Mutations in SOD1 linked to familial ALS induce the mutant protein to form intracytoplasmic aggregates. A recent study by Wang et al. in PLoS Biology used measures of several biophysical properties of mutant SOD1 to estimate aggregation rates, and then compared the data to clinical data on patient survival for each of the studied mutants. The authors suggest that the rate of mutant protein aggregation could be linked to the relative survival expectancy of individual patients. It is well established for some ALS-linked mutations that a specific point mutation, such as Ala 4 to Val, is associated with a very rapid disease course from onset to death; usually less than two years. The A4V mutation is one of the most common with well over 100 affected individuals having been examined and clinically described. However, the vast majority of SOD1-linked kindreds are much smaller with far fewer clinically evaluated patients. For this reason, one must view the association of a particular property of a given mutant protein to clinical outcomes of patients, with the same mutation, with...  Read more

Mutations in SOD1 linked to familial ALS induce the mutant protein to form intracytoplasmic aggregates. A recent study by Wang et al. in PLoS Biology used measures of several biophysical properties of mutant SOD1 to estimate aggregation rates, and then compared the data to clinical data on patient survival for each of the studied mutants. The authors suggest that the rate of mutant protein aggregation could be linked to the relative survival expectancy of individual patients. It is well established for some ALS-linked mutations that a specific point mutation, such as Ala 4 to Val, is associated with a very rapid disease course from onset to death; usually less than two years. The A4V mutation is one of the most common with well over 100 affected individuals having been examined and clinically described. However, the vast majority of SOD1-linked kindreds are much smaller with far fewer clinically evaluated patients. For this reason, one must view the association of a particular property of a given mutant protein to clinical outcomes of patients, with the same mutation, with considerable skepticism.

Nevertheless, the A4V mutation does appear to significantly destabilize normal structure to induce the aggregation of mutant SOD1. Whether one can definitively argue that clinical data from much smaller families, with slower clinical courses, carry the same weight in demonstrating that slower aggregation rates of mutant SOD1 underlie slower disease courses is less certain. However, if such data are informative, then the study by Wang et al. suggests that the aggregation of mutant protein is linked to the rate of patient decline. Notably, the study does not provide information regarding the pathologic manifestation of mutant SOD1 aggregation in patients, and thus does not provide significant insight as to whether specific types of pathologic features (such as cytoplasmic inclusions) are, or are not, toxic. The data do provide correlative evidence that aggregation of mutant SOD1 could be generating toxic entities that drive one aspect of disease progression. The nature of these toxic entities, however, remains unclear and could take on virtually any physical form such as small oligomeric structure to large filamentous aggregate.

View all comments by David R. Borchelt

Prof. Borchelt is correct that our study would have benefited from data regarding the pathologic manifestation of aggregates. Unfortunately, most of the primary literature contains only the following information: 1) patient's age at ALS onset, and 2) disease duration. Occasionally the patient’s sex is mentioned. Rarely is critical information such as weight, smoking, activities, drug use, occupation, pathology, SOD1 filter-trap assays, etc., included. While neurologists who publish onset and duration data are to be commended, it is our sincere hope that they (perhaps at patients’ behest) will begin to publish, or at least make available to ALS researchers, more comprehensive epidemiology data. Improved (read “shared”) epidemiology data will almost certainly contribute to our understanding of sporadic ALS.

Professor Borchelt also writes, “Whether one can definitively argue that clinical data from much smaller families, with slower clinical courses, carry the same weight in demonstrating that slower aggregation rates of mutant SOD1 underlie slower disease courses is less...  Read more

Prof. Borchelt is correct that our study would have benefited from data regarding the pathologic manifestation of aggregates. Unfortunately, most of the primary literature contains only the following information: 1) patient's age at ALS onset, and 2) disease duration. Occasionally the patient’s sex is mentioned. Rarely is critical information such as weight, smoking, activities, drug use, occupation, pathology, SOD1 filter-trap assays, etc., included. While neurologists who publish onset and duration data are to be commended, it is our sincere hope that they (perhaps at patients’ behest) will begin to publish, or at least make available to ALS researchers, more comprehensive epidemiology data. Improved (read “shared”) epidemiology data will almost certainly contribute to our understanding of sporadic ALS.

Professor Borchelt also writes, “Whether one can definitively argue that clinical data from much smaller families, with slower clinical courses, carry the same weight in demonstrating that slower aggregation rates of mutant SOD1 underlie slower disease courses is less certain.” Again, more epidemiology data may address his concern—we could only consider the >1,000 patients’ data that were available in the literature. Moreover, we did not assume that data from much smaller families carry the same weight as data from larger families. On the contrary, statistical analyses were weighted for the number of patients with a given mutation, thus accounting for (or at least attempting to) family size. For those concerned (as we were) that this weighted statistical analysis could itself result in selection bias, we also performed unweighted statistic analyses. Both sets of analyses were statistically significant.

View all comments by Jeffrey Agar

The paper by Subramaniam et al. demonstrates for the first time that chaperone-mediated copper loading is not required for mutant SOD protein to produce ALS-like disease. Mutant SOD protein-mediated motor neuron disease was not affected in the absence of the SOD copper chaperone (CCS), although copper-loading was reduced by 85 percent. However, since a residual 15 percent of copper loading remains, it cannot be ruled out for certain that copper-mediated toxicity does not contribute to mutant SOD protein-mediated disease. It is formally possible that the residual CCS-independent copper loading is insufficient for normal SOD-mediated reactions, but sufficient for the aberrant oxidative chemistry implicated with the mutant SOD protein. Although overall SOD activity in spinal cord tissue was dramatically reduced, it remains to be clarified whether aberrant SOD-mediated reactions were altered.

Some small notes on the data in the paper:

(1) The number of animals for disease onset are very small in the survival table.

(2) G85R is included in the survival table, although no...  Read more

The paper by Subramaniam et al. demonstrates for the first time that chaperone-mediated copper loading is not required for mutant SOD protein to produce ALS-like disease. Mutant SOD protein-mediated motor neuron disease was not affected in the absence of the SOD copper chaperone (CCS), although copper-loading was reduced by 85 percent. However, since a residual 15 percent of copper loading remains, it cannot be ruled out for certain that copper-mediated toxicity does not contribute to mutant SOD protein-mediated disease. It is formally possible that the residual CCS-independent copper loading is insufficient for normal SOD-mediated reactions, but sufficient for the aberrant oxidative chemistry implicated with the mutant SOD protein. Although overall SOD activity in spinal cord tissue was dramatically reduced, it remains to be clarified whether aberrant SOD-mediated reactions were altered.

