Advances in Genetics

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09 10 11 12 10 9 8 7 6 5 4 3 2 1
 Contents

Contributors vii

1 The Molecular Links Between TDP-43 Dysfunction

and Neurodegeneration 1
Emanuele Buratti and Francisco E. Baralle
I. Introduction 2
II. Main Text 5
III. Early Lessons From Animal Models 17
IV. Gain- Versus Loss-of-Function Scenarios 21
V. Conclusions 25
References 27

2 Resources and Strategies to Integrate the Study of

Ethical, Legal, and Social Implications of Genetics
into the Undergraduate Curriculum 35
Jinnie M. Garrett and Kathleen L. Triman
I. Introduction 36
II. Resources 37
III. Strategies 52
IV. Concluding Remarks 55
References 55

3 Synthetic Genetic Interactions: Allele Dependence,

Uses, and Conservation 61
Joseph V. Gray and Sue A. Krause
I. Genetic Enhancement 62
II. Expected Underlying Functional Relationships 65
III. Systematic Discovery of Genetic Enhancements 69
IV. Network Characteristics 71
V. Extracting Functional Relationships from
Global Networks 76

v
vi Contents

VI. Conservation 78
VII. Conclusions 80
References 81

Index 85
 Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Francisco E. Baralle (1) International Centre for Genetic Engineering and

Biotechnology (ICGEB), Trieste, Italy
Emanuele Buratti (1) International Centre for Genetic Engineering and
Biotechnology (ICGEB), Trieste, Italy
Jinnie M. Garrett (35) Department of Biology, Hamilton College, Clinton,
New York, NY, USA
Joseph V. Gray (61) Molecular Genetics and Integrative & Systems Biology,
Faculty of Biomedical and Life Sciences, University of Glasgow,
Glasgow, United Kingdom
Sue A. Krause (61) Molecular Genetics and Integrative & Systems Biology,
Faculty of Biomedical and Life Sciences, University of Glasgow,
Glasgow, United Kingdom
Kathleen L. Triman (35) Department of Biology, Franklin and Marshall
College, Lancaster, PA, USA

vii
 1
The Molecular Links Between
TDP-43 Dysfunction and
Neurodegeneration
Emanuele Buratti and Francisco E. Baralle
International Centre for Genetic Engineering and Biotechnology (ICGEB),
Trieste, Italy

I. Introduction
II. Main Text
A. Aggregation/nuclear localization
B. Degradation
C. Phosphorylation
D. Ubiquitination
E. Mutations
III. Early Lessons From Animal Models
IV. Gain- Versus Loss-of-Function Scenarios
V. Conclusions
Acknowledgments
References

ABSTRACT
TDP-43 nuclear protein is involved in several major neurodegenerative diseases
that include frontotemporal lobar degeneration with ubiquitin (FTLD-U) bodies
and amyotrophic lateral sclerosis (ALS). As a consequence, the role played by
this protein in both normal and diseased cellular metabolism has come under
very close scrutiny. In the neuronal tissues of affected individuals TDP-43 under-
goes aberrant localization to the cytoplasm to form insoluble aggregates. Further-
more, it is subject to degradation, ubiquitination, and phosphorylation.
Understanding the pathways that lead to these changes will be crucial to define

Advances in Genetics, Vol. 66 0065-2660/09 $35.00

Copyright 2009, Elsevier Inc. All rights reserved. DOI: 10.1016/S0065-2660(09)66001-6
2 Buratti and Baralle

the functional role played by this protein in disease. Several recent biochemical
and molecular studies have provided new information regarding the potential
physiological consequences of these modifications. Moreover, the discovery of
TDP-43 mutations associated with disease in a limited number of cases and the
data from existing animal models have strengthened the proposed links between
this protein and disease. In this review we will discuss the available data
regarding the biochemical and functional changes that transform the wild-type
endogenous TDP-43 in its pathological form. Furthermore, we will concentrate
on examining the potential pathological mechanisms mediated by TDP-43
in different gain- versus loss-of-function scenarios. In the near future, this
knowledge will hopefully increase our knowledge on disease progression and
development. Moreover, it will allow the design of innovative therapeutic
strategies for these pathologies based on the specific molecular defects causing
the disease. ß 2009, Elsevier Inc.

