Biogenesis and significance of lipofuscin in the equine thyroid gland : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology at Massey University

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BIOGENESIS AND SIGNIFICANCE OF LIPOFUSCIN IN THE EQUINE THYROID GLAND.

A thesis presented in partial fulfilment of the requirements for the degree of

Doctor of Philosophy

in

Veterinary Pathology

at

Massey University.

Rosalind Ruth Dalefield 1991

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ABSTRACT

Yel low-brown g ranules o f l ipofusc in ( age pigment ) a c cumulate in the cytoplasm of long-l ived cel ls of many euc a ryotes , inc luding man . The granules a re der i ved f rom lys os omes , and are defined by

character i s t i c mo rphol ogy , colou r , f luo rescence, and h i s t ochemi s t ry . They i n c rea s e i n number w i t h a ge i n p o s t -mi t o t i c cel l s , a nd a re rega rded a s ma r kers o f t he a g i n g p r o c es s . Lipo f u s c in i s widely a s s umed to c on s i st of produ c t s of pe r o xi di s ed f a t ty a c ids c ros s l i n k e d w i t h a m i n o g r o u p s o f p r o t e i n s , n u c l e i c a c i d s , a n d phospholipids , but this theory has not been proven .

The occur rence, histol ogy, st ruct ure, and c ompos it ion o f l ipofuscin in t hyro cytes f rom h o r ses o f a w i de range of a ges were s t udied . Granules were a bsent at b i r t h but were widespread by the age of 5 yea rs . I n young hor ses , granu les were la rges t in f o l l i c les which c ontained abnorma l c o l loid . After 5 years the amount o f l ipofus c in in thyrocytes was not age- related . Lipofu s c in was f luorescent , and s t a ined w i t h P A S , S chmo rl ' s , and Ma s s on ' s Fontana stains , a lthough t he st aining intensity va ried between horses . Lec t in h i s t ochemis t ry demo n s t r a t ed t h e p r e s e n c e o f ma n n o s e a n d / o r g l u c o s e . Sma l l s ubpopu l a t i ons o f granules in s ome sect ions contained i ron and some g ranules contained DNA .

L ipo fusc in granules were ir regu l a r in s i ze and shape, and cons i s ted o f a n e l e c t r o n - d e n s e ma t r i x a n d r e l a t i v e l y e l e c t r o n - l u c e n t ' vacuoles ' , which did not conta i n l ipid . They appeared t o fuse with c o l lo i d droplet s , and sma l l granu les were observed w i t h i n c o l l o i d droplet s .

T h y r o i d l i p o f u s c i n g r a n u l e s i s o l a t e d b y o s mo t i c s h o c k a n d

d i f ferent i a l cent r i fugat i o n s h o wed the s ame u l t ra s t r u c t u re a s in

s itu . The protein c ontent ranged f rom

15%

to

77%

w / w ( mean

⁼ 3 6 % ) .

In cont ras t , the amino acid c ompo s i t ion wa s const ant and s imi l a r t o

that o f t hyroglobu l i n . The mo st d i s t i n c t ive feature o f l ipofuscin

protein wa s t he presence of four proteins of 1 4 - 1 8 kDa . The h a lide

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concentration o f l ipofuscin wa s approximately t wice that o f thyroid t i s s ue . L i p o f u s c i n c o n t a ined n o t r i g l y c e r i des , a n d o n l y s ma l l quant i t ies o f pho sphol ipids (mean

⁼

1 . 2 5 % w / w ) I n cont r a s t , the c o n c ent r a t i o n s o f c h o les t e r o l a n d d o l i ch o l , up t o 1 9 % a n d 1 5 % respect ivel y , were high a lthough very va riable . Sma l l amo u n t s o f n u c lea r D N A were detected . The emp i r i c a l f o rmul a o f t he rest o f l ipofuscin indicated that i t w a s principa l ly c a rbohydrate, w h i c h was cons i s tent with t he PAS and lect in histochemi s t ry .

