DNA repair mechanisms in neurological diseases: facts and hypotheses

Journal of the Neurological Sciences, 112 (1992) 4-14 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00

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JNS 03832

Review article

DNA repair mechanisms in neurological diseases: facts and hypotheses Paolo Mazzarello a, Marco Poloni b, Silvio Spadari

a

and Federico Focher a

a lstituto di Genetica Biochimica ed Evoluzionistica, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy and b Clinica Neurologica l, Universit~ di Milano, Ospedale S. Paolo, Via di Rudin~ 8, 20142 Milano, Italy

(Received 21 January, 1992) (Revised, received 24 March, 1992) (Accepted 2 April, 1992) Key. words: DNA damage; DNA repair; Neurodegeneration; Amyotrophic lateral sclerosis; Alzheimer's disease; Xeroderma pigmentosum; Ataxia telangiectasia; Cockayne syndrome

Summary DNA repair mechanisms usually consist of a complex network of enzymatic reactions catalyzed by a large family of mutually interacting gene products. Thus deficiency, alteration or low levels of a single enzyme and/or of auxiliary proteins might impair a repair process. There are several indications suggesting that some enzymes involved both in DNA replication and repair are less abundant if not completely absent in stationary and non replicating cells. Postmitotic brain cell does not replicate its genome and has lower levels of several DNA repair enzymes. This could impair the DNA repair capacity and render the nervous system prone to the accumulation of DNA lesions. Some human diseases clearly characterized by a DNA repair deficiency, such as xeroderma pigmentosum, ataxia-telangiectasia and Cockayne syndrome, show neurodegeneration as one of the main clinical and pathological features. On the other hand there is evidence that some diseases characterized by primary neuronal degeneration (such as amyotrophic lateral sclerosis and Alzheimer disease) may have alterations in the DNA repair systems as well. DNA repair thus appears important to maintain the functional integrity of the nervous system and an accumulation of DNA damages in neurons as a result of impaired DNA repair mechanisms may lead to neuronal degenerations.

Introduction Four "letters" encode the whole information in DNA: they are nucleotides which are distinguishable by the base they contain. The bases are adenine (A), guanine (G), thymine (T) and cytosine ((2). The sequence of these "letters" along the DNA strand determines the "meaning" of the genetic message. Cellutar DNA is characterised by a double helix structure in which the bases on one strand pair with the bases on the other strand follow the rule of complementarity: A pairs with T and G pairs with C. The complementarity of the bases is the key for the correct maintenance of the genetic information during DNA replication: once the DNA strands are open, in order to allow the enzymatic machinery to synthesise the new DNA, each

Correspondence to: Paolo Mazzarello, MD, PhD, Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy. Tel.: 39-382-546354; Fax: 39-382422286.

strand becomes a template for the synthesis of the novel complementary strand. The accuracy of the overall DNA replication process is assured by several mechanisms illustrated in Fig. 1. Among them: nucleotide selection by DNA polymera~es, 3'-> 5'exonuclease proof-reading activity associated with DNA polymerases and post-replicative methylation-instructed mismatch repair system. These mechanisms, strictly associated with the DNA replication process, assure a final mutation rate of 10 -1° to 10 -12 errors per base pair per generation which is still acceptable for individual and species. Therefore they will no longer be included among the DNA repair systems dealing with damages caused to DNA by the variety of agents illustrated in Fig. 2. At the end of the DNA replication the cell has two copies of its genome each of them consisting of an old and a newly synthesized strand of DNA. Despite the high accuracy of the DNA replication process some errors arise during DNA replication, due to the incorporation of non complementary bases by

5 5'

3~

5'

,

3'

5*

Intercalating agents SHghUy modifiedbases

3*

IC

A* C*--A-

apurinic o r

apyrimidinicsite

MJs-pahing

2) 4--

-A

~*i

Pyrimidine dimers

Mutated DNA

i,

Base deletion

5'

\ Parental methylated DNA

3'

5'

Bulkilymodifiedbases A** T** Non-pairing Arrest D~A replication

Novel transiently rmn-methylatecl DNA

1) Methylation-instruca3dmismatchco~e~tionon newly synthesizedstrand.

t~

J Cross-link

2) Nudeotide sdection by DNApolymerases.

Doublestrand break

I

3) 3'->5'exonucleaseassociatedwith DNAlmlymerasss (proof-readingactivity).

3'

I

5'

Fig. 2. Some possible lesions to DNA.

