PERSPECTIVES OPINION
FTD and ALS—translating mouse studies into clinical trials Lars M. Ittner, Glenda M. Halliday, Jillian J. Kril, Jürgen Götz, John R. Hodges and Matthew C. Kiernan Abstract | Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are related neurodegenerative disorders, which are characterized by a rapid decline in cognitive and motor functions, and short survival. Although the clinical and neuropathological characterization of these diseases has progressed—in part— through animal studies of pathogenetic mechanisms, the translation of findings from rodent models to clinical practice has generally not been successful. This article discusses the gap between preclinical animal studies in mice and clinical trials in patients with FTD or ALS. We outline how to better design preclinical studies, and present strategies to improve mouse models to overcome the translational shortfall. This new approach could help identify drugs that are more likely to achieve a therapeutic benefit for patients. Ittner, L. M. et al. Nat. Rev. Neurol. advance online publication 5 May 2015; doi:10.1038/nrneurol.2015.65
Introduction
Frontotemporal dementia (FTD) and amyo trophic lateral sclerosis (ALS) are part of a disease continuum. FTD usually presents with either behavioural abnormalities or language dysfunction, but a considerable number of patients also develop the muscle weakness and wasting that is typical of ALS.1 Both FTD and ALS are characterized by short survival, and a rapid decline in function. There is no cure for either, and—apart from riluzole, which slightly prolongs survival in patients with ALS2—current treatments offer purely limited s ymptomatic relief. The number of ongoing clinical trials in ALS is substantial (Figure 1). These trials cover a wide range of therapeutic strat egies such as symptomatic improvement, immunom odulation, neuroprotection and others. Some trials have been aimed at specific patient populations, for example the investigation of antisense oligonucleotides in carriers of a specific mutation. Furthermore, new therapeutic approaches continue to emerge, such as targeting of superoxide dismutase 1 (SOD1),3 TAR DNA-binding protein (TDP‑43) 4 or granulin (GRN) 5 proteins (as reviewed elsewhere6). Competing interests The authors declare no competing interests.
In stark contrast to ALS, there are only a few ongoing clinical trials for FTD. Although a subset of patients present with pure forms of FTD or ALS, there is a continuum of symp toms, neuropathology and underlying genet ics between both conditions—even within families with the same pathogenetic muta tions.7 Therefore, therapeutic approaches for ALS may also benefit patients with FTD. The neuropathology of FTD presents as frontotemporal lobar degeneration (FTLD) characterized by neuronal loss, astrogliosis and proteinaceous intracellular inclusions.8 The inclusions found in the majority of patients with FTD are composed of either microtubule-associated protein tau, TDP‑43, and—less frequently—FUS or unknown ubiquitinated protein(s). 8,9 These major proteinopathy subtypes can be further divided according to the type and distribu tion of pathology.10–12 The clinical overlap in the symptoms of FTD and ALS is comple mented by overlapping neuropathology, such as the deposition of TDP‑43 and FUS in some patients with ALS. Notably, 2% of patients with ALS present with intracellular inclusions of SOD1 protein (caused by SOD1 mutations13), which are not seen in patients with FTD. Mutations in the tau-encoding gene MAPT were first identified in patients with
NATURE REVIEWS | NEUROLOGY
FTD and parkinsonism linked to chromo some 17 (FTDP‑17), which is associated with tau neurofibrillary tangles.14 By con trast, the TDP‑43 pathology in other forms of familial FTD has been linked to a range of mutations in GRN, C9orf72 and, rarely, VCP and TARDBP (which codes for TDP‑43 itself ). 15–21 Rare mutations in CHMP2B have been linked to an as yet unidentified proteinopathy.22 Mutations in patients with familial ALS were first identified in TDP‑43negative individuals. In these patients, muta tions were particularly common in the SOD1 gene, which account for 20% of familial ALS and 2% of all ALS cases.13 TDP‑43-positive familial ALS is heterogenetic, with muta tions found in TARDBP, FUS, C9orf72 and others.23 Though FTD and ALS can be modelled in several species, mice are the most com monly used model animal in translational research. 24,25 In this article, we focus on transgenic mouse models of FTD and ALS, though it is not our intention to discount the valuable contributions of other animal models to the field.26
Mouse models of FTD and ALS
Although it is impossible to authentically model every clinical feature of human neuro degenerative diseases in mice, mouse models are, by and large, able to recapitulate the key histopathological and biochemical features of both ALS and FTD. Transgenic mouse strains have thus become invaluable tools for studying disease mechanisms and for devel oping therapeutic strategies, as these models enable the research community to dissect central pathomechanisms of these diseases. The identification of SOD1 mutations in familial ALS and MAPT mutations in FTDP‑1713,14,27,28 first sparked the generation of transgenic mouse models of ALS and FTD (Box 1).29,30 The subsequent description of TARDBP mutations then led to a number of transgenic mouse models that express either wild-type or mutant TDP‑43. 31–37 These models reproduced many features of ALS and FTD, such as motor deficits, reduced survival, fragmentation and insolu bility of protein aggregates, reactive gliosis, and neuronal loss. None of these models is perfect: for example, some TDP‑43 mouse lines present ADVANCE ONLINE PUBLICATION | 1
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PERSPECTIVES FTD
I
ALS
II
Anakinra aMSC and riluzole Rasagiline hSCNSC
Onabotulinumtoxin A Incobotulinumtoxin A
hNSC Corticotropin Fasudil Fingolimod aBMSC Multiple aBMSC Pyrimethamine immunosuppressants aMSC
III
Nimodipine
Masitinib
Tolcapone
IV TRx0237 Oxytocin/ emotional mimicry
sNN0029 VM202 Dexpramipexole Cistanche glycosides MCI-186 Tirasemtiv Dextromethorphan and quinidine Mexiletine Mexiletine Vitamin E Creatine Mecobalamin
Phrenic nerve
Language therapy
Cannabis Taurourso- L-serine sativa deoxycholic acid Ozanezumab Olanzapine Tocilizumab
Arimoclomol Far infrared radiation
Lithium carbonate
YAM80 Transcranial direct current SOD1-ASO Excercise stimulation Diaphragm Diaphragm
Therapeutic approaches/targets Reduction of sialorrhea Enzyme inhibitors Immunomodulators Stem cells Neuroprotection Muscle function improvement
hSCNSC GM604
Far infrared radiation
Vitamins, amino acids and/or natural extracts Antibodies Antipsychotics Others Chaperone activation Mechanical devices
Figure 1 | Current clinical trials in FTD and ALS. Numerous clinical trials haveReviews been initiated Nature | Neurology for patients with ALS, in contrast to only four drug-based clinical trials for patients with FTD. Treatments under investigation are shown colour-coded according to therapeutic approaches or targets, and are arranged on the basis of the respective phase (I, II, III or IV) of each trial identified on ClinicalTrials.gov. Treatments in boxes represent nonblinded trials. Abbreviations: aBMSC, autologous bone-marrow-derived stem cells; ALS, amyotrophic lateral sclerosis; aMSC, autologous mesenchymal stromal cells; FTD, frontotemporal dementia; hNSC, human neural stem cells; hSCNSC, human spinal-cord-derived neural stem cells; SOD1-ASO, superoxide dismutase antisense oligonucleotide.
