Correspondence: F. Song, 2226 Elliman Building, 421 East Canfi eld Street, Detroit, Michigan 48201, USA. Fax: 313 577 7552. E-mail: fsong@med.wayne.edu

(Received 31 May 2013 ; accepted 6 October 2013 )

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2014; 15: 77–83

ISSN 2167-8421 print/ISSN 2167-9223 online © 2014 Informa HealthcareDOI: 10.3109/21678421.2013.853802

(7,8) and infl ammatory mechanisms have been hypothesized to induce motor neuron death in ALS and other neurons in multiple sclerosis (5,9 – 12).

Our laboratory has been working for a number of years to defi ne the communication between motor neurons and non-neuronal cells that they contact (13 – 16). The NRG1 gene produces both membrane-bound and secreted growth and differentiation factors that regulate glial develop-ment and survival, synaptogenesis, and microglial activation (6,16 – 20). We recently showed that aberrant NRG1 signaling on activated microglia is a common pathological change in the ventral horn of both ALS patients and SOD1 mice (6), raising the possibility that NRG1 may promote disease progression in the ventral horn in ALS. In this study, we focused on patients with UNM signs to measure NRG1 signaling in the degenerating lateral and ventral CSTs com-pared to control patients with no pathology in the spinal cord.

Introduction

ALS is a disease affecting the nervous system caus-ing a progressive degeneration of motor neurons that control voluntary muscle movement. ALS causes muscle weakness and wasting throughout the body and most patients die of respiratory fail-ure within 3 – 5 years of diagnosis (1). No cure yet exists for this debilitating disease. In many patients, the disease progresses through the nervous system and involves both upper and lower motor systems (1 – 3); however, exactly how upper and lower motor systems are linked during disease progression is unknown.

Under pathological conditions, microglia, resi-dent immune cells in the central nervous system (CNS), become activated and produce reactive oxygen and nitrogen species and pro-infl ammatory cytokines, molecules that can contribute to axonal demyelination and neuron death (4). Acti-vated microglia have been found in both the spinal cord (5,6) and the brain in ALS patients

ORIGINAL ARTICLE

Activation of microglial neuregulin1 signaling in the corticospinal tracts of ALS patients with upper motor neuron signs

FEI SONG 1,2,3 , POHUNG CHIANG 1,2 , JOHN RAVITS 4 & JEFFREY A. LOEB 1,3,5

1 Hiller ALS Center, 2 Department of Neurology and 3 The Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, 4 ALS and Neuromuscular Translational Research, University of California, San Diego, and 5 Department of Neurology and Rehabilitation, University of Illinois at Chicago, Chicago, Illinois, USA

Abstract We recently found neuregulin1 (NRG1) receptors are activated on microglia in the ventral horn of both ALS patients and SOD1 mice, suggesting a common pathological mechanism. However, it is not clear whether this signaling system also plays a role in patients with upper motor neuron (UMN) features, where patients show signifi cant pathological changes in the corticospinal tracts (CSTs). Since the connection between upper and lower motor neuron (LMN) systems in ALS patients is not readily seen in the SOD1 mouse, we examined the lateral and ventral CSTs for NRG1 receptor activation and NRG1 expression in ALS patients with UMN symptoms compared to control patients with no evidence of neurode-generative disease. We found that ALS patients with UMN symptoms showed increased microglial activation that colocal-ized with NRG1 receptor activation in the lateral and ventral CSTs. These same regions also showed increased NRG1 protein expression locally but no change in NRG1 mRNA. In conclusion, these data suggest that increased NRG1 protein accumulation could contribute to UMN disease through microglial activation in the CSTs.

Key words: Neuregulin , Microglial Activation , Corticospinal Tracts

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78 F. Song et al.

Materials and methods

Human tissue collection

Fresh frozen thoracic and lumbar spinal cords from four sporadic and two familial ALS patients with marked clinical UMN signs and six control patients with no pathological evidence for neurological dis-ease were provided by Ravits (21) and the Human Brain and Spinal Fluid Resource Center (VA West Los Angeles Healthcare Center, Los Angeles, CA) (Table I). Post mortem time-intervals ranged from 2 to 25 h.

