Review

Targeting TDP-43 in neurodegenerative diseases Mauricio Budini, Francisco E Baralle & Emanuele Buratti† 1.

Introduction

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

2.

Targeting protein-protein interactions

3.

Targeting RNA-protein

Introduction: TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed RNA-binding protein belonging to the hnRNP family of nuclear proteins. In human disease, its aberrant aggregation in brains has been shown to play a causative role in several neurodegenerative diseases, especially ALS and FTLD. Areas covered: In this work, we have highlighted what could be the most promising avenues that could be exploited in a profitable manner to modulate TDP-43 pathology. These range from its protein-protein interactions, RNA-protein interactions and its aberrant aggregation process. Recently published articles on these subjects have been reviewed in the writing up of this manuscript. Expert opinion: Targeting aberrant TDP-43 aggregation in neurodegenerative diseases should be considered both a challenge and an opportunity. The challenge is represented by the central role played by TDP-43 in the general cellular and developmental processes of higher proteins. This characteristic makes it difficult to target this protein in a generalized manner. In addition, and mostly because of this reason, we still lack reliable disease model systems that can reproduce most, if not all, characteristics of the human disease. Nonetheless, recent research is finally starting to provide potential therapeutic targets based on new findings that regard TDP-43 biology and functions.

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interactions 4.

Targeting TDP-43 aggregation and expression process

5.

Compounds that decrease TDP-43 levels and/or reduce TDP-43 aggregation

6.

Compounds that increase TDP-43 levels and/or trigger TDP-43 aggregation

7.

Conclusion

8.

Expert opinion

Keywords: amyotrophic lateral sclerosis, frontotemporal dementia, hnRNP, protein aggregation, RNA metabolism, TAR DNA-binding protein-43 Expert Opin. Ther. Targets (2014) 18(6):617-632

1.

Introduction

Alterations at the level of RNA-binding proteins (RBPs) have been recently found to lie at the heart of many Mendelian diseases, ranging from cancers to neuromuscular, to sensory diseases [1]. In particular, from the initial discovery in the mid-2000 that RBPs often play a central role in several neurodegenerative diseases a huge amount of effort has been spent at trying to use this knowledge to identify novel therapeutic target strategies [2]. At present, major RBPs that have been shown to play a direct role in the pathology of ALS/FTD and other neurodegenerative diseases are TAR DNA-binding protein-43 (TDP-43) [3-5], FUS/TLS [6,7], FET proteins [8] and very recently by the addition of two classic hnRNP A/B family members, hnRNP A1 and A2 [9]. On top of the misregulation and/or aggregation of these factors, the recent discovery of the presence of hexanucleotide expansions in the C9orf72 gene has also raised the possibility that pathological RNA foci may contribute to influence the onset and progression of diseases such as ALS or FTLD, either by giving rise to aberrant polypeptides and/or by sequestering additional RBPs [10,11]. For this reason, the recent advances in ALS/FTD research that look for the causes of disease are pointing toward a major role being played by alterations at the level of two broad but well-defined processes: RNA metabolism and protein homeostasis. Since this topic has been recently reviewed in detail by

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Article highlights. .

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TAR DNA-binding protein-43 (TDP-43) is not an easily targeted protein for therapeutic intervention because of its importance to the general metabolism of the cell. Moreover, we still lack accurate disease models that can recapitulate all aspects of disease as observed in humans. Basic research to improve our knowledge of TDP-43 functional properties and regulatory pathways will therefore be necessary to improve our knowledge of the role it plays in disease. Even so, research avenues in recent years have already allowed the identification of several targets that could be exploited to develop novel therapeutic approaches. At present, these targets include selected proteinprotein interactions (e.g., TDP-43-FUS, TDP-43-SMN or TDP-43-Hsp factors), various interactions with cellular complexes (e.g., stress granules, Drosha complex) and specific RNA-protein interactions (e.g., 3¢UTR region of TDP-43 itself). Moreover, they include cellular pathways directly involved in TDP-43 clearance such as the general proteasome/autophagy processes.

This box summarizes key points contained in the article.

several articles, the interested readers can be referred directly to these publications [12,13]. Having reached these conclusions, it follows that targeting these two processes in a specific manner may represent one of the most promising ways to prevent, slow down or even reverse TDP-43 proteinopathies. Before we get to this point, however, there are also several hurdles that should be considered before this possibility becomes a reality. From the point of view of RNA metabolism, one of the major problems encountered when looking proteins such as TDP-43 as a target is actually represented by their complex functional properties in the regulation of RNA metabolism [14]. The reason is that with few exceptions, RBPs don’t regulate small subsets of RNA transcripts or are involved in only one specific metabolic pathway. Most RBPs, in fact, act to regulate RNA metabolism on a global scale, often following the entire life cycle of coding and noncoding RNAs from their very beginning to their eventual degradation [15]. This is also particularly evident in the brain, where a highly intricate network of RNA processing events is known to play a major role in regulating development and normal functioning [16]. Because of these global effects, it is quite clear that targeting these proteins in a blunt fashion can probably have more adverse consequences than anything else. This is especially true for TDP-43, where also UV cross-linking and immunoprecipitation and other high-throughput studies have shown us how important TDP-43 is for the general functioning of the cell. Moreover, this has been clearly highlighted by the development of animal models. In these models, TDP-43 knockout animals have all been shown to be invariably lethal at very early embryonic stages, while 618

