Journal of the Neurological Sciences 288 (2010) 112

Contents lists available at ScienceDirect

Journal of the Neurological Sciences

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Review

The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS)
Michael J. Strong
Molecular Brain Research Group, Robarts Research Institute, London, Ontario, Canada
Department of Clinical Neurological Sciences, The University of Western Ontario, London, Ontario, Canada

a r t i c l e

i n f o

Article history:
Received 7 July 2009
Received in Revised form 27 August 2009
Accepted 25 September 2009
Available online 18 October 2009
Keywords:
RNA binding proteins
TDP-43
FUS/TLS
RNA metabolism
Neurodegeneration

a b s t r a c t
In this review, the role of aberrant RNA metabolism in ALS is examined, including the evidence that a
majority of the genetic mutations observed in familial ALS (including mutations in TDP-43, FUS/TLS, SOD1,
angiogenin (ANG) and senataxin (SETX)) can impact directly on either gene transcription, pre-mRNA
splicing, ribonucleoprotein complex formation, transport, RNA translation or degradation. The evidence that
perturbed expression or function of RNA binding proteins is causally related to the selective suppression of
the low molecular weight subunit protein (NFL) steady state mRNA levels in degenerating motor neurons in
ALS is examined. The discovery that mtSOD1, TDP-43 and 14-3-3 proteins, all of which form cytosolic
aggregates in ALS, can each modulate the stability of NFL mRNA, suggests that a fundamental alteration in
the interaction of mRNA species with key trans-acting binding factors has occurred in ALS. These
observations lead directly to the hypothesis that ALS can be viewed as a disorder of RNA metabolism, thus
providing a novel pathway for the development of molecular pharmacotherapies.
2009 Elsevier B.V. All rights reserved.

Contents
1.
2.
3.

Introduction . . . . . . . . . . . . . . . . . . . . . .
Evolving concepts in RNA metabolism . . . . . . . . . .
The case for alterations in RNA metabolism in ALS . . . .
3.1.
Gene transcription . . . . . . . . . . . . . . . .
4.
RNA granule formation and mRNA stabilization . . . . . .
4.1.
Altered NFL mRNA stability in ALS . . . . . . . . .
4.2.
The role of TDP-43, a dual DNA/RNA binding protein
4.3.
RGNEF the human homologue to p190RhoGEF . .
4.4.
FUS/TLS . . . . . . . . . . . . . . . . . . . . .
5.
RNA transport . . . . . . . . . . . . . . . . . . . . . .
6.
RNA translation . . . . . . . . . . . . . . . . . . . . .
7.
Conclusions . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
Although amyotrophic lateral sclerosis (ALS) has been recognized as
a clinical entity since the mid 1800s, the biological basis of this disorder
has remained an enigma. The classical view of ALS is that of a
degenerative process restricted to motor neurons, including those of
the descending supraspinal motor pathways and their respective target
Room C7-120, University Hospital, LHSC, 339 Windermere Road, London, Ontario,
Canada N6A 5A5. Tel.: +1 519 663 3874; fax: +1 519 663 3609.
E-mail address: mstrong@uwo.ca.
0022-510X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jns.2009.09.029

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1
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6
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9

lower motor neurons in the brainstem and spinal cord. The contemporary view of ALS is however of a multisystems degenerative disorder [1].
This is exemplied by the observation that degeneration of the frontal
and temporal lobes occurs in a signicant percentage of ALS patients and
gives rise to one or more syndromes of frontotemporal dysfunction [2,3].
This phenotypic heterogeneity of the clinical face of ALS pales in
comparison to its biological heterogeneity, reected in part by the
extensive number of genetic variants of the disease (familial ALS; fALS)
that are now recognized (Table 1). In part, this also reects the
limitations of utilizing tissue from end stage disease for biological
studies, thus providing a single snap shot of a disease process when

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

Table 1
Genetic mutations associated with familial ALS (fALS).
Name

Locus

Gene

Onset

Inheritance

Alternative phenotype

Reference

ALS1
ALS2
ALS3
ALS4
ALS5
ALS6
ALS7
ALS8
ALS9
ALS10
FTD-ALS

21q22.1
2q33
18q21
9q34
15q15.121.1
16q12
20p13
20q13.33
14q11
1p36.2
17q21.1

SOD1
Alsin
Unknown
SETX
Unknown
FUS/TLS
Unknown
VAPB
ANG/VEGF
TARDBP
MAPT

Adult
Juvenile
Adult
Juvenile
Adult
Adult
Adult
Adult
Adult
Adult
Adult

Dominant and recessive

Recessive
Dominant
Dominant
Recessive
Dominant
Dominant
Dominant
Dominant
Dominant
Dominant

none
Infantile spastic, juvenile PLS
None
CMT or dHMN
None
None
None
Late onset SMA
None
None (?FTD)
Disinhibitiondementia

[150]
[151154]
[155]
[80,82]
[156]
[19,20,157,158]
[159]
[160,161]
[6871,79]
[37,112119]
[162165]

Abbreviations: ANG (angiogenin); CMT (Charcot Marie Toothe); FTD (frontotemporal dementia); dHMN (distal hereditary motor neuropathy); MAPT (microtubule associated tau
protein); PLS (primary lateral sclerosis); SMA (spinal muscular atrophy); SOD1 (copper/zinc superoxide dismutase); SETX (senataxin); FUS/TLS (fused in sarcoma/translated in
liposarcoma); VAPB (vesicle-associated membrane protein); VEGF (vascular endothelial growth factor).

