arisla.3 – oligoals – new strategies to remove protein aggregates in als

ARISLA.3 – OLIGOALS – NEW STRATEGIES
TO REMOVE PROTEIN AGGREGATES IN ALS
Responsabile scientifico del progetto
MARIA TERESA CARRÌ
Università di Roma Tor Vergata – Fondazione Santa Lucia
Agenzia di Ricerca per la Sclerosi Laterale Amiotrofica
Finanziamento 2012
Sezione III: Attività per progetti
BACKGROUND, RATIONALE
Protein aggregates are frequently found in spinal motor neurons of all types
of ALS patients and may result toxic for motor neurons because they entrap
proteins critical for their viability, or because they cause a mechanical hindrance
and impairment of axonal transport, or because they affect specific organelles
such as mitochondria. The formation of such intracellular aggregates may
depend on the accumulation of misfolded proteins generated either as a direct
consequence of mutation, or as a consequence of oxidative stress.
In the case of SOD1, there is now general consensus that the toxic function
gained by the mutant proteins is to be related by their propensity to aggregate
and to mislocalize, thus jeopardizing neuronal viability. However, it is still
debated: 1) whether small oligomers or large aggregates represent the toxic
species; 2) the timing of appearance of these species in the course of the disease;
3) whether their localization is relevant for their toxic/protective function
[Cozzolino et al., 2012].
Similarly to SOD1, also TDP-43 and FUS/TLS are found aggregated in
cytoplasmic inclusions that are positive for ubiquitin in tissues from ALS patients
and models. ALS-related mutations seem to enhance the rate of aggregation
although aggregation may be secondary to other pathology relevant effects (e.g.
impairment of nuclear localization). As suggested by studies on SOD1, aggregates
may spread from cell to cell in a prion-like mechanism in which aggregates are
released by cells and uptaken by cells nearby, establishing transmission and selfpropagation of the pathology. Interestingly, prion-like domains have been identified
in FUS/TLS and TDP-43, but whether seeds of aggregation can propagate further
their aggregation is to be established. Thus several facets of the anomalous
behaviour of mutant SOD1 may be shared by mutant TDP-43 and FUS/TLS.
Previous studies have already demonstrated that alteration of the
GSH/GSSG ratio and downstream modulation of protein cysteines redox state
is a crucial event in aggregation of mutant SOD1 and oxidized wild-type SOD1,
and a similar mechanism may be acting for TDP-43 [Zhang et al., 2011]. Not
much is known about FUS/TLS, but an aberrant localization of this protein is
associated with misfolding and oxidation of SOD1 in ALS spinal cords, and with
ER stress and colocalization with protein disulfide-isomerase, two facts that
suggest a redox regulation also for the aggregation of this protein [Farg et al.,
2012]. The rationale of this project is that modifying the intracellular disulfide
status and the cell compartment-specific redox environment, may prevent
aggregation of ALS mutant proteins other than SOD1, thus facilitating their
degradation. If the toxic function of these proteins is linked to aggregation, this
may result in restoring a healthy phenotype.
OVERALL OBJECTIVES
It is still not clear whether large protein aggregates with toxic properties
play a central role in ALS initiation and progression, or if they represent a
defensive response aimed at protecting cells from more toxic oligomeric species.
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ARISLA.3 – Oligoals - New strategies to remove protein aggregates in ALS
Aggregation may also result in sequestration and mis-localization of crucial
proteins in a loss-of-function mechanism of motoneuronal toxicity. Furthermore,
the mechanisms of aggregation are understood only for mutant SOD1.
We have demonstrated that removal of mutant SOD1 oligomers in
motoneuronal cell cultures is possible through the modulation of the ratio
between reduced and oxidized glutathione [Cozzolino et al., 2009], through the
overexpression of glutaredoxins, that modulate the redox state of cysteine
residues in specific cell compartments [Ferri et al., 2010], or by treatment with
cisplatin, that is able to remove pre-formed mutant SOD1 oligomers in neuronal
cells [Banci et al., 2012].
In the present project, we propose to extend our previous results for SOD1
in order to better define the steps in the process of mutant TDP-43 and FUS/TLS
aggregation, mislocalization and toxicity, and to analyze by different approaches
whether removal of aggregates through the redox modulation of protein thiols
is feasible and beneficial in models for the expression of these proteins.
