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Laminin and Agrin Stimulate Nitric Oxide Release in Differentiated C2C12 Cells

by

Foo Wing Li

A thesis submitted in partial fulfillment of the requirements for the degree of

Masters of Science

Adelphi University

2014

Approved by: __________________________________________ Thesis Committee: Dr. Benjamin Weeks Dr. Alan Schoenfeld Dr. Deborah Cooperstein

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ABSTRACT

This purpose of this paper is to explain the relationship of agrin and laminin and its importance

to the formation of the neuromuscular junction. Laminin is a basement membrane glycoprotein

that is very powerful in inducing neurite outgrowth in PC12 cells. When inducing neurite

outgrowth, PC12 cells are producing nitric oxide (NO) via nitric oxide synthase. However, when

the nitric oxide inhibitor, L-NAME was added with the laminin to PC12 cells, nitric oxide

production was inhibited, yet it still induced acetylcholine receptor (AChR) clustering suggesting

that acetylcholine receptor clustering acts on a pathway completely independent of NO

production. Agrin, like laminin, is an extracellular matrix protein that also induces AChR

clustering by inducing signaling pathways that are mediated by G-proteins, Rac and Cdc42.

Dominant negative mutants of Rac and Cdc42 inhibited clustering when coupled with agrin and

laminin suggesting that Rac and Cdc42 are necessary and acts synergistically to induce AChR

clustering. This paper also makes the claim that agrin induces NO production in C2C12 cells

using an amperometric probe.

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TABLE OF CONTENTS

Figure 1- NO production from PC12 cells stimulated by laminin, AG73, AG73T (scrambled sequence),

and C16.. ................................................................................................................ 2

Figure 2: Overview of how the clustering of AChR aggregation plays a role in the function of the

neuromuscular junction. Proposed mechanism at which rapsyn increases postsynaptic AChR

aggregation……………………………………………………………………….. …4

Figure 3: Effect of dominant interfering Rac and Cdc42.. ....................................... 5

Figure 4: Wnt proteins induce AChR clusters in muscle cells.. ............................... 7

Figure 5: Myotubes were stimulated with increasing concentrations of agrin by itself or with 1 nM Wnt9a

or Wnt11.. .............................................................................................................. 8

Figure 6: Aberrant neuromuscular junction innervations in muscle. ........................ 9

Figure 7: Summary of effects of NO and vasodilators on soluble guanylyl cyclase….. 11

Figure 8: NO output in correlation to chronic kidney disease (CKD) and end stage renal disease

(ESRD). .……………………………………………………………………………….12

Figure 9- NOS inhibitors reduce the AChR aggregation in vivo in Xenopus embryo.. ..13

Figure 10- Dose-dependent assay of NOS inhibitors measuring the number of AChR aggregates

remaining. ........................................................................................................... 14

Figure 11- Overexpression of NOS increases AChR aggregation. ......................... 15

Figure 12: Effect of NMJ/AChR density with addition of transgenic and non-transgenic (non-Tg) neural

nitric oxide synthase (nNOS). ............................................................................... 16

Figure 13: Effect of AChR clustering with or without the presence of L-NAME........... 17

Figure 14: “SNAP and NOC-18 (NO donors) can produce higher fluorescence readings ……………...18

Figure 15: NMJ of individual muscle fibers. ...................................................... 19

Figure 16: Dose-dependent assay for three different NOS inhibitors and its effect on metamorphosis of

Herdmania momus. ............................................................................................ 20

Figure 17: Treatment of myotubes with Agrin, L-Name, and/or Laminin ........... ..21

Figure 18: Measurement of levels of NO using an amperometric probe.. .............. 23

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Acknowledgments

I wish to express sincere appreciation to Professor Benjamin Weeks for his assistance in the

preparation of this manuscript and defense. A special thanks also to Dr. Alan Schoenfeld, Dr.

Benjamin Weeks, and Dr. Deborah Cooperstein for taking time out of your busy schedules to give

your valuable inputs into putting this manuscript together. I would also like to extend a special

appreciation to all the friends and family that has supported me on my journey to be the best I can

be.

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I n t ro d uc t io n

C ha pt e r 1 - La mi n i n

La mi n i n i s a b as e men t m em b r an e g l yco p r o t e in t h a t i s a p o w er fu l

s t im ul a t o r o f n eu r i t e o u t gr o wt h t h a t f o r m s th e p e r i ph e r a l a nd cen t r a l

n e r vo u s s ys t em as w e l l a s o th e r n eu r on a l c e l l l i n e s s u ch as t h e r a t

p h eo c h ro m o c yt o m a -d e r i v ed PC 12 l i n e . I t i s so po w er fu l t h a t i t s t i mu la t es

n i t r i c ox id e (N O ) r e l e as e i n a m a t t e r o f s eco n ds i n P C1 2 ce l l s . Mul t i p l e

l am in in d o m ain s t ha t can s t im u la t e n eu r i t e o u t gr o w th h av e b e en i d en t i f i ed

u s i n g s yn t h e t i c p ep t id es d e r i ve d f ro m t h e l am in i n s e qu e nc e . RN IA E II KDI ,

I KVA V , an d LQ V QLS IR a r e ex a mp les o f l am in in s yn th e t i c p ep t i d es t h a t

p r o m ot es n eu r i t e ou t gr o w th . M ul t i p l e a c t i v e s i t es fo r n e u r i t e ou t gr o wth

h a v e b een i d en t i f i e d t h a t b in d d i f f e r en t ce l l s u r face r ec ep to r s an d

i n c r eas e t h e l e v e l o f t r a ns c r i p t i o n (R ia l a s , e t a l 2 00 0 ) .

Fi gu r e 1 sh ows R ia l as e t a l ex p e r i m en t sh o wi n g t h a t l a mi n i n an d

A G7 3 , wh i ch i s a s yn t h e t i c p ep t id e t h a t i nd u ce s ne u r i te o u t gr o w th an d

s t im ul a t e s n i t r i c ox id e s yn t h es i s . C1 6 , a s yn t h e t i c p ep t i de d er iv ed f ro m

l am in in -1 ga mm a ch a in , a t t ach es t o PC 12 b u t d o es no t i n du ce n e u r i t e

o u t gr o w t h o r n i t r i c o x id e r e l ea se . T he s c ra mb led s eq u enc e A G7 3t d id n o t

i nd u ce n i t r i c o x id e syn t h e s i s .

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Figure 1- NO production from PC12 cells stimulated by laminin, AG73, AG73T (scrambled

sequence), and C16. The NO production was monitored using a NO selective probe. The PC12

cells were pelleted in 1.0 mL of serum-free DMEM. The cells were then treated with 20 µg/mL of

laminin or 200 µg/mL of the other peptides (Rialas et al, 2000).

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C ha pt e r 2 - N eu ro mus cu l a r j unc t io n f o r ma t i o n

N eu ro mu sc u l a r j un c t io n (N MJ) f o rma t io n o ccu r s i n t h r e e s t ep s . T h e re

m u s t b e n e r v e -m u s c l e r ec o gn i t i on , syn a p t i c d i f f e r en t i a t i on , an d

m a t u r a t i o n o f t h e s yn a p s e . B e fo re an y i n n e r v a t i on c an h a p p en , m usc l es

h a v e t o b e “p r ep a t t e rn ed ” o r p r e - d es t i n ed (p ro b ab l y b y ex t e r n a l s i gn a l s )

a n d AC hRs a r e l o ca l i z ed (p e r ha ps b y c l us t e r i n g) i n t h e cen t r a l b a nd o f t h e

m u s c l e . S t ro ch l i c ’ s l ab f o cu sed on t h e g l yc o p r o t e in Wn t 4 . D u r i n g t h e

f o r ma t io n o f t h e n eu ro mu sc u l a r j u nc t i o n ( N MJ ) , mo t or n eur on s s e c r e t e

a gr i n t h a t c aus e s ace t y l ch o l i n e r e cep to r s ( ACh Rs ) t o c lus t e r (S t ro ch l i c e t

a l , 2 00 5 ) . Th e Rho f am i l y o f G T Pa se s , n a me l y R ac a n d Cd c4 2 , w hen

c o up l ed w i t h a gr i n , ca us es a c l us t e r i ng o f A Ch Rs . I t h a s b een fo un d w hen

R ac o r Cd c4 2 i s mu ta t ed , ag r i n - i n d u c ed AChR c l us t e r in g i s b lo c ke d

t h e r e f o r e , h i gh l i gh t in g t h e i mp or t an c e o f R a c a nd Cd c 42 a s a c r i t i ca l s t ep

i n t h e s i gn a l in g p a t h wa y t h a t c au s es AC hR s t o c lu s te r . In c o n t r as t ,

c o ns t i t u t i v e ex p res s i on o f R ac an d Cd c4 2 mu tan t s cau se ACh Rs t o

a ggr e ga t e w i th ou t t h e p r e se n ce o f ag r in .

