Abstract

Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration.

Introduction

Neurodegenerative diseases are characterized by the selective vulnerability of specific neuronal populations to toxic processes of genetic and/or environmental origin. Somatic motor neurons degenerate in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. However, not all somatic motor neurons are equally affected by the events leading to degeneration. While ventral spinal motor neurons are affected in all three diseases, and motor neurons of the lower cranial nerves (CNs) [e.g. hypoglossal (CN12)] degenerate in amyotrophic lateral sclerosis and spinobulbar muscular atrophy, upper CNs [e.g. oculomotor/trochlear (CN3/4)] are generally spared in spinal muscular atrophy, amyotrophic lateral sclerosis and spinobulbar muscular atrophy (Leveille et al., 1982; Gizzi et al., 1992; Reiner et al., 1995; Sobue, 1995; Nimchinsky et al., 2000; Haenggeli and Kato, 2002). While spinal muscular atrophy is recessively inherited and caused by loss of functional survival of motor neuron (SMN1) protein (Bussaglia et al., 1995; Lefebvre et al., 1995), spinobulbar muscular atrophy is an X-linked disorder, caused by the expansion of CAG repeats in the androgen receptor gene (Kennedy et al., 1968; La Spada et al., 1991). The vast majority of amyotrophic lateral sclerosis cases appear sporadic (∼90%). However, amyotrophic lateral sclerosis can be inherited (familial) (∼10%) due to mutations in e.g. superoxide dismutase 1 (SOD1) (Rosen et al., 1993), angiogenin (Greenway et al., 2006) or the DNA/RNA-binding proteins transactivation response element DNA binding protein (TDP-43) (Kabashi et al., 2008; Sreedharan et al., 2008) or FUS (Kwiatkowski et al., 2009; Vance et al., 2009). Importantly, differential vulnerability among motor neurons appears independent of the cause of disease, since the pathology and pattern of selective motor neuron vulnerability is similar in familial and sporadic amyotrophic lateral sclerosis (Shaw et al., 1997).

Neurodegeneration in many diseases appears to involve cell-autonomous and non-cell-autonomous events (Garden et al., 2002; Boillee et al., 2006; Jaarsma et al., 2008; Chung et al., 2009). Familial amyotrophic lateral sclerosis model data indicate that factors intrinsic of motor neurons are crucial for initiation of degeneration, while non-cell-autonomous events are instrumental for disease progression (Beers et al., 2006; Boillee et al., 2006; Nagai et al., 2007; Di Giorgio et al., 2008; Hedlund and Isacson, 2008; Marchetto et al., 2008; Yamanaka et al., 2008). Furthermore, neuronal death in Huntington’s disease, a polyglutamine expansion disease like spinobulbar muscular atrophy, involves intrinsic and exogenous events (Ross, 2004; Gu et al., 2005), suggesting that spinobulbar muscular atrophy could be due to a combination of these two.

We therefore hypothesized that dissecting the intrinsic molecular code underlying the normal physiology of motor neurons that display differential vulnerability to disease could provide a basis for revealing why one motor neuron subpopulation is more vulnerable to degeneration than another. Previous analysis of the differential vulnerability of substantia nigra and ventral tegmental area dopamine neurons to degeneration in Parkinson’s disease showed that the differential gene expression pattern of neuronal populations in the normal animal can be used to elucidate mechanisms that can protect vulnerable neurons from disease (Chung et al., 2005). Here we used laser capture microscopy (LCM) to isolate motor neurons from CN3/4, CN12 and the lateral motor column of the cervical spinal cord of the normal rat and performed an analysis of the entire rat transcriptome. Differential expression of selected genes with implications for motor neuron vulnerability was confirmed by localization of the resulting proteins. Functional in vitro analysis revealed that the CN3/4-specific proteins insulin like growth factor (IGF)-II and guanine deaminase, could protect motor neurons from glutamate-induced toxicity.

We believe that this report provides insight into the intrinsic properties of different motor neuron subpopulations and gives important clues to mechanisms of relative vulnerability. Therefore, our extensive expression analysis could provide a basis for understanding why degeneration in amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy involve some, but not all, motor neuron populations and may hopefully be used to develop treatments for these diseases.

Materials and methods

Animal procedures

All animal procedures were performed in accordance with the National Institute of Health guidelines and were approved by the Animal Care and Use Committee at McLean Hospital, Harvard Medical School. Animals were housed according to standard conditions, with access to food and water ad libitum and a dark/light cycle of 12 h.

Analysis of disease onset in the SOD1G93A rat model of amyotrophic lateral sclerosis

Transgenic rats over-expressing mutant SOD1 (SOD1G93A) were used as a model of amyotrophic lateral sclerosis (Howland et al., 2002). Disease onset in these animals was carefully monitored (for details, see Supplementary material).

Tissue preparation

Presymptomatic, 60-day-old female SOD1G93A transgenic and wild-type litter-mates (Taconic) and symptomatic SOD1G93A rats and age-matched wild-type litter-mates were anaesthetized with sodium pentobarbital (150 mg/kg i.p.). For LCM and real-time polymerase chain reaction, tissues were removed, snap-frozen in 2-methylbutane (−60°C), sectioned (12 µM coronal sections), mounted onto LCM slides (Arcturus Engineering, Inc, Mountain View, CA) and stored at –70°C. For immunohistochemistry, animals were perfused intracardially with 100 ml heparinized saline and 200 ml 4% paraformaldehyde. Brains, brain stems and spinal cords were dissected, post-fixed for 6 h, cryoprotected in 20% sucrose, sectioned (30–40 µm) and stored at −70°C.

Quantification of differential motor neuron loss in the SOD1G93A rat

The number of motor neurons present in the CN3/4, trigeminal nucleus (CN5), facial nucleus (CN7), CN12 and in the lateral motor column across C2 and C3 segments (Kakinohana et al., 2004) in the cervical spinal cord from 60-day-old SOD1G93A rats, 60-day-old wild-type litter mates and symptomatic SOD1G93A rats and age-matched wild-type litter mates were quantified. Sections were incubated with blocking buffer (phosphate buffered saline, 10% normal donkey serum or normal goat serum and 0.1% Triton-X100) for 1 h. Sections were incubated overnight at 4°C with primary antibody against choline acetyltransferase (1:750, Millipore). Sections were washed in phosphate buffered saline and incubated with a biotinylated secondary antibody (1:300; Vector Laboratories, Burlingame, CA) for 1 h at room temperature, followed by incubation in streptavidin–biotin complex (Vectastain ABC kit Elite, Vector laboratories) for 1 h and visualized by incubation in 3,3′-diaminobenzidine solution (Vector Laboratories). The number of cranial and cervical spinal cord choline acetyltransferase positive motor neurons was quantified (for additional details see Supplementary material).

Quick immunostaining and dehydration of sections for laser capture microdissection

To visualize motor neurons for LCM, sections on slides were fixed in 75% ethanol for 1 min, washed in distilled water for 2 min, stained for 4 min in HistoGene staining (Arcturus) and washed again for 30 s in distilled water. The sections were dehydrated for 30 s in 75% ethanol, 2 min in 95% ethanol, 1 min in 100% ethanol and 5 min in xylene, air dried and placed into the Veritas LCM (Arcturus).

Laser capture microdissection of motor neurons

An Arcturus Veritas LCM System was used to isolate motor neurons from the CN3/4, CN12 and the lateral motor column of the cervical spinal cord (C2 and C3 segments) of normal rats onto CapSure Macro LCM caps (Arcturus). Motor neurons (500–1000) were isolated from each subpopulation and each individual animal (n = 4–5). Settings were optimized to capture nucleus and cytosol from the motor neurons, while minimizing inclusion of surrounding tissues.

RNA preparation, amplification and oligo-microarray analysis

RNA was purified from 250 to 500 motor neurons isolated from CN3/4, CN12 or cervical spinal cord (PicoPure RNA isolation kit, Arcturus), and amplified (aRNA) (RiboAmp RNA amplification kit, Arcturus). Amplified RNA quality was analysed (Agilent 2100 Bioanalyser, Agilent technologies) and hybridized to whole rat genome oligo-microarrays (Rat Genome 230 2.0 Array, Affymetrix, for raw microarray data, see Supplementary Table 4). The microarray study consisted of a comparison between motor neurons isolated from CN3/4, CN12 or cervical spinal cord. Each group constituted 4–5 replicates (arrays). The data set was analysed using Gene Pattern (http://www.broad.mit.edu/cancer/software/genepattern/). The MultiExperiment Viewer of TM4 (http://www.tm4.org) was used for correspondence analysis, utilizing the K-nearest neighbour algorithm (number of neighbours = 10). For the hierarchical clustering and gene distance matrix analysis based on Euclidian distance, genes with significant differential expression (1968 genes) were extracted by ANOVA, P ≤ 0.01 (based on F-distribution). Genes differentially expressed between CN3/4, CN12 and cervical spinal cord, P ≤ 0.05, were selected and visualized in heat maps, gene lists and annotations (Fig. 2, Table 1; Supplementary Fig. 3, Supplementary Tables 1 and 2). All gene lists were annotated using the DAVID Bioinformatics Database Gene Id Conversion Tool (http://david.abcc.ncifcrf.gov/conversion.jsp) and NCBI Entrez gene database and BLAST tool. The functional annotation chart tool in DAVID (http://david.abcc.ncifcrf.gov/) was utilized to detect differences in gene groups between motor neuron subpopulations (Huang da et al., 2009) (Fig. 2F–H, Supplementary Table 3), with classification stringency set using the medium parameter and the similarity threshold to 0.5.

Table 1

Genes differentially expressed in motor neurons of the cervical spinal cord, hypoglossal nucleus (CN12) and the oculomotor/trochlear complex (CN3/4), selected based on fold change and function

