Neuroprotective approaches in experimental models of β-Amyloid neurotoxicity: Relevance to Alzheimer's disease

F’mq. Neu~psYchophannacoL

& Bid

Pqjchiat. 1999, Vol. 23, pp. 963-1008 CopyriSht 0 1999 Elsevier Science Inc. Printed in the USA. 0278-5846/99/$-m

ELSEVIER

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front matter

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APPROACHES

a

ExpERQolERTAL

p-AlKYLOID HEUROTOXICITYi RELEVANCE TO -8

MODELS OF DISEASE

TIBOR HARKANY’.2, TIBOR HORTOBAGY?, MARIA SASVk11*2, CSABA KGNYA’, BOTOND PENKE4, PAUL G.M. LUITEN’ AND CSABA NYAKAS’

Central Research Division of Clinical and Experimental Laboratory Medicine, Haynal Imre University of Health Sciences, 2Trace-Element Research Center, B&es Co. Ltd. and %ational Stroke

Center, National Institute of Psychiatry and Neurology, Budapest, Hungary; 4Dept. of Medical Chemistry, Szent-Gy6rgyi Albert Medical University, Szeged, Hungary and ‘Dept. of Animal Physiology, University of Groningen, Haren, The Netherlands.

(Final form, July 1999)

2. 2.1 2.2 3. 4. 4.1 4.2 4.3 4.4 5. 6.

Abstract Introduction: A5 Accumulation and APP Processing in Alzheimer’s Disease Molecular Cascades Involved in AP Neurotoxicity Ca”-Mediated Excitotoxic Cascades: Disturbances of Cellular Ion Homeostasis Free Radical Generation, Lipid Peroxidation and Cellular Energy Depletion Animal Models and Impact of Conformationa.lly Modified AP Derivatives in viva Pharmacological Prevention of A5 Toxicity in vivo Blockade of Intracellular Ca” Entry: A Proposed Role for NMDA Receptors Inhibition of Free Radical Generation: Antioxidant8 and Trace Metals Steroid Hormones P-Sheet Breaker Conformational AP Antagonists Combined Therapeutic Approaches Conclusions Acknowledgements References

Abstract

Harkany, Tibor, Tibor Hortobigyi, Maria Sasv&ri, Csaba Kenya, Botond Penke, Paul G.M. Luiten and Csaba Nyakas: Neuroprotective approaches in experimental Relevance to Alzheimer’s disease. Prog. Neuro-Psychopharmacol.

models of P-amyloid neurotoxicity, & Biol. Psychiat. 1999,2& PP. 96%

1008.01999 Elawier science Inc. I.

P-Amyloid peptides (ADS) accumulate abundantly in the Alzheimer’s disease (AD) brain in areas subserving information acquisition and processing, and memory formation. A5 fragments are produced

963

964

T. Harkany

et al.

m a process of abnormal proteolytic cleavage of thetr precursor, the amyloid precursor protein (APP). While conflicting data exist in the literature on the roles of Ags in the brain, and particularly in AD, recent studies have provided firm experimental evidence for the direct neurotoxic properties of Ag. 2.

Sequence analysis of ABs revealed a high degree of evolutionary conservation and inter-species homology of the Ag amino acid sequence. In contrast, synthetic Ag fragments, even if modified fluorescent or isotope-labeled derivatives, are pharmacological candidates for in vrtro and in viva modeling of their cellular actions. During the past decade, acute injection. prolonged mini-osmotic brain perhtsion approaches or Ag in&ions into the blood circulation were developed in order to investigate the effects of synthetic Ags, whereas transgenic models provided insight into the distinct molecular steps of pathological APP cleavage. The hippocampus, caudate putamen, amygdala and neocortex all formed primary targets of acute neurotoxicity screening, but functional consequences of Ag infusions were primarily demonstrated following either intracerebroventricular or basal forebrain (medial septum or mugnocellular basal nucleus (MBN)) infusions of AB fragments. In vivo investigations confirmed that, while the active core of Ag is located within the g(25-35) sequence, the flanking peptide regions influence not only the folding properties of the A@ fragments, but also their in viva neurotoxic potentials. It has recently been established that Ag administration deranges neuron-glia signaling, affects the glial glutamate uptake and thereby induces noxious glutamatergic stimulation of nerve cells. In fact, a critical role for N-methyl-D-aspartate (NMDA) receptors was postulated in the neurotoxic processes. Additionally, A@ might become internalized, either after their selective binding to cell-surface receptors or atIer membrane association in consequence of their highly lipophilic nature, and induce free radical generation and subsequent oxidative injury. Ca”-mediated neurotoxic events and generation of oxygen free radicals may indeed potentiate each other, or even converge to the same neurotoxic events, leading to cell death. Neuroprotection against AJ3 toxicity was achieved by both pre- and post-treatment with NMDA receptor channel antagonists. Moreover, direct radical-scavengers, such as vitamin E or vitamin C, attenuated Ag toxicity with high efficacy. Interestingly, combined drug treatments did not necessarily result in additive enhanced neuroprotection. Similarly to the blockade of NMDA receptors, the neurotoxic action of ABs could be markedly decreased by pharmacological manipulation of voltage-dependent Ca2+-channels, serotonergic IA or adenosine Al receptors, and by drugs eliciting membrane hyperpolarization or indirect blockade of Ca2’-mediated intracellular consequences of intracerebral Ag infusions. Ag neurotoxicity might be dose-dependently modulated by trace metals. In spite of the fbct that zinc (Zn) may act as a potent inhibitor of the NMDA receptor channel, high Zn doses accelerate Ag fibril formation, stabilize the g-sheet conformation and thereby potentiate Ag neurotoxicity. Combined trxe element supplementation with Se, Mn, or Mg, which prevails over the expression of detoxifying enzymes or counteracts intracellular elevations of Ca2+,may reduce the neurotoxic impact of A@ Alterations in the regulatory functions of the hypothalamo-pituitary-adrenal axis may contribute significantly to neurodegenerative changes in the brain. Furthermore, AD patients exhibit substantially increased circadian levels of steroid hormones, as well as baseline cortisol concentrations. In fact, a dose-dependent regulatory action of corticosterone on Ag or NMDA excitotoxicity has recently been demonstrated on MBN neurons, yielding a reversed bell-shaped dose-response profile. Furthermore, characteristic neuroprotective properties were postulated for estrogen both in vitro and in viva.

Neuroprotection against Ap toxicity in vivo

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A novel approach in which ‘psheet breaker’ peptide analogs are applied for the elimination of AS fibriUogenesis/aggregation, or for the prevention of the direct binding of Ags to possible selective cellsurihce recognition sites ($3 receptors) provides promising in viva tools for the prevention of AS toxicity.

Kevwords: S-amyloid, calcium, N-methyl-D-aspartate receptor, neuroprotection, oxidative stress Abbreviations: acetylcholinesterase (AChE, EC 3.1.1.7), Alzheimer’s disease (AD), g-amyloid peptide (A/3), amyloid precursor protein (APP), apolipoprotein (APO E), blood-brain barrier (BBB), ciliary neurotrophic factor (CNTF), cyclic adenosine monophosphate (CAMP), cholineacetyl-transfrase (ChAT, EC 3.2.1.6) dizocilpine maleate (MK-801) glial fibrillary acidic protein (GFAP), hypothalamo-pituitary-adrenal axis (HPA), intracellular t%ee Ca” concentration ([Ca”],), intracerebroventricular (i.c.v.), medial septum (MS), magnocellular basal nucleus (MBN), metabotropic glutamate receptor (mGluR), nerve growth factor (NGF), nitric oxide synthase (NOS), N-methyl-D-aspartate (NMDA), nuclear transcription factor rcB (NF-rcB), reactive oxygen species (ROS), receptor for ‘advanced glycation end-products’ (RAGE), scavenger receptor (SR), serotonin (5-HT), substance P (SP), superoxide dismutase (SOD), trace metal/element (TM), tau protein (r), voltage-dependent Ca*+channel (VDCC).

1.

The amyloid peptides comprise a heterogeneous group of proteins sharing a high propensity to form

insoluble aggregates in the body, which have a discrete tertiary peptide structure withstanding proteolytic degradation. Amyloid peptides or mutant amyloid variants (Castano and Frangione, Castano et al., 1996) from different

1995;

sources (e.g. liver or chorioid plexus) are the underlying causes of

numerous familial or sporadic systemic amyloidoses (Garzuly et al., 1996; Vidal et al., 1996) associated with cognitive deficits or memory disturbances (Garzuly et al., 1996). In fact, g-amyloid (AP) of neuronal or glial origin accumulates in Alzheimer’s disease (AD), and similarly in Down’s syndrome; the latter being characterized by a trisomy on chromosome 21 affecting the expression of the AP precursor protein (APP; Korenberg et al., 1989), the precursor of A$. APP, which resembles a GTP-binding protein G,-coupled cell-surface receptor (Kang et al., 1987; Nishimoto et al., 1993) occurs predominantly as a 695 amino acid-long transmembrane protein in the brain, while minor APP isoforms include 717, 751 or 771 amino acid residue proteins (Checler, 1995; Selkoe, 1991). As an interesting facet in the enigmatic development of AD, the AB sequence is located partly in the extracellular and partly in the transmembrane domains of APP (Selkoe, 1991). APP processing and cleavage at the membrane surface under physiological conditions precludes the generation of intact AS, as the cleavage site producing naturally occurring secreted APP isoforms (sAPP) is located within the PA sequence at residues L17Nl8

(Sisodia et al., 1990; Fig 1). On the other hand, the secretory

T. Harkany et al.

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APP processing is apparently disturbed in AD and shifts towards a pathological processing pathway with the abundant generation of A@ utilizing as yet unidentified secretory enzymes, termed ysecretases (Checler, 1995; Haass and Selkoe, 1993). Whereas Haass and his colleagues demonstrated that Al3 is produced during the normal cell metabolism (Haass et al, 1992, 1993), pathological APP processing, or deficiencies of AD degradation and clearance may lead to an enhanced production of Al3 in the AD brain. Clinical, neuropathological and experimental studies led to the conclusion that repeated, long-lasting inflammatory mechanisms in the brain may be of critical importance in the development of AD (McGeer et al., 1994; McGeer and McGeer, 1998; Rogers, 1995). Clinical trials demonstrated that long-term administration of anti-inflammatory

drugs, such as corticosteroids, significantly decrease

the prevalence of AD in selected populations (Paganini-Hill and Henderson, 1996). Moreover, it is known for decades that intrinsic immunoresponsive cells of the brain, such as astrocytes or microglia, become highly activated in response to head trauma, or to bacterial or viral infections (McGeer and McGeer, 1998; Rogers, 1995). Interestingly, it has recently been determined that microglial cells exhibit highly inducible APP expression profiles as a consequence of immugenic transformation, and release significantly elevated quantities of intact Aj3 (Du Yan et al., 1997; Grifftn 1998). Indeed, inflammation-induced

accumulation of intact AI3 in the brain might lead to the development of AD.