SPECIFIC OBJECTIVES
Specific aims of this project will be to assess:
1) Whether aggregated (cytosolic?) mutant TDP-43 and FUS/TLS are sufficient
to induce mitochondrial damage and neuronal death. Mitochondrial damage
seems to be a central feature in all forms of ALS; however, which may be the link
between these RNA-binding proteins and mitochondrial dysfunction is not known.
2) Whether aggregation of mutant TDP-43 and FUS/TLS is driven by a
cysteine-mediated or redox-dependent mechanism. TDP-43 has six cysteine
residues (including one in the C-terminal domain) and FUS/TLS has four (all in the
C-terminal domain); thus, in line of principle, a mechanism similar to that acting
for mutant and oxidized wild type SOD1 may be acting in the process of formation
of their oligomers and large aggregates.
3) Whether aggregation of these proteins may be prevented or reverted by
directly or indirectly targeting protein disulfides, and whether abolishing
aggregation is beneficial. These studies will be paralleled by an attempt to better
define the size and localization of aggregates and which are their molecular
partners inside the cell. This will help understanding.
4) Whether: (a) redistribution of the mutant proteins causes a depletion of the
same proteins from the nucleus and thus the loss of a nuclear function, or (b)
accumulation of a nuclear protein in other subcellular compartments, particularly
in an aggregated form, can endow these proteins with a new, toxic function.
DESCRIPTION
In this study, expression of wild type and mutant TDP-43 and FUS/TLS will
be obtained by transfection of mouse motoneuronal NSC34 cells or human
neuronal SH-SY5Y cells with plasmids coding for the mutant proteins or by
infection with adenoviral vectors also coding for those proteins.
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These cellular models will be used to study the formation of mutant protein
aggregates and their localization, and several markers of cell damage (i.e.
impairment of proteasomal function, induction of autophagy and mitochondrial
dysfunction) in basal conditions or in a context where either glutaredoxin level
is genetically altered or the ratio GSH/GSSG is chemically modulated. We will
also validate the observed effect of cisplatin to prevent SOD1 aggregate formation
in neuronal cultures overexpressing mutant TDP-43 or mutant FUS/TLS and
study whether aggregation impairs their interaction with crucial proteins.
More specifically, the study will be articulated in four workpackages.
In WP1 we will first complete the set up of necessary experimental models
in vitro constructing NSC34 cells that are stably transfected with wild-type
FUS/TLS or mutant forms of the protein that are representative of the mutations
so far identified in ALS patients. In particular, we plan to use some mutants in
the C-terminal predicted nuclear localization sequence, as well as mutants in the
Arg/Gly rich region, in the Gly rich region and in the Glu/Gly/Ser/Tyr rich region.
We will use these models to test whether, analogously to mutant SOD1:
a. Mutant TDP-43 and FUS/TLS form oligomers or large aggregates in these
cells and whether aggregation is dependent on the level of expression of the
mutant proteins. This may be relevant since the (artificial) level of expression
has been claimed to be significant for the toxic effect of mutant SOD1.
b. They induce a toxic phenotype such as mitochondrial damage, ER stress,
impairment of the UPR system, induction of autophagy and induction of
apoptosis. This may be relevant, since aggregates of these proteins that are
found in patients may simply represent a defence strategy and not a primary
determinant of neuronal impairment.
A second set of experiments (WP2) will be devoted to understand whether
aggregation of mutant TDP-43 and FUS/TLS is driven by a cysteine-mediated or
redox-dependent mechanism, as suggested by previous reports and by the
observation that both proteins do have Cys residues that may mediate aggregation
similarly to what described for SOD1. It is worth noting that both wild type
TDP-43 and wild type FUS/TLS aggregate, and this may depend by their state of
oxidation, as demonstrated for wild type SOD1 in a pro-oxidant cell environment.
Recent data suggest that oxidative stress is linked also to expression of mutant
TDP-43 and FUS/TLS. Mutant TDP-43 may be itself an inducer of oxidative stress,
as suggested by studies in neuronal cells in vitro in which this protein was shown
to down-regulate heme oxygenase-1 [Duan et al., 2010] and in yeast, in which
TDP-43 expression increased markers of oxidative stress [Braun et al., 2011].