GeneBank No Gene name Gene symbol Fold change P-value Gene function/category References 
Genes upregulated in CN3/4 versus CN12 and cervical spinal cord with a P < 0.05, sorted by fold 
NM_012560.1 Forkhead-like transcription factor bf-1 Fkhr 48.5 0.002 Transcription, development, anti-apoptosis, neuronal survival/degeneration Yuan et al., 2008 
AA818342 Guanine deaminase Gda 41.5 0.018 Purine metabolism, CNS development, post synaptic protein sorting, dendritic branching Firestein et al., 1999; Akum et al., 2004 
NM_017110.1 Cocaine and amphetamine regulated transcript Cart 41.2 0.008 Neurotransmitter/hormone, regulation of appetite and stress Rogge et al., 2008 
BG374268 Orthodenticle homologue 2 (predicted) Otx2 33.8 0.010 Transcription factor, CNS development Broccoli et al., 1999; Millet et al., 1999 
BI285485 Dermatopontin Dpt 24.5 0.028 Cell adhesion Okamoto and Fujiwara, 2006 
AF322217.1 Immunoglobulin superfamily, member 1 Igsf1 23.7 0.002 Signal transduction, transcription, cell recognition Mazzarella et al., 1998 
AI412117 Serine protease inhibitor, kunitz type 2 Spint2 22.9 0.010 Transcription Kawaguchi et al., 1997 
BI296460 Laminin b1 subunit 1 Lamb1 21.4 0.002 Cell adhesion and migration, axon outgrowth and growth cone behaviour Hopker et al., 1999 
BM390227 B-cell cll/lymphoma 11b Bcl11b 18.3 0.002 Transcription, immune system Wakabayashi et al., 2003 
BF386692 Insulin receptor substrate 4 Irs4 17.7 0.046 Signal transduction, development Bohni et al., 1999 
AB028461.1 Leucine-rich repeat, immunoglobulin-like and transmembrane domains 1 Lrit1 17.6 0.008 Retina specific receptor (morphogenesis), binding Gomi et al., 2000 
AW920064 Cathepsin C Ctsc 17.3 0.032 Degradation of proteins, immune response McDonald et al., 1969 
BI278379 Reticulocalbin 3, EF-hand calcium binding domain Rcn3 15.2 0.010 Calcium binding protein involved in protein folding and sorting (endoplasmic reticulum) Honore, 2009 
AA943808 Cyclic AMP-regulated phosphoprotein, transcript variant 1 Arpp-21 14.9 0.002 Regulation of calmodulin-dependent enzymes e.g. calcineurin in neurons Rakhilin et al., 2004 
BE105492 Forkhead box p2 (predicted) Foxp2 10.3 0.002 Transcription, development Lai et al., 2001 
NM_017090.1 Guanylate cyclase 1 soluble alpha 3 Gucy1A3 9.6 0.002 Nitric oxide-signalling, regulation of cGMP biosynthesis Zabel et al., 1998 
NM_016989.1 Adenylate cyclase activating polypeptide 1 Adcyap1 9.3 0.002 Cell-cell signalling, cell cycle regulation, neuroprotection Morio et al., 1996 
AI059914 Ets variant gene 1 Etv1 8.2 0.002 Transcription, formation of sensory-motor connections Arber et al., 2000 
M15481.1 Insulin-like growth factor 1 Igf1 6.7 0.004 DNA-replication, anti-apoptosis, cell motion, signal transduction, development, motor neuron survival, axonal regeneration Nachemson et al., 1990; Powell-Braxton et al., 1993; Kaspar et al., 2003 
NM_012743.1 Forkhead box a2 Foxa2 6.6 0.008 Transcription, development, neuronal survival Lee et al., 2005; Kittappa et al., 2007 
NM_012551.1 Early growth response 1 Egr1 5.2 0.002 Transcription, inhibition of Fas expression Dinkel et al., 1997 
NM_031511.1 Insulin-like growth factor II Igf2 2.5 0.006 Cell proliferation, development, motor neuron survival and axon regeneration Caroni and Grandes, 1990; DeChiara et al., 1991; Near et al., 1992 
Genes upregulated in CN12 versus CN3/4 and cervical spinal cord with a P < 0.05 sorted by fold 
AI511432 Similar to tripartite motif-containing 58/olfactory receptor Olr1433 Trim58/Olr1433 14.0 0.002 Protein and metal ion binding/signal transduction  
BI297651 Dopamine receptor 1a/msh homeobox 2 Drd1A/Msx2 9.1 0.002 Signal transduction/transcription, cell proliferation, development Liu et al., 1992; Satokata et al., 2000 
AI236118 Phospholipid scramblase 4 Plscr4 8.0 0.002 Phospholipid scrambling Wiedmer et al., 2000 
BE101670 Neurotensin (predicted) Nts 7.5 0.002 Signal transduction Mai et al., 1987; Marondel et al., 1996 
BF389738 Similar to Homeobox B4 (M. musculusHoxb4 6.9 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity McGinnis and Krumlauf, 1992; Dasen et al., 2005 
BE095733 Homeobox B5 (predicted) Hoxb5 6.7 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Krumlauf, 1994; Dasen et al., 2005 
NM_031598.1 Phospholipase a2, group iia (platelets, synovial fluid) Pla2G2A 5.0 0.042 Phospholipid metabolism, Ca-dependent Birts et al., 2008 
AW532697 Myeloid ecotropic viral integration site 1 homologue (predicted) Meis1 4.9 0.002 Transcription, development Moskow et al., 1995; Mercader et al., 1999 
NM_031752.1 Lutheran blood group (auberger b antigen included Bcam 4.9 0.002 Cell adhesion, signal transduction Parsons et al., 1995 
BE116745 Wingless-type mmtv integration site 5a Wnt5A 4.7 0.010 Signal transduction, cell–cell signalling, development, cell polarity, motor neuron columnar specification Witze et al., 2008; Agalliu et al., 2009; Mosimann et al., 2009 
AI030806 Similar to Slit and Ntrk-like family, Member 3/butyrylcholinesterase Slitrk3/ Bche 4.7 0.002 Neurite outgrowth/neurite outgrowth, axotomy-regulated, adhesion Flumerfelt and Lewis, 1975; Aruga and Mikoshiba, 2003; Paraoanu et al., 2006 
AI549199 Prostaglandin e receptor 4 (subtype ep4) Ptger4 4.6 0.002 Immune response, signal transduction Bastien et al., 1994 
AI170441 Homeobox A5 Hoxa5 4.5 0.006 Transcription, development, positional identity, motor neuron identity and target connectivity Krumlauf, 1994; Dasen et al., 2005 
AI556803 Pleckstrin Plek 4.1 0.024 Intracellular signalling cascade, actin assembly Abrams et al., 1995; Lian et al., 2009 
AI598945 Gastrulation brain homeobox 2 Gbx2 4.0 0.002 Transcription, development, cell proliferation, CNS development Kowenz-Leutz et al., 1997; Waters and Lewandoski, 2006 
BE108648 Eph receptor a3 Epha3 3.2 0.002 Signal transduction, separation of axial motor and sensory pathways Boyd et al., 1992; Gallarda et al., 2008 
BM385237 Annexin A4 Anxa4 2.9 0.002 Ca-dependent phospholipid-binding Barrow et al., 1994 
NM_017238.1 Vasoactive intestinal peptide receptor 2 Vipr2 2.6 0.002 Cell–cell signalling, circadian function, CNS development Xia et al., 1996; Basille et al., 2000; Harmar et al., 2002 
Genes upregulated in cervical spinal cord versus CN3/4 and CN12 with a P < 0.05, sorted by fold  
AI235507 Homeobox C8 (mapped) Hoxc8 71.9 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; McGinnis and Krumlauf, 1992 
BE096332 Crystallin, gamma N Crygn 50.3 0.002 Visual perception, cellular response to reactive oxygen species Wistow et al., 2005 
AI177143 Similar to Homeobox D8/D4 Hoxd8/d4 31.6 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; Krumlauf, 1994 
AA956024 Homeobox C5 Hoxc5 26.5 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; Krumlauf, 1994 
AA996507 Homeobox A9 Hoxa9 14.6 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Krumlauf, 1994; Dasen, 2005, p. 140 
AI501494 Homeobox C6 Hoxc6 11.6 0.016 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; Krumlauf, 1994 
AW530378 Potassium voltage-gated channel, shaker-related subfamily, member 1 Kcna1 7.8 0.010 Ion transport, synaptic transmission Adelman et al., 1995; Gu et al., 2003; Raab-Graham et al., 2006 
BE118557 Supervillin (predicted) Svil 6.6 0.004 Cytoskeleton organization, skeletal muscle development, differentiation Pestonjamasp et al., 1997 
BE108523 Solute carrier family 35, member D3 Slc35d3 6.0 0.002 Carbohydrate transport Chintala et al., 2007 
BF388562 Similar to Homo sapiens seizure related 6 homologue (mouse)-like 2 Sez6L2 6.0 0.032 Signal transduction Shimizu-Nishikawa et al., 1995 
U36899.1 Vomeronasal receptor 2 Vnr2 5.9 0.046 Olfactory sensory perception Speca et al., 1999 
AI146158 Protein phosphatase 3, catalytic subunit, alpha isoform Ppp3Ca 5.7 0.028 Protein amino acid dephosphorylation Muramatsu and Kincaid, 1993 
BF555051 Growth arrest specific 6 Gas6 5.6 0.002 Cell proliferation, signal transduction Varnum et al., 1995 
AA859669 Neuropilin 2 Nrp2 4.8 0.002 Axon guidance, CNS development Kolodkin et al., 1997 
AI060247 N-terminal EF-hand calcium binding protein 3 Necab3 4.0 0.002 Protein metabolic process, regulation of amyloid precursor protein and beta-amyloid generation Lee et al., 2000 
AI177304 Fibroblast growth factor 7 Fgf7 3.7 0.038 Cell proliferation, development, signal transduction, presynaptic organization Rubin et al., 1989; Umemori et al., 2004 
NM_012633.1 Peripherin Prph 2.7 0.002 Structural protein, axotomy-regulated, motor neuron degeneration, amyotrophic lateral sclerosis related Beaulieu et al., 1999; Robertson et al., 2003 
BF281271 Placental growth factor Pgf 1.9 0.006 Cell proliferation, signal transduction, cell–cell signalling, angiogenesis Maglione et al., 1991 
GeneBank No Gene name Gene symbol Fold change P-value Gene function/category References 
Genes upregulated in CN3/4 versus CN12 and cervical spinal cord with a P < 0.05, sorted by fold 
NM_012560.1 Forkhead-like transcription factor bf-1 Fkhr 48.5 0.002 Transcription, development, anti-apoptosis, neuronal survival/degeneration Yuan et al., 2008 
AA818342 Guanine deaminase Gda 41.5 0.018 Purine metabolism, CNS development, post synaptic protein sorting, dendritic branching Firestein et al., 1999; Akum et al., 2004 
NM_017110.1 Cocaine and amphetamine regulated transcript Cart 41.2 0.008 Neurotransmitter/hormone, regulation of appetite and stress Rogge et al., 2008 
BG374268 Orthodenticle homologue 2 (predicted) Otx2 33.8 0.010 Transcription factor, CNS development Broccoli et al., 1999; Millet et al., 1999 
BI285485 Dermatopontin Dpt 24.5 0.028 Cell adhesion Okamoto and Fujiwara, 2006 
AF322217.1 Immunoglobulin superfamily, member 1 Igsf1 23.7 0.002 Signal transduction, transcription, cell recognition Mazzarella et al., 1998 
AI412117 Serine protease inhibitor, kunitz type 2 Spint2 22.9 0.010 Transcription Kawaguchi et al., 1997 
BI296460 Laminin b1 subunit 1 Lamb1 21.4 0.002 Cell adhesion and migration, axon outgrowth and growth cone behaviour Hopker et al., 1999 
BM390227 B-cell cll/lymphoma 11b Bcl11b 18.3 0.002 Transcription, immune system Wakabayashi et al., 2003 
BF386692 Insulin receptor substrate 4 Irs4 17.7 0.046 Signal transduction, development Bohni et al., 1999 
AB028461.1 Leucine-rich repeat, immunoglobulin-like and transmembrane domains 1 Lrit1 17.6 0.008 Retina specific receptor (morphogenesis), binding Gomi et al., 2000 
AW920064 Cathepsin C Ctsc 17.3 0.032 Degradation of proteins, immune response McDonald et al., 1969 
BI278379 Reticulocalbin 3, EF-hand calcium binding domain Rcn3 15.2 0.010 Calcium binding protein involved in protein folding and sorting (endoplasmic reticulum) Honore, 2009 
AA943808 Cyclic AMP-regulated phosphoprotein, transcript variant 1 Arpp-21 14.9 0.002 Regulation of calmodulin-dependent enzymes e.g. calcineurin in neurons Rakhilin et al., 2004 
BE105492 Forkhead box p2 (predicted) Foxp2 10.3 0.002 Transcription, development Lai et al., 2001 
NM_017090.1 Guanylate cyclase 1 soluble alpha 3 Gucy1A3 9.6 0.002 Nitric oxide-signalling, regulation of cGMP biosynthesis Zabel et al., 1998 
NM_016989.1 Adenylate cyclase activating polypeptide 1 Adcyap1 9.3 0.002 Cell-cell signalling, cell cycle regulation, neuroprotection Morio et al., 1996 
AI059914 Ets variant gene 1 Etv1 8.2 0.002 Transcription, formation of sensory-motor connections Arber et al., 2000 
M15481.1 Insulin-like growth factor 1 Igf1 6.7 0.004 DNA-replication, anti-apoptosis, cell motion, signal transduction, development, motor neuron survival, axonal regeneration Nachemson et al., 1990; Powell-Braxton et al., 1993; Kaspar et al., 2003 
NM_012743.1 Forkhead box a2 Foxa2 6.6 0.008 Transcription, development, neuronal survival Lee et al., 2005; Kittappa et al., 2007 
NM_012551.1 Early growth response 1 Egr1 5.2 0.002 Transcription, inhibition of Fas expression Dinkel et al., 1997 
NM_031511.1 Insulin-like growth factor II Igf2 2.5 0.006 Cell proliferation, development, motor neuron survival and axon regeneration Caroni and Grandes, 1990; DeChiara et al., 1991; Near et al., 1992 
Genes upregulated in CN12 versus CN3/4 and cervical spinal cord with a P < 0.05 sorted by fold 
AI511432 Similar to tripartite motif-containing 58/olfactory receptor Olr1433 Trim58/Olr1433 14.0 0.002 Protein and metal ion binding/signal transduction  
BI297651 Dopamine receptor 1a/msh homeobox 2 Drd1A/Msx2 9.1 0.002 Signal transduction/transcription, cell proliferation, development Liu et al., 1992; Satokata et al., 2000 
AI236118 Phospholipid scramblase 4 Plscr4 8.0 0.002 Phospholipid scrambling Wiedmer et al., 2000 
BE101670 Neurotensin (predicted) Nts 7.5 0.002 Signal transduction Mai et al., 1987; Marondel et al., 1996 
BF389738 Similar to Homeobox B4 (M. musculusHoxb4 6.9 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity McGinnis and Krumlauf, 1992; Dasen et al., 2005 
BE095733 Homeobox B5 (predicted) Hoxb5 6.7 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Krumlauf, 1994; Dasen et al., 2005 
NM_031598.1 Phospholipase a2, group iia (platelets, synovial fluid) Pla2G2A 5.0 0.042 Phospholipid metabolism, Ca-dependent Birts et al., 2008 
AW532697 Myeloid ecotropic viral integration site 1 homologue (predicted) Meis1 4.9 0.002 Transcription, development Moskow et al., 1995; Mercader et al., 1999 
NM_031752.1 Lutheran blood group (auberger b antigen included Bcam 4.9 0.002 Cell adhesion, signal transduction Parsons et al., 1995 
BE116745 Wingless-type mmtv integration site 5a Wnt5A 4.7 0.010 Signal transduction, cell–cell signalling, development, cell polarity, motor neuron columnar specification Witze et al., 2008; Agalliu et al., 2009; Mosimann et al., 2009 
AI030806 Similar to Slit and Ntrk-like family, Member 3/butyrylcholinesterase Slitrk3/ Bche 4.7 0.002 Neurite outgrowth/neurite outgrowth, axotomy-regulated, adhesion Flumerfelt and Lewis, 1975; Aruga and Mikoshiba, 2003; Paraoanu et al., 2006 
AI549199 Prostaglandin e receptor 4 (subtype ep4) Ptger4 4.6 0.002 Immune response, signal transduction Bastien et al., 1994 
AI170441 Homeobox A5 Hoxa5 4.5 0.006 Transcription, development, positional identity, motor neuron identity and target connectivity Krumlauf, 1994; Dasen et al., 2005 
AI556803 Pleckstrin Plek 4.1 0.024 Intracellular signalling cascade, actin assembly Abrams et al., 1995; Lian et al., 2009 
AI598945 Gastrulation brain homeobox 2 Gbx2 4.0 0.002 Transcription, development, cell proliferation, CNS development Kowenz-Leutz et al., 1997; Waters and Lewandoski, 2006 
BE108648 Eph receptor a3 Epha3 3.2 0.002 Signal transduction, separation of axial motor and sensory pathways Boyd et al., 1992; Gallarda et al., 2008 
BM385237 Annexin A4 Anxa4 2.9 0.002 Ca-dependent phospholipid-binding Barrow et al., 1994 
NM_017238.1 Vasoactive intestinal peptide receptor 2 Vipr2 2.6 0.002 Cell–cell signalling, circadian function, CNS development Xia et al., 1996; Basille et al., 2000; Harmar et al., 2002 
Genes upregulated in cervical spinal cord versus CN3/4 and CN12 with a P < 0.05, sorted by fold  
AI235507 Homeobox C8 (mapped) Hoxc8 71.9 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; McGinnis and Krumlauf, 1992 
BE096332 Crystallin, gamma N Crygn 50.3 0.002 Visual perception, cellular response to reactive oxygen species Wistow et al., 2005 
AI177143 Similar to Homeobox D8/D4 Hoxd8/d4 31.6 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; Krumlauf, 1994 
AA956024 Homeobox C5 Hoxc5 26.5 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; Krumlauf, 1994 
AA996507 Homeobox A9 Hoxa9 14.6 0.002 Transcription, development, positional identity, motor neuron identity and target connectivity Krumlauf, 1994; Dasen, 2005, p. 140 
AI501494 Homeobox C6 Hoxc6 11.6 0.016 Transcription, development, positional identity, motor neuron identity and target connectivity Dasen et al., 2005; Krumlauf, 1994 
AW530378 Potassium voltage-gated channel, shaker-related subfamily, member 1 Kcna1 7.8 0.010 Ion transport, synaptic transmission Adelman et al., 1995; Gu et al., 2003; Raab-Graham et al., 2006 
BE118557 Supervillin (predicted) Svil 6.6 0.004 Cytoskeleton organization, skeletal muscle development, differentiation Pestonjamasp et al., 1997 
BE108523 Solute carrier family 35, member D3 Slc35d3 6.0 0.002 Carbohydrate transport Chintala et al., 2007 
BF388562 Similar to Homo sapiens seizure related 6 homologue (mouse)-like 2 Sez6L2 6.0 0.032 Signal transduction Shimizu-Nishikawa et al., 1995 
U36899.1 Vomeronasal receptor 2 Vnr2 5.9 0.046 Olfactory sensory perception Speca et al., 1999 
AI146158 Protein phosphatase 3, catalytic subunit, alpha isoform Ppp3Ca 5.7 0.028 Protein amino acid dephosphorylation Muramatsu and Kincaid, 1993 
BF555051 Growth arrest specific 6 Gas6 5.6 0.002 Cell proliferation, signal transduction Varnum et al., 1995 
AA859669 Neuropilin 2 Nrp2 4.8 0.002 Axon guidance, CNS development Kolodkin et al., 1997 
AI060247 N-terminal EF-hand calcium binding protein 3 Necab3 4.0 0.002 Protein metabolic process, regulation of amyloid precursor protein and beta-amyloid generation Lee et al., 2000 
AI177304 Fibroblast growth factor 7 Fgf7 3.7 0.038 Cell proliferation, development, signal transduction, presynaptic organization Rubin et al., 1989; Umemori et al., 2004 
NM_012633.1 Peripherin Prph 2.7 0.002 Structural protein, axotomy-regulated, motor neuron degeneration, amyotrophic lateral sclerosis related Beaulieu et al., 1999; Robertson et al., 2003 
BF281271 Placental growth factor Pgf 1.9 0.006 Cell proliferation, signal transduction, cell–cell signalling, angiogenesis Maglione et al., 1991 