I. INTRODUCTION

Nuclear factor TDP-43 is a multifunctional RNA binding protein that has been
described to play a role in a great variety of cellular processes such as transcrip-
tion, pre-mRNA splicing, stability, transport, and translation. In a recent review
we have tried to be as comprehensive as possible (Buratti and Baralle, 2008) but
the fast pace of research makes it desirable to look at the field again especially
with regards to its involvement in neurodegenerative diseases. From a human
disease point of view, TDP-43 was first described to participate in the develop-
ment of monosymptomatic and full forms of cystic fibrosis through its inhibitory
effects on the recognition of CFTR exon 9 (Buratti et al., 2001). More recently,
its major claim to fame comes from the observation that TDP-43 is the main
protein component of the intracellular inclusions found in the neuronal tissues of
patients affected by a series of neurodegenerative diseases which include fronto-
temporal lobar degeneration ubiquitin (FTLD-U), amyotrophic lateral sclerosis
(ALS) (Arai et al., 2006; Geser et al., 2009b; Neumann et al., 2006), and
Alzheimer disease (Amador-Ortiz et al., 2007; Bigio, 2008; Rohn, 2008). The
number of neurodegenerative diseases involved, the distribution/morphology of
inclusions in different brain regions, and the associated clinical characteristics
have already been reviewed in several recent publications (Bugiani, 2007; Cook
et al., 2008; Dickson, 2008; Dickson et al., 2007; Elman et al., 2008; Forman et al.,
2007; Kwong et al., 2008; Liscic et al., 2008; Mackenzie and Rademakers, 2007,
2008; Neumann et al., 2007b; Tolnay and Frank, 2007; Wang et al., 2008b).
Indeed, the impact of TDP-43 in the neurodegeneration field has been so
 1. The Molecular Links Between TDP-43 Dysfunction and Neurodegeneration 3

pervasive that disease nomenclature consensus are currently being modified to

better reflect the new clinical and pathological findings originating from recent
research (Geser et al., 2009a; Mackenzie et al., 2009).
In this review we will rather concentrate on available data regarding
the biochemical and functional changes of TDP-43 that lead to the human
pathology. In particular we will focus on the potential pathological mechanisms
mediated by TDP-43 mutations (both natural and artificial) and the results
obtained so far in simple or existing animal models of disease.
From a classification point of view, TDP-43 is a member of the hnRNP
protein family (Krecic and Swanson, 1999), a family that includes many of the
most common and powerful splicing modulators known so far, such as hRNP
A1/A2, PTB (hnRNP I), and hnRNP H (Martinez-Contreras et al., 2007).
A characteristic of many of these factors, however, is the role they play in
numerous diverse functions depending on their relative abundance, cellular
localization, and the interactions with themselves or other components of the
cellular machinery (Carpenter et al., 2006; Dreyfuss et al., 2002; Glisovic et al.,
2008). TDP-43 is no exception and Fig. 1.1 shows a schematic diagram of the
protein and the best characterized functions reported previously. These include
the ability of TDP-43 to regulate splicing of several exons such as CFTR exon 9
(Buratti et al., 2001), ApoAII exon 3 (Arrisi-Mercado et al., 2004; Mercado et al.,
2005), and SMN exon 7 (Bose et al., 2008) or its role in regulating transcription
of the HIV-1 genome (Ou et al., 1995) and of the SP-10 mouse promoter
(Abhyankar et al., 2007; Acharya et al., 2006).
In addition to its role in transcription/splicing TDP-43 may also possess
other functions that are still at a very early stage of characterization. These
include some very diverse properties such as acting as a neuronal response
activity factor and an in vitro mRNA translational repressor (Wang et al.,
2008a), or an mRNA stability factor for neurofilaments (Strong et al., 2007).
Finally, it is also important to mention the possibility of some additional
properties, such as microRNA processing, that up to this moment are simply
based on its association with both the human and mouse microprocessor
complexes (Fukuda et al., 2007; Gregory et al., 2004). The list of potential
functions is not likely to end here, as according to protein–protein association
studies that use high-throughput methodologies several other candidates have
been put forward (Wang et al., 2008b). In particular, two proteomics studies
(Lehner and Sanderson, 2004; Stelzl et al., 2005) involving yeast two hybrid
systems found XRN2 and PM/Scl100 (mRNA decay), ZHX1 (transcriptional
repressor), SETDB1 (chromatin remodeling regulator), and NSFL1C and ARF6
(both involved in membrane trafficking) as potential TDP-43 binding partners.
In this respect, it is important to note that TDP-43 has been described to be part
of RNA granules responsible for trafficking, sequestering, and degrading RNA
species (Elvira et al., 2006) and has been observed to colocalize strongly with
4 Buratti and Baralle

