Genetic brain polypeptide variants in inbred mice and in mouse strains with high and low sensitivity to alcohol

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Brain Research, 34 / ! ! ~85 ) 130- t 3~ Elsevier

BRE 10924

Genetic Brain Polypeptide Variants in Inbred Mice and in Mouse Strains with High and Low Sensitivity to Alcohol DAVID GOLDMAN 1, ROBERT NELSON 2, RICHARD A. DEITRICH 3, RODNEY C. BAKER 3, KAREN SPUHLER 3, HERBERT MARKLEY 4, MICHAEL EBERT 5 and CARL R. MERRIL 6

1Laboratory on Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, A CRF, 3Co215, Bethesda, MD 20205; 2Molecular Biology Institute, Rm. 555, UCLA, Los Angeles, CA 90024; 3Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262; 4Division of Neurology, Memorial Hospital, 119 Belmont St., Worcester MA 01605; 5Department of Psychiatry, Vanderbilt Medical Center North, Nashville, TN 37232; and 6National Institute of Mental Health, Bethesda, MD, 20205 (U.S.A.) (Accepted November 20th, 1984)

Key words: polymorphism - - two-dimensional electrophoresis - - alcoholism - - sedation - - LS mouse - - SS mouse

Twelve genetically determined brain polypeptide charge variants were identified by comparing cerebeUar vermis of 7 inbred mouse strains and of mice selectively bred from 8 strains closely related to these 7 ancestral strains and one other for acute behavioral sensitivity to the sedative effects of ethanol. The selectively bred ethanol-sensitive (LS, long sleep) and insensitive (SS, short sleep) mice exhibited different allelic variants at 6 of these 12 gene loci expressed in the cerebellum. Variant polypeptide A1 (81 kdaiton, pI 5.6) was shown to be associated with the membrane of synaptosomal mitochondria and to exhibit a basic variant in SS mice Chat is determined by a dominant allele. Other variant polypeptides showed codominant inheritance in F1 crosses. However. the phenotype of no single one of these brain polypeptides consistently correlated with the ethanol behavioral sensitivity of the 7 inbred mouse strains nor of 8 recombinant inbred (B x D. C57BL x DBA) strains. This finding supports the hypothesis that a substantial amount of inbreeding, leading to random fixation of alleles independent of selection for ethanol sensitivity, occurred during the breeding of the SS and LS mice. The present findings of a lack of a strong association between sleep time and a brain polypeptide variant do not preclude the existence of a major gene effect contributing to variation in acute sensitivity to ethanol but are consistent with reports that multiple loci are responsible for the difference in ethanol sensitivity between SS and LS mice. The genetically variant brain polypeptides described here are among the first examples of genetically determined variant polypeptides in mouse brain.

INTRODUCTION

times

were

designated

alcohol-insensitive

(short

sleep, SS). This successful p r o c e s s of s e l e c t i v e b r e e d T h e m e c h a n i s m by w h i c h e t h a n o l e x e r t s its a c u t e

ing used a g e n e t i c a l l y h e t e r o g e n e o u s p o p u l a t i o n of

central n e r v o u s s y s t e m effects is largely u n k n o w n .

mice, H S , as its base stock. T h e H S line was d e v e l -

L i k e w i s e . the g e n e t i c b i o l o g i c factors which c o n t r i b -

o p e d by c r o s s - b r e e d i n g 8 s e p a r a t e i n b r e d strains21.

ute to a l c o h o l i s m r e m a i n u n d e f i n e d d e s p i t e family 7, twin18,20, 26 and a d o p t i v e studies 2-4-14-16.27 which sup-

lished that the d i f f e r e n c e m t i m e to r e g a i n the right-

port their e x i s t e n c e . O n e a p p r o a c h to i d e n t i f y i n g the genetic factors i n v o l v e d has b e e n to selectively b r e e d

ing r e s p o n s e ( ' s l e e p t i m e ' ) can not be e x p l a i n e d by m e t a b o l i s m o f e t h a n o l in t h e liver11A 7 b u t d o e s c o r r e -