overexpression studies have shown that even slight changes in TDP-43 dosage can lead to the development of very aggressive ALS-type phenotypes even in the absence of TDP-43 aggregates [17]. Nonetheless, high throughput screening studies have provided us with information on its properties and RNA interactions and although they have not pointed to a specific target [18], they have already gone some way in explaining TDP-43 role in disease. In addition, the role played by other factors in the pathology such as the C9orf72 expansion should be considered [19]. In comparison to RNA metabolism, a slightly better situation can be found with regard to the involvement of protein homeostasis in causing disease. It is in fact quite clear that dysfunctions at the level of the cellular protein degradation and prolonged reaction to oxidative stress play an active role in many neurodegenerative diseases, including ALS and FTD [20]. In this case, the clearance of misfolded proteins or the disruption/inhibition of their aggregation processes represents a very promising field in which to search for therapeutic targets. To this day, several compounds have been moderately successful to rescue the toxic effects of TDP-43 overexpression and/or to reduce the aggregation properties [2,14]. However, the major difficulty in investigating this particular line of research is represented by the lack of suitable TDP-43 aggregation model systems to test the eventual efficacy of these compounds in the human setting. In this work, we will look at all these aspects and try to identify what are the best TDP-43 targets that need to be moved forward in our search for novel therapeutic effectors. Target identification based on TDP-43 properties can include all aspects of its biological interactions within the cell. At its most basic level, this can involve the modulation of TDP-43 protein-protein interactions (PPIs), proteinRNA interactions and its expression levels (by affecting either its production, which includes autoregulatory mechanisms, or its degradation/turnover by the cellular machinery). In this work, we plan to look at these three aspects separately and identify what could be the most likely targets for therapeutic intervention. 2.

Targeting protein-protein interactions

In normal cellular conditions, TDP-43 shuttles continuously from the nucleus to the cytoplasm and tends to assemble in distinct multimeric complexes that include many proteins and are associated with RNAs [21,22]. It is the variation in composition of these complexes that allows RBPs to adopt different functional properties and thus act as a bridge among the different steps regulated by a particular complex [23,24]. Indeed, this situation is well reflected in all the proteomic analyses that have been performed on TDP-43, where hundreds of potential interactors were identified in culture cell lines such as HeLa [25] and HEK-293T [26]. Of course, because of the sheer physical impossibility of binding several hundred factors at the same time, it is probable that only a subset of

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Targeting TDP-43 in neurodegenerative diseases

these proteins will be bound by TDP-43 in particular conditions or localization within the cell. Furthermore, it is not clear how many of these associated factors are directly bound by TDP-43 or just associated by RNA-mediated interactions. At the biochemical level, therefore, it is important to validate these interactions using conventional techniques such as co-immunoprecipitation and co-localization analysis. From a therapeutic point of view, this is extremely important because some of these interactions might result to be protective against the natural tendency of TDP-43 to aggregate or might define key properties that could be directly connected with the observed neuropathological effects when expression of this protein is disrupted. A better knowledge of these interactions might thus be important for the screening of compounds, commonly referred to as PPIs modulators, aimed at modulating TDP-43 PPIs. This possibility has recently received considerable attention in the biomolecular screening field and certainly will greatly contribute in the near future [27,28]. Before these strategies can be used, however, it is necessary to have a clear idea of what could be the disease-relevant interactors of TDP-43 and the functions they mediate. For this reason, we have compiled a full list of validated TDP-43 PPIs that are described in detail in Table 1 and schematically represented in Figure 1. Several of these potential interactions may be quite important from a disease point of view, as they involve proteins already known to play a role in neurodegeneration such as various hnRNPs A/B, FUS and SMN1, as well as others that play a role in axonal transport and general neuronal well-being. In addition, there is a direct connection with several Hsp proteins that could well represent a link with autophagosomal and proteasomal processes. In all these cases, therefore, mapping the interacting regions of these factors with TDP-43 may open the way to find small effector molecules or compounds that better stabilize this protein in its native condition. This work has already begun for the interaction of TDP-43 with the hnRNP A1 and A2 [29] and has made it possible to identify a short sequence that mediates this binding event as well as TDP-43 interaction with itself and whose structure has been recently solved by NMR [30,31]. Interestingly, it was found that this segment normally adopts a disordered conformation but can also adopt a b sheet rich conformation with time. Based on these findings, it has been possible to engineer a reporter system based on this sequence that is capable of inducing TDP-43 aggregation in a very efficient manner that will be used in future studies to screen for molecules/factors capable of affecting TDP-43 aggregation [32]. At the macromolecular level, another potentially important target is represented by the interaction of TDP-43 with components of the microRNA processing machinery localized in both the human and the mouse Drosha complexes (Table 1). In keeping with this hypothesis, depletion/aggregation of TDP-43 has been recently shown to affect the cellular levels of several selected microRNAs in a variety of systems [33-36] and even to affect the general efficiency of Drosha itself