AI3 exists in two mature isoforms in the AD brain. Whereas IeptomeningeaVcerebrovascular

AI3

consists of 39 amino acid residues, senile plaque-forming AI3 comprises primarily 42(43) (Ag(l-42)) or 40 amino acids (A13(1-40)); Glenner and Wong, 1984a; Masters et al., 1985) and these peptides have been demonstrated to be homologous to those found in Down’s syndrome (Glenner and Wong, 1984b; Masters et al., 1985). As a major pathological hallmark of AD, AP forms profuse insoluble aggregates, termed senile plaques, in the brain. Plaque formation is a long development, which begins as amorphous, largely non-filamentous

aggregates of Ag (Glenner and Wong, 1984). Following

maturation and Al3 condensation such ‘diffuse’ plaques become increasingly fibrillar and acquire the classical features of senile plaques, As the latest phase of plaque development, highly dense A/3 deposits become associated with dystrophic neurites and are rimmed by reactive microglial cells and astrocytes, termed ‘neuritic’ plaques. High densities of AP-containing plaques can predominantly be observed in association areas of the neocortex and in the entorhinal cortex - hippocampus complex (Braak and Braak, 1997), while basal forebrain cholinergic nuclei become afflicted in later phases of the disease process with lower plaque densities.

Neurcprotection against Ap toxicity in vivo

967

Ag deposits appear not only in the brain of aged humans but also in that of numerous aged mammalian species, such as primates, dog, bovine, sheep, rabbit, guinea pig and polar bear (Johnstone et al., 1991; Fig 1). In fact, among these species APP and particularly the Afl sequence exhibit > 95% and 100% sequence homology, respectively (Fig 1). Distinctively, both rat and mouse (De Strooper et al., 1991; Yamada et al., 1987) Ag sequences contain amino acid substitutions at positions 5, 10 and 13 in the extracellular flanking region of the protein, which may alter its folding structure and thereby account for the fact that these species do not develop Ag deposits during aging (Fig 1). The high evolutionary conservation of the peptide sequence is indicated by the Ag amino acid chain found in the African clawed frog, bearing five amino acid substitutions (at positions 2, 4, 8, 9 and 23; Okado and Okamoto, 1992). Hence, none of these alterations occur within the presumptive active center of AD, the 25-35 peptide region. These genetic differences might become of substantial importance during in vivo neurotoxicity testing, as they might influence the subjects’ sensitivity (e.g. rodents vs. primates) to Ag toxicity based on cellular protein recognition. Indeed, Geula et al. (1998) have recently demonstrated that AP exerts dramatic neurotoxicity in the primate, but not in the rodent brain. Cognitive and learning deficits in AD are predominantly attributed to the extensive (70-90%) loss of cholinergic basal forebrain neurons located in the medial septum, the diagonal bands of Broca or in the magnocellular basal nucleus (BMN), the latter being termed the basal nucleus of Meynert in humans (Bartus et al., 1982). Cholinergic neurons of the basal forebrain innervate the hippocampus, amygdala, olfactory bulb and the entire neocortex. Lesions of cholinergic basal forebrain nuclei result in the loss of cholinergic subcortical innervation in their target areas. From a mnctional viewpoint, loss of the subcortical chohnergic input to these structures, and thereby that of the tonic cholinergic regulation direct hamper learning mechanisms, memory formation and storage (Schliebs et al., 1996). During the past decade, numerous experimental and histopathological studies were undertaken to assess the role of accumulating Ags in AD. Whereas a long debate existed on the f.mctions of Ag in the brain, in parallel with the improvement of in vivo model systems, pivotal experimental data indicate the direct neurotoxic properties of these peptides. The major goal of the present review is to summarize experimental data on (I) the proposed central role of Ag in AD-related excitotoxic cascades leading directly to cell death, (2) currently available in vivo model systems, and (3) pharmacological intervention targeted at ameliorating AD neurotoxicity, with special emphasis on possible therapeutic strategies derived from experimental data. (4) Additionally, it is shown how synthetic modjiied AD fragments may contribute to an extension of our understanding neurotoxic cascades/altered intracellular signaling mediating the cellular actions of AD

of the

T. Harkany et al.

968

Extracellular region

Transmembrane region

Human

HzKl-

Polar bear

VGGVVIA-----

Clawed frog

Fig 1 Summary of known Ap sequences of different mammalian species and African clawed frog. The Ag sequence is located partly in the extracellular and partly in the transmembrane domains of APP. The vertical bar indicates the cell surface and physiological cleavage site of the protein precursor. Whereas the human, dog, bovine, sheep, rabbit, pig, guinea pig and polar bear ADS exhibit 100% amino acid homology, single amino acid substitutions (tilled boxes) are present in the rat, mouse, and clawed frog Ag sequences.

2. Molecular Cascades Involved in Al3 Neurotoxicity

Whereas the (patho)physiological role of AD in the brain and in particular in the etiology of AD remains to be elucidated, our knowledge on the fundamental cellular action of Ag, and its fragments,

and their effects

on neuron-neuron

or neuron&a

signaling,

has broadened

substantially during the past decade. In vitro investigations provided firm pharmacological data on the dose- (Mattson et al., 1992; Whiston et al., 1989; Yankner et al., 1990) and conformationdependent (Pike et al., 1993) neurotoxic potentials of Aj3s. In this respect, Yankner et al. (1990) reported that A&s act as potent neurotoxins

above a threshold concentration.

Interestingly,

similarly to the proposed role of APP during synaptogenesis (Mattson, 1994; Morimoto et al., 1998; Moya et al., 1994), picomolar concentrations of Ag enhance neurite outgrowth or nerve cell survival in vitro (Whiston et al., 1989; Yankner et al., 1990). Moreover, besides its direct neurotoxicity, a role as a potent cholinergic neuromodulator has been suggested for Ag (Auld et al., 1998). The proposed capacity of Aj3 as an inhibitor of various cholinergic neurotransmitter functions without apparent neurotoxicity might provide plausible explanation for the vulnerability of selected cholinergic cell groups in AD (Auld et al., 1998).

Neuroprotection against Ap toxicity in viva

969

Recently, two major molecular cascades became evident which may mediate the cellular neurotoxic action of APs; (1) a Cd’-mediated pathway

involving the activation of Ca”-permeable

cell surface receptors, and the activation of nitric oxide synthases (NOS) and proteolytic enzymes, leading to an altered gene expression and apoptotic cell death; and (2) oxidative processes with ‘advanced glycation end-product’ receptor (RAGE) activation, enhanced free radical generation and an oxidant-sensitive,

nuclear transcription factor ICB (NF-xB)-dependent

pathway. In fact,

various studies indicate that these pathways may converge to the same nuclear targets and induce cell death in a concerted action (Dutrait et al., 1995; Mattson et al., 1995).

2.1 Ca2’-Mediated Excitotoxic Cascades: Disturbances of Cellular Ion Homeostasis

Intracellular free Ca” exerts bi-directional functions in the central nervous system. Ca2’ plays cardinal roles in physiological neural functioning, e.g. a Ca” mflux is a pivotal constituent of membrane potential generation,

a Ca”

elevation in synaptic terminals

neurotransmitter release, Ca*’ fluxes via N-methyl-D-aspartate

facilitates vesicular

(NMDA) receptors are involved in

long-term potentiation or long-term depression (Bliss and Collingridge, 1993) and Ca2’ may act as an intracellular messenger, activates second messenger signaling or contributes to altered protein phosphorylation (Kostyuk and Verkhratsky, 1995). On the other hand, extensive elevation or dysregulated oscillations of the intracellular free C$’ concentration ([Ca*‘]i) may lead directly to cellular dysfimctioning, neuronal overexcitation and cell death (Luiten et al., 1996). Recent in vitro and in vivo data from several laboratories (Gray and Patel, 1995; Harkany et al., 1997; Hartmann et al., 1994; Ishikawa et al., 1998; Kimura and Schubert, 1993; Koh et al., 1990; Laskay et al., 1997; Mattson, 1994; Mattson et al., 1992, 1993; Mogensen et al., 1998; O’Mahony et al., 1998; Stix and Reiser, 1998) provided compelling neuroanatomical and neuropharmacological

evidence

that sustained elevations of [Ca*‘]i and the appearance of Ca2’-triggered neurotoxic cascades might be of pivotal importance in mediating the cellular neurotoxic action of Afls. In this regard, loading both of cultured neurons (Mattson et al., 1992) or of astroglia (Laskay et al., 1997) with the intracellular fluorescent Ca*’ indicator, fura 2AM demonstrated that even a brief stimulation with Ag elicits extensive membrane depolarization, and induces rapid but persistent increases in [Ca”]i, shifting the cell cycle towards neurodegeneration (Laskay et al., 1997; Mattson et al., 1992, Stix and Reiser, 1998). However, a particular binding site for Ag remains to be identified. To resolve the phenomenon of the selective interaction of Ag with cell-surface molecules, Arispe et al. (1993, 1996) proposed