Fig. 1. Enzymatic reactions allowing error avoidance during DNA synthesis.

DNA polymerases. Furthermore chemical and physical hits to DNA cause a variety of other structural and chemical lesions to the genome. The chemical compounds which react with nucleic acids range from activated oxygen species produced during oxidative metabolism to metals, alkylating agents, polycyclic hydrocarbons, aromatic amines, azo dyes and to several other molecules such as mitomycin C, aflatoxin B~, urethane etc., whereas among physical agents most common are ultraviolet and ionizing radiations (Fried-

Singlestrand break

!

berg 1985). The major kinds of DNA damage are shown in Fig. 2 and can be categorized as (1) base adducts; (2) apurinic-apyrimidinic (AP) sites; (3)single and double strand breaks; (4) DNA intra- or interstrand cross-links; (5) DNA-protein cross-links (Robison and Bradley 1984; Friedberg 1985; Sancar and Sancar 1988). If not repaired they lead to defects in subsequent replication cycles and to abnormalities both in transcription and in translation of the information coded in DNA.

TABLE 1 DNA REPAIR SYSTEMS System

Mechanism

Examples

I. Direct reversal of damage

single enzymatic reaction which removes the damage

enzymatic photoreactivation, repair of O6-alkylguanine, ligation of DNA strand break, purine insertion.

first step: cleavage of the glycosylicbond between base and sugar. first step: cleavage of the sugar-phosphate backbone

repair of deaminated bases.

II. DNA excision repair (A) base excision repair

(B) nucleotide excision repair (1) short-patch repair (2) long-patch repair IIL Daughter strand gap repair IV. Inducible SOS response

recombination multistep process

X- or "),-rayinduced repair, repair of alkylated bases. UV-induced repair. bulky lesions bulky lesions

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DNA repair The variety of DNA damage is mirrored by a variety of DNA repair systems which are able to remove the damage and to restore the normal nucleotide sequence. To date several DNA repair mechanisms have been described in mammals. They are roughly grouped in four categories (Table 1): the first one includes systems characterised by the direct reversal of DNA damage in which a single enzymatic step is able to restore the normal state of DNA. In this group fall the enzymatic photoreactivation of UV-induced pyrimidine dimers, the repair of O6-alkylguanine, ligation of DNA strand breaks and purine insertion. The second category includes: the base excision repair and the nucleotide excision repair. In the base excision repair system the unusual or modified base is removed by the action of a specific DNA glycosylase that catalyzes the hydrolysis of the N-glycosylic bond between the deoxyribose and the base. The subsequent basic steps of this process are incision of the sugar-phosphate backbone of DNA by the action of an AP endonuclease, excision of the nucleotide containing the AP site by the action of an excision exonuclease, DNA repair synthesis by a DNA polymerase that synthesises a new DNA chain a few nucleotides long which is then joined to the adjacent preexisting nucleotides by the action of a

DNA excision repair Nucleotide excisionrepair

Bane excision repair

DNA ligase (Fig. 3). In the nucleotide excision repair system the first step is the cleavage of the sugar-phosphate backbone by a damage-specific incision endonuclease, followed by the excision of the oligonucleotide containing the wrong base and then by DNA repair synthesis and ligation as previously described for base excision repair (Fig. 3) (Lindahl 1982; Friedberg 1985; Kuenzle 1985; Sancar and Sancar 1988). In mammalian cells DNA excision repair has been subdivided into short-patch and long-patch repair depending on the number of nucleotides polymerized by DNA polymerase (2-10 for short patch and 30-100 for long patch). X- and y-rays as well as T-like chemicals (such as alkylating agents) induce damage which requires the activation of short-patch repair. On the contrary, UV and UV-like chemicals induce DNA damage which is subjected to long-patch repair (Edenberg and Hanawalt 1972; Regan and Setlow 1974; Th'ng and Walker 1983). The third category includes DNA repair systems that occur when bulky lesions are left in the DNA template that have escaped the previously described DNA repair systems and therefore stop the DNA replication machinery. The synthesis of one strand is interrupted in front of the lesion and reinitiated at some points in the undamaged region beyond the lesion. The deriving gap cannot be handled by excision repair which requires an intact complementary strand. The gap in front of the damage is repaired by recombination and the damage can subsequently be repaired by many pathways that are altogether referred as postreplicative repair or "daughter strand gap repair". This DNA repair mechanism allows bypassing of some lesions by the replication enzymatic complex (replicon) and can be defined as the process that eliminates "gaps-opposite" lesions in the template strands to form

continuous daughter strands (Kaufmann 1989). DNA glycoaylaa¢

IIncision'endonuclease

I

..