Furthermore, oxidative stress promotes TDP-43 insolubilization and cytoplasmic
accumulation, probably through cross-linking via cysteine oxidation and
disulphide bond formation [Cohen et al., 2011]. Wild-type TDP-43 is capable to
assemble into stress granules (SGs) in response to oxidative stress, and this could
be associated with subsequent formation of TDP-43 ubiquinated protein
aggregates [Colombrita et al., 2009], although the relevance of mutant TDP-43
in the assembly and maintenance of SGs in response to oxidative stress has been
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questioned [McDonald et al., 2011]. FUS/TLS also localizes into SGs in response
to oxidative stress in HEK-293 cells and in zebrafish embryo spinal cords [Bosco
et al., 2010], but the relevance of this response in patients is not known.
Following this rationale, in WP3 we will then attempt to transfer our
previous successful approaches on SOD1 aggregates to TDP-43 and FUS/TLS,
i.e. study whether aggregation of these proteins may be prevented or reverted
by directly or indirectly targeting protein disulfides, and whether abolishing
aggregation is beneficial. We will use three different approaches:
1. Targeting protein disulfides through the overexpression of glutaredoxins
(Grx) – Grxs are thiol-disulfide oxidoreductases specifically involved in the
reduction of protein-glutathione (or protein-protein) mixed disulfides to protein
thiols in the presence of GSH, using reducing equivalents of NADPH.
Mammalian cells contain three Grxs that differ in size, subcellular localization,
and catalytic properties. In the brain, Grx1 is present with different abundance
in specific regions and a predominant neuronal localization. Grx1 is cytosolic
and also found in the IMS, while Grx2A is mitochondrial and Grx2B is
(peri)nuclear. Grx1 and Grx2A differ for their ability to respond to oxidative and
nitrosative stress, a fact that most likely reflects adaptations to their different
subcellular localizations and also to different regulatory functions in vivo.
Because of their different function, in principle all Grxs may prove useful in
preventing aggregation of mutant proteins in vivo, under conditions of oxidative
stress, or to favour their degradation.
2. Targeting directly free cysteine residues with cisplatin – Cisplatin (cisDiamminedichloroplatinum(II)) is a well-known chemotherapy drug that binds
to DNA and causes crosslinking in vivo, which ultimately triggers cell death.
However, cisplatin is also able to interact with cellular proteins forming stable
complexes with free cysteine residues, and it is able to prevent formation of
SOD1 aggregates. This effect is relatively specific, since another cysteine reactive,
anti-cancer molecule (Imexon) has no significant effect in the inhibition of SOD1
oligomerization, probably because of its larger molecular size [Banci et al., 2012].
Although a drug already approved for clinical use, cisplatin has a number of
adverse side effects when used acutely for cancer treatment and thus would
probably find no application in ALS treatment. Nonetheless, this part of the
study may provide the proof of principle that it is worthwhile searching for other
molecules sharing similar biochemical properties and effective at low doses in
chronic or prolonged treatment.
3. Modulating the overall thiol redox state by increasing the ratio GSH/GSSG
– Although there are several redox couples, which collectively establish the cellular
redox state, GSH/GSSG is the most abundant pair. GSH is oxidized to GSSG by
ROS and by glutathione peroxidases; glutathione reductase continually recycles
GSSG back to GSH keeping the GSH/GSSG ratio high; however, this ratio is
decreased during oxidative stress and this constitutes one of the mechanism by
which ROS alter the overall cellular redox state, through oxidation of free and
protein-bound accessible thiols. Maintenance of thiol homeostasis is important
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for normal cell activity, and physiological GSH/GSSG ratios affect the redox
state of mitochondrial membrane protein thiols including (and noticeably)
Complex I, whose inactivation is described in ALS together with an increased
ROS production. That the GSH/GSSG ratio is relevant to age-related neurodegeneration is suggested also by the fact that this ratio in ageing tissues shifts
progressively towards oxidation and the thiol redox state is a correlate of life
span in mice [Rebrin et al., 2007].
Through these different approaches, we will thus establish if there is a direct
relation between aggregation and toxicity and if such a strategy may be worth
further investigation in vivo.
