Neuron 52, 39–59, October 5, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.neuron.2006.09.

018

ALS: A Disease of Motor Neurons Review

and Their Nonneuronal Neighbors

Séverine Boillée,1 Christine Vande Velde,1 similar pathological hallmarks, including progressive
and Don W. Cleveland1,* muscle weakness, atrophy, and spasticity, each of which
1
Ludwig Institute for Cancer Research and reflects the degeneration and death of upper and lower
Departments of Medicine and Neuroscience motor neurons. Denervation of the respiratory muscles
University of California, San Diego and diaphragm is generally the fatal event. Despite
9500 Gilman Drive intense effort, very limited therapeutic options have
La Jolla, California 92093 emerged for slowing disease course, although advances
have been made in palliative therapy.
We review here what has been learned about new
Amyotrophic lateral sclerosis is a late-onset progres- genes, the current models of pathogenesis, and summa-
sive neurodegenerative disease affecting motor rize directions now being undertaken for promising new
neurons. The etiology of most ALS cases remains therapies.
unknown, but 2% of instances are due to mutations
in Cu/Zn superoxide dismutase (SOD1). Since spo- Genetics of ALS
radic and familial ALS affects the same neurons with Most incidences of ALS are sporadic but w10% of pa-
similar pathology, it is hoped that therapies effective tients have a familial history. The identified chromo-
in mutant SOD1 models will translate to sporadic somal loci containing the mutations leading to ALS-like
ALS. Mutant SOD1 induces non-cell-autonomous human motor neuron diseases (Table 1) have been de-
motor neuron killing by an unknown gain of toxicity. fined as ALS1 through ALS8, as well as ALS with fronto-
Selective vulnerability of motor neurons likely arises temporal dementia (ALS-FTD) and ALS-FTD coupled
from a combination of several mechanisms, including with Parkinson’s disease (ALS-FTDP). Mutations in
protein misfolding, mitochondrial dysfunction, oxida- genes encoding angiogenin (ANG) and VEGF and se-
tive damage, defective axonal transport, excitotoxic- quence variants in neurofilament genes have also been
ity, insufficient growth factor signaling, and inflamma- reported (for more detailed consideration of the genet-
tion. Damage within motor neurons is enhanced by ics, see Gros-Louis et al. [2006]). Although all have in
damage incurred by nonneuronal neighboring cells, common disease that affects motor neurons, the no-
via an inflammatory response that accelerates disease menclature is misleading since only ALS1, ALS3, ALS6,
progression. These findings validate therapeutic ap- ALS7, mutations in angiogenin and VEGF, and a small
proaches aimed at nonneuronal cells. proportion of incidences of ALS8 represent the classic
late-onset neurodegenerative disease with selective kill-
Introduction ing of upper and lower motor neurons leading to pro-
It has been over 135 years since the great French neuro- gressive paralysis. ALS-FTD and ALS-FTDP are likely
biologist and clinician Jean-Martin Charcot (Charcot appropriately classified as bona fide ALS, but patients
and Joffroy, 1869) first described the progressive, late- have additional disease features including dementia
onset motor neuron disease Amyotrophic lateral sclero- and dystonia. For all of these disease incidences, only
sis (ALS). With premature degeneration and death of up- the genes responsible for ALS1, ALS8, and ALS-FTDP
per and lower motor neurons provoking fatal paralysis have been identified.
as its salient clinical features, the name of the disease
derives from Charcot’s observation in the lateral por- ALS1—Mutation in SOD1
tions of the spinal cord of a distinct ‘‘myelin pallor’’ rep- The lion’s share of work has focused on ALS1 caused
resenting degeneration and loss of the axons of upper by mutations in Cu/Zn superoxide dismutase (SOD1).
motor neurons as they descend from the brain to con- Mutations in the SOD1 gene are the most common
nect directly or indirectly onto the lower motor neurons form of inherited ALS, accounting for w20% of all the fa-
within the spinal cord. Initially known as Charcot’s scle- milial ALS forms and corresponding to 1%–2% of all ALS
rosis and now more familiarly known in the United States cases. Since the first SOD1 missense mutations were re-
as Lou Gehrig’s disease, onset of disease is in midlife ported in 1993 (Rosen et al., 1993), the number of known
(usually between age 45 and 60), with a typical disease mutations has increased to more than 114. These are
course of one to five years. Consideration of incidence distributed throughout all five exons encoding the 153
(frequency of new cases per year) and prevalence (the amino acid protein, with no region of the polypeptide es-
proportion of affected individuals in the population; caping disease causing mutation. With the exception of
1–2 and 4–6 per 100,000, respectively) understates the a few instances, all SOD1 mutations are dominant.
impact of ALS, with the lifetime risk at about 1 in 1000 Sporadic and SOD1 mutant-mediated familial ALS are
(Mitsumoto et al., 1998; Yoshida et al., 1986). clinically indistinguishable and affect the same neurons,
Most incidences (90%) of ALS are sporadic, that is, but even within the familial forms, the time of onset and
without an obvious genetic component. Approximately length of disease vary. The alanine-to-valine substitu-
10% are inherited in a dominant manner (and referred tion at position 4 of SOD1 (SOD1A4V) is the most promi-
to as familial ALS). Sporadic and familial ALS produce nent mutation in North America, responsible for w50%
of cases, and has a uniformly aggressive disease course
with a mean survival of only 1 year after onset. Mice and
*Correspondence: [email protected] rats expressing mutant forms of human or mouse SOD1
Neuron
40

Table 1. Genetics of Human ALS and Other Motor Neuron Diseases Sometimes Referred to as ALS

Disease Locus Gene (Protein/Function) Heredity Onset Reference

Typical ALS
ALS1 21q22.1 SOD1 (Cu/Zn superoxide Dominant Adult (Rosen et al., 1993)
dismutase). Converts
superoxide to water
or hydrogen peroxide.
ALS3 18q21 Unknown Dominant Adult (Hand et al., 2002)
ALS6 16q12 Unknown Dominant Adult (Abalkhail et al., 2003;
Ruddy et al., 2003;
Sapp et al., 2003)
ALS7 20p13 Unknown Dominant Adult (Sapp et al., 2003)
ALS with Dementia
ALS-FTD 9q21–22 Unknown Dominant Adult ALS with (Hosler et al., 2000;
frontotemporal Ostojic et al., 2003)
dementia (FTD)
ALS-FTDP 17q21.1 MAPT (Tau) Microtubule- Dominant Adult ALS with FTD and (Clark et al., 1998;
associated protein. Parkinson’s Disease Hutton et al., 1998)
Atypical ALS
ALS8 20q13.3 VAPB (VAMP-associated Dominant Adult. Heterogeneous disease (Nishimura et al., 2004a,
protein B). May be involved (most cases are ALS with 2004b)
in vesicular trafficking. tremor; few cases are
Interacts with itself, typical ALS; 25% of
VAMP-A, and cases are late-onset
synaptobrevins. spinal muscular atrophy)
Progressive 2p13 DCTN1 (Dynactin subunit Dominant Adult. Vocal-fold paralysis; (Puls et al., 2003)
lower motor p150glued). Axonal transport weakness and muscle
neuron of cellular organelles atrophy of hands and
disease and proteins. facial muscles
Other Motor Neuron Diseases Sometimes Referred to as ALS
ALS2 2q33 ALS2 (ALS2/Alsin). Endosomal Recessive Juvenile Heterogeneous (Hadano et al., 2001;
dynamics. Guanine exchange disease (95% of cases Hentati et al., 1994;
factor for Rab5 and Rac1. are infantile-onset) Yamanaka et al., 2006;
Yang et al., 2001)
ALS4 9q34 SETX (Senataxin). Putative Dominant Juvenile (Recessive (Chance et al., 1998;
DNA/RNA helicase. mutations cause Chen et al., 2004)
ataxia-oculomotor
apraxia type 2)
ALS5 15q15.1– Unknown Recessive Juvenile (Hentati et al., 1998)
q21.1

develop progressive motor neuron degeneration (Bruijn synthesis, found no link between HRE variants and dis-
et al., 1997; Gurney et al., 1994; Howland et al., 2002; ease (Gros-Louis et al., 2003; Lambrechts et al., 2003).
Jonsson et al., 2004; Nagai et al., 2001; Ripps et al., Two haplotypes in other regions of the promoter, how-
1995; Wang et al., 2003, 2005; Wong et al., 1995), and ever, showed an increased risk of developing ALS in
most of our knowledge on mechanisms of motor neuron a Belgian, Swedish, and a British/Birmingham popula-
pathology in ALS is from studies using these models tion (Lambrechts et al., 2003), but this has not been con-
(see below). firmed in other populations (Chen et al., 2006; Terry
et al., 2004; Van Vught et al., 2005).
Vascular Endothelial Growth Factor (VEGF)
Vascular endothelial growth factor (VEGF), an estab- Angiogenin (ANG)
lished regulator of developmental, hypoxia-induced, Mutations in ANG have also been linked to ALS, empha-
and tumor-induced angiogenesis, gained interest as sizing a potential link between altered angiogenesis and
a contributor to ALS when deletion of the hypoxia re- motor neuron degeneration. Seven missense mutations
sponse element (HRE) in the murine VEGF promoter re- were identified in 15 patients with familial ALS and 11 ap-
sulted in ALS-like disease in mice (Oosthuyse et al., parently sporadic ALS patients (Greenway et al., 2006).
2001). VEGF is widely expressed throughout the central However, these mutations seem restricted to Irish and
nervous system (CNS) and can function as a neurotro- Scottish populations, as they were not found in English,
phic factor for multiple neuronal cell types, including Swedish, and North American populations and therefore
motor neurons (Oosthuyse et al., 2001; Van Den Bosch suggest that ALS-linked ANG mutations are rare. The
et al., 2004). Screening of ALS patient DNAs in promoter manner in which the identified mutations affect angioge-
regions of the VEGF gene, including the HRE and re- nin function and provoke motor neuron disease is still
gions known to correlate with downregulation of VEGF unknown. Angiogenin, which is expressed within the
Review
41