Histopathology

Fresh frozen human spinal cords were fi xed in 4% paraformaldehyde for 24 h, washed overnight in PBS, and immersed in 30% sucrose until saturated, all at 4 ° C. The spinal cords were processed and embedded in OCT (Tissue-Tek, Sakura Finetek USA, Inc., Torrance, CA) . Frozen sections were cut transversely at 20 μ m thickness and placed on Superfrost slides (Thermo Fisher Scientifi c), then stained with luxol fast-blue/periodic acid Schiff (LFB/PAS) (Poly Scientifi c, Bay Shore, NY) for the presence of myelin. For some experiments, formalin-fi xed tissue slides were used.

Immunostaining

Identifi cation and quantifi cation of microglia in human spinal cord tissue sections was performed using antibodies for human CD68 (mouse IgG1, 1:20, DAKO Cat #N1577, Carpinteria, CA). Activa-tion of NRG1 ’ s erbB2 receptor was performed with a rabbit anti-phospho-erbB2 antibody (p-Neu, Tyr1248, 1:50 Santa Cruz Biotechnology, CA). Each primary antibody was diluted in blocking solution (10% normal goat serum, 0.05% Triton X-100 in PBS) overnight at 4 ° C, followed by incubation with goat anti-mouse or rabbit Alexa fl uor 488 (1:100, Invitrogen, Carlsbad, CA). For phospho-erbB2 and CD68 immunostaining, biotin-conjugated goat anti-mouse or rabbit was used as a secondary antibody,

and the signal was amplifi ed using a tyramide signal amplifi cation kit (1:250, Invitrogen) following the manufacturer ’ s instructions. There were no signifi -cant differences in the CD68 and p-erbB2 signals between frozen/4% paraformaldehyde fi xed vs. formalin-fi xed human samples.

Microglia (CD68) and activated NRG1 receptor phospho-erbB2 (p-erbB2)-positive cells were quanti-fi ed by the presence of both a glial cell shape and a nucleus labeled with DAPI. The percent of overlapping signal was determined by the percentage of CD68 � p-erbB2 � cell number vs. CD68 � cell number in a given high-powered fi eld by manually counting.

The NRG1 rabbit polyclonal antibody AD03 was raised against the entire extracellular domain of NRG1 (18,22,23). ADO3 antibody (1:300, Assay Designs Inc., Ann Arbor, MI) was incubated overnight at 4 ° C followed by a biotinylated goat anti-rabbit IgG secondary at 1:200, streptavidin-HRP, and DAB Vectastain reagents according to the manufacturer’s recommendations (Vector Laboratories, Burlingame, CA). Use of the type III (cysteine-rich domain) specifi c antibody (clone N126B/31, UC Davis/NIH NeuroMab facility) was as described previously (6).

RNA isolation and real-time quantitative RT-PCR (qPCR)

Human thoracic and lumbar spinal cords were rapidly frozen and stored at � 80 ° C. RNA was extracted using the Qiagen RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) (24). Quantifi cation of RNA was carried out using a NanoDrop ND-1000 spectropho-tometer (Thermo Scientifi c, Wilmington, DE, USA). The quality of RNA was determined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) using an RNA 6000 Nano chip kit, RNA ladder and Agilent analysis software (Agilent Tech-nologies). All samples had RIN (RNA integrity num-ber) values above 6.0 and 260/280 ratios near 2.0.

The relative expression of human type I and III NRG1 were measured relative to GAPDH using Taq-man Assays-On-Demand primers (Applied Biosys-tems, Foster City, CA, USA); 1.5 μ g of total RNA

Table I. ALS patients with upper motor neuron signs and controls.

