Perspectives on models of spinal muscular atrophy for drug discovery

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DISEASEMODELS

Drug Discovery Today: Disease Models Vol. 1, No. 2 2004

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Denis Noble – University of oxford, UK

Central nervous system

Perspectives on models of spinalmuscular atrophy for drug discoveryMatthew E.R. Butchbach1, Arthur H.M. Burghes1,2,3,*1Department of Molecular and Cellular Biochemistry, The Ohio State University, 363 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA2Department of Neurology, College of Medicine and Public Health, The Ohio State University, Columbus, OH, USA3Department of Molecular Genetics, College of Biological Sciences, The Ohio State University, Columbus, OH, USA

Spinal muscular atrophy (SMA) is a progressive motor

neuron disease, which is one of the most common

forms of inheritable infant death in the world. SMA

results from reduced levels of SMN protein. Various

models systems used in SMA research are available and

it is possible to assess their strengths and weaknesses.

Although each of these models has its limitations, they

must be used together so as to effectively and effi-

ciently identify potential SMA therapeutics.

*Corresponding author: (A.H.M. Burghes) [email protected]

1740-6757/$ � 2004 Elsevier Ltd All rights reserved. DOI: 10.1016/j.ddmod.2004.07.001

Section Editors:Philip C. Wong, Donald L. Price—The Johns HopkinsUniversity School of Medicine, Baltimore, MD, USA.

This review describes the in vitro and animal models that are currently

being used to identify and characterize drug compounds that couldpotentially ameliorate the neurodegenerative phenotype of spinal

muscular atrophy.

Introduction

Spinal muscular atrophies (SMAs) are a family of neurological

diseases arising from the degeneration of a motor neurons in

the ventral horn of the spinal cord resulting in muscle

atrophy. Proximal autosomal recessive SMAs (incidence =

1:10,000) have been classified into three distinct groups based

on age of onset and clinical milestones (Table 1). The survival

motor neuron (SMN) protein is encoded by two genes in

humans: the centromeric SMN gene (SMN2; GenBank acces-

sion number NM_017411) and the telomeric SMN gene

(SMN1; GenBank accession number NM_000344) [1]. Loss

or mutation of the SMN1 gene but not SMN2 causes SMA.

SMN1 and SMN2 are nearly identical with the exception of a

single nucleotide that modulates the inclusion of exon 7 into

SMN transcripts. The majority of transcript from the SMN2

gene lacking exon 7. The loss of exon 7 results in a protein

that does not self-oligomerize efficiently and, therefore, is

rapidly degraded (Fig. 1). The SMN2 gene does produce some

full-length message (10–15%) and, therefore, more copies of

SMN2 positively modulate the SMA phenotype.

In vitro models of SMA

Because SMN is a ubiquitously expressed protein, cells

derived from most types of tissues can be used to study the

biology of SMN. However, SMA is a motor neuron disease

caused by deficiency of SMN; low levels of SMN are of no

consequence to tissues other than motor neurons. Although

interesting, it is not clear that the functions identified by

studies in other cell types are crucial for the progression of

SMA. It is also important to distinguish between functions

affected by low expression of (hypomorphic allele) and com-

plete absence of (null allele) SMN. Cultures of various cell

types can be used to screen for drug molecules that activate

expression of SMN2 or supplement functional defects in

those cells. Secondary or tertiary screens in motor neuron

cultures or animal are then used to determine their effects.

These molecules can be used to unravel the crucial biology

of the disease as well as can be of potential use for the

treatment of SMA. Screens of cell lines have been instrumen-

tal in identifying many compounds that can increase the

expression of SMN2 and the inclusion of exon 7 in SMN2

www.drugdiscoverytoday.com 151

Drug Discovery Today: Disease Models | Central nervous system Vol. 1, No. 2 2004

Table 1. International SMA consortium clinical classification of autosomal recessive proximal spinal muscular atrophy

Type Synonym OMIM

number

Age of onset

(months)

Ability

to sit

Ability

to stand

Ability

to walk

Age at death

(years)

I Werdnig-Hoffmann 253300 <6 No No No <2

II Chronic childhood SMA 253550 <18 Yes No No >2

III Kugelberg-Welander 253400 >18 Yes Yes Yes Adult

transcripts. These compounds include interferons-b and -g

(INFb and INFg; [2]), forskolin [3], ortho-vanadate [4], aclar-

ubicin [5], butyrate [6], 4-phenylbutyrate [7] and valproic

acid [8,9]. These are the first compounds identified from

screens and, in most cases, have subopitmal therapeutic

properties, that is, they are toxic, require high doses or are

rapidly metabolized. It is important to obtain better com-

pounds with the desired properties or to consider the use

of pro-drugs so as to obtain sufficient serum levels of the

drug in vivo.

