ALS Research Journal News - July/August 2012

This summary includes some of the most recent advances in ALS research. If you would like certain news items featured, or have questions, please contact researchgrants@alsa-national.org.

Quick Summary: This month brings promising news of a potential peripheral biomarker for early disease. In addition, new research highlights the role of gene mutations in sporadic ALS, potential loss-of-function mechanisms for FUS and TDP-43, and the roles of microglia and oligodendrocytes in helping and harming motor neurons.

Contents:

Feature News Story

Blood Biomarker for Early Disease Implicates Immune System

Clinical Updates:

Blood Protein May Provide a Biomarker for Late-Stage Progression and Response to Treatment
C9ORF72 Mutation in 37% of Familial, 5% of Sporadic ALS in United States
High Frequency of Gene Mutations in Sporadic ALS
ALS Online Database (ALSoD): Bioinformatics Tool for ALS Genes Is Available
Lack of BDNF Response in ALS May Be Due to Alternative Receptors
No Benefit from Pioglitazone
Risk of ALS Associated with Smoking but Not Drinking

Cellular, Molecular and Genetic Research

Microglia Are Protective Early but Harmful Late in ALS
Knockout and Overexpression of TDP-43 Affect Different Genes
Excess Normal TDP-43 is Toxic and May Drive Neurodegeneration
Ataxin 2 Expansion Activates Caspase 3, Cleaving TDP-43
Loss of FUS Causes Motor Neuron Disease in Flies
FUS Mutations Reduce Nuclear Binding Signal
FUS Binds to Tau RNA, and Loss of FUS Is Pathogenic
Loss of TDP-43 Causes a Motor Neuron Phenotype in Mice
Metabolic Support from Oligodendrocytes Is Reduced in ALS
Newest ALS Gene Impairs Axon Growth

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Feature News Story

Blood Biomarker for Early Disease Implicates Immune System

SOD1 mice and ALS patients have increased levels of inflammatory white blood cells, or monocytes, in the peripheral circulation early in the disease, and their detection may provide a biomarker for early disease progression, according to a new study published in The Journal of Clinical Investigation. The study also shows that blocking the inflammatory molecules with antibody was therapeutic in the SOD1 mouse, suggesting a potential new avenue for therapy.

The inflammatory signature, characterized by surface expression of LY6C and a suite of gene expression changes, was seen in splenic monocytes in SOD1 mice two months before symptom onset and persisted during disease progression. At the same time, microglia expressed complementary receptors that served to attract the monocytes into the spinal cord and not the brain in the SOD1 mouse model. Treatment with anti-LY6C antibody reduced monocyte infiltration into the central nervous system and extended survival.

In both familial and sporadic ALS patients, a similar inflammatory signature was found in peripheral monocytes, including a set of microRNAs that were upregulated in both ALS patients and SOD1 mice.

“These results support the involvement of immune/inflammatory responses in motor neuron degeneration in ALS,” the authors said. “Based on our results in the SOD1 mouse, we postulate that treatment to modulate inflammatory monocytes may slow ALS disease progression.” Finally, the researchers note that the monocyte profile may serve as a biomarker for disease onset and progression, currently an unmet need in ALS clinical studies. Further validation of this as a biomarker will be required in a larger cohort of familial and sporadic ALS patient samples.

Butovsky O, Siddiqui S, Gabriely G, et al. Modulating inflammatory monocytes with a microRNA gene signature ameliorates murine ALS. J Clin Invest 2012;122

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Clinical Research

Blood Protein May Provide a Biomarker for Late-Stage Progression and Response to Treatment

Levels of neurofilament heavy chain (NfH) in the blood correlate with disease progression and response to treatment in the SOD1 mouse, according to a new study published in PLoS One. If replicated in humans, the protein may provide a convenient and sensitive biomarker for ALS disease progression and response to treatment. The lack of such a biomarker has been a central deficit in ALS clinical research and the search for new therapies.

NfH is one component of the cytoskeleton, a protein framework that supports cells, including motor neurons. Previous work has shown that levels of NfH in the cerebrospinal fluid vary in ALS (Ganesalingam et al, 2011; Boylan et al, 2009) and have potential as a biomarker, but routinely accessing the CSF is problematic. In this study, researchers examined the level of NfH in blood plasma in SOD1 mice after disease onset and in response to treatment with arimoclomol. Arimoclomol has been shown to improve mouse survival and is currently in clinical trials in ALS patients.

