Бесплатный автореферат и диссертация по биологии на тему

Липофусцин в старении и патологии

ВАК РФ 03.00.02, Биофизика

Текст научной работыДиссертация по биологии, доктора биологических наук, Татарюнас, Антанас Бернардович, Вильнюс: Publishing Hause of the Seimas

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ANTANAS TATARIÜNAS

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LIPOFUSCIN IN AGING AND PATHOLOGY

antanas tatariunas LIPOFUSCIN IN AGING AND PATHOLOGY

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Vilnius 1998

TK in Tatariünas, Antanas

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UDK 577 2/ 3 Lipofuscin in aging and pathology.

Vilnius: The Publishing House of the Seimas, 1998.-210 p.

In this book we have a description of Ceroid-Lipofuscin cytosomes which were examined by the prism of discovered photophenomenon of increased intrinsic fluorescense. The book is illustrated with 84 figures, 11 tables and one scheme. Includes 417 bibliographical references.

ISBN 9986-18-039-2

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© A. Tatariünas, 1998. © The Publishing House of the Seiraas, 1998.

All rights reserved. No part of this book may be reproduced in any form or by any means without permissions in writing from the author or publishers.

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The Lithuanian Academy of Science

The monograph has been approved at the Institute of Experimental and Clinical Medicine of the Academy of Sciences of Lithuania

Reviewers:

Dr. V. N. Karnaukhov

Laboratory of Cellular Microspectrofluorometry/Photometry, Institute of Cell Biophysics of Russian Academy of Sciences, 142292 Pushchino City, Moscow Region, Russia

Dr. K. Muckus

Laboratory of Heart Electrophysiology, Institute of Biomedical Investigations, Kaunas Medical University, LT-3007 Kaunas, Lithuania

Dr. Suresh I. S. Rattan

Laboratory of Cellular Aging, Aarhus University, DK-8000 Aarhus-C, Denmark

ABSTRACT

Human physiological aging and the relationship of this process with calendar aging and human pathology and the ability to work belong to the most important problems not only of medical gerontology but also of biology. The main law of biological aging on cellular level is accumulation of the so-called "age pigments" (retmoids/carotenoids) in Ceroid-Iipofuscin Granules (CLG). For this reason, investigations of CLG may reveal new ways of prolonging the active life and working period of man.

The aims of this work are as follows: 1) investigations of CLG genesis in cells, 2) investigations of their intrinsic fluorescence, 3) investigations of the lipofuscinolytic ability of the drug Centrophenoxine (CP).

The work is written in conformity with a standard scheme: Preface, Review of Literature Data, Methods, Results, Summaiy, and References. In the first chapter CLG investigations in cells in situ are presented, while in the second chapter investigations of CLG isolated from tissues are described.

In this work CLG properties were investigated on light and electron microscopy levels in different human, animal, mollusk (,LymnaeastagnaSs) andtermite (Anaccmthotermes ahngeriamtsJacobson) tissues. The accumulation of CLG was studied also in the myocardium of young rabbit in case of acute infarction. The intrinsic fluorescence of CLG was investigated microspectrofluorometrically. The discovered phenomenon ofthe increase of intrinsic fluorescence of CLG in living cells caused by fluorescence-exciting ultraviolet (^=365nm) irradiation was confirmed on isolated CLG. The conclusion on cyclic changes of excited fluorophore in native CLG was drawn. The mentioned above effect was modeled in the test-tube. It was shown that the mechanism of photoinflamed intrinsic fluorescence of CLG could be explained by the photodecomposition of retinoids.

The process of agingwas modeled in nondividing cells in culture (apoptosis of sympathetic spinal superior cervical ganglion of rabbit and differentiated mouse neuroblastoma cells) and in a continuously dividing retrovirus-transfoimed hybridoma cell culture. Experimental acute infarction ofthe myocardium of young rabbit was examined as a natural model of cellular culture in vivo.

The main conclusion of the work is that CLG may be defined as cellular organelles. CLG are the product of the regulation of cell metabolic activity. They are formed from cell endoplasmic reticulum. Intrinsic fluorophores of CLG have polyisoprenoidic (retinoid/ carotenoid) nature and consist at the minimum of nine different retinoids. In native CLG the existence of a covalent-bound fluorophore-protein complex was detected. The IipofusrinoIytic,cationicamphiphilicd

asignalat the cellular receptor level. CPwas noticed to slow downthebudding of retroviruses on cellularmembrane.

