RNAi Therapy Works in Animal Models
Scott Harper et al. recently demonstrated that RNA interference (RNAi) can treat Huntington disease in an animal model (Harper et al., 2005). This work, together with a previous published experiment from the same group on treatment of spinal cerebellar ataxia (Xia et al., 2004), and two other experiments on treatment of ALS (Ralph et al., 2005; Raoul et al., 2005), demonstrates the concept of RNAi therapy for neurodegenerative diseases.

The common approach in these experiments was to deliver RNAi using viral vectors. All showed in vivo knockdown of the target gene and phenotypic improvement. These are very encouraging developments that bring RNAi one step closer to clinical application. Here I provide some background about these experiments and discuss some challenges that we still need to meet in order to realize the full therapeutic potential of RNAi.

In general, genetic disorders can be caused by two types of genetic mutations. One causes the gene to lose its function and the other causes the gene to gain a...  Read more

RNAi Therapy Works in Animal Models
Scott Harper et al. recently demonstrated that RNA interference (RNAi) can treat Huntington disease in an animal model (Harper et al., 2005). This work, together with a previous published experiment from the same group on treatment of spinal cerebellar ataxia (Xia et al., 2004), and two other experiments on treatment of ALS (Ralph et al., 2005; Raoul et al., 2005), demonstrates the concept of RNAi therapy for neurodegenerative diseases.

The common approach in these experiments was to deliver RNAi using viral vectors. All showed in vivo knockdown of the target gene and phenotypic improvement. These are very encouraging developments that bring RNAi one step closer to clinical application. Here I provide some background about these experiments and discuss some challenges that we still need to meet in order to realize the full therapeutic potential of RNAi.

In general, genetic disorders can be caused by two types of genetic mutations. One causes the gene to lose its function and the other causes the gene to gain a function—the gain could be either a novel function or an enhancement of an existing function. The treatment strategy for the loss-of-function type of mutations is to deliver to the patient a normal copy of the mutated gene so that its function can be replaced. But for a long time the conceptual framework for treatment of gain-of-function mutations was less obvious. However, RNAi has provided a conceptual basis for this kind of treatment (see ARF Live Discussion).

RNAi is a cellular function that is widely conserved in eukaryotes. Triggered by double-stranded RNA (dsRNA), RNAi destroys the target RNA that shares sequence homology with the dsRNA. The mechanism of RNAi is not understood completely, but a key executor is a protein complex called RNA-induced silencing complex, or RISC, which contains proteins and a single-stranded RNA (guide strand) derived from the dsRNA that the cells are originally exposed to. This guide strand is capable of recognizing the target RNA by Watson-Crick base pairing. After the pairing, RISC cleaves the target RNA. Thus, RNAi can destroy the target RNA specifically. When the target is an mRNA, RNAi can selectively knock down the expression of the target protein (Tomari and Zamore, 2005). This makes RNAi ideal for treatment of diseases caused by gain-of-function type of genetic mutations (see ARF Live Discussion).

A serious challenge to the success of RNAi therapy is the delivery of RNAi in vivo. Authors of the above four papers met this challenge by demonstrating that viral vector-delivered RNAi can treat neurodegenerative diseases in animal models. These experiments used viral vectors that express short hairpin RNA (shRNA) that can be processed to short RNA duplexes called small interfering RNA (siRNA) (Shi, 2003). The siRNAs mediated RNAi against mutant ataxin-1, SOD1, and huntingtin mRNAs, which cause spinal cerebellar ataxia 1 (SCA-1), amyotrophic lateral sclerosis (ALS) and Huntington disease, respectively. These successes are not surprising since all of these diseases are caused by gain-of-function gene mutations. Although the nature of the gained function is not clear, it is clear that disease severity correlates with the levels of mutant protein. Therefore, lowering the mutant protein levels will protect neurons from the toxic effects of these proteins.

What are the future challenges in developing RNAi therapy? Here I raise a few: First, how long is shRNA expressed after the viral delivery and to what extent and for how long is the target gene silenced? These questions have not been answered in these experiments. Therefore, it is not clear whether the observed therapeutic effects are a result of transient silencing or a sustained silencing of the target genes. A related question is, what is the best time to administer the RNAi therapy, before or after the disease onset? In all experiments the RNAi therapy was administered before the disease onset. It will be important to determine whether, by administration of RNAi therapy after the onset of the disease, the disease can be reversed.