Apurinic/apyrimidinic ~ ~donucl~e

Excision exonucleue

I

DNA

polymer:me ~ DNA ligase

Fig. 3. Mechanisms of DNA excision repair (from S. Linn (1982) modified).

The fourth category includes an inducible DNA repair mechanism called the SOS response. A signal transmitted by the damaged genome is thought to induce the response. The SOS response has largely been studied and elucidated in bacteria but analogous phenomena have been reported in mammalian cells (Herrlich et al. 1984). Apart from the direct reversal of DNA damage, the DNA repair processes require several enzymes probably working in a variety of mutually interacting proteins, coded by a large family of genes, that recognize and remove the lesion from the DNA. Some of these genes have been cloned, e.g. the genes for human uracil DNA glycosylase (Vollberg et al. 1989; Muller and Caradonna 1991), DNA ligase (Barnes et al. 1990) and other genes probably controlling DNA repair pathways (ERCC-1, ERCC-2, ERCC-3, ERCC-5, ERCC-6) (Hoeijmakers and Bootsma 1990) some of which are implicated in the pathophysiology of the DNA repair defect diseases xeroderma pigmentosum and Cockayne

7 syndrome. Recently it has been proposed (Mellon et al. 1986; Bohr et al. 1987; Mullenders et al. 1991) that mammalian cells exhibit heterogeneous levels of DNA repair in different genomic regions and in various types of DNA damage. This means that the whole genome is not homogeneously repaired by DNA repair systems. At least three gerarchic levels of DNA repair rate have been proposed to be present for UV-induced eyclobutane pyrimidine dimers (Mullenders et al. 1991): (1) slow repair in inactive genes; (2) fast ~'epair in active housekeeping genes, and (3) accelerated repair in the transcribed strand of active genes. Thus poorly expressed DNAregion can be repaired slowly and consequently DNA lesions might accumulate in these sequences of the genome. The different ability to perform DNA repair in active or inactive genes could be a key factor in the clinical expression of DNA repair ,deficiency diseases and particularly for neurodegenerative disorders.

DNA repair enzymes in the brain

There are some indications suggesting that postmitotic cells contain lower levels of DNA repair enzymes and repair damaged DNA slower than proliferating cells (Alexander 1967). Evidence of low levels of DNA repair activity in the brain came from the study of Korr and Schultz (1989) who found a reduced in vivo DNA repair activity in various types of cells from mouse brain. For instance the repair of O6-alkylguanine lesions as well as the damage from UV and ionizing radiations is slower in nerve cells than in other cultured cell types (Liebermann and Forbes 1973; Goth and Rejewsky 1974; Margison and Kleihues 1975; Dresler and Liebermann 1983; Gibson-D'Ambrosio et al. 1983; Wheeler and Wierowski 1983; Woodhead et al. 1985; DeSousa et al. 1986). Some studies have shown an age accumulation of single-strand breaks as well as alkali-labile sites in the brain, in the heart and also in slowly dividing cells such as hepatocytes even if other studies did not confirm such results (Mullaart et al. 1990; Bernstein and Bernstein 1991). The decline of the DNA repair capacity of post-mitotic cells seems to be related to the development and achievement of their terminal differentiated state (Alexander 1967; Bernstein and Bernstein 1991). A link between DNA replication and induction of DNA repair activity has been suggested (Sirover 1979; Gupta and Sirover 1980; Gupta and Sirover 1981; Focher et al. 1990). To this purpose it was demonstrated that post-mitotic cells reduce or "switch off' the activity levels of some enzymes and proteins directly or indirectly involved in DNA replication. However it is worthwhile to stress that some of the enzymes required for semiconservative DNA synthesis are also