Some small notes on the data in the paper:

(1) The number of animals for disease onset are very small in the survival table.

(2) G85R is included in the survival table, although no copper loading was detected even in the presence of CCS (figure 2b).

(3) No histology of early disease stage is shown for comparison, only late-disease stage is presented.

View all comments by Li-Huei Tsai

Contrary to assumptions that mutant superoxide dismutase (mSOD) causes ALS via peroxynitrite-mediated oxidative injury, Subramaniam et al. provide compelling evidence that mSOD toxicity is not due to the copper-dependent catalytic activity of peroxynitrite-mediated tyrosine nitration. Although a great deal of basic research and drug discovery in ALS has been predicated on the former assumption, the work of several researchers in the last several years has cast doubt on it. Studies have indicated that neuronal nitric oxide synthase is not directly involved in ALS pathogenesis, and the lack of evidence for the efficacy of NOS inhibitors in vivo supports this view. In addition, Doroudchi et al. have demonstrated that inhibiting nitrotyrosine production and protein nitration has little effect on the lifespan of motor neurons carrying the mSOD gene. Subramaniam's article provides more concrete and elegant evidence that the toxic gain of function of mSOD is not related to its pro-oxidative capacity.

It can be argued that the small amount of altered SOD activity still present in...  Read more

Contrary to assumptions that mutant superoxide dismutase (mSOD) causes ALS via peroxynitrite-mediated oxidative injury, Subramaniam et al. provide compelling evidence that mSOD toxicity is not due to the copper-dependent catalytic activity of peroxynitrite-mediated tyrosine nitration. Although a great deal of basic research and drug discovery in ALS has been predicated on the former assumption, the work of several researchers in the last several years has cast doubt on it. Studies have indicated that neuronal nitric oxide synthase is not directly involved in ALS pathogenesis, and the lack of evidence for the efficacy of NOS inhibitors in vivo supports this view. In addition, Doroudchi et al. have demonstrated that inhibiting nitrotyrosine production and protein nitration has little effect on the lifespan of motor neurons carrying the mSOD gene. Subramaniam's article provides more concrete and elegant evidence that the toxic gain of function of mSOD is not related to its pro-oxidative capacity.

It can be argued that the small amount of altered SOD activity still present in the CCS/SOD1 mice can cause peroxynitrite formation. If the catalyst SOD is not the limiting factor in the reaction, small changes in catalyst concentration should not affect the rate of disease progression. We know that both the low- and high-copy G93A mice possess sufficient SOD activity. However, the onset of disease development is dramatically altered with levels of SOD protein. This suggests that a simple enzymatic process may not be involved in ALS pathogenesis.

This and other studies merely suggest that the modus operandi of mSOD is not peroxynitrite-induced oxidative injury: the oxidative process may still be involved in ALS pathogenesis, as oxidative stress has long been acknowledged to be a key step in neuronal death in the neurodegenerative diseases.

A number of questions pertaining to the role of SOD and mSOD in ALS remain unanswered. For example, studies indicate that prion protein (PrP) affects SOD activity, and dysregulation of PrP expression appears to alter Cu Zn-SOD activity. In addition, PrP expression is altered in mSOD mice. It remains to be seen how the two are related and how these two seemingly disparate diseases are linked.

Alternate models have been suggested to incorporate these latter questions into the story of the toxic gain of function of SOD in ALS. "Conformational disease" models that involve non-native protein structures, such as protein aggregation and high-molecular weight protein complexes, suggest that altered protein conformation may play a role in cellular toxicity. Such models are supported by the growing evidence of a pathogenic role of protein aggregates in Huntington's disease, Alzheimer's disease, alpha1-antitrypsin deficiency, and CJD. These diseases also show the involvement of the ubiquitin-proteasome system in disease pathogenesis. Future efforts to understand the properties of the SOD protein in various conformations will enable an investigation of the possible link between structural properties, enzymatic activity, and ALS pathogenesis, and will ultimately aid in drug discovery efforts in ALS.

View all comments by Tennore Ramesh

The Science article by Clement (Don Cleveland, University of California) and other US and Canadian researchers is a masterpiece in demonstrating the prominent influence of non-neuronal cells on the ALS pathogenesis, which can be extrapolated to other neurodegenerative diseases. I take this opportunity to add a piece of evidence we observed indicating phenotypic changes within astrocytes located in the vicinity of the axons belonging to damaged (and dying) motoneurons from both SOD1 transgenic mice model and human sporadic ALS (see ref. below).

References:
Hoyaux D, Boom A, Van den Bosch L, Belot N, Martin JJ, Heizmann CW, Kiss R, Pochet R. S100A6 overexpression within astrocytes associated with impaired axons from both ALS mouse model and human patients, J Neuropathol Exp Neurol. 2002 Aug;61(8):736-44. Abstract

View all comments by Roland Pochet

In the recent papers from the groups of Przedborski and Eggan, provocative evidence is reported that spinal motor neurons may die in SOD1 mutant mice because of soluble toxic factors released by SOD1 mutant astrocytes. This result is surprising because previous studies with chimeric SOD1 mutant mice have shown that expression of mutant SOD1 in microglia but not astrocytes is implicated in the neuron death. However, profound reactive astrocytosis occurs very early in mouse and human motor neuron diseases. This is true in the SOD1 mutant mice, where reactive astrocytosis is a dramatic feature of the disease, with prominent reactive astrocytosis occurring long before much motor neuron death occurs (Carlos Pardo, personal communication).

The new studies provide striking evidence that astrocyte-conditioned medium from SOD1 mutant astrocytes is toxic, as wild-type spinal motor neurons survive longer in culture when cultured alone or with wild-type astrocyte conditioned medium than with mutant astrocyte- conditioned medium. Thus, the lower survival of the spinal motor neurons...  Read more

In the recent papers from the groups of Przedborski and Eggan, provocative evidence is reported that spinal motor neurons may die in SOD1 mutant mice because of soluble toxic factors released by SOD1 mutant astrocytes. This result is surprising because previous studies with chimeric SOD1 mutant mice have shown that expression of mutant SOD1 in microglia but not astrocytes is implicated in the neuron death. However, profound reactive astrocytosis occurs very early in mouse and human motor neuron diseases. This is true in the SOD1 mutant mice, where reactive astrocytosis is a dramatic feature of the disease, with prominent reactive astrocytosis occurring long before much motor neuron death occurs (Carlos Pardo, personal communication).