Most o f the l ip o f uscin mas s was a n a l ys ed and there wa s n o evi dence t ha t it a r o s e f rom l ip i d pe r o x i da t i o n . The p r o t e i n o f thyroid l ipo fuscin probably cons i s t s o f proteolyt i c intermediates o f t h y r o g l o bu l i n . Comp onen t s f r om o t h e r s ou rces , a n d t u r n o ver o f granule content s , cont ribute t o the chemical heterogeneit y .

T h e c ompo s i t i o n o f l i p o f u s c i n p r ob a b l y v a r i es bet w een t i s s ues . However a l l l ipofuscins a re likely t o share lysosoma l funct ions such as st orage of p roteo l yt i c intermediates , met a l s a nd do l i c ho l . As w i t h thy r o i d l ip o f u s c i n , they may be more s o luble and more readily ana lysed t han is genera lly assumed in the l iterature . They may also be act ive o rg a ne l les o f met abo l i c s ig n i f i cance, rather than inert indicators o f the aging proces s . On the b a s is o f t h i s s t udy, they should be re-eva luated .

•

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i ACKNOWLEDGEMENTS

I would like to thank my supervisors, Dr. D.N. Palmer and Professors R.D. Jolly and B.W. Manktelow, for their practical and intellectual contributions to this thesis.

I wish to acknowledge the help of many people who assisted with this study. Mrs. P. Slack and Mrs. P. Davey prepared tissues for light and electron microscopy. Mr. D. Hopcroft and Mr. R. Bennett of the Department of Scientific and Industrial Research gave advice on electron microscopy and printed the electron micrographs, and Dr. A.

Craig, also of the Department of Scientific and Industrial Research, supervised the immunogold labelling. Miss S. Bayliss electroblotted the proteins for sequencing. The metal analyses were performed by Dr. R. Reeves of the Department of Chemistry and Biochemistry, and Mr. J. Reid and Dr. G. Midwinter, of the same department, supplied the quantitative amino acid analyses. Dr. G. Ionas of the Department of Microbiology and Genetics assisted with, and instructed me on, the nucleic acid analyses. Sequencing of proteins was undertaken by J.E Walker, J.M. Skehel and I.M. Fearnley at the M.R.C. Laboratory of Molecular Biology, Hills Road, Cambridge, UK. The staff of the Microanalytical Laboratory, Department of Chemistry, University of Otago supplied the analyses of nonmetallic elements. Mr. F. Sharpe and other staff of the Department of Veterinary Pathology and Public Health assisted with horses submitted for post mortems, and many others assisted with the supply of tissues from horses.

I would also like to thank Mr. T. Law for the production of the photographs, Dr. H. Varela-Alvarez for the production of Fig.

4: 14

and Mr. S. Grant for the production of Fig.

7: 1 .

Finally I would like to thank my husband Wes for his patience and support and my son Martin for being a delightful diversion.

This work was supported by the New Zealand Medical Research Council grant

8 7 / 9 1 .