Next, as a general rule, is it necessary to administer mutant-allele-specific RNAi? In these four cases the siRNAs were not specifically designed to silence the mutant gene because the mutant genes are human transgenes that differ in sequences from the mouse endogenous genes. Silencing the transgenes does not affect expression of endogenous mouse proteins. In human patients, however, shRNAs would silence both the mutant and the wild-type gene expression. This may be problematic because the wild-type functions of these genes are necessary for normal cellular function, and animal experiments show that in some cases they are essential (Cattaneo et al., 2001; Ding et al., 2003). In humans, the phenotypes for lack of these genes are not known, but they could be serious, given the animal data. To overcome this problem, allele-specific silencing or RNAi with replacement gene therapy may be necessary (Ding et al., 2003; Miller et al., 2003; Xia et al., 2005).

Third, are the viral vectors the best way for delivery of RNAi therapy? Delivery using viral vectors carries risks associated with gene therapy (Kimmelman, 2005). An alternative is to deliver siRNA directly to patients. A recent study has demonstrated that chemically modified siRNAs can be systemically administered and can knock down specific genes in multiple tissues (Soutschek et al., 2004; see ARF related news story). However, siRNAs with chemical modifications that allow the siRNA to penetrate the blood−brain barrier have not been reported. Nevertheless, siRNA or chemically modified siRNA may be directly delivered to the CNS to induce specific knockdown (Thakker et al., 2004). Direct delivery to the CNS does carry disadvantages, such as risks and costs associated with surgery and constant consumption of drugs. Long-term risks of toxicity with chronic administration of siRNA or chemically modified siRNA are yet to be determined.

In conclusion, the new results are exciting, and they are the first steps toward developing RNAi therapy. There is little doubt that most, if not all, mammalian cells possess the RNAi mechanism. The key to therapy rests on the development of highly efficient and safe delivery methods. If this can be achieved, RNAi can not only be used to treat genetic disorders that are caused by dominant, gain-of-function mutations, but also sporadic diseases where the disease pathways are understood. For example, if the amyloid hypothesis for Alzheimer disease is correct (Hardy and Selkoe, 2002), RNAi can be used to downregulate BACE and/or other proteins that are involved in the production of Aβ, a substrate for β amyloid that is postulated to cause neuronal degeneration.

References:
Cattaneo E, Rigamonti D, Goffredo D, Zuccato C, Squitieri F, Sipione S. Loss of normal huntingtin function: new developments in Huntington's disease research. Trends Neurosci. 2001 Mar;24(3):182-8. Review. Abstract Ding H, Schwarz DS, Keene A, Affar el B, Fenton L, Xia X, Shi Y, Zamore PD, Xu Z. Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Aging Cell. 2003 Aug;2(4):209-17. Abstract Hardy J, Selkoe DJ. The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics. Science. 2002 Jul 19;297(5580):353-6. Review. Erratum in: Science 2002 Sep 27;297(5590):2209. Abstract Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q, Yang L, Kotin RM, Paulson HL, Davidson BL. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A. 2005 Apr 5; [Epub ahead of print] Abstract Kimmelman J. Recent developments in gene transfer: risk and ethics. BMJ. 2005 Jan 8;330(7482):79-82. Review. No abstract available. Abstract Miller VM, Xia H, Marrs GL, Gouvion CM, Lee G, Davidson BL, Paulson HL. Allele-specific silencing of dominant disease genes. Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7195-200. Epub 2003 Jun 2. Abstract Ralph GS, Radcliffe LA, Day DM, Carthy JM, Leroux MA, Lee DCP, Wong L-F, Bilsland LG, Greensmith L, Kingsman SM, Mitrophanous KA, Mazarakis ND, Azzouz M. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat Med. 2005 Apr;11(4):429-33. Epub 2005 Mar 13. Abstract Raoul C, Abbas-Terki T, Bensadoun J-C, Guillot S, Haase G, Szulc J, Henderson CE, Aebischer P. Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat Med. 2005 Apr;11(4):423-8. Epub 2005 Mar 13. Abstract Shi Y. Mammalian RNAi for the masses. Trends Genet. 2003 Jan;19(1):9-12. Review. Abstract Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, John M, Kesavan V, Lavine G, Pandey RK, Racie T, Rajeev KG, Rohl I, Toudjarska I, Wang G, Wuschko S, Bumcrot D, Koteliansky V, Limmer S, Manoharan M, Vornlocher H-P. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004 Nov 11;432(7014):173-8. Abstract Thakker DR, Natt F, Husken D, Maier R, Muller M, van der Putten H, Hoyer D, Cryan JF. Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference. Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17270-5. Epub 2004 Nov 29. Abstract Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev. 2005 Mar 1;19(5):517-29. Review. Abstract Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT, Paulson HL, Yang L, Kotin RM, Davidson BL. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med. 2004 Aug;10(8):816-20. Epub 2004 Jul 4. Abstract Xia XG, Zhou H, Zhou S, Yu Y, Wu R, Xu Z. An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase. J Neurochem. 2005 Jan;92(2):362-7. Abstract