T. Harkany et al.

970

cation (Ca”)-selective

channel-forming properties of Ag fragments; this characteristic feature of

Ag(25-35) or Ag(l-40) has recently been confirmed by others (Kawahara et al., 1997; Mirzabekov et al., 1994). On the basis of the extensive pharmacological data from predominantly

in vivo

experiments indicating the involvement of common excitotoxic pathways in Ag-induced brain injury (Morimoto et al., 1998a; O’Mahony, 1998) and pathways similar to that of NMDA neurotoxicity (Harkany et al., 1997, 1998a), it seems likely that Al3 binds to selective cell-surface recognition sites mediating intracellular Ca*+ entry. In this respect, Sties and Penke (personal communication) found selective, high-affinity binding of 1[~H]Ag(25-35) to membrane fractions of

the rat hippocampus, while Lambert et al. (1998) reported that non-fibrillar oligomer derivatives of Ag bind to trypsin-sensitive

cell-surface structures with high efficacy. Interestingly, radioligand

binding studies support the selective direct interaction of Al3 with the glutamate and glycine recognition sites of the NMDA receptor (Cowbum et al., 1994, 1997) whereby the [3H]dizocilpine maleate ([‘HIMK-801)

[‘HIglutamate or [3H]glycine binding is significantly displaced in the

presence of pM concentrations of Ab(25-35). A third hypothetical binding site exists for Ag, termed the receptor for advanced glycation end-products (RAGE, Yan et al., 1996) which is believed to act as a signal transducer molecule for an ‘oxidative stress’ pathway (for details, see Chapter 2.2). In summary, independently

of the particular (direct or indirect) cell-surface

recognition molecule for Al3 or its derivatives, all these pathways converge to a pathological enhancement of intracellular Ca2+ entry via Ca’+-permeable channels and subsequent elevation of [Ca”]i (Fig 2A). AP(25-35) or AP(140)-induced

elevation of [Ca’+]i, in turn, enhances Ca”

release from intracellular Ca2+ stores, such as the endoplasmatic reticulum, via Ca” mobilization by inositol-1,4,5_triphosphate signaling (Cowbum et al., 1995). The above data indicate (Fig 2A) that the altered APP processing in the microglia may result in the enhanced production and subsequent extracellular release of intact A(3 in response to noxious stimuli, e.g. head trauma, inflammation, metabolic insults or cerebrovascular abnormalities. An increased extracellular Ag concentration

compromises

both nerve cells and astrocytes in close proximity to activated

microglia (Fig 2A). The AP may then interact with one or more of the postulated recognition sites, thereby inducing membrane depolarization (Harkany et al., 1997; Laskay et al., 1997). Profound changes in glial membrane conductances

are known to impede the glial glutamate uptake

significantly by a marked blockade of Na’-dependent glial glutamate transporters (Harris et al., 1996, Keller et al, 1997). Indeed, a net decrease of 42% in the astroglial glutamate uptake was recorded after continuous exposure to Ag(25-35) in vitro (Toth and Madarasz, unpublished data).

Neuroprotection against AP toxicity in viva

icroglia (activated)

Inflammation, complement activation, noxious stimuli

icroglia (activated)

Increased proliferation, increased expression ofApo E

Fig 2 Proposed molecular cascades involved in mediation of the neurotoxic actions of Ag fragments. Whereas Fig 2A shows particular steps of a Ca”-mediated neurotoxic cascade, Fig 2B depicts a neurotoxic pathway involving free radical generation and activation of NF-KB. Both pathways involve distinct neurotoxic mechanisms in neurons and astrocytesImicroglia1 cells. While these events involve substantially different factors as effecters of AB toxicity, enhanced Ag production is dependent on microglial activation (see the detailed description in Chapters 2.1 and 2.2) and these cascades ultimately lead to cell death. Symbols and abbreviations: 0 = APP, Apo E = apolipoprotein E, I = Ap, Ghr = glutamate, 1 = glutamate transporter, Hz02 = hydrogen peroxide, IL-lg, IL-6 = interleukins 1B and 6, = membrane depolarization, @ = mGluR, MCSF = macrophage-colony stimulating factor, 0 = NMDA receptors, NF-KB = nuclear transcription factor-xB, NO = nitric oxide, NOS = nitric oxide synthase, @ = RAGE, TNFcx = tumor necrosis factor a, = VDCCS.

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Harkany et al.

Pathological elevations in the extracellular glutamate concentration elicit excitotoxic neuronal death (Fig 2A) by (I) activating NMDA receptors which are known to play critical roles in the initiation

and/or propagation of action potentials. (2) Subsequently,

voltage-dependent

Ca2’

channels (VDCC) become permeable to cations, with a significant enhancement of intracellular Ca” entry. Moreover, (3) extracellular glutamate also binds to both synaptic and extrasynaptic metabotropic glutamate receptors (mGluR), which may act in part as triggers of synaptic excitatory neurotransmitter

release or as extrasynaptic mediators of Ca2+ entry. Consequently,

continuous excitatory stimulation of nerve cells or terminals results in a pathological Ca2+ overload afflicting protein hyperphosphorylation,

such as that of the cytoskeletal protein tau (t; Shea et al.,

1997) activation of proteolytic enzymes and DNA breakdown, leading to (apoptotic) neuronal loss. Abnormal phosphorylation of cytoskeletal proteins, such as T, and thus pathological changes in cytoarchitecture elicits the degeneration of nerve cells. AD-induced intracellular accumulation of polyphosphorylated r may therefore be a critical trigger of neuronal death in AD.

2.2 Free Radical Generation. Lipid Peroxidation and Cellular Enerav Denletion

Unique metabolic processes occur in the brain, e.g. high-level glucose utilization and glucosedriven metabolic rate, low activity levels of radical scavenger enzymes, and the accumulation of enzymatically active trace metals (TMs) which may potentially catalyze free radical generation (Stadtman and Oliver, 1991) and contribute to the activation of antioxidant defense mechanisms. Accordingly, the brain is particularly accumulation

vulnerable

to metabolic disturbances

and subsequent

of highly reactive organic radicals, in particular of oxygen species, inducing

oxidative stress (Gil&rest and Bohr, 1997; Halliwell, 1987). Indeed, brain aging is accompanied by the enhancement

of mechanisms underlying

the chronic oxidative stress of nerve cells.

Characteristic features of these processes involve enhanced DNA breakdown and decrements of repair mechanisms (Gilchrest and Bohr, 1997; Mecocci et al., 1994) a decline in the expression and activity of antioxidant defense enzymes, such as superoxide dismutase (SOD) and catalase (Ames et al., 1993). Consequently,

mitochondrial

enzymatic uncoupling

occurs followed by

leakage of free radicals into the cytosol, enhanced lipid peroxidation and the accumulation of advanced glycation endproducts (AGES; Smith et al., 1995). Since aging is a major factor predisposing to AD, and an accelerated and clearly enhanced profile of oxidative cytotoxic mechanisms is evident in AD (Frolich and Riederer, 1995; Furuta et al., 1995) an oxidative stress

Neuroprotection hypothesis

against A8 toxicity in viva

973

was proposed to describe the molecular events leading to selective neuronal loss in AD

(Behl, 1997). As one of the determining

neuropathological

plaques has been directly

related

hallmarks of AD, the accumulation

to a potent production

However the critical step of how AB fragments mechanisms

is still to be ascertained.

experimental

evidence

indicates

et al., 1992, Harkany

concentration

exhibited

that AB cytotoxicity

compelling,

might effectively

such as exogenous

et al., 1998a). Moreover,

a close correlation

(Behl et al., 1994b). Nevertheless,

stress in the AD brain directly via oxidative

induce cellular disintegration

In this respect,

superoxide or hydroxyl radical scavengers, (Behl

of oxidative

they do not provide firm support for the hypothesis

though

mainly

be antagonized

indirect by either

SOD (Suo et al., 1997) or vitamin E elevations

with the neurotoxic

the aforementioned

of AB in senile

in the intracellular

properties

of AD or its fragments

data may be of secondary that AP generation

Hz02

importance

ultimately

since

and primarily

leads to an enhanced formation of free radicals. Identification

of a receptor termed as receptor for AGES (RAGE; Yan et al., 1996) that can act as

a mediator of the cellular action of Afls on neurons or endothelial Binding

of AP or probably

subsequent

of AD-related

oxidative stress. Furthermore,

peptides

of cell-surface

binding,

al., 1998a). It may be suggested recognition

membranes

that internalized

enhances the production Moreover,

of intracellular

regulate

Additionally,

the expression

glial/immune

surface

such as those involved in

transcription

The severity and complexity

of the nuclear

molecules

neurons

and translation

of these processes

of AP to a free radical state is considered

(Hensley et

of the source of HzOz, free radicals transcription

the expression and cytokines.

of

of the peptide and AB itself can act as a

factor-r&% (NF-r&l)

of several structural In this regard,

genes,

may effectively

in both

cells (Akama et al., 1998; Barger, 1995; O’Neill and Kaltschmidt,

recognition

(Mackic et

to the model reported by Hensley et al. (1994) distortions

independently

1997); in turn, this influences

binding

1993). Damage to mitochondrial

with fine-tuned

the electronic structure of Ag may result in ‘radicalization’ free radical.

intracellularly

of reactive oxygen species (ROS) and compromises

become multifold if the suggested transition

and

(SR; Mackic et al., 1998a). As a

organelles,

(Pike and Cotman,

ROS may interfere

1995). According

activation

Afl, through intimate contacts with intracellular

with a net decrease in protein synthesis.

al., 1994; Mattson,

receptors

intact [“%]AB( l-40) accumulates

chain of the mitochondria

by energy depletion. processes

scavenger

sites, directly damage membranes

the respiratory

to RAGE results in receptor

AB( l-40) has been shown to exert high-affinity

not only to RAGE, but also to macrophage consequence

cells fulfills the above criteria

nerve

and

1997; Yan et al..

including

Yan et al. (1997)

NOS, cellproposed

a

T. Harkany et al.

974

molecular pathway in which the AB-induced activation of RAGE enhances the NF-a-triggered production of macrophage-colony stimulating factor (M-CSF; Fig ZB).