T h e f o r ma t i on o f agr i n i s r e gu l a t ed b y f ac t o r s gen e r a t ed f r o m mo tor

n e u ro n s . In o th e r wo rd s , n e u r a l a gr i n b i nd s LR P4, whi ch i s p a r t o f t h e

l o w- d en s i t y l i po p ro t e in r e cep to r ( LD LR) f am i l y t h a t ac t i v a t e s th e t yro s in e

k i n ase M u SK ( Mu sc l e S p ec i f i c K in ase ) t h a t l ead s t o t h e c l us t e r i n g o f

A Ch Rs b y w a y o f m ed ia t o r p r o t e i ns t h a t i n c l ud e s t h e cy t os ke l e t a l p r o t e i n

α - a c t i n in . D u r i n g d e v e lo p m en t i n t h e e v e n t o f m us c l e f i b e r p r e - p a t t e rn in g

o r n on -n e r v o us t i s s u e AC hR c l us t e r f o r ma t io n r eq u i r es Mu SK an d LR P4 ,

b u t n o t agr i n b u t c l us t e r s w i th n e rv ou s t i s su e r eq u i r e a l l . Th i s su gge s t s

t h a t Mu S K ma y b e r egu l a t ed b y a gr i n - in d ep en d en t , bu t n o t i den t i f i e d

l i ga n ds ( Wes t on , e t . a l 2 0 00 ) .

D u r i n g e mb r yo ge n e s i s , n e rv e -d e r i v ed a gr i n s i gn a l s (v i a Mus c l e

Sp ec i f i c Kin as e o r Mu S K) s t ab i l i z e a n d gr o w s AC h R c l us t e r s a t n ew

n e u ro m us cu l a r syn a p s e . R ap syn i s a c yt o p l a sm i c AC hR a s so c i a t e d p ro t e in

t h a t a c t s a s a s t ab i l i z e r o f A ChR aggre ga t e s . Th e y s t a b i l i ze b y

c ro ss l i n k in g A C hRs an d t e t he r in g t o t h e cyt o sk e l e to n . In tu i t i v e l y, r ap s yn

a l so ac t s a s an i nh ib i t o r o f d i s a ss emb l y m ec h a n i sms . Fi gu r e 2 a i s t h e

o v e rv i e w o f t h e neu r o mu scu la r ju n c t io n . Fi gu r e 2 b s h o ws a p ro po sed

m ec h an i sm a t wh i ch r ap syn p r o m o t es t h e i n c re as e o f ACh R a ggr e ga t i o n .

( B ro ck h au sen e t a l , 2 0 0 8) .

In Wes t on e t a l 2 00 0 , i t w as fo un d th a t ag r in a c t i v a t e s R ac /C d c4 2 ,

w h ich su p po r t e d t h a t Rh o G T Pase s c an me di a t e t h e e f f ec t s o f a gr in

s i gn a l i n g o n A ChR c l u s t e r in g . I t w as a l so fo un d t h a t do m i na n t n e ga t iv e

m u t an t s o f R a c and Cd c4 2 b lo ck t h e i n d u c t i on o f a gr i n su gge s t i n g t h a t

R ac an d C d c4 2 a r e n ec es s ar y an d su f f i c i e n t f o r AC hR c l u s t e r s . Fi gu r e 3

s ho w s t h e e f f e c t o f d o mi n an t i n t e r f e r in g o f s ma l l G T Pas es R ac a nd C d c4 2 .

T h ese GT P ases h av e b e en s ho wn t o r eo r ga n i za t i o n t h e ac t i n c yt o sk e l e t o n

i n r es po n se t o ex t r ace l l u l a r cu es i n s ev e ra l ce l l t yp es . R ac an d Cd c4 2

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p r od u ces c h an ge s i n t h e o r gan i z a t i on o f t h e c yt o s k e l e to n b y co n t r o l l i n g

ac t i n p o l yme r i z a t i o n t h r ou gh mu l t i p l e r egu l a t o r y p a t h w ays ( We s t o n , e t a l

2 0 0 0 ) . T he d a t a su gge s t s t h a t R a c an d Cd c 42 a r e s u f f i c i en t and n e ces s a r y

f o r t h e a gr i n - in d u ce d c l us t e r i n g o f AC hR.

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Figure 2: a) Overview of how the clustering of AChR aggregation plays a role in the function of the

neuromuscular junction and how it ultimately leads to action potential (muscle contraction). b)

Proposed mechanism at which rapsyn increases postsynaptic AChR aggregation. The proposed

mechanism suggests that rapsyn crosslinks AChRs and tethers them to the cytoskeleton

(Brockhausen, et al 2008).

Figure 3: Effect of dominant interfering Rac and Cdc42. This graph shows that Rac and Cdc42

need to be coupled to agrin to induce AChR clustering (Weston et al, 2000).

b

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C ha pt e r 3 - Ro l e o f W nt pa thwa y i n c l us t er in g o f a ce ty l cho l i ne

r ece pt o rs

Wn t i s a f am i l y o f g l yco p r o t e i n s t h a t p l a y a v e r y i m p o r t an t ro l e i n t h e

d e v e l op m en t o f ma n y o r gan i s ms . Wn t b i n d s to a r ec ep t o r co m plex

F r i zz l ed ( Fz ) a n d LR P5 / 6 . Fz t h e n b i n d s t o t h e a d a p t e r p r o t e i n ,

D ish ev e l l ed ( D vl ) t o t u rn on i n t r ac e l l u l a r can o n i ca l an d n o n- c an o n i ca l

p a th w ays . T h e r e h av e b een r ecen t s t u d i e s t h a t s u g ge s t t h a t Wn t p l a ys a

r o l e i n s yn ap s e f o r m a t io n . Fo r e x am p l e , i n C . e l ega n s , Wn t s i gn a l i n g

d e t e r m in es po s i t i o n i n g o f N MJ s b y b l o ck i n g s yn a p to ge n es i s . In

D ro so p h i l a , Wn t p ro m o tes N MJ f o rm at io n . In ze b r af i s h , Wn t 1 1 r b i nd s t o

t h e M u SK h o m ologu e , u n p lu g g ed , t o gu i d e m ot or ax o ns . In ma mm al i a n

m u s c l e ce l l s , a gr i n - in du ced ACh R c l us t e r i n g i s b o os t ed b y Wn t 3 , b u t

r ed u ce d b y Wn t 3 a . T h e r e a r e 19 d i f fe r en t Wn t s i d e n t i f i ed i n h u m ans an d

m i ce b u t i t i s u n c l ea r w hi ch Wn t i s s u f f i c i en t t o s t i mu l a t e A ChR

c lu s t e r in g wi t ho u t a gr i n . In Z h an g’ s ex p e r i m en t i n 20 1 2 i n Mo le cu l ar

B r a i n , t h e y i d en t i f i ed 5 Wn t s t h a t w e re ab l e t o s t imu l a t e A Ch R c l u s t e r i n g

w i t ho u t a gr i n ( Wn t 9 a , Wn t 9 b , Wn t 1 0b , Wn t 1 1 , an d Wn t1 6 ) . I t was fo un d

t h a t Wn t 9 a an d Wn t 1 1 s h o w ed t h e h i gh es t ex pr es s io n i n d ev e l op in g

m u s c l e s . Th e s e r e s u l t s i n d i c a t ed t h a t t h e Wn t p a th way l i k e l y p l a ys a n

i m p or t an t ro l e i n t h e c l us t e r i n g o f AC hRs (Z h an g e t a l , 2 0 12 ) .