Patient Diagnosis Age GenderSpinal

cord levels SourceFrozen

PFA fi xedFormalin

fi xedFresh frozen

Pt1 SALS 41 M T Ravits Yes Yes YesPt2 SALS 60 M T Ravits Yes Yes YesPt3 SALS 64 F T/L UCLA Yes No YesPt4 FALS 74 F T/L UCLA Yes No YesPt5 FALS 45 M T/L UCLA Yes No YesPt6 SALS 58 F T Ravits No Yes NoCon1 Control 84 M T Ravits Yes Yes YesCon2 Control 67 M T Ravits Yes Yes YesCon3 Control 76 F T Ravits Yes Yes YesCon4 Control 68 M T Ravits Yes Yes YesCon5 Control 58 F T Ravits Yes Yes YesCon6 Control 79 M T/L UCLA Yes No Yes

SALS: sporadic ALS; FALS: familial ALS; C: cervical; T: thoracic; L: lumbar.

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Microglial neuregulin1 in the corticospinal tract 79

was used in a 20- μ l reverse transcription synthesis reaction primed with oligo-dT primers (Superscript First Strand Synthesis System, Invitrogen, Carlsbad, CA, USA). PCR was performed in triplicate using 1X Taqman Universal PCR master mix (Applied Biosystems) with the DNA Engine Opticon Con-tinuous Fluorescence Detection System (MJ Res-earch, Waltham, MA, USA) utilizing the following primers and Taqman probes: type I NRG1: Hs01108479_m1; type III NRG1: Hs01103792_m1; GAPDH: Hs02786624_g1. Cycle threshold (Ct) val-ues were calculated using Opticon monitor software, with the threshold set at 50 standard deviations above background. The relative expression was calculated by normalizing the expression of individual genes to GAPDH and using the 2 � Δ Δ Ct method (24,25).

Results

Myelin loss is associated with NRG1 receptor activation on microglia in the corticospinal tracts (CSTs) in ALS patients with UMN signs

One of the greatest pathological lesions seen in human ALS is the presence of a marked loss of

myelin in the CSTs of ALS patients. However, this is not readily seen in the CSTs of the ALS-SOD1 mouse (6). Figure 1 shows the marked loss of luxol fast-blue staining in the lateral and ventral CSTs compared to normal staining in the dorsal columns of ALS patients with UMN signs. This was seen in six out of six ALS patients we studied.

Microglia have important, though controversial, roles in neurodegenerative diseases and recently have been shown to be increased in the CST of ALS patients with UNM disease (26). NRG1 can promote microglial activation in a chronic spinal cord pain model (19) through binding to either erbB3 or erbB4 receptors that then heterodimerize with erbB2 to mediate signal transduction through tyrosine phosphorylation (19,27). We previously observed a marked increase in NRG1 receptor acti-vation (phosphorylated erbB2) on microglia in the ventral horns of ALS patients suggesting a novel mechanism of lower motor neuron disease progres-sion (6). Here, we asked whether increased erbB2 receptor activation is also present in the CSTs of patients with UMN signs and demyelination. While there is some staining in the control patients, the

Figure 1. Myelin loss in the lateral and ventral CSTs in ALS patients with clinical UMN signs, but not in the dorsal column. Spinal cords from ALS and control patients stained with luxol fast-blue PAS demonstrated myelin loss (pink areas) in ALS spinal cord within the lateral corticospinal tract (LCST) and ventral corticospinal tract (VCST). Scale bar � 20 μ m on all images. The images are representative for six ALS patients and six control patients.

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80 F. Song et al.

pattern and extent is quite different and there is no staining without the primary antibody on either control or ALS tissues (data not shown). Figure 2 shows a marked increase in erbB2 receptor activa-tion that colocalizes with activated microglia in the CST. This was seen in six ALS patients with UMN signs when compared to these ALS patients ’ dorsal columns and six control patients ’ CSTs as well as ALS patients without UMN signs (data not shown). Interestingly, erbB2 receptor activation is present at multiple levels of degenerated lateral CST (cervi-cal, thoracic and lumbar) in ALS patients with UNM signs.