CNS including the motor neurons, has a known intranu- disrupting frameshift mutation has been reported in one
clear RNase activity that can facilitate rRNA synthesis, at sporadic ALS case (Gros-Louis et al., 2004). A proposal
least in endothelial cells. Interestingly, the majority of the for the involvement of a peripherin variant, in which an
mutations found in ALS patients are located within the intronic sequence is retained in the final mRNA that
catalytic core, and one of them is in the nuclear localiza- is produced during the disease course, may also con-
tion signal, both predicting a loss of function of the tribute to human disease (Robertson et al., 2003), albeit
mutated angiogenin, although this remains to be con- deletion of the peripherin gene has no affect on disease
firmed. Whether angiogenin is endowed with neurotro- course in mice having an ALS-causing SOD1 mutation
phic properties like VEGF, in addition to its angiogenic (Lariviere et al., 2003).
activity, is not yet established.
Motor Neuron Death from Toxicity of Mutant SOD1
ALS8—VAMP-Associated Protein B (VAPB) Not Loss of Dismutase Activity
A dominant missense mutation on chromosome 20 in SOD1 is an abundant, ubiquitously expressed cytosolic
a set of seven kindreds has been linked to a heteroge- enzyme. Since the known activity of SOD1 is to dismu-
neous disease course, with most instances of disease tate (or convert) superoxide, a natural byproduct of
representing an atypical ALS that is accompanied by respiration, to water or hydrogen peroxide, the first
an unusual tremor (Nishimura et al., 2004a). About one- proposed pathogenic mechanism was the loss or
fourth of affected individuals in the same families de- diminution of this detoxifying activity. However, animals
velop a late-onset spinal muscular atrophy, and a small expressing dismutase active (hSOD1G37R [Wong et al.,
proportion of patients develop symptoms of classic 1995] and hSOD1G93A [Gurney et al., 1994; Howland
ALS. All instances apparently derive from a common et al., 2002]) as well as inactive (hSOD1G85R [Bruijn et al.,
mutation in the gene encoding vesicle-associated 1997], mSOD1G86R [Ripps et al., 1995], hSOD1G127X
membrane protein B (VAPB), also known as synaptobre- [Jonsson et al., 2004], hSOD1Quad [Wang et al., 2003],
vin-associated protein B. VAPB has been implicated in hSOD1H46R [Nagai et al., 2001]) forms of the enzyme de-
endoplasmic reticulum to Golgi transport, albeit other velop comparable disease pathologies similar to that
roles in membrane transport, including axonal transport seen in patients, including motor neuron synapse retrac-
of membrane components, are likely as well. It is un- tion (Fischer et al., 2004; Frey et al., 2000; Pun et al.,
known how the corresponding proline-to-serine substi- 2006), mitochondrial alterations (Higgins et al., 2003;
tution at position 56 affects the functional properties of Kong and Xu, 1998; Liu et al., 2004; Wong et al., 1995),
the ubiquitously expressed VAPB. and microglial activation (Boillée et al., 2006; Engelhardt
and Appel, 1990; Hall et al., 1998; Henkel et al., 2006).
Neuronal Intermediate Filaments Furthermore, SOD1 gene deletion in mice does not
The genes encoding the three neurofilament subunits lead to motor neuron disease (Reaume et al., 1996). In
have long been suspected as causative for ALS because addition, deletion of the endogenous mouse SOD1 in
of their link with motor neuron pathology in mice and mice expressing dismutase inactive hSOD1G85R does
humans. Initial evidence fueling this suspicion was the not affect disease course (Bruijn et al., 1998). Mice
realization that aberrant neurofilament accumulations expressing high levels of wild-type human SOD1
are a pathological hallmark of both familial and sporadic (hSOD1WT) transgene are healthy (Gurney et al., 1994;
ALS (Hirano, 1991; Hirano et al., 1984a, 1984b) and are Wong et al., 1995). Indeed, increased hSOD1WT, accom-
also seen in ALS mice expressing mutant SOD1 (Bruijn panied by chronic elevation of dismutase activity, has
et al., 1997; Dal Canto and Gurney 1994). In addition, either no effect on disease (Bruijn et al., 1998) or accel-
mice with increased levels of wild-type NF-H or NF-L erates it (Deng et al., 2006; Jaarsma et al., 2000). Taken
subunits develop age-dependent motor neuron pathol- together, these experiments have argued heavily
ogy (Cote et al., 1993; Xu et al., 1993), while expression against the simple loss-of-function hypothesis.
of a point mutation in NF-L at levels corresponding to SOD1 activity is dependent on a catalytic copper.
that expected for dominantly inherited disease pro- Since free copper is highly reactive and toxic, it must
duces fatal, progressive paralysis (Lee et al., 1994). be loaded onto SOD1 by a copper chaperone for
Despite this, exhaustive screening for mutations in the SOD1 (CCS; Corson et al., 1998; Wong et al., 2000b)
three neurofilament genes in patient DNAs failed to yield and subsequently held in place by a conserved disulfide
conclusive linkage to either sporadic or familial ALS bond whose formation is catalyzed by CCS (Furukawa
patients (Garcia et al., 2006), although dominant point et al., 2004). Since CCS is abundantly expressed in mo-
mutations in NF-L have been linked to a milder motor tor neurons (Rothstein et al., 1999) and motor neurons of
neuron disease, Charcot-Marie-Tooth (CMT) disease CCS-deleted mice have an increased sensitivity to axot-
(De Jonghe et al., 2001; Jordanova et al., 2003). Never- omy-induced death (Subramaniam et al., 2002), it was
theless, in-frame insertions or deletions within the nor- postulated that inefficient incorporation of copper into
mal array of 44 or 45 KSP repeats in the tail domain of SOD1 and/or a decreased shielding of copper (due to
the NF-H subunit have been reported in w1% of spo- changes in SOD1 structure as a result of mutation) could
radic ALS patients (Al-Chalabi et al., 1999; Figlewicz provide an opportunity for aberrant copper-dependant
et al., 1994; Tomkins et al., 1998). Collectively, it is likely oxidative chemistry, yielding cellular damage and motor
that these neurofilament variants are risk factors for ALS neuron degeneration. This hypothesis was tested using
and/or a primary event with low penetrance. mice in which the incorporation of copper into mutant
A final potential contribution of neurofilaments to ALS SOD1 was significantly reduced by disruption of the
comes from errors in the expression of an additional CCS gene. This did not slow disease course from either
intermediate filament subunit, peripherin. An assembly- dismutase active or inactive mutants (Subramaniam
Neuron
42

et al., 2002). Furthermore, mice in which all four copper-

binding histidines have been eliminated (hSOD1Quad;
resulting in dismutase inactive SOD1) still develop typi-
cal ALS-like motor neuron disease (Wang et al., 2003).
Taken together, it is clear that the toxic property (or
properties) acquired as a result of SOD1 mutation (1)
are independent of dismutase and CCS activities and
(2) can be generated without catalysis by the active
site copper.