The level of NfH in plasma increased in the late stage of the disease in mice and was correlated with declines in muscle strength and loss of motor neurons. NfH fell in response to treatment with arimoclomol. “The findings of this study therefore may represent an important step in the development of an easily accessible, comprehensive marker for ALS that may have utility in
future clinical trials, particularly since patients are usually diagnosed and recruited into trials well after the onset of symptoms,” the authors said. They also note that the level of NfH increases in a number of neurodegenerative diseases and so may not be useful as a diagnostic test for ALS.

Lu CH, Petzold A, Kalmar B, Dick J, Malaspina A, Greensmith L. Plasma Neurofilament Heavy Chain Levels Correlate to Markers of Late Stage Disease Progression and Treatment Response in SOD1(G93A) Mice that Model ALS. PLoS One. 2012;7(7):e40998.

http://dx.plos.org/10.1371/journal.pone.0040998

http://www.ncbi.nlm.nih.gov/pubmed/22815892

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C9ORF72 Mutation in 37% of Familial, 5% of Sporadic ALS in United States

Analysis of 617 patients from across the United States with ALS and related disorders indicates that the C9ORF72 mutation is found in 37% of familial and 4.9% of sporadic cases. The mutation was

associated with a slightly higher incidence of bulbar onset. Higher mutation frequency was found in FTD-ALS cases (45%) than in pure ALS (11.3%).

Rutherford NJ, Dejesus-Hernandez M, Baker MC, Kryston TB, Brown PE, Lomen-Hoerth C, Boylan K, Wszolek ZK, Rademakers R. C9ORF72 hexanucleotide repeat expansions in patients with ALS from the Coriell Cell Repository. Neurology. 2012 Jul 18. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22815561

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High Frequency of Gene Mutations in Sporadic ALS

Among 480 ALS patients with sporadic (non-familial) ALS, 11% had mutations in a known ALS gene, including TARDBP (2.7%), C9ORF72 (2.5%) and SOD1 (2.1%). “A considerable proportion of patients with [sporadic ALS] harbored mutations in major ALS genes,” the authors said. “This result has relevant implications in clinical practice, namely in genetic counseling,” since a proportion of patients with no family history of the disease are likely to carry gene mutations.

Lattante S, Conte A, Zollino M, Luigetti M, Del Grande A, Marangi G, Romano A, Marcaccio A, Meleo E, Bisogni G, Rossini PM, Sabatelli M. Contribution of major amyotrophic lateral sclerosis genes to the etiology of sporadic disease. Neurology. 2012 Jul 3;79(1):66-72. Epub 2012 Jun 20.

http://www.ncbi.nlm.nih.gov/pubmed/22722621

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ALS Online Database (ALSoD): Bioinformatics Tool for ALS Genes is Available

The ALS Online Database http://alsod.iop.kcl.ac.uk , funded by The ALS Association, The MND Association and The Therapy Alliance is available for ALS researchers to query and contribute to. The authors note: “ALSoD is not only a central repository for storing genetic information on the more than 100 ALS-related genes reported to date but also shows graphs of the gender, age of onset, phenotype, and family history distributions of patient data stored on the database broken down by gene, mutation or phenotypic group. The various analytical devices include a comparison tool, a pathogenicity prediction tool, and a credibility tool to enable ALS researchers objectively assess the evidence for association with ALS,” along with feedback tools and systems for linking to other databases.

Abel O, Powell JF, Andersen PM, Al-Chalabi A. ALSoD: A user-friendly online bioinformatics tool for amyotrophic lateral sclerosis genetics. Hum Mutat. 2012 Jul 2. doi: 10.1002/humu.22157. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22753137

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Lack of BDNF Response in ALS May Be Due to Alternative Receptors

The neurotrophic factor BDNF has failed in clinical trials in ALS despite its beneficial effect on motor neurons in model systems. One reason may be that the administered BDNF is tied up with a receptor that can’t send a neurotrophic signal, according to a new study in PLoS One. The receptor, called TrkB.T1, is a truncated form of the full-length BDNF receptor, called TrkB.FL. The truncated form is found widely in motor neurons and is believed to have independent signaling functions. Researchers found that deleting the truncated receptor delayed disease onset and development of muscle weakness in SOD1 mice, though it did not increase lifespan. The same benefit was achieved with a drug that activates the full-length receptor in the absence of BDNF, suggesting that the truncated receptor limited the responsiveness of motor neurons to the neurotrophic factor.  “Our data suggests that a better understanding of the signal transduction pathways downstream of neurotrophins and of modalities to activate these pathways may be more critical than identifying strategies for delivering exogenous neurotrophins,” such as gene therapy, the authors said.