involved in DNA repair. In rat brain, for instance, enzymes and proteins involved in the replication of nuclear DNA, such as DNA polymerases t~, 8 and E, uracil DNA-glycosylase, thymidine kinase or proliferating cell nuclear antigen decrease at almost undetectable levels after birth (Hiibscher et aL 1978; Spadari et al. 1988; Focher et al. 1990; Verri et al. 1992). Among these enzymes uracil DNA-glycosylase has the main reparative role in the removal of uracil from DNA (either deriving from cytosine deamination or uracil misincorporation) and replicative DNA polymerase a, at least in proliferating cells, plays some roles also in DNA repair (Friedberg 1985). This suggests that adult neurons could lack some DNA repair pathways normally present in other tissues. Not all the enzymes involved in neuronal DNA synthesis are "switched off' at birth, some maintain constant activity levels during the life span of the nerve cell, for example, deoxyuridine-triphosphatase (dUTPase) which is involved in the reduction of the intracellular pool size of deoxyuridine triphosphate (dUTP) (Focher et al. 1990) and DNA polyrnerase/3, which, in adult neurons, is responsibk for the whole nuclear DNA polymerasing activity (l-t(ibscher et al. 1978, 1979; Waser et al. 1979; Subba Ra:~ et al. 1985). Analogous findings regarding several DNA enzymes and in particular DNA polymerase /3 activity have been reported in rat glial cells during development (Verri et al. 1992). Among the mammalian DNA polymerases, DNA polymerase 13 is the more error-prone in the selection of the complementary base and its error frequency does not change with age. In fact DNA polymerase/3 obtained from neurons of young mice behaves like that purified from old mice (Subba Rao et al. 1985). Its low accuracy in DNA reparative synthesis suggests the possibility of an accumulation of errors in DNA during the lifespan of a neuron. Besides DNA polymerase /3 and dUTPase, other enzymes playing a role in DNA repair have been identified in the brain. O6-alkylguanine-DNA alkyl transferase which is responsible for the removal of alkyl groups from guanine, has been found in human (Krokan et al. 1983; Grafstrom et al. 1984; Wiestler et al. 1984) and rat brain (Grafstrom et al. 1984) even though its activity was much lower than in liver, lung, colon and oesophagus (Grafstrom et al. 1984). O6-alkylguanine-DNA alkyl transferase has also been found in human fetal brain cells but tissue-specific differences were in this case less pronounced than in the adult. DNA ligase, the key enzyme in the joining reaction of DNA fragments during DNA replication, is present in neuron and glial cells and its activity is about 11-fold higher in neuron than in glia (lnoue et al. 1979, 1980). Its activity in rat brain is comparable with those found

8 in kidney, lung and liver, but is several times less pronounced than that observed in thymus. Developmental studies have shown that DNA ligase activity remains constant in pooled non cerebellar nervous tissue, while, in cerebellar tissue, it increases postnatally until day 6 and then declines markedly (Nakaya et al. 1977). An exo- and an endonuclease, enzymes involved in the cut (endo-) and in the removal (exo-) of short pieces of DNA, have also been found in the brain (Healy et al. 1963; Ivanov et al. 1983). Although most of the enzymes involved in DNA repair are present in the brain, their low activity levels (probably a consequence of the lack of DNA replication which seems to favour and enhance the DNA repairing rate), could render nerve cells prone to an accumulation of DNA damage during their lifespan. Thus DNA repair mechanisms should have a more subtle influence in the brain, since neurons, once lethally damaged, are irreplaceable. In the following sections we summarize the main findings supporting the concept that unrepaired damaged DNA is responsible for neurodegeneration in certain systemic diseases or may be implicated in the pathogenesis of some primary degenerations of the nervous system.

DNA repair deficiency and neurodegenerative diseases

Xeroderma pigmentosum (XP) XP is an autosomal recessive inherited disease characterized by hypersensitivity of the skin to UV irradiation resulting in more than 1000 fold increased incidence of skin cancer and in an increased frequency of neoplasia in other tissues. Furthermore, a progressive neurological degeneration has been observed in some patients. XP was the first human disorder associated with a defective DNA repair mechanism. This disease is determined by the alteration of the DNA repair pathways involved in the removal of DNA lesions induced by the harmful ultraviolet radiation (Cleaver 1968). XP has been subdivided into seven complementation groups defective in nucleotide excision repair (indicated with the letters from A to G) on the basis of cell fusion studies in which cells from different groups of patients can restore each others' defect. In addition, an eighth form of XP (so-called "variant") has been described in which the molecular mechanism of the DNA repair defect is different being considered a consequence of severe deficiency in post-replicational repair. XP with neurological abnormalities was first noted in 1883 by Neisser but the first detailed clinical and pathological description of the XP neurological disease was due to De Sanctis and Cacchione (1932)who found severe