Misfolded SOD1 as a Common Feature

of ALS-Causing Mutations
Aggregates are a pathological hallmark of many differ-
ent neurodegenerative disorders including Alzheimer’s,
Parkinson’s, and Huntington’s diseases and ALS. Cyto-
plasmic protein aggregates are observed in both spo-
radic and familial ALS cases as well as in mutant SOD1
transgenic mice (Bruijn et al., 1997, 1998; Gurney et al.,
1994; Watanabe et al., 2001; Wong et al., 1995). That Figure 1. Proposed Toxicities of ALS-Causing SOD1 Protein Aggre-
said, use of the term ‘‘aggregate’’ is imprecise. In gates
some instances, it refers to abnormal accumulations of Cell machinery that might be affected by misfolded, mutant SOD1
intermediate filaments, including neurofilaments and includes coaggregation of essential cytoplasmic components, poi-
peripherin, as detected by immunostaining of spinal soning of the proteasome thereby inhibiting timely degradation of
cord tissue (Hirano et al., 1984b; Mendonca et al., many cellular proteins, saturation of cytoplasmic chaperones that
catalyze essential protein folding and refolding, and damaging
2005; Mizusawa et al., 1989; Rouleau et al., 1996; Sobue mitochondria by aggregation onto the cytoplasmic surface and/or
et al., 1990; Wong et al., 2000a). It has also been used to transport into the mitochondrial intermembrane space.
define accumulations of detergent-insoluble forms of
proteins, including SOD1, that are detected by immuno-
blotting of filter-trappable material, as well as small
SOD1- or ubiquitin-positive fibrillar inclusions in spinal activity, deregulation of organelle function including
cord sections (Deng et al., 2006; Furukawa et al., 2006; Golgi, endoplasmic reticulum, and mitochondria, and
Johnston et al., 2000; Jonsson et al., 2004, 2006; Wang axonal transport defects possibly linked to aberrant
et al., 2003, 2005). Detergent-insoluble species are de- accumulations of intermediate filaments.
tectable only in affected tissues of mutant SOD1 mice Aggregates found in ALS patients, as well as mouse
and are most prominent at symptomatic stages (Furu- models, contain ubiquitin (Ince et al., 1998; Wang
kawa et al., 2006; Jonsson et al., 2006). A propensity to et al., 2003; Watanabe et al., 2001), a protein adduct
form aggregates following synthesis of mutant SOD1 which typically targets proteins for disposal via the pro-
in primary cells is selective to motor neurons, as aggre- teasome. Misaccumulation of ubiquitinated, misfolded
gates were not produced in dorsal root ganglion (DRG) proteins might adversely affect the proteasome machin-
or hippocampal neurons expressing similar levels of ery and impair normal protein degradation. Indeed, the
mutants (Durham et al., 1997). The most misfolded un- degradation of SOD1 itself and aggregates of SOD1 is
stable SOD1 mutants (with the shortest in vivo half-lives) proteasome mediated (Basso et al., 2006; Niwa et al.,
are most prone to aggregation (Wang et al., 2003). 2002; Urushitani et al., 2004; Urushitani et al., 2002).
A very curious and unexplained aspect of disease is that While proteasome malfunction has been implicated in
the very unstable mutant hSOD1A4V (Sato et al., 2005), motor neuron death, it is not yet established if it is a
which provokes a very aggressive disease course in hu- cause or consequence of aggregate formation. Contra-
mans, neither induces aggregates nor ALS-like disease dictory results have been reported. In hSOD1G93A mice
in mice, except in the context of high levels of hSOD1WT which accumulate mutant protein to high levels, protea-
(Deng et al., 2006). Similarly, increased hSOD1WT acceler- some activity is downregulated in the lumbar spinal cord
ated disease from a second unstable mutant hSOD1L126Z. well before the development of symptoms (Kabashi
In both cases, accelerated toxicity may be mediated et al., 2004), while in a different line of mice with lower
by stabilization of the mutant via heterodimerization accumulated levels of the same mutant, increased pro-
with hSOD1WT protein. Indeed, in both cases, increased teasome activity in the spinal cord has been reported
levels of hSOD1WT generated detergent-insoluble forms at symptomatic stages (Puttaparthi and Elliott, 2005).
of SOD1 that were not seen in spinal cord extracts of Protein misfolding is a probable initiator of aggregate
hSOD1A4V or hSOD1L126Z mice alone. formation. Thus, many efforts have examined the protein
folding chaperone machinery, the heat-shock proteins
Are SOD1 Aggregates Toxic? (HSPs), in the context of disease. In spinal cord extracts
Regardless of their definition or composition, the contri- of presymptomatic ALS mice, an overall decrease of
bution of aggregates to motor neuron toxicity remains to chaperone activity has been reported which persists
be established. Indeed, it is unknown whether any of throughout disease course, and multiple recombinant
these aggregates are indeed toxic. As introduced in Fig- SOD1 mutants inhibit chaperone function in vitro (Bruen-
ure 1, proposals include aggregate-mediated inhibition ing et al., 1999; Tummala et al., 2005). Paradoxically,
of the proteasome machinery, decreased chaperone some HSPs, such as aB-crystallin and Hsp27 are
Review
43

elevated in the spinal cords of hSOD1G37R and (Higgins et al., 2003). However, vacuoles inferred to de-
hSOD1G93A mice, but Hsp27 is predominantly present rive from degeneration of mitochondria have also been
in glial cells in late disease stages (Vleminckx et al., described in mice with very high accumulated levels of
2002; Wang et al., 2003). Hsp70, Hsp40, and Hsp90 are hSOD1WT protein (Jaarsma et al., 2000, 2001) which do
also elevated but only in the spinal cords of hSOD1G85R not develop disease and are absent in mice that develop
mice (Liu et al., 2005; Vleminckx et al., 2002; Wang disease from dismutase inactive mutants (Bruijn et al.,
et al., 2003). 1997; Jonsson et al., 2006). Thus, vacuolation cannot
Motor neurons have a high threshold for induction of be a primary/common pathogenic mechanism for motor
the stress response, at least in vitro (Batulan et al., neuron degeneration.
2003), which may contribute to their selective vulnerabil- A proportion of the predominantly cytosolic SOD1
ity in ALS. Mutant SOD1-mediated depletion of HSPs is localizes to mitochondria in certain contexts. In both ro-
a plausible possibility given that Hsp70 and Hsp25 pref- dent models and patient samples, mutant SOD1 is pres-
erentially bind mutant SOD1 (Okado-Matsumoto and ent in fractions enriched for mitochondria derived from
Fridovich, 2002). Expression of several different HSPs affected, but not unaffected tissues (Bergemalm et al.,
(Hsp70, Hsp40, Hsp27) in cultured cells and primary mo- 2006; Deng et al., 2006; Liu et al., 2004; Vijayvergiya
tor neurons decreases aggregate content and apoptosis et al., 2005). Mitochondrial localization of SOD1 has
and improves axonal outgrowth (Bruening et al., 1999; been confirmed by electron microscopy in both isolated
Patel et al., 2005; Takeuchi et al., 2002). Unfortunately, mitochondria (Liu et al., 2004) and motor neurons in situ
applying this strategy in vivo by increased expression (Higgins et al., 2002; Sasaki et al., 2004). SOD1 mutants
of Hsp70 in four different mutant SOD1 mouse lines that cause disease at the lowest accumulated levels
did not ameliorate disease or pathology (Liu et al., (hSOD1G85R and hSOD1G127X; all dismutase inactive)
2005). Induction of a broader stress response extended have the highest relative proportions that are mito-
life span in a small cohort of hSOD1G93A mice after treat- chondrially associated (Liu et al., 2004). There is dis-
ment with arimoclomol, a drug which induces the phos- agreement in defining the submitochondrial compart-
phorylation-mediated activation of the HSP inducing ment(s) with which SOD1 is localized (or potentially
factor HSF-1, thereby leading to increased levels of aggregated). Mutant SOD1 has been reported in both
Hsp70 and Hsp90 in spinal cords (Kieran et al., 2004). In- the intermembrane space and the matrix, as well as
terestingly, missense mutations in the gene encoding both the inner and outer membranes of spinal cord
Hsp27 have been identified in a number of families and brain mitochondria (Bergemalm et al., 2006; Deng
with distal hereditary motor neuropathies and CMT neu- et al., 2006; Higgins et al., 2002; Liu et al., 2004; Pasinelli
ropathies (Evgrafov et al., 2004). et al., 2004; Vijayvergiya et al., 2005). While these appar-
ent contradictions remain to be resolved, all reports
Mitochondrial Dysfunction as a Proximal Cause agree that SOD1 mutant proteins of divergent biochem-
of Motor Neuron Death ical characteristics localize to mitochondria, consistent
A common feature of many neurodegenerative diseases with a common contribution to pathogenesis.
is damage to mitochondria that contributes to the de- Key questions are unresolved. Is there some intrinsic
generative phenotype. Mitochondria were implicated feature of spinal cord mitochondria which permits SOD1
as a possible target for toxicity in ALS by histopatholog- association and/or increases mitochondrial vulnerability
ical observations of vacuolated and dilated mitochon- to mutant SOD1? Alternatively, is it the cytoplasmic
dria with disorganized cristae and membranes in the components of chaperone or proteasome activities
motor neurons (and muscle) of both sporadic and famil- which confers some selectivity and facilitates mitochon-
ial ALS patients (Afifi et al., 1966; Hirano et al., 1984a, drial association (Figure 1)? In which cell type(s), if any,
1984b). Mitochondrial defects have been reported in does mitochondrial association of the mutant play a cen-
spinal cords and muscle biopsies of patients ranging tral role in pathogenesis? Of relevance here, the endog-
from histopathological observations to impaired mito- enous wild-type SOD1 protein is largely excluded from
chondrial respiration and increased levels of uncoupling spinal cord mitochondria, but all human SOD1 mutants
proteins (Chung and Suh, 2002; Dupuis et al., 2003; examined to date are mitochondrially associated. The
Echaniz-Laguna et al., 2002; Vielhaber et al., 1999; same human mutant proteins are largely excluded
Wiedemann et al., 2002). However, since all patients from similar preparations of liver mitochondria (Liu
tested were obligatorily symptomatic, it is difficult to de- et al., 2004) despite a proportion of endogenous mouse
termine if these defects are primary or secondary to SOD1 localized to the intermembrane space of those
muscle atrophy. mitochondria (Liu et al., 2004; Okado-Matsumoto and
Mitochondrial abnormalities, especially multimem- Fridovich, 2002). This discordance highlights that not
brane-containing vacuoles, are observed in the motor all mitochondria are created equally, as supported by
neurons of mice that develop disease from accumula- recent proteomic efforts profiling mitochondrial hetero-
tion of dismutase active mutants (hSOD1G37R [Wong geneity across tissues (Kislinger et al., 2006; Mootha
et al., 1995] and hSOD1G93A mice [Dal Canto and Gurney, et al., 2003).
1994; Higgins et al., 2003; Kong and Xu, 1998]) at very How mutant SOD1 affects mitochondrial function is
early, presymptomatic stages (prior to any detectable not yet clear, but differences in protein composition be-
motor neuron loss or other observable damage (Higgins tween mutant and nonmutant mitochondrial popula-
et al., 2003; Kong and Xu, 1998; Wong et al., 1995). It has tions have been reported (Fukada et al., 2004; Kirby
been proposed that vacuole formation is due to expan- et al., 2005; Lukas et al., 2006). It is important to note
sion of the mitochondrial intermembrane space and that the mitochondrial genome itself encodes for only
consequent distention of mitochondrial membranes thirteen of the estimated 1500 proteins required for
Neuron
44

