Desarrollo de sistemas de derribo sacudidores de copa específicos

Reports from nearly 10 years ago first showed aberrant exon and intron splicing o f astroglial

glutamate transporter EAAT2 as the cause o f abnormal RNA metabolism in sALS (Lin 1998). During

the last decade, the discovery o f alterations in TDP-43, FUS and C9orf72 firm ly established ALS as

a disease o f impaired RNA processing. Nevertheless, the role o f RNA metabolism in ALS is further

underscored by disease-causing mutations in ANG, SETX, TAF15 and ELP3, encoding fo r proteins

involved in RNA processing, as well as by the recognition o f intermediate length polyglutamine

expansions in ATXN2, a RNA binding protein, as a risk factor fo r ALS (see ALS genetics section).

TDP-43 and FUS, are both abnormally aggregated and mislocalised in ALS and FTLD. They display

structural similarities and participate in multiple levels o f mRNA processing, such as transcription,

splicing, transport and translation (Buratti 2008; Janknecht 2005) (Figure 10). W ith the exception 74

o f a mutation located in a RRM, all TDP-43 mutations identified are located in the C-terminal

domain o f the protein, which is im portant fo r interactions w ith members o f the hnRNP family of

splicing regulators (Buratti 2005; D'Ambrogio 2009; Kabashi 2008; Lagier-Tourenne 2009). Recent

technologies coupled w ith high-throughput sequencing have improved the ability in identifying

RNA targets. Thus, over 6000 TDP-43 RNA targets have been identified, including transcripts of

genes relevant to neurodegeneration, such as FUS, VCP, TARDBP, progranulin and choline

acetyltransferase, transcripts encoding proteins involved in RNA metabolism, synaptic function

and CNS development (Sephton 2011; Polymenidou 2011; Tollervey 2011), suggesting that

disrupting the function o f a single RNA-binding protein can affect at the same tim e many

alternative spliced transcripts (Licatalosi 2008). Moreover, multiple RNA targets o f TDP-43 are

deregulated in ALS, when TDP-43 is mutated or depleted, suggesting tha t a nuclear loss-of-

function is likely to contribute to the pathophysiology o f ALS (Xiao 2011).

TDP-43 was initially proposed to repress transcription by binding to the TAR DNA sequence o f

HIV-1 (Ou 1995) and to mouse SP-10 gene prom oter (Abhyankar 2007). FUS can influence the

general transcriptional machinery associating w ith RNA polymerase II and TFIID complex

(Bertolotti 1996; Yang 2000). In response to DNA damage it can also be recruited by noncoding

RNAs transcribed in the 5' regulatory region o f the gene encoding cyclin D l, leading to the

repression o f cyclin D l transcription (Wang 2008).

Beyond transcription, these tw o proteins have also a role in splicing regulation. They associate

w ith other splicing factors and their depletion or overexpression affects the splicing pattern o f

specific targets (Buratti 2005; Freibaum 2010). In particular, TDP-43 regulates tha alternative

splicing o f the cystic fibrosis transmembrane regulator (CFTR) (Buratti 2001), and promotes the

inclusion o f SMN exon 7 (Bose 2008). Abnormal expression o f peripherin splice variants (Xiao

2008), mRNA-editing errors o f the GluR2 AMPA receptor subunit (Kawhara 2004) and other

splicing alterations (Rabin 2010) have been reported in sALS patients.

Although TDP-43 and FUS are mainly nuclear proteins, they are also present in the cytosol, where

their binding onto RNA 3'UTRs has been linked w ith other aspects o f RNA metabolism, like

stabilization, transport, translation or degradation. Indeed, TDP-43 binding sites were identified

on the 3'UTR o f several genes involved in ALS pathogenesis including FUS, the glutamate

transporter EAAT2 and the light chain o f neurofilament (NFL) (Polymenidou 2011). In particular,

TDP-43 stabilises NFL mRNA, whose levels are decreased in degenerating spinal MNs in ALS

(Strong 2007). Moreover, in neurons TDP-43 and FUS are found in RNA-transporting granules

which translocate to dendritic spines following different neuronal stimuli (Fujii 2005; Wang 2008;