growth dysfunction, immature sexual development and progressive neurological degeneration beginning at 2 years of age in three affected Italian brothers. Neurological abnormalities have mainly been described in compIementation group A, few in complementation group D and rarely in other groups. The progressive nervous system impairment of these patients ranges from severe forms which start in early childhood, to mild forms in which the earliest signs of the disease (absence of tendon reflexes) appear at the end of the first decade of life. In the most severe cases, belonging to complementation group A, microcephaly, ataxia, ventricular dilatation, sensorineural deafness, cerebellar disturbances, cerebral cortical atrophy, dementia, corticospinal tract abnormalities, spasticity, peripheral neuropathy and choreoathetosis have been observed. Neuropathological examination revealed in these cases diffuse neuronal involvement without signs of inflammation or evidence of regeneration. All reported cases of patients affected by neurological forms of XP indicate that this disease becomes symptomatic before age 21 years. According to the age of symptomatic onset of the disease, neurological XP was classified by Robbins (1989) and Robbins and al. (1991) in (i) early onset juvenile form (< 7 years), (ii) intermediate onset juvenile form (between 7 and 12 years) and (iii) late onset juvenile form (> 12 years). In addition, 2 XP patients belonging to complementation group C, were found to have in their fourth decade only slight neurological signs of the disease suggesting the existence of a presymptomatic form of neurological XP possibly evolving into an adult onset form of the disease (Robbins et al. 1991). XP-C patients have normal repair levels of actively transcribed genes but are unable to perform normal repair of inactive genes (Mullenders et al. 1991). No neurological abnormalities have yet been observed in complementation groups E and F even though in 2 cases the patients were 64 and 50 years old, respectively. Two out of 3 patients belonging to complementation group G have neurological abnormalities (one with an early and the other with an intermediate onset juvenile form of XP). XP-B is represented by an unique patient in which there is an association b~tween XP and Cockayne syndrome (CS) as shown by complementation group studies. Another association with CS has been observed in some XP-D patients (previously consideced as cases of the new complementation group XP-H but subsequently demonstrated to fall into complementation group D; V~rmeulen et al. 1991). These patients showed the neurological abnormalities of Cockayne syndrome that are distinct from those observed in XP. Cell lines derived from XP patients with neurological abnormalities were found to be the most radiosensitive (Andrews et al. 1978). To explain the neurode-

9 generation in XP it has been proposed that transcribed genes in neuronal cells are continuously damaged by UV-mimetic metabolites normally produced inside the cells and that these lesions are not repaired because of the biochemical defect of XP. Thus, when the accumulation of UV-mimetic DNA damage impairs the transcription and consequently the synthesis of proteins necessary to neuronal survival the cells degenerate and die prematurely (Robbins 1989; Robbins et al. 1991). Different-neuronal groups probably possess various degrees of DNA damage (and of DNA repair machinery) and this could account for the selective loss of certain neuronal systems.

Ataxia-telangiectasia (AT) This is an autosomal recessive inherited disease affecting many organs and tissues, particularly the nervous system, immune system and skin. Neuropathological examination reveals degeneration and atrophy of the cerebellar cortex and in older patients a widespread neuronal degeneration, especially in the spinal cord. Laboratory investigations have shown an impaired T-cell-mediated immunity and often low levels of IgA. The two marked symptoms of this disease are cerebellar ataxia and the presence of telangiectasia particularly in the eyes and skin. Many patients suffer from recurrent severe pulmonary infections due to immunological abnormalities. Furthermore, a high incidence of lympho-reticulo-endothelial system tumors has been observed with this disease. Cell lines derived from AT patients displayed many features of genetic instability such as increased spontaneous or X-irradiation-induced chromosomal alterations; increased cellular sensitivity to ionizing radiations and to the radiomimetic chemical bleomycin. Contrary to XP, AT cell lines do not have an increased sensitivity to UV and UV-mimetic substances. Hybridization studies allowed subdivision of AT cell lines into five complementation groups (Jasper et al. 1988) which probably reflect mutations in different genes or gene complexes. An involvement of the topoisomerase II in the pathophysiology of AT has been suggested (Thacker and Debenham 1988). This enzyme controls the superhelical stress of the DNA in chromatin domains and seems to' be required for DNA repair of the genome overall.