--_-i--.---r RAGE, SR I

Sustained interaction with the NMDA receptor channel

Direct membrane damage/ Intemalization

Lipid peroiidation NF-KB activation

Sustained CaZ+ overload e

Aotease NOS activation

+ Oxidative damage

activatiorf Mitochondrial

)

energy depletion J

DNA fragmentation,

Apoptotic gene activation

Fig 3 Possible interactions of distinct molecular cascades, in particular free radical-triggered neurodegeneration, mediated by RAGE and SR receptors, or direct membrane damage, and Ca”mediated neurotoxicity. Whereas distinct signal transduction pathways mediate the cellular effects of Ag, these cascades converge to the same intracellular effector molecules/events conveying to cell death. Note that sustained elevation of [Cazf]i and excess generation of free radials are closely related processes and directly regulate each other. Arrows indicate successive steps of the Aginduced cellular cascade ultimately leading DNA fragmentation and neurodegeneration. Abbreviations: NF-KB = nuclear transcription factor-r&, NOS = nitric oxide synthase, RAGE = receptor for advanced glycation endproducts, SR = scavenger receptor.

Neuroprotection against AP toxicity in vivo

975

M-CSF interacts with its receptor, activates microglia and thereby initiates an inflammatory cascade. Moreover, recent reports indicate that NOS expression and NO production also occur through an NF-i&dependent reactive peroxynitrite

mechanism (Akama et al., 1998). Since NO forms the highly

radical with superoxide. enhanced NO formation may enhance ROS-

mediated Al3 toxicity. The relevance of NF-xB activation to AD is supported by the fact that activated NF-xB was visualized in neurons and glia situated in close vicinity to early plaques (Kaltschmidt et al.. 1997). It is interesting to note that mutation-induced increases in baseline NFKB expression mediate resistance to oxidative stress in vitro (Lezoualc’h

et al., 1998) a

phenomenon which might indicate a bi-directional regulatory role for NF-rcE3 in intracellular signaling. It is likely, however, that an intracellular Ca” entry-coupled radical-generating

cascade also

exists (Fig 3). This hypothesis is derived from morphological data depicting that exposure of cultured neurons to Al3 triggers a rapid disintegration of plasma membranes and membranous structures. indicated by fragmented microtubuli and damage to inner mitochondrial membranes (Behl et al., 1994a). An extensive enhancement of lipid peroxidation supports the membranedamaging properties of Al3 (Keller and Mattson, 1998). Furthermore, ligand-binding data indicate that Al3 selectively interacts with the NMDA receptor channel (Cowburn et al.. 1997). The Aginduced activation of NMDA receptors and subsequent Ca” entry result in a conformationdependent 3- to 6-fold increase in Ca”/calmodulin-activated

NOS activity (O’Mahony et al.,

1998) providing a possible coupling of Ca2’- and ROS-mediated neurotoxic pathways

3. Animal Models and Imoact of Conformationallv Modified AB Derivatives in vivo

Neurotoxic properties of the full-length Al3 sequence (AP( I-42)) truncated SA fragments and AD homologs were initially demonstrated on cultured neural cells (Yankner et al. 1989, 1990) and a dose-response

relationship

was identified

(Yankner et al., 1990). Discovery of the direct

neurotoxic potential of Al3 boosted follow-up experiments to confirm and extend these data. In vitro studies in a multitude of laboratories profoundly improved our understanding of (I) selected

signal transduction mechanisms (e.g. receptor or recognition site-mediated cell-surface action of AP, internalization

of AB(l-40), accumulation of [Ca”]i, enhanced production of ROS), (2)

possible secondary pathways towards apoptosis/necrosis, and (3) pharmacological interactions of drugs enhancing or diminishing AD neurotoxicity (e.g. Al3 aggregation enhancers or blockers, receptor agonists/antagonists

or channel blockers; for reference, see Chapters 4.1 and 4.4).

T. Harkany et al.

976

However, in vitro models on selected cell lines of neuronal or glial origin have firm limitations, since they cannot provide data on (I) the interactions of nerve ceils with glia, (2) the influence of the extracellular matrix on cell-surface interactions and on Ag diffusion, (3) the modulatory influence or interactions of excitatory and inhibitory neurotransmitter systems on target cells, (4) the circadian rhythm of hormones or (5) the age-dependent expressions of receptors or cytokines, which differ extensively from those found in the embryonic brain. To resolve these diffrcuhies, in vivo

models

were

introduced

which

allow thorough

behavioral

testing,

indicating

the

phenotypically appearing outcome of neurotoxicity screening. Initially, animal models designed to determine the hypothetical toxicity of AS or its derivatives focussed on the extent of AJ3-induced neuronal loss or memory disturbances in rats (Frautschy et al., 1991; Kowall et al., 1991) or mice (Flood et al., 1991). From this point on, Ag toxicity research became bifocal: (I) a vast majority of in vivo experiments aimed at identifying the precise neuroanatomical

neurotransmitter

substrates

of A(3 toxicity,

systems involved,

conformation-dependent

the time-course

and the morphometric

of neuronal

damage,

the

properties (state of aggregation,

deposit formation, Giovannelli et al., 1998, Shin et al., 1997) of Ag

deposits (Tables 1 and 2) or (2) the functional

consequences

of A@induced brain injury and

possible neuroprotection against AB excitotoxicity (Table 3). Whereas a long debate existed on AP toxicity in the rat brain (Clemens and Stephenson, 1992; Emre et al., 1992; Games et al., 1992; Stein-Behrens et al., 1992, Winkler et al., 1994) previous neuroanatomical

and neurochemicaf

studies in our laboratory revealed that AS( l-42) and AS(25-35) induce a significant decline in the activity of cholinergic MBN neurons as indicated by marked decreases in AChE and ChAT activities and in the B,

of Mz muscarinic acetylcholine receptors in the somatosensory cortex

(Harkany et al., 1995b). The existence of degenerating projection fibers in cortical sections became evident following silver impregnation of axonal debris (Harkany et al., 1995b). These findings accord with those of Giovannelli et al. (1995) who reported persistent behavioral deficits and an extensive loss of ChAT-positive chohnergic projection neurons following AS infusion into the MBN. AS lesions in the rat medial septum (MS) provided in vivo evidence for Ca”-mediated excitotoxic

events,

as

parvalbumin-containing

y-aminobutyric

acid

(GABA)ergic

septal

interneurons exhibited resistance to AS(l-42) toxicity (Harkany et al., 1995a). The survival of septal GABAergic interneurons confirmed the previous findings of Pike and Cotman (1993, 1995) who demonstrated intact GABAergic, Ca2’-binding protein (calretinin, parvalbumin)-containing interneurons selectively withstanding Ag application in vitro. These data are also supportive of earlier observations indicating excite-protective roles for Ca” -binding proteins such as calbindinD28k (Mattson et al., 1991).

Neuroprotection against AP toxicity in vivo

Long-lasting

behavioral

consequences

of Ag(Phe24(S0sH))25-35

977

infusion afford behavioral

evidence for the selective cholinotoxicity of Ag in the MBN (Harkany et al., 1998b). While the cholinergic activity significantly decreased and resulted in pronounced cognitive deficits, sensory information

misprocessing,

or overt mobility

under

dark-phase

conditions,

the unaltered

GABAergic functions maintained the normal circadian rhythms of the animals (Harkany et al., 1998b). Ag(1-42)

Ag(25-35)

and Ag(Phe24(S0sH))2S-35-induced

behavioral

dysfunctions

(Harkany et al., 1998a,b) are in concert with those found by others in rats (Delobette et al., 1997; Giovannelli et al., 1995; Nitta et al., 1994) or in mice (Flood et al., 1991, 1994a), suggesting that Ap may contribute directly to the progression of memory disturbances. Underlying mechanisms for in viva A@toxicity may involve both Ca*’ and ROS-mediated mechanisms. However a primary role for a pathological Ca*’ overload has recently been postulated (Abraham et al., 1998; Harkany et al., 1997, 1998a; O’Mahony et al., 1998). In this respect, the activation of NMDA receptors and NMDAhreceptor

activation-coupled

Ca*‘/calmodulin-dependent

neuronal

NOS

has

been

demonstrated (O’Mahony et al., 1998). Pharmacological data (Harkany et al., 1998a; Morimoto et al., 1998a; O’Mahony et al., 1998) point to a primary role of Ca”-mediated neurotoxic events in the course of in vivo Ag toxicity, which might be connected directly to ROS-generating pathways and NP-KB activation by the enhanced production of NO and peroxynitrite (see also Chapter 2.2). It is worthy of attention dat