with severe gastrointestinal pathology that is not a typical symptom of FTD or ALS.38,39 This symptom is caused by trans gene expression in the myenteric plexus of the gut, probably due to promoter activity outside the CNS.39,40 Treating the gastro intestinal problems of TDP‑43 transgenic mice prolongs their survival, which allows sufficient time for TDP‑43 pathology to develop in the CNS. 41 However, nuclear depletion and cytoplasmic deposition of TDP‑43 (common in human FTD–ALS) is rare in TARDBP transgenic mice (Box 2). Although TDP‑43 mouse models present with motor symptoms, at most 25% of
motor neurons are lost,32 which is in stark contrast to the 90% losses reported in SOD1 transgenic mice and in patients with ALS.29 Mutations in GRN also result in TDP‑43 neuropathology in humans, but Grn knockout mice show little pathologically phosphorylated TDP‑43. 42–44 Thus, Grn knockouts have not been able to elucidate how GRN mutations lead to TDP‑43 patho logy in humans. Interestingly, Grn-deficient mice do display behavioural changes remi niscent of FTD, such as social deficits, aggression, depression-like behaviour, and disinhibition.44–46 TDP‑43-immunoreactive (but VCP-negative) aggregates have also
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been reported in a series of VCP transgenic mice,47–51 with some strains even showing the nuclear depletion of endogenous TDP‑43 that is typical of FTD–ALS.47,49,52 Transgenic mice that homozygously express wild-type human FUS develop ALS-like symptoms, with hindlimb paraly sis and shortened life span, together with cytop lasmic FUS aggregation. 53 How FUS expression causes these deficits, and whether expression of mutant FUS would have similar consequences, remains to be determined. Finally, expression of truncated CHMP2B results in ubiquitinated inclusions that are negative for both TDP‑43 and FUS, thereby recapitulating the histopathology seen in some patients with FTLD.54 This finding suggests a toxic gain of function by pathogenetic CHMP2B mutations that cause C‑terminal truncation of CHMP2B. Taken together, the recent past has seen a wealth of new genetic mouse models of FTD and ALS, yet the replication of clini cal and neuropathological findings from human patients remains incomplete.
Difficulties with translation
Interventions for progressive diseases should start at an early stage; therefore, understand ing the early pathomechanisms is vital. This type of investigation can best be achieved in animal models that develop an authentic pathology as they age. Accordingly, SOD1 transgenic mouse models—which remain the most accurate models of ALS—have been used extensively to study disease devel opment. Unfortunately, these mice are likely to reflect only familial SOD1 ALS, which— together with poor clinical trial design (Box 3)—might partly explain why the trans lation from mice to human patients with ALS has been so disappointing.55,56 Minocycline is a good example of the failure to translate. This agent is an inhibi tor of microglial activation, and increased the lifespan of SOD1-Gly93Ala transgenic mice when administered before the onset of symptoms.57 However, in clinical trials, minocycline accelerated ALS progression relative to placebo.58 More recently, mino cycline was shown to have no therapeutic effect in SOD1-Gly93Ala mice when given after the onset of symptoms. 59 Similarly, ceftriaxone, which activates the glutamate transporter EAAT2 and thereby reduces excitotoxicity, increases survival in SOD1Gly93Ala mice only when administered before symptom onset. 60,61 A phase III c linical trial of ceftriaxone showed no efficacy.62 www.nature.com/nrneurol
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PERSPECTIVES These examples illustrate the impor tance of clinically relevant preclinical study design when testing novel compounds in mouse models of ALS and FTD.63 In par ticular, a detailed understanding of the age at onset in mouse models is required: it was recently shown that disease onset is >2 months earlier in SOD1 transgenic mice than previously reported.64 A substantial number of compounds that modulate pathology have been identified in mouse models of FTD and ALS, most of which await thorough confirmation in com prehensive human studies. The poor trans lational success to date requires us to refine models and preclinical study design. Many potential therapeutics have only been tested in models of tau-transgenic FTD models or SOD1-transgenic ALS models, which rep resent only a small fraction of the human disease spectrum.55 Given the number of genetic mouse models available, compre hensive studies including several models are advisable before advancing a compound to clinical trials. Additionally, compounds should be tested in small numbers of mice with different genetic backgrounds, to determine the effects of genetic variability on therapeutic efficacy.65 This approach to preclinical testing should identify promis ing candidate therapies that have wide applicability and efficacy, thereby increasing potential effectiveness in humans. In turn, this approach might reduce failure rates of clinical trials, and re-establish confidence in animal models of ALS and FTD.