I n Z h an g e t a l 2 01 2 , a l l 19 Wn t s we r e s t ud i ed t o ex ami n e th e i r

e f f ec t s o n ACh R c lu s t e r i n g . Wn t9 a , Wn t9 b , Wn t 1 0b , Wn t1 1 , an d Wn t 1 6

a l l i n du c ed c l u s t e r in g . Ho w eve r , Wn t9 a a nd Wn t 1 1 s h o wed th e mos t

ex p r es s io n , wh i ch p r ob a b l y m ea n s i t i s p a r t o f man y p a th w a ys c an on i ca l

an d no n -ca no n i ca l . T h e r egu l a t i on o f t h e i r c lu s t e r i n g f r o m t h e Wn t

p a th w ay s u g ge s t s t h a t i t p l a ys a v e r y i m p or t an t r o l e i n th e n eur o m us c u l a r

j un c t io n ( N MJ ) f o rm a t io n . Fi gu r e 4 (Z h an g, e t a l 2 0 12 ) s ho ws an a s s a y

w h er e t h a t Wn t 9 a an d Wn t1 1 w e re s h o wn t o be d o s e -d ep end en t . T h e

f i gu r e s h o ws t h a t t h e c lu s t e r in g o f A Ch R in m yo t u bes wi t h Wn t 9 a an d

Wn t 1 1 a r e co n c en t ra t i on d ep en d en t . Fi gu r e 4 s h ow s t h a t Wn t 9 a an d Wn t

1 1 a re no t s u f f i c i e n t t o i n d u c e ACh R c lu s t e r in g a n d t h a t i t r eq u i r es

co up l in g w i t h ag r i n t o i nd u ce c lu s t e r i n g s u gges t i ng t h a t a gr i n i s

n e ce ss a r y. T h i s d a t a su gge s t e d t h a t t h e s e Wn t p ro t e i ns a r e s u f f i c i en t t o

a l t e r AChR c lu s t e r in g i n m us c l e ce l l s . Fi gu re 5 b e lo w s h o w s 5 o f t h e 1 9

Wn t s w er e a b l e to ex p r es s A chR c lu s t e r s i n t h e ab s en c e o f a gr i n .

T he Wn t 4 i n v o l v em en t i n mu s c l e d e v e l op m en t p ro mp t ed Sh i a o’ s l ab

t o i nv es t i ga t e i t s r o l e i n v e r t eb ra t e d ev e l op me nt . Wn t p r o t e i n s a re

ex t e r n a l c u es t h a t t e l l c e l l s wh e r e t o gr o w, w h er e t o m i gr a t e , w h e r e t o

a s s e mb l e , e t c . In t h e ca s e fo r Dr os op h i la , Wn t 4 c o n t ro l s mu s c l e

i nn e rv a t i o n t o po gr ap h y. In ma mm a l i a n N MJ , v e r y l i t t l e i s k n o wn in t h e

ea r ly s t ages o f n erv e - mus c l e r e co gn i t i o n . Th e r e i s ev ide n c e s u gge s t i n g

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Wn t 4 i n v o l v em en t . F i r s t , t emp o r a l exp re s s i on p a t t e rn s sh o w th a t mR NA

an d Wn t 4 a r e c on s i s t en t b e twe en i n v i v o an d i n v i t ro . Se co n d , l o s s o f

Wn t 4 f u n c t i on c au s e s d e fec t s i n ax o n p a t h f in d i n g an d s ec t i o n s o f AChR

c l u s t e r s wo ul d n o t b e i nn e rv a t e d . Th i rd , Wn t 4 i s on l y f o u n d ex p r ess e d i n

mu s c l e c e l l s , n o t i n mo to r n eu ro n s (S t ro ch l i c , e t a l 2 01 2 ) .

Figure 4: Wnt proteins induce AChR clusters in muscle cells. The arrows in the pictures in panel

A shows clusters. Panel B shows the count of AChR clusters per millimeter. Panel C was an assay

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that determined which Wnt(s) maybe involved in the regulation of neuromuscular junction

formation by measuring the amount of mRNA using qRT-PCR (Zhang et al, 2012).

Figure 5: Myotubes were stimulated with increasing concentrations of agrin by itself or with 1 nM

Wnt9a or Wnt11. The data shows that Wnt9a and Wnt11 are not sufficient for AChR clustering

indicating that agrin is necessary for AChR clustering (Zhang et al, 2012).

Fi gu r e 6 b e l o w s h ow s t h e e f f ec t s o f d o mi n an t n ega t iv e Wn t 4 m ut a n t

o n N MJ fo rm a t io n . T h e d a t a sh o ws th a t Wn t 4 mu t an t s i n d u c ed a b e r r a n t

n e u ro m us cu l a r i n ne rv a t i o n . A l l t h e Wn t 4 - / - mi c e d i ed wi t h i n 2 4 h ou rs

b e ca us e t h e k id n eys d o n o t d ev e l o p c o r r ec t l y . T h i s a l s o su gge s t s t h a t

Wn t4 p l ays a r o l e i n k i dn e y f o r m a t i on a s w el l o r t h e do m i na n t n e ga t i v e

m u t an t i s i n t e r f e r in g i n o th e r as pe c t s o f k i dn e y f o r m a t io n . Wn t 4 mu t an t s

c au s e c l us t e r s t o b e l a r ge r c o m p a re d t o t h e wi ld - t yp e . T h e wi d t h o f t h e

b a nd s o f c lu s t e r s a re l a r ge r co m p a r ed t o t h e wi ld t yp e ( ap p rox i m a te l y 1 2 0

µ m wi ld t yp e a n d a p p rox i ma t e l y 1 6 0 µ m in t h e m u ta n t ) . ( S t ro c h l i c e t a l ,

2 0 05 )

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Figure 6: Aberrant neuromuscular junction innervations in muscles- Confocal images that are

showing the effects of mutant Wnt4 on the diaphragm, intercostal muscles, and limb muscle

sections. The white arrows (merged images) indicate nerve terminals that are protruding beyond

the central band of AChR clusters in the diaphragm and intercostals muscles. The “stars” indicate

non-innervated synapse in limb muscles. The graphs underneath the confocal images (G) show that

dominant negative Wnt4 increases the endplate band width, increases the AChR cluster size,

intensity, and increase in non-innervated synapses (Strochlic et al, 2005).

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C ha pt e r 4 - R ol e o f N i tr i c O xi de i n a ce t y l c ho l i ne re ce pto r c l us te r i ng

N i t r i c ox i d e (N O ) i s kn o wn t o b e i n vo lv ed i n ma n y b i o lo gi c a l

f u n c t i on s s uch a s b l oo d p r es su re re gu l a t i on , p l a t e l e t ad h es io n , n eu t r op h i l

a ggr e ga t i o n , as w e l l a s s yn ap t i c p l as t i c i t y i n t h e b r a in . N i t r i c ox id e an d

o th e r ox id an t s a re a l s o c yt o t o x i c agen t s ma d e b y m ac r o ph a ge s an d

n e u t ro p h i l s . Ni t r i c o x i d e i s a t ox i c gas b u t t h e c he mi ca l p ro p e r t i es t h a t

c au s e N O t o b e t o x i c ca n a l s o ex p l a i n w h y i t i s v e r y u s e fu l a s a r ap id ,

l o c a l m es s en ge r . S i n c e N O i s a hyd ro p h o b i c ga s , i t c a n e a s i l y c ro ss

t h ro u gh th e c e l l mem b r a n e l i ke ox yg e n an d c a rb o n d i ox id e ca n w i th o u t

a n y ch an n el s o r r ece p to r s t o gu i d e i t t h ro u gh . Wh y i s NO s o f av o rab l e?

T h e d i f fu s i o n co ef f i c i en t i s wa t e r i s h igh e r t h a n ox yge n , ca rb on d i ox id e ,

o r ca rb on mon ox id e s o i t i s i d e a l fo r ca r r yi n g i n f o r m at io n . Th e s i gn a l i s

s ho r t l i v e d an d s p on t an e ou s l y d ec o m po s es w h en i t i s r eac t e d wi t h ox yge n .

N i t r i c o x i d e i s a d i f f us ib l e , un s t a b l e ga s mo le cu l e t ha t p l a ys a r o l e i n

n e u ro n a l d ev e lo p me n t , p l as t i c i t y, an d n eu r i t e r em o d e l in g . N i t r i c ox id e

s yn t h as e i s ex p r es s ed in th e b r a i n i n d i f f e r en t c e l l t yp es d u r i n g d i f f e r en t

t i mes o f d ev e lo p men t i n mi ce an d h u ma n s (R ia l as e t a l 2 00 0 ) .

N i t r i c ox id e s yn t h a s e (N OS ) co m es i n t h ree d i f f e r e n t i s o f o r ms :

n e u ro n a l (n N O S or N O S1 ) , i n d u c i b l e ( i NO S o r NOS 2 ) , an d end o t h e l i a l

( eN O S o r NO S3 ) . A l l o f t h es e c an b e f o un d i n t he ad u l t s ke l e t a l mus c l e

a n d f e t a l mo u s e m od e l s .

