for the FGF-2 knock out. Against our previously assumed hypothesis we observed significant neuroprotective effects of FGF-2 deficiency in SOD1G93A mice. We could detect a significant delay in disease onset, less impairment of motor function and prolonged survival in SOD1G93AFGF-2+/- and SOD1G93AFGF-2-/- mice in comparison to mutant SOD1G93A mice with normal FGF-2 expression (SOD1G93AFGF- 2+/+). Behavioural and survival data correlated well with histological data: motoneuron
Discussion
numbers were significantly higher in the double mutants and astrocytosis was diminished at disease endstage.
Several mechanisms could underlie this rather unexpected protective effect: Previous studies have already demonstrated that FGF-2 does not only support survival of neurons but can also induce neuronal death, dependent on activation of different FGF receptors. Lesion-induced neuronal cell death was prevented in FGFR3- and FGF-2 knock out mice, indicating FGFR3-mediated cell death induced by FGF-2 binding (GROTHE et al. 2006). FGF-2 deficient SOD1G93A mice may therefore have benefitted from a reduction of pro-apoptotic effects of FGF-2. FGF-2 inhibits the expression of glutamine synthetase and can therefore increase excitotoxic effects of glutamate (KRUCHKOVA et al. 2001) so that FGF-2 deficiency may have indirectly prevented glutamate-mediated excitotoxicity. Furthermore FGF-2 is expressed not only in neurons but also in astrocytes (GONZALEZ et al. 1995) and induces astroglial reactivity (GODDARD et al. 2002). Diminished astrocytosis in ALS mice lacking FGF- 2 could reflect that FGF-2 indeed plays a role in astrocyte activation and therefore contributes to motoneuron degeneration in ALS. Most interestingly, we detected changes in mRNA expression levels of other neurotrophic factors in our double mutant animals: there was a significant up-regulation of CNTF mRNA in muscle tissue and of GDNF mRNA in lumbar spinal cord and muscle tissue in FGF-2 deficient SOD1G93A mice. As mRNA expression levels of other FGFs and FGF receptors and of other neurotrophic factors important for motoneurons did not differ between all groups, the significant up-regulation of these two genes may represent the putative compensatory effects of the FGF-2 knock out on expression levels of GDNF and CNTF. Both for CNTF and GDNF protective effects have been observed
Discussion
in mouse models of ALS. Moreover, interactions in mRNA-expression levels between FGF-2 and GDNF and also CNTF have been described before (SUTER- CRAZZOLARA u. UNSICKER 1996).
In the FGF-2 deficient mutant mouse used in this study, all FGF-2 isoforms (low and high molecular weight) were deleted. Further studies will need to address specific functions of the different FGF-2 isoforms in ALS as well as their subcellular localization. A more thorough characterization of disease-specific interactions between different growth factors will possibly enable novel therapeutic strategies based on combined administration of different neurotrophic factors.
Based on previously described alterations in PGC-1α expression and function in animal models of Huntington’s and Alzheimer’s disease (CUI et al. 2006; WEYDT et al. 2006; SHI u. GIBSON 2007; CHATURVEDI et al. 2009; QIN et al. 2009) and on studies in ALS transgenic mice which revealed protective effects of PGC-1α activation (LIANG et al. 2011; ZHAO et al. 2011; DA CRUZ et al. 2012), the aim of the second study was the analysis of mRNA and protein expression of PGC-1α and several PGC-1α-regulated factors in spinal cord and muscle tissue of SOD1G93A ALS mice and non-transgenic controls as well as in human post mortem brain and spinal cord tissue and in muscle biopsy specimens of ALS patients and controls.
Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction and impaired antioxidant defense both in the ALS mouse model and in human sporadic ALS. It is supposed that mitochondria are key players in the pathogenesis of ALS (BEAL 2000; BRUIJN et al. 2004; MANFREDI u. XU 2005) and it is known that PGC-
Discussion
metabolism and biogenesis (J. LIN et al. 2005; SCARPULLA 2006). In relation to this we found mRNA and protein reduction of PGC-1α together with Tfam which sustains and promotes mitochondrial biogenesis in human spinal cord and motor cortex tissue of ALS patients. In SOD1G93A ALS mice, we similarly detected significant PGC-1α and Tfam down-regulation in spinal cord and muscle tissue. We further observed decreased mRNA levels of NRF-1 and NRF-2 in human ALS tissues. In SOD1G93A mice, NRF-1, which is mostly important for the regulation of mitochondrial transcription factor, was downregulated at the mRNA and protein level.
Besides its direct effects on mitochondrial gene expression, PGC-1α is also involved in the regulation of genes of the antioxidant defence system, including the reactive oxygen species (ROS)-detoxifying enzyme mnSOD. We found reduced mRNA and protein expression of mnSOD in mouse spinal cord and muscle tissue in endstage disease SOD1G93A mice, consistent with reduced mnSOD expression in tissue from sporadic ALS patients, most apparent in the primary motor cortex. It is not clear if oxidative stress is a primary or a secondary cause of neurodegeneration in ALS, but research in both human tissue and transgenic animal models suggests that it is a critical factor leading to motoneuron death (BARBER u. SHAW 2010).
