Workpackage 8: Functional Studies




Full behavioural, histopathological and molecular characterisation of mice which overexpress mutant and wildtype TDP43 and FUS (become available in 2010);


Development of cell culture assays to characterize specific cellular defects in motor neurons of these mice on the cellular level. These studies include comparison of primary motoneurons from mouse models with mouse and human and ES cell derived motoneurons. This development includes techniques for enrichment of ES cell derived motoneurons so that these cells can be used in WP2-6 for –omics work.


Full behavioural, histopathological and molecular characterisation of mice which conditionally overexpress mutant and wildtype TDP43 and FUS (TDP-43: available in 2010; FUS: to be generated). We will express the transgene ubiquitously and in motor neurons selectively


To undertake a full characterisation and analysis of the pathology of the mouse strains with a mutation in the endogenous TDP-43 by in vivo investigation of their neuromuscular physiology and histopathology in order to compare this to human neurodegenerative conditions particularly ALS, in parallel with the in vitro analysis of the molecular cell biology of these mouse mutants compared to normal animals. This will include the analysis of the transcriptome of these mice, as well as the study of the effects of the mutations on the normal cellular functions of this protein in primary motor neuron cultures


To characterize the phenotype of a mutant and wildtype TDP-43 overexpressing zebrafish model and to study the effect of factors known to affect TDP-43 location such as PGRN. To characterize the phenotype of mutant and wildtype FUS overexpressing zebrafish.


To establish in vitro models for selected mutations in the dynactin P150 subunit which are found in humans (position 34, 59, 63, 196, 1049, 1248 and others).


To determine the phenotype of mouse models over expressing the DCTN1 63 and 196 mutation and compare it to the mutant SOD1 phenotype.


To elucidate whether tubulin over- or underacetylation affects neurodegeneration of motor neurons and in particular the motor neuron degeneration induced by mutant SOD1.


To elucidate the molecular basis for the increased susceptibility to ALS that is associated with low Elp3 expression.


To elucidate the molecular basis for the increased susceptibility to ALS that we identified to be associated with UNC13A polymorphisms


Provide a collection of samples from motor neuronal cell models of ALS, expressing G93A SOD1, and exposed to risk factors inducing changes of energy metabolism (hypoxia), and from transgenic SOD1G93A mice and rats fully phenotypically characterized and at different stages of the disease, to the consortium for multilevel -omics analyses.


Validation of biomarkers based upon metabolic and oxidative stress (nitroproteins) previously identified by proteomic studies in models in vitro and in vivo.


To characterize the role of the PHDs in motor neuron degeneration. We will determine the expression patterns as well as the downstream targets of the different PHD isoforms and factor inhibiting HIF (FIH) in the spinal cord in healthy mice and in SOD1G93A mice during the disease course. We will evaluate the effect of PHD loss- and gain-of-function studies in mouse models of ALS. We will dissect the cell-specific role of PHD1 in further detail. Additionally, we will define the mechanisms via which PHDs modify motor neuron disease, i.e. via regulation of neurotrophic growth factors such as VEGF, vascular effects, mitochondrial respiration and oxidative stress (hypoxia tolerance), and other possible mechanisms. Moreover, as PHD enzymes are druggable targets, we will also evaluate the therapeutic potential of modulating PHD activity in preclinical models of ALS.


To delineate the contribution of altered lipid metabolism and SCD1 to ALS. We will study the effects of SCD1 loss on motor and metabolic phenotype. The identification of upstream factors responsible for SCD1 loss will be also undertaken, in order to isolate specific causes of lipid metabolism dysfunction in ALS muscle.


To develop therapeutic strategies based on lipid metabolism handling and SCD1 function. We will determine whether restoring SCD1 levels in skeletal muscle by nutritional, pharmacological and genetic means is sufficient to rescue mutant SOD1 mouse hypermetabolism and delay motor neuron death.


To perform a morpholino-based genetic screen of the mutant SOD1 zebrafish model we described previously.


To validate the hits (WP8-1)


To validate the hits identified in the newly generated mutant TDP-43 model and in a mouse model for motor neuron degeneration (mutant SOD1)


To explore the therapeutic significance of the hits identified in the mutant SOD1 based model for ALS in the zebrafish


To validate the results of WP2-6 by modelling the factors identified in the zebrafish and to study the interaction of previously known ALS-causing or –modifying genes with the newly identified factors from these WPs.