Figure 2. Mutant SOD1-Mediated Damage to Mitochondria

Mutant SOD1 is preferentially imported into or deposited onto mitochondria in affected tissues. The mechanisms by which mutant protein
might damage mitochondrial function are drawn. Mutant SOD1 may interfere with the elements of the electron transport chain, thereby disrupting
ATP-generating oxidative phosphorylation. Mutant SOD1 may also disrupt mechanisms by which mitochondria buffer cytosolic calcium levels.
Mutant SOD1 aggregates may interfere with components of mitochondrial-dependent apoptotic machinery, such as Bcl-2, thereby triggering
premature activation of an apoptotic cascade including cytochrome c release into the cytosol. Mutant SOD1 may indirectly affect similar path-
ways linked to mitochondria by physically blocking the protein import machines, TOM and TIM. Oxidative damage incurred by various mitochon-
drial proteins may also contribute to overall mitochondrial dysfunction. Collectively, these mechanisms (or a combination thereof) are predicted
to disturb cellular homeostasis (within glial and/or motor neurons), ultimately triggering motor neuron death.

function. The majority of mitochondrial proteins are nu- mechanisms that may be central to neuronal damage
clear encoded and thus are synthesized in the cytosol (see Figures 2 and 3 and below).
and subsequently transported into mitochondria. Import
of the majority of mitochondrial proteins is accom- Mitochondria: The Gatekeepers of Cell Death
plished by membrane-spanning multisubunit transloca- Mitochondria are the gatekeepers of apoptosis, with
tors of the outer and inner membrane (TOM and TIM, opening of the permeability transition pore and release
respectively). Mutant SOD1 associated with or aggre- of cytochrome c central to the cascade of caspase acti-
gated onto the mitochondrial surface could impede the vation. A final apoptotic mechanism is probably a central
import machinery (Liu et al., 2004). aspect of neuronal death, as activation of the execu-
Many aspects of the electron transport chain, which tioner caspase-3 is found in mouse models contempo-
generates ATP via oxidative phosphorylation, have raneous with neuronal death (Li et al., 2000; Pasinelli
been reported as either unchanged or altered, although et al., 2000) and lowered levels of the antiapoptotic
there is little or no consensus as to whether these activ- Bcl-2 have been reported in spinal cord motor neurons
ities are accentuated or attenuated. For example, ATP of ALS patients (Ekegren et al., 1999; Mu et al., 1996)
synthesis has been reported as unchanged in aged and mutant SOD1 mice (Gonzalez de Aguilar et al.,
hSOD1G85R mice (Damiano et al., 2006) or depleted in 2000; Vukosavic et al., 2000). Intriguingly, it has recently
late symptomatic hSOD1G93A mice (Mattiazzi et al., been demonstrated that caspase-3 activation in glial
2002), or ATP levels are depleted in presymptomatic cells proteolytically inactivates the glutamate trans-
mice (Browne et al., 2006). Creatine, which extended porter EAAT2 (Boston-Howes et al., 2006). Since
survival in hSOD1G93A mice by alleviating presumed en- EAAT2 is selectively lost during the disease process
ergy deficits (Browne et al., 2006; Klivenyi et al., 1999), and is considered to be a main participant in excitotox-
was ineffective in human clinical trials (Groeneveld icity (which can trigger additional mitochondrial dys-
et al., 2003; Shefner et al., 2004). Complex I activity function), this provides an attractive mechanism by
has been reported as elevated (Bowling et al., 1993; which to unite mitochondrial dysfunction with excitotox-
Browne et al., 1998), reduced (Jung et al., 2002; Kirkine- icity (see later).
zos et al., 2005; Mattiazzi et al., 2002), and unchanged An alternate mechanism, in which SOD1 is proposed
(Damiano et al., 2006). Complex IV activity is reportedly to interact with Bcl-2 and possibly interferes with Bcl-2
reduced, but only at very late disease stages (Kirkinezos antiapoptotic activity (Pasinelli et al., 2004), is attractive
et al., 2005; Mattiazzi et al., 2002). Early impairment in but has recently been disputed (Gould et al., 2006). How-
mitochondrial calcium-buffering capacity in mutant ever, both constitutively increased expression of Bcl-2
SOD1 spinal cord prior to symptoms and only in dis- (Kostic et al., 1997) and virally driven overexpression of
ease-relevant tissues in two different mutant SOD1 Bcl-2 in motor neurons (Azzouz et al., 2000) protect
models (Damiano et al., 2006) is perhaps the most per- motor neurons, delaying disease onset but not progres-
suasive of the efforts, since mitochondrial control of cal- sion. Bcl-2 is unable to similarly rescue motor neuron
cium homeostasis is fully compatible with excitotoxic death in other models, such as wobbler and transgenic
Review
45

Figure 3. Schematic of the Evolution of Motor Neuron Degeneration and Glial Activation during the Course of SOD1 Mutant-Initiated ALS Disease
Four stages are defined (normal, early phase, symptomatic, and end stage). Toxicity is non-cell-autonomous, produced by a combination of
damage incurred directly within motor neurons that is central to disease initiation and damage within nonneuronal neighbors, including astroc-
tyes and microglia, whose actions amplify the initial damage and drive disease progression and spread. Selective vulnerability of motor neurons
to ubiquitously expressed mutant SOD1 is determined by the unique functional properties of motor neurons (e.g., they are very large cells with
large biosynthetic loads, high rates of firing, and respond to glutamate inputs) and damage to their supporting cells in the neighborhood.