neurons are severely impaired. However, even given this limitation, it is

evident that motor neuron death in ALS is the culmination of multiple
aberrant biological processes, including derangements in cytoskeletal
protein composition, mitochondrial dysfunction, impaired calcium and
glutamate homeostasis, and enhanced oxidative injury. The involvement
of non-neuronal cells in the disease process, including both microglia
and astrocytes, has signaled a need to be viewing ALS as a disorder in
which the motor neurons bear the brunt of the disease process, but do
not do so in isolation [48].
Pathological intracellular protein aggregates are amongst the
neuropathological hallmarks of ALS and can consist of one or more of
the intermediate lament proteins (neurolament proteins (NF) [9],
-internexin [10] or peripherin [11]), 14-3-3 proteins [12,13],
copper/zinc superoxide dismutase (SOD1) [1416], TAR DNA
binding protein of 43 kDa (TDP-43) [17,18] and the recently described chromosome 16 linked fused in sarcoma/translated in
liposarcoma gene product (FUS/TLS) (Fig. 1) [19,20].
In the case of NF aggregates, their formation is associated with a
selective disturbance in the steady state levels of the individual NF
subunit protein mRNA levels such that low molecular weight NF
protein (68 kDa; NFL) steady state mRNA levels are suppressed
relative to those of either the intermediate molecular weight
(160 kDa; NFM) or high molecular weight (200 kDa; NFH) proteins
[2123]. It is thus of considerable interest that the repertoire of mRNA
binding proteins that are capable of binding to and modulating NFL
mRNA stability include several of the proteins not only known to be
associated with intraneuronal protein aggregates in ALS, but are
themselves associated with specic variants of fALS (Fig. 1, Table 2).
Because alterations in RNA metabolism and disturbances in cytoskeletal protein expression are increasingly being seen as a key biological
aspect of ALS, it is timely to examine the evidence that perturbations
in the processing of RNA from gene transcription through degradation
(RNA metabolism) may contribute to the motor neuron degeneration
in ALS.
2. Evolving concepts in RNA metabolism
To understand the potential role of altered RNA processing in ALS, it
is helpful to rst examine evolving concepts in the regulation of RNA
metabolism. The dynamic regulation of gene expression and its linkage
to protein expression is increasingly seen to be governed by
determinants of RNA metabolism that regulate the post-transcriptional
processing of mRNA, including pre-mRNA splicing and mRNA processing, editing, transport, stabilization and translation, and degradation
(Fig. 2) [24]. The ability of a neuron to somatotopically direct protein
synthesis to the sites of greatest need is embodied in the model of
asymmetrical protein translation in which the site-specic synthesis of
protein is dependant on the transport of translationally quiescent mRNA

within transport RNA granules and then the unbundling of the granules
to yield a translationally active polysome at the site at which protein
is needed [25]. While important to neuronal development, this
process is increasingly seen as key to the mechanisms underlying
neuronal plasticity, neuroregeneration, and when perturbed, neurodegeneration. Moreover, while the model allows for a localized
rapid induction of protein synthesis with spatial precision, it also
provides for a considerable level of post-transcriptional regulation of
gene expression.
The ability to direct protein synthesis to specic somatotopic targets in
response to cellular needs is further enhanced by the coordinated
expression of multiple nascent mRNAs into functionally related bundles
through macromolecules termed ribonucleoprotein (RNP) complexes
(also termed RNA granules). This is the core of the RNA operon theory in
which genes whose products are functionally related are expressed in the
same temporal and spatial patterns [24,26,27]. RNP complexes contain
ribosomal subunits, translation factors, decay enzymes, helicases, scaffold
proteins, molecular motors and RNA binding proteins (RBPs) that control
the localization, stability and translation of their RNA cargo [28]. Their
composition is thus critical to the regulation of RNA metabolism. RBPs are
themselves subject to considerable post-translational modication and
provide a dynamic linkage between RNA processing and intracellular
signaling pathways [27,29].
Three main types of RNA granules are present in the mature neuron,
including transport granules (maintaining mRNA in a translationally silent
state until transport to the site of nascent protein synthesis), stress
granules (sequestering mRNA in a translationally silent state at times of
neuronal injury) and cytosolic processing bodies (also known as P-bodies
or degradative granules) [3032]. In general, it has been held that each of
the three granules is discretely synthesized. However, both stress granules
and P-bodies share a common origin (Fig. 3). Both are simultaneously
assembled in response to cellular stress on untranslated mRNA derived
from disassembled polysomes, and they share a number of proteins,
including FAST, XRN-1, eIF4E, TTP and BRF2. Both exist in a state of
dynamic ux that can be modied in an activity dependant manner with
interchange between the two [3335].
In mammalian cells, RNA degradation can be initiated by deadenylation followed by 3-5 exonucleolytic cleavage or by decapping
followed by 5-3 exonucleolytic cleavage. 3-5 exonucleolytic cleavage is
performed in a multiprotein complex termed the exosome, the catalytic
domains of which are orientated to the internal cavity thus preventing
non-specic RNA degradation. In order to gain access to this catalytic
domain, RNA is unwound by helicases. Degradation also occurs in Pbodies, characterized by the presence of GW182 RNA binding protein,
decapping enzymes, decapping associated proteins and the 5-3
exonuclease XRN-1.
It is increasingly evident that mRNA degradation is also regulated by
microRNAs (miRNAs) highly abundant, highly-conserved non-coding