Cockayne syndrome Inherited as autosomally recessive and characterized by premature aging, this disorder was described by Cockayne in 1936. CS patients are characterized by arrested growth with onset between 1 and 2 years of age. Their general aspect is that of an old person. The main clinical findings are dwarfism, microcephaly,

deafness, retinal degeneration and spasticity. In addition, other symptoms include sunken eyes, hypertension, osteoporosis, dementia, intracranial calcifications, optic atrophy, primary central and peripheral demyelination and the triad of normal pressure hydrocephalus. Skin fibroblasts from CS patients are more sensitive to killing by UV and this suggests that CS individuals may be defective in the repair of UV-induced damages. Different CS strains were assigned to three complementation groups, named A, B and C (Lehmann 1982). Group C is represented by an unique subject that showed an association with XP and was at the same time the sole representative of XP-B. CS cell strains derived from patients of complementation groups A and B were less able than normal cells to repair pyrimidine dimers from the transcriptionally active housekeeping genes adenosine deaminase and dihydrofolate reductase. On the contrary, XP-C cells are unable to perform a normal repair of inactive genes but have a normal repair rate of actively transcribed genes. CS patients are characterized by neurodegeneration while XP-C subjects rarely suffer from neurological abnormalities (or perhaps they have only a slight neurological invelvement; see above). A wide fraction of the neuronal genome is actively transcribed so that the neurodegeneration characteristic of CS has been considered the consequence of a DNA repair defect of these actively transcribed genes. The differences in the repair mechanisms found in these two diseases probably account for the specific neurological involvement of CS. The lack of efficient repair of transcribed genes has been so far considered a possible key factor in neuronal degeneration (Venema et al. 1990; Mullenders et al. 1991).

Down syndrome (DS) The basis of this disease is trisomy of the distal part of chromosome 21. Clinically the patients have premature aging of the features and neuropathological investigations have shown many findings typically present in old and/or demented brain cells. Some evidence substantiates the idea that in DS there is increased oxidative damage and an increased level of peroxidation of the cellular structure. Other experimental data suggest that the pathophysiology of this disease could be dependent on DNA repair deficiency. As reviewed by Bernstein and Bernstein (1991) chromosome aberrations induced by X-rays in lymphocytes were higher in DS than in normal diploid subjects. Single-strand DNA breaks induced by X-rays in lymphocytes were less efficiently repaired in DS than in normal individuals and the decreased DNA repair capability has been correlated with the increased risk of developing leukemia in this disease.

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Amyotrophic lateral sclerosis (ALS) ALS is a neurodegenerative disease leading to death of motor neurons both at cortical and spino-bulbar levels. In the majority of patients the survival is limited to 2-3 years after the onset of the symptomatology. The etiology remains unknown and the role of several factors, which have beea implicated (such as traumatic, toxic, infective, met~_b:~lic, immunologic, endocrinologic, and genetic), remains largely speculative. In motoneurons of ALS patients nuclear and nucleolar changes are common (Mann and Yates 1974) with a concomitant loss of cytoplasmic RNA (Mann and Yates 1974; Davidson et al. 1981; Davidson and Hartmann 1981a; Murakami 1990) as well as modified base composition of RNA. There is a lower percentage of adenine and a reduced ratio of adenine to uracil in respect to controls (Davidson and Hartmann 1981b). These biochemical and neuropathological data indicate the nucleus as the first site where the degenerative process takes place, even though a recently published study failed to find a generalized impairment of neuronal gene expression in the pathogenesis of ALS (Clark et al. 1990). Bradley and Krasin (1982a,b) have postulated that an accumulation of abnormal DNA, due to defective repair mechanisms, can occur in this disease. Accordingly, the impaired transcription and translation and the consequent decrease of protein synthesis causes the death of motor neurons. Lambert et al. (1986) have observed an increased sensitivity of ALS lymphoblasts to methyl methane sulphonate (MMS). Tandan et al. (1987) have studied the survival and the DNA repair capacity in cultured fibroblasts of ALS and control patients and have observed a significant reduction of mean survival and mean unscheduled DNA synthesis following exposure to MMS. Analogous results were obtained in monocytes (Tandan et al. 1988). On the other hand the DNA repair response to other DNA damaging agents (UV light, mitomycin C, X-rays) was found to be normal (Tandan et al. 1987). This suggests a specific defective repair of AP lesions in ALS. Since AP site formation is a naturally occurring phenomenon due to spontaneous depurination (Friedberg 1985), a defect in DNA repair at this level might be the biochemical basis of neurodegeneration in ALS. According to an alternative DNA damage hypothesis which will be fully discussed below, an accumulation of uracil into neuronal DNA in ALS during DNA repair synthesis can occur as a consequence of an intracellular dUTP/deoxythymidine triphosphate (dTrP) pool imbalance (Mazzarello and Poloni 1988; Mazzarello et al. 1990). Motor neurons have an especially high need for uridine nucleotides (Engel 1991); thus an elevated concentration of such nucleotides in these cells could be expected which may predispose these cells to a dUTP/dTTP pool imbalance. The

presence of this incorrect base in DNA could produce a misunderstanding in the specific DNA/protein recognition as it was demonstrated in vitro for the somatostatin gene promoter (Verri et al. 1990). High levels of uracil in DNA could then lead to abnormal gene transcription with a consequent alteration of intraneuronal protein synthesis.