Cross comparisons of genes differentially expressed between motor neurons of the cervical spinal cord, CN12 and CN3/4. Displayed genes were selected based on their high differential expression between the motor neuron populations showing differential vulnerability to degeneration and their functions (two-sided t-test using 1000 permutations, P ≤ 0.05).

Preparation of primary spinal cord cultures and in vitro analysis of neuroprotection

Time-pregnant Sprague Dawley wild-type rats were anaesthetized, decapitated and embryonic day (E) 15.5 embryos collected, decapitated and spinal cords isolated in Hanks balanced salt solution (Invitrogen). Dissections of spinal cords were done carefully to avoid the inclusion of somites or other external tissues (Hedlund et al., 2004). Cells were dissociated by gentle trituration and incubation with papain (Worthington Biochemical Corporation). Cells were cultured for 6 days in Neurobasal media (Invitrogen) containing 10% foetal bovine serum (Fisher scientific), 1 × B27 supplement (Invitrogen), 500 µM glutamine (Invitrogen), 25 µM mercaptoethanol (Invitrogen), penicillin–streptomycin (Invitrogen) or in Dulbecco’s modified Eagle’s medium/F12 (Invitrogen) containing 5% foetal bovine serum, 1 × N2 supplement A (Stem Cell Technologies), glucose (0.36%, Sigma), bovine serum albumin (0.25%, Invitrogen) and penicillin–streptomycin (Invitrogen). Either culture media could maintain spinal cord cultures containing motor neurons. This 6-day culture period was developed to allow astrocytes time to proliferate in vitro and motor neurons to form an interconnected network prior to exposure to glutamate and the glutamate uptake blocker, l-trans-2,4-pyrrolidine-2,4-dicarboxylic acid (PDC). After the 6 days of culture, glutamate toxicity was induced by the addition of 20 µM glutamate and 100 µM PDC for 4–7 days. For analysis of neuroprotection, the glutamate challenge was preceded by a 2–4 h pretreatment with 1–100 ng/ml recombinant IGF-II (R&D Systems) or 100 ng/ml guanine deaminase (MP Biomedicals, LLC, Solon, OH). Cultures were subsequently maintained for an additional 4–7 days. Identification of motor neurons was done by staining fixed cultures using antibodies against islet-1/2 [1:500 when antibodies were used in combination and 1:100 when used separately, 39.4D5, 40.2D6, Developmental Studies Hybridoma Bank (DSHB), University of Iowa], Neurofilament (150 kDa) (1:500), choline acetyltransferase (1:750, Millipore) and homeodomain protein (MNR2)/homeobox 9 (1:100, 81.5C10, DSBH).

Immunofluorescent staining and stereological procedures

For immunofluorescent staining, coverslips/sections were rinsed with phosphate buffered saline and incubated with blocking buffer (see above) for 1 h. Coverslips/sections were then incubated overnight at 4°C with primary antibodies in blocking buffer. The following antibodies were used: mouse anti-islet-1/2 and rabbit anti-neurofilament (see above), rabbit anti-peripherin (1:100), mouse anti-tyrosine hydroxylase (1:1000), mouse anti-NeuN (1:1000, Millipore), rabbit anti-G protein-coupled inwardly rectifying potassium channel 2 (1:80, Alomone Laboratories), rabbit anti-guanylate cyclase soluble subunit alpha-3 (Gucy1a3) (1:60, Abgent), rabbit anti-placental growth factor (1:30, Proteintech group), rabbit anti-IGF-II (1:100, R&D systems), rabbit anti-early growth response protein 1 (1:100), goat anti-cypin (Guanine Deaminase) (A-20, 1:100, Santa Cruz Biotechnology) and mouse anti-glial fibrillary acidic protein (1:1000, Sigma). Localization of the proteins was done on multiple sections along the cervical spinal cord in multiple animals. The coverslips/sections were then incubated with Alexafluor secondary antibodies for 1 h and rinsed. Hoechst 33342 (4 µg/ml) was used for counterstaining. Confocal analysis was performed using a Zeiss LSM510/Meta Station (Thornwood, NY, http://www.zeiss.com), with optical thickness kept to a minimum and orthogonal reconstructions obtained. The effect of glutamate, IGF-II and/or guanine deaminase on motor neuron survival was carefully evaluated, as was the co-localization of motor neuron markers (see Supplementary material for details).

Quantitative polymerase chain reaction

Quantitative polymerase chain reaction on mRNA extracted from LCMed motor neurons, utilizing SYBR green I (Molecular Probes, Eugene, OR) or specific Taqman probes was used to confirm differential gene expression. For details, see Supplementary material.

Statistical analysis

All experimental data was analysed using Student’s t-test and ANOVA. InStat3 software (GraphPad software inc.) was used for the statistical analyses.

Results

Microarray analysis of motor neurons showed high reproducibility and specificity to the anatomical nuclei

Based on the differential motor neurons loss in motor neuron diseases, we isolated individual motor neurons from CN3/4 (do not degenerate in amyotrophic lateral sclerosis, spinobulbar muscular atrophy or spinal muscular atrophy), CN12 (show vulnerability in amyotrophic lateral sclerosis and spinobulbar muscular atrophy) and from the lateral motor column of the cervical enlargement of the spinal cord (degenerate in all three diseases) using LCM in wild-type rats (Fig. 1A–N). The RNA isolated from motor neurons was hybridized to whole genome rat arrays. The gene expression data showed that individual replicates within a motor neuron nucleus were highly reproducible with an average Pearson’s correlation of 0.93 for CN3/4, 0.94 for CN12 and 0.93 for the cervical spinal cord. When motor neurons from different groups were compared, the average Pearson’s correlation was 0.91 for CN12 versus cervical spinal cord, 0.89 for CN3/4 versus CN12 and 0.86 for CN3/4 versus cervical spinal cord cross comparisons (Supplementary Fig. 2). Additionally, correspondence analysis, hierarchial clustering and gene distance matrix confirmed that individual samples within each nucleus clustered together and showed that motor neurons located in CN12 and in the lateral motor column of the cervical spinal cord had the most similar transcriptomes, whereas motor neurons of CN3/4 showed a more different gene regulation pattern (Fig. 2A and B; Supplementary Fig. 3A). Within the heat map, the locations of peripherin, which was predominantly expressed in motor neurons of the cervical spinal cord, and IGF-II and guanine deaminase, which were restricted to CN3/4 motor neurons, have been indicated (Fig. 2B).