The book is illustrated with 95 figures and tables, more than 400 references are cited It can be recommended to basic gerontologists, cell molecular biologists, biophysicists, biochemists andwide-scopepathologists.

CONTENTS

PREFACE..................................................................................................................................................................................................8

ABBREVIATIONS............................................................................................................................................................11

INTRODUCTION..................................................................................................................................................................13

Terminology and historical remarks....................................................................................13

Distribution of CLG in the cells..................................................................................................19

Morphology of CLG..........................................................................................................................................24

Physicochemical characteristics of isolated CLG........................................2 8

AIMS OF THE STUDY............................................................................................................................................43

MATERIALS AND METHODS................................................................................................................44

RESULTS AND DISCUSSION......................................................................................................................52

A. INVESTIGATIONS OF CLG IN SITU............................................................52

Al. Chronological (calendar) aging. Retinoid/carotenoid nature of intrinsicfluorophore in CLG........................................................................................................52

A.1.1. Intrinsic fluorescence spectra of tissues of aged

animals and some retinoids/carotenoids..........................................................5 2

A.1.2. CLG investigations in human and animal tissues........................................................................................................................................................................................58

A.1.3. CLG investigations in mollusk Lymnaea

stagnalis..........................................................................................................................................................................70

A1.4. CLG investigations in termites A nacanthotermes

ahngetianus Jacobson..............................................................................................................................78

A2. Models of aging. Establishment of CLG genesis from

cellER..............................................................................................................................................................................................83

A.2.1. Nondividing cells in culture. Apoptosis of symphathetic spinal superior cervical ganglion from the

rabbit......................................................................................................................................................................83

A2.2. Nondividing cells in culture. Apoptosis of differentiated mouse neuroblastoma cell........................................................................87

A2.3. Natural cell culture model. Myocardial infarction of young rabbit....................................................................................................................................90

A.2.3.1. External control of acute myocardial infarction process................................................................. 90

A.2.3.2. Investigations of myocardial infarction on

the visible light microscopy level.................................... 95

A.2.3.3. Investigations of myocardial infarction on

the EM level......................................................................... 103

A2.4. Rapidly dividing cells in culture. Hybridoma cells

for A, phage antibodies.................................................... 109

A.2.4.1. CLG genesis in the retrovirus-transformed

hybridoma cell..................................................................... 109

A.2.4.2. The action of Centrophenoxine on the

retrovirus-transformed hybridoma cell.............................. 118

A.2.5. Natural (physiological) model of aging. Hibernating ground squirrel Citelus undulatus........................... 131

B. INVESTIGATIONS OF ISOLATED CLG. ESTABLISHMENT OF REHNOID-PROTEIN COMPLEX IN CLG AND CONFIRMATION OF RETINOID/CARO-TENOID NATURE OF FLUOROPHORE........................... 140

GENERALIZATION AND CONCLUSIONS...................... 151

APPENDIX..............................................................................................................................................................................................161

A retinoid model of the effect of increased intrinsic fluorescence of CLG............................................................................................................................................161

Materials and methods......................................................................................................................161

Results and discussion..........................................................................................................................163

SUMMARY.............................................................................................. 178

REFERENCES.

180

PREFACE

In 1969, being a student of Kaunas Medical Institute (Lithuania), I started at the former Institute of Biophysics of USSR Academy of Sciences in Pushchino City, Moscow Region. It was the time of common increase of knowledge in biology after the dark Lysenko's period. We were young, and we all dreamed of winning our Nobel Prizes in a purely biophysical field of electrophysiology. I started also with electrophysiological investigations at the Laboratory of Neuron Biophysics (headed by late Prof. B. N. Veprintsev) of that Institute. After some time I was deeply charmed by another purely biophysical method -microspectrofluorometrical investigations of living cell. The importance of living cell investigations stood out in all discussions of late Academician G. M. Frank, Director of Institute of Biophysics. The first microspectrofluorometer was made at that time at the Laboratory of Dr. V N. Karnaukhov. And so, I was included in that Lab's team. I worked at this Lab all the time till the renewed Independence of Lithuania in 1990 as attached Senior Scientist from Kaunas Medical Academy. I can say clearly that I am a follower of the Department of Living Cell of the former Institute of Biophysics of the Academy of Sciences of USSR that has developed into the separate Institute of Cell Biophysics of Russian Academy of Sciences. I am extremely thankful to late Academician G. M. Frank and Dr. V. N. Karnaukhov (my supervisors for PhD Thesis) for molding my scientific way of thinking and logic just from the living cell point of view. Especially, I am very thankful to my informal tutor-guide Prof. E. A. Burstein. He taught me honesty and persistence in science and life.