Current and future directions
The onsets of sporadic FTD or ALS are typically in late adulthood, yet virtually all transgenic mouse models express patho genic proteins at a very early stage in brain development (postnatally or even during embryogenesis). 24 Developing neurons probably cope differently with pathologies than do mature cells. For example, when TDP‑43 expression is induced during brain development, mice show marked brain atro phy and loss of neurons, but no overt fea tures of the human disease.66 However, if TDP‑43 expression is initiated when mice are >3 weeks old, mice develop progressive features similar to human disease, with slow neuronal loss.66 At present, few mouse models allow for transgenic protein expression to be induced in late life.34,66–69 These models do not directly reveal new therapeutic options, but they might be an appropriate model for determining the ideal timing of putative
Box 1 | Tau and SOD1 transgenic mouse models paved the way Mutations in SOD1 were first identified in patients with familial ALS in 1993,13 and expressed in transgenic mice 1 year later.29 Several mutant SOD1 mouse lines show progressive ALSlike motor neuron degeneration that leads to paralysis and death.29,111,112 The SOD1-Gly93Ala mouse line remains the most commonly used ALS model. These mice show disease onset and survival correlated with the level of mutant protein expression.29 A long list of interventions prolong survival and/or delay onset of paralysis in mice, but very few of these have successfully translated into clinical trials.113 This paucity could owe to the fact that SOD1-ALS represents a small (2%), pathomechanistically distinct, subset of all ALS cases. Nevertheless, there are examples of treatments (for example, riluzole) where effects seen in SOD1 mice match the clinical experience.114 In 1998, MAPT mutations were identified in patients with FTDP‑17,14 prompting the generation of the first mutant tau transgenic mouse with neurofibrillary tangles in 2000. 30 In addition to recapitulating neuropathological features of FTD, such as hyperphosphorylation and insolubility of tau protein, these mice develop behavioural and motor deficits24,115 that can be used as measures of drug efficacy.110 Several therapeutic interventions, including a substantial number of active and passive tau-vaccination studies, are effective in these mice. 77,85,116–119 These strategies are being extensively explored by several pharmaceutical companies, and are likely to soon enter clinical trials. Tau and SOD1 transgenic mice are commonly used models for FTD and ALS, and they have substantially elucidated disease pathomechanisms, and helped to develop new treatment strategies. As such, these models are excellent examples of successful translation of human conditions into animals. Furthermore, we have seen the first ‘back-translations’ of therapies into clinics, with hopefully many more to come. Abbreviations: ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; FTDP‑17, FTD with parkinsonism linked to chromosome 17.
Box 2 | Modelling neuropathology Neuropathology can provide only limited insight into early disease mechanisms, as it is difficult to speculate what has happened to dead or damaged neurons. Mouse models can help uncover the fate of cells that are lost during disease progression. For example, transgenic overexpression of TDP‑43 is toxic to neurons despite the fact that key features of the human pathology are not reproduced.120 Though one could argue that this mismatch suggests that these models are inaccurate, little is known about whether dead neurons in patients with FTD or ALS underwent a similar pathological process as seen in mice (that is, cytoplasmic accumulation of TDP‑43). Alternatively, mice could lack essential mechanisms of coping with abnormal TDP‑43, which might be present—though not yet recognized—in humans. Furthermore, it is not clear whether the protein inclusions in patients’ remaining neurons are pathogenic, or represent protein aggregation that is necessary for reducing the toxicity of the pathological form of TDP‑43. Future transgenic TARDBP mouse models might reveal whether the presence of TDP‑43 in cytoplasm is toxic per se, or if it simply reflects a successful survival strategy. The value of currently available TARDBP transgenic mice for developing targeted treatments will only become clear when clinical trials are initiated on the basis of data generated in these mice. Abbreviations: ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; TDP‑43, TAR DNA-binding protein 43.
‘pathogen’-reducing therapies, which aim to restore neuronal function.67 Thus, additional studies with existing models are desirable, as is the introduction of new models with advanced induction systems.70 Notably, the currently available inducible systems are notoriously leaky (that is, some transgenic proteins are still expressed when switched ‘off ’), but next-generation inducible systems should overcome this shortfall.70 The notion of prion-like spreading of pathology between brain areas in FTD– ALS could affect the clinical translation of findings in animal models.71,72 For tau, spreading has been partially reproduced
NATURE REVIEWS | NEUROLOGY
in cell cultures73,74 and transgenic mouse models. 74–79 A similar pathomechanism might apply for TDP‑43 in FTD–ALS55,72,80 and SOD1 in familial ALS.81 Arguably the current in vivo approaches to prion-like spreading seem crude, requiring intra cerebral injections of proteins into geneti cally manipulated models.75 A more elegant approach is to use focal mutant tau expres sion to reproduce disease-like spread ing among connected brain areas in vivo. However, this approach requires great lengths be taken to rule out possible expres sion of aberrant transgenes82,83 or spreading of viral particles84 in recipient cells. ADVANCE ONLINE PUBLICATION | 3
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PERSPECTIVES Box 3 | What we need for successful clinical trials Efficient phase II trials Although phase III clinical trials are essential for drugs to be approved by regulatory authorities, phase II trials are critical for identifying agents for further development. Unfortunately, this key distinction between phase II and phase III trials is becoming blurred: given the difficulties associated with providing evidence of drug efficacy in small phase II studies, many positive trends are not sustained in larger phase III trials. Two possible refinements to phase II design are to employ refined statistical strategies that minimize trial duration and sample size, and to conduct phase III clinical trials tailored as if they were phase II trials, with smaller patient cohorts and safety used as an outcome measure.121 Appropriate primary endpoints The choice of the ‘correct’ primary endpoint for trials in patients with FTD and/or ALS continues to be the subject of debate. Trials using functional scales as primary endpoints, such as the ALS Functional Rating Scale Revised, have dominated the field, while trials principally concerned with patient survival have become less common. The methodological benefits of choosing functional endpoints over survival endpoints include smaller required sample sizes, shorter trial duration, and clinically meaningful treatment effects. However, it is plausible that measurement of survival is the only means to determine whether a treatment effect truly exists. Biomarkers There is a dearth of biomarkers in FTD–ALS, and no diagnostic test currently exists for either condition.122 A biomarker would clearly facilitate diagnosis, which, in turn, might expedite the initiation of neuroprotective therapies. Furthermore, biomarkers could also have a role in selecting patients for enrolment in clinical trials, or for identifying subgroups that will benefit most from certain medications. The proximity of CSF to disease-affected regions of the brain makes CSF a logical place to search for biochemical signatures of FTD–ALS. Abbreviations: ALS, amyotrophic lateral sclerosis; CSF, cerebrospinal fluid; FTD, frontotemporal dementia.