In vivo models of SMA

Mice have long been used as animal models to understand the

pathophysiology of SMA. Before the identification of SMN as

the SMA gene, many spontaneously generated mutant mice

were used to understand the mechanisms underlying motor

neuron disease and to test various neuroprotective therapies

[10]. Unlike humans, there is one Smn gene in mice (mSmn;

GenBank accession number NM_011420). Removal of mSmn

by gene ablation results in embryonic lethality [11,12].

Disruption of one mSmn allele (mSmn+/�) results in a 40%

Figure 1. Difference between SMN1 and SMN2 genes and the molecular basis o

within exon 7. This change disrupts the activity of an exon splice enhancer within

Translation of these SMN2D7 mRNAs produces a truncated protein (shown by t

SMN protein can also disrupt oligomerization of full-length SMN. SMA results fro

less SMN protein than SMN1. Modified from [1].

152 www.drugdiscoverytoday.com

loss of motor neurons in the spinal cord of mSmn+/� mice at

six months of age [13]. These studies demonstrate that SMN is

essential for survival which is what one would expect given

the important role of SMN in snRNP biogenesis.

To generate mouse models of human SMA, one needs to

overcome the embryonic lethality that results from the loss of

mSmn. Two strategies have been used to circumvent this

lethality: (1) insertion of human SMN2 in a mSmn null back-

ground and (2), conditional ablation of mSmn in specific

tissues. Two groups [12,14] independently generated trans-

genic mice that harbor genomic DNA containing the com-

plete human SMN2 gene and interbred these mice onto a

mSmn null genetic background. The expression of human

SMN2 rescues the embryonic lethality resulting from the loss

of mSmn [12,14]. SMN2;mSmn�/� mice carrying one to two

copies of SMN2 from both groups develop clinical pheno-

types indicative of severe SMA and survive for 6–10 days. The

loss of motor neurons in severe SMA is a late-onset phenom-

enon because a significant loss of spinal motor neurons

occurs at three to five days of age in severe SMA mice whereas

there is no significant reduction in the number of motor

f SMA. The main difference between SMN1 and SMN2 is a C!T conversion

exon 7, thereby reducing the incorporation of exon 7 into the SMN2 mRNA.

he incomplete circles) which is unstable and rapidly degraded. The truncated

m the loss of SMN1 but retention of SMN2; SMN2, however, produces much

Vol. 1, No. 2 2004 Drug Discovery Today: Disease Models | Central nervous system

neurons in the spinal cord of severe SMA mice at one day of

age [14]. Both severe SMA transgenic mice produce low levels

of SMN protein [12,14]. We have also observed that intro-

duction of 8–16 copies of human SMN2 into an mSmn null

background completely rescues the embryonic lethality phe-

notype [14]. These high-copy SMN2;mSmn�/� mice live a

normal lifespan and show no overt motor neuron degenera-

tion and muscle weakness. These studies suggest that increas-

ing the expression of SMN2 to compensate for the loss of

SMN1 might be beneficial for the treatment of SMA in

humans. However, two important questions arising from

these studies remain to be answered: when during develop-

ment does SMN expression need to be increased; and in

which tissues is increased expression required for ameliora-

tion of disease severity?

A conditional knockout of mSmn was created using the

Cre–LoxP system whereby mSmn exon 7 was deleted [15,16].

The resultant double transgenic mice produced mRNA and

protein that lacked exon 7 (SMND7) in those tissues that

expressed the Cre recombinase. Two different types of mice

were generated: ablation of mSmn in neurons by placing Cre

under the control of the neuron-specific enolase (NSE) pro-

moter [15] and ablation of mSmn in mature skeletal muscle by

placing Cre under the control of the human skeletal muscle a-

actin (HSA) promoter [16]. Motor deficits were observed in

NSE:Cre;mSmnF7/D7 mice at around two weeks after birth and

these mice survived for 17–36 days [15]. There was a signifi-

cant reduction in the number of motor neurons in the ventral

horns of NSE:Cre;mSmnF7/D7 mice [16]. Do these mice truly

mimic the situation that occurs in SMA? First, it is important

to consider the time at which high levels of SMN are required

to prevent SMA. It would be hard for this model to mimic the

development profile of low SMN expression. Second, the

expression of Cre in all neurons will result in the removal

of exon 7 and no full-length SMN being produced. This

contrasts with what is observed in SMA where low amounts

of full-length SMN are produced. A knockin of the human

SMN2 exon7 would more closely resemble the situation in

SMA but these mice would have to be crossed onto SMN2

transgenic lines so as to modulate copy number of SMN2.