Increased NRG1 protein accumulation but not mRNA expression in the CST

We measured the expression of NRG1 protein in the spinal cords of these same patients to identify the source of NRG1 production using NRG1 anti-bodies directed against different domains of the protein. Using an antibody that recognizes the entire extracellular domain of both soluble (type I) and membrane-bound (type III) NRG1 forms (ADO3) (18,22,23), there was a signifi cant increase in staining of both the lateral and ventral CSTs in ALS patients with UMN signs, but not in controls (Figure 3A). The pattern of staining with this anti-body was mostly extracellular and outside of the myelinated axons (shown with the arrows), suggest-ing that much of this immunoreactivity is in the extracellular matrix as we have shown extensively for soluble forms of NRG1 that strongly associate with heparan sulfate proteoglycans (17,28). In addition, we used an antibody specifi c for mem-brane-bound, type III NRG1 isoforms that also show marked staining of the CSTs. In control tis-sues, this antibody showed more marked axonal staining that was also present in the ALS patients (arrows), but most of the signal was again outside the myelinated axons and included the staining of small cells, as we have reported previously for the ventral horn (6) (Figure 3B).

Exactly which cells are producing the NRG1 pro-teins seen in the CSTs is not clear. During develop-ment, the major source of both soluble (type I) and membrane-bound (type III) NRG1 isoforms is from the lower motor neurons in the ventral horn (28 – 30). We therefore compared NRG1 isoform (types I and III) mRNA expression levels from the spinal cords from ALS and control patients. No signifi cant differ-ence in mRNA levels for types I and III NRG1 was seen, suggesting that the increase in NRG1 protein levels is due either to increased transcription at higher cortical levels or to an accumulation of NRG1 proteins both in the extracellular matrix and non-neuronal cells between the axons (Figure 4).

Discussion

NRG1-induced microglial activation as a possible therapeutic target for ALS patients with UMN signs

Our observation of microglial activation in the CSTs is confi rmed by other recent fi ndings showing that microglial activation in the lateral CST correlates with disease progression in ALS patients with UMN signs (7,26). Microglia could contribute to myelin loss in the CSTs and LMN loss in the ventral horn in ALS spinal cord through infl ammatory mecha-nisms (1,6,7,10,11,26, 31 – 33). Consistently, anti-infl ammatory drug treatments have had promising effects on survival in ALS animal models (34,35), but human ALS clinical trials have not been encour-aging (36,37). Therefore, a better understanding of

Figure 2. NRG1 receptor activation is increased in activated microglia in the CSTs of ALS patients with clinical UMN signs, but not in the dorsal column. NRG1 receptor activation measured with specifi c phosphor-antibodies against its receptor (p-erbB2, green) colocalized with activated microglia (CD68, red) in the LCST and the VCST of ALS but not in the dorsal column and control patients. Nuclei are stained blue with DAPI (blue). Quantitation of the percent p-erbB2 overlap with CD68 staining in each high-powered fi eld revealed a 44 � 7% overlap in the LCST compared to the dorsal column of 17 � 2% in ALS patients. No signifi cant difference between the LCST (8 � 3%) and dorsal column (12 � 3%) was seen in control patients. Scale bar � 500 μ m on the top two rows, and 10 μ m on the other images. Similar results were seen in six ALS ( p � 0.05) and six control patients in two different experiments.

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Microglial neuregulin1 in the corticospinal tract 81

the local signals between CST axons and different subtypes of microglia will be important to modulate their putative neurodegenerative and neuroprotec-tive roles in ALS. It is also possible that these acti-vated microglia serve a phagocytic function based on

their rounded morphology that could be indepen-dent of NRG1 signaling.

Our present data are consistent with a hypothesis that NRG1 released from central axons or other cell types in the CST induces microglial activation,

Figure 4. No changes in type I and III NRG1 mRNA levels were seen in ALS spinal cords. Quantitative PCR was performed on full transverse spinal cord sections in triplicate for ALS ( n � 5) and control ( n � 6) patients using NRG1 isoform-specifi c primers against types I and III NRG1.