Molecular biologists are continuously searching for new objects of investigations to show all the possibilities of their favourite methods. I hope that I have opened here a new theme in Biophysical Gerontology and showed a new object for them, because human physiological aging and the relationship of this process with calendar aging, human pathology and the ability to work is one of the most important problems not only in Medical Gerontology but in Molecular Biology too. The main law of biological aging on cellular level is accumulation of the so-called "age pigments" (retinoids/carotenoids) in Ceroid-Lipofuscin Granules (CLG). For this reason, the investigations of CLG may also reveal new ways of prolonging the active life and working period of man.

While preparing a draft of this monograph, my view of the phenomenon of increased intrinsic fluorescence of CLG in case of fluorescence-exciting UV irradiation considerably broadened. I can say that my eyes opened in 1995 during my Study Visit as Invited Research Professor at the Tokyo Metropolitan Institute of Gerontology (TMIG). The period of thinking over the mechanism of this phenomenon which I found in 1980 at the former Institute of Biophysics in Pushchino City ended. I am very glad to write now that I discovered the phenomenon of increased intrinsic fluorescence of CLG exactly in Dr. V. N. Karnaukhov's Lab. In Tokyo, together with Dr. Sh. Matsumoto from Department of Biochemistry & Isotopes of TMIG, we modeled in the test-tube the above-mentioned effect to whom all this monograph is mainly dedicated. Thus, I decided to include here also the results of my Tokyo period. I am sure the data presented in Appendix are like a small solid granade flying straight to the gate of Aging.

I would be extremely happy if this monograph consisting mainly of my results obtained over the period of a quarter of a century would be serve a large circle of cellular molecular biologists and not only a close professional enclave of gerontologists. I sincerely hope that my results will stimulate a new round of wide investigations on CLG isolated from tissues.

I hope also that scientists will be able to understand how many stereotypical and psychological barriers I broke in my scientific way developing this theme merely from descriptive medical observations. Looking back, I can say that it was a kind of fighting between old histological findings of CLG in cells of different age and my biophysical thinking with the understanding of the metabolic regulation of living cell. As a scientist, I am really proud that during one lifetime (Thanks Fortune who was so generous to me!) I found the mechanism of a physical phenomenon and explained it on biological objects. I think this phenomenon will serve in future investigations of CLG as a background and a key instrument for understanding deeper the cell activity regulation and cell transduction system by retinoids in aged living cells.

I want to say many thanks to all my friends and colleagues all around the world, without whom this work would be impossible. I would like to express my gratitude to all my co-authors in published or still unpublished papers.

I would like to say many thanks to Dr. Akira Kobata, Director of Tokyo Metropolitan Institute of Gerontology (TMIG). He invited

me and kindly prolonged my stay at TMIG until I got the final result. I am so thankful to Dr. M. Matsuo, former Director of Department of Biochemistry & Isotopes of TMIG, because he undertook a special trip to Linkôping (Sweden), where I then worked as Invited Research Professor at Prof. U. Brunk's Lab of Department of Pathology (Linkôping University), for our first interview. His support and encouragement were so helpful at the period of hard experimental work in his former laboratory in Tokyo.

I greatly appreciate my wife Aldona and sons Audrius and Julius for their patience during my absence from home, since in Lithuania there were no equipments that I needed for the investigations of CLG.

My late parents Anastasia and Bernardas did all they could for my education. I think, I justified their hopes by this monograph.

I highly appreciate the grant from Government of Lithuania and the Lithuanian Academy of Sciences which covered the costs of publishing this monograph.

AG AO ATP b.t.