mice expressing a point mutation in the NF-L gene A spontaneous dominant mouse mutant Wlds (Waller-
(Coulpier et al., 1996; Houseweart and Cleveland, ian degeneration slow) significantly delays the degener-
1999). Moreover, consistent with a similar outcome in ation of axonal segments located distal of an axonal cut
which intracerebroventricular (ICV) infusion of a broad (axotomy; Glass et al., 1993; Perry et al., 1990). This is
spectrum caspase inhibitor (Li et al., 2000) slowed mu- due to a unique fusion protein containing the first 70 res-
tant SOD1-mediated neuronal death, disease onset idues of the ubiquitination factor UFD2/E4 joined to
was also delayed in the absence of Bax, but progression mononucleotide adenylyltransferase (Nmnat), an en-
was unaffected (Gould et al., 2006), casting doubt on the zyme that facilitates nicotinamide adenine dinucleotide
utility of antiapoptotic therapies. (NAD) synthesis (Conforti et al., 2000; Mack et al.,
2001). Despite conferring robust axonal protection in
Loss of the Neuromuscular Synapse as the First neuropathy mouse models, including the peripheral
Cellular Phenotype in ALS neuropathy model (pmn/pmn; Ferri et al., 2003) and
The axon is the only means by which a motor neuron a model of myelin-related axonopathy (P0-deficient;
communicates with its downstream target muscle, Samsam et al., 2003), Wlds failed to provide a benefit
both sending output information and receiving signaling in three different mutant SOD1 models (Fischer et al.,
inputs from the muscle and other cells. Connection to 2005; Vande Velde et al., 2004).
the muscle at the neuromuscular junction is lost in ALS
mouse models long before motor neuron degeneration Damage within the Axon: Compromised Axonal
or death and the initiation of symptoms (Fischer et al., Transport in ALS
2004; Frey et al., 2000; Kong and Xu, 1998; Pun et al., Motor neurons are among the most asymmetric cells in
2006). It has been long established in humans (Kawa- nature, extending axons in humans that can be more
mura et al., 1981) and in mice (Bruijn et al., 1997; Wong than a meter in length. Alterations in axonal structure
et al., 1995) that it is the motor neurons that generate are well documented in both patients (Hirano et al.,
large caliber (>5 mm) myelinated axons that are selec- 1984a, 1984b; Kawamura et al., 1981) and mutant
tively vulnerable in ALS. This important distinction has SOD1 mice (Bruijn et al., 1998; Kong and Xu, 1998;
now been refined with the demonstration that different Wong et al., 1995), especially the inappropriate misac-
types of motor neurons, innervating different subsets cumulation of neurofilaments, the most abundant struc-
of muscle fibers, have different susceptibilities to mu- tural components of large myelinated axons. Neurofila-
tant SOD1 toxicity. Specifically, in two different mutant ments are obligate heteropolymers of NF light (NF-L),
SOD1 models, the fast-fatiguable motor neurons were NF medium (NF-M), and NF heavy (NF-H) subunits and
shown to be affected first, with denervation of their tar- interestingly, transgenic mice expressing a point muta-
get muscles well before symptoms. Retraction of the tion in NF-L develop motor neuron disease (Lee et al.,
fast-fatigue-resistant motor neurons followed next, 1994). Neurofilament accumulations are seen early in
with the slow type partially resistant to mutant SOD1 mutant SOD1 mice (Kong and Xu, 1998). Removal of all
and actually attempting to reinnervate previously dener- axonal neurofilaments by deletion of NF-L substantially
vated regions (Frey et al., 2000; Pun et al., 2006). prolonged survival of hSOD1G85R and hSOD1G37R mice
Neuron
46

(Nguyen et al., 2001; Williamson et al., 1998), as did re- the known inherited forms are caused by mutations in
moval of most axonal neurofilaments by excessive genes that are ubiquitously expressed (SOD1 and
levels of NF-H (Couillard-Despres et al., 1998; Kong VAPB) or expressed in multiple cells types (VEGF and
and Xu, 2000; Nguyen et al., 2001). Since neurofila- ANG). The initial evidence that damage within more
ment-dependent slowing of slow axonal transport is it- than one cell type was required for disease came from
self directly dependent on the phosphorylation state of expression of mutant SOD1 only within motor neurons
neurofilament tail domains (Ackerley et al., 2003), the in- (Lino et al., 2002; Pramatarova et al., 2001) or astrocytes
creased survival benefit likely derives primarily from re- (Gong et al., 2000). None of these efforts produced mo-
ducing the neurofilament-dependent burden on the ax- tor neuron degeneration or death (albeit the expression
onal transport machinery. This was demonstrated to of mutant SOD1 selectively within motor neurons might
be the case by use of gene replacement to remove the have been at levels too low to initiate disease).
phosphorylated ‘‘tail’’ domains of NF-M and NF-H that More definitive evidence emerged from construction
normally provide intra-axonal crosslinking of adjacent and analysis of chimeric mice that were mixtures of
filaments. Absence of those tail domains sharply slows hSOD1 mutant-expressing cells and normal cells (Clem-
SOD1 mutant-induced disease (Lobsiger et al., 2005). ent et al., 2003). Some animals with only a small minority
Consistent with this, a presymptomatic deficit of slow of normal cells escaped disease, despite having high
axonal transport has been described in mutant SOD1 proportions of mutant motor neurons. These efforts
mice (Ackerley et al., 2003; Williamson and Cleveland, conclusively demonstrated that expression of mutant
1999, Zhang et al., 1997). SOD1 within individual motor neurons, even at levels
Additional evidence for a link between transport and that cause early onset, rapidly progressing disease
motor neuron disease arose from the discovery of a mu- when expressed ubiquitously, is not sufficient to pro-
tation in the p150Glued subunit of dynactin in a family af- voke cell-autonomous degeneration or death of individ-
fected with a slowly progressive, autosomal dominant ual motor neurons of comparable ages.
form of lower motor neuron disease (Puls et al., 2003). Construction and analysis of mice carrying a deletable
p150Glued is responsible for providing processivity by (‘‘floxed’’) mutant SOD1 gene that can be excised by the
bridging between microtubules and cytoplasmic dynein action of the Cre recombinase has identified differential
(Vaughan and Vallee, 1995; Waterman-Storer et al., contribution of mutant damage within motor neurons
1995), a motor powering retrograde axonal transport and nonneuronal neighbors in triggering disease onset
along microtubules (Goldstein and Yang, 2000). Mice and progression (Boillée et al., 2006). Excision of the
heterozygous for either of two point mutations in dynein floxed mutant SOD1 gene exclusively within motor neu-
have mispositioned hindlimbs, a phenotype reflected in rons (by action of Cre recombinase expressed under
the names of the mutations, Legs at odd angles (Loa) control of the motor-neuron-specific promoter Islet-1)
and Cramping1 (Cra1; Hafezparast et al., 2003). Surpris- extended survival by slowing onset and an early phase
ingly, introducing either the Loa (Kieran et al., 2005) or of disease progression. Similar findings by altering com-
Cra1 (Teuchert et al., 2006) mutation to hSOD1G93A ponents expressed only within neurons (i.e., gene dis-
mice significantly improves survival. This unexpected ruption to remove all neurofilaments [Williamson et al.,
amelioration of SOD1 mutant toxicity by a mutation ex- 1998] or gene replacement to alter axonal structure
pected to alter retrograde axonal transport is indeed and volume after elimination of the tail domains of the
a conundrum that remains to be resolved. NF-M and NF-H subunits [Lobsiger et al., 2005]) provide
Further genetic evidence implicating axonal transport extended survival of SOD1 mutant mice but only by
defects as a bona fide pathogenic mechanism in motor slowing disease onset.
neuron degeneration stem from additional mutations in In contrast, diminishing mutant SOD1 levels within mi-
multiple genes relevant to transport in both human dis- croglia and peripheral macrophages (using a Cre trans-
ease and mouse models. These include loss of the kine- gene with a CD11b promoter that is expressed only
sin motor protein KIF1Bb as causative for CMT type 2A within the microglia and peripheral macrophage line-
(Zhao et al., 2001), loss of KIF5A in hereditary spastic ages) had little effect on early disease but sharply
paraplegia (Reid et al., 2002), and loss of KIF21A in slowed later disease progression, extending overall sur-
a rare disorder affecting the oculomotor nerve (Yamada vival by 99 days. Similarly, in a complementary approach
et al., 2003). Mutations in genes involved in transport of the entire myeloid lineage was replaced by transplanta-
membranous vesicles include the ALS8 gene VAPB tion of normal bone marrow cells into SOD1 mutant mice
(Nishimura et al., 2004b), Rab7 in CMT type 2B (Verho- that were themselves unable to synthesize their own
even et al., 2003), and Vps54 (vacuolar-vesicular protein myeloid cells due to deletion of the transcription factor
sorting 54) which is responsible for cervical motor neu- PU.1 (Beers et al., 2006). Transplantation at birth with
ron degeneration in the wobbler mouse (Schmitt-John hSOD1G93A mutant-expressing myeloid cells (which
et al., 2005). populate both the CNS and the periphery) produced
onset and survival typical of the hSOD1G93A mutant
The Neighborhood Matters: Non-Cell-Autonomous line. Replacement of the microglial, monocyte, and
Death of Motor Neurons macrophage lineages with normal cells had no effect
Although progressive paralysis in ALS arises from de- on disease onset but slowed disease progression after
generation and death of motor neurons, evidence from onset.
several directions has converged to demonstrate that Thus, both approaches demonstrated that mutant
toxicity is non-cell-autonomous. That is, toxicity to mo- SOD1 within macrophages/microglial cells accelerates
tor neurons derives from damage developed within cell disease progression, while mutant action within the
types beyond the motor neurons. Consistent with this, motor neurons is a primary determinant of onset and
Review
47