Figure 3. NRG1 protein expression is increased in the CSTs of ALS patients with UMN signs. NRG1 antibodies against two different NRG1 epitopes showed increased staining in the LCST and VCST. Whereas AD03 targets NRG1 ’ s extracellular domain (A), the CRD antibody is a type III specifi c antibody that stains membrane-bound type III forms (B). Each antibody has a different staining pattern suggesting both the presence of matrix-bound (AD03) and membrane-bound (CRD) forms. The arrows show punctuate axonal staining within myelinated axons, suggesting a central source of NRG1 both in controls and ALS patients. Scale bar � 500 μ m on the top images, and 20 μ m on the other images. These images are representative for six ALS patients with clinical UMN signs and six control patients.

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82 F. Song et al.

which, in turn, leads to progressive axonal degenera-tion. This is similar to our previous fi ndings in the ventral horn of ALS patients and SOD1 mice (6), where we saw a robust activation of NRG1 ’ s erbB2 receptors on activated microglia in the CSTs. Taken together, these fi ndings suggest that NRG1 may be a common therapeutic target that could potentially slow disease progression in both the upper and lower motor systems in ALS by disrupting NRG1 signaling. We developed a NRG1 antagonist shown to block micro-glial activation in the spinal cord of rats that develop chronic pain following peripheral nerve injury which could be tested in ALS models as well (18,19).

Source of NRG1 in ALS patients with clinical UMN signs

The NRG1 gene produces both secreted (type I) and membrane-bound (type III) forms that are highly expressed in spinal motor neurons (28,29). While the mRNA expression of both of these forms did not change in the spinal cords of ALS and con-trol patients, using antibodies against two unique NRG1 epitopes we saw a marked increased in NRG1 protein expression in the CSTs. One of these antibodies (AD03) recognizes all extracellular domains and secreted forms, but its mostly extracel-lular staining pattern suggests a build-up of soluble forms of NRG1 in the extracellular matrix. The sec-ond antibody is highly specifi c for type III NRG1 forms that are thought to be mostly membrane-bound. This antibody stains central axons in both controls and ALS patients, but has additional stain-ing between axons, perhaps also within small cells present between axons.

The presence of increased soluble and membrane bound NRG1 forms in the absence of any change in NRG1 mRNA levels, raises the interesting possibili-ties that either NRG1 protein is transported down central motor axons where it accumulates in the extracellular matrix, or is expressed in other cell types within the CST, and that this increase in pro-tein expression is responsible for sustained activation of erbB2 receptors in microglia. Because most secreted NRG1 forms bind specifi c heparan-sulfate proteoglycans in the extracellular matrix (38), the degenerative disease process in the CSTs could be propagated through changes in the extracellular matrix composition leading to increased NRG1 accumulations and signaling in the CSTs.

Acknowledgements

We thank W. Kupsky for expert neuropathological advice, S. Dettloff for reviewing the manuscript, J. Liu and M. Baughn for technical support.

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

The work was supported by grants from the Hiller ALS Center at Wayne State University and the NIH NINDS NS059947.