I t h a s l on g b e en i mp l i ca t ed t h a t t h e l os s o f e nd o t he l i u m-d e r iv ed NO

i s a c r i t i ca l f a c to r a t t h e o n s e t o f ca rd i ov a s cu l a r d i s e as e i n c lu d in g

h yp e r t e ns io n an d a t h e r os c l e ro s i s . In 1 9 77 , Fe r i d Mu r rad ’ s l ab o ra to r y a t

t h e U ni v er s i t y o f T ex as H e al t h Sc i en c e C en te r i n H ou s to n r e po r t ed t h a t

v a so d i l a to r s s u ch a s n i t r o g l yc e r i n and n i t ro p ru s s id e i n d u ce d s mo o th

m u s c l e r e l ax a t i on b y ge n e ra t i n g NO , w h ic h i n t u r n a c t i v a t ed gu an yl yl

c yc l a s e a nd i n c r eas e d p ro du ct io n o f c GM P. T h e e f f ec t o f t h e i n c r eas ed

p r od u c t io n p ro m ote d v as o d i l a t i on . Be ca u s e o f t h i s f i nd in g, i t e v en t u a l l y

w as p ro po s ed t o b e a s ec o nd m es s en g e r mo l ecu l e . In ad d i t i on t o b e in g a

v a so d i l a to r , N O ha s s ev e r a l p ro t ec t i v e fu n c t i on s , s uch a s i n h i b i t i n g:

n e u t ro p h i l ac t i v a t i o n a nd a dh es io n , p l a t e l e t ad h e s io n an d a ggr e ga t i o n ,

v a s cu l a r s m o oth m u s c l e p r o l i f e r a t i o n , a n d ex p r es s i on o f p ro - in f la m ma t or y

c yt o k i n es . S i n c e t h e e nd o th e l i u m r e tu rn s t o a no rm a l s t a t e w i t h n o r m al

n i t r i c ox id e p r od uc t i o n , i t wo u ld b l ock a t h e ro s c l e ro s i s . Fi gu r e 7 b e low

s u m ma ri z es t h e e f f e c t s o f NO an d v a so d i l a to rs o n s o l ub l e gu an yl yl

c yc l a s e ( Ka B i an e t a l , 2 00 8 ) .

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Figure 7: Summary of effects of NO and vasodilators on soluble guanylyl cyclase (Ka Bian et

al, 2008).

N i t r i c ox id e h as i mm u n o lo g ic a l i mp l i c a t i on s a s we l l . P r io r t o

1 9 87 , i t w as kn own th a t i n ce r t a i n ev en t s th a t t h e i n j ec t i o n o f b ac t e r i a l

b y-p r o d u ct s o r p a th o gen i c a ge n t s can i n du ce s o m e k i nd o f r es i s t a n c e t o

o th e r p a th o ge n s an d s to pp i n g t h e p ro gr es s i on o f t um o r s . Th e f i r s t

o b s e r v a t i o n s w e re ma d e u s in g h u ma n p a t i en t s , wh er e i t w as obs e rv ed t h a t

i n j e c t i on o f pa th o ge n i c b ac t e r i a l ex t r ac t s ( i n d u c es a s t ro ng im m u n o lo gi c a l

r e s po ns e ) i n to p a t ie n t s w i th t e rm in a l can ce r r e s u l t ed i n a d r a ma t i c

n e c r os i s o f t u m or s i n m an y ca s e s ( n o t a l l ) . T h e i d ea f o r u s e i n t h e r ap y

l os t i n t e re s t i n t h e 1 9 9 0 s b ec aus e o f i t s t ox i c i t y an d t h e b egi n n in g o f t h e

r i se o f ch em ot he r ap y ( Lan c as t e r , 1 9 96 ) .

T h e fo l l o win g f i gu r e s ( i n c l u d in g o r i g i n a l r e s ea r c h d a t a f r o m We eks

e t a l co n t a in d a t a t h a t s u gges t a gr i n a n d l am in i n s t imu l a t e n i t r i c ox i d e

r e l e as e ev en t ho u gh t h e re a r e m an y o th e r p ro p o s ed m ec h an i s m s t ha t a r e

r e l a t ed an d ma yb e r e l ev an t to t h a t c on c l us i on .

T h e l ac k o f N O i s imp l i ca t e d i n a v a r i e t y o f k i d n e y d i s eas e s s u ch a s

c h r on i c k i d n e y d i s ea s e (C KD) , g l o mer u l a r h yp e r t en s io n , an d en d s t age

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r en a l d i s e as e (E SR D ) . F i gu r e 8 be l ow d e s cr ib e s t h e a mo un t o f NO

p r e s en t a s s o c i a t e d w i th d i f f e re n t k id n e y a i l me n t s . In p an e l A, n i t r i t e +

n i t r a t e o u tp u t i s m ea s u r ed th r ou gh u r in a r y ex c r e t i on . T h e co n t ro l i s a

p a t i en t wi th n o rma l r en a l fu n c t i on . T h i s i s m eas u re d aga i n s t p a t i en t s

w i t h E SR D wh o un d e r w en t p e r i t o n ea l d i a l ys i s (P D) o r h em o d ia l ys i s ( HD ) .

A l l t h e p a t i e n t s h e r e we r e s u b j ec t ed t o l o w NO x d i e t s . T h e d a t a s ho ws

t h a t N O p r o d uc t i on i s v as t l y r ed u ce d i n t h e p r e s en ce o f C KD o r E SRD

( B ayl i s , 20 07 ) .

Figure 8: NO output in correlation to chronic kidney disease (CKD) and end stage renal disease

(ESRD). This describes the amount of NO present associated with different kidney ailments. “In

panel A, nitrite + nitrate output is measured through urinary excretion. The control is a patient

with normal renal function. This is measured against patients with ESRD who underwent

peritoneal dialysis (PD) or hemodialysis (HD). All the patients here were subjected to low NOx

diets. The data shows that NO production is vastly reduced in the presence of CKD or ESRD

(Baylis, 2007).”

I n a r e s e ar c h by Sch wa r t e , e t a l , 20 0 4 , t h e y d i d a s s a ys t h a t d e t e rmi n ed

w h e th e r N OS a c t i v i t y i s r e qu i r e d fo r p os t -s yn ap t i c d i f f e r en t i a t i on du r i n g

t h e f o r m at i on o f th e n eu r o mu s cu l a r j un c t io n . In Fi gu r e 9 be lo w, t h e y

ex po s ed X en o p us em b r yo s t o N OS i n h ib i t o r s t o se e i f t h e y b l o c k

a ce t y l ch o l i n e r e cep to r ( ACh R) ag gr ega t i o n . T he N OS i nh ib i t o r s u se d

w er e 7 -n i t ro in da zo l e ( Pa n e l B ) , v in yl - L-N IO (P an e l C ) , an d 1 4 00 W ( Pan e l

D ) . Pan e l s B ,C , an d D w er e co m p are d t o an u n t r ea t ed c o n t ro l e mb r yo in

Pa n e l A. Co n fo ca l mi c ro s co p y i m a ge s w er e t a ke n a nd t he am o un t o f

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a ggr e ga t i o n a rea i s m ea su r ed us in g i ma ge an a l ys i s s o f t w ar e t h a t

c o nv e r t s i t t o a p e rce n t age . T h e y r ed u ce d ag gr e ga t i o n by 7 7 %, 9 9 %, an d

8 9 %, r es p e c t i v e l y c o m p are d t o t h e u n t r ea t ed co n t ro l . Th e mi c ro s co p e

p i c tu r e s s h ow ed ACh R in h i b i t i o n i n - v i v o .

Figure 9- NOS inhibitors reduce the AChR aggregation in vivo in Xenopus embryo.

Immunostaining for AChR was performed using Green Fluorescent Protein (GFP). Panel A is the

untreated embryo. Panels B, C, and D were exposed to NOS inhibitors at 26 h of development.

Panel B was exposed to 7-nitroindazole. Panel C was exposed to vinyl-L-NIO . Panel D was

exposed to 1400W (Schwarte et al 2004).