m i c e for e i t h e r v e r y high o r v e r y low sensitivity to the

late well with the d i f f e r e n t i a l sensitivity o f c e r e b e l l a r

a n a e s t h e t i c and ataxic effects of e t h a n o l 21. In t h e se-

P u r k i n j e cells (i.e. inhibition of firing r a t e ) t o locally

lection p r o t o c o l , a n i m a l s w i t h l o n g r e c o v e r y times

applied ethanolS.28 -30. In c o n t r a s t , p y r a m i d a l cells o f

f o l l o w i n g a s t a n d a r d d o s e of e t h a n o l a d m i n i s t e r e d in-

the h i p p o c a m p u s , w h i l e d e p r e s s e d by local applica-

t r a p e r i t o n e a l l y (i.p.) w e r e d e s i g n a t e d alcohol-sensitive (long sleep, LS) a n d t h o s e with short r e c o v e r y

tion of e t h a n o l , s h o w the s a m e sensitivity in LS a n d SS m i c e 28. T h e differential sensitivity of LS and SS

N u m e r o u s studies using t h e s e animals h a v e estab-

Correspondence: D. Goldman. Laboratory on Clinical Studies. National Institute on Alcohol Abuse and Alcoholism. ACRF. 3C-215, Bethesda. MD 20205, U.S.A.

131 cerebellar Purkinje cells is retained even if cerebellar buds from fetal mice are transplanted to the anterior chamber of the eye of adult animals and allowed to develop25. The differential sensitivity is also retained in cerebellar slices 1. Presumably, the mechanism by which the LS and SS animals are genetically differentially sensitive to ethanol resides in genetically variant proteins involved in physiological and metabolic processes affected by ethanol. Because of the observed difference in the in vitro sensitivity of cerebellar Purkinje cells to ethanol, a logical site to search for the genetic protein differences underlying differential sensitivity was the cerebellum. To identify the protein variants, we elected to use two-dimensional electrophoresis because it can be used to simultaneously screen a large number of loci for genetic polypeptide charge variants12.13.2~.31.32. The charge variants resolved by this method probably comprise about a third of the genetic variant polypeptides present; isoelectrically neutral substitutions usually do not result in well-resolved variants. Nevertheless, Comings, in a study of human brain polypeptides, observed that a polymorphic variant of a 19 kdalton brain protein was increased in frequency in alcoholics, depressives and patients with multiple sclerosis 6. MATERIALS AND METHODS Male mice of the SS and LS lines from generation 28 to generation 37, male F1 and F2 progeny from LS x SS crosses, males from inbred strains closely related to 7 of the original 8 inbred strains intercrossed to develop the heterogeneous stock (HS) and males from 8 recombinant inbred strains ( B x D , C57BL x DBA; Jackson Laboratory, Bar Harbor, ME) were used. The 7 inbred strains used included 1SBI/Crgl (from Cancer Research Laboratory, Berkeley, CA), C3H/2Ibg (from IBG, Boulder, CO), A/J, AKR/J, DBA/2J and BALB/cJ (from Jackson Laboratory, Bar Harbor, ME). The C57BL, ISBI and C3H/2 strains are direct descendents of the lines originally cross-bred to derive the HS mice. The remaining lines are closely related to the original ancestral substrains. SS and LS lines were bred by a mass selection procedure 2~. Animals with the shortest latency to righting response were selected for the next SS generation