References

Kiernan MC , Vucic S , Cheah BC , Turner MR , Eisen A , 1. Hardiman O , et al . Amyotrophic lateral sclerosis . Lancet. 2011 ; 377 : 942 – 55 . Gordon PH , Cheng B , Katz IB , Mitsumoto H , Rowland LP . 2. Clinical features that distinguish PLS, upper motor neuron-dominant ALS, and typical ALS . Neurology. 2009 ; 72 : 1948 – 52 . Eisen A , Weber M . The motor cortex and amyotrophic lateral 3. sclerosis . Muscle Nerve. 2001 ; 24 : 564 – 73 . Colton CA . Heterogeneity of microglial activation in the 4. innate immune response in the brain . J Neuroimmune Pharmacol. 2009 ; 4 : 399 – 418 . Yiangou Y , Facer P , Durrenberger P , Chessell IP , Naylor A , 5. Bountra C , et al . COX-2, CB2 and P2X7-immunoreactivi-ties are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord . BMC Neurol. 2006 ; 6 : 12 . Song F , Chiang P , Wang J , Ravits J , Loeb JA . Aberrant 6. neuregulin 1 signaling in amyotrophic lateral sclerosis . J Neuropathol Exp Neurol. 2012 ; 71 : 104 – 15 . Turner MR , Cagnin A , Turkheimer FE , Miller CC , 7. Shaw CE , Brooks DJ , et al . Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: an [11C](R)-PK11195 positron emission tomography study . Neurobiol Dis. 2004 ; 15 : 601 – 9 . Brettschneider JD , Libon J , Toledo JB , Xie SX , 8. McCluskey L , Elman L , et al . Microglial activation and TDP-43 pathology correlate with executive dysfunction in amyotrophic lateral sclerosis . Acta Neuropathol. 2012 ; 123 : 395 – 407 . Sargsyan SA , Monk PN , Shaw PJ . Microglia as potential 9. contributors to motor neuron injury in amyotrophic lateral sclerosis . Glia. 2005 ; 51 : 241 – 53 . Kriz J , Nguyen MD , Julien JP . Minocycline slows disease 10. progression in a mouse model of amyotrophic lateral sclero-sis . Neurobiol Dis. 2002 ; 10 : 268 – 78 . McGeer PL , McGeer EG . Infl ammatory processes in amyo-11. trophic lateral sclerosis . Muscle Nerve. 2002 ; 26 : 459 – 70 . Rosen DR , Siddique T , Patterson D , Figlewicz DA , Sapp P , 12. Hentati A , et al . Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclero-sis . Nature. 1993 ; 362 : 59 – 62 . Loeb JA , Fischbach GD . Neurotrophic factors increase neu-13. regulin expression in embryonic ventral spinal cord neurons . J Neurosci. 1997 ; 17 : 1416 – 24 . Loeb JA , Hmadcha A , Fischbach GD , Land SJ , Zakarian VL . 14. Neuregulin expression at neuromuscular synapses is modu-lated by synaptic activity and neurotrophic factors . J Neuro-sci. 2002 ; 22 : 2206 – 14 . Esper RM , Loeb JA . Rapid axoglial signaling mediated by 15. neuregulin and neurotrophic factors . J Neurosci. 2004 ; 24 : 6218 – 27 . Ma Z , Wang J , Song F , Loeb JA . Critical period of axoglial 16. signaling between neuregulin-1 and brain-derived neuro-trophic factor required for early Schwann cell survival and differentiation . J Neurosci. 2011 ; 31 : 9630 – 40 . Esper RM , Pankonin MS , Loeb JA . Neuregulins: versatile 17. growth and differentiation factors in nervous system devel-opment and human disease . Brain Res. 2006 ; 51 : 161 – 75 . Ma Z , Li Q , An H , Pankonin MS , Wang J , Loeb JA . Target-18. ing human epidermal growth factor receptor signaling with the neuregulin’s heparin-binding domain . J Biol Chem. 2009 ; 284 : 32108 – 15 . Calvo M , Zhu N , Tsantoulas C , Ma Z , Grist J , Loeb JA , 19. Bennett DL . Neuregulin-ErbB signaling promotes microglial

Am

yotr

ophi

c L

ater

al S

cler

osis

and

Fro

ntot

empo

ral D

egen

erat

ion

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Way

ne S

tate

Uni

vers

ity o

n 04

/15/

14Fo

r pe

rson

al u

se o

nly.