Figure 1

LCM of motor neurons from subpopulations showing differential vulnerability to degeneration in amyotrophic lateral sclerosis. (A) Approximately 40% of cervical spinal motor neurons had degenerated in the SOD1G93A rats at the time of disease onset, as defined by grip strength analysis, while the number of motor neurons in the different brain stem nuclei remained unchanged. (B) Schematic figure depicting the rat brain, brainstem and spinal cord, displaying three nuclei of motor neurons along the rostrocaudal axis of the CNS which show differential vulnerability to degeneration in amyotrophic lateral sclerosis: CN3/4, CN12 and the lateral motor column of the cervical spinal cord. Motor neurons in the (C–F) CN3/4, (G–J) CN12 and (K–N) the cervical spinal cord, visualized by (C, G and K) choline acetyltransferase (ChAT) staining or (D, H and L) HistoGene staining were isolated by LCM (E, F, I, J, M and N).

Figure 1

LCM of motor neurons from subpopulations showing differential vulnerability to degeneration in amyotrophic lateral sclerosis. (A) Approximately 40% of cervical spinal motor neurons had degenerated in the SOD1G93A rats at the time of disease onset, as defined by grip strength analysis, while the number of motor neurons in the different brain stem nuclei remained unchanged. (B) Schematic figure depicting the rat brain, brainstem and spinal cord, displaying three nuclei of motor neurons along the rostrocaudal axis of the CNS which show differential vulnerability to degeneration in amyotrophic lateral sclerosis: CN3/4, CN12 and the lateral motor column of the cervical spinal cord. Motor neurons in the (C–F) CN3/4, (G–J) CN12 and (K–N) the cervical spinal cord, visualized by (C, G and K) choline acetyltransferase (ChAT) staining or (D, H and L) HistoGene staining were isolated by LCM (E, F, I, J, M and N).

Figure 2

Global gene expression analysis of motor neurons isolated from CN3/4, CN12 and the cervical spinal cord of normal rats. (A) Correspondence analysis of the gene expression data (22 911 genes) showed that individual samples within each nucleus clustered together and that motor neurons of the CN12 and the cervical spinal cord were closely associated, whereas motor neurons of CN3/4 were less associated with the other motor neuron nuclei. (B) Hierarchial clustering, using Euclidean distance, of the 1968 genes (extracted by ANOVA using a P-value set to 0.01) that were differentially expressed between the three nuclei, showed that individual samples within each somatic motor neuron nucleus clustered closely together, and that motor neurons of CN12 shared more genes with those of the cervical spinal cord compared to CN3/4. Within the heat map, the location of peripherin, which was predominantly expressed in spinal motor neurons, and the CN3/4-restricted genes IGF-II and guanine deaminase (GDA) have been indicated. (C) Heat map displaying the differential expression pattern of Hox genes in motor neuron subpopulations (CN3/4, CN12 and cervical spinal cord) according to their anterior–posterior position within the brain and spinal cord (extracted using two-sided t-test with 1000 permutations, and a P-value set to ≤ 0.05). (D–F) Examples of gene groups that showed a high differential expression level between the three different motor neuron subpopulations. ALS = amyotrophic lateral sclerosis.

Figure 2

Global gene expression analysis of motor neurons isolated from CN3/4, CN12 and the cervical spinal cord of normal rats. (A) Correspondence analysis of the gene expression data (22 911 genes) showed that individual samples within each nucleus clustered together and that motor neurons of the CN12 and the cervical spinal cord were closely associated, whereas motor neurons of CN3/4 were less associated with the other motor neuron nuclei. (B) Hierarchial clustering, using Euclidean distance, of the 1968 genes (extracted by ANOVA using a P-value set to 0.01) that were differentially expressed between the three nuclei, showed that individual samples within each somatic motor neuron nucleus clustered closely together, and that motor neurons of CN12 shared more genes with those of the cervical spinal cord compared to CN3/4. Within the heat map, the location of peripherin, which was predominantly expressed in spinal motor neurons, and the CN3/4-restricted genes IGF-II and guanine deaminase (GDA) have been indicated. (C) Heat map displaying the differential expression pattern of Hox genes in motor neuron subpopulations (CN3/4, CN12 and cervical spinal cord) according to their anterior–posterior position within the brain and spinal cord (extracted using two-sided t-test with 1000 permutations, and a P-value set to ≤ 0.05). (D–F) Examples of gene groups that showed a high differential expression level between the three different motor neuron subpopulations. ALS = amyotrophic lateral sclerosis.

Several known motor neuron markers were expressed at similar levels within motor neurons of all three anatomical nuclei (see Supplementary material).

Genes of the Hox cluster provide positional information needed for spatial and temporal pattering of the vertebrate body axis. The known differential expression of the Hox genes, along the anterior posterior axis of the developing hindbrain and spinal cord, was used to validate the microarray data further. Hox A genes in positions 1 and 2, with anterior limits within the midbrain/hindbrain (Carpenter, 2002) were expressed in all three motor neuron nuclei, as would be expected (Fig. 2C). Hox genes in positions 3–5 were identified only in CN12 and the cervical spinal cord as expected based on their known expression in brain stem and spinal cord, but lack thereof in midbrain (Fig. 2C). Finally, Hox genes in position 6–9, with anterior limits at cervical spinal cord levels (Carpenter, 2002), were consequently only identified in motor neurons of the cervical spinal cord (Fig. 2C). The 90 most differentially expressed genes for each cross comparison between the three groups are displayed in heat maps (Supplementary Fig. 2B–G; Supplementary Table 1). There were large differences in the number of regulated genes involved in endoplasmatic reticulum function, mitochondria, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, cell adhesion and growth (Fig. 2E–F; Supplementary Tables 1 and 2). There were also large differences in the number of genes involved in transcription, RNA metabolic and biosynthetic processing, RNA binding, RNA splicing and regulation of translation (Fig. 2D; Supplementary Tables 2 and 3), functions which have recently been implicated in amyotrophic lateral sclerosis. Furthermore, we identified IGF-I to be preferentially expressed in motor neurons of the CN3/4 (Table 1; Supplementary Table 1). IGF-I can protect spinal motor neurons from degeneration in a mouse model of amyotrophic lateral sclerosis (Kaspar et al., 2003). Our novel finding that IGF-I is preferentially expressed within CN3/4 motor neurons could perhaps further explain the resistance of these cells to degeneration. Functional annotation and pathway analysis showed that several genes, including catalase, neurofilaments, protein phosphatase 3 and tumour protein p53, shown to be involved in amyotrophic lateral sclerosis pathogenesis, were more highly expressed in motor neurons of the cervical spinal cord (Fig. 2F; Supplementary Fig. 4). Furthermore, investigation of ubiquitin mediated proteolysis, a process thought to be involved in the pathogenesis of motor neuron diseases showed that multiple genes were expressed at higher levels in spinal cord motor neurons (Fig. 2F; Supplementary Fig. 5).

Confirmation of differential expression in specific anatomical motor neuron nuclei

Immunofluorescence of the resulting proteins of genes identified as differentially expressed among motor neuron subpopulations displaying differential vulnerability to degeneration confirmed their specific localization and differential expression. The intermediate neurofilament peripherin protein showed a preferential expression within spinal motor neurons (Fig. 3A–C), consistent with the mRNA expression (Fig. 2; Supplementary Fig. 6A). Placental growth factor protein was predominantly localized to spinal motor neurons (Fig. 3D–F), consistent with their microarray data. IGF-II mRNA (Fig. 2; Supplementary Fig. 6B) and protein (Fig. 3G–I) were restricted to motor neurons of CN3/4. Guanine deaminase mRNA and protein were restricted to CN3/4 motor neurons (Fig. 5B–E, Table 1; Supplementary Table 1). Guanine deaminase was also expressed within the striatum, as previously shown (Firestein et al., 1999). The soluble protein Gucy1a3’s mRNA and protein were mainly expressed in motor neurons of CN3/4, but were also detectable in spinal motor neurons. Gucy1a3 protein was also expressed in non-motor neurons within and surrounding CN3/4 (Fig. 3J–L, Supplementary Fig. 7). Early growth response 1 protein was mainly expressed in CN3/4 motor neurons (Fig. 3M–O), consistent with the mRNA expression. The G protein-coupled inwardly rectifying potassium channel 2 mRNA and protein were predominantly expressed in CN3/4 motor neurons. In the cervical spinal cord, the expression appeared more variable, with some neurons displaying a high and others a somewhat lower level of the protein (Supplementary Fig. 8A–D).

Figure 3

Confirmation of protein expression of genes differentially expressed in motor neurons of the cervical spinal cord, CN12 and the CN3/4. Immunofluorescent analysis using confocal microscopy was used to analyse the protein expression of genes found to be differentially expressed between different groups of motor neurons using microarray. Consistent with the microarray RNA data, the protein expression of peripherin in motor neurons of CN3/4 (A) and CN12 appeared low (B), whereas in cervical spinal cord it was high (C and inset). Placental growth factor (PGF) was localized to motor neurons of the spinal cord (F and inset) and to a lesser extent to motor neuron of CN3/4 (D) and CN12 (E). IGF-II protein was expressed in motor neurons of CN3/4 (G and inset), but was below the detection level in motor neurons of CN12 (H) and the cervical spinal cord (I). Gucy1a3 protein was present in motor neurons of the CN3/4. Gucy1a3 was also localized to additional cell types within and surrounding the CN3/4 (J and inset). The level of Gucy1a3 in CN12 was very low (K) whereas it was more easily detected in motor neurons of the cervical spinal cord (L). Early growth response 1 (Egr-1) protein was highly expressed in motor neurons of CN3/4 (M), while the expression level in motor neurons of CN12 and the cervical spinal cord was much lower (N and O). Scale bar: 100 µm (O, applies to A–N).

Figure 3

Confirmation of protein expression of genes differentially expressed in motor neurons of the cervical spinal cord, CN12 and the CN3/4. Immunofluorescent analysis using confocal microscopy was used to analyse the protein expression of genes found to be differentially expressed between different groups of motor neurons using microarray. Consistent with the microarray RNA data, the protein expression of peripherin in motor neurons of CN3/4 (A) and CN12 appeared low (B), whereas in cervical spinal cord it was high (C and inset). Placental growth factor (PGF) was localized to motor neurons of the spinal cord (F and inset) and to a lesser extent to motor neuron of CN3/4 (D) and CN12 (E). IGF-II protein was expressed in motor neurons of CN3/4 (G and inset), but was below the detection level in motor neurons of CN12 (H) and the cervical spinal cord (I). Gucy1a3 protein was present in motor neurons of the CN3/4. Gucy1a3 was also localized to additional cell types within and surrounding the CN3/4 (J and inset). The level of Gucy1a3 in CN12 was very low (K) whereas it was more easily detected in motor neurons of the cervical spinal cord (L). Early growth response 1 (Egr-1) protein was highly expressed in motor neurons of CN3/4 (M), while the expression level in motor neurons of CN12 and the cervical spinal cord was much lower (N and O). Scale bar: 100 µm (O, applies to A–N).