The new studies provide striking evidence that astrocyte-conditioned medium from SOD1 mutant astrocytes is toxic, as wild-type spinal motor neurons survive longer in culture when cultured alone or with wild-type astrocyte conditioned medium than with mutant astrocyte- conditioned medium. Thus, the lower survival of the spinal motor neurons cannot be attributed to less production of neurotrophic factors by the mutant astrocytes. Together, these in-vitro and in-vivo findings directly implicate reactive astrocytes in the pathophysiology of spinal motor neuron death in the SOD1 mutant mice. One caveat is that in the in-vitro studies, the astrocytes that were studied were obtained from neonatal spinal cords long before any reactive gliosis actually occurs. However, neonatal astrocytes in culture have a similar phenotype to reactive astrocytes in vivo, and may in fact be comparable.

So how can these new observations of Przedborski and Eggan be reconciled with previous studies that found that chimeric SOD1 mutant mice with mutant SOD1 in microglia but not astrocytes is implicated in the neuron death? For one thing, it is unclear if mutant microglia were actually present in the astrocyte cultures used in these new studies. Steps were taken to minimize microglial contamination, but because astrocytes secrete high levels of microglial mitogens such as colony stimulating factor-1 (CSF1), microglia almost always heavily contaminate neonatal astrocyte cultures prepared by the commonly used methods of McCarthy and DeVellis. It would be good to repeat the study using more stringent methods of microglial elimination, such as immunopanning, and it would be important to confirm by immunostaining that microglia are in fact absent from the astrocyte-conditioned medium at the time it is harvested. It is also possible that mutant astrocytes release factors in culture that are toxic to motor neurons but that these factors are not actually secreted in vivo or are not toxic to the neurons in vivo. The only way to find out for sure, of course, will be to identify this toxic astrocyte factor. At the present time, it is difficult to think of a model that reconciles the previous in-vivo observations implicating mutant microglia with the present in-vitro observations implicating mutant astrocytes.

Assuming astrocytes make a toxic factor, what could be its nature? The possibility that it is glutamate has already been ruled out, as have been the obvious cytokine candidates. Moreover, the motor neurons undergo apoptosis. One possibility is that it is a factor that binds to, inhibits, or proteolyzes required trophic factors or culture substrates present in the culture medium that are required for long-term motor neuron survival.

Another possibility is that the toxic astrocytes alter the pH of the culture medium or lower antioxidant levels, which are both crucial parameters for good neuronal survival. Alternatively, a toxic factor could be released, such as a cytokine of some sort or an excitotoxin. Glutamate agonists have not been ruled out. For instance, homocysteine is an NMDA agonist that is exclusively made by astrocytes; other possibilities are aspartate and N-acetyl-aspartylglutamate, which all act on NMDA receptors. In addition, astrocytes have previously been shown to secrete high levels of NMDA potentiators such as L-glycine or D-serine, and it is possible that the mutant astrocytes secrete higher levels of these. Glutamate excitotoxicity can lead to apoptosis, so it would be important in future experiments to test whether the toxic astrocyte factor can be blocked by APV or other NMDA receptor blockers, as so far only kainate and AMPA receptor blockers have been tested.

A very interesting new paper by Don Cleveland’s group provides evidence, using laser capture studies of mRNA expression, that spinal motor neurons in the SOD mutant mice have elevated levels of several complement proteins. This raises the possibility that there is complement-induced toxicity. However, microglia and serum, which are both rich sources of the complete set of complement proteins required for the complement cascade to function, were not present in the motor neuron cultures; therefore, this seems an unlikely possibility. Moreover, complement-mediated toxicity would be expected to cause lysis and not necessarily apoptosis (though mild toxic insults are well documented to lead to apoptosis in neurons). Interestingly, the new Cleveland work also provides evidence for a strong upregulation of the serine biosynthetic pathway. Astrocytes have previously been shown to preferentially use the serine synthetic pathway, whereas neurons do not (a result we have recently confirmed by gene profiling of purified neural cell populations; Cahoy and Barres, unpublished observations). It is possible that SOD1 mutant neurons upregulate these pathways as they die, but a more likely possibility is that there was some contamination by reactive glial genes in these studies, a possibility that is suggested by the presence of other upregulated well-described astrocyte genes such as CD44 and aquaporin 4. This latter possibility again raises the possibility that the toxic factor being released by mutant astrocytes is D-serine or L-glycine.

Whatever the case, these new papers call attention to the important but still poorly understood roles of neuron-glial interactions in the pathophysiology of neurodegenerative disease.

View all comments by Ben Barres

These two papers by Nagai et al. (2007) and Di Giorgio et al. (2007) independently provide strong evidence that glial cells, and perhaps specifically astrocytes, bearing SOD1 mutations are responsible for degeneration and death of motor neurons in embryonic stem cell (ESC)-based co-cultures of primary neurons and glial cells. Motor neurons bearing SOD1 mutation did not degenerate in the absence of mutant glial cells.

While these elegant findings provide important insights into the interdependency between neurons and glial cells, and provide key data concerning the pathogenesis of human ALS associated with SOD1 mutation, their relevance to sporadic and other non-SOD1 related forms of human ALS is uncertain. Increasingly, it is becoming recognized that SOD1- associated ALS, and non-SOD1 forms of ALS may be driven through different pathogenetic cascade mechanisms. In SOD1 ALS, the accumulated protein within the conglomerated ubiquitinated inclusion bodies is mutated SOD1. In other, non-SOD1 forms of familial ALS, and sporadic ALS, the filamentous or skein-like ubiquitinated...  Read more

These two papers by Nagai et al. (2007) and Di Giorgio et al. (2007) independently provide strong evidence that glial cells, and perhaps specifically astrocytes, bearing SOD1 mutations are responsible for degeneration and death of motor neurons in embryonic stem cell (ESC)-based co-cultures of primary neurons and glial cells. Motor neurons bearing SOD1 mutation did not degenerate in the absence of mutant glial cells.