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TABLE

OF CONTENTS

Page

Acknow1edgements ⁱ

Tab1e of Contents i i

List o f Figures

^vi

List of Tab1es

^ix

Abbreviations xi

Chapter 1: Introduction

1

Characte rist ics of lipofuscin 2

D i s t r ibut ion of l ipofuscin 6

Int racellular s ignificance of lipofusci n

9

Othe r lipopigment s

10

Theo r ies of lipofuscin biogene s i s

14

Age p igment a n d the aging proce s s

18

Othe r theories of aging

20

Glycat ion 2 2

Concluding comments 2 3

Chapter 2 : The thyroid gland : a review o f its gross and

microscopic anatomy, phys io1ogy, and l ipofuscin accumulation

2 6

Gross anatomy 2 6

Hist ology 2 6

Normal thyroid ultrast ructure 2 7

Thyroglobulin

28

Thyroglobulin endocyt o s i s and catabolism

3 0

Regulat ion o f thyrocyte act ivity

31

Lipofuscin in thyrocytes 32

Othe r age-related changes in the thy r o id g land 36

Chapter 3 : General materials and methods

38

H i s t ology Horses

Fixation and l ight microscopy Stat ist ical analys i s

Concanava l in A labell ing Fluorescence micro scopy Frozen sect ions

Electron micro scopy

3 8 3 9 3 9 3 9 4 0 4 0 4 0

ii

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Product i on of ant ibody for immunocytochemi s try

Electroblott ing and irnrnunostaining Imrnunogold labelling of equine thyroid t i s sue

Isolat i on and analyses of lipofuscin

i i i

4 0 4 1

4 2

Horses

4 2

Is olat ion o f lipofus cin

4 3

Dens ity determination

4 3

Amino acid analysis

4 3

Tryptophan analysis

4 4

Soluble protein extraction

4 4

Lithium dodecyl s ulphate-polyacrylamide

ge l e lectrophore sis

4 4

Silver sta ining of ge ls

4 4

Electroblotting from ge ls

4 5

Protein sequencing

4 5

Lectin affinity labe l ling of

electroblotted proteins

4 6

Proteolipid analysis of equine thyroid

lipofuscin

4 6

Element a l analysis

4 6

Lipid extract io n and analysis

4 7

DNA concentrat ion

4 7

DNA characteris ation

4 8

Phenol-sulphuric acid a s s ay for

neutral sugars

4 9

Sample preparation for

Gas-l iquid chroma t ography of s ugars

4 9

Derivatis ation of s ample s for

Gas-liquid chroma t ography of s ugars

4 9

Gas-liquid chromat ography of s ugars

4 9

Chemicals

5 0

Chapte r 4 : Subclinical pathology and lipofuscin accumulation

in the equine thyroid gland

5 1

Introduction

5 1

Spec ial methods

5 1

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Re sults

Thyroid s ize and co lour Foll icle s ize and act ivity Lipofuscin

Changes in colloid

Thyrocyte l ipofuscin re lated to co lloid abnorma l it ies

Lipofuscin granule numbers pe r cel l in

5 1 5 2 5 2 5 5

5 8

sect i on

5 9

Lipofuscin and fol l icle act ivity

61

Lipofuscin and f o l l icle s ize

62

Lipofuscin and frequency of abn o rma l

co lloid

62

D i scus s ion

62

Summa ry

6 9

Chapter

5:

Lipofuscin in the equine thyroid gland :

histochemical and morphological heterogeneity 7 1

Int roduct ion

7 1

Re sult s

Fluorescence and hist ochemi s t ry Compa rison of s t a i n affinities Hist ochemi stry of l ipofuscin i n the

5

year old horse

Ult ra s t ructure of equ ine thyroid lipofuscin

Immunogold elect ron microscopy D i scu s s i on

Summary

Histochemi s t ry of l ipofuscin Ultra st ructure

Chapter

6:

Lipofuscin in the equine thyroid gland : i s o lation and chemical analys^es

Int r oduct ion Special methods Res u lt s

7 1 7 4

7 6

7 6 7 7

7 8 8 2 8 4

8 6 8 6 8 7

Dry matter content of equine thyroid

8 9

Isolat ion of l ipofuscin

8 9

iv

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Density determination

Elementa l analysis of lipofuscin Proteins

Lipids

Ca rbohydrates Nucleic acids Discussion

S umma ry

Chapter 7 : Genera1 Discussion Experimenta l

Equine thyroid lipofuscin and the lipid p e roxidation theory

Lysosomes, residual bodies and lipofuscin Possible mechanisms for biogenesis o f equine thyroid lipo fuscin

Avenues of further study

Chapter 8 : References

V

9 1 9 1 9 6 1 0 3 1 0 4 1 0 7 1 0 8 1 1 8 1 20 1 20

1 27 1 29

1 3 2

1 3 5

1 3 8

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LIS T OF FIGURES

Figure 4 : 1 .