In the present study, we investigated not only advanced stages of sporadic ALS but also SOD1G93A mice in presymptomatic and early symptomatic stages, because several studies suggest that mitochondrial abnormalities occur long before disease onset (M. T. LIN u. BEAL 2006). Accordingly, we found reduction of PGC-1α gene expression already before disease onset in skeletal muscle tissue of SOD1G93A mice.
Similarly, PGC-1α mRNA was reduced in muscle biopsy specimens from ALS patients which dated from much earlier time points associated with less severe
Discussion
functional impairment than the post mortem brain and spinal cord material. This is of particular interest as there is evidence that degeneration of motor endplates occurs before the loss of motoneuron cell somata in the spinal cord and that neurodegeneration in ALS therefore is a dying back process which begins in the muscle (KENNEL et al. 1996; FREY et al. 2000; FISCHER et al. 2004). Similar to the assumption of Chartuvedi et al. in Huntington’s disease mice (CHATURVEDI et al.
2009), one can therefore postulate that the abnormalities in PGC-1α related mitochondrial gene transcription which we observed in pre-symptomatic ALS mice and sALS biopsy specimens contribute to muscular and distal axonal pathology.
PGC-1α plays a specific role in the skeletal muscle in the neuromuscular junction gene program by co-activation of NRF-2. Infection of C2C12 myotubes with PGC-1α- enconding adenoviral vectors led to upregulation of AChRε, Utrn and NRF-2 mRNA, which was shown before in a mouse model of Duchenne muscular dystrophy. In transgenic animals with either muscle specific PGC-1α-expression or muscle-specific PGC-1α knockout, it was demonstrated that PGC-1α levels directly correlate with the number of AChR clusters in the skeletal muscle (HANDSCHIN et al. 2007). However, we could not show a specific correlation between PGC-1α reduction and changes in expression of AChR and Utrn in the ALS mouse model or in human sporadic ALS.
ALS is associated with several defects in energy metabolism, including weight loss, hypermetabolism, and hyperlipidemia (DUPUIS et al. 2004; DUPUIS et al. 2008).
Muscle mitochondrial defects might lead to hyperlipidemia and impaired energy expenditure (DUPUIS et al. 2011). We observed decreased expression of PGC-1α, Tfam and NRF-1 which in muscle tissue of both SOD1G93A mice and sALS patients
Discussion
Activation or overexpression of PGC-1α could be a useful approach to compensate mitochondrial loss and dysfunction and to counteract oxidative stress and therefore protect against neurodegeneration in ALS.
Dysregulation or loss of neurotrophic factors or transcriptional regulators certainly is not the main cause of ALS, but seems to play an important role and to contribute to neuronal death.
As a multifunctional factor FGF-2 might be connected with diverse ALS-related mechanisms and processes like neuroinflammation, astrocytosis, excitotoxicity and of course dysregulation of other neurotrophic factors. The same diversity holds true for PGC-1α, which can influence energy metabolism and oxidative stress.
More precise knowledge of the molecular interactions in specific disease scenarios will allow for more efficient combined therapeutic strategies targeting more than one pathomechanism. Our results illustrate the need to analyze not individual factors separately but to identify the fine adjustment between their expression levels and characterize distinct disease-related interactions.
Summary
Summary
Nadine Thau
Transcriptional regulators and neurotrophic factors in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) Histopathological and biochemical studies in the G93A ALS mouse model and in ALS post mortem tissue
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motoneurons in the primary motor cortex, brainstem and spinal cord, ultimately leading to death within 3-5 years due to respiratory failure. The cause for the selective death of motoneurons is still unclear, but many different pathomechanisms are discussed including loss of neurotrophic factors, oxidative stress, and mitochondrial alterations.
We assumed that the neurotrophic factor FGF-2 plays a decisive role in ALS pathogenesis. To clarify the involvement of FGF-2 in chronic motor neuron loss we generated double mutant mice overexpressing the human ALS related SOD1G93A mutation and lacking endogenous FGF-2. While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1G93A animals, our results revealed a protective effect of FGF-2 reduction: double mutants showed a delay in disease onset, less impaired motor performance, prolonged survival, attenuated motoneuron loss and astrocytosis. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors (CNTF and GDNF) with proven protective effects in the ALS mouse model.
Summary
In the second study, we investigated the mRNA and protein expression of the transcriptional co-activator PGC-1α and of PGC-1α-regulated factors which play a central role in the regulation of mitochondrial metabolism and biogenesis. Disease- related alterations at the mRNA and protein level were identified in brain, spinal cord and muscle tissue of both SOD1G93A mice and ALS patients. Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction both in the ALS mouse model and in human sporadic ALS.