Belly 2005; Elvira 2006). TDP-43 depletion reduces dendritic branching and synaptic form ation in

drosophila neurons (Lu 2009; Feiguin 2009); while cultured neurons from FUS-/- mice show

abnormal spine morphology (Fujii 2005). Collectively, these works suggest that both proteins

could play a role in the modulation o f synaptic plasticity by influencing mRNA stabilization,

transport and local translation in neuronal cells, and as a consequence o f the mislocalisation and

aggregation could thereby contribute to MN injury. In agreement w ith this hypothesis, other

studies have shown that TDP-43 and FUS are components o f cytoplasmic RNA stress granules,

cytoplasmic foci containing mRNA and ribonucleoprotein (RNP) complexes in which the

translation is stalled under stress conditions (Colombrita 2009; Moisse 2009; Nishimoto 2010).

Hence it is plausible that the physiologic TDP-43- or FUS-containing stress granules may transform

into pathogenic inclusions during neurodegeneration. Therefore, sequestration o f specific cellular

RNAs w ithin cytoplasmic TDP-43 and FUS inclusions may deplete the cell o f essential RNA

components, contributing to pathogenesis, and possibly explaining the observation that TDP-43

and FUS binding to RNA is linked to their cytotoxicity independently o f their propensity to

aggregate (Elden 2010; Sun 2011; Voigt 2010). Indeed, an interesting possibility is that

aggregation might cause TDP-43 to bind RNA more avidly; alternatively, or in addition, RNA might

stabilize or divert TDP-43 to adopt specific misfolded forms that are highly toxic (King 2012).

The high expression level o f microRNAs (miRNAs) and the exclusive expression o f certain miRNAs

in the CNS highlights their biological importance at all stages o f neural development as well as in

differentiated neurons. In particular, miRNA activity is essential fo r long-term survival o f

postm itotic spinal MNs and fo r the bidirectional signaling between MNs and skeletal muscle fibers

76

at neuromuscular synapses. Several miRNAs potentially implicated in skeletal muscle and

neuromuscular junction regeneration were de-regulated in ALS brains and specific miRNAs

disease-related changes were detected at an earlier stage o f sALS (Williams 2009; Russell 2012;

De Felice 2012). TDP-43 and FUS may also be involved in these pathways, since they participate in

miRNA biogenesis through association w ith the Drosha complex (Buratti 2008). In addition, TDP-

Major steps in RNA processing and relative DNA/RNA targets. Modifed from Fiesel 2011.

Another mechanism o f RNA toxicity in ALS and FTLD is caused by pathological (G4C2)3o-i6oo

hexanucleotide repeat expansions in nontranslated regions o f the C90RF72 gene. As established

in other neurological diseases, especially myotonic dystrophy types 1 and 2 or Fragile X-associated

trem or ataxia syndrome (FXTAS), pathogenicity is initiated at the RNA level through tw o possible

pathways, (i) Consistent w ith a loss-of-function mechanism and supported by the 50% reduction

in C9orf72 transcript levels in patients w ith expansions, repeats prevent the expression of the

normal protein (haploinsufficiency o f C90RF72). (ii) In addition, expanded RNA forms pathogenic

nuclear RNA foci that trap specific RNA-binding proteins w ith affinity for the expanded RNA,

resulting in their depletion and loss-of-function (protein sequestration mechanism) (DeJesus-

Hernandez 2011). hnRNP A2/B1 is a potentially interesting RNA-binding protein in this context,

since it interacts w ith the C/G-rich repeats that form RNA foci in another neurodegenerative

condition (FXTAS) (Sofola 2007) and is also a direct interactor o f TDP-43 (Buratti 2005). Thus,

these observations could possibly explain the presence of TDP-43-based pathology in expanded

repeats carriers and add evidence to RNA misprocessing as a common pathogenic mechanism in

ALS and FTD.

The RNA-based mechanisms described are extensively coupled w ith protein misfolding and

aggregation, suggesting that these defects are inevitably interconnected in neurodegenerative

processes. Moreover, both contribute to neurogegeneration broadly, thus, regardless which one

is the primary or secondary event, they represent key steps in the pathological cascade, further

supporting the hypothesis (Dormann 2011) o f a "m ultiple hit" pathogenesis o f ALS.

1.5.7 Insufficientneurotrophic/growthfactors