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In f i gu re 1 0 b e low , e mb r yo s we re ex p o s ed t o N O S i nh ib i t o r s f ro m 26

h r s o f de v e lo p m en t ( s t age 2 4 ) t o s t a ge 3 1 o v e rn igh t a t 1 6- 1 8 ºC . T h e

f i gu r e sh ow s th e nu mb er o f r em ai n i n g a gr i n - in du ced AChR ag gr e ga t e s l e f t

o v e r a f t e r t r e a t m ent o f N O S in h i b i t o r s . T he p e r cen t a ge s w er e o b t a i n ed i n

t h e sa me m ann e r a s t h e co nf o c a l m ic ros co p y i m age a n a l ys i s so f t w a re . T h e

t r ea t me nt o f t h e se m u s c l e s ce l l s c o m pl e t e l y b lo ck ed a gr i n - in d u ced ACh R

c lu s t e r in g . T h e IC 5 0 v a lu e s fo r 7 -Ni n t r o in d azo l e (7 - NI ) an d 14 00 W we r e

2 5 µ M a nd 2 n M, r e s p e c t i v e l y. L- A r g i n i n e w as ad d e d b ack t o t h e 7 -NI

t r ea t ed c e l l s an d i t r ev e r s e d t h e i n h ib i t i on o f a gr i n . Th e da t a s u g ges t s

t h a t t h e s e i n h ib i t o rs b lo cke d a gr i n - in du ce d AC hR ag gr e ga t io n an d no t a n

e f f e c t o f t ox i c i t y. ( Sc h w ar t e , e t a l , 2 00 4 ) .

Figure 10- Dose-dependent assay of NOS inhibitors measuring the number of AChR aggregates

remaining. The number of aggregates remaining is dose-dependent and is not caused by toxicity

(Schwarte et al, 2004)

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C o n v er s e l y, o v e r ex p re ss io n o f NO S i n c r ea s es AChR a ggr e ga t i o n as

s ho w n b e l ow . In t h i s ex p e r i m en t , t h ey i n j ec t ed d i f f e ren t i s o fo r m s o f N O S

i n t o t h e o n e -c e l l s t a ge o f Xe no p u s . As a r e s u l t , t h ey go t a 1 00 % -2 00 %

i n c r eas e i n ACh R agg r e ga t i o n ( Fi gu r e 1 1 ) . A t r un ca t ed fo r m o f N OS 1 an d

N OS 2 we r e i n j ec t ed an d th e s e d i d n o t i n c r e as e ACh R a ggr e ga t io n

s u gges t i n g t h a t e nz ym at i ca l l y ac t i v e N O S i s n ec es s a r y. (S ch w a r t e e t a l ,

2 0 04 ) .

Figure 11- Overexpression of NOS increases AChR aggregation. Immunostaining for AChR was

performed using Green Fluorescent Protein (GFP). Panel A (control), Panel B (nNOS treatment),

Panel C (iNOS treatment), Panel D (eNOS treatment) (Schwarte et al, 2004).

I n f i gu r e 1 2 b e l ow, i t w as f ou n d th a t a n u l l m u t a t i o n o f n NO S ca us es a

s i gn i f i can t d ec r ease i n A ChR d e ns i t y c o m p a re d t o t h e C 57 c o n t ro l . N u l l

m u t an t s o f α - s yn t ro p h in a l s o s h o wed t h e s a me a f f ec t s , wh i ch s u gges t s t h a t

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n - N O S an d α - s yn t r o ph in a r e i n v o lv ed i n s i mi l a r p a th wa ys an d a r e

n e ce ss a ry fo r AC h R c lu s t e r i n g (Sh a i o e t a l 2 00 4 ) .

Figure 12: Effect of NMJ/AChR density with addition of transgenic and non-transgenic (non-Tg)

neural nitric oxide synthase (nNOS). “it was found that a null mutation of nNOS causes a

significant decrease in AChR density compared to the C57 control. Null mutants of α-syntrophin

also showed the same affects, which suggests that n-NOS and α-syntrophin are involved in similar

pathways and are necessary for AChR clustering (Shaio et al 2004).”

I n f i gu r e 1 3 be lo w , Pan e l A s h o ws tha t wi t ho u t t h e p r e s en ce o f L- N A ME ,

V VAB 4 (V isc i a V i l l os a a gg l u t i n i n B4 ) , wh i ch i s a m yo tu b e s t i mu lan t o r

l am in in c au s es a n i n c re as e i n A ChR c lu s t e r in g . In co n t ra s t , t h e p r e s en c e

o f L-N A ME r ed u ce s c l u s t e r i n g r e ga r d l es s o f t h e p r e s en ce o f V VAB 4 or

l am in in . In Pan e l B , N O r e l ea s e i n c re as es w i th VVA B4 o r l a min i n

c o mp ar ed t o t h e DME M c o n t ro l ( Sh a io e t a l 2 0 04 ) .

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Figure 13: Effect of AChR clustering with or without the presence of L-NAME. “Panel A shows

that without the presence of L-NAME, VVAB4 (Viscia Villosa agglutinin B4), which is a myotube

stimulant or laminin causes an increase in AChR clustering. In contrast, the presence of L-NAME

reduces clustering regardless of the presence of VVAB4 or laminin. In Panel B, NO release

increases with VVAB4 or laminin compared to the DMEM control (Shaio et al 2004).”

In f i gu r e 1 4 b e lo w s ho w s t h e SN A P a nd N O C-1 8 ( NO d o no rs ) can p ro d u ce

h i gh e r f l uo r es c en ce r ead in gs , wh i ch i n d i ca t es t h a t NO ca n p r od u c e

s u r face ex p r es s io n o f A ChR s d u r i n g m yo t u b e gr o w t h bu t i s no t n ec es s a r y.

L- N AM E h ad no e f f ec t o n m yo t u b e g ro w th whi ch ca n f u r t h e r p ro v e th a t L-

N AM E d oe s n o t wo rk i n t h e s am e p a t h wa y a s A C hR c l u s t e r in g . Sh a io e t

a l 2 00 4

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Figure 14: “SNAP and NOC-18 (NO donors) can produce higher fluorescence readings, which

indicates that NO can produce surface expression of AChRs during myotube growth but is not

necessary. L-NAME had no effect on myotube growth which can further prove that L-NAME does

not work in the same pathway as AChR clustering (Shaio et al 2004)”.

Sh iao ’ s l ab o ra to ry w an t e d t o f i nd o u t wh e t h e r r e s to r i n g t h e NO

p r od u c t io n i n d ys t r o p h in - d ef i c i en t mu s c l e ( m dx ) co u l d “ re s cu e ” N MJ

d e f ec t s i n a r ch i t ec t u r e a nd ACh R co n cen t r a t i o n . In f i gu r e 1 5 , t h e

f l uo r e s c en ce m ic ros co p y p i c t u re s b e lo w ( Pa n e l G) sh ow s th a t t he p o s t -

s yn a p t i c m e mb r a ne r e t u r n ed t o n o r ma l . Ad d i t i on a l l y, t h e A Ch R d en s i t y a t

N OS /T g m d x f i be r me mb r an e s s h ow ed a d r a ma t i c in c r eas e co m p ar ed t o

n o n- T g m dx mi ce .

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Figure 15: NMJ of individual muscle fibers. Adding NOS to deficient muscle fibers “rescues” NMJ

defects. AChR were labeled with fluoroscein isothionate α-bungarotoxin. A= wild type C57

muscle. B= mdx EDL (extensor digitorum longus). C= α-syn -/- EDL muscle. D= α-syn -/- muscle

STM (superior tarsal muscle). E= NOS -/- EDL muscle. (F) NOS Tg EDL muscle, (G) NOS

Tg/mdx EDL muscle, (H) NOS Tg/a-syn 2/2 EDL muscle, (I) NOS Tg/a-syn

2/2 STM muscle, (J) C57 SOL muscle, (K) 2-week-old C57 EDL muscle and (L) 2-week-old mdx

EDL muscle (Shaio et al 2004).

I n t h e gr a p h b e low ( f i gu r e 1 6 ) , s ho wi n g d os e -d e p en d en t a s s a ys o f t h r e e

d i f f e r en t NO S i nh ib i t o r s a nd i t s e f f e c t on la r v a l m e t a mo rp ho s i s o n

H er dm an ia mo m us , w h ic h i s a so l i t a ry a s c i d i an t h a t i s co m mo nl y r e f e r r e d

t o a s s ea s qu i r t s t h a t a r e f i l t e r f e ed e r s . T h e t h r ee d i f f e r en t N OS

i nh ib i t o r s u s e d i n t h e ex p e r i m en t b e l o w a re L- NA ME , S MIS , an d AG S L-

N AM E s i gn i f i c an t ly i n h ib i t ed l a r v a l m e t a mo rp ho s i s a t 1 m M an d 1 0 m M

c o n c en t r a t i o n c o mp a re d t o f i l t e r ed s e a wa t e r (n ega t i ve co n t r o l ) . T h e

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o th e r NOS in h i b i t o r s , A GS a nd S MIS d id n o t p ro d u ce s ign i f i can t e f f ec t s

c o mp ar ed t o f i l t e r e d s eaw at e r r es u l t s . B u t i n a l l t h r ee ex p e r i men t s , t h e

p o s i t i v e con t ro l , 4 0 mM o f KCl i n d uc ed s i gn i f i c an t l y h i gh e r p e r cen t a ges

o f m e ta mo rp h os i s T h e d a t a su g ge s t s t h a t N O i s a p o s i t i ve r egu l a to r r a th e r

t h an a nega t i v e on e a nd t h a t N OS in h ib i t o r s do n o t i n d u ce m e t a mo rp h os i s .