View all comments by Zuoshang Xu

From a clinical perspective, the identification of TDP-43 protein represents a major breakthrough in our understanding of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The TDP-43 is the mystery protein that is associated with the ubiquitin-positive inclusions that are commonly found in many patients with FTLD and in most, if not all, patients with ALS.

This finding is particularly important because several recent papers suggest that patients who have FTLD with ubiquitin inclusions at autopsy (FTLD-U) account for approximately 50 percent of all autopsy-confirmed FTLD cases (1-3). The remaining majority of FTLD cases are associated with the tau protein, but other neuropathological diagnoses exist. The finding that possibly one-half of all FTLD patients may have ubiquitin-positive neuropathology means that any breakthroughs in the biology of this protein could potentially translate into helping a large proportion of FTLD patients.

In addition, the finding that the TDP-43 protein is also found in patients with ALS further supports...  Read more

From a clinical perspective, the identification of TDP-43 protein represents a major breakthrough in our understanding of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The TDP-43 is the mystery protein that is associated with the ubiquitin-positive inclusions that are commonly found in many patients with FTLD and in most, if not all, patients with ALS.

This finding is particularly important because several recent papers suggest that patients who have FTLD with ubiquitin inclusions at autopsy (FTLD-U) account for approximately 50 percent of all autopsy-confirmed FTLD cases (1-3). The remaining majority of FTLD cases are associated with the tau protein, but other neuropathological diagnoses exist. The finding that possibly one-half of all FTLD patients may have ubiquitin-positive neuropathology means that any breakthroughs in the biology of this protein could potentially translate into helping a large proportion of FTLD patients.

In addition, the finding that the TDP-43 protein is also found in patients with ALS further supports the overlap between FTLD and ALS. Future research on the TDP-43 protein will likely also benefit ALS patients and help us understand how these two very different clinical phenotypes are related.

References:
1. Lipton AM, White CL 3rd, Begio EH. Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration. Acta Neuropathol (Berl). 2004 Nov;108(5):379-85. Abstract

2. Johnson JK, Diehl J, Mendez MF, Neuhaus J, Shapira JS, Forman M, Chute DS, Roberson ED, Pace-Savitsky C, Neumann M, Chow TW, Rosen HJ, Forstl H, Kurz A, Miller BL.. Frontotemporal lobar degeneration: demographic characteristics of 353 patients. Archives of Neurology. 2005;62:925-930. Abstract

3. Forman MS, Farmer J, Johnson JK, Clark CM, Arnold SE, Coslett HB, Chatterjee A, Hurtig HI, Karlawish JH, Rosen HJ, Van Deerlin V, Lee V M-Y, Miller BL, Trojanowski JQ, & Grossman M. (2006). Frontotemporal dementia: Clinicopathological correlations. Annals of Neurology. 2006;59:952-962. Abstract

View all comments by Julene K. Johnson

In this paper, Drs. Lee and Trojanowski and colleagues have at long last identified the mystery protein hiding within the ubiquitinated inclusions that characterize certain histological forms of frontotemporal lobar degeneration (FTLD), termed FTLD-U. This task has challenged neuroscientists for well over a decade, with all prior attempts at identification using immunohistochemical or biochemical methods proving fruitless. The culprit protein is a TAR DNA-binding protein, known as TDP-43. This protein is present within all the ubiquitinated structures in FTLD-U, viz., the neuronal cytoplasmic inclusions, the neuronal intranuclear inclusions, and the neuritic changes, though whether this is the sole component of these structures (other than ubiquitin) remains uncertain. Some previous studies reported the presence of p62 protein within neuronal cytoplasmic inclusions, but such findings have been inconsistent. Moreover, Lee and Trojanowski have shown that the ubiquitinated neuronal cytoplasmic inclusions seen within spinal and cranial nerve nuclear motor neurons in motor neuron...  Read more