Lastly, given that NSE is a pan-neural promoter it would be

interesting to know if neurons other than those in the motor

system were affected in these mice.

The use of Cre-mediated conditional gene knockout has

been extended to selective deletion of SMN in muscle, which

resulted in a dystrophic phenotype and, more recently,

the liver where it appears to be lethal. In the muscle mutant

mice (HSA:Cre;mSmnF7/D7), paralysis was observed within

three weeks after birth and these mice survived for 28–37

days [16]. Ablation of mSmn in skeletal muscle resulted

in a pathology similar to that seen in muscular dystrophy

(i.e. variability in muscle fiber size, fibrosis, presence of

inflammatory cells and centrally-nucleated regenerating

myofibers). The authors of these studies believe that skeletal

muscle in addition to motor neurons might have primary

roles in the pathogenesis of SMA. We feel that SMN is an

essential protein for all cell types; therefore its depletion by

Cre-mediated ablation will essentially be lethal to that cell

type, cause its destruction and a phenotype that would be

consistent with loss of an essential gene in that tissue. In

SMA, there is sufficient SMN for most cell types but not in

motor neurons. We are not certain whether high levels of

SMN are needed throughout the lifetime of a motor neuron or

only during a specific time. It would be interesting to cross-

breed the SMN2 transgene onto the NSE:Cre;mSmnF7/D7 mice.

If SMN2 rescues the phenotype of these mice then it would

indicate that high levels of SMN are not required later in

motor neuron maintenance but are required early. These

experiments would provide important information about

the mechanism of SMN2-induced amelioration of phenotype

severity in SMA mice.

It has been suggested that SMND7 is detrimental in SMA.

Those compounds that would activate the SMN promoter

would then be problematic for the treatment of SMA because

SMND7 levels would be increased in addition to full-length

SMN levels. To determine definitively if SMND7 is either

beneficial or detrimental to SMA, transgenic mice were gen-

erated that express SMND7 and these mice were then interbred

onto a severe SMA genetic background (SMN2;mSmn�/�; T.T.

Le, L.T. Pham, D.D. Coovert, U.R. Monani, MERB, T.O. Gavri-

lina, and AHMB, unpublished). Introduction of SMND7 to

severe SMA mice, in fact, extended survival by 160% (from

5.2 to 13.3 days). The SMND7;SMN2;mSmn�/� mice develop a

progressive degenerative phenotype starting at around five

days of age characterized by difficulty in righting themselves,

ambulation and gait abnormalities (Le et al., unpublished).

These mice lose ~20% of their spinal motor neurons at nine

days of age whereas younger mice (four days) have no sig-

nificant loss of motor neurons. Immunoblot analysis shows

that SMND7;SMN2;mSmn�/� mice produce SMND7 protein but

the amount of SMND7 produced is, at most, equivalent to the

amount of full-length SMN protein produced by the human

SMN2 transgene. SMND7 produced at those levels found in

SMND7;SMN2;mSmn�/� mice appears to be beneficial and not

detrimental to SMA mice. These observations suggest that

SMND7 protein is unstable and rapidly degradable. The

amount of SMND7 produced in these mice might more closely

approximate the levels of SMN protein that would be pro-

duced upon induction of SMN2 expression in vivo. These mice

are particular advantageous in SMA for testing drug com-

pounds because the drugs can be introduced by oral gavage

or subcutaneous injection as early as two days. The progression

of the motor neuron disease can be monitored easily with

survival being used as an outcome measure.

Expression of a SMN1 missense mutation (A2G) that

occurs in type III SMA patients on a severe SMA genetic



Links

� National Institute of Neurological Diseases and Stroke

(www.ninds.nih.gov/health_and_medical/disorders/sma.html)

� The SMA Project (www.smaproject.org)

� Families of SMA (www.fsma.org)

� Andrew’s Buddies (www.fightsma.org)

� SMA Foundation (www.smafoundation.org)

background partially rescues the severe SMA phenotype

in the mouse [17]. The SMN(A2G) transgene, however,

cannot rescue the embryonic lethality resulting from the

ablation of mSmn without the presence of SMN2. The

SMN2;SMN(A2G);mSmn�/� mice have a delayed onset of

motor neuron loss and display motor characteristics indica-

tive of type III SMA in humans [17]. Furthermore, adult

SMN2;SMN(A2G);mSmn�/� mice show significant loss of

motor neuron cell bodies in the lumbar spinal cord and

a consequent loss of motor axons in the ventral roots.