during neuronal differentiation [37]. Interestingly, many of these microRNAs regulated by TDP-43 have also been found to be dysregulated in ALS patients [38]. Therefore, from a target point of view these particular microRNAs and the interaction between TDP-43 and Drosha should be considered therapeutically promising. Finally, and perhaps most importantly, TDP-43 has been found to be localized and participate in stress granule (SG) formation under different stress stimuli [39]. SGs are cytoplasmic ribonucleoprotein complexes that form when cells are exposed to stress. Their purpose is to transiently store mRNAs that encode housekeeping proteins and thus promote the selective translation of stress-response proteins. When the stress is removed, SGs dissolve and mRNA translation goes back to normal. Formation of SGs, however, could be extremely important for the pathology as one likely hypothesis for its origin is that a sustained SG condition within cells could culminate in irreversible TDP-43 aggregation, as recently reviewed [40-42]. Because of this connection, SG formation represents a likely target for therapeutic intervention. In keeping with this view, Kim et al. have recently observed that the toxicity of TDP-43 in Saccharomyces cerevisiae and Drosophila melanogaster models, as well as in mammalian neurons, was due to a prolonged phosphorylation of eIF2a factor that is responsible for SG formation and arrest of translation [43], supporting the idea that a sustained SG situation could be participating in the progression of these neurodegenerative diseases. 3.

Targeting RNA-protein interactions

Just like with PPIs, several high-throughput analyses have been published that had the aim to identify specific TDP-43 RNA targets and how these targets could be related to neurodegeneration. The result of all these studies has suggested that overexpression or depletion of TDP-43 can lead to the misregulation of several hundred targets, at both the coding and the noncoding mRNA level. Considering that several reviews have been written on the subject, interested readers can be referred to them in order to see a more detailed list of all RNA targets of TDP-43 [2,13]. At present, however, there are several major difficulties in using these candidate genes as a potential therapeutic target. First of all, there is very little overlap between the different published studies, raising the possibility that TDP-43 activities may be very dependent on the cellular context, experimental design and specific technical issues [18]. This means that at the moment we are still at the stage where all these results should be thoroughly validated before they can be considered potential targets. At present, in fact, there is really no indication of which ones can be particularly active in a disease setting and, therefore, which should be prioritized for intervention at the therapeutic level. In addition, because TDP-43 is a very aggregation-prone protein [44] it has also been very hard to obtain high-resolution

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Direct interaction mediated by NFL mRNA has been reported ex vivo but in vivo these two proteins do not normally co-localize ATXN2 and TDP-43 associate in a complex that is dependent on RNA and can act as powerful disease modifier. Interaction with TDP-43 is dependent on RNA. Increased ataxin-2 levels may impair the assembly of TDP-43 (and FUS) into mRNP granules, leading to increased cytoplasmic distribution BRDT regulates transcription and splicing in the 3¢UTR region of TDP-43 in the testis The TDP-43/FMRP/STAU1 complex binds to the 3¢UTR of SIRT1 mRNA and controls its expression. Depletion of TDP-43/FMRP/STAU1 sensitizes cells to apoptosis and DNA damages. Co-localization of TDP-43 with STAU1 and FMRP has also been reported in primary motor neurons Wild-type TDP-43 can bind to a low extent to FUS/TLS, but binding is enhanced with disease-associated mutations (Q331K and M337V). FUS direct interaction with SMN also links ALS with SMA Several hnRNP A/B family members have been shown to interact with TDP-43 in the C-terminal region (residues 342 -- 365). This interaction is important for the functional splicing properties of TDP-43, its aggregation and autoregulation processes hnRNP H is a nuclear splicing factor that binds to G-repeated elements and can act as enhancer or silencer of splicing depending on local context. Interaction with TDP-43 is dependent on RNA Overexpression of the HSP40 chaperone can suppress heat-induced TDP-43 aggregation All these proteins are involved in axonal mRNA transport and translation and co-IP with TDP-43. FMRP was also tested for co-IP but was observed just to co-localize, because the interaction is either weak or not direct MECP2 is part of a family of proteins that binds to methylated DNA and regulates gene expression. Mutations in this gene cause Rett syndrome NAC1 mediates recruitment of the UPS to dendritic spines and may thus cause recruitment of TDP-43 by the proteasome p62 physiologically binds to TDP-43 and likely is involved in degradation of TDP-43 with 35-kDa, but not full-length TDP-43. It also co-localizes with TDP-43-positive cytoplasmic inclusions, ubiquitin and UBQLN2 in patients with FTLD-MND TDP-43 acts as a co-activator of p65 PABPC1 is a predominantly cytoplasmic protein that associates with poly(A) mRNA stabilizing it. It also regulates RNA translation. Interaction with TDP-43 is dependent on RNA Immunoprecipitation from human A315T transgenic mice has detected the presence of a multiprotein complex between TDP-43, Parkin and HDAC6 Co-IP studies showed that PTEN forms a protein complex (but does not bind directly) with TDP-43 in the nucleus to negatively regulate TDP-43 expression These paraspeckle markers are found in insoluble TDP-43 artificial aggregates together with stress granule (SG) markers Mutations in the murine homologue of this protein cause altered NFL mRNA stability and lead to NF aggregate formation and a motor neuronopathy. RGNE can also interact by IP with FUS/TLS and p62 proteins

Notes

BBs: Bunina bodies; ER: Endoplasmic reticulum; SG: Stress granule; TDP-43: TAR DNA-binding protein-43; UPS: Ubiquitin proteasome system.

