• SUMMARIZING DISCUSSION
Myoto nic d ystro ph y type 1 (DM1) is caused by the expansion of a (CTG) n t r i p l e t re peat in exon 15 of the DMPK gene (175). Since the repeat sequence is not situ ate d in the protein-coding region of the gene, the le ng th in creas e of the t r a n s c r i p t does not r e s u l t in produ ction of a m u t a n t protein. R a th e r un c o n ve n tio n a l m e c h a n is m s m u s t t h u s be in volv ed in diseas e m a n if e s ta tio n of DM1. N owad ay s it has been co m m o n ly accepted th a t a d o m inan t RNA pat ho ge ne sis m e c h a n is m -a proce ss by w hic h a b n o r m a l t r a n s c r i p t s induce pathology- may be the main caus e of the m a jo r i t y of s y m p t o m s (129, 300). The RNA to xicity m e c h a n is m has received in creas ed atten tio n since the dis c ove ry t h a t a second fo rm of myoto nic dystrophy, DM type 2 (DM2), is caused by a (CCTG)n re peat expansion in intron 1 of the ZNF9 gene (225). T ra n s c rip ts bearing an expanded (C/CTG) n re peat ca pture p ro tein s invol ved in splic ing, t r a n s c r ip t i o n or m R N A ex por t and th e re b y induce m is re g u la ti o n of s e v e r a l genes in t r a n s (65, 181).
So is t h e re no need to invoke DMPK protein p ro du cts in our t h e o ry ab out the co m p le x c li n i c a l m a n if e s t a t io n of DM1? Based on c u rr e nt knowledge, we fe e l this p o s s ib il it y can s t i l l not be excluded. For example, expansion of the (CTG)n re peat may affect the rate of DMPK t ra n s c r ip ti o n or the mode and a c t i v it y of a lt e r n a t i v e sp lic in g or t r a n s p o r t of DMPK t r a n s c r ip t s in cis and th us d ir e c tly influence the le v e l of e xpre ssio n or the com positio n of the DMPK isoform re p e rtoire. Any a b e r r a n c y in the e xpress io n of DMPK protein is ofo rm s could have an ad d it iv e ro le in one or more of the s y m p t o m s in the m u lt is y s t e m i c p atte rn of DM1 p a t h o l ogy. Only v e ry few w e ll - c o n t r o lle d in-depth stud ie s have a c t u a lly dea lt w it h this m a t t e r (135, 297). A ls o s t i l l r e la t i v e l y l it t le is known ab out the e xpress io n and b io lo gical function of a lt e r n a t i v e protein v a r i a n t s of the DMPK gene. This PhD st ud y aim ed to im p ro ve our le v e l of kn o w le d g e on DMPK protein s t r u c t u r e and function by in ve stig atin g the lo ca liza tio n and f u n c t io n a l p ro p e rtie s of d if fe re nt DMPK sp lic e is ofo rm s in more detail. • DMPK splicing and isoform expression
P r i m a r y t r a n s c r i p t s of the DMPK gene in both man and mo us e are sub jec t to a lt e r n a t i v e splicing. Mos t DMPK sp lic e mode s are e v o l u t i o n a r i ly co n se rv e d and r e s u l t in the e xpre ssio n of m u lt ip le isof orms, six of w hich are con sidered m a jo r is ofo rm s (95). One of the d if fe re nc es betw ee n is ofo rm s is the p resence or abse nc e of a five-amino- aci d VSGGG m o tif C- te rm inal of the kin ase domain. The re la t iv e e xpress io n of VSGGG-encoding t r a n s c rip t s s li g h tl y in c re a s e s during myogenic d if fe re n tia tio n (S. Mulders, PhD Thesis, Radboud U n iv e rs it y Nijmegen). For our studies, we decided to co n ce ntra te on the protein p ro du cts encoded by th e se m o st ab und an t m R N A s (DMPK A, C and E), w hic h are the is ofo rm s w it h hi gh est kinase
123 6 • S u m m a ri z in g D is c u s s io r
6 • S u m m a ri z in g D is c u s s io n a c ti v it y (296).
A l t e r n a t i v e splicing, by w hic h d if fe re n t C-termini are gen erated, co n fers d if fe re nt m e m b r a n e ass oc ia tio n p ro p e rtie s to DMPK A, C and E, in t h a t DMPK A and C t y p i c a ll y bind to i n t r a c e l l u l a r m e m b r a n e s and DMPK E r e m a i n s in the cytosol. Species-specific m e m b r a n e ta rg e ti n g occurs for the DMPK A isoform. DMPK A fr om mo us e lo c a li z e s at the ER m e m b r a n e and DMPK A fr om hum a n lo c a li ze s at the MOM (286). DMPK C pro tei ns fr om hum an and mo us e both bind to the MOM.
Becaus e of the ty p i c a l C - te rm in a l associatio n to m e m b ra n e s , long DMP K is o f o rm s A-D belong to the group of t ail - anc ho re d (TA) p ro te in s (18). TA p ro tein s c h a r a c t e r i s t i c a l l y expose t h e ir N - t e rm in a l protein do m ain into the cy to so l and contain a se g m e nt of hyd ropho bic am in o acids in the C te rm in u s , w hich in s e rt s in a m e m b rane . V ari ou s i n t r a c e l l u l a r m e m b r a n e s including the n u c le a r m e m b r a n e and p la s m a m e m b r a n e are p o t e n t ia l t a r g e t s for TA p ro te in s (21, 223). DMP K is the f i r s t and only TA kin ase rep orted in lit e r a t u r e to date.