While these elegant findings provide important insights into the interdependency between neurons and glial cells, and provide key data concerning the pathogenesis of human ALS associated with SOD1 mutation, their relevance to sporadic and other non-SOD1 related forms of human ALS is uncertain. Increasingly, it is becoming recognized that SOD1- associated ALS, and non-SOD1 forms of ALS may be driven through different pathogenetic cascade mechanisms. In SOD1 ALS, the accumulated protein within the conglomerated ubiquitinated inclusion bodies is mutated SOD1. In other, non-SOD1 forms of familial ALS, and sporadic ALS, the filamentous or skein-like ubiquitinated inclusions contain the TAR DNA binding protein, TDP-43 (Neumann et al., 2006; Davidson et al., 2007). Pertinently, the inclusions in SOD1-associated ALS are not TDP-43 immunoreactive (Tan et al., 2007). These latter morphological and immunohistochemical data reinforce the concept that SOD1 and non-SOD1 ALS are separate disorders even though they share a common clinical phenotype. The data, moreover, imply that a role for glial cells, as described in the work of Nagai et al., (2007) and Di Giorgio et al. (2007), may not pertain in the more common forms of ALS that are not associated with SOD1 mutation.

Nonetheless, a potential role for glial cells in non-SOD1 ALS could, perhaps, be tested in ESC-based studies using the Q342X stop codon mutation in the intraflagellar transport protein 74 (IFT74) gene, which has been associated in one family with a frontotemporal dementia and motor neuron disease (FTD+MND) clinical phenotype (Momeni et al., 2006). One patient from this family with this mutation showed ubiquitinated pathological changes within cerebral cortex and brain stem and spinal cord detectable by TDP-43 immunohistochemistry (Cairns et al., 2007). These were typical of those seen in FTD+MND, and in ALS alone (Neumann et al., 2006; Davidson et al., 2007).

References:
Cairns NJ et al (2007) Amer J Pathol (in press).

Davidson Y, Kelley T, Mackenzie IR, Pickering-Brown S, Du Plessis D, Neary D, Snowden JS, Mann DM. Ubiquitinated pathological lesions in frontotemporal lobar degeneration contain the TAR DNA-binding protein, TDP-43. Acta Neuropathol (Berl). 2007 May;113(5):521-33. Epub 2007 Jan 12. Abstract

Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci. 2007 May;10(5):608-614. Epub 2007 Apr 15. Abstract

Momeni P, Schymick J, Jain S, Cookson MR, Cairns NJ, Greggio E, Greenway MJ, Berger S, Pickering-Brown S, Chio A, Fung HC, Holtzman DM, Huey ED, Wassermann EM, Adamson J, Hutton ML, Rogaeva E, St George-Hyslop P, Rothstein JD, Hardiman O, Grafman J, Singleton A, Hardy J, Traynor BJ. Analysis of IFT74 as a candidate gene for chromosome 9p-linked ALS-FTD. BMC Neurol. 2006 Dec 13;6:44. Abstract

Nagai M, Re DB, Nagata T, Chalazonitis A, Jessell TM, Wichterle H, Przedborski S. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci. 2007 May;10(5):615-622. Epub 2007 Apr 15. Abstract

Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. Abstract

Tan CF, Eguchi H, Tagawa A, Onodera O, Iwasaki T, Tsujino A, Nishizawa M, Kakita A, Takahashi H. TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation. Acta Neuropathol (Berl). 2007 May;113(5):535-42. Epub 2007 Feb 27. Abstract

View all comments by David M.A. Mann

Astrocytes in brain as a form of extracellular matrix. As evolution proceeded, animals developed more astrocytes relative to neurons. Annelids have equal numbers, monkeys already have more astrocytes than neurons, humans 3 times more. We need more investigations of astrocytes in brain diseases. I predict that astrocytes will be the cells of 21 century.

View all comments by Soraya Valles

On Computers, Flies, and Alzheimer Disease
Two recently published papers address the fundamental question of how amyloid proteins form neurotoxic assemblies (see Luheshi et al., 2007 and Cheon et al., 2007). Pat McCaffrey has written an informative and insightful news report that summarizes their key findings and implications. The work reported extends efforts by the ”Cambridge group” (broadly defined, and including those in Firenze, Italy; Busan, Korea; and Jülich, Germany) to explore ”generic” protein folding pathways and their biological consequences. In these latest publications, the group extends the idea of generic protein structures to generic toxicity, meaning that protein assemblies that share structural features also share toxic activity. Importantly, algorithms have been developed that allow prediction of assembly state and neurotoxicity from protein primary structure.

The technical rigor of the two studies is excellent. Thus, within the contexts of the...  Read more

On Computers, Flies, and Alzheimer Disease
Two recently published papers address the fundamental question of how amyloid proteins form neurotoxic assemblies (see Luheshi et al., 2007 and Cheon et al., 2007). Pat McCaffrey has written an informative and insightful news report that summarizes their key findings and implications. The work reported extends efforts by the ”Cambridge group” (broadly defined, and including those in Firenze, Italy; Busan, Korea; and Jülich, Germany) to explore ”generic” protein folding pathways and their biological consequences. In these latest publications, the group extends the idea of generic protein structures to generic toxicity, meaning that protein assemblies that share structural features also share toxic activity. Importantly, algorithms have been developed that allow prediction of assembly state and neurotoxicity from protein primary structure.

The technical rigor of the two studies is excellent. Thus, within the contexts of the experimental systems employed, namely in silico and in vivo (in Drosophila), one may have great confidence in the results. However, now comes the more philosophical and difficult question of meaning. Specifically, how do these results contribute to our understanding of diseases of protein folding?

In this brief discussion, I consider this question and raise a number of others for consideration by the reader. My goal in playing the metaphorical ”Devil's advocate” is to stimulate scientific discourse.