Ag fragments exert conformation-dependent

demonstrated by (I) transient toxicity profiles for Ag(25-35), while Ap(l-42)

neurotoxicity,

as

exerts persistent

neurodegenerative effects (Giovannelli et al., 1995) (2) the conformation-dependent

activation of

neuronal NOS and (3) the different neuroprotection profiles of NMDA receptor antagonists for Ag(25-35), or Ag(l-42) (O’Mahony et al., 1998). It may therefore be claimed that the secondary and tertiary peptide structure predisposes Ag fragments to proteolytic degradation and clearance. Another possible classification of in vivo Ag toxicity studies can be based on animal species lesioned with Ag or Ag-related peptides. While rats are commonly used as target animals in the majority of in vivo studies (Tables 1 and 3) Ags elicit similar behavioral disturbances neurochemical/neuroanatomical

or

changes in the mouse brain (Flood et al., 1991, 1994a,b)

Moreover, whereas Podlisny et al. (1993) noted a lack of neurotoxicity following synthetic Ab infusions in the monkey cerebral cortex, Geula et al. (1998) recently reported significant data on age-dependent Ap neurotoxicity in the primate, and particularly in the marmoset or rhesus monkey brain. In fact, while no significant Ag neurotoxicity was found in the young rhesus monkey or in the aged rat brain, plaque-equivalent

concentrations of Ap induced marked neurotoxicity in aged

rhesus monkeys or marmosets (Table 2).

T. Harkany et al.

978

Table I

Neuroanatomical and Neurochemical Studies on in vivo Neurotoxicity of Ap Fragments in Rat Brain

Author

Target brain region

A$sequence(s)

l-28,25-35, l-40

Abe et al.. 1994

Major conclusion

Decreased hippocampal AC%release ..-__.-Clemens and Stephenson, 19922-------- l-40 ~ Lack of Aj3toxicity Hippocampus, skiatum following implantation ._ -I___.__.... _. Delobette et al., 1997 l-2825-35, l-42 i.c.v. _- Conformation-dependent memory impairment l-40 Acute neurotoxicity ~-_ Emre et al., 1992 ._. -.-.__I_-._.Cortex-~ -_-.Neuronal loss and tau Frautschy et al., 1991 Hippocampus, cortex PA cores phosphotylation -_I__ ._..-_ .-.- -.25-35, l-38, l-40 Lack of AD-related Games et al., 1992 Hippocampus, cortex pathology 25-35, l-40 MBN Conformation-dependent _ Giovannelli et al., 1995 toxii _--._-_ _.._ .--__ ---.. .._ _-_ _I_ Giovannelli et al., 1998 l-40 Cholinergic recovery MBN correlated with the

Jancd et al., 1998

l-42

Nitta et at-i?94

l-40

_.__

..___ ._1_40

MS

. ..._

Nitta et al., 1997.

--.-_.

P,uta.et al:11997_-._

-_ - ~.-_.__... Shin et al., 1997

..__..i~_--.__. .._____..

--..

I-40, l-42 25-35

Stein-Behrens et al., 1992 ___ __ _ ._____. Winkler et al., 1994

.-_. ._I___ -_.-._-_-

UarnadaeKl~~~-

.__-. -l-42

---_-

_ l-40

release --__. Deterib-. brain barrier function --_. -transport-_ i.c.v. Decreased ChAT activity associated with learning ____ deficits .-i.c.v. Learning deficits and neuronal loss a&r _-.. - _. continuous infusion ..Blood_brainbanier Extravasation of AS--~~~ transport enhanced by cerebral ischemia Hippocampus, cortex DiiTerentiaJfibril formation _ __zr!!!! _ Lack of hippocampal Hippocampus neuronal loss and At3 toxicity ______--_.--. Lack of long-term i.c.v., hipp&upus neurotoxicity_ .---&Tected CNTF production i.c.v. dopaminergi~i~ms -.. Blood-brain barrier

Summary of in vivo studies directed towards the identification of Aj3 neurotoxicity in rat brain. These studies include solely behavioral, neuroanatomical or neurochemical experiments designed to determine the effects of acute or prolonged (minutes or days) PA infusion into selected rat brain areas (see details in the text).

Neuroprotection

against AP toxicity in vivo

979

The situation relating to the in vivo Ag toxicity models becomes even more complex when the most common carrier systems for the infusion of Ag into the brain are considered, such as acute peptide injections, microdialysis, prolonged perfusion by using osmotic minipump systems, or intracarotid infusions. Acute Iniections: Ag is most commonly delivered into the rodent or primate brain by means of acute microinjections (Tables 1, 2 and 3). Freshly dissolved or ‘aged’ AP is injected into target areas such as the hippocampus (Games et al., 1992). cerebral cortex (Emre et al., 1992) or basal forebrain nuclei (Giovannelli et al., 1995; Harkany et al., 1995a,b, 1998a,b) in a volume of l-3 pl. The small quantities of infL.sed Ags permit a consequent appraisal of Ag-induced local neuronal damage or functional consequences in target areas. In this regard, we have reported a standardized model for consequent determination of Ag neurotoxicity in the rat MBN and assessment of cholinotoxicity/cholinergic

fiber degeneration in the somatosensory cortex (Abraham et al., 1997,

Table 2

Investigations on in vivo Ag Neurotoxicity in Mouse or Primate Brain

Species Mouse - .---.

Author Flood et al., 1991 Flood et al., 1994a - _-. Flood et al., 1994b

ADsequence Target brain region l-28, 12-28, 18i.c.v. 28,12-20 .-____ 12-28 i.c.v. 12-28

Limbic strwtmw

Drug(s)

VFFsequence -

Major conclusion Disturbed memory processing Amnesic effects, peptide antagonists Altered K+-chan&i iiUlCtiOll

%mfu2

Geula et al., 1998

Ma&c et al., 1998b

l-40

Age- and _-

SW-40

CifItiOll

--.--_ McKee et al., 1998 Podlisny et al., 1993

1-40,40-l l-40

Frontal cortex cortex

-

dependent toxicity Age-dependent metabolism of blood-borne PA Dose-dependent cortical lesions Lackofacutc’ neurotnxicitv

Summary of Ap neurotoxicity studies in the mouse or primate brain and possible modes of neuroprotection. It is noteworthy that the data reported by Podlisny et al. (1993) were obtained after acute peptide injections into the adult rhesus monkey brain, and were partly confirmed by Geula et al. (1998) who found age- and species-dependent Ag neurotoxicity in primates.

T. Harkany et al.

980

B

Fig 4 Acute and intruccwotid perfusion models of in viva A$ toxicity. Fig A shows a schematic outline of the lesion site, needle track and degenerating cortical cholinergic projection fibers following neurotoxic lesions in the MBN. Fig B shows distinct Evans blue extravasation in damaged areas of the right hemisphere of the rat brain I h after a 10411 intracarotid A&Phe(S@H)*‘)25-35 infbsion according to Jan& et al. (1998).

981

Neuroprotection against Ap toxicity in vivo 1998; Harkany et al., 1995b; Harkany et al., 1998a.b; O’Mahony et al., 1998; Fig 4A). As Fig 4A shows, one of the major advantages of this model is the relatively extensive parietal cortical cholinergic

fiber loss following

lesioning

of the MBN. Loss of cortical projection

fibers

originating in the MBN can directly be localized on the basis of previously established detailed projection patterns of MBN subdivisions to neocortical structures (Luiten et al., 1987). Moreover, this model prevents biased cell counting-related errors as quantitative cortical acetylcholinesterase (AChE)

densitometry

is independent

of local

neurotoxic

injury-induced

morphological

perturbations of identified neurons. Microdialvsis: Although microdialysis infusion of Al.3or its fragments has pronounced technical limitations, based on a considerable aggregation probability in the microdialysis probe of the Ag analogs applied, this model deserves special attention since it may provide (I) a link between acute and long-lasting peptide infusions, and (2) information on the time-course-related

changes in the

extracellular

or

concentrations

of

released

neurotrophic

factors,

excitatory

inhibitory

neurotransmitters or neuromodulators (Harkany et al., 1997). Perfusion models: Ag accumulation and deposition in neuritic plaques and plaque maturation span decades in the human brain (Selkoe, 1991). In this respect, an intriguing question arises of whether long-term Ag infusion into the rat brain elicits gradual formation of Ag deposits and/or hyperphosphorylation of tau, neuropathological changes similar to those obsetved in the AD brain. It may also be hypothesized that extensive peptide accumulation over prolonged periods in the brain and the formation of fibrillar amyloid deposits might influence Ag neurotoxicity. To test these hypotheses, Nitta et al. (1994, 1997) and others (Yamada et al., 1995) introduced a long-term perfbsion system by using mini-osmotic pumps. These studies revealed that intracerebroventricular (i.c.v.) administration of Ag disrupts the cerebrospinal fluid-brain barrier, leads to the formation of extensive Ag deposits in the brain and to long-lasting behavioral disturbances (Nitta et al., 1994). Interestingly, persistent Ag infusions can significantly alter cytokine expressions, among which ciliary neurotrophic factor (CNTF) concentrations (Yamada et al., 1995). Intracarotid Infusion: Although the neurotoxic action of Ag has now been firmly established, there is a lack of direct evidence as to whether Ag toxicity only results from glial/neuronal APP misprocessing or possibly also from extremely low concentrations