Gro s s appearance o f fresh thyroid glands .

Figure 4 : 2 .

His t o logy o f equine thyroid tissue.

Figure 4 : 3 .

Bas ophil lic zone in the colloid.

Figure 4 : 4.

Solid co l loid, divided into f ragments.

Figure 4 : 5 .

Fluo rescence microscopy.

Figure 4 : 6.

Fluo rescence microscopy . Figure 4 : 7 .

Shreds o f col loid and e rythrocytes in f o l licle s . Figure 4 : 8 .

Sphe rites.

Figure 4 : 9 .

Mul t iple abnorma litie s in the colloid . Figure 4 : 1 0 .

Multiple abno rma lities in the col loid.

Figu res 4 : 1 1 and 4 : 12 .

Compa rison of H&E and Schmo r l's stain . Figure 4 : 1 3 .

H&E-stained section o f the five yea r old horse.

Figure 4 : 14.

Lipo fuscin granu les pe r ce l l.

Figure 5 : 1

Lipofuscin granules by light microscopy .

F i gure 5 : 2

Thyroid tis sue s t ained with PAS.

F i gure 5 : 3

Thyroid tis sue s t ained with Ma s s on's Fontana.

F i gure 5 : 4

Thyroid t is s ue s t ained with AFIP stain for lipofuscin .

vi

Following page

5 2

52

55

5 5

5 6

58

5 8

6 1

7 2

7 4

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Figure 5 : 5

Thyroid tis sue st ained with Pe r l's stain for iro n . Figure 5 : 6

Thyroid tis sue stained with Feulgen-NAH s t ain .

Figure 5 : 7

Thyroid ti ssue s t ained with Methyl Green-Pyronin (MGP) . Figure 5 : 8

Frozen section of thyroid s t ained with Sudan B lack B.

Figure 5 : 9

Frozen section of thyroid tis s ue s tained with Oil Red 0.

Figure 5 : 1 0

Thyroid tissue labe l led with Concanavalin A / Horseradish peroxidase .

Figure 5 : 1 1

Ultrastructure of equine thyroid tis sue . Figure 5 : 12

Variations in e lectron density of lipofuscin and col loid droplets.

Figure 5 : 13

Lipofuscin granule with vacuole of high e lect ron densit y . Figure 5 : 14

Fine detail of lipofuscin granules.

Figure 5 : 15

Thyroid tis sue proce s s ed without o smium and n ot stained.

Figure 5 : 16

Appa rent fusion between a lipofuscin g ranule and a co l l oid droplet .

Figure 5 : 17

Va riations in contents of col loid droplets . Figure 5 : 1 8

Anti-thyroglobulin irnrnunogold labelled thyroid tis sue . Figure 6 : 1 .

I s o lated lipofuscin g ranules.

Figure 6:2

Montage of lipofuscin prote ins sepa rated by LDS-polyacryl arnide electropho resis .

Figure 6 : 3

vii

75

76

77

8.9

9

7

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Lipofuscin granules afte r Folch extraction . Figure 6 : 4

Thin l ayer chromatogram of pho spho lipids . F igure 6 : 5

Thin l ayer chromatogram of neutral l ipids . Figure 6 : 6

Aga ro s e ge l electrophore s is o f nucle ic acid from thyroid lipofuscin .

Figure 6 : 7

Aga ro s e gel electrophore s i s of nucle ic acids after incubation with nuclea ses .

Figure 6: 8

Aga rose gel electrophores i s of nucleic acids after incubation with restriction enzyme s . Figure 7 : 1

Proposed pathways o f lipofuscin biogenes i s .

viii

1 0 3

1 0 7

1 3 2

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Tabl.e 3 : 1

Horses u sed fo r h i s t ology Tabl.e

4 : 1

LIST OF TABLES .