and those with the longest latency to righting response for the next LS generation. The only restrictions on mating pairs were that neither brother-sister matings nor cousin matings were permitted whenever possible 21. For assessment of sensitivity to the hypnotic/sedative effect of ethanol (sleep time), male mice were administered 3.3 g ethanol/kg b.wt., i.p., as a 30% (w/v) solution in 0.9% saline. Mice were tested for sensitivity at 60-65 days of age. For two-dimensional electrophoresis of cerebellar proteins, the vermis from mouse brains was quickly dissected following decapitation and frozen on dry ice. All tissue samples were frozen within one minute of decapitation to avoid post-mortem changes. For two-stage electrophoresis, the procedure of O'Farrel124 was used with minor modifications. A 4:1 mixture of 5/7 and 3/10 ampholytes was used for isoelectric focusing and 10% polyacrylamide gels (0.8 mm thick) were used for second stage electrophoresis in the presence of SDS. Approximately 100/zg of protein was electrophoresed. For silver staining 22,23, gels were sequentially: (1) fixed with 10% methanol/5% acetic acid for at least one hour; (2) equilibrated with 5% glutaraldehyde for 30 rain; (3) washed twice for 30 rain each time with deionized water; (4) washed overnight with deionized water; (5) soaked with dithiothreitol (5 rag/l) 30 rain; (6) soaked in 0.2% silver nitrate for 30 rain; (7) image developed in 3% sodium carbonate with 0.5 ml formaldehyde/l; (8) stopped 10 rain with 3% acetic acid; and (9) washed twice for 10 rain with deionized water. RESULTS Twelve genetic brain polypeptide charge variants were identified in SS, LS and inbred mouse strains among approximately 200 polypeptides of cerebellar vermis analyzed in two-dimensional electrophoretograms. SS and LS mice differed at 6 of these 12 loci. The positions of these charge mobility variants is indicated in Fig. 1 and their coordinates are listed in the legend. The phenotypes of each variant protein in LS and SS mice and in the ancestral inbred mouse strains are given in Table I. ' A ' indicates that the acidic charge phenotype was observed, 'B' indicates that the basic charge phenotype was observed and ' H ' indicates that at least one of four animals tested

132

~ ~!ii ~

~!!~!i'

L

t

0

"

10 9 AC I D I C

B A S IC

Fig. ]. Location of genetic variant brain polypeptides on an electrophoretogram of m o u s e cerebellar vermis protein t I00 .ug). The pI scale is approximately 4.5 on the left to 6.0 on the right and the molecular weight gradient is from approximately 15 kdalton at bottom to 200 kdalton at top. Two-stage electrophoresis and silver-staining were performed as described in Materials and Methods. Po[ymorphic brain protein loci located in inbred, LS and SS mice had the following coordinates:

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 All A12

Mol. wt. (kdalton) 81 48 30 112 122 72 71 31 33 23 65 28

lsoelectric point (acidic variant) 5.6 5.6 5.6 5.7 5.8 5.8 5.4 5.2 5.3 5.4 5.5 5.6

133 TABLE I Phenotypes of12 polypeptide loci for SS, LS and inbred mice Strain

AI

A2

A3

A4

A5

A6

A7

A8

A9

AIO

All

AI2

Sleep time*

SS C57BL/crgl C3H/2IBg AKR/J IS/BiCrgl BALB/cJ a/J RIIIS/J DBA/2J LS

B B A B B B B ND A A

B B B B A B B ND B A

H A A A B B B ND A B

H B A B ND B A ND A A

B B B B ND B B ND A B

H A A A ND A A ND A A

A A B A ND A A ND A A

A B A A ND A A ND A A

A A B B ND B B ND B A

A A A B ND A A ND A A

B A A B ND A B ND A A

A B A B ND A B ND A A

0 28 37 54 64 72 74 77 83 145

ND, not determined; A, acidic phenotype only; B, basic phenotype only; H, at least one heterozygous individual observed. * Time to regaining of righting response after ethanol 3.3 g/kg, i.p., ref. 30. For SS and LS fines, 10 animals were tested. For inbred strains, 5 animals were tested.

was h e t e r o z y g o u s . E a c h of the 7 i n b r e d strains was

these F1 p r o g e n y s h o w e d two p r o t e i n spots of e q u a l

m o n o m o r p h i c at all 12 loci. H e t e r o z y g o t e s w e r e ob-

density s e p a r a t e d by a unit c h a r g e (as i n d i c a t e d by

s e r v e d a m o n g 4 SS m i c e at 3/12 of t h e s e v a r i a n t brain

the e x t e n t of shift in c h a r g e p o s i t i o n caused by post-

loci but no h e t e r o z y g o t e s w e r e o b s e r v e d a m o n g LS

translational p r o c e s s e s such as p h o s p h o r y l a t i o n and

mice at the 12 loci.

c a r b a m y l a t i o n which result in a single c h a r g e shift).