Stud y of the o r g a n e lle - s e le c t iv e m e m b r a n e b eh av io r of DMPKs w ith n a t u r a l and a r t i f i c i a l sequence v a ri a t io n in t h e ir C - te rm in a l domain i m proved our un d e rs ta n d in g of ta rg e ti n g r e q u ir e m e n t s and sp e cif ic ity for m e m b r a n e binding (Cha p ter 3; see below).
• DMPK expression profile
In o rd e r to identify a co n tr ib ut io n of a lte re d DMPK e xpre ssio n to D M l's co m p le x pathology, it is im p o r t a n t to obtain d eta iled kn o w le d g e on the DMPK expre ssio n p atte rn in d if fe re nt c e ll type s and tissues. A c o n tr o v e rs y ab o ut DMPK e xpre ssio n in n o r m a l and DM1 pa tient t is s u e s has existed eve r since the DMPK gene w a s fi rs t describ ed ab out tw o decades ago, p a r t ly because a p p ro p ria te too ls and w e ll - m a t c h e d tis s ue s a m p le s w e r e lacking. An tib o d ie s used in e a rly stud ie s often recognized protein p ro du cts ranging in size fr om 42-54 kDa, w h e r e a s DMPK has a predicted m o l e c u l a r w eigh t of 68-72 kDa (297). Unfortun ately, an tibo d ie s ag a in st protein d om a in s s e le c t iv e ly p re se n t in so me DMPK is ofo rm s are dif fic ult to m ake ( W a n s in k et al. u n pub li she d r e s u l ts ) and t h e re fo re s t i l l not exist.
In situ hyb ri diz atio n a n a ly s is on mouse tis s u e s d em o n stra te d DMPK RNA e xpre ssio n in a ran ge of t is s u e s t h a t show p at ho logy in DM1 (244), but p atte rn in g in the DMPK s p lic e isoform p ro fi le s or a c t u a l protein l e v els w e r e not w e l l studied. Here, we chose a W e s t e rn blotting approach using a high -af fin ity p o ly c lo n a l a n t i s e r u m spe ci fic for the DMPK protein core to obtain a r e lia b le p ictu re of the ra tio s of e xpress io n and t i s sue d is t rib u ti o n of long (A-D) and s h o r t (E-F) DMPK isoforms. A lt h o u g h w it h this tec hn ique in fo rm atio n on ce ll- type specificity, in t ra t is s u e and in t r a c e l l u l a r pat te rn in g is lost, W e s t e rn blo tting is q u a n t it a t iv e ly more r e lia b le and e n a b le s easy d is c rim in a t io n betw ee n m e m b r a n e - a s s o c ia te d (lo ng ) and c y t o s o lic ( s h o rt ) isoforms. We found t h a t the rati o betw een st e ad y st at e co n c e n tra tio n s of long and s h o r t DMPK is ofo rm s v a r i e s be tween t is s u e s and ch an g es during m yo ge n esis (Cha p ter 2). This suggest s d if fe re nt f u n c t io n a l re q u ir e m e n t s for DMPK protein during d ev elo p m en t in d if fe re nt tissue s, because the long and s h o r t DMPK is ofo rm s pro bably d isp lay d if f e r e n t ia l s u b s t ra te sp e cif ic ity at the d if fe re nt lo catio n s in the c e ll (296).
n re peat ex pa ns ion and DMPK gene p ro du ct dosage (16, 101, 211). We found t h a t long and s h o r t is ofo rm s e xis t in ab out e q u a l co n c e n tra tio n s in card iac tis s ue and HL-1 h e a rt c e ll s in cultu re. If this su g g e sts that tig ht r e g u la t io n has c a rd io - p h ys io lo g ic a l rele vanc e , a b e r r a n t e xpr ess ion l e v e ls of sp lic e is ofo rm s as m ay occur in DM1, could p ossibly c o n tr ib ute to cardio pathology.
W e a k n e s s and w a s t in g of d is t a l s k e l e t a l m u s c le s and my otonia are o th er h a l l m a r k s of DM1 (101), w h e re m o s t ly type I ( slo w tw itc h ) fibers are affected (289). S e v e r a l of these disease fe a t u r e s are also found in DMPK KO mice and Tg o v e r e x p r e s s o r mice, again sug gest ing t h a t tight c o n tr o l of DMPK protein e xpr ess io n may be n e c e s s a ry to p re ve n t disease d eve lo p m en t (126, 211). We found t h a t in s k e l e t a l m usc le tis s ue long DMPK is ofo rm s p re do min at e, being e xpr ess ed ab o ut three-f old more than s h o r t isoforms. Stri kin g ly , DMPK protein w a s e xp ress ed at c o m p a ra b le le v e l s in both type I and II musc le s: in s o le u s m u s c le ( p r e d o m in a n t ly type I) only s li g h t l y high er am o u n ts of DMPK w e r e found tha n in t ib ia lis a n te r i o r ( m a in ly type II) and g a s t ro c n e m iu s (mixed type I and II muscle). These o b s e r v a t io n s c o rrob o ra te find in gs re por ted in an ISH stu d y (244). Co m p aris on of e xpress io n le v e ls of DMPK protein or RNA betwee n m usc le types, therefore , gives not a s im p le e xplanatio n for the pre dis p o sitio n to type I m usc le patho lo g y in DM1.
G a s t r o i n t e s t in a l d is t u r b a n c e s m ig ht be rela te d to DMPK e xpr ess ion le v e l s in s m o o th m u s c le tissue , a lt h o u g h no d ire c t e vid enc e for DMPK