Ultimately, the best models will have naive recipient cells and not need surgi cal intervention. Development of these models will require expression vectors that achieve accurate temporal expression in strictly defined brain areas and neuron populations. Nevertheless, current genetic– surgical models offer the opportunity to study key processes in disease propagation in vivo, and to develop treatments tailored to preventing release, passage and/or uptake of prion-like proteins. Tau-targeted immu nization is an early example of a strategy to interfere with this pathological spreading in mice.77,85 In addition to improving the accuracy of ALS and FTD mouse models, methods for analysing phenotypes are becoming increas ingly sophisticated. For example, multipho ton microscopy of the living brain provides detailed insight into disease processes, such as the surprisingly normal signalling activity of neurons bearing neurofibrillary tangles observed in mutant tau transgenic mice.86 Similarly, simple behavioural testing has evolved into sophisticated touchscreen-based paradigms, which enable precise cognitive assessment using proto cols with direct relevance for translation into clinical practice.87 Also, brain network function can now be assessed in freely moving mice via telemetric EEG, which has been used to demonstrate Aβ-induced hypersynchronicity in mouse models of
Alzheimer disease.88,89 Many of these tech nologies have yet to be applied to ALS and FTD mouse models, but their relevance is clear. The identification of intronic polyGGGGCC (G 4C 2) hexanucleotide inclu sions in C9orf72 as the most common genetic cause of FTD–ALS has been a significant advancement. 17,18,90,91 In vitro studies have proposed several patho genetic mechanisms, including the for mation of RNA foci, 17 repeat-associated non-ATG (RAN) translation92,93 and loss of function,17,18,94 all of which are awaiting confirmation in vivo.95 Recently, the first transgenic C9orf72 repeat-expansion mouse model with ubiquitin-positive inclusions was reported, but phenotypic analysis is pending.96 A C9orf72 knockout mice was also reported as part of a technical article on TALEN-based gene editing,97 but without a phenotypic description. The high prevalence of C9orf72 muta tions in familial FTD–ALS suggests that accurate mouse models of C9orf72 muta tions are likely to contribute to drug devel opment and testing. Unfortunately, cloning of transgenic expression constructs with large numbers of G4C2-repeats remains a challenge, and currently available models express 80 repeats in mice 96 and up to 103 repeats in flies,98 contrasting the >1,000 repeats in humans. This technical limita tion could be overcome by using bacterial
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artificial chromosome clones from human C9orf72 poly‑G4C2 carriers. Alternatively, chemical stabilization of poly‑G4C2 repeats (for example, via modified DNA back bones) can be used during synthesis. Yet another approach could be to use alterna tive codons to obtain the dipeptide products (such as poly-GlyAla) of poly‑G4C2 RAN translation. This approach could elucidate the pathogenetic relevance of RAN transla tion products versus RNA foci or C9orf72 loss of function. Metabolic and other systemic alterations have been described in a range of neuro degenerative conditions (as reviewed else where99). Furthermore, metabolic changes have been found in variants of FTD. 100 Therefore, a systemic-level appreciation of current and future mouse models of ALS and FTD might provide a new perspec tive on translation. On the other hand, when exploring translatable approaches for ALS and FTD, it is important not to limit studies to mice. Neurons derived from induced pluripotent stem cells (iPSCs) also offer a platform to model patho logy and test therapeutic candidates. 101 Accordingly, in iPSC-derived neurons from patients with C9orf72-linked ALS, oligonucleotides targeting hexanucleotide repeat transcripts reduced RNA foci forma tion and other pathological features.102–104 Oligonucleotide-targeting of these repeat transcripts might prevent the formation of dipeptide repeat inclusions,105 although this effect has not been achieved in all iPSC-based approaches.102 iPSCs are likely to become a standard in the field, complementing, but not repla cing, genetic animal models. The emer gence of powerful gene-editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR) reduces the necessity of sourcing cells from patients, and enables the introduction of specific mutations into well characterized iPSC lines and into endogenous genes in mice.106–108 The use of endogenous mouse genes might help overcome problems arising from the expression of human rather than mouse proteins. CRISPR gene editing also allows parallel gene loci to be simultaneously targeted,109 opening up new possibilities for multifactorial models both in iPSCs and mice.