Figure 16: Dose-dependent assay for three different NOS inhibitors and their effect on

metamorphosis of Herdmania momus. “In all three experiments, the positive control, 40 mM of

KCl induced significantly higher percentages of metamorphosis The data suggests that NO is a

positive regulator rather than a negative one and that NOS inhibitors does not induce

metamorphosis (Ueda et al, 2013).”

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Chapter 5: Original research from Weeks et al 2013 (unpublished)

In p r ev io us s t u d i e s , l am i n in h as sh ow n t o i n d u c e AC hR c lu s t e r in g w i th

t h e me d i a t i o n o f NO b u t no p ap e r h as d i s c us s e d i f ag r i n in d uc es t h e

p r od u ct i o n o f N O. T h e fo l l o wi n g ex pe r i m en t s w i l l d i s c us s t h e i m pac t o f a

n i t r i c ox id e i n h i b i t o r o n t h e c lu s t e r ing o f A Ch R an d wi l l a ns we r t h e

q u es t i o n w h e th e r o r n o t ag r i n in du c es N O s yn t h es i s .

Fi gu re 1 7 s ho ws t h e t r e a t m en t o f m yo t u b e s wi th a gr in , L- N A ME ,

an d / o r l am in in . L- N A ME i s a kn o wn n i t r i c o x id e s yn t h as e i nh i b i t o r . T h e

d a t a s u gges t s t h a t L- NA ME i s ab l e t o i n h i b i t n i t r i c o x ide s yn t h e s i s bu t i s

u n ab l e t o i n h ib i t AC hR c lu s t e r in g f u r th e r s u gge s t i n g t h a t t h e p a t h wa y t o

b lo c k AC hR c lu s t e r in g i s i nd e p en de n t o f n i t r i c o x id e s yn t h e s i s ( Wee ks ,

u n p u b l i s h ed r es ea rch )

Figure 17: Treatment of myotubes with Agrin, L-Name, and/or Laminin (Weeks, unpublished).

AChRs are stained to show that clustering was not inhibited suggesting that NO synthesis and

AChR clustering act on independent pathways.

A = u n t r e a t e d m y o t ub e s B = A g r i n C = Ag r i n + L n a m e D = L a m i n i n E La m i n i n + L N a m e

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Panel

# of AChR

clusters/cell

A 10

B 150

C 300

D 100

E 200

Table 1: Number of clusters per cell from Figure 17

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T h e g r ap h s i n Fi gu r e 1 8 b e l ow a r e s ho w in g l e v e l s o f n i t r i c ox id e r e l e a s e

f ro m a gr i n o r l ami n in ( d a t a co u r t e s y o f D r . Wee ks ) . T h e p l o t s a r e

me as u r i n g c u r r en t ( p A ) v s . t i me ( m in) u s i n g an a mp e r om e t r i c p r ob e t h a t

i s sp e c i f i c t o N O . T h e c o n t r o l ( p an e l 1 ) i s sh ow in g n o n i t r i c ox id e

r e l ea s e . T h e a dd i t i on o f a gr i n ( p an e l 2 ) sh o ws a v er y p r o n o un ce d s i gn a l

s h o wi n g s t r on g r e l ea s e an d t h e l a min in ( p an e l 3 ) i s sh owi n g s o m e r e l e a s e .

T h i s l i k e l y s u gges t s t ha t l a min in a nd a gr i n a r e i nv o l v ed i n t wo d i f f e r en t

p a th w ays a n d th a t l am i n i n i s no t n eces s a r y t o i n d u c e n i t r i c o x id e r e l ea s e .

Figure 18: Measurement of levels of NO using an amperometric probe. The plots are measuring

current (pA) vs. time (min) using an amperometric probe that is specific to NO. The control (panel

1) is showing no nitric oxide release. The addition of agrin (panel 2) leads to very strong release

and the laminin (panel 3) is showing some release.

Panel 1- Control

Panel 2- Agrin

Panel 3 -

Heated

Laminin

Current (pA)

Time (minutes)

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Discussion

A gr i n an d l a min in b o t h i n i t i a t e t ra n s me mb ran e s ign a l in g c as ca des t h a t

i nd u ce AC hR c l u s t e r in g i n cu l t u r ed mu sc l e c e l l s . Wes t on ’ s s tu d i e s

s ho w e d so me n o v el f i n d in gs . Lam i n in h as s ho wn t o ac t iv a t e R ac , Cd c4 2 ,

a n d Rh o i n C2 ce l l s an d t h a t t h i s a c t i v a t i o n i s r eq u i r ed fo r t h e c lu s t e r i n g

f o r A ChR . Mu ch o f t h e d a t a p re se n t ed he r e h av e su gge s te d t h a t AC hR

c lu s t e r in g t h r o u gh l a m in in a nd a gr i n ac t t h r ou gh a s i mi l a r p a th w a y.

H o w ev er , t h e re a r e a f ew d i f f e r en ce s b e tw e en t h e tw o p a t h wa ys . AChR

c lu s t e r in g i n r e sp on s e t o ag r i n mus t go t h ro u gh t h e Rac /Cd c4 2 p a th w ay t o

f o r m mi c r o c lu s t e r s , t h e n t h ro u gh a Rho - d ep e nd e n t c on d en s a t i o n o f t he

m i c r o c lu s t e r s t o fo rm f u l l c lu s t e r s . Fo r l a m in i n , R ho ac t i v a t i on i s

b lo cke d d ue t o t h e ex p ress io n o f do m in a n t n e ga t i ve R h o o r C 3 t r an s f e ra s e

a s t he y b o th i m p ai r t h e f o r ma t io n o f AC hR c lu s t e r s o r mic ro c l us t e r s . I t

w as sh o wn th a t when agr i n was b lo c ked a t t h e l ev e l o f Rh o , C d c4 2 a nd

R ac s ign a l in g w as n o t a f f ec t e d . M ic ro c lu s t e r s w er e s t i l l a b l e t o fo rm b u t

n e v e r d ev e l op ed i n t o f u l l s ca l e c lu s t e r s ( We s t on , e t a l 2 00 7 ) .

I n Zh a n g’s s t u dy , a l l 19 d i f f e r en t Wn t s w e re s tu d i e d f o r e f f ec t s o n

A Ch R c l u s t e r in g . Wn t9 a , Wn t 9 b , Wn t1 0 b , Wn t 1 1 , an d Wn t1 6 w er e a b l e

t o i n du c e AC hR c lu s t e r in g w i t h ou t ag r i n . In t h e d ev e l op me n t o f m u sc l e s ,

Wn t9 a a nd Wn t 1 1 w e re ex p res s ed t h e m o s t hea v i l y . I t was s ho wn t h a t

A Ch R c l u s t e r in g w as d o se -d ep en d en t a n d no n -ad d i t i v e , su gges t i n g t h a t

Wn t s a c t on s i m i l a r p a t hw a ys t o i n d u ce c lus t e r in g . Th e fu n c t i on o f Wn t 9 a

a n d Wn t 1 1 r eq u i r e s Mu SK (M u s c l e S pec i f i c K in ase ) a nd LR P4 ( Z h an g e t

a l , 2 0 12 ) .

N i t r i c ox id e i s a d i f fu s i b l e gas t h a t p l a ys a r o l e i n n eu r on a l

d e v e l op m en t , p l as t i c i t y, a n d n eu r i t e r em o d e l in g . D ur in g em b r yo n i c

d e v e l op m en t , NO S h as b een f ou nd t o be ex p res sed i n b ra in a t d i f f e r en t

s t age s i n r od en t s an d h u m an s . Kn o cko u t mi ce sh o w ed no p h en o t yp i ca l o r

p a th w ay ab n o r ma l i t i e s an d t r ea t men t o f N OS in h ib i t o r L-N A ME d id n o t

a l t e r t h e o v e ra l l s t ru c tu r a l mo r ph o l o gy o f t h e b r a in o r n eu r o t r ans m i t t e r -

r e l a t ed m ar k e r s i n ce r eb e l l u m , h i pp oca mp u s , o r co r t ex . H o w ev er , nN O S

kn o ck o u t mi ce sh ow ed a m u ch red u ced d end r i t i c b r an ch-o u t s .