TDP-43
Localization RNA and protein binding

RRM-1 RRM-2
N-term. (103–183) (190–265) C-term.
RNA binding
1 321 366 414 1 321 366 414
N C N C
147FF 149
D169
82 98 187 192 239 250
KRK....VKR PL....RK IAQ....LII

NES Nuclear UBQLN hnRNP A/B

NLS Intranuclear localization
localization
Properties

mRNA mRNA
Transcription pre-mRNA splicing Other processes
stability translation

Cell Nuclear
HIV-1 TAR microRNA
CFTR Apo AII SMN hNFL processing shape
DNA element death
exon 9 exon 3 exon 7 mRNA maintenance

Neuronal CDK6
Mouse SP-10 activity expression
promoter response levels
factor

Figure 1.1. DP-43 protein. Schematic diagram of TDP-43 sequences important for its nuclear/
cytoplasmic localization (left) and its RNA and protein binding properties (right).
The lower part of the diagram reports some of the described and proposed functions
of TDP-43 in cellular metabolism.

Staufen (a protein involved in mRNA transport in dendrites), moderately with

TIA-1 (a splicing factor) and weakly with XRN1, an exoribonuclease involved
in mRNA decay (Moisse et al., 2009). TDP-43 has also been shown to colocalize
with wild-type and mutant forms of valosin-containing protein (VCP) in culture
cells (Gitcho et al., 2009) and with UBAP1 in the neuronal cytoplasmic inclu-
sions of a familial FTD case (Rollinson et al., 2009). Partial colocalization has
also been observed with GW182 and eIF4E (both markers of P-bodies) (Wang
et al., 2008a). Finally, in sporadic ALS patient samples TDP-43 has also been
found to colocalize with the phosphorylated Smad2/3 factors (pSmad2/3)
(Nakamura et al., 2008), which are central mediators of the TGF-beta signal
transduction pathway, essential for maintaining the survival of neurons. In this
respect, it should also be noted that TDP-43 has been originally suggested to play
a central role in organizing the higher order structures of eukaryotic nuclear
bodies (Wang et al., 2002). In conclusion, although for a complete list of TDP-43
properties and functions we will certainly have to wait for a long time these
 1. The Molecular Links Between TDP-43 Dysfunction and Neurodegeneration 5

observations are already sufficient to support the view that TDP-43 is involved in
more than one cellular process, possibly through interactions of different parts of
its molecule.
Interestingly, TDP-43 is not the only DNA/RNA binding protein
recently found to be involved in ALS. In fact, the recent reports regarding the
involvement of mutations in the FUS/TLS protein in a series of patients affected
by familial forms of ALS suggests that alterations in DNA/RNA processing
mechanisms may be a fertile area for research to further our understanding
of neurodegenerative diseases (Kwiatkowski et al., 2009; Vance et al., 2009).
Hopefully, this will place us in the best prospective position to design viable
therapeutic strategies.
Before tackling this issue, however, it is best to summarize what happens
to the predominantly nuclear, wild-type TDP-43 in a pathological setting.

II. MAIN TEXT

As clearly highlighted by the two pioneering studies in this field, the pathological
TDP-43 protein analyzed in patients displays some very clear modifications with
respect to the wild-type protein (Arai et al., 2006; Neumann et al., 2006). These
include an increased cytoplasmic localization under the form of insoluble aggre-
gates, ubiquitination, phosphorylation, and degradation to yield C-terminal
fragments (schematically reported in Fig. 1.2). In the past 2 years, several
advances have been made with regards to explaining these aspects. In the
following paragraphs, we will briefly review what is currently known about
these aberrant events.