Electromyography shows abnormal spontaneous electrical

activity – in the form of fibrillation potentials and biphasic

positive sharp waves – in the skeletal muscles of adult

SMN2;SMN(A2G);mSmn�/� mice indicative of denervation

injury [17]. This denervation injury leads to muscle atrophy

as evidenced by the presence of atrophied fibers in affected

muscles from SMN2;SMN(A2G);mSmn�/� mice. The milder

phenotypes of the SMN2;SMN(A2G);mSmn�/� mice along

with the SMND7;SMN2;mSmn�/� mice relative to the

SMN2;mSmn�/� mice demonstrate the importance of the

expression level of SMN (even mutated SMNs) in modulating

the severity of SMA. These mice with less severe SMA phe-

notypes permit a more detailed study of the effect of drugs

using behavioral and electrophysiological parameters to

monitor disease progression. Breeding of SMA mice onto

different genetic backgrounds can result in a modified phe-

notype that could be useful for certain experiments.

Studies in zebrafish (Danio rerio) would supplement or

provide an alternative to those studies in mice and should

allow rapid screening of drug molecules in vivo. Although this

organism is particularly advantageous for developmental

studies, it could also be adapted for in vivo screening of drug

compounds. Using antisense morpholino oligonucleotides

(MOs) directed against zebrafish SMN, the levels of SMN

can be reduced in zebrafish so as to mimic SMA [18]. Only

motor neurons are affected in SMN MO-injected zebrafish.

There is initial abnormal outgrowth of axons followed by

excessive branching around the second choice point (inter-

mediate target). Coinjection of human SMN mRNA with

zebrafish SMN antisense MOs rescues the truncation and

branching defects [18]. Interestingly, motor neurons cultured

from severe SMA (SMN2;mSmn�/�) mice show reduced axon

length and a reduction in the amount of b-actin at axonal

growth cones [19]. SMN has also been shown to travel along

axons in discrete particles [20]. These findings taken together

begin to underscore the importance of SMN-containing

macromolecular complexes in the motor neuron axons in

SMA. The SMN knockdown experiments in zebrafish demon-

strate the importance of SMN in motor axon development.

This study also indicates that the deficiency of SMN acts in a

cell-autonomous manner suggesting that low levels of SMN in

muscle is of minimal importance in the pathogenesis of SMA.

The further development of zebrafish SMA models might


allow rapid, in vivo screens for phenotypic correction mole-

cules These putative compounds identified in the zebrafish

models could then be tested with the mouse models of SMA.

In silico models of SMA

It is currently difficult to conceive in silico models of SMA as

we are uncertain about the function of SMN. Certainly, it is

important to determine the targets of the identified drugs. In

the case of SMN promoter activation, INFb, INFg [2] and

forskolin [3] were identified as potential SMN2 inducers by

analyzing the promoter region of human SMN2 (GenBank

accession number: AF187725). Putative drugs that induce

SMN2 expression might be identified by identifying novel

transcription factor binding sites within the SMN2 promoter.

Of course, these agents would have to be tested in both the

primary culture and animal models of SMA. We also do not

fully understanding the alternative splicing of SMN tran-

scripts in humans. Specifically, we have not completely

identified the intrinsic and extrinsic factors required for

the inclusion of exon 7 in SMN transcripts. Only after a

comprehensive characterization of the splicing factors impor-

tant for the inclusion of exon 7 is complete can an in silico

model be generated that truly represents SMA.

Model comparison

In silico identification of SMN2-inducing agents is based on

available information about the transcription factors that

bind to the SMN2 promoter, but not all of the transcription

factors found in human, or in animal models, have been

characterized. Additionally, a truly effective therapeutic

agent against SMA has to increase the efficiency of inclusion

of SMN2 exon 7 as well as increase SMN2 transcript levels.

There are currently no in silico means of identifying factors –

and ultimately, drugs – that affect the splicing of transcripts.

The use of SMA-derived cultured cells can provide valuable

initial data regarding the effectiveness of drugs that increase

the levels of SMN. These in vitro models can easily be used for

high-throughput candidate drug screening. Once identified

in a high-throughput-based screen, the candidate molecules

can be tested with secondary screens to ensure they have the

desired properties and, in most cases, developed more che-

mically to have the required potency and properties.