The CN3/4-restricted genes IGF-II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity

For analysis of possible neuroprotective properties of differentially expressed candidate genes on somatic motor neurons we developed a primary embryonic spinal cord culture system. The cultures initially contained a majority of neurons, but also a smaller population of astrocytes, which continuously profilerated and thereby constituted the majority of cells at the later parts of the culture time (Hoechst staining in Supplementary Fig. 10 and data not shown). The presence of cell types other than motor neurons provided trophic support, enabling culturing without the addition of growth factors that are necessary if motor neurons are to be cultured alone (Henderson et al., 1993). Motor neurons were present at all times in the culture and displayed large neuritic networks as the culture time progressed. The motor neurons had a healthy appearance and expressed neurofilament and islet-1 (Supplementary Fig. 10). Islet-1 positive cells also expressed homeobox 9 (98.4 ± 1.5% of islet-1 positive cells were homeobox 9 positive) and choline acetyltransferase (Supplementary Fig. 11), confirming their motor neuron identity. At Day 13 of the culture, 9.7 ± 5.3% of all the cells in the culture were motor neurons (homeobox 9 positive, islet-1 positive). Glutamate toxicity could be a general downstream event of degeneration in motor neuron disease. Addition of glutamate (20 µM) and a general glutamate uptake blocker (PDC, 100 µM) induced motor neuron toxicity (Figs. 4A and 5F; Supplementary Fig. 12; *P < 0.001, ANOVA). We selected the CN3/4-restricted genes IGF-II and guanine deaminase for analysis of neuroprotective properties, based on their high differential expression and predominant expression in protected motor neurons and specific cellular functions (Table 1; Supplementary Tables 1 and 2). We hypothesized that the endogenous expression of IGF-II and/or guanine deaminase within CN3/4 motor neurons might protect these cells from glutamate toxicity. IGF-II is a survival factor for motor neurons in some instances and guanine deaminase is a protein important for dendritic branching and synaptic function, but it was not known if either of these proteins could help motor neurons resist high levels of glutamate. Because IGF-II and guanine deaminase are both present extracellularly, which may be of significance and benefit for therapeutic development, we added either of these proteins exogenously to primary spinal cord cultures prior to glutamate insult. Pretreatment with IGF-II at 10–100 ng/ml concentrations protected motor neurons from glutamate-induced toxicity (Fig. 4A–D; Supplementary Fig. 12; *P < 0.001, ANOVA). Confocal analysis of IGF-II pretreated cultures exposed to glutamate for 7 days show healthy motor neurons expressing neurofilament and islet-1 (Fig. 4B–D). Preincubation of the spinal cultures with guanine deaminase (Table 1, Fig. 5A–E) at 100 ng/ml concentrations also protected motor neurons from glutamate-induced toxicity (Fig. 5F–I, P < 0.001, ANOVA).

Figure 4

IGF-II protected spinal motor neurons in primary culture from glutamate-induced toxicity. (A) The number of spinal motor neurons in primary culture was significantly decreased after the addition of 20 µM glutamate (Glu) and 100 µM of the glutamate uptake blocker PDC (P < 0.001, ANOVA). Pretreatment of the cultures with IGF-II (10–100 ng/ml) for 2–4 h prior to glutamate insult protected motor neurons against the toxicity (P < 0.001, ANOVA). Confocal analysis of (B and D) 150 kD neurofilament (NF) and (C and D) islet-1 expression in primary spinal cord cultures pretreated with IGF-II show the presence of large numbers of motor neurons in the cultures after 7 days of combined IGF-II and glutamate treatment. Scale bar: 50 µm (D, applies to B and C). Asterisk signifies a statistically significant difference with a P < 0.01, by ANOVA.

Figure 4

IGF-II protected spinal motor neurons in primary culture from glutamate-induced toxicity. (A) The number of spinal motor neurons in primary culture was significantly decreased after the addition of 20 µM glutamate (Glu) and 100 µM of the glutamate uptake blocker PDC (P < 0.001, ANOVA). Pretreatment of the cultures with IGF-II (10–100 ng/ml) for 2–4 h prior to glutamate insult protected motor neurons against the toxicity (P < 0.001, ANOVA). Confocal analysis of (B and D) 150 kD neurofilament (NF) and (C and D) islet-1 expression in primary spinal cord cultures pretreated with IGF-II show the presence of large numbers of motor neurons in the cultures after 7 days of combined IGF-II and glutamate treatment. Scale bar: 50 µm (D, applies to B and C). Asterisk signifies a statistically significant difference with a P < 0.01, by ANOVA.

Figure 5

Guanine deaminase was expressed within CN3/4 motor neurons and exogenous delivery protected primary spinal motor neurons from glutamate toxicity. Immunofluorescent analysis using confocal microscopy was used to analyse the protein expression of guanine deaminase (GDA). (A) Striatal tissue was used as a positive control for the antibody staining. (B and E) Consistent with the microarray RNA data, guanine deaminase protein was present in motor neurons of the CN3/4 as well as within other surrounding cell types within this nucleus. The level of guanine deaminase in CN12 and cervical spinal cord was very low (C and D). (F–I) The addition of glutamate (Glu, 20 µM) and the glutamate uptake blocker PDC (100 µM) to primary spinal cord culture decreased the number of motor neurons (P < 0.001, ANOVA). Pretreatment of the cultures with guanine deaminase (100 ng/ml) for 3–4 h prior to glutamate insult protected motor neurons against the toxicity (P < 0.001, ANOVA). Scale bars: 100 µm (D, applies to A–C), 50 µm (E) and 50 µm (I, applies to G and H). ChAT = choline acetyltransferase. Asterisk signifies a statistically significant difference with a P < 0.01, by ANOVA.

Figure 5

Guanine deaminase was expressed within CN3/4 motor neurons and exogenous delivery protected primary spinal motor neurons from glutamate toxicity. Immunofluorescent analysis using confocal microscopy was used to analyse the protein expression of guanine deaminase (GDA). (A) Striatal tissue was used as a positive control for the antibody staining. (B and E) Consistent with the microarray RNA data, guanine deaminase protein was present in motor neurons of the CN3/4 as well as within other surrounding cell types within this nucleus. The level of guanine deaminase in CN12 and cervical spinal cord was very low (C and D). (F–I) The addition of glutamate (Glu, 20 µM) and the glutamate uptake blocker PDC (100 µM) to primary spinal cord culture decreased the number of motor neurons (P < 0.001, ANOVA). Pretreatment of the cultures with guanine deaminase (100 ng/ml) for 3–4 h prior to glutamate insult protected motor neurons against the toxicity (P < 0.001, ANOVA). Scale bars: 100 µm (D, applies to A–C), 50 µm (E) and 50 µm (I, applies to G and H). ChAT = choline acetyltransferase. Asterisk signifies a statistically significant difference with a P < 0.01, by ANOVA.

Discussion

Difference in vulnerability of specific cellular populations is a prominent feature of neurodegenerative diseases. Understanding such differences by analysing normal function and gene expression within particular neuronal populations could lead to a better understanding of the molecular events underlying degeneration as well as protection.

Relative vulnerability and comparative analysis of differential gene expression among motor neuron subpopulations

Factors intrinsic to motor neurons appear crucial for initiation of motor neuron degeneration in amyotrophic lateral sclerosis (Boillee et al., 2006; Jaarsma et al., 2008) and perhaps also in spinobulbar muscular atrophy and spinal muscular atrophy. To understand why disease is initiated in some, but not all motor neurons we analysed the gene expression profiles of motor neurons isolated from CN3/4 (unaffected in amyotrophic lateral sclerosis, spinobulbar muscular atrophy and spinal muscular atrophy), CN12 (affected in amyotrophic lateral sclerosis and spinobulbar muscular atrophy) and the lateral motor column of the cervical spinal cord (highly affected in amyotrophic lateral sclerosis, spinobulbar muscular atrophy and spinal muscular atrophy). We studied motor neurons using LCM, which allows for isolation of neurons from adult animals and does not require any genetic manipulation.

Our analyses demonstrated that motor neurons in CN12 and the lateral motor column of the cervical spinal cord had more commonality in gene expression levels than those in CN3/4. This higher degree of clustering of CN12 and cervical spinal cord motor neurons could be related to parameters such as the size of the neurons, the lengths of their projections and their respective muscle targets. We believe that our analysis of ‘gene expression dosage’ can give important information about the normal function of a cell and its disease responses. Differences in actual gene dosage are highly linked to selective vulnerability and neurodegenerative diseases. Familial Parkinson’s disease can be caused by point mutations in the α-synuclein gene (Polymeropoulos et al., 1997) or increased gene dosage (Singleton et al., 2003). Data from animal models indicate that Down’s syndrome occurs due to an increased dosage of genes located in a critical region on the triplicated chromosome 21 (Belichenko et al., 2009). Interestingly, in respect to the lack of degeneration of motor neurons in CN12 and CN3/4 in spinal muscular atrophy, genes identified as common expressors between these groups could provide clues to protective mechanisms in this disease where degeneration is caused by a deficiency of SMN1 protein, with altered stoichiometry of small nuclear RNAs and pre-mRNA splicing defects (Zhang et al., 2008). On the other hand, genes that are differentially expressed between these groups, and genes that are shared between the spinal cord and CN12 motor neurons, could reveal why motor neurons in CN12 and spinal cord degenerate in amyotrophic lateral sclerosis and spinobulbar muscular atrophy, while those in CN3/4 do not.

Critically, our analyses revealed that all three motor neuron subpopulations displayed distinct profiles and exhibited genes with unique expression. Our novel identification of differences in Hox gene expression levels in motor neurons isolated from the three anatomical nuclei along the anterior–posterior (A–P) axis of the adult rat matched that of the developing embryo (Carpenter, 2002) and validated the microarray data. This differential Hox gene expression pattern in the adult nervous system indicates that these genes might be important for maintenance of phenotype in addition to providing positional information during development. Consistent with such a role, adult expression of the Hox-like homeoprotein pancreatic and duodenal homeobox 1 (Pdx1) is necessary for the maintenance of pancreatic cells (Holland et al., 2002) and prospero homeobox protein 1 for lymphatic endothelial cells (Johnson et al., 2008). Comparison of groups of genes revealed differences in regulation of genes involved in endoplasmatic reticulum and mitochondrial functions, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport and growth; cellular pathways that have been implicated in the pathogenesis of amyotrophic lateral sclerosis (Collard et al., 1995; Raoul et al., 2002, 2006; Inoue et al., 2003; Pasinelli et al., 2004; Jiang et al., 2005; Kirkinezos et al., 2005; Obal et al., 2006; Nishitoh et al., 2008). The importance of RNA processing for motor neuron survival is evident since loss of SMN1 causes spinal muscular atrophy (Bussaglia et al., 1995; Lefebvre et al., 1995) and mutations in TDP-43 and FUS cause amyotrophic lateral sclerosis (Kabashi et al., 2008; Sreedharan et al., 2008; Kwiatkowski et al., 2009; Vance et al., 2009). Our expression analysis showed large differences in the number of genes involved in transcription, RNA metabolic and biosynthetic processing, RNA binding, RNA splicing and regulation of translation in the different motor neuron subpopulations, further indicating that these processes could in part be responsible for the differential vulnerability observed.

While it is beyond the scope of this article to go into detail for all these pathways, it illustrates the usefulness of a cell phenotype analysis with subsequent verification and exploration of a few differentially expressed genes with implications for motor neuron degeneration.

Differential expression of genes with implications for motor neuron vulnerability

We identified peripherin to be predominantly expressed in spinal motor neurons. Over-expression of peripherin, results in defective axonal transport of neurofilament proteins (Millecamps et al., 2006) and late-onset motor neuron degeneration (Beaulieu et al., 1999). Elevated levels of peripherin splice forms have been detected in spinal cords of patients with familial (Robertson et al., 2003) and sporadic amyotrophic lateral sclerosis (He and Hays, 2004; Xiao et al., 2008). Mutations in the peripherin gene are associated with a small percentage of amyotrophic lateral sclerosis cases (Gros-Louis et al., 2004; Leung et al., 2004). Consequently, a higher level of peripherin within specific motor neurons might predispose these cells to degenerative events.

We identified several genes as selectively expressed within motor neurons of CN3/4, which could play protective roles, e.g. Gucy1a3, early growth response protein 1, IGF-II and guanine deaminase. Gucy1a3 functions as the main receptor for nitric oxide (Zabel et al., 1998). Motor neurons from mutant SOD1 mice show increased susceptibility to exogenous nitric oxide, through upregulation of Fas ligand and subsequent Fas receptor activation. The activation of Fas receptor leads to further nitric oxide synthesis and it has been proposed that chronic low-level activation of the Fas/nitric oxide feedback loop may underlie the progressive motor neuron loss that characterizes familial amyotrophic lateral sclerosis (Raoul et al., 2006). The presence of Gucy1a3 within motor neurons of CN3/4 suggests that these cells will contain less unbound nitric oxide and, as a consequence, might show a lower level of Fas activation. Furthermore, the CN3/4-restricted gene early growth response protein 1 can confer resistance to apoptotic signals by inhibiting Fas expression, and thereby leading to insensitivity to Fas ligand (Dinkel et al., 1997). The higher expression of early growth response protein 1 within motor neurons of CN3/4 could help to explain further why these cells are not affected by degeneration in amyotrophic lateral sclerosis. In addition, the restricted expression of IGF-II to CN3/4 motor neurons could prove beneficial to these cells. IGF-II can act as a survival factor for motor neurons and can support regeneration of motor axons after nerve injury and during normal development (Caroni and Grandes, 1990; Near et al., 1992; Pu et al., 1999). Guanine deaminase catalyses the conversion of guanine to xanthine. Analysis in hippocampal neurons has shown that guanine deaminase can regulate post-synaptic sorting (Firestein et al., 1999) and promote dendritic branching (Akum et al., 2004). Interestingly, the gene TDP-43 can promote dendritic branching, but amyotrophic lateral sclerosis-associated mutations in TDP-43 attenuates the dendritic function (Lu et al., 2009). We therefore reasoned that a high expression of guanine deaminase, a protein important for dendritic branching and synaptic function, could be protective to motor neurons.