In this paper, Drs. Lee and Trojanowski and colleagues have at long last identified the mystery protein hiding within the ubiquitinated inclusions that characterize certain histological forms of frontotemporal lobar degeneration (FTLD), termed FTLD-U. This task has challenged neuroscientists for well over a decade, with all prior attempts at identification using immunohistochemical or biochemical methods proving fruitless. The culprit protein is a TAR DNA-binding protein, known as TDP-43. This protein is present within all the ubiquitinated structures in FTLD-U, viz., the neuronal cytoplasmic inclusions, the neuronal intranuclear inclusions, and the neuritic changes, though whether this is the sole component of these structures (other than ubiquitin) remains uncertain. Some previous studies reported the presence of p62 protein within neuronal cytoplasmic inclusions, but such findings have been inconsistent. Moreover, Lee and Trojanowski have shown that the ubiquitinated neuronal cytoplasmic inclusions seen within spinal and cranial nerve nuclear motor neurons in motor neuron disease (amyotrophic lateral sclerosis) also contain TDP-43.

This is an immensely important study with huge implications for neurobiology.

Firstly, it pinpoints a key biochemical constituent in the pathogenesis of FTLD-U and motor neuron disease (MND), and one which previous work would never have regarded as a likely candidate protein. Secondly, although an association between FTLD and MND had long been known on account of some cases showing defined clinical features of both disorders, sharing pathological features of both disorders, and families being known where some members had FTLD, others MND, and others the combined disorder, it was never clear whether this association was coincidental or causal. Now we can see causality, and the implication that FTLD and MND are part and parcel of the same disease spectrum will have major ramifications for understanding pathogenesis, and eventual treatment. Thirdly, the finding of TDP-43 pathological changes in FTLD patients with mutations in the newly identified progranulin (PGRN) gene, who typically show FTLD-U pathological changes, firmly brings together a causal relationship in these two fundamental proteins in driving the pathogenesis of the disorder, and opens up untapped vistas of neurobiological research.

Therefore, in rapid time, two major (protein) pieces in the jigsaw puzzle of FTLD have been identified. The challenge now will be to fit the pieces around these and eventually identify the linking processes that bring these together into the fuller picture. Nonetheless, it is clear that even within FTLD-U there are different histological and clinical phenotypes, and it will be necessary to dissect out biochemical or other factors that might determine where the TDP-43 pathological changes take place in the brain to produce the clinical phenotype. That is, why is it that in some patients the most common clinical manifestation of FTLD-U, frontotemporal dementia, is present in association with bilateral involvement of the frontal and temporal lobes, yet in others only the temporal lobes are affected—producing semantic dementia—and in others the left hemisphere is preferentially affected to give progressive non-fluent aphasia. Also, what determines whether TDP-43 changes will be in the brainstem and spinal cord to give MND, or in the cerebral cortex to give FTLD? Lastly, in all this flurry of excitement, it should not be forgotten that tauopathy is still a major cause of FTLD, and it is not immediately apparent how pathological changes in the expression or function of tau might link in with progranulin and TDP-43. Clearly, changes in all three molecules can produce the same disorder of FTLD either separately or collectively: it is not possible to unequivocally discriminate FTD patients with MAPT mutations from those with PGRN mutations, or others without mutations in either. Interrelationships within this Bermuda triangle of tau, progranulin, and TDP-43 will need to be addressed.

The identification of TDP-43 as a (major/sole) component of the ubiquitinated protein of FTLD and MND, in conjunction with the identification of mutations in PGRN, have opened up huge new fields within the neurobiology of neurodegenerative disease with tentacles that may stretch far wider than these two disorders themselves. Whether there is a role for either or both of these proteins in other disorders like Alzheimer disease and Parkinson disease remains to be seen. The gauntlet has been cast down—it is up to the neuroscience community to pick this up and address these issues. What is certain is that there will be a major change in the focus of neurobiological research as groups worldwide seek to investigate the implications of changes in proteins such as progranulin and TDP-43 in terms of health and disease. We can look forward within the near future to major advances in our understanding of how the brain works in respect of these molecules and why neurodegenerative disease occurs when they fail to function properly. Maybe even a treatment for neurodegenerative disease may come a little closer.