Alzheimer disease (AD) AD was described in 1910 by Alois Alzheimer and represents the most common neurodegenerative affection. AD is characterized by senile and presenile dementia, diffuse cerebral atrophy and reduced volume and weight of the brain. Extensive investigations have shown reduced levels of mRNA, the presence of translational alterations in neurons from AD patients (Mann and Sinclair 1978; Sajdel-Sulkowska and Marotta 1984, 1985) as well as nuclear and nucleolar morphological changes (Mann et al. 1981). These observations have substantiated the hypothesis that in AD neurons, similar to that seen in ALS, there is an impaired protein synthesis due to a reduced concentration and to a lowered translational activity of RNA (Mann et al. 1981; Sajdel-Sulkowska and Marotta 1984, 1985). Along the same line of evidence are the findings of Guillemette et al. (1986) and Clark et al. (198.9) who showed an altered gene expression in AD. One plausible hypothesis is that in AD there is a neuronal DNA repair deficiency leading to an accumulation of altered DNA with consequent impaired transcription and altered protein synthesis. Cells from AD patients have an increased sensitivity to DNA damaging agents bleomycin, mitomicyn C and 4-nitroquinoline-l-oxide (Hirsch et al. 1982; Li and Kaminskas 1985). AD cells were found to be less able to repair X-ray-induced DNA lesions (Robbins et al. 1985), y-ray-induced DNA lesions (Chen et al. 1991) and DNA damages induced by MMS (Tandan et al. 1988), N-methyl-N'-nitro.N. nitrosoguanidine and ethyl-methanesulfonate (see Bernstein and Bernstein 1991). On the basis of fusion studies using a ,/-induced chromosome aberration assay, late-onset AD patients have been subdivided into four complementation groups (Chen et al. 1991). These findings provide additional support to those obtained from genetic analysis and clinical evaluation indicating that AD represents a heterogeneous disorder. Thus experimental evidence suggests that DNA repair deficiency could play a role in the pathobiology of AD.

Other neurological diseases DNA repair deficiencies were found in cell lines from patients suffering from Parkinson's disease (see Robison and Bradley 1984; Bernstein and Bernstein 1991), Friedreich ataxia (Chamberlain and Lewis 1982)

11 and Huntington disease (Moshell et al. 1980; Scudiero et al. 1981).

DNA damage, DNA repair and neurodegeneration Some of the neuropathological and biochemical features of neuronal loss in many degenerative neurological diseases could be explained by a reduced flow of genetic information required for normal cell functions. The accumulation of abnormal DNA and the increase in the number of transcriptional errors, with the consequent synthesis of altered proteins, would cause the premature death of a functionally related population of neurons that occur in many degenerative diseases of the nervous system. Unrepaired DNA lesions could so far arise from a decreased rate of repair and/or increased damage to DNA molecules. DNA repair alterations have been demonstrated in diseases affecting many organs and tissues including the nervous system (see above). Oxidative DNA stress has been proposed to be a major factor of the aging process (Bernstein and Bernstein 1991) but its role in neurodegeneration remains questionable. The hypothesis that an accumulation of unrepaired DNA arising from decreased levels of DNA repair could produce neurodegeneration has been originally proposed by Robbins and coworkers in many publications (Robbins et al. 1974; Andrews et al. 1976, 1978; Moshell et al. 1980; Scudiero et al. 1981) and is based on a number of observations indicating that some diseases are coupled with DNA repair deficiency. Many of these diseases are characterized by features of neurodegeneration. These are xeroderma pigmentosum, ataxia telangiectasia, Cockayne syndrome and have offered support to the hypothesis that other degenerative diseases of the nervous system including the primary neuronal degeneration may have a DNA repair defect (Robbins et al. 1974, 1983, 1985; Moshell et al. 1980; Scudiero et al. 1981; Bradley and Krasin 1982a,b; Li and Kaminskas 1985). To explain the molecular mechanisms of aging and degenerative diseases another hypothesis was recently proposed (Linnane et al. 1989). This hypothesis, which can be called the "mitochondrial DNA damage hypothesis", was based on the following evidence: (1) the high frequency of mitochondrial DNA (mtDNA) mutations, and (2) the low levels of DNA repair mechanisms for mtDNA. Given the physiological role of mitochondria, mtDNA is strongly subjected to oxidative stress particularly in the brain which is a major site of oxidative metabolism. In fact the amount of oxidatively modified base 8-hydroxy-2'-deoxyguanosine (produced by the attack of oxygen radicals to DNA) in rat mitochondrial DNA was found about to be 16 times higher than in nuclear DNA (Richter et al. 1988). An estima-

