Martina Wiedau-Pazos

Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis

A subset of individuals with familial amyotrophic lateral sclerosis (FALS) possesses dominantly inherited mutations in the gene that encodes copper-zinc superoxide dismutase (CuZnSOD). A4V and G93A, two of the mutant enzymes associated with FALS, were shown to catalyze the oxidation of a model substrate (spin trap 5,5′-dimethyl-1-pyrroline N-oxide) by hydrogen peroxide at a higher rate than that seen with the wild-type enzyme. Catalysis of this reaction by A4V and G93A was more sensitive to inhibition by the copper chelators diethyldithiocarbamate and penicillamine than was catalysis by wild-type CuZnSOD. The same two chelators reversed the apoptosis-inducing effect of mutant enzymes expressed in a neural cell line. These results suggest that oxidative reactions catalyzed by mutant CuZnSOD enzymes initiate the neuropathologic changes in FALS.

Human Wild-Type Tau Interacts with wingless Pathway Components and Produces Neurofibrillary Pathology in Drosophila

Pathologic alterations in the microtubule-associated protein tau have been implicated in a number of neurodegenerative disorders, including Alzheimers disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). Here, we show that tau overexpression, in combination with phosphorylation by the Drosophila glycogen synthase kinase-3 (GSK-3) homolog and wingless pathway component (Shaggy), exacerbated neurodegeneration induced by tau overexpression alone, leading to neurofibrillary pathology in the fly. Furthermore, manipulation of other wingless signaling molecules downstream from shaggy demonstrated that components of the Wnt signaling pathway modulate neurodegeneration induced by tau pathology in vivo but suggested that tau phosphorylation by GSK-3beta differs from canonical Wnt effects on beta-catenin stability and TCF activity. The genetic system we have established provides a powerful reagent for identification of novel modifiers of tau-induced neurodegeneration that may serve as future therapeutic targets.

Directed differentiation of human induced pluripotent stem cells generates active motor neurons

The potential for directed differentiation of human‐induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS‐derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS‐derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS‐derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. STEM CELLS 2009;27:806–811

Induction of Nrf2 and xCT are involved in the action of the neuroprotective antibiotic ceftriaxone in vitro.

In amyotrophic lateral sclerosis, down‐regulation of the astrocyte‐specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine‐antiporter system xc−. Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system xc− and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2), a known inducer of system xc−, and the specific xc− subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone‐stimulated changes in Nrf2, system xc−, and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell‐derived human motor neurons. We conclude that ceftriaxone‐mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system xc− than to excitatory amino acid transporter induction.

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration.

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimers disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.

IL-17A is increased in the serum and in spinal cord CD8 and mast cells of ALS patients

The contribution of inflammation to neurodegenerative diseases is increasingly recognized, but the role of inflammation in sporadic amyotrophic lateral sclerosis (sALS) is not well understood and no animal model is available. We used enzyme-linked immunosorbent assays (ELISAs) to measure the cytokine interleukin-17A (IL-17A) in the serum of ALS patients (n = 32; 28 sporadic ALS (sALS) and 4 familial ALS (fALS)) and control subjects (n = 14; 10 healthy subjects and 4 with autoimmune disorders). IL-17A serum concentrations were 5767 ± 2700 pg/ml (mean ± SEM) in sALS patients and 937 ± 927 pg/ml in fALS patients in comparison to 7 ± 2 pg/ml in control subjects without autoimmune disorders (p = 0.008 ALS patients vs. control subjects by Mann-Whitney test). Sixty-four percent of patients and no control subjects had IL-17A serum concentrations > 50 pg/ml (p = 0.003 ALS patients vs. healthy subjects by Fishers exact test). The spinal cords of sALS (n = 8), but not control subjects (n = 4), were infiltrated by interleukin-1β- (IL-1β-), and tumor necrosis factor-α-positive macrophages (co-localizing with neurons), IL-17A-positive CD8 cells, and IL-17A-positive mast cells. Mononuclear cells treated with aggregated forms of wild type superoxide dismutase-1 (SOD-1) showed induction of the cytokines IL-1β, interleukin-6 (IL-6), and interleukin-23 (IL-23) that may be responsible for induction of IL-17A. In a microarray analysis of 28,869 genes, stimulation of peripheral blood mononuclear cells by mutant superoxide dismutase-1 induced four-fold higher transcripts of interleukin-1α (IL-1α), IL-6, CCL20, matrix metallopeptidase 1, and tissue factor pathway inhibitor 2 in mononuclear cells of patients as compared to controls, whereas the anti-inflammatory cytokine interleukin-10 (IL-10) was increased in mononuclear cells of control subjects. Aggregated wild type SOD-1 in sALS neurons could induce in mononuclear cells the cytokines inducing chronic inflammation in sALS spinal cord, in particular IL-6 and IL-17A, damaging neurons. Immune modulation of chronic inflammation may be a new approach to sALS.