of aggregated Al3 in the

cerebrospinal fluid and in the general circulation (Pischke et al., 1998). To shed light on this question, Pluta et al. (1997) and Jancd et al. (1998) devised a new model involving administration of small quantities of Ag into the peripheral circulation. Aps significantly impaired the integrity of endothelial cells and hence’blood-brain

barrier (BBB) Iunction (Table 1). Furthermore, PA was

T. Harkany et al.

982

transported across the BBB (Mackic et al., 1998a.b) and full-length Ap(1-42) accumulated in the brain, indicating that APP misprocessing and thus, the generation of blood-borne Ag at the periphery may contribute directly to neuronal damage (Banks et al., 1997). Synthetic modification of Ap(I-42) and the application of water-soluble oligo/monomeric Al3 derivatives, or those of the active center (Ag(25-35))

may become valuable tools in in viva

modeling of the neurotoxic properties of Ag. We have recently demonstrated (Harkany et al., 1998b) that a water-soluble Ag derivative, Ag(Phe(SO&)24)25-35, generated by coupling of the A(3(25-35) sequence with a phenylalanine

sulfonate residue at position 24, elicits long-lasting

behavioral disturbances and induces the loss of basal forebrain cholinergic neurons. Delivery of this modified peptide analog into the circulation via the carotid artery resulted in extensive BBB damage as indicated by Evans blue extravasation (Fig 4B). The BBB injury involved basal brain regions intimately surrounding the circle of Willis,but also occipito-temporal neocortical areas. In fact, animals exposed to amyloid infusions in the general circulation exhibited similar behavioral dysfunctions

(e.g. lack of passive avoidance

discrimination

environment;

Harkany et al., 1998b) to those of h4BN-lesioned rats (a&a not shown). It is

therefore plausible to speculate that artificial modifications

or hypoactivity

in a novel

of the Al3 sequence - e.g. the

substitution of amino acid residues within the g(25-35) region or the coupling of inorganic constituents to short Afl sequences - may act as potent tools for in vivo modeling of AD toxicity or even the pharmacological characteristics of conformational Al3 antagonists (Laskay et al., 1997). Point mutations or synthetic variations of the native Al3 peptide chain, such as substitutions of amino acid residues both in the active core (A/3(25-35)) and in the flanking peptide regions, the coupling of inorganic ‘protective cap-like’ groups, such as Phe(S0sI-I) or the generation of peptide libraries of truncated A$ fragments, may eventually lead to substantial changes in the chemical properties and the biological activity of AP (Forloni et al., 1996, 1997; Giovannelli et al., 1998; Harkany et al., 1998b). Interestingly, the presence of an aspartate residue at position 7 was established to be pivotal for the activation of a classical complement pathway (Velazquez et al., 1997) as it was demonstrated by using truncated Al3 fragments. Recently, amidation of Al3(25-35) has been reported to reduce the tibril-forming neurotoxicity neurotoxicity,

properties of the peptide without infhtencing its

(Forloni et al., 1997). In support of the role of fibril formation in amyloid Giovannelli

immunoreactivity

et

al.

(1998)

reported

a

concomitant

recovery

of

ChAT

in the MBN with concomitant loss of amyloid fibrils 6 months after MBN

lesions. However, fibril formation was suggested not to be a prerequisite of A/3 toxicity, as both monomer and oligomer Ag exerted neurotoxicity (Harkany et al., 1998b), and exhibited selective

983

Neuroprotection against A8 toxicity in viva binding to cell-surface recognition sites (Lambert et al., 1998). Recently, [‘z11/‘2sI]or [3H]-labeled AS Fragments were employed to prove selective BBB transport (Saito et al., 1995) cell-surface binding mechanisms (Sties

and Per&e, unpublished &a),

and AS uptake and intact peptide

internalization (Mackic et al., 1998a). Interestingly, oligopeptide derivatives of Ag( I-42) commonly coupled to inorganic structurestabilizing groups, have been demonstrated to act as ‘peptide antagonists’ or ‘P-sheet breaker peptides’ either by preventing A(3 aggregation and tibril formation or by counteracting with proposed cell-surface recognition pathways of A$ (see Chapter 4.4).

4. Pharmacolonical Prevention of Al3 Toxicitv in vivo

Whereas ample in vitro and in vivo experimental data indicate well-defined intracellular changes in response to AS administration,

little is known about pharmacologically

inducible cascades,

which prevent or block Ag toxicity. Recently, numerous albeit controversial in vitro studies indicated that AR excitotoxicity can be antagonized by tachykinins (e.g. substance P (SP); Kowall et al., 1991; Yankner et al., 1990) by Ca”-channel blockade (Ueda et al., 1997; Weiss et al., 1994; Whiston and Appel, 1995), by inhibition of free radical generation and lipid peroxidation (Mark et al., 1997b), by hormone, primarily estrogen, replacement (Goodman et al., 1996; Gridley et al., 1997; Mook-Jung et al., 1997). or by g-sheet breaker peptide fragments analogous to the AS sequence (Soto et al., 1998). In spite of the convincing data yielded by in vitro pharmacological studies, non-transgenic in vivo models are sparingly used (I) to elucidate particular steps of Afl action, or (2) to evaluate the neuroprotective potentials of candidate molecules in the prevention or inhibition of AS toxicity in the brain. The authors feel, however, that animal models of Ag neurotoxicity have contributed significantly to a better understanding of the molecular pathways initiated by infusion of synthetic Ag tiagments (Table 3). In the following chapters we summarize novel pharmacological approaches (Pig 6) which might contribute to the development of effective therapeutic strategies of AD.

T. Harkany

984

et al.

Table 3

Neuropharmacological Studies on AD Toxicity and its Prevention in vivo

Author A$sequewe(s) Abrahhm et al., 1998 l-42

Target brain region ME!N

Major conclusion

Drug(s) Cmne

Dose-dependent

_...*;d~_. Harkaoy et al., 1997

1-42

MBN

et al., 1998a

1-42

MBN

Harkany

ML801 ...__ -_ MK-801, vitmnin E

Neuroprotectionby. __-....- *f_d”Lg_trealment

__---~-. Kowall et al., 1991

l-40

COltCX

Mamice et al., 1996

25-35

i.c.v.

Morimoto et al., 1998a

25-35, l-40

Hippocampus

O’Mahony et al., 199

25-35, I-42

MBN

Rush et al., 1992

25-35, I-40

Hippocampus

Soto d al., 1998

l-42

Yamada et al., 1998

l-40

neurotoxic pathway -_

Substance P ____._

Amygdala -..---

Nearotoxicity prevented by substance P D+diW~ine, Reversal of behavioral mik!em& deficits C&~ns wiJ% -- tiIG&GzG&-MK-801, enhanced ibotenic acid, MKneuro-toxicity with 801 iboteoic acid -_ _. --~ ...-... NMDA receptor and &tlp?odil NOS activation ___ Neorotoxic damage and Subs&e P neuropg&ion by_.SP -- no.-_ __~.~~b~~ Prevention of Afl ._.&_

_. !!@~and~0~-t;v Improved learnmg and memory processes

i.c.v.

Recent in vitro data indicate that neurotrophic factors, which directly stimulate cholinergic fimctioning (e.g. nerve growth factor (NGF)) or counteract molecular neurotoxic cascades such as basic fibroblast growth factor (bFGF), attenuate AD-induced oxidative stress, the impairment of mitochondrial functions, and destabilization of [Ca”]i (Mark et al., 1997a; Mattson et al., 1993). In spite of the promising evidence that growth factots, and particularly NGF, or brain-derived neurotrophic

factor,

can

protect

cholinergic

neurons

against

IgG192-saporin-induced

immunolesions in the rat basal forebrain (Schliebs et al., 1996), AD neurotoxicity in both young and aged animals was only marginally decreased by continuous i.c.v. administration of NGF (Fig 5). The limited efticacy of NGF-mediated neuroprotection is probably due to the limited diffision of the neurotrophin to the damaged MBN structures. NGF delivery to basal forebrain cholinergic ceil groups must therefore be re-examined to improve the extent of cholinergic nerve cell survival against PA toxicity.

Neuroprotection against AP toxicity in vivo

0

Shsm-opcrated n pAp(I-42)

985

l ml-42)+NGF

Fig 5 Loss of AChE-positive cholinergic projection fibers 14 days after unilateral Ag(l-42) infusion (freshly dissolved, 0.2 nmol/l pl) into the MBN, and the efficacy of continuous icv. administration of NGF (7 day pre-treatment, 1.2 ug/NGF/day). NGF infusion did not attenuate AD-induced cholinergic fiber loss. Sham-operated animals received vehicle solution only. * p < 0.01 vs. sham-control animals (one-way analysis of variance), NS = non-significant difference, n = 7 - 10 per group. l

4.1 Blockade of Intracellular Ca” Entrv: A Pronosed Role for NMDA Recenters Ag attacks nerve cells by a rapid and sustained activation of Ca’+-permeating mechanisms (Mattson et al., 1992), or by the formation of bivalent cation-selective transmembrane

pores

(Arispe et al., 1993, 1996). Increased intracellular Ca*’ entry results in a pathological Ca” overload, leading to a widespread derangement of Rmdamental cellular functions (Luiten et al., 1996). Ag-induced pathological elevations of [Ca”]i occur within minutes and persist for several hours (Laskay et al., 1997; Mattson, 1994). depending on the chemical nature of the actual AS fragment. It is therefore likely that neurons undergo excitotoxic cell death, which involves the exhaustion reticulum

of intracellular

Ca2’-storage

mechanisms,

in mitochondria

and endoplasmatic

the high-efftcacy binding of Free intracellular Ca” to Ca2’-binding proteins, like

parvalbumin, calretinin, or calbindinD28k transmembrane

Ca”

extrusion.