Zusammenfassung
Zusammenfassung
Nadine Thau
Transkriptionale Regulatoren und neurotrophe Faktoren in der Pathogenese der Amyotrophen Lateralsklerose (ALS) Histopathologische und biochemische Untersuchungen im G93A ALS Mausmodell und in ALS post mortem Gewebe Amyotrophe Lateralsklerose (ALS) ist eine bisher nicht kurativ behandelbare und unweigerlich tödlich verlaufende neurodegenerative Erkrankung, die zu rasch progredienter Degeneration motorischer Nervenzellen im primär-motorischen Kortex, Hirnstamm und Rückenmark führt. Die Patienten entwickeln fortschreitende Paresen und Muskelatrophien und versterben in der Regel 3 bis 5 Jahre nach Erkrankungsbeginn an Atemlähmung. Der genaue Grund für das selektive Absterben der Motoneurone ist bislang noch nicht geklärt, Verlust neurotropher Faktoren, oxidativer Stress und mitochondriale Veränderungen konnten jedoch als wesentliche Pathomechanismen identifiziert werden.
Basierend auf der Hypothese, dass der neurotrophe Faktor FGF-2 eine Rolle in der ALS-Pathogenese spielt, wurden im ersten Teil des Dissertationsprojektes transgene Mäuse generiert, die die ALS-auslösende humane SOD1G93A-Mutation überexprimieren, bei nur heterozygoter oder komplett fehlender Expression des FGF- 2-Gens. Entgegen der Erwartung, dass ein Verlust von FGF-2 den Phänotyp von SOD1G93A Tieren zusätzlich verschlechtern würde, zeigten unsere Ergebnisse einen protektiven Effekt der FGF-2-Reduktion: Die Doppelmutanten zeigten einen
Zusammenfassung
Verhaltenstests, eine höhere Lebenserwartung, weniger Motoneuronverlust und Astrozytose. Bei der Suche nach dem zugrundeliegenden Mechanismus konnte eine Hochregulierung anderer neurotropher Faktoren (CNTF und GDNF) mit nachgewiesenen protektiven Effekten im ALS-Mausmodell gezeigt werden.
In der zweiten im Rahmen der Dissertation durchgeführten Studie wurde die mRNA- und Proteinexpression des transkriptionalen Ko-Aktivators PGC-1α sowie PGC-1α- regulierter Faktoren, die eine zentrale Bedeutung für mitochondrialen Metabolismus und Biogenese haben, untersucht. Die Studie ergab krankheitsassoziierte Veränderungen auf mRNA- und Proteinebene in Gehirn-, Rückenmarks- und Muskelgewebe von SOD1G93A Mäusen und von ALS-Patienten. Diese Ergebnisse weisen auf eine Rolle von PGC-1α in der mitochondrialen Dysfunktion sowohl im ALS Mausmodell als auch in der humanen sporadischen ALS hin.
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Acknowledgements
Acknowledgements
First of all, I would like to thank my main supervisor Prof. Dr. Susanne Petri for scientific advises, continued support, confidence and patience throughout every phase of my PhD study and for always encouraging and helping me to achieve my aims.
Great acknowledge to my co-supervisors Prof. Dr. Claudia Grothe and Prof. Dr.
Baumgärtner for their excellent advice and unrestricted support.
Further I would like to thank Prof. Dr. Reinhard Dengler for giving me the opportunity to perform my PhD thesis in the department of Neurology of the Hannover Medical School.
Special thanks to Dr. Sarah Knippenberg for great support, motivation and constructive discussions (not only on the “main research stuff”).
I kindly thank Dr. Julia Jungnickel and Dr. Andreas Ratzka for their support and academic advice.
For excellent technical assistance I would like to thank especially Christiane Hotopp-Herrgesell, Carola Kassebaum, Kerstin Kuhlemann, Silke Fischer, Natascha Heidrich, Gesa Hellmich and Hella Brinkmann.
Also I would like to thank Andreas Niesel and Dr. Darius Moharregh-Khiabani for their perfect technical support in the matter of problems with computer, microscope, etc..
I kindly thank all members of the Neurology for the excellent working conditions and my colleagues from the “Hühnerstall” for constructive discussions and great support and especially for the fun moments in between.
Finally I thank my family and friends and in particular Patrick Eisentraut for boundless support, ongoing reinforcement, complete confidence and continuous patience throughout every period of my PhD study.
Declaration
Declaration
I herewith declare that I autonomously carried out the PhD-thesis entitled
“Transcriptional regulators and neurotrophic factors in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) Histopathological and biochemical studies in the G93A ALS mouse model and in ALS post mortem tissue”.
No third party assistance has been used.
I did not receive any assistance in return for payment by consulting agencies or any other person. No one received any kind of payment for direct or indirect assistance in correlation to the content of the submitted thesis.
I conducted the project at the following institution:
Department of Neurology Hannover Medical School
The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a similar context.
I hereby affirm the above statements to be complete and true to the best of my knowledge.
_______________________________
date, signature