RGNEF

RBM14, PSF, NonO

PTEN

Parkin/HDAC6

p65 (NF-kB) PABPC1

p62/SQSTM1

NAC1

MECP2

HSP40/HSP70 IMP1, HuD, SMN

hnRNP H

hnRNP A/Bs

FUS/TLS

BRDT FMRP/STAU1

ATXN2

14-3-3

Interacting proteins

Table 1. TDP-43 interacting proteins.

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[104]

[103]

[102]

[101]

[100] [26]

[98,99]

[97]

[21]

[95] [90,96]

[26]

[29,32,47,93-95]

[25,91,92]

[88] [85,89,90]

[86,87]

[85]

Ref.

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Expert Opin. Ther. Targets (2014) 18(6)

SGs represent a protective mechanism to bypass the cellular insult, as the majority of mRNAs are silenced in these macromolecular structures in stalled 48S ribosomal complexes, while only specific and essential transcripts (i.e., Hsp70) are maintained in active translation Components of the Drosha microRNA processing complex that may interact directly with TDP-43 include ILF2/NF45, ILF3/NF90, DDX5, DDX17 and DDX3X TDP-43 co-localizes with Gems and can interact with the tudor domain of SMN and Gemin 8. The 321 -- 366 region is important for this interaction. RNA-binding activity is partially required. Interaction with Gemin components, however, was not reported in a second study that nonetheless showed a connection between the presence of TDP-43 and number of gems BBs are eosinophilic inclusions originated from the ER and immunostained by Abs against cystatin C, transferrin and peripherin

Notes

Optineurin is highly expressed in skeletal muscle and mutations in the gene, OPTN, have been reported to be causative for familial ALS RBM45 co-localizes with TDP-43 in inclusion bodies and is especially present in ALS-FTLD patients with C9orf72 expansions Following heat shock, TDP-43 transiently co-localizes with these two nuclear shock factors. The prion domain (266 -- 414) is responsible for this co-localization

Notes

TDP-43 has been suggested to interact with mutant but not wild-type TDP-43. Probably indirectly, SOD1 misfolding has also been observed with cytosolic accumulation of mutant FUS or TDP-43 in FUSFALS and TDP-43-FALS, respectively, as well as of wild-type TDP-43 in SALS. Conversely, mislocalization of TDP-43 has been observed in transgenic SOD1 mice and patients. Co-localization in ALS samples has also been reported TIA-1 is a strong translational repressor. As part of its interaction with SGs, TDP-43 can bind directly to TIA-1 TDP-43 can dimerize and oligomerize like many other hnRNP proteins. The domains involved in this process have been proposed include the N-terminus, RRM-2 and the Q/N rich 342 -- 365 C-terminal region. Oxidative stress can also promote TDP-43 dimerization and oligomerization via disulfide bond formation through its Cysteine residues. Homodimer formation can also be impaired following removal of the extreme 10 residues at its N-terminus Binding of TDP-43 to Ubiquilin 2 occurs through the C-terminus and can be inhibited by 4-aminoquinolines

Notes

BBs: Bunina bodies; ER: Endoplasmic reticulum; SG: Stress granule; TDP-43: TAR DNA-binding protein-43; UPS: Ubiquitin proteasome system.

Bunina bodies (BBs)

GEMS

Drosha/Dicer complex

SGs

Macromolecular complexes

HSF1/SFB

RBM45

Optineurin

Co-localizing proteins

UBQLN2

TDP-43

TIA-1

SOD1 (G93A/A4T)

Interacting proteins

Table 1. TDP-43 interacting proteins (continued).

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Ref.

Ref.

[124]

[122,123]

[21,25,34]

[39,109,120,121]

[95]

[119]

[118]

[76]

[94,110-117]

[109]

[85,105-108]

Ref.

Targeting TDP-43 in neurodegenerative diseases

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p62

TIA-1, G3BP PABPC1 EIF4G FUS/TLS Stress granules

Unmapped co-IP interactions

Parkin HDAC6 SOD1 (G96A/A4T)

TDP-43 RNA

IMP1, HuD, SMN Drosha + Dicer

IMP1, HuD Expert Opin. Ther. Targets Downloaded from informahealthcare.com by University Of South Australia on 05/24/14 For personal use only.

268 324 316

FMRP/ STAU1

NES261

NLS RRM1

N 1

31

107 81 98 106

RRM2

401 401 C

Prion-domain GRR

176

Q/N 321

MECP2

366

414 C-term

N-term

p65 (NF-KB)

UBQLN2

PTEN

FUS SMN

Indirect Binding mediated by RNA

NAC1

Gemin 8

Self-interaction hnRNP A/Bs

Ex vivo

GEMS

TIA-1 NFL mRNA HSP40/ HSP70 BRDT

14-3-3

U1snRNP

ATXN2 PABPC1 hnRNP H

Figure 1. Schematic representation of validated TDP-43 interactions with various cellular proteins and macromolecular complexes. The central diagram shows a scheme of the TDP-43 protein with its various functional regions. Immunoprecipitation-validated interactions are shown on the left. When mapped, the region of TDP-43 that is involved in the interaction is also shown. More detail regarding each interaction is reported in Table 1.