Yanpallewar SU, Barrick CA, Buckley H, Becker J, Tessarollo L. Deletion of the BDNF Truncated Receptor TrkB.T1 Delays Disease Onset in a Mouse Model of Amyotrophic Lateral Sclerosis. PLoS One. 2012;7(6):e39946. Epub 2012 Jun 27.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0039946

http://www.ncbi.nlm.nih.gov/pubmed/22761934

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No Benefit from Pioglitazone
The diabetes drug pioglitazone is not beneficial in ALS despite positive results in the mouse model of the disease. Two hundred-nineteen ALS patients were randomized to receive placebo or 45 mg/day of pioglitazone. The study was stopped approximately two years after the first patients were enrolled due to lack of efficacy. Treatment was no better than placebo at increasing survival, preventing functional decline, slowing loss of vital capacity, or improving quality of life. The lack of benefit, despite positive results in mice, argues strongly for development of new animal models, the authors said.

Dupuis L, Dengler R, Heneka MT, Meyer T, Zierz S, Kassubek J, Fischer W, Steiner
F, Lindauer E, Otto M, Dreyhaupt J, Grehl T, Hermann A, Winkler AS, Bogdahn U,
Benecke R, Schrank B, Wessig C, Grosskreutz J, Ludolph AC; the GERP ALS Study
Group. A Randomized, Double Blind, Placebo-Controlled Trial of Pioglitazone in
Combination with Riluzole in Amyotrophic Lateral Sclerosis. PLoS One. 2012;7(6):e37885. Epub 2012 Jun 8.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0037885

http://www.ncbi.nlm.nih.gov/pubmed/22715372

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Risk of ALS Associated with Smoking but Not Drinking

A comparison between almost 500 ALS patients and 1,600 controls for current and past smoking and drinking habits indicates an increased risk of ALS among current smokers (odd ratio 1.38), who also had reduced survival. In contrast, current alcohol consumption was associated with a decreased risk (odds ratio 0.52).

de Jong SW, Huisman MH, Sutedja NA, van der Kooi AJ, de Visser M, Schelhaas HJ, Fischer K, Veldink JH, van den Berg LH. Smoking, Alcohol Consumption, and the Risk of Amyotrophic Lateral Sclerosis: A Population-based Study. Am J Epidemiol. 2012 Jul 11. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22791740

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Cellular, Molecular, and Genetic Research

Microglia are Protective Early, but Harmful Late in ALS

Microglia are immune cells of the central nervous system that can be either protective or toxic to motor neurons. In a new study featured in Pub Med, researchers show in SOD1 mice that microglia play a protective role at the beginning of the disease but later develop a toxic phenotype that injures motor neurons. Because individual microglia can switch between these roles, the authors suggest that therapies to promote a more protective phenotype for microglia may be beneficial in ALS.

Liao B, Zhao W, Beers DR, Henkel JS, Appel SH. Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS. Exp Neurol. 2012 Jun 23;237(1):147-152. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22735487

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Knockout and Overexpression of TDP-43 Affect Different Genes

To further understand how mutations in the TDP-43 gene cause ALS, researchers funded by The ALS Association examined the effects of both preventing gene expression and overexpressing it in the fruit fly. They looked at the effects of these two changes on the level of expression of other genes and found that reducing expression of the fly version of TDP-43 increased expression of a wide array of genes, while overexpressing it had the opposite effect. They concluded that TDP-43 acts as a negative regulator, probably through direct binding to DNA or RNA. But the genes affected in each case were mostly different, “suggesting that different cellular programs are being activated in these two situations,” they write. Affected genes implicate pathways involving nerve transmission, nerve process outgrowth and synaptic function.

Dennis J. Hazelett, Jer-Cherng Chang, Daniel L. Lakeland, David B. Morton. Comparison of Parallel High-Throughput RNA Sequencing Between Knockout of TDP-43 and Its Overexpression Reveals Primarily Nonreciprocal and Nonoverlapping Gene Expression Changes in the Central Nervous System of Drosophila. G3 July 2012 2:789-802

http://www.g3journal.org/content/2/7/789.full.pdf+html

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Excess Normal TDP-43 is Toxic and May Drive Neurodegeneration

Working in the roundworm, researchers have discovered that the worm version of TDP-43 functions in the insulin growth factor pathway in response to stress. They found that ALS-causing mutations in TDP-43 or FUS lead to excess amounts of the normal worm protein and reduce lifespan. They propose that this may lead to a positive feedback loop in which elevated protein results in even more protein. While the short-term elevation of normal protein may be an adaptive response to stress, over the long term, it may be toxic, they suggest.