”Meaning” is a nebulous and malleable term for which a definition invariably depends upon the system of evaluation one employs. The goal of the studies of Luheshi et al. and Cheon et al. is to answer the questions of “... the molecular basis of amyloid formation and the nature of the toxic species.” In this context, it is reasonable to ask whether simulating the self-association of Aβ(16-22) or Aβ(25-35) has any relevance (meaning) to Alzheimer disease (AD) or any other disease. Why? Neither peptide is found in vivo. Historically, the former has been a favorite of theorists (including this writer), as its size makes it amenable to in silico study and it forms fibrils in vitro. The latter has been studied since 1990, when the suggestion was made that it was homologous to the tachykinin family of neuropeptides (Yankner et al., 1990). However, the homology relationship was tenuous (as are many when sequence length is so short), authentic tachykinin peptides had no trophic or toxic effects on neurons, and significant evidence supporting the tachykinin connection has not emerged in the subsequent 17 years. Thus, without compelling biological precedent, one must ask what study of these peptides can reveal. For example, are these peptides proxies for holo-Aβ? Clearly, the answer must be ”no,” as the critical determinant of peptide pathogenicity lies at the Aβ C-terminus in the form of the Ile-Ala dipeptide.

Why are people studying what may be irrelevant peptides, and why is such irrelevance not recognized? An answer may come from what, until recently, has been one of the most controversial and contentious fields of modern biology, i.e., prions. The prion theory postulates that the causative agent of a variety of neurodegenerative diseases in animals and humans is composed entirely of protein (no nucleic acid). In the last three decades, the status of this ”protein only” hypothesis in the scientific community has moved from heresy to orthodoxy. However, questions about the scientific appropriateness of this changing perspective have led some, including Laura Manuelidis, to suggest that a re-examination is warranted of ”the objectivity of science and whether it is a myth vanished.” Manuelidis opines that the acceptance of the theory reflects "the peculiarly American sport of betting on popular momentum” (Manuelidis, 2000). A more apropos metaphor, considering that one prion disease is bovine spongiform encephalopathy (“Mad Cow” disease), might have been that of “following the herd.”

Much research on AD could be subject to the same type of criticism. Consider the example of what may be called the "generic” herd. This herd believes that amyloid structure is "generic” because many (most? all?) proteins form amyloids with some common structural organization. Although amyloids, by definition, do share a number of biophysical and spectroscopic features, great structural diversity may be found in the assemblies formed by classical and non-classical amyloid proteins and peptides (e.g., see Sawaya et al., 2007). Importantly, no generic structure outside of the cross-β core of the amyloid fibril has been shown to exist, for obvious reasons. Regions outside of the core, which can be quite extensive in protein, as opposed to peptide amyloids, are likely to influence the biological behavior of the assemblies significantly.

Now, Cheon and colleagues suggest that amyloid formation involves a second generic process, a two-step mechanism of “collapse” of monomers and their subsequent rearrangement into amyloid fibrils. This idea appears to invoke known processes of globular protein folding in the context of amyloid formation, specifically the classical idea of hydrophobic collapse into a molten globule followed by proper arrangement of secondary structure elements to form the native tertiary structure. The idea that some peptides bypass this two-step pathway if they can immediately form hydrogen bonds in their eventual cross-β organization is quite interesting. However, although plausible for short, disordered peptides of the sort studied here, what happens in the common case of natively folded proteins forming amyloid? Here, and as the authors themselves suggest implicitly, factors other than the intrinsic properties of the protein monomer likely moderate amyloid assembly. This increased complexity requires me to question the value of this suggestion of generic mechanisms. Scientists, especially medicinal chemists, need targets. Does a “generic amyloid target” exist? Could a single compound directed at such a target be of value in the treatment of the greater than two score amyloid diseases defined thus far?

Maybe a generic target does exist. In Luheshi et al., studies of the effects of expression of human Aβ42 in Drosophila suggest that protofibril formation correlates with neuronal dysfunction and neurodegeneration. In addition, in a kind of Anfinsen redux (Anfinsen, 1973), an algorithm has been created to predict from primary structure alone the propensity of a protein to form toxic protofibrils. My question: Does the experimental assessment of Aβ-induced locomotor and longevity effects in flies, and its correlation with the toxicity metric, have any relevance to the consideration of Aβ-induced disease in humans? Granted, the same question is sometimes raised as gratuitous criticism of work in a variety of non-human animal models, and it is an easy concern to raise, but that does not diminish the significance of the question.

In closing, it may appear to some that the answers to the questions I have asked are implicit in the construction of the questions themselves. This certainly was not my intention. From a purely academic perspective, I found the publications rigorous, enjoyable to read, and quite thought-provoking. It is the provocation aspect of the experience that operates here, particularly with respect to establishing the meaning of the results and their impact on our shared efforts to understand and treat diseases of aberrant protein folding and assembly.

View all comments by David Teplow

Reply by Leila M. Luheshi, Giorgio Favrin, Damian C. Crowther, Michele Vendruscolo, and Christopher M. Dobson to Teplow Comment
We are pleased to have the opportunity of adding further observations to a recent commentary by David Teplow about the “generic hypothesis” of amyloid fibril formation (1). According to this hypothesis, the ability to form amyloid structures is an inherent property of polypeptide chains, although the propensity to form such structures can vary dramatically with their sequences (2).

This hypothesis is supported by a growing body of experimental evidence that has been summarized in a number of recent reviews (3). The generic nature of amyloid fibrils resides in their core cross-β structure, which is stabilized predominantly by backbone hydrogen bonding interactions (4). It has also been recently discovered that the range of proteins capable of forming toxic oligomers, that may well be precursors to mature amyloid fibrils, is very large and includes those with no known association with disease (5-7). Of course, there are many...  Read more

Reply by Leila M. Luheshi, Giorgio Favrin, Damian C. Crowther, Michele Vendruscolo, and Christopher M. Dobson to Teplow Comment
We are pleased to have the opportunity of adding further observations to a recent commentary by David Teplow about the “generic hypothesis” of amyloid fibril formation (1). According to this hypothesis, the ability to form amyloid structures is an inherent property of polypeptide chains, although the propensity to form such structures can vary dramatically with their sequences (2).

This hypothesis is supported by a growing body of experimental evidence that has been summarized in a number of recent reviews (3). The generic nature of amyloid fibrils resides in their core cross-β structure, which is stabilized predominantly by backbone hydrogen bonding interactions (4). It has also been recently discovered that the range of proteins capable of forming toxic oligomers, that may well be precursors to mature amyloid fibrils, is very large and includes those with no known association with disease (5-7). Of course, there are many additional complexities involved in misfolding diseases, and it is also clear that the aggregation of different proteins in vitro does result in fibrils of different morphologies, and the aggregation of different proteins in vivo causes diseases with very different characteristics.