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

Fig. 1. Protein aggregates in spinal motor neurons in ALS. Neurolament aggregates assume a variety of morphologies, including discrete cytoplasmic aggregates (A; arrow),
skein-like structures (B, arrow) and neuroaxonal spheroids (C) (SMI 31, 1:30,000 titre, antigen retrieval). Neuroaxonal spheroids are also intensely immunoreactive to peripherin
(D), while affected spinal motor neurons demonstrate either a more diffuse pattern of immunoreactivity or intense peripherin aggregation to the periphery of the cytosol
(E) (polyclonal anti-peripherin antibody, 1:4000 titre). Less frequently, prominent intraneuronal -internexin immunoreactive aggregates are observed, often obliterating the
nucleus (F) (monoclonal anti--internexin antibody, 1:1000 titre). A prominent upregulation of TDP-43 expression with intense nuclear immunoreactivity is amongst the most
common nding for TDP-43 pathology (G, arrow directed towards a motor neuron nucleus). Pathognomic of degenerating motor neurons in ALS is however a dramatic increase in
cytosolic TDP-43 immunoreactivity with the formation of dense aggregates and skein-like structures (I; sALS). In contrast, pathological FUS/TLS immunoreactivity is only
observed in fALS cases harbouring mutations in FUS/TLS and generally appears as a loss of nuclear staining with prominent upregulation of cytosolic staining (H; fALS with c.1561
C > T missense mutation). 14-3-3 protein expression appears predominantly as a marked increased in diffuse cytosolic staining (J), although dense intracellular aggregates have
been previously described. (All colorimetric photographs (AF) 100 magnication before reproduction; all confocal images (GJ), 10 m scale bar included with Hoescht
staining for nuclei (blue)).

RNAs that are derived from endogenous genes [36]. miRNAs are single
stranded RNAs that play a critical role in the regulation of neuronal
development [37,38], have been shown to regulate the development of
dendritic synaptic spines [39], and are increasingly recognized to
participate in neuronal degeneration [4042] including roles in
regulating the expression of -site APP-cleaving enzyme (BACE) in
Alzheimer's disease [43] and in prion-induced neurodegeneration [44].
The aberrant expression of miRNAs has also been linked to several
human malignancies [36,45].
It is estimated that a third of all human RNA expression is controlled
by miRNAs that are produced at extremely high copy number (estimates

range from 1000 to 30,000 copies per cell compared to <100 copies per
cell for mRNA). They are transcribed by RNA polymerase II from either
non-coding DNA or, less commonly, from the introns of protein coding
genes [46] (Fig. 4). These pri-miRNAs, which can be kilobases long, form
imperfect stemloop structures (hairpins) that are cleaved into 7085 nt
precursor miRNAs (pre-miRNAs) within the nucleus in a process that is
mediated by the RNase III enzyme Drosha and its binding partner DGCR8.
Both TDP-43 and FUS/TLS, mutations of which are linked to fALS,
associate with Drosha in the microprocessor complex [47].
The pre-miRNA is then exported from the nucleus by Exportin-5
which also serves to protect it from degradation [48]. In the cytoplasm,

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

Table 2
RNA metabolic pathways of importance to ALS.

Gene transcription

Process

Outcome

ALS/MND relevant example

Reference

Alternative splicing

Altered protein function (ligand interactions,

subcellular localization, activity)
Preferential expression of specic variant
Altered spliceosome assembly
Altered rRNA transcription
Altered modulation of RNARNA and DNARNA
interactions
Aberrant expression of, or function, of normally
expressed RNA stability determinant
Pathological gain of function of a mutant protein
Altered interaction of normal mRNA binding protein
with cis-acting binding domain

peripherin (Per 28)

[53,54]

EAAT2
SMN 1, 2 (SMA type I/II)
Angiogenin
Senataxin, IGHMBP2

[57]
[61]
[74]
[80,82,83]
[92]
[19,20]
[94,166,167]
[138]

Pre-mRNA splicing
Ribosomal (rRNA) transcription
DNA/RNA helicase
RNA granule formation

mRNA stability

mRNA compartmentalization

Altered miRNA signature

TDP-43
FUS/TLS
mtSOD1 (NFL, IL-8, VEGF)
RGNEF (the human homologue of
murine p190RhoGEF)
14-3-3
PGRN 3UTR mutation with altered
miRNA interaction
miR 215 (mt SOD1)

Axonal transport of
translationally quiescent mRNA
RNA oxidation

Failure of transport to site of synthesis greatest needs

dynactin

Altered 3UTR cis-acting element

RNA transport
RNA translation

Reduced protein expression

[93]
[168]
Unpublished
observation
[143,144]
[149]

IGHMBP2 (Immunoglobulin Mu-Binding Protein 2).

the pre-miRNA is further cleaved by Dicer and its binding partner TRBP
into a transient miRNA duplex containing a mature miRNA sequence of
22 nt and the complementary 2125 nt miRNA. The association of this
miRNA duplex with Argonaute (Ago) proteins (the catalytic component

of the RNA-induced silencing complex (RISC) [49,50]) leads to the

generation of the functionally mature and active miRNA (the remaining
strand is degraded) which, with Ago, becomes incorporated into
ribonucleoprotein particles (RNPs). This complex is then guided to its