Microglial neuregulin1 in the corticospinal tract 83

proliferation and chemotaxis contributing to microgliosis and pain after peripheral nerve injury . J Neurosci. 2010 ; 30 : 5437 – 50 . Falls D . Neuregulins: functions, forms, and signaling strate-20. gies . Experimental Cell Research. 2003 ; 284 : 14 – 30 . Ravits J , Laurie P , Fan Y , Moore DH . Implications of ALS 21. focality: rostral-caudal distribution of lower motor neuron loss post mortem . Neurology. 2007 ; 68 : 1576 – 82 . Pankonin MS , Gallagher JT , Loeb JA . Specifi c structural 22. features of heparan sulfate proteoglycans potentiate neureg-ulin-1 signaling . J Biol Chem. 2005 ; 280 : 383 – 8 . Pankonin MS , Sohi J , Kamholz J , Loeb JA . Differential dis-23. tribution of neuregulin in human brain and spinal fl uid . Brain Res. 2009 ; 1258 : 1 – 11 . Rakhade SN , Yao B , Ahmed S , Asano E , Beaumont TL , 24. Shah AK , et al . A common pattern of persistent gene activation in human neocortical epileptic foci . Annals of Neurology. 2005 ; 58 : 736 – 47 . Livak KJ , Schmittgen TD . Analysis of relative gene 25. expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method . Methods. 2001 ; 25 : 402 – 8 . Brettschneider J , Toledo JB , van Deerlin JB , Elman L , 26. McCluskey L , Lee VM , Trojanowski JQ . Microglial activa-tion correlates with disease progression and upper motor neuron clinical symptoms in amyotrophic lateral sclerosis . PLoS One. 2012 ; 7 : e39216 . Carraway KL , Cantley LC . A neu acquaintance for erbB3 27. and erbB4: a role for receptor heterodimerization in growth signaling . Cell. 1994 ; 78 : 5 . Loeb JA , Khurana TS , Robbins JT , Yee AG , Fischbach GD . 28. Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development . Development. 1999 ; 126 : 781 – 91 . Meyer D , Yamaai T , Garratt A , Riethmacher-Sonnenberg E , 29. Kane D , Theill LE , Birchmeier C . Isoform-specifi c

expression and function of neuregulin . Development. 1997 ; 124 : 3575 – 86 . Corfas G , Rosen KM , Aratake H , Krauss R , Fischbach GD . 30. Differential expression of ARIA isoforms in the rat brain . Neuron. 1995 ; 14 : 103 – 15 . Ince PG , Evans J , Knopp M , Forster G , Hamdalla HH , 31. Wharton SB , Shaw PJ . Corticospinal tract degeneration in the progressive muscular atrophy variant of ALS . Neurology. 2003 ; 60 : 1252 – 8 . Kawamata T , Akiyama H , Yamada T , McGeer PL . Immuno-32. logic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue . Am J Pathol. 1992 ; 140 : 691 – 707 . Henkel JS , Engelhardt JI , Siklos L , Simpson EP , Kim SH , 33. Pan T , et al . Presence of dendritic cells, MCP-1, and acti-vated microglia/macrophages in amyotrophic lateral sclerosis spinal cord tissue . Ann Neurol. 2004 ; 55 : 221 – 35 . Drachman DB , Frank K , Dykes-Hoberg M , Teismann P , 34. Almer G , Przedborski S , Rothstein JD . Cyclo-oxygenase 2 inhibition protects motor neurons and prolongs survival in a transgenic mouse model of ALS . Ann Neurol. 2002 ; 52 : 771 – 8 . Zhu X , Hill RA , Nishiyama A . NG2 cells generate oli-35. godendrocytes and gray matter astrocytes in the spinal cord . Neuron Glia Biol. 2008 ; 4 : 19 – 26 . Mosley RL , Gendelman HE . Control of neuroinfl ammation 36. as a therapeutic strategy for amyotrophic lateral sclerosis and other neurodegenerative disorders . Exp Neurol. 2010 ; 222 : 1 – 5 . McGeer EG , McGeer PL . Pharmacologic approaches to the 37. treatment of amyotrophic lateral sclerosis . BioDrugs. 2005 ; 19 : 31 – 7 . Li Q , Loeb JA . Neuregulin-heparan sulfate proteoglycan 38. interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle . J Biol Chem. 2001 ; 276 : 38068 – 75 .

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