early disease. Importantly, introducing mutant SOD1- Cyclooxygenase-2 (COX-2), produced in abundance
expressing microglial cells into control animals did not by microglia and other inflammatory cells (but also by
give rise to motor neuron disease, demonstrating that neurons and astrocytes), plays a key role in stimulating
mutant SOD1-expressing macrophages/microglial cells production of proinflammatory cytokines, and COX-2
themselves are not sufficient to cause motor neuron expression is induced in spinal cords of ALS patients
death. (Yasojima et al., 2001; Yiangou et al., 2006). In mice,
It should be emphasized that a contribution of periph- use of a COX-2 inhibitor (celecoxib) prolonged survival
eral macrophages to accelerating disease progression by slowing disease onset (Drachman et al., 2002) but
cannot be excluded. Indeed, a proportion of macro- disappointingly did not alter progression after onset.
phages/microglial cells in the spinal cord of mutant Subsequent test of celecoxib in a human trial failed to
SOD1 mice have been shown to enter from the periphery provide a benefit in ALS (Cudkowicz et al., 2006).
during the course of disease in mice (albeit this contribu- Additional evidence implicating microglia in patho-
tion seems minor [Solomon et al., 2006]). There is dis- genesis of ALS arose from forcing activation of the im-
agreement whether replacing all of the peripheral, but mune system using chronic administration of lipopoly-
only a few of the spinal cord, myeloid derived cells by saccharide (LPS), a well-known microglial activator.
bone marrow transplantation following irradiation is ben- Such treatment exacerbated SOD1 mutant-mediated
eficial in slowing disease (Corti et al., 2004; Solomon disease in mice (Nguyen et al., 2004). Similarly, inducing
et al., 2006). However, there is no uncertainty that replac- microglial activation by deleting the fractalkine receptor,
ing both the peripheral and CNS microglial pools slows a cytokine receptor expressed by microglia, mildly ac-
disease progression after onset (Beers et al., 2006). celerated neuronal loss in mutant SOD1 mice (Cardona
Contributions to toxicity developed within astrocytes, et al., 2006). Increased levels of several proinflammatory
the myelinating Schwann cells, or the target muscles factors have been described in the spinal cords of mu-
have not yet been established, although the ‘‘floxed’’ tant SOD1 mice even before motor neuron loss (Elliott,
mutant transgene approach is well suited for such tests. 2001; Hensley et al., 2003; Yoshihara et al., 2002). Cere-
brospinal fluid (CSF) and serum of ALS patients also
contain increased levels of inflammation related factors,
Targets for Therapy: Microglial Cells including complement proteins and monocyte chemoat-
as Determinants of Disease Progression tractant protein-1a (MCP-1a; Goldknopf et al., 2006;
Microglial cells, the macrophages of the CNS, have been Simpson et al., 2004).
long suspected as central components in neurodegen- One particularly interesting cytokine upregulated in
erative diseases where their role may include secretion mutant SOD1 mouse spinal cords which could play
of trophic or toxic molecules. Their role in neuronal de- a role in motor neuron degeneration is tumor necrosis
generation during development is well established factor-a (TNFa). TNFa has been shown to be produced
(Marin-Teva et al., 2004). In ALS, microglial activation in higher levels by adult hSOD1G93A microglial cells
has been described in the brain and spinal cord of pa- when stimulated with LPS compared to nontransgenic
tients (Engelhardt and Appel, 1990; Henkel et al., 2004; microglial cells (Weydt et al., 2004). Administration of
Kawamata et al., 1992; McGeer et al., 1991; Troost a TNFa antagonist yielded a mild increase in survival in
et al., 1993; Turner et al., 2004) and in the spinal cord mutant SOD1 mice (West et al., 2004). A direct toxic ef-
of different mutant SOD1 mouse models (Hall et al., fect of activated microglial cells on motor neurons has
1998; Henkel et al., 2006; Kriz et al., 2002). The microglial been shown in a coculture system and seemed to impli-
reactivity is initiated before motor neuron loss (Henkel cate nitric oxide (Zhao et al., 2004). Microglial cells could
et al., 2006). In addition, increased numbers of dendritic therefore be an important player in a Fas-ligand (FasL)-
cells, potent antigen-presenting cells implicated in the induced apoptosis pathway within motor neurons that is
immune response, have been reported in spinal cords driven by nitric oxide synthesis (discussed in more detail
of ALS patients and hSOD1G93A mice during the disease below). In addition, mutant SOD1, which has now been
course (Henkel et al., 2006; Henkel et al., 2004). reported to be released by motor neurons, is a potent
Several groups have tried to treat motor neuron dis- activator of microglial cells (Urushitani et al., 2006), em-
ease by using minocycline, a tetracycline derivative pre- phasizing the likely crosstalk between motor neurons,
viously shown to inhibit microglial activation (Yrjanheikki microglial cells, and potentially other nonneuronal cells
et al., 1999). Minocycline was potent in increasing sur- that may cooperate to drive disease progression.
vival of ALS mice and reduced microglial activation
(Kriz et al., 2002; Van Den Bosch et al., 2002; Zhu An Outside-In Cascade Triggering a Cell-Death
et al., 2002). The primary effect of minocycline treatment Pathway Intrinsic to the Motor Neuron
initiated in very young mice is a delay in disease onset At least a partial explanation for the selectivity of motor
(Zhu et al., 2002), while administration closer to disease neurons to SOD1 mutant toxicity has arisen from identi-
onset results in a slowing of disease progression after fication of a motor-neuron-specific cell-death pathway.
onset (Kriz et al., 2002). Regardless, both treatment par- Not surprisingly, embryonic motor neurons extracted
adigms provided a comparable extension of survival from spinal cords of mutant SOD1 mice are more sus-
(Kriz et al., 2002). Since minocycline also exerts an anti- ceptible to toxic insults (Kruman et al., 1999; Raoul
apoptotic property to neurons (Zhu et al., 2002), it is un- et al., 2002; Spalloni et al., 2004b; Van Den Bosch
clear in which cells the minocycline effect was active. et al., 2004) and have modified electrophysiology and al-
Either way, supported by its beneficial effect in ALS tered excitability (Pieri et al., 2003; Spalloni et al., 2004a)
mice, minocycline has been proposed for clinical trial compared to neurons from normal mice or mice ex-
(Gordon et al., 2004; Pontieri et al., 2005). pressing high levels of hSOD1WT protein. These neurons
Neuron
48