structural insight with regard to the way it binds to RNA target sequences. This is of course a prerequisite knowledge if one wants to look into the search for small molecule or other types of effectors capable of modulating its binding to particular RNAs. It is only recently, in fact, that a highresolution solution structure has been published of TDP-43 bound to a UG-rich sequence [45]. Hopefully, future studies will address the task of characterizing in depth the way TDP-43 binds to specific RNA sequences of clear functional importance (see below). A detailed knowledge of these interactions at the molecular level will allow the design of specific inhibitors or stabilizers capable of modulating TDP-43 binding to these sites. 622

For example, one particular interaction that has been well characterized at both the binding and the functional level is represented by the specific binding of TDP-43 to its own mRNA in the 3¢UTR region [46,47]. Binding of TDP-43 to this 3¢UTR region induces the activation of a normally silent intron in its 3¢UTR sequence that results in use of alternative polyadenylation signals (PAS) through RNA Pol II stalling and competition with the binding to polyadenylation factors (Figure 2) [48]. Recently, the importance for the autoregulatory process of the intron-removal event versus the intrinsic quality of the various PAS sites has been further compared and results have shown that TDP-43 binding and spliceosomal complex assembly on the normally unprocessed 3¢UTR

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Targeting TDP-43 in neurodegenerative diseases

TDP-43 Ex6

70K U1 snRNP

U2AF 35 U2AF65

pA2

pA1 Intron 7

CI HN

N

2

AS oligos

NH

70K U1 snRNP

Optimized snRNPs

Small molecules

2

O

NH

2

NH

AF U2

U2 AF 6

K U1 P RN sn

70

5

35

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H N

N

pA2

Ex6

Ex6

70K U1 snRNP

U2AF 35 U2AF65

pA1

pA2

TDP-43 Ex6

70K U1 snRNP

pA1

Intron 7

Intron 7 Modulated intron 7 processing

Increased intron 7 processing

Ex6 Ex6

pA1 Ex6

Increased TDP-43 production

pA2

pA1

Intron 7

Reduced intron 7 processing

U2AF 35 U2AF65

pA2

Decreased TDP-43 production

Ex6

pA1 pA2

Regulated TDP-43 production

Figure 2. RNA-based techniques that could be used to modulate the autoregulatory process of TDP-43. As shown in the upper diagram, the key event in TDP-43 autoregulation process is represented by the assembly of a spliceosomal complex on a specific intron in the 3¢UTR region of TDP-43. Using antisense nucleotides to block the recognition of the 5¢ and 3¢ss sites of this intron (left panels) would block its recognition leading to an increased protein production. On the other hand, a modified U1snRNP molecule (middle panels) that was better capable of recognizing the donor site of the intron would be expected to increase spliceosomal complex formation and have the opposite effect of reducing TDP-43 protein production. Finally, it should be considered that intron processing is often a co-operative process where many splicing factors play a role. Therefore, it could be possible to screen for compounds capable of affecting this splicing event in either a positive or negative manner depending on the way they affect the expression of these auxiliary factors. AS: Antisense; Ex: Exon; pA: Polyadenylation site.

intron are the two key events that cause the pre-mRNA to be retained in the nucleus and degraded [49]. As a result, when TDP-43 is in excess, only few TDP-43 mRNAs are exported to the cytoplasm, translated and are the source for the reduced protein production. Considering the central role played by this intron processing event, there are several possibilities to exploit this knowledge to affect TDP-43 protein production in either a positive or a negative manner, as has already been described to occur in many other pathologically important splicing events [50]. As shown in Figure 2, for example use of antisense nucleotides directed against the splice sites of the intron would be expected to lead to an inhibition of its recognition and thus a higher protein

production. Conversely, use of optimized U1snRNP molecules that better recognize the 5¢ splice site sequence of this intron, an approach that has already been used successfully in other splicing systems to rescue pathological mutations [51], would be expected to increase its recognition by the splicing machinery and lead to decreased TDP-43 protein production. Most importantly, use of small molecules that can modulate intron recognition by affecting the expression of basic splicing factors would also be a viable possibility to affect the level of intron processing. Enhancing or inhibiting this specific splicing event, in fact, would allow the possibility of modulating protein production in either a positive or negative manner.

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4.

Targeting the abnormal aggregation processes in neurodegenerative diseases has always represented one of the most desirable ‘target’ options for all those neurodegenerative diseases that display abnormal protein aggregation processes. This is especially true for RBPs that tend to self-assemble, such as TDP-43, in part because of their characteristic of containing prion-like domains within their sequence that promotes the formation of aggregates [52]. Like many other proteins prone to aggregate, TDP-43 must therefore be subject to regulatory mechanisms that control not just its protein levels, like the autoregulatory loop described above, but also its folding, trafficking, localization and degradation. It is the correct cooperation of all these processes that is essential to maintain the equilibrium between solubility and aggregation [53]. As previously mentioned, in neurons from patients affected by these disorders, TDP-43 is found to be present in an abnormal, aggregated form that localizes in the cytoplasm and less frequently in cell nuclei [54]. Additionally, these aggregates are characterized by the presence of C-terminal fragments and full-length TDP-43, positive to posttranslational modifications like ubiquitination and phosphorylation [3]. According to the evidence that we have available at present, two most likely hypotheses might be able to explain the role of the aggregates in the pathology. One is that aggregates may exert a toxic gain-of-function, whilst the other (loss-of function) consider that they may work like a ‘sink’. These sinks would act by trapping the nuclear and cytoplasmic TDP-43 in unfunctional aggregates and thus affect the cellular processes where this protein is involved [55,56]. A third hypothesis that still remains to be investigated is the possibility that aggregates could be protective at least during the early stages of the disease (this is a situation that has been observed for other diseases like Huntington [57]). From a therapeutic point of view, the identification of molecules capable of modulating TDP-43 expression and/or aggregate formation/clearance is a very promising target option. Several of these compounds and their effectiveness have already been discussed elsewhere [14,58]. Nevertheless, here we will discuss some of the most promising ones, their effects, their action mechanism and the models used for their identification.