Fig. 2. Schematic illustration of RNA metabolism and potential points of impact for ALS associated mutations or disease processes. Following gene transcription, pre-mRNA is
modied by the spliceosome into mRNA which is then bundled in RNP complexes (RNA granules) and actively transported to the cytoplasm. In the mature neuron, RNA granules
can exist in three forms, including transport granules in which the mRNA is translationally quiescent and transported to the sites of nascent protein synthesis. At these sites, an active
polyribosome is reconstituted and translation proceeds. For many proteins, retrograde transport of either intact protein or degradation products allows for further regulation of gene
expression. The RNA granule can also be modied in response to stalled initiation to give rise to either a stress granule, in which instance the mRNA is held until such time as
needed, or a processing body (P-body) in which mRNA is degraded. The process of mRNA degradation within the P-body is governed in part by the specic association between the
mRNA and miRNAs. The potential sites, or pathways, at which the interaction of RNA binding proteins or proteins associated with RNA processing, and which have been identied as
disease modifying or causative agents in ALS, are indicated.

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

Fig. 3. Schematic illustration of the 5 stages of stress granule (SG) formation (modied from Anderson and Kedersha [34]). In response to cellular injury or stress, phosphorylation of
eiF2 results in abortive initiation complexes with stalled initiation and the conversion of polysomes into 48S ribosomes. Primary aggregation and SG nucleation is dependant on the
presence of free 48S complexes and can be initiated by multiple proteins which then become part of the SG they nucleate. Secondary aggregation through protein:protein
interactions (e.g., PABP-1 mediated) results in the progressive fusion of SG to form larger aggregates (visible at the m size). In the next step, proteins that lack mRNA binding
properties are recruited which then integrate the SG with specic signaling pathways (e.g., TIA-1 binding proteins SRC3, FAST, PMR1; G3BP binding protein plakophilin 3) and
provide the SG with an ability to integrate aspects of cellular metabolism with the translational response to stress. At this stage, RNA is triaged either into a translationally quiescent
compartment which protects the RNA from degradation until translation is reinitiated, or into a pathway in which the RNA is destabilized and degraded in P-bodies. The interchange
between SGs and P-bodies is dynamic with reversible exchange of mRNA, with the current view being that the determination of whether a mRNA is stabilized or degraded being in
part dependant on the nature of the miRNA interaction with its respective MRE.

target mRNA through base pairing interactions of 67 nt at the 5 end of

the miRNA with its target (the seed region). If the miRNA has complete
complementarity to its target mRNA recognition element (MRE), the
mRNA is preferentially directed to P-bodies and the mRNA targeted for
degradation [51]. However, the majority of miRNAs base pair with

imperfect complimentarity, leading to translational silencing, possibly

by the inhibition of translational initiation [40]. The complexity of this
process is further highlighted by the observation that multiple MREs (for
the same, or different, miRNAs) within the same 3UTR can function cooperatively to enhance repression [41]. To add to this, miRNAs are

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

and thus, not surprisingly, an increasing number of degenerative

disorders are being linked to disturbances in RNA metabolism. In this
context, it is possible to examine alterations in RNA metabolism in the
context of ALS and the related motor neuron diseases by considering
perturbations in RNA processing in several broad categorizations,
including gene transcription, RNA granule formation with mRNA
stabilization, transport and translational regulation.
3.1. Gene transcription

Fig. 4. miRNA. The processing of miRNA includes both nuclear and cytoplasmic
components. The primary miRNA transcript is predominantly transcribed by RNA
polymerase II from intronic DNA, may be kilobases in length, and forms stemloop
structures that are cleaved by a microprocessor complex, the composition of which
includes Drosha, TDP-43 and FUS/TLS (highlighted in red as fALS associated proteins).
Following nuclear export, the resultant pre-miRNAs are cleaved by Dicer/TRBP to yield
transient miRNA duplexes which are then associated with argonaute proteins (Argo),
the catalytic component of the RNA-induced silencing complex (RISC), gives rise to the
functionally mature miRNA. With Argo, the miRNA becomes associated with RNPs. If
the miRNA has complete complimentarity to its mRNA recognition element, the mRNA
is directed to P-bodies for degradation. However, if the complementarity is incomplete,
then translation inhibition is induced and the mRNA preferentially targeted to stress
granules.

promiscuous (a single miRNA can interact with many different mRNA

species) and some miRNAs can lead to increased translational activity.
3. The case for alterations in RNA metabolism in ALS
As can be seen from the preceding discussion, there are signicant
opportunities to pathologically alter RNA metabolism, ranging from the
site of nascent RNA synthesis, through transport, reassembly into
translationally active polyribosomes, to enhanced rates of degradation.
While this complexity is critical to the site-specic regulation of gene
expression, it also allows for considerable opportunity for misfortune