also have what has been proposed as a motor-neuron- tissues of ALS patients (Rothstein et al., 1992), and
specific cell-death pathway downstream of the Fas levels of EAAT2 are reduced in the motor cortex and spi-
death receptor. Increased susceptibility to death trig- nal cord of ALS patients (Fray et al., 1998; Maragakis
gered by the Fas receptor requires nitric oxide, the Fas et al., 2004; Rothstein et al., 1995; Sasaki et al., 2000),
death domain-associated protein Daxx, and p38 kinase spinal cords of mutant SOD1 mice (Bruijn et al., 1997)
activation, which subsequently drives nitric oxide pro- and rats (Howland et al., 2002), as well as in a model of
duction, thus generating a feed-forward amplification Sindbis virus-induced motor neuron disease (Darman
loop (Raoul et al., 1999, 2002, 2006). Cultured motor neu- et al., 2004). This is functionally of consequence for
rons from transgenic mutant SOD1 mice have an in- familial ALS in that hSOD1G93A mice heterozygous for
creased susceptibility to activation of this pathway, EAAT2 develop earlier-onset disease (Pardo et al.,
which is apparently also activated in presymptomatic 2006), while drugs that increase EAAT2 activity extend
ALS mice (Raoul et al., 2002, 2006; Wengenack et al., survival (Ganel et al., 2006; Rothstein et al., 2005). In-
2004). Given the acceleration of disease by mutant mi- deed, screening of FDA-approved drugs for those
croglia, it is plausible that SOD1 mutant-induced release that could elevate EAAT2 activity has identified a
of nitric oxide by microglia drives this death cascade CNS-penetrating b-lactam antibiotic, ceftriaxone, as a
within motor neurons. transcriptional inducer that modestly extends survival
in hSOD1G93A mice (Rothstein et al., 2005). An inhibitory
Astrocytes in ALS: Gatekeepers of Synaptic effect on quantal glutamate release is also believed to
Glutamate and Excitoxicity underlie the mild slowing of disease course in human
Astrocytes are essential partners of motor neurons, pro- ALS from the only FDA-approved treatment (riluzole;
viding them with trophic support and mediating rapid re- Doble, 1996).
covery of synaptic glutamate through the action of the Additional implication for an astrocytic contribution in
glial glutamate transporter EAAT2 (also referred to as ALS is demonstration in cell culture that activated astro-
GLT-1 in rodents). Astrocytes insert finger-like pro- cytes induce embryonic motor neuron degeneration via
cesses loaded with this transporter into and surrounding their production of NGF, which in the presence of low
synapses, thereby facilitating rapid removal of synaptic concentrations of nitric oxide can induce apoptosis of
glutamate following its release from the presynaptic motor neurons through the p75NTR receptor. Indeed, rel-
terminal (as detailed in Figure 3). Glutamate-mediated ative to resting astrocytes, activated astrocytes in the
excitotoxicity can induce neuronal damage and death spinal cord of hSOD1G93A mice have increased NGF syn-
as a consequence of repetitive firing, which in turn thesis (Pehar et al., 2004).
drives calcium entry through calcium-permeable AMPA
glutamate receptors. Within the spinal cord, preventing Muscle Involvement in ALS: Targets for Therapy?
glutamate-driven excitotoxicity is primarily the job of as- Among the earliest events in the human ALS- and SOD1-
trocytes. Motor neurons have an inherently high sensitiv- mediated disease is withdrawal of the motor axons from
ity to excitotoxicity since their glutamate receptors have the neuromuscular synapse (Fischer et al., 2004; Frey
a lower proportion of the GluR2 subunit (Van Damme et al., 2000; Pun et al., 2006; Schaefer et al., 2005). This
et al., 2002) relative to many other types of neurons. Reg- denervation generates the progressive paralysis of ALS,
ulation of the Ca2+ permeability properties of GluR2 is a consequence of which is muscle atrophy (Cifuentes-
due to posttranscriptional RNA editing, with only AMPA Diaz et al., 2001). Mutant SOD1 is also expressed by
receptors containing edited GluR2 resistant to Ca2+ en- muscle, but whether its presence there contributes to
try. Already with low GluR2 levels, the efficiency of pathogenesis is not yet established.
mRNA editing is reduced in the spinal cord motor neu- A test of whether enhanced muscle mass and strength
rons of ALS patients compared to controls (Kawahara per se can be beneficial in ALS has been attempted by
et al., 2004; Takuma et al., 1999). Altered efficiency of ed- repetitive injection into hSOD1G93A mice of an antibody
iting was not found in hSOD1G93A or hSOD1H46R rats (Ka- to myostatin, a secreted protein whose action inhibits
wahara et al., 2006), thereby demonstrating that change muscle growth. Initiated before disease onset, this pro-
in mRNA editing is not required for familial disease. duced enhanced muscle mass initially, but the effect on
Astrocytes also respond to damage in many settings limb muscles was short lived, yielding neither a survival
by what is referred to as activation. This includes an in- benefit nor preservation of muscle mass throughout dis-
crease in assembly of their intermediate filaments (as- ease progression (Holzbaur et al., 2006).
sembled from glial fibrillary acidic protein, GFAP) and On the other hand, muscle hypertrophy induced by
an increase in the number and size of processes ex- agents such as insulin-like growth factor-1 (IGF-1) or
tended from the cell body (Figure 3). Astrocyte activa- growth hormone (Dobrowolny et al., 2005; Kaspar
tion is seen in spinal cords of ALS patients and SOD1 et al., 2003, 2005) has led to significant life extensions
mutant mice (Hall et al., 1998; Levine et al., 1999; Schiffer in ALS transgenic mice. For the IGF-1 studies, not only
et al., 1996). Indeed, for mice expressing the dismutase was there muscle hypertrophy but also concomitant
inactive mutant hSOD1G85R, a very prominent early pa- stimulation of muscle satellite cell proliferation and an
thology that increases markedly during disease course increase of centrally nucleated muscle fibers, indicating
is the presence of SOD1-containing inclusions within regeneration. Although in one instance IGF-1 synthesis
activated astrocytes (Bruijn et al., 1997). was mediated by a transgene expressed only by the
Altered glutamate handling is one of the few firm muscle (Dobrowolny et al., 2005), it has not been estab-
mechanistic links between sporadic and SOD1 mutant- lished if the secreted IGF-1 acts on the muscle, the mo-
caused ALS. Diminished glutamate transport has been tor neuron, or both. At a minimum, these efforts illustrate
reported in synaptosomes obtained from affected CNS the possibility of using trophic factor synthesis by
Review
49

muscle (see section below) as a way of rescuing and an ensuing excitotoxicity that is accompanied by
motor neurons. Added to this, a number of studies excessive Ca2+ entry that further damages mitochondria
have shown that exercise is beneficial in ALS transgenic and finally triggers a caspase-dependent cell suicide
animals (Kirkinezos et al., 2003; Veldink et al., 2003), pathway within motor neurons.
with exercise and IGF-1 exhibiting a synergistic effect Thus, selective sensitivity of motor neurons to ubiqui-
resulting in an increase in median life span by 83 days tously expressed SOD1 mutants derives from the com-
(Kaspar et al., 2005). The mechanism underlying this bination of risk factors shared by those neurons and
synergism is not known, and the relevance for human their most intimately associated cellular neighbors.
ALS unclear, as no consensus has emerged from several Seen this way, it is clear that the question is not whether
efforts to evaluate a benefit of exercise in human the mitochondrial damage and ensuing oxidative dam-
disease (Drory et al., 2001; Pinto et al., 1999; Scarmeas age hypothesis is right, any more than it is whether the
et al., 2002). hypotheses for glutamate-linked excitotoxicity, axonal
transport clogging, loss of trophic support, or excessive
A Model for Selective Vulnerability of Motor release of toxic species are right. All these mechanisms
Neurons in ALS (which have also been invoked in sporadic ALS) are right
Combining the lessons from multiple animal models and all combine to underlie selective vulnerability. This
used a determine pathogenic mechanisms in inherited is very good news for the design of therapies: inter-
ALS, the central insight is that although motor neuron vention at any of these steps can, in principle, disrupt
degeneration and death is the primary cause of the pro- the toxic cascade between the motor neuron and its
gressive paralysis, toxicity is produced by damage neighbors.
developed not only within motor neurons, but also by
other nonneuronal neighbors (Figure 3). Growth Factor Therapies in ALS
Onset and Early Phase Use of trophic factors has dominated recent efforts in
Damage directly within motor neurons is an important clinical trials in ALS. All of these efforts were predicated
determinant of disease onset. The earliest event, prior on the hope that whatever the primary disease provok-
to disease symptoms, is retraction of motor axons ing insult, provision of increased levels of factors that
from their synapses onto muscles. During this phase, could be trophic for motor neurons would be of benefit.
mutant SOD1 primarily acts directly within motor neu- These have lead to nearly uniform disappointment, pro-
rons, with aggregation of misfolded SOD1 damaging viding little or no benefit for IGF-1 (Borasio et al., 1998;
cellular machinery, especially mitochondria, so as to in- Lai et al., 1997) and no benefit for BDNF (BDNF Study
hibit one or more normal functions. Anterograde axonal Group, 1999) or CNTF (ALS CNTF Treatment Study
transport is also inhibited, as the consequence either of Group, 1996). Aside from the absence of evidence that
mitochondrial damage or of disorganization of axonal growth factors were limiting in ALS, the weakness of
neurofilaments. SOD1 mutant action within the motor these approaches was insuring adequate delivery to
neuron is amplified by action within other cell types, the right cells. One method for eliminating blockage of
especially the microglia which respond to the initial drug delivery by the blood-brain barrier is direct, contin-
damage by amplifying it, thereby driving more rapid dis- uous infusion into either the brain (ICV) or spinal cord
ease progression and spread. (intrathecal; Figure 4B). Continuous intrathecal adminis-
Symptomatic tration of BDNF was found to be without benefit, but ICV
The symptomatic phase is characterized by a massive infusion of IGF-1 extended survival in mice (Nagano
activation of microglia and astrocytes, in addition to et al., 2005a), and a phase I clinical trial using the same
continuing damage within motor neurons themselves. strategy showed a modest benefit to patients without
Misfolded SOD1 mutant within astrocytes, as well as adverse effects (Nagano et al., 2005b).
their activation in response to neuronal damage, in- In animal models, efficacy in growth factor delivery
duces loss of the EAAT2 glutamate transporter, reduc- has been achieved by either of two approaches: viral-
ing rapid recovery of synaptic glutamate and driving mediated gene delivery to convert target cells into facto-
an excitotoxic response that includes excessive influx ries that secrete trophic factors and infusion of trophic
of Ca2+ during repetitive firing of glutamate receptors molecules directly into the brain or spinal cord (Figure 4).
on motor neurons. SOD1 mutant action directly within For the first approach (Figure 4A), an IGF-1-encoding
microglial and astrocytic cells may provoke reduced se- gene was inserted into adenoassociated virus (AAV),
cretion of trophic molecules or increased release of a small replication-defective virus that is episomally
toxic factors (e.g., nitric oxide, TNFa, or mutant SOD1 maintained within the nucleus of a host cell. Injection
secreted or released by cell leakage or lysis). The latter into muscle yielded viral expression within motor neu-
molecules drive spread of toxicity to neighboring cells. rons for at least 1 year. Use of this strategy slowed
Mutant SOD1 can itself activate microglia. In return, disease progression in hSOD1G93A mice even when initi-
microglial cells, through secretion of toxic factors, ated after disease onset (Kaspar et al., 2003). Not sur-
harm their environment, including motor neurons. prisingly, success required the right trophic factor. The
End Stage same AAV producing GDNF used in the same manner
At end stage, denervation has produced paralysis ac- provided only a very modest benefit (Kaspar et al.,
companied by muscle atrophy. Activated microglia or 2003), consistent with prior efforts using GDNF encoded
astrocytes produce diffusible toxic products, including by adenovirus (Acsadi et al., 2002) or AAV (Wang et al.,
nitric oxide and/or TNFa, which accelerate disease 2002) delivered intramuscularly.
spread. Loss of EAAT2 glutamate transporters from as- With the potential that variants in the VEGF gene con-
trocytes drives repetitive firing of glutamate receptors tribute to some examples of ALS (Table 1), delivery of an
Neuron
50