A n gi o t en s in I I ac t i v a t e s n i t r i c ox id e syn t h es i s , wh i ch a c t s as a m e d i a t o r

i n f o r m in g n ew b lo o d v es se l s an d n eu r i t e o u t g ro wt h . B es i d es t h e ro l e N O

h a s i n n e ur a l d e v e lo p m en t , n i t r i c ox ide a nd n i t r i c ox id e syn t h as e m a y

h a v e i mp l i c a t i o n s i n r ep a i r i n g d a m age d ne r v es ( r egen e r a t i on ) ( R i a l a s , e t

a l 2 00 0 ) . T he s tu d y o f N O h as ad v a n ced v e r y r ap id l y i n t h e l a s t t wo

d e ca d es . T h e p a thw a ys o f NO s yn t h es i s , s i gn a l i n g , a nd me t abo l i sm we re

m a j o r s t u d i e s t h a t l ed t o a No b el P r i z e i n 1 99 8 ( Ka B i an , e t a l 2 0 07 ) .

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D e f ec t s i n t h e dys t r o p h in - gl yc o p r o t e i n c o m pl ex (D GC) a r e k n o wn to

d i s r up t t h e fo r ma t io n o f t h e n eu r o mu sc u l ar j un c t io n an d A Ch R c l us t e r in g .

T h i s i s e v i d en c e t h a t t h e DGC p l a ys an i mp o r t an t ro l e i n r egu l a t i n g t h e

s t ru c t u re o f t h e NM J . T h e f r agm e n t ed ap p ea ra n c e o f t h e N MJ in m dx

( d ys t r op h i n - d e f i c i en t ) m i ce i s e v id e n ce o f a d e fe c t i n NM J s t r u c t u re .

E a r l y s t ud i e s h av e s ho wn t h a t s u g ge s t e d t h e f r a gm en ta t i on o f t h e N MJ

w o ul d ge t wo r s e wi t h a ge an d i t wo u ld o ccu r s e co nd a r y t o n e c ro s i s an d

r egen e r a t i o n r a t h e r t h an a d i r e c t r e su l t o f d ys t r op h in d e f i c i e nc y ( Sh i a o , e t

a l 2 00 4 ) .

I n Sc w ar t e ’ s l ab , N O S r o l e in t h e c l us t e r i n g o f A Ch Rs w er e an a l yz ed .

T h e i r d a t a su gge s t ed t h a t N OS i s a med i a to r o f agr i n s i gn a l i n g t h a t l e ad s

t o p o s t s yn ap t i c ACh R c l u s t e r in g d u r i n g s yn a p t o gen es i s . Ex po su r e o f

d e v e l op i n g em b r yo s t o v ar io u s N O S in h i b i t o r s 7 -n i t ro ind azo l e , v in yl - L-

N IO, a nd 1 40 0 W d ra ma t i ca l l y i n h ib i t ed ACh R ag gr e ga t i on a t e mb r yo n i c

N MJ s . H ow e v er , h igh e r c on cen t r a t i on s a re r eq u i r ed t o b lo c k a ggr e ga t i o n

i n v i vo t h an i n v i t ro . O t he rwi s e , N O S in h i b i t o r s s t i l l b l oc ke d ag gr e ga t i o n

b y 5 0 - 90 % s u gges t i n g NO S a c t i v i t y i s n e ce ss a ry fo r agr i n - i nd uc ed

a ggr e ga t i o n . C on c lu s i v e l y , t h e r e su l t s su gges t ed t h a t N O p ro d u ce d b y

N OS i s n eces sa r y a n d su f f i c i en t fo r a g r i n - in d u ced c l us t e r i n g o f ACh Rs i n

m u s c l e c e l l s . Th e d a t a a l so s u g ges t ed t h a t NO p r od u c t i o n i s n eces s a r y f o r

n e r v e- in d u c ed AChR c lu s t e r i n g a t t h e em b r yo n i c N MJ ( Scw a r t e , e t a l

2 0 04 ) .

I n t h e u n p ub l i sh e d w or k b y We eks , h i s ex p e r i m en t s m ad e t he c l a i m

t h a t ag r i n p r od u ces n i t r i c ox id e , a s t a te m en t n o t ma d e i n a n y p u b l i ca t i o n

t o d a t e . T h e ex p e r i me n t s sh ow ed th a t L- N AM E w as ab l e t o i n h ib i t n i t r i c

ox i d e i n l ami n i n and a gr i n m yo tu b es b u t w as u n ab le t o i nh ib i t AC hR

c lu s t e r in g , su gges t i n g t h a t NO s yn t h es i s an d A ChR c lu s t e r i n g a r e s e pa ra t e

p a th w ays .

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References:

Ando A, Yang A, Nambu H, Campochiaro PA. Blockade of nitric-oxide synthase reduces

choroidal neovascularization. Mol Pharmacol. 2002 Sep;62(3):539-44.

Barber, A.J., Lieth, Erich. Agrin Accumulates in the Brain Microvascular Basal Lamina During

Development of the Blood-Brain Barrier. Developmental Dynamics 1997 208:62-74

Baylis, C. Nitric Oxide Deficiency in Chronic Kidney Disease. Am J Physiology Renal

Physiology 2008 294:F1-F9

Bian, Ka. Vascular System: Role of Nitric Oxide in Cardiovascular Diseases. The Journal of

Clinical Hypertension 2008 10(4):304-310

Blottner D, Luck G. Just in time and place: NOS/NO system assembly in neuromuscular

junction formation. Microsc Res Tech. 2001 Nov 1;55(3):171-80. Review.

Borges LS, Ferns M. Agrin-induced phosphorylation of the acetylcholine receptor regulates

cytoskeletal anchoring and clustering. J Cell Biol. 2001 Apr 2;153(1):1-12.

Brockhausen, J., Cole, R. N., Gervasio, O.L., Ngo, Shyuan T., Noakes, P.G., Phillips, W.D.

Neural Agrin Increases Postsynaptic ACh Receptor Packing by Elevating Rapsyn Protein at the

Mouse Neuromuscular Synapse. Developmental Neurobiology 2008 1153-1169.

Brown, MD, Cornejo, BJ, Kuhn, TB, Bamburg, JR. Cdc42 Stimulates Neurite Outgrowth and

Formation of Growth Cone Filopodia and Lamellipodia. Journal of Neurobiology 2000 43:352-

364.

Burkin DJ, Gu M, Hodges BL, Campanelli JT, Kaufman SJ. A functional role for specific

spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering. J Cell Biol. 1998

Nov 16;143(4):1067-75.

Christova T, Grozdanovic Z, Gossrau R. Nitric oxide synthase (NOS) I during postnatal

development in rat and mouse skeletal muscle. Acta Histochem. 1997 Aug;99(3):311-24.

Cornish T, Chi J, Johnson S, Lu Y, Campanelli JT. Globular domains of agrin are functional

units that collaborate to induce acetylcholine receptor clustering. J Cell Sci. 1999 Apr;112 (Pt

8):1213-23.

27

27

Ferns M, Deiner M, Hall Z. Agrin-induced acetylcholine receptor clustering in mammalian

muscle requires tyrosine phosphorylation. J Cell Biol. 1996 Mar;132(5):937-44.

Gairthwaite, J. Concepts of neural nitric oxide-mediated transmission. European Journal of

Neuroscience 2008 27:2783-2802.

Gautam, M., Noakes, P.G., Moscoso, L., Rupp, F., Scheller, R.H., Merlie, J.P., Sanes, J.R.

Defective Neuromuscular Synaptogenesis in Agrin-Deficient Mutant Mice. Cell 1996 85:525

535.

Gloe T, Riedmayr S, Sohn HY, Pohl U. The 67-kDa laminin-binding protein is involved in

shear stress-dependent endothelial nitric-oxide synthase expression. J Biol Chem. 1999 Jun

4;274(23):15996-6002.

Godfrey EW, Schwarte RC. The role of nitric oxide signaling in the formation of the

neuromuscular junction. J Neurocytol. 2003 Jun-Sep;32(5-8):591-602. Review.