The zebrafish and mouse models allow drugs to be screened

for in vivo activity. The zebrafish model could be used for the

Vol. 1, No. 2 2004 Drug Discovery Today: Disease Models | Central nervous system

Table 2. Summary of models of SMA

In vitro models In vivo models In silico models

Pros Easy to grow and maintain Mimic the genetics and

pathogenesis of SMA

Inexpensive information

is readily available

Fairly inexpensive Can test multiple aspects

of SMA pathology

Amenable to

high-throughput

drug screening

Can observe any adverse

drug interactions

Cons Does not mimic motor

neuron disease

Expensive to produce

and maintain

Based on promoter predictions

of known transcription factor binding sites

Assumes same magnitude

of inducibility between

fibroblasts and neurons

Large number of animals

for proper screening

No information about factors

which regulate splicing

Need to determine

best route of delivery

Based on promoter predictions

of known transcription factor binding sites

Best use of model Initial screen of

SMN2-inducing

compounds

Identification of candidate

SMN2-inducing compounds

Preliminary identification

of potential SMN2-induced compounds

How to get access

to the model

Through the investigators Through the investigators; some

have been deposited into the

Jackson Laboratory (www.jax.org)

NCBI (GenBank accession number AF187725)

Relevant patents n/a n/a n/a

References [3–11] [14–20] [4,5]

identification of candidate drugs that alter a particular phe-

notype in a medium-throughput manner. Inducers of SMN or

molecules that complement function with the desired prop-

erties can be tested in the available SMA mice. It is important

to select the correct animal model of SMA for drug screening.

The complete absence of functional SMN in a specific tissue

(i.e. the conditional gene ablation models [15,16]) does not

accurately mimic SMA in human. SMA results from the loss of

SMN1 and low levels of SMN protein resulting from SMN2.

Table 2 summarizes the strengths and weaknesses of in silico,

in vitro and in vivo models of SMA. To effectively and effi-

ciently identify potential SMA therapeutics, each of these

models must be used in concert.

Related articles

Chang, J.G. et al. (2001) Treatment of spinal muscular atrophy by sodium

butyrate. Proc. Natl. Acad. Sci. USA 98, 9808–9813

Lesbordes, J.C. et al. (2003) Therapeutic benefits of cardiotrophin-1 gene

transfer in a mouse model of spinal muscular atrophy. Hum. Mol. Genet.

12, 1233–1239

Haddad, H. et al. (2003) Riluzole attenuates spinal muscular atrophy

disease progression in a mouse model. Muscle Nerve 28, 432–437

Mercuri, E. et al. (2004) Pilot trial of phenylbutyrate in spinal muscular

atrophy. Neuromuscul. Disord. 14, 130–135

Russman, B.S. et al. (2003) A phase 1 trial of riluzole in spinal muscular

atrophy. Arch. Neurol. 60, 1601–1603

Model translation to humans

Data obtained from model systems have been extremely

valuable in developing and testing potential therapies

against SMA. In fact, some of the drugs tested using in vitro

and in vivo SMA models have been used in early clinical

drug screenings (see ‘Related articles’). Although all

SMA models have provided useful information, those

in vivo models based on the insertion of human SMN2 into

a mSmn null background most closely mimic SMA in

humans. These in vivo models should be used for future

testing of drugs that can induce SMN levels as well as for

other SMA therapies.

Conclusions

In summary, we have model systems of SMA that are

founded on computer-based screening of the SMN2 promo-

ter (in silico), primary cultures derived from SMA patients

(in vitro) and genetically engineering animal models

(in vivo). Although each model system has its strengths

and weakness, we have learned a great deal about SMA from

these models. These model systems can be used to examine

unresolved issues regarding the pathobiology of SMA and

the role of SMN in SMA (Outstanding issues). These different

models can also be used to identify potential candidate

drugs that would ameliorate the progression of SMA in

patients.



Outstanding issues

� What is (are) the function(s) of SMN in motor neurons?

� Which type(s) of cells is (are) primarily involved in the pathogenesis of

SMA?

� How does motor neuron degeneration occur in SMA? Is it different

from other types of motor neuron disease?

� For SMN2 inducer-based therapies, where and when does SMN have

to be induced to ameliorate the SMA phenotype?

� What is (are) the best mode(s) of increasing SMN levels in SMA (i.e.

drug, gene therapy)?

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Perspectives on models of spinal muscular atrophy for drug discovery