So far the generic hypothesis has provided the inspiration for a number of innovative studies, including the two mentioned by David Teplow (8, 9). We have been astonished by the way that the principles observed in the test tube and in the computer are able to explain the behavior and lifespan of living organisms such as Drosophila. We are therefore optimistic that the insights such studies have given us will lead, in the next few years, to the development of effective therapeutic strategies to combat the debilitating and increasingly prevalent diseases associated with protein misfolding.

References:
1. Dobson CM. Protein misfolding, evolution and disease. Trends Biochem Sci. 1999 Sep;24(9):329-32. Review. No abstract available. Abstract

2. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM. Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature. 2003 Aug 14;424(6950):805-8. Abstract

3. Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 2006;75:333-66. Review. Abstract

4. Knowles, T. P. J. et al. The Role of Inter-Molecular Forces in Defining the Material Properties of Fibrillar Protein Nanostructures. Science (2007) in press.

5. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature. 2002 Apr 4;416(6880):507-11. Abstract

6. Baglioni S, Casamenti F, Bucciantini M, Luheshi LM, Taddei N, Chiti F, Dobson CM, Stefani M. Prefibrillar amyloid aggregates could be generic toxins in higher organisms. J Neurosci. 2006 Aug 2;26(31):8160-7. Abstract

7. Kayed R, Glabe CG. Conformation-dependent anti-amyloid oligomer antibodies. Methods Enzymol. 2006;413:326-44. Abstract

8. Luheshi LM, Tartaglia GG, Brorsson AC, Pawar AP, Watson IE, Chiti F, Vendruscolo M, Lomas DA, Dobson CM, Crowther DC. Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity. PLoS Biol. 2007 Oct 30;5(11):e290. Abstract

9. Cheon M, Chang I, Mohanty S, Luheshi LM, Dobson CM, Vendruscolo M, Favrin G. Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. PLoS Comput Biol. 2007 Sep 14;3(9):1727-38. Abstract

View all comments by Leila Luheshi

VAPs (VAMP/synaptobrevin associated proteins) are evolutionarily conserved proteins comprising an amino-terminal domain with significant homology to the major sperm proteins (MSPs), a central coiled-coil domain, and a membrane anchor at the carboxy-terminal domain. MSPs are the most abundant proteins in the amoeboid nematode sperm, where they perform both cytoskeletal and signaling functions. In C. elegans, MSPs signal by antagonizing ephrin/Eph receptor pathway to promote oocyte meiotic maturation, ovarian sheath cell contraction, and oocyte microtubule reorganization. In 2004, Nishimura et al. reported a mutation substituting a conserved proline with a serine in a Brazilian family affected by a heterogenous group of motor neuron diseases ranging from amyotrophic lateral sclerosis (ALS) to atypical ALS and spinal muscular atrophy (1). In Drosophila, dVAP modulates number and size of boutons at neuromuscular junctions (2). Loss of function in dVAP disrupts microtubule cytoskeleton and causes an increase in miniature excitatory post-synaptic potentials that...  Read more

VAPs (VAMP/synaptobrevin associated proteins) are evolutionarily conserved proteins comprising an amino-terminal domain with significant homology to the major sperm proteins (MSPs), a central coiled-coil domain, and a membrane anchor at the carboxy-terminal domain. MSPs are the most abundant proteins in the amoeboid nematode sperm, where they perform both cytoskeletal and signaling functions. In C. elegans, MSPs signal by antagonizing ephrin/Eph receptor pathway to promote oocyte meiotic maturation, ovarian sheath cell contraction, and oocyte microtubule reorganization. In 2004, Nishimura et al. reported a mutation substituting a conserved proline with a serine in a Brazilian family affected by a heterogenous group of motor neuron diseases ranging from amyotrophic lateral sclerosis (ALS) to atypical ALS and spinal muscular atrophy (1). In Drosophila, dVAP modulates number and size of boutons at neuromuscular junctions (2). Loss of function in dVAP disrupts microtubule cytoskeleton and causes an increase in miniature excitatory post-synaptic potentials that correlates with an increase in post-synaptic glutamate receptor clustering. It has also been shown that hVAPB, the causative gene of ALS8, rescues the lethality and the neuromuscular junction phenotype associated with loss of DVAP, clearly indicating that the fly protein and human VAP perform homologous functions (3).

Recently, reports from two independent labs (Tsuda et al. and Ratnaparkhi et al.) have provided new and exciting insight on the normal function of VAP proteins and their possible role in the pathogenesis of VAP-induced ALS. Comments on these papers can be summarized as follows.

The paper by Tsuda et al. reports that VAP proteins are cleaved, and an N-terminal fragment of a size compatible with the size of the MSP domain is secreted and binds to the Eph receptors. The pathogenic allele induces the accumulation of the mutant and the wild-type (wt) protein into the ER and a failure to secrete the cleaved MSP domain. Non cell-autonomous effects of the mutant and wt proteins have been reported both at the level of the Drosophila nervous system and the nematode reproductive system. The ability of dVAP to be cleaved and secreted has been shown with an elegant experiment in which the expression of dVAP has been driven in a subset of cells in the wing imaginal discs. A diffusion of dVAP MSP beyond the protein expressing cells was observed. However, there is no direct evidence that this process of cleavage and secretion of VAP proteins is occurring in neurons, in muscles, or in any other tissue that would be more relevant to the human disease.

The ability of the pathogenic allele to induce the formation of aggregates has been previously reported in cell culture (1,4,5) and Drosophila model systems (3). Tsuda and colleagues report that expression of the mutant protein in a null background induces the formation of detergent-insoluble aggregates. Despite the mutant allele being inherited in a dominant manner in humans, these data lead to the important conclusion that the wild-type protein is not necessary for the formation of aggregates. However, an intriguing question arises: how can the presence of these aggregates be reconciled with the ability of the mutant protein to rescue the phenotypes associated with null mutations in dVAP as shown by three independent studies (3, Ratnaparkhi et al., Tsuda et al.). Are these aggregates different from the ones observed when the mutant protein is expressed in the presence of the wt protein?

Other outstanding questions will need to be addressed: which is the protease or proteases responsible for the cleavage? Is the secretion of the MSP domain of VAP proteins occurring through an unconventional mechanism as already proposed for the MSP proteins in C. elegans? Which is the subcellular compartment in which the cleavage occurs?