Conclusions
To facilitate translation from mouse models to clinical trials and elucidate under lying disease mechanisms, it may become www.nature.com/nrneurol
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PERSPECTIVES important to rethink our expectation that a ‘good’ mouse model of disease exactly reflects the symptoms seen in patients. This conceptualization has and will continue to fail us, given that mice and humans would probably present different overt phenotypes even if the underlying mechanisms were identical. For example, motor deficits are a prominent feature of tau transgenic FTD mouse models, yet only a fraction (
FTD and ALS—translating mouse studies into clinical trials Lars M. Ittner, Glenda M. Halliday, Jillian J. Kril, Jürgen Götz, John R. Hodges and Matthew C. Kiernan Abstract | Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are related neurodegenerative disorders, which are characterized by a rapid decline in cognitive and motor functions, and short survival. Although the clinical and neuropathological characterization of these diseases has progressed—in part— through animal studies of pathogenetic mechanisms, the translation of findings from rodent models to clinical practice has generally not been successful. This article discusses the gap between preclinical animal studies in mice and clinical trials in patients with FTD or ALS. We outline how to better design preclinical studies, and present strategies to improve mouse models to overcome the translational shortfall. This new approach could help identify drugs that are more likely to achieve a therapeutic benefit for patients. Ittner, L. M. et al. Nat. Rev. Neurol. advance online publication 5 May 2015; doi:10.1038/nrneurol.2015.65
Introduction
Frontotemporal dementia (FTD) and amyo trophic lateral sclerosis (ALS) are part of a disease continuum. FTD usually presents with either behavioural abnormalities or language dysfunction, but a considerable number of patients also develop the muscle weakness and wasting that is typical of ALS.1 Both FTD and ALS are characterized by short survival, and a rapid decline in function. There is no cure for either, and—apart from riluzole, which slightly prolongs survival in patients with ALS2—current treatments offer purely limited s ymptomatic relief. The number of ongoing clinical trials in ALS is substantial (Figure 1). These trials cover a wide range of therapeutic strat egies such as symptomatic improvement, immunom odulation, neuroprotection and others. Some trials have been aimed at specific patient populations, for example the investigation of antisense oligonucleotides in carriers of a specific mutation. Furthermore, new therapeutic approaches continue to emerge, such as targeting of superoxide dismutase 1 (SOD1),3 TAR DNA-binding protein (TDP‑43) 4 or granulin (GRN) 5 proteins (as reviewed elsewhere6). Competing interests The authors declare no competing interests.
In stark contrast to ALS, there are only a few ongoing clinical trials for FTD. Although a subset of patients present with pure forms of FTD or ALS, there is a continuum of symp toms, neuropathology and underlying genet ics between both conditions—even within families with the same pathogenetic muta tions.7 Therefore, therapeutic approaches for ALS may also benefit patients with FTD. The neuropathology of FTD presents as frontotemporal lobar degeneration (FTLD) characterized by neuronal loss, astrogliosis and proteinaceous intracellular inclusions.8 The inclusions found in the majority of patients with FTD are composed of either microtubule-associated protein tau, TDP‑43, and—less frequently—FUS or unknown ubiquitinated protein(s). 8,9 These major proteinopathy subtypes can be further divided according to the type and distribu tion of pathology.10–12 The clinical overlap in the symptoms of FTD and ALS is comple mented by overlapping neuropathology, such as the deposition of TDP‑43 and FUS in some patients with ALS. Notably, 2% of patients with ALS present with intracellular inclusions of SOD1 protein (caused by SOD1 mutations13), which are not seen in patients with FTD. Mutations in the tau-encoding gene MAPT were first identified in patients with
NATURE REVIEWS | NEUROLOGY
FTD and parkinsonism linked to chromo some 17 (FTDP‑17), which is associated with tau neurofibrillary tangles.14 By con trast, the TDP‑43 pathology in other forms of familial FTD has been linked to a range of mutations in GRN, C9orf72 and, rarely, VCP and TARDBP (which codes for TDP‑43 itself ). 15–21 Rare mutations in CHMP2B have been linked to an as yet unidentified proteinopathy.22 Mutations in patients with familial ALS were first identified in TDP‑43negative individuals. In these patients, muta tions were particularly common in the SOD1 gene, which account for 20% of familial ALS and 2% of all ALS cases.13 TDP‑43-positive familial ALS is heterogenetic, with muta tions found in TARDBP, FUS, C9orf72 and others.23 Though FTD and ALS can be modelled in several species, mice are the most com monly used model animal in translational research. 24,25 In this article, we focus on transgenic mouse models of FTD and ALS, though it is not our intention to discount the valuable contributions of other animal models to the field.26
Mouse models of FTD and ALS
Although it is impossible to authentically model every clinical feature of human neuro degenerative diseases in mice, mouse models are, by and large, able to recapitulate the key histopathological and biochemical features of both ALS and FTD. Transgenic mouse strains have thus become invaluable tools for studying disease mechanisms and for devel oping therapeutic strategies, as these models enable the research community to dissect central pathomechanisms of these diseases. The identification of SOD1 mutations in familial ALS and MAPT mutations in FTDP‑1713,14,27,28 first sparked the generation of transgenic mouse models of ALS and FTD (Box 1).29,30 The subsequent description of TARDBP mutations then led to a number of transgenic mouse models that express either wild-type or mutant TDP‑43. 31–37 These models reproduced many features of ALS and FTD, such as motor deficits, reduced survival, fragmentation and insolu bility of protein aggregates, reactive gliosis, and neuronal loss. None of these models is perfect: for example, some TDP‑43 mouse lines present ADVANCE ONLINE PUBLICATION | 1
© 2015 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES FTD
I
ALS
II
Anakinra aMSC and riluzole Rasagiline hSCNSC
Onabotulinumtoxin A Incobotulinumtoxin A
hNSC Corticotropin Fasudil Fingolimod aBMSC Multiple aBMSC Pyrimethamine immunosuppressants aMSC
III
Nimodipine
Masitinib
Tolcapone
IV TRx0237 Oxytocin/ emotional mimicry
sNN0029 VM202 Dexpramipexole Cistanche glycosides MCI-186 Tirasemtiv Dextromethorphan and quinidine Mexiletine Mexiletine Vitamin E Creatine Mecobalamin
Phrenic nerve
Language therapy
Cannabis Taurourso- L-serine sativa deoxycholic acid Ozanezumab Olanzapine Tocilizumab
Arimoclomol Far infrared radiation
Lithium carbonate
YAM80 Transcranial direct current SOD1-ASO Excercise stimulation Diaphragm Diaphragm
Therapeutic approaches/targets Reduction of sialorrhea Enzyme inhibitors Immunomodulators Stem cells Neuroprotection Muscle function improvement
hSCNSC GM604
Far infrared radiation
Vitamins, amino acids and/or natural extracts Antibodies Antipsychotics Others Chaperone activation Mechanical devices
Figure 1 | Current clinical trials in FTD and ALS. Numerous clinical trials haveReviews been initiated Nature | Neurology for patients with ALS, in contrast to only four drug-based clinical trials for patients with FTD. Treatments under investigation are shown colour-coded according to therapeutic approaches or targets, and are arranged on the basis of the respective phase (I, II, III or IV) of each trial identified on ClinicalTrials.gov. Treatments in boxes represent nonblinded trials. Abbreviations: aBMSC, autologous bone-marrow-derived stem cells; ALS, amyotrophic lateral sclerosis; aMSC, autologous mesenchymal stromal cells; FTD, frontotemporal dementia; hNSC, human neural stem cells; hSCNSC, human spinal-cord-derived neural stem cells; SOD1-ASO, superoxide dismutase antisense oligonucleotide.