View all comments by David M.A. Mann

Neumann, Sampathu, Kwong, and colleagues have resolved a long-standing issue in the research field of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). These authors have identified TDP-43 as a major component of ubiquitin-positive inclusions that characterize these disorders. They first extracted a fraction from the patients' brains using monoclonal antibodies and then analyzed it by mass spectrometry. Their findings have greatly facilitated the understanding of the molecular pathogenesis of FTLD and ALS.

Independently, we have also found TDP-43 as a component of the inclusions in FTLD [1]. Following electrophoresis of the sarkosyl-insoluble brain extracts from FTLD, Alzheimer disease (AD) and dementia with Lewy bodies (DLB), we have done exhaustive analyses by mass spectrometry. Following identification of each molecule that is more abundant in FTLD than AD/DLB, we have studied FTLD brain samples immunochemically and immunohistochemically. The antibodies to TDP-43 have immuno-stained neuronal inclusions and dystrophic neurites in the...  Read more

Neumann, Sampathu, Kwong, and colleagues have resolved a long-standing issue in the research field of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). These authors have identified TDP-43 as a major component of ubiquitin-positive inclusions that characterize these disorders. They first extracted a fraction from the patients' brains using monoclonal antibodies and then analyzed it by mass spectrometry. Their findings have greatly facilitated the understanding of the molecular pathogenesis of FTLD and ALS.

Independently, we have also found TDP-43 as a component of the inclusions in FTLD [1]. Following electrophoresis of the sarkosyl-insoluble brain extracts from FTLD, Alzheimer disease (AD) and dementia with Lewy bodies (DLB), we have done exhaustive analyses by mass spectrometry. Following identification of each molecule that is more abundant in FTLD than AD/DLB, we have studied FTLD brain samples immunochemically and immunohistochemically. The antibodies to TDP-43 have immuno-stained neuronal inclusions and dystrophic neurites in the hippocampus and the temporal cortex in FTLD, and skein-like inclusions in the spinal cord in FTLD and ALS. Immunoblotting of the sarkosyl-insoluble fraction has shown abnormal changes in TDP-43 including hyperphosphorylation, fragment formation, and smear-like staining, all of which are similar to abnormal tau in AD and suggest a central role for the formation of abnormal aggregates. These findings are comparable with those by Lee's group. This is not surprising, since both groups have employed principally the same polyclonal antibody which is the only commercially available rabbit polyclonal.

In addition, however, we have found TDP-43-positive glial inclusions in the spinal cord in FTLD and ALS. These inclusions were also positive for tau. The distribution of glial inclusions was consistent with the degenerating areas, suggesting that glial abnormalities are involved in the pathological processes of ALS and FTLD. A difference between our results and theirs is the TDP-43-positive staining of some, but not all, tau-positive structures including Pick bodies and neurofibrillary tangles. The significance of these findings remains to be established, since immunoblot analysis did not show any abnormality in TDP-43 in Pick disease and Alzheimer disease. Our paper will appear shortly in Biochem Biophys Res Commun [1].

In the case of tau and α-synuclein, detection of abnormally modified molecules has revealed far more extensive pathology than that seen by ubiquitin immunohistochemistry. While lesions immunohistochemically labeled for TDP-43 are a little more numerous than those labeled for ubiquitin, the difference is far less than that we have experienced for tau and α-synuclein immunohistochemistry. This may be a point that remains to be cleared up. Another issue that is open for further investigations is to prove, by protein chemistry, the ubiquitination of TDP-43.

In any event, it has to be emphasized that two different approaches have come to the same conclusion, establishing with certainty that TDP-43 is the major component of the inclusions in FTLD and ALS. This further strengthens the hypothesis that these disorders are part of a clinicopathological spectrum that shares similar pathogenesis, and suggests the possibility that TDP-43 may be a common therapeutic target for these disorders. It is now necessary to investigate the relationship of TDP-43 to other molecules that have been reported to be associated with familial FTD, FTD with motor neuron disease, or ALS. Such molecules include progranulin, charged multivesicular body protein 2B (CHMP2B), valosin-containing protein, dynactin, and an unidentified protein in familial disease linked to chromosome 9.