All but o n e (locus A 1 ; see Fig. 2 A ) of the c h a r g e -

T h e p r o t e i n spots in the F1 w e r e of e q u a l density and

shift p r o t e i n p o l y m o r p h i s m s t e s t e d s h o w e d c o d o m i -

half the density of the p r o t e i n spots in the h o m o z y -

nant i n h e r i t a n c e . F o r e x a m p l e , A 2 (mass 48 k d a l t o n ,

gotes. A l s o consistent with c o d o m i n a n c e is the find-

pI 5.6; see Fig. 2B) was m o n o m o r p h i c in LS m i c e

ing that F2 p r o g e n y of LS and SS w e r e 10/29 h o m o z y -

(acidic), SS m i c e (basic) and in the 7 i n b r e d strains

gous acidic, 13/29 h e t e r o z y g o u s and 6/29 h o m o z y -

tested. F1 p r o g e n y of SS and LS m i c e w e r e all h e t e r o -

gous basic (a ratio of 0 . 3 5 : 0 . 4 4 : 0 . 2 1 ) .

zygous at this locus ( l U l l

p r o g e n y t e s t e d ) . E a c h of

A1 (mass 81 k d a l t o n , a p p r o x i m a t e pI 5.6; Fig. 2 A )

TABLE II Densitometric analysis" o r A l (pl 5.6, 81 kdalton), a triad of polypeptides which shift in charge mobility ND, not detectable. Position density* Basic

Acidic Strain SS LS F1

1 ND 0.027-0.004 ND

2 0.028-0.002 0.056-0.005 0.033-0.004

3

4

Ratio

0.058-0.004 0.092-0.003 0.083-0.005

0.102-0.008 ND 0.145-0.006

0.27 0.57 1.00 0.29 0.62 1.00 (I.23 0.57 1.00

* Polypeptide densities were measured from the 3 positions occupied by the triad of polypeptides on any one gel. Densities are given as means + S.E.M. and are in units of optical density × mm 2. Six SS mice, 5 LS mice and 4 FI mice were measured. Measurements were obtained using an image analysis system developed for two-dimensional electrophoretograms. For this computerized densitometry, stained gels are dried between sheets of cellophane or photographic negatives of gels are taken next to a calibrated density standard. Images were scanned using a high resolution densitometer (Optronics, Chelmsford, MA) at 100-200 ,zm resolution. Image data were analyzed using software written for an array processor (DeAnza IP5000) and DEC 1100 series computers 12,13. For measurements, a local background was computed using the mode of all picture value intensities in the immediate vicinity of each polypeptide spot. Density of each polypeptide spot was computed as (average spot density-background) x area. Spot densities from each gel were normalized using a constant derived from the densities of other protein spots which did not show variation in concentration.

134 is a triad of 3 polypeptide spots, spaced at intervals of a single charge. Each of the three A1 polypeptides has a more acidic mobility in LS than in SS animals. That this triad of polypeptides shifts as a group is indicated by the fact that the relative quantities of the three A1 polypeptides remain constant in SS, LS and F1 animals, despite the shift in charge, as demonstrated in Table II. A1 is genetically fixed in both the LS and the SS mice and also in the 7 inbred strains tested, showing the acidic phenotype in 2/7 inbred strains. Dominance of the allele producing the SS (basic) variant is indicated because 11/11 F1 progeny of SS and LS mice manifested the basic variant and 8/33 F2 progeny showed the acidic phenotype (a proportion of 0.24 compared to 0.25 expected). Subcellular fractionation studies revealed that A1 is associated with the membrane fraction of synaptosomal mitochondria (Table III). The relative concentration of A1 is 3 times greater in that fraction than in whole cellular homogenate. The protein is almost undetectable in non-synaptosomal mitochondria, cytosolic and non-membrane synaptosomal fractions. We observed a weak association between sleep time and a cerebellar protein variant for only one locus, A1 (Table I). At this locus, the acidic phenotype was observed in LS mice and generally in the alcoholsensitive inbred lines whereas the basic phenotype was observed in SS mice and the less sensitive strains. However, C3H/2IBg, a sensitive strain, showed the acidic phenotype. Eight recombinant inbred (B x D, C57BL x DBA) strains were studied to test whether this association could be strengthened. Three of the 8 RI strains showed the acidic phenotype at the A1 locus that was also observed in LS mice and these 3 RI strains were among the 4 least sensitive RI strains tested. Therefore, it is unlikely that any of the 6 cerebellar genetic polypeptide differences that we have observed between LS and SS mice represent major loci determining the difference in sensitivity to the hypnotic effects of ethanol.