Vaccaro A, Tauffenberger A, Ash PE, Carlomagno Y, Petrucelli L, Parker JA. TDP-1/TDP-43 Regulates Stress Signaling and Age-Dependent Proteotoxicity in Caenorhabditis elegans. PLoS Genet. 2012 Jul; 8(7):e1002806. Epub 2012 Jul 5

http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002806

http://www.ncbi.nlm.nih.gov/pubmed/22792076

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Ataxin 2 Expansion Activates Caspase 3, Cleaving TDP-43

Intermediate polyglutamine expansion of ataxin 2, a recently identified risk factor for ALS, increases cleavage and phosphorylation of TDP-43 in response to stress, according to a new study in human cells published in the Journal of Neuroscience. Ataxin 2 is the same gene that, when fully expanded, causes spinocerebellar ataxia type 2. The cleavage of TDP-43 was due to activation of caspase 3 and could be mitigated by inhibitors of the enzyme. An increase in activated caspase 3 was seen in ALS patients with the intermediate ataxin 2 expansion but not in ALS patients with normal-length ataxin 2. “Given its important role in stress granule assembly and function, ataxin 2 could serve as a linchpin linking signaling between cellular stress responses and apoptotic cascades via its interactions within stress granules,” the authors write They propose that activation of moderately expanded ataxin 2 occurs at lower levels of stress, which may in turn set off a pathological cascade leading to the accumulation of TDP-43. 

Hart MP, Gitler AD. ALS-Associated Ataxin 2 PolyQ Expansions Enhance Stress-Induced Caspase 3 Activation and Increase TDP-43 Pathological Modifications. J Neurosci. 2012 Jul 4;32(27):9133-42.

http://www.ncbi.nlm.nih.gov/pubmed/22764223

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Loss of FUS Causes Motor Neuron Disease in Flies

A decrease in the amount of the fly version of the FUS protein in fruit flies leads to reduced climbing ability and abnormalities in the structure of motor neurons. The results of this study published in PLoS One suggest that mutations in FUS may cause ALS through loss of function of the normal protein, rather than a toxic gain of function of the mutant protein. A similar proposal for TDP-43 was highlighted in the June edition of the Journal News

Sasayama H, Shimamura M, Tokuda T, Azuma Y, Yoshida T, Mizuno T, Nakagawa M, Fujikake N, Nagai Y, Yamaguchi M. Knockdown of the Drosophila Fused in Sarcoma (FUS) Homologue Causes Deficient Locomotive Behavior and Shortening of Motoneuron Terminal Branches. PLoS One. 2012;7(6):e39483. Epub 2012 Jun 19.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0039483

http://www.ncbi.nlm.nih.gov/pubmed/22724023

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FUS Mutations Reduce Nuclear Binding Signal

ALS-causing mutations in the FUS nuclear localization signal weaken its ability to bind to its nuclear import receptor, according to a new structural and modeling study. The loss of affinity for the receptor of the various mutations correlates with both cytoplasmic mislocalization and disease severity, according to the authors.

Zhang ZC, Chook YM. Structural and energetic basis of ALS-causing mutations in the atypical proline-tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS). Proc Natl Acad Sci U S A. 2012 Jul 9. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22778397

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FUS Binds to Tau RNA, and Loss of FUS is Pathogenic

Mutations in FUS cause some cases of ALS, but whether this is due to a loss of function or a toxic gain of function of the FUS protein is unknown. To study this question, and to learn more about the normal role of FUS, researchers first showed that the FUS protein binds to the RNA transcript of the tau gene. Tau protein is a critical part of the cytoskeleton, which provides structure to axons and helps move materials along them. The interaction between FUS and tau RNA suggests that FUS influences exons of the RNA used to make protein, thus controlling the final structure of tau. In support of that view, they showed that reducing the level of FUS in neuronal cells in culture caused cells to make more of a specific form of tau, so-called 4R tau, a form of the protein linked to frontotemporal dementia and Parkinsonism. Reduction in FUS led to cytoskeletal abnormalities and shortened axons. These findings suggest that mutation of FUS may cause ALS through its effect on tau-related changes in cytoskeleton structure.