A. Aggregation/nuclear localization
With some exceptions, TDP-43 aberrant aggregation and cellular localization
are usually linked with each other. In normal conditions, independent studies
agree with the conclusion that TDP-43 is a predominantly a nuclear protein with
a very small amount being present in the cytoplasm (Ayala et al., 2008b; Winton
et al., 2008a). In rat hippocampal neurons the percentage of cytoplasmic TDP-43
has been recently estimated as 15% (Wang et al., 2008a). The small amount
present in the cytoplasm, however, should not be considered simply a cellular
background. In fact, using heterokaryon assays it has been noted that TDP-43 is
continuously shuttling between the nucleus and the cytoplasm unlike exclusively
nuclear factors (Ayala et al., 2008b). This finding suggests that TDP-43 cyto-
plasmic levels may play a functional role also in normal conditions. On the other
hand, a typical hallmark of diseased cells is represented by the almost complete
absence of nuclear TDP-43 and staining for this protein can be observed
6 Buratti and Baralle

TDP-43
cytoplasmic
Dendrites aggregates:
-ubiquitinated
-phosphorylated
-cleaved to generate
C-terminal fragments (CTFs)

Neuron
Axon

Aberrant cleavage sites: Aberrant phosphorylation sites

85 88
DETD 403/4
cleavage sites

SS
Caspase-3

P P

N C 43 kD N C
P P P
DEND DVMD 379 409/10
10 13 216 219
S SS
Caspase-3

N C 42 kD
fragments

N C 35 kD
Ubiquitination (sites unknown):
N C 25 kD
95 136
79 97 114 137 145 176 224 251 263
K KK K KK K K K K K

N C
fragment

N C 22 kD
FTLD-U

82 102 121 140 160181 192 408

84 K K K K K K K
P P
R KK
208 409/10
SS

Figure 1.2. Pathological TDP-43 characteristics. Characteristic features of pathological TDP-43 in

neurodegeneration. The normally nuclear TDP-43 is transported to the cytoplasm of
the neuron (upper diagram), it’s cleaved by caspases to generate
35 and
25 kDa
fragments (lower left) or by unknown proteases in the
22 kDa fragment. The frag-
ments and the full length protein become aberrantly phosphorylated at serine residues
379, 403/404, and 409/410 (right diagram). In addition to all these modifications, it is
ubiquitinated in still unknown residues (all lysine residues, which represent the normal
substrate for ubiquitination, are shown in the lower right picture).

predominantly in the cytoplasm, where it is often found in insoluble aggregate

form. In this respect, it is worthy to point out that TDP-43 aggregations in brain
tissues has not been detected in response to metabolic insults such as anoxia or
ischemia (Lee et al., 2008). One important general observation concerning
aggregation is that TDP-43 tends to be a very “sticky” protein even in normal
or slightly altered conditions. In fact, some reports describe aggregation of wild-
type TDP-43 in common laboratory culture cell lines or yeast cells (Johnson
et al., 2008; Winton et al., 2008a). Moreover, a recent report has also reported
the observation that isolated RRM-2 domains can assemble in higher order
particularly thermal-stable assemblies in physiological (in vitro) conditions
(Kuo et al., 2009). As formation of these structures does not occur with the full
protein itself this observation suggests that the degradation processes produces
TDP-43 fragments with increased tendency to aggregation.
 1. The Molecular Links Between TDP-43 Dysfunction and Neurodegeneration 7