The paper by Ratnaparkhi et al. focuses on another important aspect, which is the determination of the disease mechanism. In humans, the pathogenic mutation is inherited in a dominant manner. Dominant mutations are due to a gain of function (hypermorphs and neomorphs), dominant-negative interactions (antimorphs) or haplo-insufficiency. Understanding the patho-mechanism of the disease is important as it can indicate new possible strategies for therapeutic interventions. Several lines of evidence support a possible dominant-negative effect of the pathogenic allele. The formation of aggregates, the depletion of the wild-type protein from its normal localization (3,4,5), and the sequestration of the wt protein in the aggregates clearly suggest a dominant-negative effect (4). Moreover, the fact that the pathogenic allele acts as a dominant-negative can be proven if the overexpression of the mutant protein in the presence of the wt protein leads to a phenotype similar to the loss-of-function mutation. Indeed, it has been reported that transgenic expression of the mutant protein induces a reduction in number of boutons (3), a disruption of the presynaptic cytoskeleton (Ratnaparkhi et al.) and a reduction in miniature excitatory post-synaptic potentials (Tsuda et al.). Ratnaparkhi et al. attempt to further support this statement by performing a systematic analysis of mutant phenotypes in different functional contexts. They compared the effect of overexpressing the wt protein with the overexpression of the mutant protein in transgenic lines expressing comparable amounts of transgenes. The expression levels of the proteins were estimated only for the full-length VAP. Although the mutant allele impairs the secretion of the MSP domain, the cleaved product is still produced as shown in several Western blots reported by Tsuda et al. The same Western blots suggest that the levels of the full-length protein and the cleaved MSP domain are not stoichiometrically similar; therefore, restricting the analysis to the expression levels of the full-length protein may be misleading. A cleaved, non-secreted MSP domain could still be responsible for the intracellular, cell-autonomous effects of the protein.

Although there are several lines of evidence supporting a possible dominant-negative effect, there is other evidence suggesting different mechanisms for the disease. Mutant VAP proteins still retain some functional properties of the wt protein such as the ability to self-oligomerize (3,4) and the ability to rescue, at least in part, the mutant phenotype due to the loss of the endogenous protein. The mutant allele has also acquired new functional properties that are not shared by the normal version of the protein such as the propensity to form aggregates and the “floating active zones” phenotype reported by Ratnaparkhi et al. In one report it has also been shown that the mutant protein has an increased ability of inhibiting the activity of ATF6, a transcription factor involved in UPR (6).

We propose that the mutant allele may cause the disease by a combination of mechanisms that include dominant-negative interactions and toxic effects due to gain of new functions. Although a lot still remains to be done, studies published over the last six months have convincingly shown that the variety of genetic tools available in Drosophila can now be exploited to foster our understanding of the patho-mechanisms responsible for motor neuron diseases in humans.

References:
1. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

2. Pennetta G, Hiesinger PR, Fabian-Fine R, Meinertzhagen IA, Bellen HJ. Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron. 2002 Jul 18;35(2):291-306. Abstract

3. Chai A, Withers J, Koh YH, Parry K, Bao H, Zhang B, Budnik V, Pennetta G. hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum Mol Genet. 2008 Jan 15;17(2):266-80. Abstract

4. Kanekura K, Nishimoto I, Aiso S, Matsuoka M. Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8). J Biol Chem. 2006 Oct 6;281(40):30223-33. Abstract

5. Teuling E, Ahmed S, Haasdijk E, Demmers J, Steinmetz MO, Akhmanova A, Jaarsma D, Hoogenraad CC. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates. J Neurosci. 2007 Sep 5;27(36):9801-15. Abstract

6. Gkogkas C, Middleton S, Kremer AM, Wardrope C, Hannah M, Gillingwater TH, Skehel P. VAPB interacts with and modulates the activity of ATF6. Hum Mol Genet. 2008 Jun 1;17(11):1517-26. Abstract

View all comments by Giuseppa Pennetta

Amyotrophic lateral sclerosis is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within five years. About 10 percent of ALS cases are familial; 20 percent of these are caused by mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1). More recently, it has been shown that mutations in the TDP-43 gene are also causative for familial ALS (1-3). The VAPB P56S mutation was originally observed in a large Brazilian family of Portuguese descent that displayed a pattern of dominantly inherited ALS/motor neuron disease across four generations (4). Subsequent studies identified the mutation in at least seven different families, all of Portuguese-Brazilian origin, each displaying a different clinical course ranging from late-onset spinal muscular atrophy (SMA) to typical and atypical ALS (4). Our previous work identified only a single case of a VAPB mutation (P56S) in a screen of 80 familial ALS samples, demonstrating that VAPB mutations are extremely rare (5). As such, why is it important...  Read more

Amyotrophic lateral sclerosis is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within five years. About 10 percent of ALS cases are familial; 20 percent of these are caused by mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1). More recently, it has been shown that mutations in the TDP-43 gene are also causative for familial ALS (1-3). The VAPB P56S mutation was originally observed in a large Brazilian family of Portuguese descent that displayed a pattern of dominantly inherited ALS/motor neuron disease across four generations (4). Subsequent studies identified the mutation in at least seven different families, all of Portuguese-Brazilian origin, each displaying a different clinical course ranging from late-onset spinal muscular atrophy (SMA) to typical and atypical ALS (4). Our previous work identified only a single case of a VAPB mutation (P56S) in a screen of 80 familial ALS samples, demonstrating that VAPB mutations are extremely rare (5). As such, why is it important to study a mutation which is only responsible for a small percentage of ALS cases?

One reason is due to the fact that from a clinical point of view, familial and sporadic ALS cases are virtually identical. As such, it is not unreasonable to postulate that although ALS may be caused by different genetic factors, they all may lead to common sets of pathways that eventually result in the ALS phenotype. Thus, a high level of importance should be placed on understanding the common features of all known ALS genes since they may shed light on these pathways. Therefore, even though VAPB mutations are indeed rare, characterizing their effects may provide insight on how cases of ALS develop overall.