There is increasing evidence for alterations in RNA splicing as being

associated with not only ALS, but related forms of motor neuron diseases.
Alternative splicing allows for an intricate control over the protein
isoforms expressed by any given cell, and in the central nervous system, is
a key process in determining the properties of individual neurons [52]. The
process of intronic excision and exon ligation is catalyzed by the
spliceosome, a large RNP complex containing small nuclear RNAs
(snRNA) and upwards of 50 associated proteins, including small nuclear
RNPs (snRNPs), helicases, and survival of motor neuron protein (SMN),
critical to splicing regulation and RNP trafcking. The consequence of
alternative splicing is to not only increase the repertoire of functionally
related protein isoforms, but it can also alter ligand-binding properties,
subcellular localization, activity, or interactions with signaling pathways
[52].
An example of the expression of a toxic splice variant in ALS is the
expression of the Per 28 isoform of peripherin. Peripherin is a type III
intermediate lament that is associated with a variety of intraneuronal
inclusions in spinal motor neurons in ALS, including ubiquitinated
inclusions, Lewy body like inclusions and axonal spheroids [53] as well as
a generalized increase in peripherin immunoreactivity [54]. Transgenic
mice expressing mtSOD1 also express a toxic splice variant of peripherin
(Per 61) which is generated by the in-frame retention of intron 4,
introducing a 32 amino acid insertion [54]. In ALS, a peripherin transcript
retaining exons 3 and 4 is expressed, with the exon 3 encoding for a
premature stop codon and a truncated protein of 28 kDa (Per 28) [55].
The expression of Per 28 in SW13vim() cells induces the formation of
peripherin inclusions, suggesting that the expression of a peripherin
splice variant is directly related to the induction of disease pathology.
This is further supported by the nding that TDP-43 (discussed below)
co-localizes to peripherin immunoreactive inclusions in ALS [56].
Although the preferential expression of alternative splice variants of
the astrocytic glutamate transporter (EAAT2) through intron-retention
and exon-skipping was postulated to be associated with the alterations
in astrocytic glutamate uptake observed in ALS, the specicity of
expression of EAAT2 splice variants to ALS has been called into question
[5760]. However, it is possible that it is not the altered expression of an
individual splice variant that is pathogenic per se, but rather it's effect
upon associated mRNAs and RBPs. Until this is further claried, the role
of altered EAAT2 splice variant expression remains to be claried.
Reduced expression of SMN is causal to a severe form of spinal
muscular atrophy (SMA), an autosomal recessive disease that gives rise to
a progressive loss of spinal motor neurons. The SMN gene on chromosome
5q13 is present in multiple copies in the human: one telomeric copy
(SMN1 or SMNt; encoding a 39 kDa protein) and several centromeric
copies (SMN2 or SMNc) [61]. The vast majority of patients with SMA have
a homozygous disruption of SMN1 (deletion, rearrangement or mutation)
giving rise to a deciency of SMN1. SMN2 undergoes alternative splicing,
yielding a truncated mRNA isoform lacking exon 7 (SMN7) and a
nonfunctional protein. However, about 10% of SMN2 is properly spliced,
yielding a full-length functional protein. SMN, in association with several
SMN-associated proteins (Gemins), participates in the assembly of
uridine-rich small nuclear RNPs (U snRNPs) which are the principal
components of the spliceosome. SMN also associates with RNP granules
that are actively transported into both motor neuron neurites and growth
cones in association with hnRNP R and Q [6264]. The interaction of
hnRNP R with the 3UTR of -actin mRNA and the reduced levels of both

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

SMN protein and -actin mRNA in axons and growth cones in SMA,
has suggested a key role for SMN protein in the formation and
transport of -actin mRNA containing RNP complexes. The observation that antisense morpholino induced reductions in SMN levels in
zebrash is associated with motor neuron pathnding defects
(motor axon truncations) in the absence of enhanced motor neuron
death supports the critical role of SMN in motor neuron biology [65].
Mutations in the ANG gene at chromosome 14q11 encoding
angiogenin (ANG), associated with fALS type 9, are postulated to give
rise to alterations in ribosomal transcription. Angiogenin, a 14.1 kDa
protein member of the pancreatic ribonuclease A superfamily, is expressed within both the cytoplasm and nucleus of motor neurons [66]. In
endothelial cells, it is actively taken up and transported to the nucleus [67].
It can promote ribosomal RNA transcription and act as a stress-activated
ribonuclease that cleaves tRNA and inhibits protein translation [68]. An
association between mutations in the ANG gene and ALS has been
observed across a number of populations [6973]. Although the
mechanism by which ANG mutations induce motor neuron degeneration
are not yet clearly dened, diminished ribonucleolytic activity or impaired
nuclear translocations are a consideration [71]. In this context, the ability
of ANG to enhance ribosomal RNA (rRNA) transcription through binding
to an ANG-binding element (ABE) consisting of CTCT repeats is integral to
regulating the expression of vascular endothelial growth factor (VEGF)
[74]. Mice with VEGF deletions develop a progressive motor neuronopathy, suggesting a role for this pathway in motor neuron degeneration
[75]. Both VEGF and ANG enhance survival in the G93A mtSOD1
transgenic mouse model of ALS [7678] while G93A mtSOD1 mice
crossbred with mice expressing reduced levels of VEGF have a more
aggressive phenotype of motor neuron degeneration [79], further
supporting a role in motor neuron biology.
Mutations in the SETX gene at chromosome 9q34 encoding senataxin
(SETX) are associated with both cerebellar and motor neuron degeneration. When inherited in an autosomal recessive pattern, a severe
cerebellar ataxia syndrome with oculomotor apraxia (AOAO2) ensues,
while autosomal dominant inheritance gives rise to a slowly progressive
motor neuronopathy with pyramidal signs but sparing of both bulbar
and respiratory systems [80,81]. A novel SETX mutation (Thr1118Ile) has
been recently associated with a single sALS case, potentially expanding
the phenotype of SETX mutations to include classical ALS [82]. The
mechanism by which SETX mutations give rise to disease is unknown,
although dysfunction of helicase activity or other steps in RNA
processing are postulated [80]. This is supported in part by the homology
of SETX to the DNA/RNA helicase Immunoglobulin Mu-Binding Protein 2
(IGHMBP2), mutations which lead to a recessively inherited variant of
progressive spinal muscular atrophy with respiratory distress (SMARD1)
[83]. RNA helicases are members of the DEAD box family of enzymes,
named according to one of their conserved motifs. They play a critical
role in multistep nuclear actions requiring sequential rearrangements of
RNA interactions including the ability to unwind folded double-stranded
RNA (dsRNA) or modify RNARNA interactions [84]. RNA helicases are
also involved in the modication of chromatin structure, key to the
initiation of transcription, either directly through modulation of the
chromatin structure, or through interactions with the assembly of the
transcriptioninitiation complex. As alluded to earlier, RNA helicases are
associated with the spliceosome where they are postulated to be key to
regulating the base pairing between snRNAs and pre-mRNA. A role for
RNA helicases in translation initiation and termination has also been
proposed.
4. RNA granule formation and mRNA stabilization
While transcriptional rates are key regulatory components of gene
expression, the modulation of mRNA half-life is also key to regulating
gene expression [24,85]. A wide range of mRNA half-lives exist,
including short-lived mRNAs involved with signaling through to
housekeeping mRNAs with longer half-lives. This variable rate of turn-