Thyroid activity, p resence of colloid abno rma l i ties

ix

Page

3 8

a n d numbe rs of lipofu s c i n granules counted i n p lane o f sect i on

5 3

Tabl.e 4 : 2

Presence of cold follicles

5 4

Tabl.e 4 : 3

F requencies of type s of colloid abnormalities observed Tabl.e 4 : 4

Lipofus cin granu le s per

1 0 0

cells i n diffe rently stained sections from ho rse 2 5G

Tabl.e 5 : 1

His t o chemistry of equine thyroid lipofus cin Tabl.e

6 : 1

Dry matter content of equine thyroid tis s ue Tabl.e

6 : 2

Yields of lipofu s cin granules i s olated from equine thyroid glands

Tabl.e 6 : 3

Den sity of lipofu s cin granules Tabl.e

6 : 4

Elemental compo s it ion of equine thyroid lipofuscin ( I) : non-met allic elements (% by weight)

Tabl.e 6 : 5

Elemental composition of equine thyroid lipofus cin ( II) : met a llic elements, boron and phospho rus ( ug / g)

Tabl.e

6 : 6

Metal content o f thyroid tissue and thy r o id l ipofuscin Tabl.e

6 : 7

Rat io of stored e lements in lipofuscin c ompa red to thyroid t i ssue

Table 6 : 8

Ami n o acid comp o s i t ion of thyroid t i s sue, i s olated lipofus c in,

5 7

6 0

7 3

8 9

9 0

9 1

92

93

9 4

9 5

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and e xt racted proteo l ipid compared t o thyroglobulins Table 6 : 9

9 8 X

Ami no a c i d c ompo s i t i on o f l i p o f u s c i n p r o te i n, l ipo f u s c in prote i n fract ion s oluble in LDS / 2 -mercaptoethano l , a nd lipofusc i n

prote i n f raction insoluble in LDS / 2 -merc aptoethanol Table 6 : 1 0

P ro t e i n content o f lyophilised equine thyroid l ipofuscin

Table 6 : 1 1

Protein content o f equine thyroid l ipofusc i n i n

98%

f o rmic acid Table 6 : 12

10 0

102

Neu t r a l l ipids o f equine thyroid l ipofusc i n 10 4 Table 6 : 1 3

Rat i o s o f nonprotein ca rbon, hydrogen a nd oxygen in lipofusc i n 10 6

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AF I P BHT BSA D I T EDTA

Feu lgen-NAH HPLC

H & E I gG kDa LDS LDS -P AGE M I T

NMR

PAS P B S T P VDF

TBS TLC TRH T r i s T r izmaTM T S H TTBS T3 T 4

ABBREVIATIONS

Armed F o rces Institute of P a t h o logy Buty lat ed hydroxyto luene

Bovine s erum a lbumin d i iodotyrosine

ethylenediaminet et raacet i c a c i d Feu lgen-Naphthoic acid Hydra z i ne

high performance liquid c hromatography haematoxylin a nd eosin

immunoglobulin G k i loDalt o n

Lithium dodecyl sulphate

LDS - polyacrylamide& i �� i!!!�ii��t � a �

monoiodo t yrosine

nuc lea r magnet i c resonanc e per iodic acid-Schiff

phosphat e-buffered s a line/ Tween 20 P o lyviny lidene difluoride ( Immob i lon™, M illipo re Corporat ion, MA.)

T r i s -buffered s a line t h i n layer chromatography T S H - relea s i ng hormone

T r i s ( hydroxymethyl) aminomet h a ne

T r i s ( hydroxymethyl) aminometh a ne ( Sigma ) Thyroid stimulating hormone

Tween 20 /Tris + Cat ions T r i iodothyronine

Tet ra iodothyro nine, thyroxine

xi