tion of the rate of mtDNA mutation, based on studies of restriction fragment length polymorphism of mammalian mtDNA, revealed a number of mutations ten time higher than that observed in nuclear DNA (Brown et al. 1979; Giles et al. 1980). Furthermore, no organized DNA repair system seems to occur in mitochondria, even though the presence of mtDNA associated uracil-DNA glycosylase (Anderson and Friedberg 1980; Domena et al. 1988) suggests that base excision repair can function in these organelles. According to this data mtDNA mutations accumulate during life. Cells with varying degrees of bioenergetic capacity will develop due to the random cytoplasmic segregation of mtDNA, and as a consequence cells which accumulate a large number of mtDNA mutations will be severely impaired while those which accumulate few or no mutations would be less affected or not at all. In this perspective it may be speculated that the rate and the distribution of endogenous and/or exogenous sources of mtDNA damage in the tissues as well as the energy demand of the tissues could account for the different features of degeneration including the selective death of some neuronal populations. In addition it has been proposed that the physiological DNA repair process per se could produce an accumulation of uracil in the DNA which might be involved in the normal process leading to aged brain and/or neurodegeneration (Mazzarello and Poloni 1988; Focher et al. 1990, 1992; Mazzarello et al. 1990). The bases for this hypothesis are: (1) cytosine spontaneously deaminates to uracil in DNA. As suggested by in vitro studies it is plausible to consider that over a period of 30 years between 0.01% and 1% of cytosines in the DNA would be deaminated (Lindahl 1979) or by the action of toxins, oxidative agents and other chemicals (such as nitrous acid and sodium bisulfite); (2) the only DNA polymerase present in adult neurons, DNA polymerase//, cannot discriminate between dTTP and dUTP (Focher et al. 1990) during reparative DNA synthesis. This suggests that the incorporation of dUTP into DNA is solely dependent on its relative intracellular concentration (normally less than 1% of dTTP). To this purpose numerous studies have shown that DNA precursor pool imbalances are mutagenic (Kunz 1988; Meuth 1989). Thus alteration of enzymatic activities that control the pathways of deoxynucleoside triphosphate biosynthesis could produce a d'ITP/dUTP pool imbalance responsible for an increased misincorporation of uracil in the place of thymine in the DNA; (3) at birth, when neurons stop proliferating, uracil-DNA glycosylase, the enzyme responsible for the removal of uracil from DNA, disappears (Focher et al. 1990); (4) uracil in DNA either deriving from misincorporation or from cytosine deamination dramatically alters the specific recognition of DNA sequences by regulatory and/or by DNA binding proteins (Verri et al. 1990).

12 At present only one study has attempted to measure the amount of uracil in a small fragment of D N A extracted from an aged brain and liver obtained at autopsy but the result was negative. However, the sensitivity of the method employed was too low to draw any definitive conclusion (Weiss et al. 1983). In conclusion, D N A repair deficiency produces neurodegeneration in some systemic diseases with neurological involvement. Furthermore a massive amount of evidence ~uggests that alteration in some D N A repair steps could be implicated also in the pathobiology of some degenerative diseases although it still remains to elucidate whether these abnormalities are a cause or an effect. However, in these diseases the D N A repair defect theory could explain: (1) the reduced RNA and protein synthesis observed in some neuronal systems, thus when a gene product essential to cell survival is lost cell death follows; (2) why no immunological nor viral involvement has been found; (3) the multifactorial pathogenesis since D N A and its repair enzymes may be affected by several endogenous or exogenous factors; (4) the rise of incidence of these diseases with age; (5) the presence of both the sporadically occurring and genetically inherited forms. Acknowledgement This work was supported by the PF-CNR Biotecnologie e Biostrumentazione and Chimica Fine If. The invaluable advice and the useful criticism of Prof. P. Pinelli are gratefully acknowledged.

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