None of these gene products have, to our knowledge, previously been identified to have a differential expression within subpopulations of motor neurons.

Functional analysis revealed neuroprotective properties of the CN3/4-restricted genes IGF-II and guanine deaminase

Motor neuron toxicity and protection in response to glutamate was assayed in a system containing neurons and astrocytes. Glutamate toxicity was utilized since it is considered a downstream event in motor neuron degeneration. Motor neurons are usually protected from high levels of glutamate in vivo by surrounding astrocytes. However, astrocytes in the spinal cords of patients with amyotrophic lateral sclerosis and lower motor neuron disease (Rothstein et al., 1995; Sasaki et al., 2000) and in mutant SOD1 mice (Bruijn et al., 1997) and rats (Howland et al., 2002), have been shown to lose the expression of the focal glutamate transporter excitatory amino acid transporter 2, which could decrease their ability to sequester glutamate. In our assay we blocked excitatory amino acid transporters to mimic glutamate over-load in motor neuron disease and to hopefully create a reliable tool in predicting substances that can protect motor neurons in vivo. We believe that the predicted value for finding neuroprotective genes by analysing neurons displaying differential vulnerability to disease for their expression profiles in the normal animal is high, based on our previous approach in Parkinson’s disease models (Chung et al., 2005). Subsequently, we tested the effects of the CN3/4-specific genes IGF-II and guanine deaminase in our assay and found that IGF-II blocked glutamate-induced motor neuron loss completely, while guanine deaminase considerably decreased the loss of motor neurons. Future studies will analyse the utility of additional CN3/4-specific genes for neuroprotective properties in vitro and evaluate the protective properties of IGF-II and guanine deaminase in animal models of motor neuron disorders. These data are promising for selection of future gene therapy and/or drug targets for motor neuron diseases based on a specific gene expression within protected motor neurons. These findings also suggest that future drug screening profiles could analyse the up- or down-regulation of a handful of genes (identified as differentially expressed among motor neurons showing differential vulnerability to degeneration) to enable an evaluation of possible neuroprotective properties of drug candidates and hopefully of potential clinical success.

We believe that our report provides insight into the intrinsic properties of different motor neuron subpopulations and gives important clues to the mechanisms of relative vulnerability. Therefore, this extensive expression analysis could provide a basis for understanding why degeneration in amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy involve some, but not all, motor neuron populations and hopefully be used to develop treatments for these diseases.

Funding

This work was supported by a young investigator award from the ALS Association (E.H.), ALS Research Program Therapeutic Development Award/DOD USAMRAA W81XWH-08-1-0496 (O.I.) the Consolidated Anti-Aging Foundation (O.I.) and the training grant award number T32AG000222-17 from the National Institute On Aging (T.O.).

Supplementary material

Supplementary material is available at Brain online.

Abbreviations

    Abbreviations
  • CN

    cranial nerve

  • Gucy1a3

    guanylate cyclase soluble subunit alpha-3

  • IGF

    insulin-like growth factor

  • LCM

    laser capture microdissection

  • PDC

    l-trans-2,4-pyrrolidine-2,4-dicarboxylic acid

  • SMN1

    survival of motor neuron protein

  • SOD1

    superoxide dismutase 1

References

Abrams
CS
Wu
H
Zhao
W
Belmonte
E
White
D
Brass
LF
Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains
J Biol Chem
 , 
1995
, vol. 
270
 (pg. 
14485
-
92
)
Adelman
JP
Bond
CT
Pessia
M
Maylie
J
Episodic ataxia results from voltage-dependent potassium channels with altered functions
Neuron
 , 
1995
, vol. 
15
 (pg. 
1449
-
54
)
Agalliu
D
Takada
S
Agalliu
I
McMahon
AP
Jessell
TM
Motor neurons with axial muscle projections specified by Wnt4/5 signaling
Neuron
 , 
2009
, vol. 
61
 (pg. 
708
-
20
)
Akum
BF
Chen
M
Gunderson
SI
Riefler
GM
Scerri-Hansen
MM
Firestein
BL
Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly
Nat Neurosci
 , 
2004
, vol. 
7
 (pg. 
145
-
52
)
Arber
S
Ladle
DR
Lin
JH
Frank
E
Jessell
TM
ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons
Cell
 , 
2000
, vol. 
101
 (pg. 
485
-
98
)
Aruga
J
Mikoshiba
K
Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth
Mol Cell Neurosci
 , 
2003
, vol. 
24
 (pg. 
117
-
29
)
Barrow
LL
Simin
K
Jones
JM
Lee
DC
Meisler
MH
Conserved linkage of early growth response 4, annexin 4, and transforming growth factor alpha on mouse chromosome 6
Genomics
 , 
1994
, vol. 
19
 (pg. 
388
-
90
)
Basille
M
Vaudry
D
Coulouarn
Y
Jegou
S
Lihrmann
I
Fournier
A
, et al.  . 
Comparative distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites and PACAP receptor mRNAs in the rat brain during development
J Comp Neurol
 , 
2000
, vol. 
425
 (pg. 
495
-
509
)
Bastien
L
Sawyer
N
Grygorczyk
R
Metters
KM
Adam
M
Cloning, functional expression, and characterization of the human prostaglandin E2 receptor EP2 subtype
J Biol Chem
 , 
1994
, vol. 
269
 (pg. 
11873
-
7
)
Beaulieu
JM
Nguyen
MD
Julien
JP
Late onset of motor neurons in mice overexpressing wild-type peripherin
J Cell Biol
 , 
1999
, vol. 
147
 (pg. 
531
-
44
)
Beers
DR
Henkel
JS
Xiao
Q
Zhao
W
Wang
J
Yen
AA
, et al.  . 
Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis
Proc Natl Acad Sci USA
 , 
2006
, vol. 
103
 (pg. 
16021
-
6
)
Belichenko
NP
Belichenko
PV
Kleschevnikov
AM
Salehi
A
Reeves
RH
Mobley
WC
The “Down syndrome critical region” is sufficient in the mouse model to confer behavioral, neurophysiological, and synaptic phenotypes characteristic of Down syndrome
J Neurosci
 , 
2009
, vol. 
29
 (pg. 
5938
-
48
)
Birts
CN
Barton
CH
Wilton
DC
A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal
J Biol Chem
 , 
2008
, vol. 
283
 (pg. 
5034
-
45
)
Bohni
R
Riesgo-Escovar
J
Oldham
S
Brogiolo
W
Stocker
H
Andruss
BF
, et al.  . 
Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4
Cell
 , 
1999
, vol. 
97
 (pg. 
865
-
75
)
Boillee
S
Yamanaka
K
Lobsiger
CS
Copeland
NG
Jenkins
NA
Kassiotis
G
, et al.  . 
Onset and progression in inherited ALS determined by motor neurons and microglia
Science
 , 
2006
, vol. 
312
 (pg. 
1389
-
92
)
Boyd
AW
Ward
LD
Wicks
IP
Simpson
RJ
Salvaris
E
Wilks
A
, et al.  . 
Isolation and characterization of a novel receptor-type protein tyrosine kinase (hek) from a human pre-B cell line
J Biol Chem
 , 
1992
, vol. 
267
 (pg. 
3262
-
7
)
Broccoli
V
Boncinelli
E
Wurst
W
The caudal limit of Otx2 expression positions the isthmic organizer
Nature
 , 
1999
, vol. 
401
 (pg. 
164
-
8
)
Bruijn
LI
Becher
MW
Lee
MK
Anderson
KL
Jenkins
NA
Copeland
NG
, et al.  . 
ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions
Neuron
 , 
1997
, vol. 
18
 (pg. 
327
-
38
)
Bussaglia
E
Clermont
O
Tizzano
E
Lefebvre
S
Burglen
L
Cruaud
C
, et al.  . 
A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients
Nat Genet
 , 
1995
, vol. 
11
 (pg. 
335
-
7
)
Caroni
P
Grandes
P
Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors
J Cell Biol
 , 
1990
, vol. 
110
 (pg. 
1307
-
17
)
Carpenter
EM
Hox genes and spinal cord development
Dev Neurosci
 , 
2002
, vol. 
24
 (pg. 
24
-
34
)
Chintala
S
Tan
J
Gautam
R
Rusiniak
ME
Guo
X
Li
W
, et al.  . 
The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet-dense granules
Blood
 , 
2007
, vol. 
109
 (pg. 
1533
-
40
)
Chung
CY
Koprich
JB
Siddiqi
H
Isacson
O
Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy
J Neurosci
 , 
2009
, vol. 
29
 (pg. 
3365
-
73
)
Chung
CY
Seo
H
Sonntag
KC
Brooks
A
Lin
L
Isacson
O
Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection
Hum Mol Genet
 , 
2005
, vol. 
14
 (pg. 
1709
-
25
)
Collard
JF
Cote
F
Julien
JP
Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis
Nature
 , 
1995
, vol. 
375
 (pg. 
61
-
4
)
Dasen
JS
Tice
BC
Brenner-Morton
S
Jessell
TM
A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity
Cell
 , 
2005
, vol. 
123
 (pg. 
477
-
91
)
DeChiara
TM
Robertson
EJ
Efstratiadis
A
Parental imprinting of the mouse insulin-like growth factor II gene
Cell
 , 
1991
, vol. 
64
 (pg. 
849
-
59
)
Di Giorgio
FP
Boulting
GL
Bobrowicz
S
Eggan
KC
Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation
Cell Stem Cell
 , 
2008
, vol. 
3
 (pg. 
637
-
48
)
Dinkel
A
Aicher
WK
Haas
C
Zipfel
PF
Peter
HH
Eibel
H
Transcription factor Egr-1 activity down-regulates Fas and CD23 expression in B cells
J Immunol
 , 
1997
, vol. 
159
 (pg. 
2678
-
84
)
Firestein
BL
Firestein
BL
Brenman
JE
Aoki
C
Sanchez-Perez
AM
El-Husseini
AE
, et al.  . 
Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting
Neuron
 , 
1999
, vol. 
24
 (pg. 
659
-
72
)
Gallarda
BW
Bonanomi
D
Muller
D
Brown
A
Alaynick
WA
Andrews
SE
, et al.  . 
Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling
Science
 , 
2008
, vol. 
320
 (pg. 
233
-
6
)
Garden
GA
Libby
RT
Fu
YH
Kinoshita
Y
Huang
J
Possin
DE
, et al.  . 
Polyglutamine-expanded ataxin-7 promotes non-cell-autonomous purkinje cell degeneration and displays proteolytic cleavage in ataxic transgenic mice
J Neurosci
 , 
2002
, vol. 
22
 (pg. 
4897
-
905
)
Gizzi
M
DiRocco
A
Sivak
M
Cohen
B
Ocular motor function in motor neuron disease
Neurology
 , 
1992
, vol. 
42
 (pg. 
1037
-
46
)
Gomi
F
Imaizumi
K
Yoneda
T
Taniguchi
M
Mori
Y
Miyoshi
K
, et al.  . 
Molecular cloning of a novel membrane glycoprotein, pal, specifically expressed in photoreceptor cells of the retina and containing leucine-rich repeat
J Neurosci
 , 
2000
, vol. 
20
 (pg. 
3206
-
13
)
Greenway
MJ
Andersen
PM
Russ
C
Ennis
S
Cashman
S
Donaghy
C
, et al.  . 
ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis
Nat Genet
 , 
2006
, vol. 
38
 (pg. 
411
-
13
)
Gros-Louis
F
Lariviere
R
Gowing
G
Laurent
S
Camu
W
Bouchard
JP
, et al.  . 
A frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis
J Biol Chem
 , 
2004
, vol. 
279
 (pg. 
45951
-
6
)
Gu
C
Jan
YN
Jan
LY
A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels
Science
 , 
2003
, vol. 
301
 (pg. 
646
-
9
)
Gu
X
Li
C
Wei
W
Lo
V
Gong
S
Li
SH
, et al.  . 
Pathological cell-cell interactions elicited by a neuropathogenic form of mutant Huntingtin contribute to cortical pathogenesis in HD mice
Neuron
 , 
2005
, vol. 
46
 (pg. 
433
-
44
)
Haenggeli
C
Kato
AC
Differential vulnerability of cranial motoneurons in mouse models with motor neuron degeneration
Neurosci Lett
 , 
2002
, vol. 
335
 (pg. 
39
-
43
)
Harmar
AJ
Marston
HM
Shen
S
Spratt
C
West
KM
Sheward
WJ
, et al.  . 
The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei
Cell
 , 
2002
, vol. 
109
 (pg. 
497
-
508
)
He
CZ
Hays
AP
Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis
J Neurol Sci
 , 
2004
, vol. 
217
 (pg. 
47
-
54
)
Hedlund
E
Isacson
O
ALS model glia can mediate toxicity to motor neurons derived from human embryonic stem cells
Cell Stem Cell
 , 
2008
, vol. 
3
 (pg. 
575
-
6
)
Hedlund
E
Karsten
SL
Kudo
L
Geschwind
DH
Carpenter
EM
Identification of a Hoxd10-regulated transcriptional network and combinatorial interactions with Hoxa10 during spinal cord development
J Neurosci Res
 , 
2004
, vol. 
75
 (pg. 
307
-
19
)
Henderson
CE
Camu
W
Mettling
C
Gouin
A
Poulsen
K
Karihaloo
M
, et al.  . 
Neurotrophins promote motor neuron survival and are present in embryonic limb bud
Nature
 , 
1993
, vol. 
363
 (pg. 
266
-
70
)
Holland
AM
Hale
MA
Kagami
H
Hammer
RE
MacDonald
RJ
Experimental control of pancreatic development and maintenance
Proc Natl Acad Sci USA
 , 
2002
, vol. 
99
 (pg. 
12236
-
41
)
Honore
B
The rapidly expanding CREC protein family: members, localization, function, and role in disease
Bioessays
 , 
2009
, vol. 
31
 (pg. 
262
-
77
)
Hopker
VH
Shewan
D
Tessier-Lavigne
M
Poo
M
Holt
C
Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1
Nature
 , 
1999
, vol. 
401
 (pg. 
69
-
73
)
Howland
DS
Liu
J
She
Y
Goad
B
Maragakis
NJ
Kim
B
, et al.  . 
Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS)
Proc Natl Acad Sci USA
 , 
2002
, vol. 
99
 (pg. 
1604
-
9
)
Huang da
W
Sherman
BT
Lempicki
RA
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources
Nat Protoc
 , 
2009
, vol. 
4
 (pg. 
44
-
57
)
Inoue
H
Tsukita
K
Iwasato
T
Suzuki
Y
Tomioka
M
Tateno
M
, et al.  . 
The crucial role of caspase-9 in the disease progression of a transgenic ALS mouse model
Embo J
 , 
2003
, vol. 
22
 (pg. 
6665
-
74
)
Jaarsma
D
Teuling
E
Haasdijk
ED
De Zeeuw
CI
Hoogenraad
CC
Neuron-specific expression of mutant superoxide dismutase is sufficient to induce amyotrophic lateral sclerosis in transgenic mice
J Neurosci
 , 
2008
, vol. 
28
 (pg. 
2075
-
88
)
Jiang
YM
Yamamoto
M
Kobayashi
Y
Yoshihara
T
Liang
Y
Terao
S
, et al.  . 
Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis
Ann Neurol
 , 
2005
, vol. 
57
 (pg. 
236
-
51
)
Johnson
NC
Dillard
ME
Baluk
P
McDonald
DM
Harvey
NL
Frase
SL
, et al.  . 
Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity
Genes Dev
 , 
2008
, vol. 
22
 (pg. 
3282
-
91
)
Kabashi
E
Valdmanis
PN
Dion
P
Spiegelman
D
McConkey
BJ
Vande Velde
C
, et al.  . 
TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis
Nat Genet
 , 
2008
, vol. 
40
 (pg. 
572
-
4
)
Kakinohana
O
Cizkova
D
Tomori
Z
Hedlund
E
Marsala
S
Isacson
O
, et al.  . 
Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study
Exp Neurol
 , 
2004
, vol. 
190
 (pg. 
122
-
32
)
Kaspar
BK
Llado
J
Sherkat
N
Rothstein
JD
Gage
FH
Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model
Science
 , 
2003
, vol. 
301
 (pg. 
839
-
42
)
Kawaguchi
T
Qin
L
Shimomura
T
Kondo
J
Matsumoto
K
Denda
K
, et al.  . 
Purification and cloning of hepatocyte growth factor activator inhibitor type 2, a Kunitz-type serine protease inhibitor
J Biol Chem
 , 
1997
, vol. 
272
 (pg. 
27558
-
64
)
Kennedy
WR
Alter
M
Sung
JH
Progressive proximal spinal and bulbar muscular atrophy of late onset. A sex-linked recessive trait
Neurology
 , 
1968
, vol. 
18
 (pg. 
671
-
80
)
Kirkinezos
IG
Bacman
SR
Hernandez
D
Oca-Cossio
J
Arias
LJ
Perez-Pinzon
MA
, et al.  . 
Cytochrome c association with the inner mitochondrial membrane is impaired in the CNS of G93A-SOD1 mice
J Neurosci
 , 
2005
, vol. 
25
 (pg. 
164
-
72
)
Kittappa
R
Chang
WW
Awatramani
RB
McKay
RD
The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age
PLoS Biol
 , 
2007
, vol. 
5
 pg. 
e325
 