over is determined in part by the interaction of specic RNA binding

proteins (trans-acting factors) with their respective mRNA binding
elements (cis-acting elements) which are generally, but not exclusively, AU-rich elements (AREs) within the mRNA 3 untranslated
region (3UTR). Trans-acting factors may be either stabilizing (e.g.,
ELAV-like (embryonic lethal and abnormal vision) proteins including
Hu proteins), destabilizing (e.g., K-homology splicing regulatory
protein (KSRP), tristetraprolin (TTP)) or in the specic example of
the A + U-rich element binding factor 1 (AUF1), exhibit a degree of
stabilization which is isoform specic and dependant on nucleocytoplasmic localization [86]. This subcellular distribution and RNA
binding activity of specic trans-acting factors is further modied by
both post-translational modications of the trans-acting factors,
including phosphorylation [8789], and the nature of the proteins
that associate with the individual trans-acting factors in forming a
RNA binding complex that then regulates mRNA degradation.
4.1. Altered NFL mRNA stability in ALS
To gain further insight into this process in ALS, we have focused on
understanding the genesis of the selective suppression of NFL mRNA
steady state levels that are observed within degenerating motor neurons
in ALS [2123]. The expression of NF proteins is under tight regulatory
control during both development and regeneration (reviewed in [90].
The rst suggestion that the stability of NFL mRNA differed between ALS
and neurologically intact (control) spinal cord arose though studies in
which NFL mRNA was incubated with homogenates of either control or
ALS-derived lumbar spinal cord. We observed that NFL mRNA degraded
more rapidly in the presence of control homogenates, and that the rate of
degradation could be made to approximate that of ALS homogenate
exposed NFL mRNA by either heat inactivation or proteinase K digestion
[91]. Although we interpreted these ndings to suggest that one or more
trans-acting proteins capable of destabilizing NFL mRNA, normally
expressed in control, were absent from ALS tissues, this left the difculty
of explaining suppressed levels of NFL mRNA in ALS spinal motor
neurons. However, our subsequent ndings that each of mutant SOD1,
14-3-3 and TDP-43 proteins could alter NFL mRNA stability [9294],
coupled with the observations (described above) that each could also
form pathological intraneuronal inclusions in spinal motor neurons,
suggest that the observations regarding mRNA stability determinants
derived from whole tissue samples (such as the preceding studies)
might not be immediately extrapolated to the subset of motor neurons.
The proposal that alterations in the interactions between NFL mRNA
trans-acting factors and their respective cis-acting elements are relevant
to the development of a motor neuron degeneration is supported by the
observations of Schlaepfer and colleagues who have extensively
characterized the destabilizing interaction between p190RhoGEF and
murine NFL mRNA [95]. Mutations in the p190RhoGEF cis-acting
element result in a motor neuronopathy in vivo and NF aggregates in
vitro [9698], in part through a mechanism mediated by the direct
interaction of p190RhoGEF with unassembled NFL protein and the
consequent dysregulation of NFL mRNA stability [99].
4.2. The role of TDP-43, a dual DNA/RNA binding protein in ALS
A signicant advance in our understanding of the pathophysiology of ALS was the discovery of TDP-43 as a key pathological
substrate for inclusion formation in both ALS and frontotemporal
lobar degeneration (FTLD) with ubiquitin immunoreactive dystrophic neurites and neuronal cytoplasmic inclusions (FTLD-U) [17,18].
Consistent with its role as a dual DNA/RNA binding protein, TDP-43 is
normally seen as punctate nuclear staining with minimal cytosolic
staining. In both ALS and FTLD-U, there is an abundance of cytosolic
TDP-43, some of which forms aggregates or bril-like structures
termed skeins. Some, but not all, of these aggregates and skeins are
ubiquitinated [17,18]. Western blots of urea solubilized protein