To generate induced pluripotent stem cell lines (iPS cells) from ALS patients and non-ALS controls for the in-vitro generation of glia and motor neurons


To perform high throughput small molecule- and siRNA screens to identify new targets for disease intervention


To use a comparative analysis approach to identify novel marker genes that could aid as diagnostic tools for early detection of ALS


To validate the factors identified in WP2-6 by studying them in vitro using cells derived from patients with sporadic and familial ALS


To validate the role of factors identified in WP2 to 6 in the biology of motor neurons in culture.


To validate the effect of these factors in the zebrafish models described.


Throughout the project, samples from the models generated as described below will be used as substrate for the approach described in WP2 to 6.  From the start, we will make use of well defined models of familial ALS (mutant SOD1 rodents and NSC34 cells) to allow to initiate WP2 to 6 using animal and in vitro samples. The advantage  of this approach in respect to what has been done so far  is the unique opportunity offered by this consortium to have a complete -omic profile of peripheral and CNS tissues from the same animals at defined stages of the disease. In a first set of experiments, groups of transgenic SOD1G93A mice with difference in  disease progression, not attributable to variance in transgene copies, will be used. Brain, lumbar spinal cord ventral/ dorsal horns, muscle , blood, will be taken from each animal at presymptomatic, symptomatic and advanced stage of disease.  All collected tissues will then be sent to the –omic units. Transgenic SOD1G93A rats will be used to examine CSF and nerves as these tissues are more easily obtained. The outcomes from the analyses will be validated in the new genetic animal models  developed within this WP8.

Results from this analysis will provide an indication of whether and how the peripheral markers are representative of the progressive pathological condition in the CNS for development of lab assays useful to monitor the disease progression and the therapy outcomes in patients.

Furthermore, SOD1G93A mice and NSC34 cell lines will be used for the functional validation of the outcome from the ALS system biology model, like genes modifier and /or relevant pathways to ALS, as described lower.

The main goal of the current WP is to generate new models which can be studied on their own, and can be used as a source of samples for WP2 to 6.

Task 1. Generation of ALS models related to RNA processing: modeling mutant TDP-43 and FUS

Models of mutant TDP-43 overexpression

 We are currently generating transgenic mouse models with mutations in TDP43 and FUS. We are focussing on the mutations M337V and G348C in TDP43, and R521C, T525L in FUS/Tls because these mutations interfere with interactions with other proteins that we have already characterized in the context of the Smn--/SMN2tgmotoneurons from the SMA mouse model {Rossoll, 2003 #39}. The group of Dr Sendtner is highly experienced in isolating highly enriched primary motoneurons from mouse models of motoneuron disease, i.e. pmn and/or Smn-/-/SMN2tg mice  and these techniques shall also be applied to investigate the specific defects in TDP43 and FUS mutant motoneurons. We would like to characterize axon and dendrite growth, to characterize spontaneous excitability, differentiation of the axonal growth cone and of the presynaptic apparatus. In addition, we have established techniques for 2-photon-microscopy that can be used to investigate neuromuscular endplate function in living mice in vivo. These techniques will be applied to the analysis of these mouse models and could also be made available for collaborative projects within this consortium.

Conditionally TDP-43 overexpressing mice have been generated already. The transgene has been cloned in the Rosa26 locus, in which a floxed stop signal is present. This means the transgene will only be expressed upon deletion of the stop sequence by Cre. Cre expression will be driven by a CMV promoter and by the VAchT promoter to obtain ubiquitous and motor neuron-specific expression respectively. All these mice are available to us and crossbreeding of the TDP-43 mice with the Cre overexpressing mice can be started in late 2010. The FUS conditionally overexpressors will be generated using the same approach.

Models with endogenous TDP-43 mutation

We will perform an in vitro analysis of the effects of the mutations we have identified in 6 mouse strains with TDP43 mutations, including 3 with mutations in exon 6. Using primary motor neurons and transfected cell lines we will examine the effects on the known cellular functions of TDP-43 including stress granule formation, neurite outgrowth, RNA binding, TDP-43 protein localisation. This study will also determine which of the remaining mutant TDP-43 strains of mice to rederive and research. We then will investigate the two mouse strains already rederived, followed by the most interesting stains identified in the in vitro work, to assess their phenotype and to understand the biology of TDP-43, particularly with respect to RNA metabolism. We will undertake a longitudinal analysis of their locomotor phenotype by rotarod and grip strength testing, and at key stages assess their neuromuscular function, histopathology (brain, spinal cord, muscle, peripheral nerve) and cell biology (stress granule formation, presence of inclusion bodies, TDP-43 protein localisation). We will also undertake molecular cell biology and RNA metabolism studies.