(Mattson et al., 1991; Pike and Cotman, 1993) or

Independently

of the

actual

Ca2’ permeating

pathway,

T. Harkany et a,!.

986

0A NMDA mcept~WmpkX

Membtane

depo&rkatbn

0B

-. Mn

-

.’ y

_--

Ca”

y --__

~a-/cy-N0Sl ;

K 0

NOSinhibitws, “6Nhb-LArSMle

In E, Anthuidantr

_---

Prevention of -ideath

Fig 6 Schematic representation of the cellular action of AP, its intracellular consequences (A), and molecular targets of novel neuroprotective approaches (B) attenuating A@excitotoxicity (for details. see chapters 4.1-4.4).

Neuroprotection against AP toxicity in vivo

987

the blockade of (I) selective target recognition involved in the transmembrane ‘receptor’ binding of Ag or (2) the net cellular Ca*’ entry might provide appealing objects to achieve significant neuroprotection (Fig 7). A probable selective cell-surface binding element for Ag was recently assumed by Cowbum et al. (1994, 1997) and Laskay et al. (1997). While ligand-binding

data

support an essential role for NMDA receptors as mediators of AD-induced cell damage (Cowburn et al., 1997) a specific, though as yet unidentified receptor with marked Ca*’ conductance was also hypothesized (Laskay et al., 1997). Identification

of a Ag-induced

glutamate-triggered

excitotoxic pathway (Fig 2; Harkany et al., 1997, 1998a) emphasized a principal role of the NMDA receptor channel. Hence. the blockade of NMDA receptors or VDCCs which become activated upon depolarization, might provide potent tools for the prevention of PA toxicity (Fig 7). Our own pharmacological studies (Harkany et al., 1997; 1998a; O’Mahony et al., 1998) and those of others (Maurice et al., 1996; Morimoto et al., 1998a) have provided compelling experimental evidence that both pre- and post-lesion administration of the non-competitive NMDA receptor antagonist MK-801 or of the extracellular o-site ligand ifenprodil significantly

attenuates Ag

toxicity. These data accord with previous findings (Barth et al., 1996; Stuiver et al., 1996) showing high-efficacy

neuroprotection

under experimental

conditions

such as ischemia

or NMDA

neurotoxicity, where persistent activation of NMDA receptor channels is a pivotal trigger of excitotoxic stimuli. Another line of investigations suggests the direct involvement of overt Ca*’ signaling via L-, but not P/N-type VDCCs in Ag neurotoxicity (Ueda et al., 1997; Weiss et al., 1994). Supportive to the selective involvement of VDCCs in Ag toxicity, treatment of cell cultures with the L-type Ca*‘channel antagonist nimodipine, or cobalt (Co), but not with the P-type Ca*‘-channel blocker oconotoxin or the N-type VDCC antagonist o-agatoxin, abolished Ag toxicity and the elevation of [Ca*‘]i (Ueda et al., 1997). It is worthy of attention that NMDA-induced degeneration of the cholinergic cells in the rat MBN was markedly decreased by chronic nimodipine administration (Luiten et al., 1995). In

summary, we conclude that NMDA receptors might act either as selective substrates of Ag

binding or as mediators of A@triggered glutamate excitotoxicity. While NMDA receptor channels are involved in action potential generation, VDCC opening is essential for spike propagation (Luiten et al., 1996). Pharmacological intervention in NMDA receptor activation might therefore be of great significance, as it rescues AD-stimulated neurons from sustained depolarization, while VDCC blockade diminishes the sequential effects of prolonged excitation (Fig 6). The latter step of our hypothesis is supported by the fact that hyperpolarizing agents, such as anticonvulsants or K’ channel openers protected hippocampal neurons against the noxious effects of Afi (Goodman

T. Ha&any et a&

988

and Mattson, 1996; Mark et al., 1995). On the basis of the aforementioned electrophysiological phenomenon, it might be postulated that the simultaneous application of NMDA receptor and VDCC antagonists results in additive neuroprotection (Stuiver et al., 1996; see Chapter 5; Fig 7).

4.2 Inhibition of Free Radical Generation: Antioxidants and Trace Metals

Al3 accumulation induces oxidative stress to neurons (Elehl, 1997). To prevent or diminish oxidative stress-induced neuronal injury, antioxidant therapy has recently been suggested and extensively reviewed elsewhere (Behl, 1997; Markesbery, 1997; Multhaup et al., 1997; de la Terre et al., 1996). In the present review, the authors focus on two aspects of the experimental inhibition of ROS generation, and particularly on the roles of direct antioxidants or trace metals (TMs). While endogenous radical scavengers such as ascorbate, glutathione and vitamin E contribute directly to ROS detoxification, the inhibition of lipid peroxidation or the maintenance of ion homeostasis (Ames et al., 1993; Halliwell, 1987; Mattson, 1998) TM (e.g. Cu, Co, Mn, MO. Ni, Se, V, Zn) and inorganic ions (e.g. Mg, Fe) play special roles in normal central nervous system activity. Whereas a strictly controlled regulation of TM metabolism and clearance is a prerequisite of a physiological brain function, extremely high or low concentrations of TMs influence neuronal excitability, neurotransmitter release and an adequate neurotransmitter-receptor

function (Kostyuk

and Verkhratsky, 1995). In fact, numerous TM, such as Zn, Ni or V, exhibit high-afftnity binding to transmembrane receptors, like VDCCs, and influence the activity of Na+/K’-ATPases (Kostyuk and Verkhratsky, 1995). Additionally, selenide ions are pivotal structural constituents of the active core of the detoxifying enzyme glutathione peroxidase, while Zn, Mn, MO and Cu may act as allosteric enzyme co-factors or modulators of peptide conformation, catalytic activity and gene function (Cuajungco and Lees, 1997). In this respect has TM malnutrition has been proposed as a factor in the etiology of human neurodegenerative disorders, such as AD or Parkinson’s disease. In spite of the theoretical relevance of antioxidants or TM to the prevention of Al3 toxicity, controversial in vitro data have been reported on the efftcacy of such therapies. In this regard, Pike et al. (1997) demonstrated the lack of significant protection by synthetic antioxidants against Aginduced oxidative stress, whereas vitamin E (Behl, 1997; Gwebu et al., 1997; Koppal et al., 1998) or ascorbate (Yallampalli et al., 1998) significantly attenuated the DA-induced ROS generation and lipid peroxidation. It is noteworthy that ascorbate, with its high-affinity binding to the redox modulatory site of the NMDA receptor, may reduce the AD-induced elevation of [Ca2’]i directly. Recently, we demonstrated ascorbate administration

(Harkany et al., 1998a) that chronic combined vitamin E and

significantly improves learning and memory deficits and also altered

Neuroprotection against AP toxicity in vivo

989

spontaneous behavior following unilateral A(3 injections in the rat MBN. Moreover, reductions in cortical ChAT, AChE and MnSOD activities were markedly attenuated in this experiment, while the CwZnSOD activity remained largely unchanged. To the best of the authors’ knowledge, this was the first report demonstrating in viva efficacy of antioxidant treatment. Follow-up studies revealed that both pre- and post-treatment with high doses of vitamin E diminished Ag-induced behavioral dystinctions

and decrements of cholinergic marker enzyme activities (da& not shown).

TM, and particularly Cu and Zn, were proposed to dramatically enhance the aggregation or

folding properties of Ag (Atwood et al., 1998; Huang et al., 1997). Zn treatment of rats 48 hrs prior to Ag infusions into the MBN resulted in the potentiation

of PA-induced behavioral

dysfunctions, such as a strong increase of immobility under elevated plus maze or open-field conditions (Harkany et al., 1998c). However, Zn neurotoxicity was significantly attenuated by coadministration of a complex containing vitamin E and other TMs, such as Cu. Mn, Mg and Se, which are known to influence Zn absorption and modulate the cellular action of Zn (e.g. by counteracting the NMDA receptor complex). Indeed, neuroprotection by vitamin E, ascorbate and high doses of Mg ameliorated Ag neurotoxicity at both the behavioral and the neurochemical level. A complete behavioral recovery indicated by the preservation discrimination

of passive avoidance

or explorative rearing activities was recorded throughout the I4-day survival

period. Subsequently, determination

of cortical AChE and ChAT activities did not reveal a

significant loss of cholinergic marker enzymes, as compared to sham-operated animals (Harkany, Kenya and Nyakas, unpublished data). In summary, the co-administration of natural antioxidants and selected TMs or minerals might provide alternative therapeutic strategies to AD

4.3 Steroid Hormones

Steroid hormones have critical roles in fundamental brain functions, as they penetrate in the brain and bind to intracellular

receptors (Joels, 1997). Distinct populations of intracellular

receptors, termed mineralocotticoid

steroid

or glucocorticoid receptors, mediate substantially different

cellular responses. In this respect, the diversity of steroid hormones implies different, commonly opposing cascade mechanisms and widespread effects on neurons. While corticosteroid hormones via mineralocorticoid

receptors yield fast neurotransmission

in the brain, glucocorticoid receptor

activation suppresses the action of amino acid neurotransmitters.