CDC13

CHC13

CLG

CLN

INCL

LINCL

JNCL

13C-NMR

CP

CT

Dol-OH

Dol-P

EM

ER

F

TTNMR

a-HPLC

p-HPLC

LDS

MF

Mr

MT

cMT-ATPe

MV

N

ABBREVIATIONS

Golgi apparatus acridine orange adenosine triphosphate body temperature deutered chloroform chloroform

ceroid-lipofuscin granules ceroid-lipofuscinosis, neuronal: infantile CLN (CLN1) late infantile CLN (CLN2) juvenile CLN (CLN3) carbon nuclear magnetic resonance centrophenoxine chlorotetracyclin dolichol

dolichyl phosphates

electron microscopy

endoplasmic reticulum

fluorescence intensity in relative units

proton nuclear magnetic resonance

analytic high performance liquid chromatography

preparative high performance liquid chromatography

lithium dodecyl sulfate

microfilaments

molecular mass

mitochondria

subunit c of mitochondrial ATP synthase

microvilli

nucleus

NAD/P-H reduced Nicotinamide-adenine dinucleotide and/or its

phosphates

NC virus nucleocapsid

NE nucleus envelope

PA Palmitic acid

PAGE polyacrylamide gel electrophoresis

PAS periodic acid-Schiff reagent

PM plasma membrane

R272, R346, R368 retinoids with main absorption maxima at 272, 346 and

368 nm in methanol, respectively

R retention of fluorescing zone on the chromatogram

RA Retinyl acetate

RNP virus ribonucleoprotein

RP Retinyl palmitate

RPE retinal pigment epithelium

SDS sodium dodecyl sulfate

TLC thin layer chromatography

TM tubulin microtubules

UV intrinsic fluorescence exciting ultraviolet light (365 nm)

V formed retrovirus

VM virus envelope membrane

WHC width of the head capsule of termite

X fluorophore in CLG

X* excited fluorophore in CLG

n quantity of X*

INTRODUCTION

Terminology and historical remarks

One of the clearly expressed and most universal morphological changes caused by age in cells is accumulation of colour granules in their cytoplasm on light microscopy level. These pigmented bodies were described in non-dividing postmitotic cells soon after the cell theory formation, and the first publication appeared in 1842 [1].

The relationship between normal aging and accumulation of these pigmented cytosomes was mentioned in 1886 by Koneff [2]. The most frequently cited paper in morphometrical correlation of these yellow granules with human calendar age is the paper by Strehler [3]. In his work a linear increase in the amount of pigmented granules with age was shown. The rate of accumulation is about 0.3% of the total heart volume per decade (or about 0.6% of the intracellular volume per decade). This amount may represent as much as 30% of the total solids of human myocardium, and by the time a human being reaches 90 years of age, as much as 6-7% of the intracellular volume may be occupied by the pigmented cytosomes (In this respect, it is of interest to note that the velocity of accumulation of the pigmented cytosomes in the Japanese in Hiroshima after nuclear bombing was nearly twice higher than in the Americans [3]).

In 1912 Hueck [4] first termed the yellow pigmented granules "lipofuscins" and made the distinction between this pigment and the tyrosine pigment melanin. The term "lipofuscin" is derived from Greek "lipo" meaning fat and from Latin "fuscus" meaning dark. Thereafter, accumulation of similar pigments called ceroids [5,6] was observed by investigators in a variety of tissues under certain pathological conditions, both clinical, e.g. Ceroid-Lipofuscinosis [7,8] and experimental, e.g. vitamin E deficiency [9,10].

The term "ceroid" was originally exploited by Lillie [5,11-13] especially for the colour granules derived in experimental hepatic cirrhosis in hepatocytes, but these inclusions, meaning wax-like from Greek "keros", were described in autopsy tissue as early as 1894 by Goebel [14]. Pearse [15] advanced an idea that ceroid pigment is a precursor of lipofuscin, and this view exists in literature now. This view is supported also by findings showing that the nuclear magnetic resonance spectra of ceroid and lipofuscin are very similar [16].

Other terms for the yellow-brown pigmented granules accumulating with age were also used, but "lipofuscin" has become the most accepted among all other synonyms, such as "age pigment", "yellow pigment", "lipopigment", "wear and tear pigment", "chromolipid", "lipochrome", etc. The term lipofuscin as a rule is associated with normal calendar human and animal aging.

Nevertheless, in the Human Gene Mapping Nomenclature [17], among the most common progressive encephalopathies of childhood [18,19] there is a special term, "Ceroid-Lipofuscinosis". These disorders are characterized by a severe psychomotor retardation, visual failure, seizures, brain atrophy and the accumulation of cytosomes in neurons and glial cells resembling pigments like ceroid derived under experimental conditions and lipofuscin in normal aging [8,19].