Kolodkin
AL
Levengood
DV
Rowe
EG
Tai
YT
Giger
RJ
Ginty
DD
Neuropilin is a semaphorin III receptor
Cell
 , 
1997
, vol. 
90
 (pg. 
753
-
62
)
Kowenz-Leutz
E
Herr
P
Niss
K
Leutz
A
The homeobox gene GBX2, a target of the myb oncogene, mediates autocrine growth and monocyte differentiation
Cell
 , 
1997
, vol. 
91
 (pg. 
185
-
95
)
Krumlauf
R
Hox genes in vertebrate development
Cell
 , 
1994
, vol. 
78
 (pg. 
191
-
201
)
Kulesh
DA
Oshima
RG
Complete structure of the gene for human keratin 18
Genomics
 , 
1989
, vol. 
4
 (pg. 
339
-
47
)
Kwiatkowski
TJ
Jr
Bosco
DA
Leclerc
AL
Tamrazian
E
Vanderburg
CR
Russ
C
, et al.  . 
Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis
Science
 , 
2009
, vol. 
323
 (pg. 
1205
-
8
)
La Spada
AR
Wilson
EM
Lubahn
DB
Harding
AE
Fischbeck
KH
Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy
Nature
 , 
1991
, vol. 
352
 (pg. 
77
-
9
)
Lai
CS
Fisher
SE
Hurst
JA
Vargha-Khadem
F
Monaco
AP
A forkhead-domain gene is mutated in a severe speech and language disorder
Nature
 , 
2001
, vol. 
413
 (pg. 
519
-
23
)
Lee
CS
Friedman
JR
Fulmer
JT
Kaestner
KH
The initiation of liver development is dependent on Foxa transcription factors
Nature
 , 
2005
, vol. 
435
 (pg. 
944
-
7
)
Lee
DS
Tomita
S
Kirino
Y
Suzuki
T
Regulation of X11L-dependent amyloid precursor protein metabolism by XB51, a novel X11L-binding protein
J Biol Chem
 , 
2000
, vol. 
275
 (pg. 
23134
-
8
)
Lefebvre
S
Burglen
L
Reboullet
S
Clermont
O
Burlet
P
Viollet
L
, et al.  . 
Identification and characterization of a spinal muscular atrophy-determining gene
Cell
 , 
1995
, vol. 
80
 (pg. 
155
-
65
)
Leung
CL
He
CZ
Kaufmann
P
Chin
SS
Naini
A
Liem
RK
, et al.  . 
A pathogenic peripherin gene mutation in a patient with amyotrophic lateral sclerosis
Brain Pathol
 , 
2004
, vol. 
14
 (pg. 
290
-
6
)
Leveille
A
Kiernan
J
Goodwin
JA
Antel
J
Eye movements in amyotrophic lateral sclerosis
Arch Neurol
 , 
1982
, vol. 
39
 (pg. 
684
-
6
)
Lian
L
Wang
Y
Flick
M
Choi
J
Scott
EW
Degen
J
, et al.  . 
Loss of pleckstrin defines a novel pathway for PKC-mediated exocytosis
Blood
 , 
2009
, vol. 
113
 (pg. 
3577
-
84
)
Liu
YF
Civelli
O
Zhou
QY
Albert
PR
Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A
Mol Endocrinol
 , 
1992
, vol. 
6
 (pg. 
1815
-
24
)
Lu
Y
Ferris
J
Gao
FB
Frontotemporal dementia and amyotrophic lateral sclerosis-associated disease protein TDP-43 promotes dendritic branching
Mol Brain
 , 
2009
, vol. 
2
 pg. 
30
 