Compounds that decrease TDP-43 levels and/or reduce TDP-43 aggregation 5.

The first two reported compounds able to reduce TDP-43 aggregation were Methylene Blue (MB) and Dimebon. In this work, Yamashita et al. found that these molecules had the capacity to reduce the aggregates generated by the overexpression of a TDP-43 mutant (DNLS/187-192) or a TDP-43 624

C-terminal fragment fused to GFP in SH-SY5Y cell line [59]. However, in vivo experiments developed later had controversial results. In fact, MB was found to reduce the neurotoxic effects caused by the overexpression of two TDP-43 mutants (A315T and G348C) in Caenorhabditis elegans and Danio rerio models [60]. However, it failed to improve cytosolic translocation, ubiquitination, inflammation and motor dysfunction in an ALS mouse model [61]. Nonetheless, from a mechanistic point of view it has just been shown that MB works by reducing the endoplasmic reticulum (ER) stress [62]. In keeping with this conclusion, also other compounds involved in the ER unfolded protein response in C. elegans and D. rerio (salubrinal, guanabenz and a structurally related compound, phenazine) could rescue the neurodegenerative phenotypes [62]. Considering this issue, therefore, the study of compounds that activate the ER unfolded protein response should become a worthwhile trend that needs further investigation. In parallel to this pathway, other compounds that activate the autophagy or ubiquitin proteasome system (UPS), like rapamycin and IU1, respectively, have also been reported to reduce TDP-43 aggregation. Rapamycin is known to act as selective inhibitor of mTOR, a serine/threonine protein kinase that in turn works as inhibitor of autophagy. The treatment of rapamycin reduced the formation of aggregates in two different cell lines overexpressing a 25-KD C-terminal TDP-43 fragment [63]. These results were confirmed in vivo by Wang et al. who showed that the administration of rapamycin to an FTLD-mouse model could reduce the presence of cytoplasmic TDP-43 positive inclusions followed by an improvement on motor function at late pathological stages [64]. Regarding IU1, it was found to be a selective inhibitor of the deubiquitylating enzyme USP14. The incubation of MEF cells with the inhibitor could enhance the degradation through proteasome of several proteins involved in neurodegeneration, including ubiquitinated TDP-43 [65]. More recent evidence regarding the involvement of the autophagy and UPS system in the degradation of TDP-43 has also pointed out that they act at different stages of TDP-43 clearance. In particular, clearance of aggregated TDP-43 requires the autophagy system while soluble TDP-43 is principally targeted by UPS [66]. This means that any therapy aimed at clearing excess TDP-43 will benefit from the use of inhibitors that target both these pathways. Other compounds that could be involved in the reduction of TDP-43 aggregation come from the study of patient iPS cells [67]. In a recent work, in fact, Burkhardt et al. reproduced the TDP-43 aggregation by the generation of motor neuronlike cells from ALS patient’s fibroblast [67]. After performing a high-content screening, they found that different compounds like Cardiac glycosides, Triptolide, CDK inhibitors and c-JNK inhibitors were able to reduce TDP-43 aggregation. The exact mechanisms by which these compounds reduce TDP-43 aggregation are unknown. However, regarding cardiac glycosides, it has been shown that they are involved

