Evaluación de los sistemas de recolección para olivar tradicional. 112

These lesions, in the form o f extra- and intra-cellular inclusions containing ubiquitin, are a

hallmark o f both sALS and fALS (see Histopathological Features section), suggesting a shared

pathogenic link. However, the role o f protein aggregation in ALS pathogenesis is unclear: they

might cause cellular toxicity, be only by-products o f the neurodegeneration process or even a mean o f cell protection by sequestering toxic proteins (Cleveland 1999; Tran 1999).

-»• SODl aggregates

The hypothesis o f toxicity from protein aggregation in ALS initially arose from the discovery o f

intracellular inclusions in MNs and astrocytes in mouse models and patients w ith mSODl

(Watanabe 2001; Kato 1999). Insoluble SODl-containing aggregates appear before or coincident

w ith onset o f disease symptoms and increase in abundance w ith disease progression, suggesting

that SODl aggregation may be an early event in disease pathogenesis (Johnston 2000; Wang

2002). Aggregates can be composed o f different proteins such as SODl, ubiquitin, dorfin,

neurofilaments, peripherin; additionally, chaperones and proteasome subunits may also be

sequestered (Watanabe 2001; Strong 2005; Basso 2009).

The oligomerisation hypothesis has been proposed as an alternative mechanism to the oxidative

damage hypothesis (see Oxidative stress section 1.5.1), to explain the toxicity acquired by mSODl

in ALS. M utant proteins are likely to cause disease through a toxic gain-of-function, possibly an

increase in the propensity o f the protein to misfold and aggregate because of conformational

instability. SODl conformational stability seems to be tightly linked to Cu2+ and Zn2+ ion

coordination, which drives the form ation o f an intramolecular disulfide bond; metallation and

disulfide bond promote the homodimerisation o f SODl subunits (Arnesano 2004). Indeed, in vitro

studies have shown that in contrast to stable, dimeric wild-type SODl, mutant proteins are more

prone to oligomerize over tim e and to form SODl-rich fibrils (similar to some forms o f p-amyloid

protein) (Ray 2004; Matsumoto 2006), suggesting that the conversion o f soluble SODl into

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amyloid fibrils may play an im portant role in the aetiology o f fALS. Generally, the mutations can

determine both local perturbations o f the secondary, tertiary or quaternary structure, diminishing

the metal coordination or altering the net charge (Shaw 2007). In addition, SODl aggregation

might also be promoted by ROS: the active site Cu2+ ion can react w ith hydrogen peroxide, leading

to enzyme inactivation (Valentine 2002) and therefore promoting the population o f metal free

• Aberrant oxidative •Aggregates • Aggregates •Aggregates chemistry from wrong -• Loss of protein • Reduced chaperone •Reduced

substrate function through activity proteasome activity co-aggregation

Figure 8 Models for SODl-mediated toxicity.

SODl-mediated toxicity linked to altered conformation and aggregation of mSODl subunits. Adapted from Cleveland 2001.

Various hypotheses have been proposed regarding the cytotoxicity o f SODl aggregates (Figure 8),

including the disruption o f axonal transport (Stokin 2005), the aberrant binding o f apoptosis

regulators (Pasinelli 2004; Tomik 2005) and chaperones proteins, such as heat shock proteins

(HSPs), tha t are crucial fo r the correct folding o f many other im portant cellular proteins (Shinder

2001; Okado-Matsumoto 2001). Indeed, the overall chaperone activity is modestly diminished in

SpC o f G93A-SOD1 mice, suggesting that insufficient levels o f chaperones can be partially

responsible fo r the accumulation o f insoluble aggregates (Bruening 1999). In addition, treatments

w ith arimoclomol, which increases HSPs expression, extend lifespan o f ALS transgenic mice

(Kieran 2004). Nevertheless, recent studies proposed that aggregation occurs despite HSPs

expression (Krishnan 2008) which anyway contribute in protecting MNs (Patel 2005).