Zebrafish models for mutant TDP-43 and FUS

We have generated a zebrafish model for TDP-43 associated ALS by overexpressing mutant (A315T) and wild type TDP-43. TDP-43A315T dose dependently induced axonal shortening and aberrant branching, similar to those induced by mutant SOD1. However, in contrast to wild type SOD1, wild type TDP-43 also induced these abnormalities albeit to a statistically significant lesser degree than TDP-43A315T  Similarily we will generate mutant FUS zebrafish by expressing wildtype and mutant FUS mRNA as we have done before with SOD1.

Using these fish we will study the pathogenic role of TDP-43 cleavage by caspase 3 and of phosphorlyation. This will be done by evaluating the phenotype induced by mutants that are caspase resistant or either are resistant to or are constitutively phosphorylated. All these constructs are available in the lab. Furthermore, these models will be used to study the interaction between the different genes involved in neurodegeneration in general motor neuron degeneration in particular. For reasons explained above, we will study the interaction between PGRN and TDP-43 by studying the effect of PGRN overexpression on the mutant TDP-43-induced fish phenotype. This will be done by rescuing the axonal abnormalities with co-injections of progranulin RNA.

Task 2. Intracellular trafficking in motor neuron degeneration

Models to study the role of dynactin in sporadic and familial ALS

Motor neurons will be virally transfected with DCTN1 constructs harbouring human mutations. In these cells neuritic transport will be studied using life cell imaging and mechanisms of peripheral and central synaptogenesis (in collaboration with  S.  Jablonka, T Böckers, T Wirth). We will study mutations 34, 59, 63, 177, 178, 196, 1490, 1248. The phenotype of mice overexpressing mutations at positions 63 and 196 will be characterized by behavioural, motor and MRI phenotyping. Phenotypes of these mice will be compared with high and low Cu/Zn SOD expressing mice.

Tubulin acetylation in neurodegeneration

If low acetylation levels of a-tubulin makes neurons susceptible to neurodegeneration, and increased levels protect from it, it can be hypothesized that increasing acetylation of a-tubulin through inhibition of the deacetylating HDAC6 or SIRT2 should protect.

We will therefore investigate whether inhibition of these enzymes in the zebrafish will rescue the axonopathy induced by mutant SOD1 and mutant TDP-43. This will be done using morpholinos against these proteins, and by evaluating the aberrant branching induced by mutant SOD1 or TDP-43.

Tubacin is a specific HDAC6 inhibitor, that was made available to us in a collaboration with dr S. Schreiber and dr. J. Bradner, Dana Farber Cancer Institute, Boston. Therefore, we will treat fish in which mutant SOD1 or mutant TDP-43 is overexpressed with tubacin and evaluate the effect on axonal length and aberrant branching. Furthermore, we will investigate whether tubacin can protect against yet another form of motor axonal damage, the one induced by mutant HSP27.

Finally we will evaluate the effect of tubulin acetylation on axonal transport by measuring axonal transport of mitochondria. Both primary motor neuron cultures and dorsal root ganglion neuronal cultures are routinely available in our lab and have been extensively studied in previous projects. We have established a method to measure axonal transport in these cultures as described by De Vos et al.{De Vos, 2007 #40}. We then will study the effect of increasing acetylation on the dysregulation of axonal transport by mutant SOD1 and HSP27 in these models by investigating the effect of HDAC6 inhibition (using tubacin) on the abnormalities of mitochondrial transport induced by SOD1G93A and by HSPB1S135F expression. These neurons will be cultured from SOD1G93A and SOD1WT E13 mice, and from HSPB1S135F and HSPB1WT mice  resp., as we routinely do in the lab.