Clearly, a first step to understand the modulation of TDP-43 cyto-

plasmic localization is to map the sequences that make TDP-43 a predominantly
nuclear protein. TDP-43 has been shown to have a bipartite nuclear localization
signal (NLS) within residues 82–98 (Winton et al., 2008a) of the N-terminal
domain. Deletion/mutation of this sequence causes a prominent nuclear to
cytoplasmic shift of TDP-43 localization, a result that has been confirmed
independently by other groups (Ayala et al., 2008b; Inukai et al., 2008). More-
over, it has to be noted that mutations in this NLS sequence may well represent a
potential risk factor for disease as exemplified by a missense A90V variation
found in a FTLD/ALS patient with a family history of dementia that has been
associated with an increased TDP-43 presence in the cytoplasm and pathological
aggregate formation (Winton et al., 2008b). Another important sequence
identified by Winton et al. (2008a) that may contribute to modulate the NLS
action is represented by the nuclear export sequence (NES) localized at residues
235–250 of TDP-43. Deletion of this sequence led to the formation of intra-
nuclear inclusions. These NLS and NES sequences, however, are not the only
determinants of TDP-43 cellular localization.
Recently, for example, the integrity of the C-terminal tail has also been
shown to be very important with regards to the localization of this protein as its
progressive deletion results in the formation of large nuclear and cytoplasmic
aggregates if the protein is overexpressed (Ayala et al., 2008b). This region can
bind other members of the hnRNP A/B family (especially hnRNP A2) (He and
Smith, 2007) and is essential for TDP-43 to function as splicing silencer in the
CFTR exon 9 and Apo AII exon 3 systems (Ayala et al., 2005; Buratti et al.,
2005) (Fig. 1.2). Furthermore, the C-terminal region is also required for the
ability of TDP-43 to act as a transcriptional insulator for the mouse SP-10
gene (Abhyankar et al., 2007). However, immunohistochemical studies
performed on cytoplasmic TDP-43 inclusions in brain tissues from patients
have observed the absence of these hnRNP proteins (Neumann et al., 2007a),
suggesting that maintenance of TDP-43 biochemical functionality may well be
a factor to keep this protein predominantly nuclear and smoothly shuttling
between nucleus and cytoplasm.
The possibility that increased transport from the nucleus to the cyto-
plasm may represent a physiologic response following neuronal injury has been
recently addressed by Moisse et al. (2009). These authors have shown that, in
both distally and proximally axotomized mice there is an increased expression
and relocalization of TDP-43 from the nucleus to the cytoplasm (which is also
reversible), suggesting that this protein may play an early role in neuronal repair.
Interestingly, Western blot analysis of TDP-43 from lumbar hemicords did not
reveal any difference from controls, also suggesting that cytoplasmic TDP-43
does not necessarily correlate with aberrant modifications. This conclusion is
consistent with the results recently obtained in a SH-SY5Y cell model of disease
8 Buratti and Baralle

where evident cytoplasmic TDP-43 inclusions of a mutant carrying a deletion in

the NLS could be observed only following addition of the proteasome inhibitor
MG132 (Nonaka et al., 2008).
Beside nuclear/cytoplasmic distribution, it is now also becoming clear that
the localization of TDP-43 within the nuclear compartment is just as tightly
regulated. For example, Ayala et al. (2008b) have shown that an extensive deletion
in the RRM-1 motif region (residues 106–175) leads to the formation of dot-like
structures within the nucleus that are associated with a substantial redistribution of
TDP-43 in chromatin fractions. This effect could also be achieved by simply
mutating the two key Phe residues (147–149) that have been described as essential
for TDP-43 correctly binding to UG-rich RNA (Buratti and Baralle, 2001), sug-
gesting that RNA binding determines TDP-43 intranuclear distribution.
Although there are differences in the morphology of the nuclear bodies, the
TDP-43 with RRM-1 deleted and the TDP-43 with the Phe 147–149 mutated are
identical regarding the chromatin fractions. From this observation it can be inferred
that RNA binding may affect TDP-43 mobility from insoluble heterochromatin
bound to a looser chromatin interaction or even nucleoplasm solubility. Interest-
ingly, the formation of apparently similar dot-like structures has also been recently
observed in a mutant carrying a 187–192 residues deletion, in which TDP-43 also
displayed a low level of aberrant phosphorylation (Nonaka et al., 2008).
Taken together, all these data suggest that TDP-43 cellular distribution
is a tightly regulated process and that numerous sequences in its primary amino
acid structure can contribute to the final picture. Furthermore, it is clear that
cellular distribution is not an exclusive function of TDP-43 sequence, but that
general cellular localization and aggregation controlling factors play a critical
role. For example, it has recently been observed that injection in culture cells of
mutant VCP associated with FTLD disease results also in the altered localization
of TDP-43 from the nucleus to the cytoplasm, in addition to other effects such as
apoptosis, ER-stress, and impairment in cell viability (Gitcho et al., 2009). The
observation that VCP and TDP-43 coimmunoprecipitate together not only from
transfected cells but also from brain homogenates may point to a functional
relationship between these two proteins (Gitcho et al., 2009).
In normal conditions, the integrity of the general cellular trafficking
and transport mechanisms of the cell may also play a role in keeping TDP-43
proper cellular distribution. First of all, it is important to note that TDP-43 has
been described to be part of RNA granules responsible for trafficking, seques-
tering, and degrading RNA species (Elvira et al., 2006). More recently, it has
been shown that its clearance by autophagic pathways is an additionally impor-
tant factor in determining its eventual aggregation in the cytoplasm. Evidence
for this observation has been first obtained by work performed on the endosomal
sorting complexes required for transport (ESCRT). These complexes are impor-
tant for recognition of ubiquitinated endocytosed integral membrane proteins,
 1. The Molecular Links Between TDP-43 Dysfunction and Neurodegeneration 9