Association of GSK3B With Alzheimer Disease and Frontotemporal Dementia

BACKGROUND Deposits of abnormally hyperphosphorylated tau are a hallmark of several dementias, including Alzheimer disease (AD), and about 10% of familial frontotemporal dementia (FTD) cases are caused by mutations in the tau gene. As a known tau kinase, GSK3B is a promising candidate gene in the remaining cases of FTD and in AD, for which tau mutations have not been found. OBJECTIVE To examine the promoter of GSK3B and all 12 exons, including the surrounding intronic sequence, in patients with FTD, patients with AD, and aged healthy subjects to identify single-nucleotide polymorphisms associated with disease. DESIGN, SETTING, AND PARTICIPANTS Single-nucleotide polymorphism frequency was examined in a case-control cohort of 48 patients with probable AD, 102 patients with FTD, 38 patients with primary progressive aphasia, and 85 aged healthy subjects. Results were followed up in 2 independent AD family samples consisting of 437 multiplex families with AD (National Institute of Mental Health Genetics Initiative AD Study) or 150 sibships discordant for AD (Consortium on Alzheimers Genetics Study). RESULTS Several rare sequence variants in GSK3B were identified in the case-control study. An intronic polymorphism (IVS2-68G>A) occurred at more than twice the frequency among patients with FTD (10.8%) and patients with AD (14.6%) than in aged healthy subjects (4.1%). The polymorphism showed association with disease in both follow-up samples independently, although only the Consortium on Alzheimers Genetics sample showed the same direction of association as the case-control sample. CONCLUSIONS To our knowledge, this is the first evidence that a gene known to be involved in tau phosphorylation, GSK3B, is associated with risk for primary neurodegenerative dementias. This supports previous work in animal models suggesting that such genes are therapeutic targets.

Charcot-Marie-Tooth disease CMT4A: GDAP1 increases cellular glutathione and the mitochondrial membrane potential

Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.

Integrative gene–tissue microarray-based approach for identification of human disease biomarkers: application to amyotrophic lateral sclerosis

Advances in genomics and proteomics permit rapid identification of disease-relevant genes and proteins. Challenges include biological differences between animal models and human diseases, high discordance between DNA and protein expression data and a lack of experimental models to study human complex diseases. To overcome some of these limitations, we developed an integrative approach using animal models, postmortem human material and a combination of high-throughput microarray methods to identify novel molecular markers of amyotrophic lateral sclerosis (ALS). We used laser capture microdissection coupled with microarrays to identify early transcriptome changes occurring in spinal cord motor neurons or surrounding glial cells. Two models of familial motor neuron disease, SOD1(G93A) and TAU(P301L), transgenic mice were used at the presymptomatic stage. Identified gene expression changes were predominantly model-specific. However, several genes were regulated in both models. The relevance of identified genes as clinical biomarkers was tested in the peripheral blood transcriptome of presymptomatic SOD1(G93A) animals using custom-designed ALS microarray. To confirm the relevance of identified genes in human sporadic ALS (SALS), selected corresponding protein products were examined by high-throughput immunoassays using tissue microarrays constructed from human postmortem spinal cord tissues. Genes that were identified by these experiments and located within a linkage region associated with familial ALS/frontotemporal dementia were sequenced in several families. This large-scale gene and protein expression study pointing to distinct molecular mechanisms of TAU- and SOD1-induced motor neuron degeneration identified several new SALS-relevant proteins (CNGA3, CRB1, OTUB2, MMP14, SLK, DDX58, RSPO2) and putative blood biomarkers, including Nefh, Prph and Mgll.

Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS

SUMMARY Human embryonic stem cell (hESC)-derived neurons have the potential to model neurodegenerative disorders. Here, we demonstrate the expression of a mutant gene, superoxide dismutase 1(SOD1), linked to familial amyotrophic lateral sclerosis (ALS) in hESC-derived motor neurons. Green fluorescent protein (GFP) expression under the control of the HB9 enhancer was used to identify SOD1-transfected motor neurons that express human wild-type SOD1 or one of three different mutants (G93A, A4V and I113T) of SOD1. Neurons transfected with mutant SOD1 exhibited reduced cell survival and shortened axonal processes as compared with control-transfected cells, which could survive for 3 weeks or more. The results indicate that hESC-derived cell populations can be directed to express disease-relevant genes and to display characteristics of the disease-specific cell type. These genetically manipulated hESC-derived motor neurons can facilitate and advance the study of disease-specific cellular pathways, and serve as a model system to test new therapeutic approaches.