UNC13A in motor neuron degeneration

Munc13-1 (UNC13A) knockout mice have been generated and are available to us through

Dr. Nils Brose (Max-Planck-Institut for Experimentelle Medizin, Gottingen, Germany). Mice lacking Munc13-1 (UNC13A) die immediately after birth and show a striking defect in neurotransmitter release {Betz, 1998 #43;Augustin, 1999 #44}. UNC13A is required for priming presynaptic vesicles before they fuse to the cell membrane and in the absence of UNC13A the synaptic vesicle cycle is arrested after docking but before fusion, preventing the release of neurotransmitters {Brose, 2000 #45}. In studies to determine the role of UNC13A in ALS we will use mice heterozygous for the Munc13-1 (UNC13A) deletion (which is analogous to the human situation where most patients will carry one affected allele). The analyses are aimed at defining the role of UNC13A in motor neuron function and survival in vivo. The experiments will be performed on 1) Munc13-1 and control (heterozygous/wildtype littermates) mice, and 2) Munc13-1 mice which have been cross-bred with G93A-hSOD-1 and hSOD1 mice. This latter group of mice will enable us to study whether a deficiency (missing one allele) in the UNC13A (Munc13-1) gene modifies (e.g. accelerates) the ALS-like phenotype of G93A-hSOD-1 mice. Brain, spinal cord and muscle tissue will be collected from 5 to 18 week old mice, sectioned and subjected to immunohistochemistry and in situ hybridization. Extensive analyses of motor neuron survival as well as clinic phenotype to define disease onset, disease progression and lifespan will be performed on 1) Munc13-1 and control (heterozygous and wildtype littermates) mice, and 2) heterozygous Munc13-1 mice which have been cross-bred with G93A-hSOD-1 and hSOD1 mice.

These experiments may provide new animal models for studying ALS, and will establish the function of UNC13A in the intact mature motor system and its potential contribution to motor neuron degeneration in ALS.

Task 3. Study metabolic dysregulation as a pathogenic factor in ALS

Oxygen sensors, oxidative stress and metabolic dysregulation in ALS

Expression profiles of PHD1-3, FIH, HIF-1a and HIF-2a in the spinal cord will be analyzed by immunohistochemistry on spinal cord sections of wild-type and SOD1 mice during disease course, complemented by RT-PCR and immunoblotting. Mice underexpressing PHDs and HIFs (all available), will be intercrossed with SOD1 mice. Phenotypical analysis (motor performance, survival, additional histological characterization) will allow to define the role of PHDs and HIFs in MND. Initial results indicate that homozygous PHD1 deficiency extends the lifespan in SOD1G93A mice. We will further unravel the cell-specific role of PHD1 using PHD1lox/lox mice crossed with cell-specific Cre lines (GFAP-Cre, PDGFb-Cre, Thy1-Cre mice; available). We will subsequently study the mechanisms of the PHD/FIH mediated modulation of MND. Vascular phenotyping, analysis of neurotrophic signaling and metabonomic studies will be performed in the PHD knock-out SOD1 mice. Both vessel structure and function will be analysed as endothelial PHD2 haplodeficiency is known to alter endothelial morphology improving vessel perfusion and oxygenation. Metabolic reprogramming will be assessed in in vitro motoneuron cultures by measuring glycolytic flux, oxygen consumption, expression profiles and activity of metabolic enzymes and oxidative stress measurement.  The downstream effectors of PHDs in different neural cell types will be determined by assessing the expression of HIF-1 vs HIF-2 in isolated motoneurons from PHD knockout mice and complementary by using RNAi techniques in neural cultures. The therapeutic potential of the PHD inhibition in MND will be explored as preliminary results indicate that PHD1 deficiency delays MND in mutant SOD1 mice.

Dyslipidemia and metabolism in ALS

For the study of dyslipidemia and metabolic dysregulation, we will determine whether loss of SCD1 induces by itself motor neuron pathology. This will be done by studying whether scd1-/- mice display neuromuscular defects and abnormal nerve regeneration after crush. We will also test whether the neuromuscular pathology of mutant SOD1 mice is dependent upon diets supplemented with monounsaturated fatty acids, the products of the enzymatic reaction of SCD1. Furthermore, we will determine the metabolic consequences of SCD1 loss in ALS mice. We will investigate whether SCD1 loss in ALS mice causes their metabolic phenotype. For this, we will rescue SCD1 expression through gene therapy in ALS mice and study their metabolic phenotype. To determine the effects of nutritional complementation on lipid metabolism, SCD1 loss and neuromuscular phenotype of ALS mice, we will test whether the already documented protective effect of high fat feeding restores normal SCD1 function, and determine whether monounsaturated fatty acid supplementation is sufficient to rescue the metabolic and motor phenotype of ALS mice. Finally, we will study the effect of therapeutic interventions on SCD1 function in mutant SOD1 mouse. We will attempt to compensate for SCD1 loss in mutant SOD1 mice, by means of pharmacological (stimulation of the transcriptional activator LXR with in vivo TO901317 treatment) and genetic (targeted overexpression of SCD1 in skeletal muscle) approaches, in order to determine whether SCD1 is a critical player in ALS-linked hypermetabolism and motor neuron death.