References:
1. T. Arai, M. Hasegawa, H. Akiyama, K. Ikeda, T. Nonaka, H. Mori, D. Mann, K. Tsuchiya, M. Yoshida, Y. Hashizume, T. Oda, TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis, Biochem Biophys Res Commun, in press

View all comments by Tetsuaki Arai

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

These papers represent exciting work describing a new genetic mutation associated with familial ALS. The results further highlight the importance for RNA processing in at least familial forms of motor neuron disease. Much work remains to determine the exact mechanisms by which FUS modulates motor neuron survival. It may be related to that of TDP-43. However, the lack of cytoplasmic aggregation of TDP-43, and rare ubiquitin inclusions in the patients with FUS mutations, suggest the mechanisms may be distinct. It is interesting that FUS protein did not accumulate in the cytoplasm of motor neurons in sporadic ALS patients, again suggestive that the pathogenic mechanisms of mutant FUS-induced motor neuron degeneration may be distinct from that in sporadic ALS.

View all comments by Robert Bowser

These studies raise interesting questions about whether one problem in ALS and perhaps other neurodegenerative diseases is that RNA trafficking proteins fail to properly deliver RNAs to dendritic spines. The paper by Kwiatkowski et al. reports evidence that wild-type FUS and TDP-43 may be involved in transporting RNA into dendrites, where it mediates local protein synthesis that can be stimulated by neural activity. The clumping of the mutant form described by both new papers could therefore perturb the transport of RNA. Local protein synthesis in dendrites plays a major role in the activity-dependent modulation of synaptic strength. Changes in synaptic activity have been recently reported in the mouse model of SOD1 mutation (van Zundert et al., 2008), so it will be worthwhile to examine this issue in the FUS mice that will certainly be developed by these investigators.

View all comments by Eric Frank

This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS (Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).

Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA...  Read more

This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS (Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).

Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA metabolism and (lower) motor neurons.

By the way, all these cases appear to predominately involve a lower motor neuron form of ALS. The hint from genetics does suggest more of a loss of function rather than gain, but cell biology will ultimately sort that out. We certainly await the generation of mouse or fly models, which are now well underway for TDP-43. However, this may be a particularly difficult target for specific, non-toxic drug therapy.

View all comments by Jeffrey D. Rothstein

These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In...  Read more

These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In addition, defects in the trafficking of mRNA may lead to depletion of key proteins that are in high demand locally for motor neuron function. But if FUS has a general function in mRNA transcription, splicing, and trafficking, why do mutations in this gene cause ALS and not other neurodegenerative diseases? What makes motor neurons more vulnerable in the presence of defective FUS? It could be true that in motor neurons FUS controls the transcription of a distinct set of mRNA that is expressed in a cell-type-specific manner in motor neurons, or that FUS controls the production of a key protein that is highly required in motor neurons when compared to other cell-types, and thus motor neurons may become vulnerable first. FUS seems to be the tip of the iceberg. Finding effectors, binding partners including mRNA, may lead to the identification of key components of both familial and sporadic ALS. More work is on the way!

References:

Kneussel M. Dynamic regulation of GABA(A) receptors at synaptic sites. Brain Res Brain Res Rev. 2002 Jun ;39(1):74-83. Abstract

Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, Clawson L, Rothstein JD. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998 Mar;20(3):589-602. 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

Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo JM, Miller CC, Shaw CE. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. Abstract

View all comments by P. Hande Ozdinler

This study characterizes the dynamic behavior of SOD1 in detail. First, it essentially reproduces previous studies including the ones from the authors' group, as it has been well known that overall structures are similar between wild-type and mutant SOD1 proteins. In addition, significant differences in the dynamic behavior have been observed between Apo and holo forms of SOD1. When the metal ions are removed from the protein, structural disorder increases particularly in the loop regions.