Gurjar MV, DeLeon J, Sharma RV, Bhalla RC. Mechanism of inhibition of matrix

metalloproteinase-9 induction by NO in vascular smooth muscle cells. J Appl Physiol. 2001

Sep;91(3):1380-6.

Herbst, R., Burden, S.J. The juxtamembrane region of MuSK has a critical role in agrin-

mediated signaling. The EMBO Journal 2000 19(1):67-77

J Biol Chem. 2003 Feb 21;278(8):6450-5. Epub 2002 Dec 06.

Jones MA, Werle MJ. Agrin-induced AChR aggregate formation requires cGMP and aggregate

maturation requires activation of cGMP-dependent protein kinase. Mol Cell Neurosci. 2004

Feb;25(2):195-204.

Jones MA, Werle MJ. Nitric oxide is a downstream mediator of agrin-induced acetylcholine

receptor aggregation. Mol Cell Neurosci. 2000 Nov;16(5):649-60.

Kim, N., Stiegler, A.L., Cameron, T.O., Hallock, P.T., Gomez, A.M., Huang, J.H., Hubbard, S.

R., Dustin, M.L., Burden, S.L. Lrp4 Is a Receptor for Agrin and Forms a Complex with MuSK.

Cell 2008 135:334-342.

Kober, M, Su, Z, Moon, H. Exogenous Nitric Oxide Enhances Synaptic Transmission at the

Crayfish Neuromuscular Junction. Pioneering Neuroscience 2010 10:63-66.

Lancaster, Jr., J. Nitric Oxide Principles and Actions pp 1-130. 1996 Academic Press

Lee NP, Cheng CY. Nitric oxide/nitric oxide synthase, spermatogenesis, and tight junction

dynamics. Biol Reprod. 2004 Feb;70(2):267-76. Epub 2003 Oct 01. Review.

28

28

Linnoila, J., Wang, Y., Yao, Y., Wang, Z. A Mammalian Homolog of Drosophila Tumorous

Imaginal Discs, Tid1, Mediates Agrin Signaling at the Neuromuscular Junction. Neuron 2008

60:625-641.

Luck G, Hoch W, Hopf C, Blottner D. Nitric oxide synthase (NOS-1) coclustered with agrin-

induced AChR-specializations on cultured skeletal myotubes. Mol Cell Neurosci. 2000

Sep;16(3):269-81.

Mascarenhas JB, Ruegg MA, Winzen U, Halfter W, Engel J, Stetefeld J. Mapping of the

laminin-binding site of the N-terminal agrin domain (NtA). EMBO J. 2003 Feb 3;22(3):529-36.

Maselli, RA, Fernandez, JM, Arrendondo, J., Navarro, C., Ngo, M., Beeson, D., Cagney, O.,

Colette Williams, D., Wollmann, RL, Yarov-Yarovoy, V., Ferns, MJ. LG2 Agrin Mutation

Causing Severe Congential Myastenic Syndrome Mimics Functional Characteristics of Non Neural

(z-) Agrin. Human Genetics 2012 131:1123-1135.

Meier T, Ruegg MA, Wallace BG. Muscle-specific agrin isoforms reduce phosphorylation of

AChR gamma and delta subunits in cultured muscle cells. Mol Cell Neurosci. 1998

Jul;11(4):206-16.

Minatel E, Santo Neto H, Marques MJ. Acetylcholine receptors and neuronal nitric oxide

synthase distribution at the neuromuscular junction of regenerated muscle fibers. Muscle Nerve.

2001 Mar;24(3):410-6.

Mittaud P, Marangi PA, Erb-Vogtli S, Fuhrer C. Agrin-induced activation of acetylcholine

receptor-bound Src family kinases requires Rapsyn and correlates with acetylcholine receptor

clustering. J Biol Chem. 2001 Apr 27;276(17):14505-13. Epub 2001 Jan 31.

Moransard M, Borges LS, Willmann R, Marangi PA, Brenner HR, Ferns MJ, Fuhrer C. Agrin

regulates rapsyn interaction with surface acetylcholine receptors, and this underlies cytoskeletal

anchoring and clustering. J Biol Chem. 2003 Feb 28;278(9):7350-9. Epub 2002 Dec 16.

Neill, S., Bright, J., Desikan, R., Hancock, J., Harrision, J., Wilson, I. Nitric oxide evolution and

perception. Journal of Experimental Botany 2008 59(1):25-35.

Nitkin RM, Smith MA, Magill C, Fallon JR, Yao YM, Wallace BG, McMahan UJ.

Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol.

1987 Dec;105(6 Pt 1):2471-8.

Oliveira, DM, Silva-Teixeira, DN., Goes, AM. Evidence for Nitric Oxide Action on in Vitro

Granuloma Formation Through Pivotal Changes in MIP-1α AND IL-10 Releases in Human

Schistosomiasis. Nitric Oxide: Biology and Chemistry 1999 3(2):162-171.

29

29

Petrov, K., Malomouzh, AI, Kovyazina, IV, Krejci, E., Nikitashina, AD, Proskurina, SE, Zobov,

VV, Nikolsky, EE. Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular

junction via N-methyl-D-aspartate receptor activation. European Journal of Neuroscience 2013

37:181-189.

Pun S, Ng YP, Yang JF, Ip NY, Tsim KW. Agrin-deficient myotube retains its acetylcholine

receptor aggregation ability when challenged with agrin. J Neurochem. 1997 Dec;69(6):2555-6

Rialas CM, Nomizu M, Patterson M, Kleinman HK, Weston CA, Weeks BS. Nitric oxide

mediates laminin-induced neurite outgrowth in PC12 cells. Exp Cell Res. 2000 Nov

1;260(2):268-76.

Schwarte RC, Godfrey EW. Nitric oxide synthase activity is required for postsynaptic

differentiation of the embryonic neuromuscular junction. Dev Biol. 2004 Sep 15;273(2):276-84.

Shiao T, Fond A, Deng B, Wehling-Henricks M, Adams ME, Froehner SC, Tidball, JG. Defects

in neuromuscular junction structure in dystrophic muscle are corrected by expression of a NOS

transgene in dystrophin-deficient muscles, but not in muscles lacking alpha- and beta1-

syntrophins. Hum Mol Genet. 2004 Sep 1;13(17):1873-84. Epub 2004 Jul 06.

Strochlic, L., Falk, J., Goillot, E., Sigoillot, S., Bourgeois, F., Delers, P., Rouviere, J., Swain, A.,

Castellani, V., Schaeffer, L., Legay, C. Wnt4 Participates in the Formation of Vertebrate

Neuromuscular Junction. PLos One 2012 7(1):1-12

Tseng CN, Zhang L, Cascio M, Wang ZZ. Calcium plays a critical role in determining the

acetylcholine receptor-clustering activities of alternatively spliced isoforms of Agrin. J Biol

Chem. 2003 May 9;278(19):17236-45. Epub 2003 Mar 05.

Ueda, N., Degnan, S. Nitric Oxide Acts as a Positive Regulator to Induce Metamorphosis of the

Ascidian Herdmania momus. PLos One 2013 8(9):1-16.

Walker, SJ., Wu, W., Cerione, RA, Brown, HA. Activation of Phospholipase D1 by Cdc42

Requires the Rho Insert Region. Journal of Biochemistry 2000 275(21):15665-15668.

Weston C, Gordon C, Teressa G, Hod E, Ren XD, Prives J. Cooperative regulation by Rac and

Rho of agrin-induced acetylcholine receptor clustering in muscle cells.

Weston C, Yee B, Hod E, Prives J. Agrin-induced acetylcholine receptor clustering is mediated

by the small guanosine triphosphatases Rac and Cdc42. J Cell Biol. 2000 Jul 10;150(1):205-12.

Wilson, ID, Neill, SJ, Hancock, JT. Nitric oxide synthesis and signalling in plants. Plant, Cell,

and Environment 2008 31:622-631.

30

30

Yin, A., Wilks, O., Mosher, P. Nitric Oxide Plays a Significant Role as a Retrograde

Messenger that Causes Long-Term Facilitation at the Crayfish Neuromuscular Junction.

Pioneering Neuroscience 2010 11:31-37.

Zhang B., Liang C., Bates R., Yin Y., Xiong W., and Mei, L. Wnt proteins regulate acetylcholine

receptor clustering in muscle cells. Molecular Brain. 2012 5:7.

Zong, Y., Zhang, B., Gu, S., Lee, K., Zhou, J., Yao, G., Figueiredo, D., Perry, K., Mei, L., Jin, R.

Structural basis of agrin–LRP4–MuSK signaling. Genes and Development 2012 26:247-258.

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