The first report about patients suffering from Ceroid-Lipofuscinosis was published in 1826 by Stengel [20]. The disease was characterized by loss of vision and mental abilities manifesting after 6 years of age in four consecutive children of the same family. Later on, patients with similar symptoms were studied by Batten [21,22], Vogt [23], Spielmeyer [24], Jansky [25], Bielschowsky [26], Kufs [27] and Sjogren [28].

The term "Neuronal Ceroid-Lipofuscinoses" (CLN) was introduced to distinguish these disorders from other types of amaurotic family idiocy [8], e.g., Tay-Sachs disease characterized by intraneuronal accumulation of gangliosides.

Human CLN are classified into four main types according to age at onset, clinical course, neurological, neurophysiological, ophthalmologi-cal and neuropathological findings:

CLN1, infantile (INCL, Santavuori-Haltia disease);

CLN2, late infantile (LINCL, Jansky-Bielschowsky disease, early onset Batten disease);

CLN3, juvenile (JNCL, Spielmeyer-Vogt-Sjogren disease, late onset Batten disease);

CLN4, adult (Kufs'disease).

Variants or nontypical forms of different CLN types have also been reported, but the common denominator always is the intraneuronal and astrocytic accumulation of Ceroid-Lipofuscin granules (CLG). It remains unknown whether these distinguishable types represent different nosologic entities.

CLN have been diagnosed worldwide, and their incidence has been estimated to be as high as 1:12500 (all types included) [18,19]. Kufs'

disease is very rare and occurs mostly in adults. All childhood forms of CLN are inherited as an autosomal recessive trait, while in Kufs' disease both recessive and dominant forms have been reported [29]. The main symptoms of CLN are blindness, behavioural abnormalities, motor dysfunction and premature death.

Massive accumulation of intraneuronal and astrocytic inclusions like CLG in children was found in homozygous inbred English Setters with cerebroretinal degeneration [30]. A similar disease also occurs in sheep, horses, cows and other animals [7].

It is necessary to note that in the coastal region of South Western Australia and Africa CLN in horses, goats, sheep and pigs occur when grazing livestock are forced to eat the onion weed Trachyandra divaricata and laxa (family Liliaceae) in large quantities over four weeks under hot, dry summer conditions inducing a scarcity of alternative food [31-34]. In these grazing livestocks intensive CLG accumulation in neurons in the brain, spinal cord and peripheral ganglia was found; in the Kupffer cells of the liver, renal tubular cells and macrophages in the spleen, lung and intestinal mucosa less CLG were found.

It is clear that animal CLN are used as a model for studies of human CLN and aging [30], e.g., it is known that ovine CLN closely resembles the JNCL of man [7].

Pigmentation of CLG from the early beginning of investigations of CLG was closely connected with naturally found plant carotenoids [35, 36]. Later it was approved that pigmentation in CLG had a polyisoprenoidic (retinoid/carotenoid) nature [37-50]. Unfortunately, for a long period of time biological science didn't know about other functions of polyisoprenoids except the vision process. Functional activity of dolicholes (Dol-OH) and dolichylphosphates (Dol-P) in the transport of oligosaccharides for glycosilation of proteins and lipids in cell endoplasmic reticulum (ER) membrane is established [51]. It is interesting to note that their biosynthesis has the same precursors as the synthesis of retinoids. It has recently been shown that Dol-OH and Dol-P content from CLG isolated post mortem from the tissues of patients with CLN is significantly higher than in the age-matched patients with other neurologic diseases [52-58]. The crucial role of retinoids in controlling the cell differentiation process in embryogenesis and neoplasia has become evident only now, along with the discovery of the nuclear receptors for retinoic acid, which belong to the

steroid/thyroid hormone receptor subfamily [59]. Thus, polyisoprenoid compounds control cell metabolism and probably CLG formation in cell also.

Intrinsic fluorescence of CLG in situ was first discovered by Stubel in 1911 [60]. He observed a dark brown fluorescence of CLG in the myocardium of animals when CLG were excited by ultraviolet (UV). This intrinsic fluorescence was immediately recognized as one of the most consistent features of CLG [4], and since then intrinsic fluorescence has been used for the visualization of CLG. The developing fluorescence microscopy and microspectrofluorometric technique [61 -81] allows not only to detect CLG in living cells, but also ensures rapid monitoring of CLG by personal computer. So, spectral analysis of them in the living cell became possible.