In both of the papers presented (6,7), the authors have each developed a Drosophila model of ALS which expresses mutant VAPB. The use of these models will undoubtedly be beneficial in future experiments to further decipher the ALS phenotype. Of great significance, though, is that each study observes in vivo that mutant VAPB is capable of inducing intracellular aggregates. This work reinforces previously published observation that in vitro expression of mutant human P56S protein results in cellular aggregates (4,5). The fact that the aggregation phenotype of this mutation is conserved down to Drosophila is quite interesting. Aggregates are commonly observed within ALS cases, as well as other neurodegenerative diseases, although whether these aggregates are pathogenic is still up for debate. The formation of intracellular aggregates has also been observed via expression of mutant SOD1 and mutant TDP-43 (3). Taken together, the observation that three different familial ALS genes all are capable of inducing intracellular aggregates reinforces the notion that understanding the activation of pathways by protein misfolding is key to understanding the pathogenic nature of ALS.

References:
1. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, Baralle F, de Belleroche J, Mitchell JD, Leigh PN, Al-Chalabi A, Miller CC, Nicholson G, Shaw CE. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008 Mar 21;319(5870):1668-72. Abstract

2. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Vande Velde C, Bouchard JP, Lacomblez L, Pochigaeva K, Salachas F, Pradat PF, Camu W, Meininger V, Dupre N, Rouleau GA. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008 May;40(5):572-4. Abstract

3. Winton MJ, Van Deerlin VM, Kwong LK, Yuan W, Wood EM, Yu CE, Schellenberg GD, Rademakers R, Caselli R, Karydas A, Trojanowski JQ, Miller BL, Lee VM. A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Lett. 2008 Jun 25;582(15):2252-6. Abstract

4. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

5. Landers JE, Leclerc AL, Shi L, Virkud A, Cho T, Maxwell MM, Henry AF, Polak M, Glass JD, Kwiatkowski TJ, Al-Chalabi A, Shaw CE, Leigh PN, Rodriguez-Leyza I, McKenna-Yasek D, Sapp PC, Brown RH Jr. New VAPB deletion variant and exclusion of VAPB mutations in familial ALS. Neurology. 2008 Apr 1;70(14):1179-85. Abstract

6. Tsuda H, Han SM, Yang Y, Tong C, Lin YQ, Mohan K, Haueter C, Zoghbi A, Harati Y, Kwan J, Miller MA, Bellen HJ. The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell. 2008 Jun 13;133(6):963-77. Abstract

7. Ratnaparkhi A, Lawless GM, Schweizer FE, Golshani P, Jackson GR. A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism. PLoS ONE. 2008 Jun 4;3(6):e2334. Abstract

View all comments by John Landers

This work builds on the work by Yamanaka and others showing that relatively short periods of exposure and/or sequential exposure to reprogramming signals is sufficient to transform cells into pluripotent cells. This raised the possibility that episomal/transient vectors, protein transduction strategies and small molecules may work.

In this manuscript the authors have shown that inducible adenovirus persists for sufficiently long periods to reprogram cells and as such minimizes risks associated with nonrandom integration and disruption of potentially important genes. These induced cells appeared similar to cells derived by other integrating methods and were capable of robust chimera formation.

While clearly an important step forward, several issues remain. The authors note that adenovirus infection efficiency is variable in different cell types. Persistence and levels of expression are variable as well, and both of these likely reduce the efficiency of reprogramming. Indeed, the authors used liver cells for their experiments as...  Read more

This work builds on the work by Yamanaka and others showing that relatively short periods of exposure and/or sequential exposure to reprogramming signals is sufficient to transform cells into pluripotent cells. This raised the possibility that episomal/transient vectors, protein transduction strategies and small molecules may work.

In this manuscript the authors have shown that inducible adenovirus persists for sufficiently long periods to reprogram cells and as such minimizes risks associated with nonrandom integration and disruption of potentially important genes. These induced cells appeared similar to cells derived by other integrating methods and were capable of robust chimera formation.

While clearly an important step forward, several issues remain. The authors note that adenovirus infection efficiency is variable in different cell types. Persistence and levels of expression are variable as well, and both of these likely reduce the efficiency of reprogramming. Indeed, the authors used liver cells for their experiments as these are much more efficiently infected with adenovirus as compared to fibroblasts. Experiments were performed with rodent cells, and it is unclear if the longer cycle time and longer period of induction to pluripotency required will represent a benefit or a hindrance to this methodology. Some of the adeno-associated viruses integrate into the genome, albeit at a very low frequency, and it will be important to test for such integration.

Nevertheless, these experiments represent an important first step in the transition to the clinic. Other episomal viruses exist (some of which persist for longer periods), several methods can be used to improve viral uptake, and some viruses appear to infect far more efficiently. We have no doubt that this group and others are already trying these alternatives.

View all comments by Mahendra Rao

In addition to the traditional funding models mentioned in this story, we would like to also mention a new funding model available for ALS research. Prize4Life is a nonprofit organization that awards two prizes of $1 million each (the ALS Biomarker Challenge and the ALS Treatment Prize). Instead of recognizing historical accomplishments, Prize4Life designs prizes that we believe are achievable in a two- to three-year timeframe and then recruits teams to compete. Prize competitions have been steadily gaining traction in a variety of domains of innovation because their emphasis on specific outcomes has the capacity to propel a field forward very quickly, and can attract creative thinking from both within a field and “outside the box.” For example, in 2009 Prize4Life awarded two $50,000 Milestone Prizes, one of which went to an established ALS researcher, and one of which went to a trained dermatologist who explored a completely novel approach towards an ALS biomarker. Visit Prize4Life to learn more or to register to compete for a...  Read more

In addition to the traditional funding models mentioned in this story, we would like to also mention a new funding model available for ALS research. Prize4Life is a nonprofit organization that awards two prizes of $1 million each (the ALS Biomarker Challenge and the ALS Treatment Prize). Instead of recognizing historical accomplishments, Prize4Life designs prizes that we believe are achievable in a two- to three-year timeframe and then recruits teams to compete. Prize competitions have been steadily gaining traction in a variety of domains of innovation because their emphasis on specific outcomes has the capacity to propel a field forward very quickly, and can attract creative thinking from both within a field and “outside the box.” For example, in 2009 Prize4Life awarded two $50,000 Milestone Prizes, one of which went to an established ALS researcher, and one of which went to a trained dermatologist who explored a completely novel approach towards an ALS biomarker. Visit Prize4Life to learn more or to register to compete for a prize.

View all comments by Meghan Kallman