Aggregated SODl has also been linked to perturbations in mitochondrial function and calcium

homeostasis (Hervias 2006; Sumi 2006), and progressive failure o f the proteasome system to

eliminate ubiquitinated aggregates (Cleveland 2001; Cheroni 2005 and 2009; Kabashi 2008).

Consistent w ith the latter hypotesis, aggregates in mSODl mice are intensely immunoreactive to

antibodies fo r ubiquitin (Stieber 2000; Watanabe 2001), whose role is to label and target

misfolded and inactive proteins to the proteasome machinery. Thus, proteasome activity could be

inhibited by SODl and this pathologic starting event could trigger the accumulation o f aberrantly

folded forms o f SODl and other proteins (Cleveland 2001). Finally, unfolded protein response,

which follows a severe ER-stress, may contribute to protein misfolding and aggregation. Indeed,

tw o ER chaperones recognized as markers o f ER stress, GRP78 and protein disulfide isomerase

(PDI), are up-regulated in G93A-SOD1 mice MNs, in sALS patients and in fALS cases linked to SODl

and TDP-43 (Tobisawa 2003; Zetterstrom 2011; Atkin 2006 and 2008; Honjo 2011). Misfolded

SODl can specifically aggregate in the ER-Golgi compartments, thus impairing also the Golgi

apparatus that has been detected fragmented in MNs o f sALS and fALS patients (Okamoto 2010).

-» TDP-43 inclusions

TDP-43 misaccumulation was observed in all variants o f ALS, w ith rare reports in ALS-mSODl

cases (Mackenzie 2007; Robertson 2007; Tan 2007; Okamoto 2011; Sumi 2009). Besides ALS and

FTLD-U, TDP-43 cytoplasmic accumulation and loss o f nuclear localisation in affected neurons are

now reported in a number o f neurodegenerative conditions, including a fraction o f AD, PD and HD

patients (reviewed in Lagier-Tourenne 2010). In most ALS cases, the cytoplasmic accumulation of

TDP-43 was seen mainly in neurons and glia o f the primary motor cortex, brainstem m otor nuclei

and SpC (Mackenzie 2007). ALS patients MNs, showing a normal TDP-43 staining, have a normal

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Golgi apparatus profile, whereas Golgi fragmentation is detectable in neurons w ith cytoplasmatic

TDP-43 inclusions, thus suggesting that aggregation o f TDP-43 in the cytoplasm can be linked to

impairm ent in the secretory pathway mediated by the Golgi (Okamoto 2010).

The C-terminal domain of this protein is crucial fo r spontaneous aggregation and several

mutations, w ithin this domain, increase the number o f TDP-43 aggregates, thus promoting

toxicity (Johnson 2009). The observation that ALS-linked mutations increase the stability o f TDP-

43 protein (Guo 2011; Johnson 2009; Ling 2010) supports the view tha t protein aggregation may

initiate the pathogenic cascade in ALS.

Moreover, pathological TDP-43 is hyperphosphorylated and cleaved to generate C-terminal

fragments o f approximately 25kDa (Neumann 2006; Arai 2006) (Figure 9). The phosphorylation of

full length TDP-43 results in a product o f 45kDa that was observed in FTLD-U and ALS brains.

Although the pathological significance o f TDP-43 phosphorylation is not yet known, it has been

proposed that this modification might promote TDP-43 aggregation. In vitro studies have

demonstrated that phosphorylation of recombinant TDP-43 leads to an increase in the level of

TDP-43 oligomers (Hasegawa 2008). Additionally, abundant filaments, immunopositive for

S409/S410 phosphorylation-specific TDP-43 antibody, have been observed by electron

microscopy (Hasegawa 2008). Although phosphorylation may enhance TDP-43 oligomerization, it

may not actually be necessary for aggregation to occur: recent findings have suggested that

phosphorylation occurs late in the conversion o f soluble to insoluble TDP-43, thus indicating that

it is not an essential prerequisite for TDP-43 aggregation (Dormann 2009) and that is not required

fo r C-terminal cleavage (Zhang 2009).