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

isolated from affected brain regions of either disease show a high

molecular weight smear, hyper-phosphorylated 4345 kDa species,
and abnormal C-terminus fragments of 2426 kDa.
Although this discovery of a novel protein as the substrate for the
ubiquitinated inclusions of ALS and FTLD-U suggested a specic disorder
of TDP-43 metabolism, there is a need to differentiate between the
physiological upregulation of TDP-43 expression in response to neuronal
injury, and that which is present as a pathological process. TDP-43
immunoreactive intraneuronal cytoplasmic inclusions, dystrophic neurites and glial cytoplasmic inclusions can be observed across a number of
disorders in addition to ALS and FTLD-U [100102], including the
Guamanian ALS-PD complex [103], FTLD-MND (the neuropathological
entity of clinical FTD and post-mortem observation of motor neuron
degeneration) [104106], corticobasal degeneration and in approximately 70% of cases with hippocampal sclerosis [107]. TDP-43 also colocalizes to neurobrillary tangles in approximately 20% of Alzheimer's
disease cases, with -synuclein inclusions in diffuse Lewy body disease,
and in FTLD associated with mutations in the valosin-containing protein
gene (VCP) [108110]. Conversely, pathological TDP-43 is not seen in
all FTLDs (for example its absence in the chromosome 3 linked FTD
(CHMP2B mutation)) [109]. It is also not seen in a host of brain
pathologies, including both anoxic injuries and malignancies, with the
exception of those with high mitotic activity [111]. However, perhaps
the most convincing evidence that there is a link between pathological
TDP-43 and neurodegeneration are the increasing number of both
familial and sporadic ALS cases in which TDP-43 mutations are found
[37,112121].
The fact that TDP-43 can function as either a DNA or a RNA binding
protein is conferred in part by the unique nature of its primary structure
that includes two RNA recognition motifs (RRM-1, RRM-2), nuclear export
and nuclear localization sequences and a glycine rich sequence enabling
protein:protein interactions [122126]. Although TDP-43 was rst
described as a regulator of HIV-1 gene expression [122], and then subsequently as a mediator of CFTR exon-9 skipping in cystic brosis [123], its
role in regulating RNA trafcking and translation is becoming increasingly
important. TDP-43 has a primary structure characteristic of the
heterogenous ribonucleoprotein (hnRNP) family of splicing regulating
factors [127], and has 11 potential splice variants [124,125]. It is
ubiquitously expressed and can regulate gene expression by several
mechanisms. TDP-43 can tether DNA to the nuclear matrix and subnuclear
membrane to maintain it in a transcriptionally inactive state [128], and
can act as a splicing regulator [129,130]. While the classic TDP-43 DNA or
RNA interactions have been described as requiring (TG)612 or (UG)612
repeat motifs, interestingly, TDP-43 regulation of HIV-1 gene expression
occurs by the recognition of a motif consisting of a polypyrimide rich
region [127]. This exception to the rule of TDP-43 binding through UG/
TG repeats is important when we consider the interaction of TDP-43 with
NFL mRNA where no UG repeats are evident [92,124].
In response to neuronal injury, TDP-43 associates with stress
granules RNA granules that form in order to maintain mRNAs
encoding for housekeeping proteins in a translationally silenced state
until such time as needed in neuronal repair [28,131]. Finally, in cases of
severe neuronal stress, TDP-43 can also be found in P-bodies [132].
We have shown that TDP-43 is a RNA binding protein that can
modulate NFL mRNA stability and that altered NFL mRNA stability
underlies the formation of NF aggregates in ALS [22,91,92]. In addition to
inuencing the stability of RNA, RNA binding proteins participate in both
translational silencing and translational enhancement (reviewed in
[133]). The nding that TDP-43 is a component of RNA granules directly
links it to the process of RNA movement within a cell and to
somatotopically driven protein synthesis [134,135]. Precise mRNA
localization within the neuron involves a close association between the
cytoskeleton and 3UTR domains of the target mRNA transcript [136] and
the ability to effectively modulate its stability until such time as the mRNA
is delivered to its nal destination [137]. The participation of TDP-43 in
such a process has been recently demonstrated [30].