Nevertheless, a number of staining methods has been used also in visualizing the CLG [15], e.g. Sudan black B, luxol fast blue, methylene blue, toluene blue, Nile blue, periodic acid-Schiff reagent (PAS), the Schmorl reaction, neutral red, thionine, gallocyanin, cresyl violet, etc.

It is known [15] that the sudanofilia reflects lipids and both lipid and hydrophobic protein complexes in CLG. Acid fastness is thought to signify the presence of unsaturated fatty acids of high molecular weight in CLG. Luxol fast blue is generally a stain for phospholipids, but in fixed tissues, especially in the paraffin sections, it typically stains the lipid-protein complexes. In PAS stain the periodic acid is an oxidant capable to break 1,2 glycol C-C bonds and to form carbonyl groups that later on can react with the Schiff reagent giving red staining. In tissue sections it is essentially a stain for 1,2 glycol groups found in the hexoses of glycoproteins. It is also possible that periodic acid can catalyze the hydrolysis of bis(monoacylglycero)phosphate which occurs with the production of a 1,2 glycol structure in CLG. The Schmorl reaction in CLG is attributed to the production of reducing groups by the oxidative process, however, this is a relatively non-specific stain for CLG. Thus, histochemistry reflects the chemical composition of CLG.

Since 1950/60s new era in CLG investigations has begun:

1. Electron microscopy (EM) became the method of choice to study the fine structure and ultrastructural histochemistry of CLG [7].

2. In 1955 Heindenreich and Siebert [82] observed the resistance of CLG to relatively strong methods of mechanical treatment. They succeeded in CLG isolation from human heart by differential and discontinuing density gradient centrifugations of tis-

sue homogenates disintegrated with a blendor. This pioneer study further was extended by other investigators [83-89]. They modified the procedure, and by their methods CLG of high purity could be obtained and their morphological and biochemical characteristics could be thoroughly studied.

3. The formation of CLG has been shown in model systems in young animals [90-98], subjecting them to hypoxia, hyperoxia, vitamin E deficiency, in case of inhibitors of lysosomal enzymes, etc., and in model systems of senescence in different cellular cultures [99-102]. All this opened new experimental horizons in investigations of aging on the cellular level.

4. It has been found also that the newly synthesized preparation ANP-235 [103,104] later named Centrophenoxine (derivative of plant natural growth factors - auxins) has a lipofuscinolytic effect. It can decrease the quantity of CLG in cells both in natural [105-111] and in model aging [99] in different cellular cultures [112-117]. Its synonyms are Meclofenoxate, Lucidril, Cerutil, Acephen, etc.

Investigations of CLG till 1980s were summarized in the following reviews:

1. Wolman M. Lipids, vol.5, Second part, Histochemistry of lipids in pathology. Gustav Fisher Verlag, Stuttgart, 1964.

2. Strehler B. J. (Ed.). Advances in gerontological research, vol.1., Acad. Press, N.Y., 1964.

3. Wolman M. (Ed.). Pigments in pathology. Acad. Press, N.Y., 1969.

4. Ordy J. M., Brizzee K. R. (Eds.). Neurobiology of aging. Plenum Press, N.Y., London, 1975.

5. Glees P., Hasan M. Lipofuscin in neuronal aging and diseases. In Bargmann W., Doerr W (Eds.). Normal and pathological anatomy, vol.32, Georg Thieme Publishers, Stuttgart, 1976.

6. Miquel J., Oro J., Bensch K.G., Johnson J.E., Jr. Lipofuscin: Fine-structural and biochemical studies. In Pryor W.A. (Ed.). Free radicals in biology, vol.3, pp. 133-182. Acad. Press, N.Y., San-Francisco, London, 1977.

7. Zeman W., Donohue S., Dyken P., Green J. The neuronal ceroid-lipofuscinoses (Batten-Vogt syndrome). In Vinken P.J., Bruyn G.W (Eds.). Leukodystrophies and poliodystrophies. Amsterdam, North-Holland Publishing Company, Handbook of Clinical Neurology, 10:588-679,1979.

8. Sohal R.S. (Ed.). Age pigments. Elsevier/North-Holland Biomedical Press, Amsterdam, N.Y., Oxford, 1981.

Since 1980s there have been five Meetings dealing with CLG and six Meetings on the inherited CLN problems.

The