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in the inhibition and alteration of Na+/K+ ATPase pump and Ca++ influx, consequently affecting the Ras, IP3 and NFK-b signaling cascades. Interestingly, Triptolide is a diterpenoid isolated from Tripterygium wilfordii and has been used as anticancer, anti-inflammatory and immunosuppressive. In addition, it has recently been shown to have significant effects on CNS such as Parkinson and Alzheimer and spinal cord [68]. Finally, regarding the topic of reducing aggregation, it should be mentioned that CDK and JNK inhibitors have been reported as modulators of TDP-43 aggregation, implying that phosphorylation pathways could also play an active role in TDP-43 aggregation process [69]. As mentioned earlier, the identification of compounds that can decrease TDP-43 levels could also be useful to try and reduce TDP-43 aggregation. Regarding this, Trehalose, a natural disaccharide and enhancer of TOR-independent autophagy, was observed to decrease the expression of TDP-43 and its 25-kDa fragment in HEK293 cells [70]. Additionally, Trehalose has been used in NSC-34 cells or in vivo mutant SOD1 transgenic mice showing that it can reduce the aggregation condition and delay the neurodegeneration features, respectively [71,72]. On the other hand, Curcumin, a dietary pigment, has been demonstrated to downregulate TDP-43 levels in MCF-7 cells [73]. These results were supported by another report where it was found that Curcumin Dimethoxy (a Curcumin derivate) was also able to reduce both the protein levels and the aggregation triggered by a 25-kDa fragment from TDP-43. These results also correlated with an improvement in neuroprotection as evaluated by oxidative stress conditions [74]. Along the same research lines, the levels of TDP-43 were also shown to decrease in the presence of 4-aminoquinolines derivatives. These compounds were able to enhance caspase-3 and caspase-7 activity and to reduce the levels of TDP-43generating TDP-43 C-fragments. Subsequently, experiments developed by the same authors showed that 4-aminoquinolines bind to the C-terminal fragment of TDP-43 at the same place that UBQLN2 protein binds, a protein promoting the degradation of TDP-43 trough proteasome system [75,76]. Additionally, working with ALS iPSC-derived motor neurons Egawa et al. showed that mRNA TDP-43 levels were increased in iPSC from patients compared with controls [77]. However, the incubation of these cells with Anacardic acid (a histone acetyltransferase inhibitor) could recover normal mRNA TDP-43 levels, reduce the insoluble protein fraction and rescue the motor neuron death previously induced by Arsenite incubation. Although the specific mechanisms by mean Anacardic acid downregulate TDP-43 mRNA levels and aggregation are unknown, the authors speculate that NF-kB or redox signaling could be involved [77]. Finally, considering the possibility that prolonged SGs formation could be the cause of irreversible aggregation, some compounds have been identified to inhibit the participation of TDP-43 in SGs. For example, Parker et al. have found that bis(thiosemicarbazonato)--copper complexes ((Cu(II)(btsc)s)

can prevent TDP-43 SGs formation in SH-SY5Y cell line by inhibiting ERK pathway [78]. Furthermore, these data were supported by the identification of a related compound, PERK inhibitor (GSK2606414), which could also revert both SG formation and the neurological toxicity related to TDP-43 in S. cerevisiae and D. melanogaster models [43]. Finally, another recent work identified several compounds (mentioned as LDN-0000020, LDN-0002741, LDN-0002590 and LDN-0000827 LDN-0001080) that have the capacity to inhibit the TDP-43 aggregation induced by SGs formation and ameliorate the neurodegeneration symptoms of a C. elegans ALS model [79].

Compounds that increase TDP-43 levels and/or trigger TDP-43 aggregation

6.

To this day, not many compounds have been identified that are able to enhance TDP-43 expression levels within cells and that correlate with an increase in aggregate formation. For example, MG-132, (a proteasome inhibitor), Pepstatin A (a protease inhibitor) and 3-MA (an inhibitor of autophagosome and lysosome) were shown to increase the TDP-43 levels in cell culture [70]. Later, van Eersel et al. showed that the incubation of primary cortical neurons with MG-132 and lactacystin triggered TDP-43 aggregation making the cells more sensitive to TDP-43 downregulation [80]. Additionally, amino acid analogs have also been used to investigate the effects on TDP-43 levels. With respect to this, azetidine2-carboxylic acid and Canavanine were observed to increase the levels of TDP-43 in neurons and astrocytes; however, the consequences of these treatments were also the proteolysis and insolubilization of TDP-43 causing cell toxicity [81]. Interestingly, one study directed to test compounds that prevent cancer colon identified that 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) can increase TDP-43 at protein and mRNA levels [82]. However, the connection between the effects of this molecule and the increased levels of TDP-43 is currently still unknown. Finally, compounds that are able to increase TDP-43 aggregation without any apparent change in TDP-43 levels have also been identified. In this respect, Dormann et al. found that Staurosporine (a protein kinase inhibitor) can trigger the degradation of TDP-43 through caspases activation, producing TDP-43 C-terminal fragments and inducing aggregation [83]. However, these data could not be supported by Leggett et al. who did not observe TDP-43 inclusion in an in vitro neural tissue model in which they also used Staurosporine to induce apoptosis [84]. In particular, in the same work the authors showed that Tunicamycin, which affects the unfolding protein response by inhibiting N-glycosylation, was efficiently able to induce TDP-43 aggregation [84]. The importance of finding compounds that can increase aggregation will have an impact on therapeutic opportunities and our understanding of disease. First of all, if aggregates are protective at least during the early stages of disease it follows

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Normal nuclear and cytoplasmic functions

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Normal degradation pathways

TIA-1, G3BP, PABPC1 EIF4G

Autophagy, UPS

Dysregulated nuclear and cytoplasmic functions

Stress, oxidizing agents, ageing, mutations, excessive production, dysregulated nuclear/cytoplasmic shutting Natural tendency of FUS/ TDP-43 TLS to form aggregates and become recruited to stress granules

Gain- and loss- of function pathological effects

IMP1, HuD, SMN

Compounds that target TDP-43 levels and aggregation processes

Figure 3. Small-molecule compounds capable of affecting the expression or aggregation process of TDP-43. This figure shows a schematic representation (left) of the normal TDP-43 life cycle: shuttling from the nucleus and cytoplasm, its eventual degradation and its potential recruitment to stress granules (that in normal conditions is a reversible process). In the pathological process, right panels, aggregate formation disrupts this shuttling process and eventually leads to the sequestration of nuclear and cytoplasmic TDP-43, causing a disruption in all the RNA processes/pathways controlled by this protein. In order to prevent this condition, several compounds (shown in the Table 1) have been found to act in a way to reduce aggregate formation, TDP-43 production or both. UPS: Ubiquitin proteasome system.

that compounds/strategies which decrease TDP-43 solubility might be useful to slow down disease progression. Second, identifying those compounds that can induce aggregation will provide additional clues with regard to prevention and avoiding exposure to potential risk factors (whether environmental, pharmacological or genetic) associated with disease onset. 7.