with severe gastrointestinal pathology that is not a typical symptom of FTD or ALS.38,39 This symptom is caused by trans gene expression in the myenteric plexus of the gut, probably due to promoter activity outside the CNS.39,40 Treating the gastro intestinal problems of TDP‑43 transgenic mice prolongs their survival, which allows sufficient time for TDP‑43 pathology to develop in the CNS. 41 However, nuclear depletion and cytoplasmic deposition of TDP‑43 (common in human FTD–ALS) is rare in TARDBP transgenic mice (Box 2). Although TDP‑43 mouse models present with motor symptoms, at most 25% of
motor neurons are lost,32 which is in stark contrast to the 90% losses reported in SOD1 transgenic mice and in patients with ALS.29 Mutations in GRN also result in TDP‑43 neuropathology in humans, but Grn knockout mice show little pathologically phosphorylated TDP‑43. 42–44 Thus, Grn knockouts have not been able to elucidate how GRN mutations lead to TDP‑43 patho logy in humans. Interestingly, Grn-deficient mice do display behavioural changes remi niscent of FTD, such as social deficits, aggression, depression-like behaviour, and disinhibition.44–46 TDP‑43-immunoreactive (but VCP-negative) aggregates have also
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been reported in a series of VCP transgenic mice,47–51 with some strains even showing the nuclear depletion of endogenous TDP‑43 that is typical of FTD–ALS.47,49,52 Transgenic mice that homozygously express wild-type human FUS develop ALS-like symptoms, with hindlimb paraly sis and shortened life span, together with cytop lasmic FUS aggregation. 53 How FUS expression causes these deficits, and whether expression of mutant FUS would have similar consequences, remains to be determined. Finally, expression of truncated CHMP2B results in ubiquitinated inclusions that are negative for both TDP‑43 and FUS, thereby recapitulating the histopathology seen in some patients with FTLD.54 This finding suggests a toxic gain of function by pathogenetic CHMP2B mutations that cause C‑terminal truncation of CHMP2B. Taken together, the recent past has seen a wealth of new genetic mouse models of FTD and ALS, yet the replication of clini cal and neuropathological findings from human patients remains incomplete.
Difficulties with translation
Interventions for progressive diseases should start at an early stage; therefore, understand ing the early pathomechanisms is vital. This type of investigation can best be achieved in animal models that develop an authentic pathology as they age. Accordingly, SOD1 transgenic mouse models—which remain the most accurate models of ALS—have been used extensively to study disease devel opment. Unfortunately, these mice are likely to reflect only familial SOD1 ALS, which— together with poor clinical trial design (Box 3)—might partly explain why the trans lation from mice to human patients with ALS has been so disappointing.55,56 Minocycline is a good example of the failure to translate. This agent is an inhibi tor of microglial activation, and increased the lifespan of SOD1-Gly93Ala transgenic mice when administered before the onset of symptoms.57 However, in clinical trials, minocycline accelerated ALS progression relative to placebo.58 More recently, mino cycline was shown to have no therapeutic effect in SOD1-Gly93Ala mice when given after the onset of symptoms. 59 Similarly, ceftriaxone, which activates the glutamate transporter EAAT2 and thereby reduces excitotoxicity, increases survival in SOD1Gly93Ala mice only when administered before symptom onset. 60,61 A phase III c linical trial of ceftriaxone showed no efficacy.62 www.nature.com/nrneurol
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PERSPECTIVES These examples illustrate the impor tance of clinically relevant preclinical study design when testing novel compounds in mouse models of ALS and FTD.63 In par ticular, a detailed understanding of the age at onset in mouse models is required: it was recently shown that disease onset is >2 months earlier in SOD1 transgenic mice than previously reported.64 A substantial number of compounds that modulate pathology have been identified in mouse models of FTD and ALS, most of which await thorough confirmation in com prehensive human studies. The poor trans lational success to date requires us to refine models and preclinical study design. Many potential therapeutics have only been tested in models of tau-transgenic FTD models or SOD1-transgenic ALS models, which rep resent only a small fraction of the human disease spectrum.55 Given the number of genetic mouse models available, compre hensive studies including several models are advisable before advancing a compound to clinical trials. Additionally, compounds should be tested in small numbers of mice with different genetic backgrounds, to determine the effects of genetic variability on therapeutic efficacy.65 This approach to preclinical testing should identify promis ing candidate therapies that have wide applicability and efficacy, thereby increasing potential effectiveness in humans. In turn, this approach might reduce failure rates of clinical trials, and re-establish confidence in animal models of ALS and FTD.