their sorting in multivesicular body (MVB), and for subsequent degradation in

lysosome/vacuoles. Depletion of ESCRT subunits in HeLa cells was shown to
correlate with endogenous TDP-43 accumulating in ubiquitin-positive cytoplas-
mic aggregates (Filimonenko et al., 2007). Another interesting potential link
between TDP-43 accumulation and defects in intracellular transports concern
Perry’s syndrome, which is characterized by rapidly progressive and fatal autoso-
mal dominant parkinsonism, central hypoventilation, depression, and severe
weight loss. In this case, TDP-43 inclusions are also quite distinguished from
those observed in FTD/ALS as they occur only in the extrapyramidal system but
are absent in the neocortex and motoneurons (Wider et al., 2008). The connec-
tion with the transport system has been recently uncovered following the
discovery that patients affected by this disease present mutations in the
DCTN1 gene that codes for the large subunit of the dynactin complex
p150glued (Farrer et al., 2009). Dynactin is a multiprotein complex that is
important for microtubule-based motility and anchoring at centrosomes.
Interestingly, dynactin complex proteins have been observed to colocalize with
TDP-43 inclusions in some cases, raising the possibility that TDP-43 aggregation
may possess a direct connection with dynactin complex disruption.
Finally, it has to be noted that Golgi apparatus fragmentation has been
reported to be a common feature of several neurodegenerative diseases including
ALS. Interestingly, disruption of the Golgi apparatus in the anterior horn cells
of neurons form ALS-affected patients has been shown to correlate with cytoplas-
mic localization of TDP-43 (Fujita et al., 2008). Considering that Golgi fragmen-
tation seems to be an early event in neurodegeneration it may well be that early
disruption of the cellular transport, targeting, and other Golgi-mediated activities
results in abnormal TDP-43 localization/aggregation, although further work
will be required to exactly determine the degree of Golgi disruption in other
neurodegenerative diseases and its pathological connection.

B. Degradation
Increased degradation is a hallmark of TDP-43 in affected neuronal tissues. The
initial studies by the groups of V. Lee and T. Arai/M. Hasegawa (Arai et al., 2006;
Neumann et al., 2006) have both described the presence of
25 and
35 kDa
fragments that are normally absent from normal controls. A recent report has
suggested that these fragments derive from the activation of caspase-dependent
cleavage and results in redistribution of the fragments in the cytoplasm (Zhang
et al., 2007). In keeping with these conclusions, the wild-type TDP-43 contains
three well-defined caspase cleavage sites that are predicted to yield fragments of a
size compatible with those observed in the patients (highlighted in Fig. 1.2, lower
left panel). In particular, caspase-3 activation has been proposed to occur
following the downregulation of the PGRN gene and considering that mutations