4.3. RGNEF the human homologue to p190RhoGEF

We have recently identied that RGNEF is the human homologue
to p190RhoGEF with near complete conservation of the NFL mRNA
interacting sequence [138]. Moreover, RGNEF is highly expressed
across the central nervous system in both control and ALS and
interacts directly with human NFL mRNA. When GSTRGNEF is incubated with extracts from either control or ALS spinal cord followed
by immunoprecipitation (anti-GST antibody) and then RT PCR of the
immunoprecipitant (technique of RNA IP), NFL mRNA is only
precipitated from ALS and not control homogenates. This suggests
that either ALS-derived NFL is conformationally altered to allow the
interaction between the RGNEF binding site and RGNEF, or there is an
absence of a second RNA binding protein that normally inhibits the
interaction.
4.4. FUS/TLS
The newest addition to the aberrant expression of RNA binding
proteins in ALS is that of FUS/TLS (fused in sarcoma/translated in
liposarcoma) [19,20]. It is associated with chromosome 16-linked fALS
and is found as extranuclear cytoplasmic aggregates in affected
individuals. FUS/TLS is a dual DNA/RNA binding protein that contains
RNA recognition motifs similar to those observed with TDP-43 and is
found as a component of the hnRNP complex involved with pre-mRNA
splicing and in the export of fully processed mRNA to the cytoplasm [139].
Given the relatively recent discovery of the linkage of FUS/TLS mutations
to ALS, little is currently known with respect to its mechanism of
pathogenicity. The association of FUS/TLS with Drosha in the microprocessor complex suggests that it will have a role to miRNA processing [47].
5. RNA transport
Consistent with the concept of asymmetrical protein translation,
there is a requirement to transport RNP complexes containing translationally quiescent mRNA to the site of nascent protein synthesis. To
accomplish this, anterograde axonal and dendritic transport is mediated
by the kinesin superfamily proteins (KIFs), while retrograde transport is
mediated by cytoplasmic dyneins [31]. The anterograde transport of RNA
granules requires the direct binding to the C-terminal tail of KIF5 [32].
More recently, the involvement of KIF4 in the anterograde axonal
transport of P0, a major structural component of ribosomes, has been
observed [140].
Alterations in RNA transport have been indirectly implicated in
motor neuron degeneration. Experimentally, mutations in the dynein
heavy chain, homopolymers of which contribute to the formation of
the microtubule motor dynein complex and thus interact with
dynactin [141], are associated with a progressive motor neuronopathy
and severe impairments in retrograde axonal transport [142].
Mutations in dynactin result in a variable motor neuron phenotype,
including a single family in which both FTD and ALS co-segregated
[143,144]. The mechanism(s) by which dynein/dynactin mutations
give rise to a motor neuron degeneration remain unknown, but likely
include impairments in retrograde axonal transport of NF or RNPs
[141]. Although to date, no kinesin mutations leading to perturbations
in anterograde RNP transport have been documented in ALS, reduced
expression of the kinesin-associated protein 3 (KIFAP3) has recently
been associated with enhanced survival in sALS [145].
6. RNA translation
Little is known with respect to the regulation of RNA translation in
ALS. However, RNA oxidation can lead to reduced mRNA translation
through either ribosomal stalling or a retardation in the rate of
translation [146], or through the induction of translational errors and
the synthesis of defective proteins [147,148]. Increasingly, this is

M.J. Strong / Journal of the Neurological Sciences 288 (2010) 112

postulated to be a mechanism key to the process of neurodegeneration [148]. In both sALS and fALS, between 6 and 10% of motor cortex
and spinal cord mRNAs are oxidized [149]. RNA oxidation precedes
the development of overt motor neuron damage in transgenic mice
expressing any one of a range of fALS associated mutations in SOD1
(SOD1G93A, SOD1G37R, SOD1G85R, SOD1G127X, and SOD1H46R/H48Q)
[149]. Amongst the mRNA species affected, RNAs associated with
mitochondrial function, ribosome composition and cytosol were the
greatest at risk, with oxidative modications also observed for mRNAs
directed towards protein biosynthesis and maintenance of the
cytoskeleton. These included mRNAs for proteins postulated to be
related to ALS pathogenesis, including SOD1, dynactin 1, vesicleassociated membrane protein 1, NF subunits and metallothioneins.
7. Conclusions
Traditionally, ALS has been viewed as either a familial or sporadic
disorder, with rare hyper-endemic foci such as those of the western
Pacic and the Kii Peninsula of Japan. Following the discovery of
mutations in SOD1 associated with a signicant proportion of fALS cases,
there was considerable enthusiasm that the pathogenesis of ALS would
rapidly unfold. This has however not been the case until the recent
identication of several additional genetic mutations, which as described
above begin to link ALS to disturbances in RNA metabolism. The
complexity of RNA metabolism, and indeed the regulation of gene
expression through the modulation of rates of RNA decay and translational activity, at rst seem daunting. However, when viewed within
the context of the RNA operon hypothesis, this conceptualization of ALS
as a RNA mediated disorder becomes clearer. While the fundamental
tenant of the hypothesis is to provide a mechanism by which the neuron
can efciently regulate the temporal expression of functionally related
genes, this can be expanded to include the provision of somatotopically
specic protein synthesis within the neuron (the concept of asymmetrical protein synthesis). In doing so, the neuron is required to assemble a
highly complex RNP complex (RNA granule) in which the interactions
and activities of multiple components are further regulated through
individual post-translational modications, and then transport the RNP
complex to the site of nascent protein synthesis.
The hypothesis offers the advantage of not being dependant upon a
single mechanism of disease induction, but rather a common pathway in
which a multiplicity of seemingly divergent biological effects can coalesce.
If the nal product is that of a fundamental disturbance in protein expression, as we have previously proposed [9], then the mechanisms
described above lend themselves well to this process either through
aberrant pre-mRNA splicing, granule formation, transport or transcriptional activation. At minimum, the hypothesis provides a conceptual
framework in which to consider novel therapies for ALS.
Acknowledgements
Research supported by the ALS Association, ALS Society of Canada,
Canadian Institutes of Health Research (CIHR), Muscular Dystrophy
Association (MDA Tuscon) and the Michael Halls Endowment. The
assistance of Wencheng Yang, MD and Jennifer Strong in the preparation
of Fig. 1, Rosa Rademakers for genotyping of the FUS/TLS associated fALS
case, and Kathryn Volkening, PhD for fruitful discussions and manuscript
review is gratefully acknowledged.
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