Figure 4. New Directions for Therapies in

ALS Using Viral Delivery or Direct Infusion
into the Brain or Spinal Cord
(A) Delivery by injection into muscle of viral
particles (AAV) encoding trophic factors
such as IGF-1 or VEGF. Viral particles are
taken up at the synapse by the motor neuron
terminal and retrogradely transported to the
motor neuron cell body in the spinal cord.
After translocation into the nucleus, the epi-
somal viral genome is stably expressed for
at least 1 year, converting motor neurons
into factories for secretion of trophic factors.
Trophic factors could also be synthesized
directly in the muscle, increasing their sup-
port of the motor neuron terminal. This ap-
proach is also suitable for viral delivery of
transcription-based siRNA approaches in
which mutant SOD1 mRNAs (or specific cel-
lular mRNAs) are targeted for degradation
selectively within motor neurons. An alterna-
tive is for direct injection of viral particles
into the spinal cord. This approach is more in-
vasive but has the advantage of targeting not
only the motor neurons but also nonneuronal
neighboring cells of the motor neurons.
(B) Direct infusion into the spinal cord (intra-
thecal) or brain (intracerebroventricular, ICV)
of trophic factors directly into the CSF yields
widespread delivery throughout the brain and
spinal cord. Alternatively, ICV infusion of anti-
sense oligonucleotides can reduce mutant
SOD1 by targeting intranuclear degradation
of its mRNA both within the motor neurons
and the surrounding microglial and astrocytic
cells.

integrating lentivirus encoding VEGF (and pseudo- Three efforts using siRNA to catalyze degradation of
coated so as to be retrogradely transported) extended the mRNA encoding SOD1 have proven successful in
survival of hSOD1G93A mice (Azzouz et al., 2004). So hSOD1G93A mice. In the most comprehensive, a lentivi-
too did continuous ICV infusion of recombinant VEGF rus encoding such an siRNA was injected into multiple
protein into the CSF (Figure 4B): disease onset was de- muscles of very young mice (an age equivalent to inject-
layed and survival extended by 22 days (Storkebaum ing infants in humans). After retrograde transport to mo-
et al., 2005). This ICV delivery of VEGF was especially ef- tor neuron cell bodies, reducing SOD1 mutant synthesis
fective in slowing forelimb paralysis, suggestive of in motor neurons had a remarkable effect in slowing dis-
a higher concentration of VEGF closer to the site of infu- ease initiation (108 days) and extending overall survival
sion. A modest benefit was seen even when VEGF treat- by 99 days (Ralph et al., 2005). Despite sharply delayed
ment was initiated after symptomatic onset. Unresolved disease onset through gene silencing within motor neu-
is which cells are targeted by this VEGF. Weekly intra- rons but not other cells of the CNS, disease progression
peritoneal injection of VEGF also has been reported to was not slowed at all, with animals reaching endstage
slow disease in hSOD1G93A mice (Zheng et al., 2004), w25% faster in the treated animals (Ralph et al., 2005).
raising the possibility that important target cells are out- Similar slowing of disease onset was seen by retrograde
side the CNS, including those in the vasculature. delivery of an siRNA-encoding AAV (Miller et al., 2005).
Direct viral injection of a similar siRNA virus into the
spinal cord (to target both motor neurons and their non-
Gene Therapies in ALS neuronal neighbors) delayed degeneration of motor
With recognition that the instances of ALS caused by neurons near the site of injection but it could not be de-
dominant mutation in SOD1 derive from a toxic property termined whether this affected disease progression
of the mutant protein and that even complete absence of (Raoul et al., 2005).
dismutase activity (by gene deletion) does not cause In considering extension of such viral efforts to the hu-
overt disease in mice (Reaume et al., 1996), strategies man setting, substantial practical issues remain. Current
to limit the synthesis of the mutant gene product have viral vectors provide no mechanism for altering dosage
become an increasingly realistic possibility. Two general or discontinuance of therapy and face significant chal-
strategies have been used: virally delivered, transcrip- lenges for reaching widespread areas of the CNS. An
tion-based RNA silencing (siRNA), and direct infusion alternative approach that surmounts both of these is
of antisense oligonucleotides (Figure 4). targeted mRNA degradation after direct ICV delivery of
Review
51

synthetic antisense oligonucleotides into the CNS. differentiated into motor neurons showed that the
Small (15–25) nucleotide sequences of DNA bind by grafted motor neurons themselves, rather than other
Watson-Crick hybridization to a target mRNA in a se- surviving derived stem cells, account for the improve-
quence-specific manner. The mRNA in such a heterodu- ment. Whether this strategy will be successful in a model
plex is a substrate for catalytic, intranuclear degradation of chronic motor neuron degeneration, such as ALS, re-
by endogenous RNase H. This approach has been suc- mains to be determined.
cessfully used for lowering mutant SOD1 levels by Other strategies with hSOD1G93A mice have used (1)
w50% throughout the brain and at all levels of the spinal human neural stem cells grafted into the spinal cord
cord of hSOD1G93A rats. Initiated near disease onset, (Yan et al., 2006), (2) human umbilical cord blood cells
this approach successfully slowed progression after on- transfused into the systemic circulation (Garbuzova-
set (Smith et al., 2006), a benchmark for a human ther- Davis et al., 2003), or (3) bone marrow transplant (Corti
apy. Similar ICV administration to nonhuman primates et al., 2004), and each has been reported to provide
demonstrated effective delivery to both the motor neu- some extension in survival (Corti et al., 2004). One study
rons and nonneuronal neighbors throughout the brains reported an early protection of motor neurons which
and spinal cords (Smith et al., 2006). These efforts es- was not sustained at end stage (Corti et al., 2004).
tablish that direct delivery of antisense oligonucleotides An alternative strategy is the idea of stimulating the
can be an effective, dosage-regulatable means of treat- endogenous pool of stem cells for replacement of de-
ing neurodegenerative diseases, including ALS. Added generating motor neurons. In hSOD1G93A mice, an in-
to this, direct infusion of siRNA duplexes, assuming is- crease in neural progenitor cell proliferation, migration,
sues of stability can be surmounted, add an additional and neurogenesis in the lumbar region of the spinal
therapeutic possibility, including the ability to target cord has been observed in response to motor neuron
a mutant-encoding mRNA without affecting the wild- degeneration (Chi et al., 2006). Furthermore, the endog-
type mRNA (Xia et al., 2006). enous recruitment of neural progenitors (as determined
by nestin staining) was initiated on the predominantly
Stem Cell Therapies in ALS symptomatic side of asymmetrically paralyzed
The selective, age-dependent killing of motor neurons hSOD1G93A rats (de Hemptinne et al., 2006). Whether
has made ALS one of the poster diseases for cell re- endogenous neuroprogenitors could be used for
placement using stem cells. Replacement of human mo- generating new motor neurons is not yet established, al-
tor neurons, however, faces daunting challenges. The though examination of the neural stem cell/neuroproge-
lower motor neurons have their cell bodies scattered nitor niches of the forebrain has revealed alterations in
throughout the length of the spinal cord, so that any hSOD1G93A mice (Liu and Martin, 2006). It must also be
cell replacement might require multiple spinal injections recognized that any new neurons produced by such an
at all levels of the cord. Further, even if the developmen- approach will obligatorily express mutant protein and
tal program can be achieved and issues of inhibition of will also be at risk.
axonal extension within the CNS are overcome, re- Finally, with the recognition of non-cell-autonomous
growth and correct targeting of meter-long axons will contributions to motor neuron toxicity (Boillée et al.,
require at least a year (assuming the maximal rate of 2006; Clement et al., 2003), other stem cell strategies
human axonal extension observed in early postnatal to replace nonneuronal precursors, including those en-
development). gineered to produce trophic factors such as IGF-1, are
These issues not withstanding, recent successes now sensible approaches.
have overcome several of the important hurdles to
the feasibility of a stem cell replacement strategy after
acute motor neuron killing induced by Sindbis virus Acknowledgments
exposure (Deshpande et al., 2006; Harper et al., 2004). S.B. is a recipient of a Fondation pour la Recherche Medicale
In a first series of experiments, mouse embryonic stem fellowship and an INSERM fellowship. C.V.V. is a recipient of a
(ES) cells were differentiated into motor neuron precur- Development Grant from the Muscular Dystrophy Association
sors and were injected into rat spinal cords paralyzed and a fellowship from the PVA Spinal Cord Research Foundation.
by Sindbis virus exposure. A quarter (w3000 cells) of D.W.C. receives salary support from the Ludwig Institute for Cancer
the in vitro differentiated, ES cell-derived motor neurons Research. This work has been supported by a grant from the NIH
(NS27036) to D.W.C.
survived within the spinal cord and even a few of them
extended axons into the ventral roots, but only when
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