Finally, in WP4 we will complete the characterization of TDP-43 and
FUS/TLS aggregates by studying their intracellular localization and if the
strategies mentioned above have any effect in preventing mislocalization of the
mutant proteins. We and others have previously demonstrated that mutant SOD1
tends to associate with mitochondria. However, not much is known about a
possible analogous behaviour of mutant FUS/TLS and TDP-43. Using subcellular
fractionation and biochemical methods, we will try to assess if this is the case,
i.e. if part of the mutant proteins accumulate in mitochondria.
We will then determine whether aggregates bind/co-localize (and sequester?)
other proteins which are essential for neuronal survival.
Indeed, the role of the wild type proteins is not clear: functions in pre-mRNA
splicing and transport of the mature mRNAs have been proposed. The
redistribution of FUS/TLS from the nucleus to the cytoplasm is related to the
onset and progression of the disease and mutations that affect aminoacid
residues responsible for the binding to Transportin receptors (thus affecting the
nuclear import of the protein) result in a more severe disease progression
[Dormann et al., 2010]. Thus, understanding which are FUS/TLS protein
interactors in subcellular compartments may help to shed light on its function
and on alterations in ALS. In this part of the project we will particularly focus on
RNA binding proteins such as SMN (Survival of Motor Neurons), that is a wellknow determinant of neuronal damage. In a set of preliminary experiments, we
have verified that FUS/TLS binds to the spliceosomal snRNAs in NSC34 cells.
Further, we find that SMN and FUS/TLS interact and also co-localize in these
cells (unpublished data). It therefore appears quite likely that the two proteins
in fact converge onto the same molecular pathway.
It is important to stress that any result (even negative) from this study would
provide valuable information. For instance, if we can prove that aggregates are
not dependent on cysteine oxidation or that they are not toxic by themselves, than
it will become clear that other functions/mechanisms of toxicity are to be sought
and the study of FUS/TLS protein interactors will partially address this issue.
PRELIMINARY DATA
We have previously studied the contribution of specific cysteine residues
in the mechanism of aggregation of mutant SOD1 and demonstrated that
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aggregates are the consequence of covalent disulfide cross-linking and noncovalent interactions. In particular, we demonstrated that the redox environment
(e.g. the main redox couple GSH/GSSG) influences the oligomerization/
aggregation pathway of mutant SOD1 and that Cys111 as a key mediator of
this process [Cozzolino et al., 2009]. Thus, we have exploited the ability of
glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols in different
cell compartments as a tool for restoring a correct redox environment and
preventing the aggregation of mutant SOD1. We have show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but
does not inhibit mitochondrial damage and apoptosis in neuronal cells.
Conversely, the overexpression of Grx2A increases the solubility of mutantSOD1 in mitochondria, preserves mitochondrial function and strongly protects
neuronal cells from apoptosis [Ferri et al., 2010].
A different approach was to target the critical Cys residues directly to prevent
aggregation. We have been able to demonstrate that cisplatin binds to Cys111
and inhibits aggregation of demetalated oxidized SOD1, and it is further able to
dissolve and monomerize oxidized SOD1 oligomers in vitro and pre-existing
mutant SOD1 aggregates in neuronal cell cultures [Banci et al., 2012]. At higher
doses, cisplatin also affects the overall amount of all SOD1 protein levels and
this effect is prevented by the treatment with the cell-permeable proteasome
inhibitor MG132, suggesting that the fraction of SOD1 that is solubilized by
cisplatin is degraded by the ubiquitin-proteasome system.
We have confirmed previous observations that FUS/TLS can aggregate [Sun
et al., 2011], and ALS-related mutations seem to enhance the rate of aggregation
as observed in transfected NSC34 cells.
Finally, we have obtained preliminary indications that FUS/TLS binds to the
spliceosomal snRNAs and that aggregated mutant FUS/TLS colocalizes with
SMN in NSC34 cells. This suggests that the two proteins in fact localize in the
same subdomain, converge onto the same molecular pathway, and that
spliceosomal snRNPs might be sequestered by cytosolic aggregates of the mutant
proteins, thus affecting the nuclear function (i.e. splicing) of this complexes.
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