tively bred from these ancestral strains differ in phenotype at 6 of these 12 brain loci. The geneticaUy variant brain polypeptides described here are among the first examples of genetically determined variant polypeptides detected in mouse brain. Elliot i~ has found an average of 8 liver polypeptide charge differences between different inbred strains utilizing twodimensional electrophoresis and has chromosomally mapped 5 loci using recombinant inbred strains. There was no strong association between any of the brain polypeptide variants and ethanol behavioral sensitivity of 7 inbred mouse strains or the 8 recom-

DISCUSSION We have detected 12 loci showing genetically determined polypeptide charge variants in the cerebellum of mouse inbred strains. Ethanol-sensitive, LS and ethanol-insensitive, SS mice which were selec-

Fig. 2. A: illustration of the acidic and basic phenotypes of polymorphic locus A1 (MW 81 kdalton pI 5.6). At the topis the acidic phenotype, as seen in an LS mouse:{also present in DBA/2J and C3FF2iBg mice). At the bottom is the bagicphenotype, as seen in an SS mouse (also present in the BALB/CJ, A/J, IS/BiCrgl, AKR/J and C57BL/Crglinbred strains).

135 binant inbred ( B x D , C57BL x D B A ) strains. A l though the lack of an association b e t w e e n any of these cerebellar p r o t e i n variants and sleep time does not preclude the possibility of a m a j o r genetic locus determining the trait, it is consistent with reports that multiple loci are responsible for the differences in

ethanol sensitivity between SS and LS mice and for the differences in inbred miceS, 9. The results of this study are consistent with the hypothesis of a substantial a m o u n t of inbreeding in the SS and LS lines, leading to r a n d o m fixation of alleles, i n d e p e n d e n t of selection for ethanol response. In the

~' i~ii~i~iiii~!i~ '

!~i ¸

Fig. 2. B: illustration of the acidic and basic phenotypes of polymorphic locus A2 (MW 48 kdalton, pI 5.6). At the top is the acidic phenotype, as seen in an LS mouse (also present in IS/BiCrgl). At the bottom is the basic phenotype, as seen in an SS mouse (also present in the DBA/2J, BALB/cJ, A/J, AKRJJ, C57BL/Crgl and C3H/2IBg inbred strains).

136 TABLE III

LS lines has been calculated (J. DeFries, personal

Subcellular distribution of protein A 1

communication) based upon the n u m b e r of mating

Subcellular fractionation19:brains were homogenized in 0.4 M sucrose containing 1 mM EDTA, pH 6.8 to make a 20% w/v homogenate. The homogenate was centrifuged for 20 min at 2000 g. The pellet was resuspended in 0.4 M sucrose to a volume equal to the original volume and centrifuged again. Supernatant fractions were then combined and centrifuged at 12,000 g for 20 min. The resulting pellet was composed primarily of mitochondria and synaptosomes. The supernatant was centrifuged for one hour at 105,000 g. The pellet from this centrifugation was labelled the microsomal fraction and the supernatant was labelled the cytosol. The mitochondrial plus synaptosomal fraction was layered on a discontinuous gradient consisting of 1.2 M, 0.8 M, and 0.4 M sucrose and centrifuged for two hours at 90,000 g. The pellet was labelled non-synaptosomal mitochondria, the material at the 0.8-1.2 interface was labelled synaptosomes and the material at the 0.4-0.8 M interface was labelled myelin. Synaptosomes were disrupted by incubating in 2 ml of water per gram of original tissue for 1.5 min at 0 degrees. This material was then layered over 1.2 M sucrose and centrifuged at 63,000 g for two hours. The pellet was labelled synaptosomal mitochondria. For isoelectric focusing and gel electrophoresis, the various fractions were diluted with equal parts 0.01 M Tris-HC1, pH 7.4 and a solution of 9.2 M urea, 4% v/v NP-40, 10% v/v mercaptoethanol and 10% v/v ampholytes. Samples were adjusted to be 9 M with urea. Protein concentration was such that 100 ~m of protein could be applied to isoelectric focusing gets in a volume of 25 bd. Quantitative analysis of a two-dimensional electrophoretogram of each subcellular fraction was performed. Gels were stained, photographed, scanned and measured using a computerized image analysis system as described. To compensate for variations in total protein and staining between gels, the densities of 61 other polypeptides were summed to compute a normalization factor. Quantities are in units of optical density x mm2. Subcellular fraction