Current and future directions
The onsets of sporadic FTD or ALS are typically in late adulthood, yet virtually all transgenic mouse models express patho genic proteins at a very early stage in brain development (postnatally or even during embryogenesis). 24 Developing neurons probably cope differently with pathologies than do mature cells. For example, when TDP‑43 expression is induced during brain development, mice show marked brain atro phy and loss of neurons, but no overt fea tures of the human disease.66 However, if TDP‑43 expression is initiated when mice are >3 weeks old, mice develop progressive features similar to human disease, with slow neuronal loss.66 At present, few mouse models allow for transgenic protein expression to be induced in late life.34,66–69 These models do not directly reveal new therapeutic options, but they might be an appropriate model for determining the ideal timing of putative
Box 1 | Tau and SOD1 transgenic mouse models paved the way Mutations in SOD1 were first identified in patients with familial ALS in 1993,13 and expressed in transgenic mice 1 year later.29 Several mutant SOD1 mouse lines show progressive ALSlike motor neuron degeneration that leads to paralysis and death.29,111,112 The SOD1-Gly93Ala mouse line remains the most commonly used ALS model. These mice show disease onset and survival correlated with the level of mutant protein expression.29 A long list of interventions prolong survival and/or delay onset of paralysis in mice, but very few of these have successfully translated into clinical trials.113 This paucity could owe to the fact that SOD1-ALS represents a small (2%), pathomechanistically distinct, subset of all ALS cases. Nevertheless, there are examples of treatments (for example, riluzole) where effects seen in SOD1 mice match the clinical experience.114 In 1998, MAPT mutations were identified in patients with FTDP‑17,14 prompting the generation of the first mutant tau transgenic mouse with neurofibrillary tangles in 2000. 30 In addition to recapitulating neuropathological features of FTD, such as hyperphosphorylation and insolubility of tau protein, these mice develop behavioural and motor deficits24,115 that can be used as measures of drug efficacy.110 Several therapeutic interventions, including a substantial number of active and passive tau-vaccination studies, are effective in these mice. 77,85,116–119 These strategies are being extensively explored by several pharmaceutical companies, and are likely to soon enter clinical trials. Tau and SOD1 transgenic mice are commonly used models for FTD and ALS, and they have substantially elucidated disease pathomechanisms, and helped to develop new treatment strategies. As such, these models are excellent examples of successful translation of human conditions into animals. Furthermore, we have seen the first ‘back-translations’ of therapies into clinics, with hopefully many more to come. Abbreviations: ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; FTDP‑17, FTD with parkinsonism linked to chromosome 17.
Box 2 | Modelling neuropathology Neuropathology can provide only limited insight into early disease mechanisms, as it is difficult to speculate what has happened to dead or damaged neurons. Mouse models can help uncover the fate of cells that are lost during disease progression. For example, transgenic overexpression of TDP‑43 is toxic to neurons despite the fact that key features of the human pathology are not reproduced.120 Though one could argue that this mismatch suggests that these models are inaccurate, little is known about whether dead neurons in patients with FTD or ALS underwent a similar pathological process as seen in mice (that is, cytoplasmic accumulation of TDP‑43). Alternatively, mice could lack essential mechanisms of coping with abnormal TDP‑43, which might be present—though not yet recognized—in humans. Furthermore, it is not clear whether the protein inclusions in patients’ remaining neurons are pathogenic, or represent protein aggregation that is necessary for reducing the toxicity of the pathological form of TDP‑43. Future transgenic TARDBP mouse models might reveal whether the presence of TDP‑43 in cytoplasm is toxic per se, or if it simply reflects a successful survival strategy. The value of currently available TARDBP transgenic mice for developing targeted treatments will only become clear when clinical trials are initiated on the basis of data generated in these mice. Abbreviations: ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; TDP‑43, TAR DNA-binding protein 43.
‘pathogen’-reducing therapies, which aim to restore neuronal function.67 Thus, additional studies with existing models are desirable, as is the introduction of new models with advanced induction systems.70 Notably, the currently available inducible systems are notoriously leaky (that is, some transgenic proteins are still expressed when switched ‘off ’), but next-generation inducible systems should overcome this shortfall.70 The notion of prion-like spreading of pathology between brain areas in FTD– ALS could affect the clinical translation of findings in animal models.71,72 For tau, spreading has been partially reproduced
NATURE REVIEWS | NEUROLOGY
in cell cultures73,74 and transgenic mouse models. 74–79 A similar pathomechanism might apply for TDP‑43 in FTD–ALS55,72,80 and SOD1 in familial ALS.81 Arguably the current in vivo approaches to prion-like spreading seem crude, requiring intra cerebral injections of proteins into geneti cally manipulated models.75 A more elegant approach is to use focal mutant tau expres sion to reproduce disease-like spread ing among connected brain areas in vivo. However, this approach requires great lengths be taken to rule out possible expres sion of aberrant transgenes82,83 or spreading of viral particles84 in recipient cells. ADVANCE ONLINE PUBLICATION | 3
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PERSPECTIVES Box 3 | What we need for successful clinical trials Efficient phase II trials Although phase III clinical trials are essential for drugs to be approved by regulatory authorities, phase II trials are critical for identifying agents for further development. Unfortunately, this key distinction between phase II and phase III trials is becoming blurred: given the difficulties associated with providing evidence of drug efficacy in small phase II studies, many positive trends are not sustained in larger phase III trials. Two possible refinements to phase II design are to employ refined statistical strategies that minimize trial duration and sample size, and to conduct phase III clinical trials tailored as if they were phase II trials, with smaller patient cohorts and safety used as an outcome measure.121 Appropriate primary endpoints The choice of the ‘correct’ primary endpoint for trials in patients with FTD and/or ALS continues to be the subject of debate. Trials using functional scales as primary endpoints, such as the ALS Functional Rating Scale Revised, have dominated the field, while trials principally concerned with patient survival have become less common. The methodological benefits of choosing functional endpoints over survival endpoints include smaller required sample sizes, shorter trial duration, and clinically meaningful treatment effects. However, it is plausible that measurement of survival is the only means to determine whether a treatment effect truly exists. Biomarkers There is a dearth of biomarkers in FTD–ALS, and no diagnostic test currently exists for either condition.122 A biomarker would clearly facilitate diagnosis, which, in turn, might expedite the initiation of neuroprotective therapies. Furthermore, biomarkers could also have a role in selecting patients for enrolment in clinical trials, or for identifying subgroups that will benefit most from certain medications. The proximity of CSF to disease-affected regions of the brain makes CSF a logical place to search for biochemical signatures of FTD–ALS. Abbreviations: ALS, amyotrophic lateral sclerosis; CSF, cerebrospinal fluid; FTD, frontotemporal dementia.