Maglione
D
Guerriero
V
Viglietto
G
Delli-Bovi
P
Persico
MG
Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor
Proc Natl Acad Sci U S A
 , 
1991
, vol. 
88
 (pg. 
9267
-
71
)
Mai
JK
Triepel
J
Metz
J
Neurotensin in the human brain
Neuroscience
 , 
1987
, vol. 
22
 (pg. 
499
-
524
)
Marchetto
MC
Muotri
AR
Mu
Y
Smith
AM
Cezar
GG
Gage
FH
Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells
Cell Stem Cell
 , 
2008
, vol. 
3
 (pg. 
649
-
57
)
Marondel
I
Renault
B
Lieman
J
Ward
D
Kucherlapati
R
Physical mapping of the human neurotensin gene (NTS) between markers D12S1444 and D12S81 on chromosome 12q21
Genomics
 , 
1996
, vol. 
38
 (pg. 
243
-
5
)
Mazzarella
R
Pengue
G
Jones
J
Jones
C
Schlessinger
D
Cloning and expression of an immunoglobulin superfamily gene (IGSF1) in Xq25
Genomics
 , 
1998
, vol. 
48
 (pg. 
157
-
62
)
McDonald
JK
Zeitman
BB
Reilly
TJ
Ellis
S
New observations on the substrate specificity of cathepsin C (dipeptidyl aminopeptidase I). Including the degradation of beta-corticotropin and other peptide hormones
J Biol Chem
 , 
1969
, vol. 
244
 (pg. 
2693
-
709
)
McGinnis
W
Krumlauf
R
Homeobox genes and axial patterning
Cell
 , 
1992
, vol. 
68
 (pg. 
283
-
302
)
Mercader
N
Leonardo
E
Azpiazu
N
Serrano
A
Morata
G
Martinez
C
, et al.  . 
Conserved regulation of proximodistal limb axis development by Meis1/Hth
Nature
 , 
1999
, vol. 
402
 (pg. 
425
-
9
)
Millecamps
S
Robertson
J
Lariviere
R
Mallet
J
Julien
JP
Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin
J Neurochem
 , 
2006
, vol. 
98
 (pg. 
926
-
38
)
Millet
S
Campbell
K
Epstein
DJ
Losos
K
Harris
E
Joyner
AL
A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer
Nature
 , 
1999
, vol. 
401
 (pg. 
161
-
4
)
Morio
H
Tatsuno
I
Hirai
A
Tamura
Y
Saito
Y
Pituitary adenylate cyclase-activating polypeptide protects rat-cultured cortical neurons from glutamate-induced cytotoxicity
Brain Res
 , 
1996
, vol. 
741
 (pg. 
82
-
8
)
Mosimann
C
Hausmann
G
Basler
K
Beta-catenin hits chromatin: regulation of Wnt target gene activation
Nat Rev Mol Cell Biol
 , 
2009
, vol. 
10
 (pg. 
276
-
86
)
Moskow
JJ
Bullrich
F
Huebner
K
Daar
IO
Buchberg
AM
Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice
Mol Cell Biol
 , 
1995
, vol. 
15
 (pg. 
5434
-
43
)
Muramatsu
T
Kincaid
RL
Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (calcineurin A alpha)
Biochim Biophys Acta
 , 
1993
, vol. 
1178
 (pg. 
117
-
20
)
Nachemson
AK
Lundborg
G
Hansson
HA
Insulin-like growth factor I promotes nerve regeneration: an experimental study on rat sciatic nerve
Growth Factors
 , 
1990
, vol. 
3
 (pg. 
309
-
14
)
Nagai
M
Re
DB
Nagata
T
Chalazonitis
A
Jessell
TM
Wichterle
H
, et al.  . 
Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons
Nat Neurosci
 , 
2007
, vol. 
10
 (pg. 
615
-
22
)
Near
SL
Whalen
LR
Miller
JA
Ishii
DN
Insulin-like growth factor II stimulates motor nerve regeneration
Proc Natl Acad Sci USA
 , 
1992
, vol. 
89
 (pg. 
11716
-
20
)
Nimchinsky
EA
Young
WG
Yeung
G
Shah
RA
Gordon
JW
Bloom
FE
, et al.  . 
Differential vulnerability of oculomotor, facial, and hypoglossal nuclei in G86R superoxide dismutase transgenic mice
J Comp Neurol
 , 
2000
, vol. 
416
 (pg. 
112
-
25
)
Nishitoh
H
Kadowaki
H
Nagai
A
Maruyama
T
Yokota
T
Fukutomi
H
, et al.  . 
ALS-linked mutant SOD1 induces ER stress- and ASK1-dependent motor neuron death by targeting Derlin-1
Genes Dev
 , 
2008
, vol. 
22
 (pg. 
1451
-
64
)
Obal
I
Engelhardt
JI
Siklos
L
Axotomy induces contrasting changes in calcium and calcium-binding proteins in oculomotor and hypoglossal nuclei of Balb/c mice
J Comp Neurol
 , 
2006
, vol. 
499
 (pg. 
17
-
32
)
Okamoto
O
Fujiwara
S
Dermatopontin, a novel player in the biology of the extracellular matrix
Connect Tissue Res
 , 
2006
, vol. 
47
 (pg. 
177
-
89
)
Paraoanu
LE
Steinert
G
Klaczinski
J
Becker-Rock
M
Bytyqi
A
Layer
PG
On functions of cholinesterases during embryonic development
J Mol Neurosci
 , 
2006
, vol. 
30
 (pg. 
201
-
4
)
Parsons
SF
Mallinson
G
Holmes
CH
Houlihan
JM
Simpson
KL
Mawby
WJ
, et al.  . 
The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver
Proc Natl Acad Sci USA
 , 
1995
, vol. 
92
 (pg. 
5496
-
500
)
Pasinelli
P
Belford
ME
Lennon
N
Bacskai
BJ
Hyman
BT
Trotti
D
, et al.  . 
Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria
Neuron
 , 
2004
, vol. 
43
 (pg. 
19
-
30
)
Pestonjamasp
KN
Pope
RK
Wulfkuhle
JD
Luna
EJ
Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily
J Cell Biol
 , 
1997
, vol. 
139
 (pg. 
1255
-
69
)
Polymeropoulos
MH
Lavedan
C
Leroy
E
Ide
SE
Dehejia
A
Dutra
A
, et al.  . 
Mutation in the alpha-synuclein gene identified in families with Parkinson's disease
Science
 , 
1997
, vol. 
276
 (pg. 
2045
-
7
)
Powell-Braxton
L
Hollingshead
P
Warburton
C
Dowd
M
Pitts-Meek
S
Dalton
D
, et al.  . 
IGF-I is required for normal embryonic growth in mice
Genes Dev
 , 
1993
, vol. 
7
 (pg. 
2609
-
17
)
Pu
SF
Zhuang
HX
Marsh
DJ
Ishii
DN
Insulin-like growth factor-II increases and IGF is required for postnatal rat spinal motoneuron survival following sciatic nerve axotomy
J Neurosci Res
 , 
1999
, vol. 
55
 (pg. 
9
-
16
)
Raab-Graham
KF
Haddick
PC
Jan
YN
Jan
LY
Activity- and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites
Science
 , 
2006
, vol. 
314
 (pg. 
144
-
8
)
Rakhilin
SV
Olson
PA
Nishi
A
Starkova
NN
Fienberg
AA
Nairn
AC
, et al.  . 
A network of control mediated by regulator of calcium/calmodulin-dependent signaling
Science
 , 
2004
, vol. 
306
 (pg. 
698
-
701
)
Raoul
C
Buhler
E
Sadeghi
C
Jacquier
A
Aebischer
P
Pettmann
B
, et al.  . 
Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL
Proc Natl Acad Sci USA
 , 
2006
, vol. 
103
 (pg. 
6007
-
12
)
Raoul
C
Estevez
AG
Nishimune
H
Cleveland
DW
deLapeyriere
O
Henderson
CE
, et al.  . 
Motoneuron death triggered by a specific pathway downstream of Fas
potentiation by ALS-linked SOD1 mutations. Neuron
 , 
2002
, vol. 
35
 (pg. 
1067
-
83
)
Reiner
A
Medina
L
Figueredo-Cardenas
G
Anfinson
S
Brainstem motoneuron pools that are selectively resistant in amyotrophic lateral sclerosis are preferentially enriched in parvalbumin: evidence from monkey brainstem for a calcium-mediated mechanism in sporadic ALS
Exp Neurol
 , 
1995
, vol. 
131
 (pg. 
239
-
50
)
Robertson
J
Doroudchi
MM
Nguyen
MD
Durham
HD
Strong
MJ
Shaw
G
, et al.  . 
A neurotoxic peripherin splice variant in a mouse model of ALS
J Cell Biol
 , 
2003
, vol. 
160
 (pg. 
939
-
49
)
Rogge
G
Jones
D
Hubert
GW
Lin
Y
Kuhar
MJ
CART peptides: regulators of body weight, reward and other functions
Nat Rev Neurosci
 , 
2008
, vol. 
9
 (pg. 
747
-
58
)
Rosen
DR
Siddique
T
Patterson
D
Figlewicz
DA
Sapp
P
Hentati
A
, et al.  . 
Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis
Nature
 , 
1993
, vol. 
362
 (pg. 
59
-
62
)
Ross
CA
Huntington's disease: new paths to pathogenesis
Cell
 , 
2004
, vol. 
118
 (pg. 
4
-
7
)
Rubin
JS
Osada
H
Finch
PW
Taylor
WG
Rudikoff
S
Aaronson
SA
Purification and characterization of a newly identified growth factor specific for epithelial cells
Proc Natl Acad Sci U S A
 , 
1989
, vol. 
86
 (pg. 
802
-
6
)
Sasaki
S
Komori
T
Iwata
M
Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis
Acta Neuropathol
 , 
2000
, vol. 
100
 (pg. 
138
-
44
)
Satokata
I
Ma
L
Ohshima
H
Bei
M
Woo
I
Nishizawa
K
, et al.  . 
Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation
Nat Genet
 , 
2000
, vol. 
24
 (pg. 
391
-
5
)
Shaw
CE
Enayat
ZE
Powell
JF
Anderson
VE
Radunovic
A
al-Sarraj
S
, et al.  . 
Familial amyotrophic lateral sclerosis. Molecular pathology of a patient with a SOD1 mutation
Neurology
 , 
1997
, vol. 
49
 (pg. 
1612
-
6
)
Singleton
AB
Farrer
M
Johnson
J
Singleton
A
Hague
S
Kachergus
J
, et al.  . 
alpha-Synuclein locus triplication causes Parkinson's disease
Science
 , 
2003
, vol. 
302
 pg. 
841
 
Sobue
G
X-linked recessive bulbospinal neuronopathy (SBMA)
Nagoya J Med Sci
 , 
1995
, vol. 
58
 (pg. 
95
-
106
)
Speca
DJ
Lin
DM
Sorensen
PW
Isacoff
EY
Ngai
J
Dittman
AH
Functional identification of a goldfish odorant receptor
Neuron
 , 
1999
, vol. 
23
 (pg. 
487
-
98
)
Sreedharan
J
Blair
IP
Tripathi
VB
Hu
X
Vance
C
Rogelj
B
, et al.  . 
TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis
Science
 , 
2008
, vol. 
319
 (pg. 
1668
-
72
)
Umemori
H
Linhoff
MW
Ornitz
DM
Sanes
JR
FGF22 and its close relatives are presynaptic organizing molecules in the mammalian brain
Cell
 , 
2004
, vol. 
118
 (pg. 
257
-
70
)
Vance
C
Rogelj
B
Hortobagyi
T
De Vos
KJ
Nishimura
AL
Sreedharan
J
, et al.  . 
Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6
Science
 , 
2009
, vol. 
323
 (pg. 
1208
-
11
)
Varnum
BC
Young
C
Elliott
G
Garcia
A
Bartley
TD
Fridell
YW
, et al.  . 
Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6
Nature
 , 
1995
, vol. 
373
 (pg. 
623
-
6
)
Wakabayashi
Y
Watanabe
H
Inoue
J
Takeda
N
Sakata
J
Mishima
Y
, et al.  . 
Bcl11b is required for differentiation and survival of alphabeta T lymphocytes
Nat Immunol
 , 
2003
, vol. 
4
 (pg. 
533
-
9
)
Waters
ST
Lewandoski
M
A threshold requirement for Gbx2 levels in hindbrain development
Development
 , 
2006
, vol. 
133
 (pg. 
1991
-
2000
)
Wiedmer
T
Zhou
Q
Kwoh
DY
Sims
PJ
Identification of three new members of the phospholipid scramblase gene family
Biochim Biophys Acta
 , 
2000
, vol. 
1467
 (pg. 
244
-
53
)
Wistow
G
Wyatt
K
David
L
Gao
C
Bateman
O
Bernstein
S
, et al.  . 
gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates
Febs J
 , 
2005
, vol. 
272
 (pg. 
2276
-
91
)
Witze
ES
Litman
ES
Argast
GM
Moon
RT
Ahn
NG
Wnt5a control of cell polarity and directional movement by polarized redistribution of adhesion receptors
Science
 , 
2008
, vol. 
320
 (pg. 
365
-
9
)
Xia
M
Gaufo
GO
Wang
Q
Sreedharan
SP
Goetzl
EJ
Transduction of specific inhibition of HuT 78 human T cell chemotaxis by type I vasoactive intestinal peptide receptors
J Immunol
 , 
1996
, vol. 
157
 (pg. 
1132
-
8
)
Xiao
S
Tjostheim
S
Sanelli
T
McLean
JR
Horne
P
Fan
Y
, et al.  . 
An aggregate-inducing peripherin isoform generated through intron retention is upregulated in amyotrophic lateral sclerosis and associated with disease pathology
J Neurosci
 , 
2008
, vol. 
28
 (pg. 
1833
-
40
)
Yamanaka
K
Chun
SJ
Boillee
S
Fujimori-Tonou
N
Yamashita
H
Gutmann
DH
, et al.  . 
Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis
Nat Neurosci
 , 
2008
, vol. 
11
 (pg. 
251
-
3
)
Yuan
Z
Becker
EB
Merlo
P
Yamada
T
DiBacco
S
Konishi
Y
, et al.  . 
Activation of FOXO1 by Cdk1 in cycling cells and postmitotic neurons
Science
 , 
2008
, vol. 
319
 (pg. 
1665
-
8
)
Zabel
U
Weeger
M
La
M
Schmidt
HH
Human soluble guanylate cyclase: functional expression and revised isoenzyme family
Biochem J
 , 
1998
, vol. 
335 (Pt 1)
 (pg. 
51
-
7
)
Zhang
Z
Lotti
F
Dittmar
K
Younis
I
Wan
L
Kasim
M
, et al.  . 
SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing
Cell
 , 
2008
, vol. 
133
 (pg. 
585
-
600
)

Author notes

*Present address: Eva Hedlund, Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, 171 77 Stockholm, Sweden.