Quantity

Relative concentration

Homogenate Microsomes Cytosol Non-synaptosomal mitochondria Synaptosomal mitochondria Supernatant Membranes

0.0084 0.0099 0.0020

1.00 1. t8 0.24

0.0019

0.23

0.0174 0.0000 0.0274

2.07 0.00 3.26

SS line, heterozygotes were observed at only 3 of 12 of the variant brain loci. A n even higher a m o u n t of inbreeding was found in the LS line, in which heterozygotes were not observed at any of the variant brain loci. The LS line experienced greater inbreeding than the SS line because of a bottleneck at generations 6 and 7 when only one mating pair was available2E The cumulative a m o u n t of inbreeding in the SS and

pairs for each generation. The equations used were: F N = FN_ 1 + F ( 1 - F N _ 0 , where F = 1/8Nm + 1/8Nf F N is the inbreeding coefficient at each generation and at generation 0 the inbreeding coefficient is taken as 0. F is the increment in inbreeding. N m is the n u m b e r of males in each generation and Nf is the n u m b e r of females in each generation. The assumption was that parents for each generation were selected at r a n d o m from the families. This results in the most conservative estimate of the inbreeding coefficient and yields, as lower limits of inbreeding, 0.60 for the SS mice and 0.79 for the LS mice at generation 33. The SS/LS mouse breeding program was designed to produce mice which genetically differed in their sedative response to ethanol and whose genetic differences would generally be secondary to the selection rather than to r a n d o m fixation of alleles at loci not involved in this response. The successful response to selection by the SS and LS lines demonstrates that the sensitivity to the hypnotic effect of ethanol is u n d e r genetic control and permitted the estimation that multiple loci are involved9. It has also stimulated studies which have demonstrated a correlation between the ethanol-induced depression of brain n e u r o n a l firing rate and the behavioral hypnotic effect of ethanol in SS and LS mice and the ancestral mouse inbred strains. To identify the genetic loci responsible for differences m sedative response to ethanol, selectively bred mice such as the SS and LS lines could be vital in pointing to particular loci which should be studied further. However. it should be noted that a large fraction of the allelic differences between LS and SS lines have arisen by r a n d o m fixation resulting from inbreeding rather than from the selection. Comparison of only LS and SS lines does not allow one to distinguish which alleles have been randomly fixed. This is particularly true for the majority of selectable loci which may individually contribute only minimally to genetic variation in the sedative response to ethanol. We have identified 12 loci expressed in brain which

137 s h o w g e n e t i c allelic v a r i a t i o n and h a v e d e s c r i b e d a

ioral loci.

p a r a d i g m for identifying b e h a v i o r a l l y significant loci by the analysis of genetic p r o t e i n v a r i a n t s in s e l e c t e d

ACKNOWLEDGEMENTS

lines and i n b r e d strains. If i n b r e e d i n g is m i n i m i z e d and c o n c u r r e n t l y s e l e c t e d r e p l i c a t e lines are also analyzed, application of this p a r a d i g m c o u l d p r o v i d e an

S u p p o r t e d in part by N I H G r a n t s A A 0 3 5 2 7 and D A 0 7 0 4 3 . A . O r r p r o v i d e d technical assistance.

efficient m e t h o d of s c r e e n i n g for significant b e h a v -

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