Ultimately, the best models will have naive recipient cells and not need surgi cal intervention. Development of these models will require expression vectors that achieve accurate temporal expression in strictly defined brain areas and neuron populations. Nevertheless, current genetic– surgical models offer the opportunity to study key processes in disease propagation in vivo, and to develop treatments tailored to preventing release, passage and/or uptake of prion-like proteins. Tau-targeted immu nization is an early example of a strategy to interfere with this pathological spreading in mice.77,85 In addition to improving the accuracy of ALS and FTD mouse models, methods for analysing phenotypes are becoming increas ingly sophisticated. For example, multipho ton microscopy of the living brain provides detailed insight into disease processes, such as the surprisingly normal signalling activity of neurons bearing neurofibrillary tangles observed in mutant tau transgenic mice.86 Similarly, simple behavioural testing has evolved into sophisticated touchscreen-based paradigms, which enable precise cognitive assessment using proto cols with direct relevance for translation into clinical practice.87 Also, brain network function can now be assessed in freely moving mice via telemetric EEG, which has been used to demonstrate Aβ-induced hypersynchronicity in mouse models of
Alzheimer disease.88,89 Many of these tech nologies have yet to be applied to ALS and FTD mouse models, but their relevance is clear. The identification of intronic polyGGGGCC (G 4C 2) hexanucleotide inclu sions in C9orf72 as the most common genetic cause of FTD–ALS has been a significant advancement. 17,18,90,91 In vitro studies have proposed several patho genetic mechanisms, including the for mation of RNA foci, 17 repeat-associated non-ATG (RAN) translation92,93 and loss of function,17,18,94 all of which are awaiting confirmation in vivo.95 Recently, the first transgenic C9orf72 repeat-expansion mouse model with ubiquitin-positive inclusions was reported, but phenotypic analysis is pending.96 A C9orf72 knockout mice was also reported as part of a technical article on TALEN-based gene editing,97 but without a phenotypic description. The high prevalence of C9orf72 muta tions in familial FTD–ALS suggests that accurate mouse models of C9orf72 muta tions are likely to contribute to drug devel opment and testing. Unfortunately, cloning of transgenic expression constructs with large numbers of G4C2-repeats remains a challenge, and currently available models express 80 repeats in mice 96 and up to 103 repeats in flies,98 contrasting the >1,000 repeats in humans. This technical limita tion could be overcome by using bacterial
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artificial chromosome clones from human C9orf72 poly‑G4C2 carriers. Alternatively, chemical stabilization of poly‑G4C2 repeats (for example, via modified DNA back bones) can be used during synthesis. Yet another approach could be to use alterna tive codons to obtain the dipeptide products (such as poly-GlyAla) of poly‑G4C2 RAN translation. This approach could elucidate the pathogenetic relevance of RAN transla tion products versus RNA foci or C9orf72 loss of function. Metabolic and other systemic alterations have been described in a range of neuro degenerative conditions (as reviewed else where99). Furthermore, metabolic changes have been found in variants of FTD. 100 Therefore, a systemic-level appreciation of current and future mouse models of ALS and FTD might provide a new perspec tive on translation. On the other hand, when exploring translatable approaches for ALS and FTD, it is important not to limit studies to mice. Neurons derived from induced pluripotent stem cells (iPSCs) also offer a platform to model patho logy and test therapeutic candidates. 101 Accordingly, in iPSC-derived neurons from patients with C9orf72-linked ALS, oligonucleotides targeting hexanucleotide repeat transcripts reduced RNA foci forma tion and other pathological features.102–104 Oligonucleotide-targeting of these repeat transcripts might prevent the formation of dipeptide repeat inclusions,105 although this effect has not been achieved in all iPSC-based approaches.102 iPSCs are likely to become a standard in the field, complementing, but not repla cing, genetic animal models. The emer gence of powerful gene-editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR) reduces the necessity of sourcing cells from patients, and enables the introduction of specific mutations into well characterized iPSC lines and into endogenous genes in mice.106–108 The use of endogenous mouse genes might help overcome problems arising from the expression of human rather than mouse proteins. CRISPR gene editing also allows parallel gene loci to be simultaneously targeted,109 opening up new possibilities for multifactorial models both in iPSCs and mice.
Conclusions
To facilitate translation from mouse models to clinical trials and elucidate under lying disease mechanisms, it may become www.nature.com/nrneurol
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PERSPECTIVES important to rethink our expectation that a ‘good’ mouse model of disease exactly reflects the symptoms seen in patients. This conceptualization has and will continue to fail us, given that mice and humans would probably present different overt phenotypes even if the underlying mechanisms were identical. For example, motor deficits are a prominent feature of tau transgenic FTD mouse models, yet only a fraction (