Significance of nitrative stress in  ALS

Nitroproteins, specific marker of nitrative stress, have been identified as candidate biomarkers of the disease in ALS sporadic patients and mutant SOD1 experimental models {Casoni, 2005 #26;Nardo, 2009 #27}. Nitroproteins will be analyzed in tissues and cells collected within this WP from the different experimental models to validate their association with disease mechanisms. Redox proteomic tools and antibodies specific for single nitrated proteins (nitroactin) will be used for quantitative analysis. Nitroactin will also be measured by large scale immunoassays in lymphoblastoid cells from a retrospective cohort of ALS patients (n=200) and controls (n=200) provided by partners 12 and 14.

Task 4. Use zebrafish as a model to study the pathogenesis and treatment of ALS

Morpholino-based screen to identify modifying genes.

The mutant SOD1 induced axonopathy we described before, will be used as a model {Lemmens, 2007 #35}. We will screen 300 morpholinos against neuronally expressed genes and genes involved in intracellular transport to identify those that rescue the phenotype. The hits will be validated in the mutant TDP-43 model. A explorative screen of 20 genes has already identified one hit. This morpholino targets the RTK2, a receptor of the ephrin family of axonal repellent factors (EphA4) {Pasquale, 2008 #48}. We have obtained mice in which EphA4 has been knocked out and will validate the findings obtained in the zebrafish by studying the effect of EphA4 deletion on mutant SOD1-induced motor neuron degeneration in the mouse. Similarly we will explore whether blocking RTK2 in the fish or mouse has therapeutic potential. Lastly, we will investigate whether polymorphisms in the EphA4 gene are associated with ALS in a large scale association study (see other work package). Newly identified hits will be studied in the same way.

Modeling factors identified in unbiased screens in the zebrafish

We will study the effect on motor axons of overexpression and knockdown of genes of which the products establish the basis of the ALS model generated based upon the results of WP2-6, as we have done before. Furthermore, we will study the interactions of these genes with those that are known to cause monogenic ALS and have been modeled in zebrafish before or in WP8-1.

Task 5.  Make induced pluripotent stem cells as a model to study familial and sporadic ALS

The Department of Neurology in Utrecht, in collaboration with its partners involved in this proposal, has been active in genetics research in ALS and has established a unique national ALS database and biobank containing cells from more than 1,500 patients. This biobank includes fibroblast cell lines of more than 150 patients with various genetic backgrounds. Furthermore, fibroblasts and keratinocytes are available from the departement of Neurology in Ulm and Leuven, which will provide us with an unprecedented toolbox of diverse patient samples. 

We have extensive experience with the generation of human patient-derived iPS cell lines, and the subsequent differentiation of these cells towards the neural lineage. We plan to generate iPS cell lines from ALS patients, as well as healthy control cell lines and use these stem cells to generate an in vitro model system of human ALS. With the UMC screening facility led by Dr. David Eggan, we plan to perform high throughput screens for small molecules or siRNA’s that positively influence ALS motor neuron degeneration. In addition, we plan to perform a comparative gene expression analysis to identify novel disease specific markers that could allow early detection of the disease.

Task 6. Validation of consortial findings in primary motor neuron cultures and zebrafish

This WP is solely devoted to the validation of the findings obtained in the unbiased approaches that have been used in WP2 to 6. We will validate those factors in two easily accessible models, one in vitro and one in vivo.

Primary motor neuron cultures have the advantage that they are immediately available and that they can be readily transfected and transduced. In addition, methods to study their survival, response to exogenously administrated factors, their intracellular (axonal) transport and mRNA profile are available. Our consortium has vast experience with these methods as pointed out earlier.
Zebrafish are in vivo models that can be easily genetically manipulated. They are a tool of choice to rapidly model interaction of newly identified factors (WP2 to 6) with known ALS causes such as mutant SOD1, TDP-43, FUS, as we have shown before {Lemmens, 2007 #35}.