Conclusion

In conclusion, when studying the role of TDP-43 in neurodegenerative diseases there are a few areas that might be 626

preferentially targeted to develop novel therapeutic or diagnostic approaches. In particular, it would be advantageous to try and modulate in a general manner the pathways through which this protein is regulated. For example, molecules capable of generally improving the efficiency of autophagic, proteome clearance systems and preferably both would certainly be beneficial in avoiding the building up of the various aggregates. With respect to this issue, however, it should be noted that most of the identified compounds found to be involved in reducing aggregation or capable of modifying TDP-43 levels are also participating in general cellular pathways. This makes it

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difficult to target specifically TDP-43 without affecting the normal cell metabolism. For this reason, additional molecular studies focused on the regulation of TDP-43 expression, on TDP-43 protein levels maintenance or on the interaction of normal TDP-43 with preformed TDP-43 aggregates will help to identify compounds that specifically target TDP-43 in these diseases. At present, a more specific approach would be to look for molecules capable of modulating TDP-43 production, either through modulating the expression of its gene or by affecting its autoregulatory mechanism at the mRNA level. In order to achieve these aims, however, priority should be given toward developing animal and cellular models that accurately mimic the pathology observed in patients. These models can then be used as ideal substrates to perform screening analysis in order to identify the genetic and environmental agents capable of modifying disease course and progression. 8.

Expert opinion

Research on TDP-43 has been often hampered by two factors: the first, is that like many RBPs the functions of TDP-43 are wide-ranging. This makes it difficult to target this protein just in a particular function and avoid unwanted consequences in other aspects of its biology. Second, we still lack TDP-43-based model systems that can recapitulate most if not all aspects of the human disease. Although considerable progress has been made in both these areas in recent years much basic research will have to be done in the future to better address these issues. For example, in vitro models of TDP-43 aggregation have been obtained by overexpressing this protein either in its wild-type/mutated form or by directly transfecting C-terminal fragments, as recently reviewed [14]. However, it is not known if these approaches actually mimic disease conditions. Furthermore, many of these models cannot be easily compared with each other as they use different cell lines and transfection conditions that can considerably affect results [18]. For this reason, it is important to set up models where TDP-43 aggregation can be triggered in more natural conditions. In this respect, the use of specific effectors based on the prion-like domain of TDP-43 [32] or molecules (Figure 3) that can trigger protein aggregation without affecting TDP-43 expression will represent an advantage over previous methods. Alternatively, even

with all its limitations the development of iPSC technology [77] has certainly offered an attractive approach to accurately model the pathology in vitro [77]. Also with regard to in vivo approaches, it is unfortunate that most models obtained so far generally fail to satisfactorily mimic human pathology and what is more important do not display TDP-43 aggregates. In the future, however, new in vivo models should take advantage of the recent advances in our knowledge of the genetics of FTD and ALS and take into account some of the new major players in determining disease origin and progression such as C9orf72 expansions, polyglutamine repeats and other genes that have been recently suggested to modify disease onset and progression. Taken together, the therapeutic potential of all these basic research approaches is extremely high. It should be considered, in fact, that therapeutical options at both the RNA level and the protein aggregation level represent a major research topic worldwide. The consequence of this situation is that once suitable TDP-43 targets are identified researchers will presumably find themselves with myriad options, already optimized in different systems, to try and regulate/modify its consequences/course of action. In conclusion, there is a very strong possibility that TDP-43 research will allow to develop novel and more specific therapeutic opportunities to slow down or even reverse disease course in a relatively brief amount of time. Finally, it should also be noted that all this newly gained knowledge may also be useful for research/therapy in other diseases that are characterized by TDP-43 aggregation, such as inclusion body myopathy with early onset Paget disease and frontotemporal dementia [11]. Even more importantly, it could also be important for research/therapy in other very common neurodegenerative diseases such as Alzheimer and Parkinson, where TDP-43 aggregations have been described and where they could play a secondary role in the pathology.

Declaration of interest The authors are supported by grants from AriSLA (TARMA to FB) and Thierry Latran Foundation (REHNPALS to EB). M Budini is a post-doctoral fellow, FE Baralle and E Buratti are senior scientists at ICGEB, Trieste, Italy. The authors state no conflict of interest and have received no payment in preparation of the manuscript.

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Affiliation Mauricio Budini, Francisco E Baralle & Emanuele Buratti† † Author for correspondence International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy Tel: +39 040 3757316; Fax: +39 040 655522; E-mail: [email protected]

Targeting TDP-43 in neurodegenerative diseases.

TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed RNA-binding protein belonging to the hnRNP family of nuclear proteins. In human diseas...
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