Orozco D, Tahirovic S, Rentzsch K, Schwenk BM, Haass C, Edbauer D.

Loss of fused in sarcoma (FUS) promotes pathological Tau splicing. EMBO Rep. 2012 Jun 19. doi: 10.1038/embor.2012.90. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22710833

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Loss of TDP-43 Causes a Motor Neuron Phenotype in Mice

By shutting down the TDP-43 gene in the motor neurons of the spinal cord, investigators showed that loss of the protein led to symptoms reminiscent of ALS. These results suggest that mutation in TDP-43 may cause ALS through loss of normal function. If this is so, therapies could be targeted at augmenting whatever functions are lost.

Wu LS, Cheng WC, Shen CK.

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis (ALS)-like Phenotypes in Mice. J Biol Chem. 2012 Jun 20. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22718760

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Metabolic Support from Oligodendrocytes is Reduced in ALS

Brain cells called oligodendrocytes provide crucial metabolic support for motor neurons, according to a new study published in Nature, and that support is diminished in ALS. The result focuses attention on the potential role of these cells in ALS and may suggest new targets for treatment. Oligodendrocytes occur throughout the central nervous system and manufacture the myelin sheath that provides electrical insulation for motor neurons. The new study shows that oligodendrocytes also bear a protein called monocarboxylic transporter 1 (MCT1), which transports lactate (an energy source) to motor neurons. When MCT1 was knocked down or inhibited, motor neurons in culture died, an effect enhanced when the neurons were stressed. Mice deficient in MCT1 developed damage to the axons of motor neurons. Patients with ALS had reduced amounts of MCT1 in their motor cortex (the portion of the brain containing upper motor neurons), but not in the frontal cortex, which was not affected in the disease. Loss of MCT1 was also seen in the spinal cord of SOD1 mice. “Taken together, these results suggest that alterations in oligodendrocyte MCT1 may contribute to motoneuron degeneration in ALS,” the authors concluded. Future experiments will determine if treatment aimed at increasing MCT1 may be therapeutic.

Lee Y, Morrison BM, Li Y, Lengacher S, Farah MH, Hoffman PN, Liu Y, Tsingalia A, Jin L, Zhang PW, Pellerin L, Magistretti PJ, Rothstein JD. Oligodendroglia metabolically support axons and contribute to neurodegeneration. Nature. 2012 Jul 11. doi: 10.1038/nature11314.

http://www.ncbi.nlm.nih.gov/pubmed/22801498

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Newest ALS Gene Impairs Axon Growth

A newly discovered ALS gene points to defects in axon growth as one cause of the disease. Mutations in the gene, called profilin 1 (PFN1), are responsible for 1% to 2% of familial ALS, according to the new study published in Nature. PFN1 helps link individual actin proteins to form actin filaments, and its loss causes defects in the actin cytoskeleton. The researchers performed whole-exome sequencing on DNA samples from two large families with dominantly inherited ALS. Other ALS genes had been ruled out in these families. They found two different mutations in the PFN1 gene located on chromosome 17. Sequencing of PFN1 in 272 additional familial ALS patients with no known genetic cause revealed five more cases.

In cell culture, non-mutant PFN1 was diffused throughout the cytoplasm, while mutant PFN1 was localized in aggregates that also contained TDP-43. PFN1 was not found in aggregates from patients with sporadic ALS, likely ruling out its involvement in that form of the disease.

The PFN1 mutations identified were all close to the actin-binding site on the protein, and four of the five mutations discovered impaired the protein’s ability to bind actin. Three of those led to decreased growth of axons.

PFN1 also interacts with the ALS gene VCP as well as the genes responsible for two other neurodegenerative diseases, Huntington’s disease and spinal muscular atrophy.

Wu CH, Fallini C, Ticozzi N, Keagle PJ, Sapp PC, Piotrowska K, Lowe P, Koppers M, McKenna-Yasek D, Baron DM, Kost JE, Gonzalez-Perez P, Fox AD, Adams J, Taroni F,  Tiloca C, Leclerc AL, Chafe SC, Mangroo D, Moore MJ, Zitzewitz JA, Xu ZS, van den Berg LH, Glass JD, Siciliano G, Cirulli ET, Goldstein DB, Salachas F, Meininger V, Rossoll W, Ratti A, Gellera C, Bosco DA, Bassell GJ, Silani V, Drory VE, Brown RH, Landers JE. Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012 Jul 15. doi: 10.1038/nature11280. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/22801503