TDP-43 truncation is supposed to be region specific; in fact an enrichment in C-terminal fragments

was observed in TDP-43 inclusions in the hippocampus and cortex o f FTLD-U and ALS cases, while

lesions in MNs consist primarily o f full-length TDP-43 (Igaz 2008). Several in vitro studies have

shown that C-terminal fragments of TDP-43 are more likely than full-length TDP-43 to form

insoluble cytoplasmic aggregates that become phosphorylated, ubiquitinated and toxic to cells

(Igaz 2009; Nonaka 2009; Arai 2010). It has been also demonstrated that the expression o f

C-term inal TDP-43 fragm ents in cultured cells leads to an aberrant sequestration o f the endogenous transcription, splicing, and mRNA stabilization. (B) In pathological conditions, TDP-43 is cleared from the nuclear compartm ent and accumulates in the cytoplasm. Pathological TDP-43 has been demonstrated to be hyperphosphorylated and/or ubiquitinated and also C-terminal cleaved. TDP-43 is also more prone to aggregation. Adapted from Chen-Plotkin 2010.

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The expression o f m utant TDP-43 in cultured cells results in increased formation o f C-terminal

fragments, w ith even more aggregation and toxic effects than the wild-type protein (Kabashi

2010; Nonaka 2009; Arai 2010; Barmada 2010). In vivo studies [conducted in fru it fly (Drosophila

melanogaster), mice and rats] have shown that overexpression o f wild-type human TDP-43 can

cause the abnormal formation o f TDP-43 cytoplasmic inclusions, w ith the associated

neurodegeneration and m otor dysfunction (Li 2010; Wils 2010; Tatom 2009). Whereas results

from transgenic animal models overexpressing mutant forms o f human TDP-43 are rather

inconsistent and difficult to interpret, indicating that the pathogenic mechanism in TDP-43

proteinopathy is still unresolved.

- > FUS/TLS inclusions

As fo r TDP-43 proteinopathies, also FUS cytoplasmic inclusions have been detected in a growing

number o f neurodegenerative disorders (Doi 2010; Woulfe 2010). Analyses o f the brains and SpCs

o f ALS patients w ith FUS mutations have revealed normal staining o f FUS in the nuclei o f many

neurons and glial cells, while it is partially cleared from those nuclei in neurons tha t contain

cytoplasmic aggregation (Neumann 2009; Tateishi 2009; Kwiatkowski 2009; Vance 2009). In vitro

expression o f C-terminal FUS mutations has revealed variable increases in cytoplasmic

concentrations o f FUS that are compatible w ith the disruption o f its nuclear localisation signal and

impaired transportin-mediated nuclear im port (Dormann 2010), suggesting tha t an altered

nuclear im port o f FUS can represent a crucial event in disease pathogenesis.

In contrast with TDP-43 proteinopathies, FUS lacks most o f the post-translational modifications

(such as hyperphosphorylation, ubiquitination and cleavage) tha t have been reported fo r TDP-43.

Thus, if common mechanisms underlie toxicity mediated by TDP-43 and FUS, this divergence in

the biochemical properties o f the proteins may suggest that these alterations are not key

contributors to pathogenesis (Da Cruz 2011).

-> p62 inclusions

The presence o f p62 in ubiquitin-positive inclusions in many different neurodegenerative

diseases, including ALS-FTD, further provides evidence for a potential common mechanism in

these disorders, namely, proteins misfolding and impaired proteasomal digestion and autophagy.

In addition, mutations in VCP, ubiquilin 2, and p62 (see ALS genetics section), further contribute

to alter or impair proteasomal digestion and different steps o f the autophagic process, leading to

accelerated neurotoxicity, as the catabolic mechanisms o f proteasomal degradation and

lysosomal-mediated autophagy are hoverwhelmed, and undigested misfolded proteins wreak

havoc in the cell (Appel 2012).

Nevertheless, it is not yet clear which one among these events is the cause and which one is the

consequence o f ALS: it can be proposed that a vicious circle where aggregation and impairment of

protein folding and degradation are both present and involved in the pathology.