We think that one of the interesting findings in this paper is the increased solvent accessibility of Cys-6 upon metal removal. Cys-6 is one of the four Cys residues (Cys-6, 57, 111, 146) in SOD1 and is buried toward the protein interior in the holo form of SOD1. In an enzymatically active form of SOD1, an intra-molecular disulfide forms between Cys-57 and 146, while Cys-6 and 111 remain reduced. In contrast, pathological inclusions purified from several ALS-model mice contain SOD1 multimers that are cross-linked via non-physiological disulfide bonds (  Read more

This study characterizes the dynamic behavior of SOD1 in detail. First, it essentially reproduces previous studies including the ones from the authors' group, as it has been well known that overall structures are similar between wild-type and mutant SOD1 proteins. In addition, significant differences in the dynamic behavior have been observed between Apo and holo forms of SOD1. When the metal ions are removed from the protein, structural disorder increases particularly in the loop regions.

We think that one of the interesting findings in this paper is the increased solvent accessibility of Cys-6 upon metal removal. Cys-6 is one of the four Cys residues (Cys-6, 57, 111, 146) in SOD1 and is buried toward the protein interior in the holo form of SOD1. In an enzymatically active form of SOD1, an intra-molecular disulfide forms between Cys-57 and 146, while Cys-6 and 111 remain reduced. In contrast, pathological inclusions purified from several ALS-model mice contain SOD1 multimers that are cross-linked via non-physiological disulfide bonds (Furukawa et al., 2006).

It is, however, still controversial which Cys residues are involved in the formation of cross-linked SOD1 multimers under pathological conditions. While we have previously reported that the disulfide formation is not absolutely required for triggering SOD1 aggregation (Furukawa et al., 2008), an important role of Cys-6 and 111 in the formation of disulfide cross-links has been also suggested in the cultured cell model (Niwa et al., 2007). In addition, ALS-causing mutations at position 6 have been reported (i.e., C6G and C6F), implying that the other Cys residues are involved in the formation of disulfide-linked multimers even when Cys-6 is unavailable for disulfide formation. Nonetheless, the increased flexibility and solvent accessibility of Cys-6 upon metal removal will be an important clue to explain a molecular mechanism of the pathological SOD1 oligomer formation.

View all comments by Yoshiaki Furukawa

This study elegantly gives a first insight on a transgenic mouse model of mutant TDP-43 (A315T) identified in familial ALS patients. For those in the field, it is clear that generating these mouse models is a mammoth task on its own. Among the many interesting findings in this paper, the first to catch my attention was that the 25-kDa TDP-43 C-terminal fragments (CTFs) were recovered from detergent-soluble fractions but not from urea fractions as observed in sporadic and familial ALS/FTLD patients. If the TDP-43 25-kDa CTFs would indeed be confirmed as the real culprit, this would yet again emphasize the importance of soluble but not aggregated protein/peptide in cellular toxicity, as has been shown for a number of other proteinopathies including Aβ, α-synuclein, polyglutamine expansion in Huntingtin, and mutant SOD1.

Another important observation made in this paper was that ubiquitin-immunoreactive (ir) inclusions observed in select neurons including motor neurons were not TDP-43-ir. Thus, the mutant TDP-43 (A315T) mice do not completely model ALS, where...  Read more

This study elegantly gives a first insight on a transgenic mouse model of mutant TDP-43 (A315T) identified in familial ALS patients. For those in the field, it is clear that generating these mouse models is a mammoth task on its own. Among the many interesting findings in this paper, the first to catch my attention was that the 25-kDa TDP-43 C-terminal fragments (CTFs) were recovered from detergent-soluble fractions but not from urea fractions as observed in sporadic and familial ALS/FTLD patients. If the TDP-43 25-kDa CTFs would indeed be confirmed as the real culprit, this would yet again emphasize the importance of soluble but not aggregated protein/peptide in cellular toxicity, as has been shown for a number of other proteinopathies including Aβ, α-synuclein, polyglutamine expansion in Huntingtin, and mutant SOD1.

Another important observation made in this paper was that ubiquitin-immunoreactive (ir) inclusions observed in select neurons including motor neurons were not TDP-43-ir. Thus, the mutant TDP-43 (A315T) mice do not completely model ALS, where ubiquitin-ir inclusions are also TDP-43-ir; nevertheless, this work does lead to a very interesting question: what are these inclusions composed of?

Knowing earlier studies (see Tatom et al., 2009 and ARF related news story), I am also not surprised at the glaring omission of wild-type TDP-43 mice as a better control than the non-transgenic mice utilized in this study. So although clearly not all is answered yet, let's see how these and other TDP-43 mouse models currently being developed will unfold the mysteries of TDP-43-led neurodegeneration.

View all comments by Samir Kumar-Singh