In fact, recent studies clearly

showed the regulatory effects of estrogen and corticosterone on AP cytotoxicity (Abraham et al., 1997, 1998; Behl et al., 1997; Goodman et al., 1996; Green et al.. 1996; Gridley et al., 1997;

T. Harkany et al.

990

Mook-Jung et al., 1997; Xu et al., 1998), while testosterone or aidosterone displayed no effect on neuronal vulnerability against excitotoxic stimuli. On the basis of clinical studies, a neuroprotective role for estrogen was proposed as prolonged estrogen administration to post-menopausal women reduced the risk and delayed the onset of AD (Paganini-Hill and Henderson, 1996). Although estrogen, in particular 17p-estradiol, significantly reduces the neuronal generation of AD (Xu et al., 1998) and exerts beneficial effects on APinduced neurotoxicity’ (Mook-Jung et al., 1997) by the suppression of ROS generation (Gridley et al., 1997) and by the stimulation of the o-secretase pathway of APP processing (Jaffe et al., 1994) in vitro, little is known about the particular molecular steps involved in the inhibition of Ag cytotoxicity. However, a structure-activity relationship was proposed for the antioxidant action of estrogen, indicating an essential hydroxyl group on C3 of ring A of the steroid skeleton (BehI et al., 1997). Interestingly,

Goodman et al. (1996) described adverse effects of estrogen and

corticosterone on hippocampal neurons exposed to AP or glutamate. Whereas estrogens protected hippocampal neurons against excitotoxicity, corticosterone

exacerbated

these neurotoxic

FeSO.+-induced injury and glucose deprivation, effects. Similarly

to the in vitro effects of

corticosterone, chronic corticosterone replacement of adrenalectomized animals revealed a dosedependent steroid action. Extremely low concentrations or highly elevated plasma corticosterone levels dramatically enhanced both A/3 and NMDA cholinotoxicity

in the rat MBN, whereas

continuous release of corticosterone from subcutaneously implanted 25% or 100% corticosteronecontaining pellets resulted in significant neuroprotection (Abraham et al., 1997; 1998). Thus, a dose-dependent

reversed bell-shaped neuroprotection

profile was achieved following

chronic

graded corticosterone replacement (Abraham et al.. 1998). It may be suggested that the in vivo regulatory effects of corticosterone might be due to a differential occupancy of glucocorticoid and mineralocorticoid receptors, and selective alterations of intracellular Ca” signaling (Joels, 1997).

Initially,

Hilbich

et al. (1991,

1992) demonstrated

that the aggregation

properties and

amyloidogenity of Ag is dependent on the integrity of a well-preserved hydrophobic core around residues 17 and 20 of the Ag sequence, and postulated that site-directed modification of PA within this region may guide the development of agents for AD therapy. Moreover, Flood et al. (1994a) reported that small biologically inactive peptides containing sequences homologous to a Val-PhePhe tripeptide residue of Ag blocked the amnesic effects of Ap( 12-28); this might open the way to design memory-modulating peptidic and non-peptidic substances which can effectively antagonize

Neuroprotection against Af3 toxicity in vivo

991

Ag neurotoxicity. Recently, two major strategies for the design of inhibitors of Ag toxicity became evident: (I) inhibitors of Al3 aggregation or tibril formation and (2) tetra- or pentapeptides which may potentially interfere with cell-surface gA binding and cellular l3A recognition. In fact, the Ag aggregation kinetics was demonstrated to be dependent’on the peptide conformation (e.g. Ag( I42) aggregated more rapidly than Ag( l-40)) the actual Ag concentration, the temperature and the ionic strength of the solvent in vitro. Moreover, Ag( l-40) was shown to retard the aggregation of A(3(l-42) in a dose-dependent

manner (Snyder et al., 1994) suggesting direct protein-protein

interactions during Ag folding. Inhibition of Ag toxicity was also achieved by site-directed Nterminal antibodies

(Solomon et al., 1997) which bind to Al3 assemblies leading to the

disaggregation of Al3 fibrils and the restoration of peptide solubility. Another approach to the utilization of N-terminal recognition was proposed by Ghanta et al. (1996) on the basis of a dimer aggregation inhibitor consisting of Ag(lS-25) residues, termed ‘a recognition element’, linked to an oligolysine ‘disrupting’/‘inhibitor’

domain. The proposed in vitro neuroprotective mechanisms

might include alterations of aggregation kinetics and higher-order structural characteristics. Such a selective inhibition of Al3 fibril formation could also be achieved with organic compounds, like hexadecyl-N-methylpiperidinum

bromide (Wood et al., 1996) or rifampicin and its derivatives

(e.g. hydroquinone; Tomiyama et al., 1996). Tjemberg et al. (1996) and Soto et al. (1998) recently reported N-terminal pentapeptide antagonists, namely a Leu-Pro-Phe-Phe-Asp

sequence derived

from Ag(16-20) (Soto et al., 1998) which might become a lead compound in Al3 aggregation inhibition, as it blocks amyloidogenesis,

disassembles peptide fibrils in vitro and significantly

reduces Ag deposition and the formation of amyloid fibrils following acute Al3 injections in vivo. Another strategy to inhibit Al3 toxicity has recently been summarized by Laskay and his colleagues (Laskay et al., 1997) who designed a lead compound derived from the (3 l-34) region of Aj3 acylated with propionic acid (Pr-Ile-Ile-Gly-Leu-NHZ).

This analog of Al3 was capable of

blocking increases in [Ca”]i of cultured astroglial cells after S-hour treatment with 1 pM concentrations of Ag( l-42) AP( l-40), Ag(25-35) or Ag(3 l-35). The propionyl-tetrapeptide

also

exerts its protective effects in viva, as a significantly reduced elevation of extracellular glutamate concentration and a diminished loss of AChE-positive cortical fiber innervation were found upon microdialysis of Pr-Ile-Ile-Gly-Leu-NH2

into the rat MBN prior to PA delivery (Harkany et al.,

1999). In summary, new strategies for the design of selective inhibitors of either Al3 fibrillogenesis or biological

activities (e.g. elevations of [Ca”]i, binding to putative receptors,

formation) of Al3 might result in drug candidates with marked therapeutic potential.

‘Ag-radical’

T. Harkany et

al.

From a therapeutic point of view, the most efficient strategies of AD therapy might involve simultaneous combined administrations

of drugs interfering

with distinct cellular signaling

pathways elicited by noxious extracellular concentrations of Al3 in the brain. As in vivo Al3 toxicity was established to share tindamental

features with NMDA excitotoxicity (Abraham et al.,

1998; Harkany et al., 1997), it is plausible to hypothesize that effective neuroprotection achieved by simultaneous administration of a non-competitive NMDA receptor antagonist (MK-801) and an L-type Ca’+-channel blocker (nimodipine) neurotoxicity.

might exert similar protective potentials against Al.3

Indeed, Stuiver et al. (1996) have demonstrated

&tive

neuroprotection

by

simultaneous blockade of NMDA receptors and L-type VDCC. The concerted action of Ca” antagonists depolarization

might

prevent

an

excitotoxin-induced

and action potential

Ca”

generation/propagation

overload,

sustained

membrane

leading to cell death (Fig 7).

Interestingly, whereas a positive interaction of Nh4DA and VDCC blockers was found, combined therapy with the SHTr* receptor agonist 8-OH-DPAT significantly

augmented additive neuroprotection

and nimodipine

did not result in

(Stuiver et al., 1997). These data might be

Fig 7 Neuroprotection by single (A) vs. combined (ES)drug administration against Al.3toxicity. While single blockade of NMDA receptors results in partial inhibition of intracellular Ca’+ entry, combined treatment with NMDA and L-type VDCC antagonists enhances the efftcacy of neuroprotection (Stuiver et al., 1996). Interestingly, Al3 toxicity can be regulated by selective Ltype VDCC ligands, whereas administration of drugs interacting with the N/P-type VDCC does not influence Ag toxicity (Ueda et al., 1997).

Neuroprotection against A8 toxicity in vivo

993

indicative of the fact that blockade of the primary source of Ca*’ entry, such as of prolonged NMDA receptor activation, is a prerequisite for an adequate pharmacological

action against

excitotoxic stimuli. As the single administration of NMDA receptor antagonists (Harkany et al., 1997, 1998a; Maurice et al., 1996; Morimoto et al.. 1998a; O’Mahony et al., 1998), VDCC ligands (Luiten et al., 1995) SHT~Areceptor agonists (Oosterink et al., 1998) or the Al adenosine receptor ligand propentofylline

(Yamada et al., 1998) revealed the potential prevention of excitotoxic

neuronal damage, further studies might explore optimal therapeutically relevant combinations of drugs abolishing the cellular toxic action of AB. Combined administration of NMDA receptor antagonists and antioxidants, such as that of MR801 and a-phenyl-tert-butyl-nitrone, window of neuroprotection

has previously been shown to increase the efficacy and time-

following transient

ischemia/hypoglycemia

in organotypic

slice

cultures (Barth et al., 1996). However, potent antioxidants might have side-effects and act as selective regulators of receptors. In this respect, recent experimental data indicate that ascorbate attenuates AD neurotoxicity (Yallampalli et al., 1998) whereas combined treatment with ascorbate and the NMDA receptor blocker MR-801 results in diminished drug action (Harkany et al., 1998a). The pharmacological counteraction might be due to drug interference on regulatory sites of the NMDA receptor channel (Harkany et al., 1998a). In contrast, aqueous-phase antioxidants or vitamin E may block ROS generation and thereby enhance the neuroprotective effects of highly selective receptor ligands. The relevance of a combined Ca” entry blockade and antioxidant therapy is supported by recent findings which demonstrated convergence and synergistic actions of Ca2’ and ROS-induced neurotoxicity (Dutrait et al., 1995; Mattson et al., 1995). Additionally, combination of vitamins (e.g. vitamin E and ascorbate) or vitamins and minerals/TMs, such as Mg or Se might envisage new vistas for effective adjuvant therapeutic considerations of AD-induced neurodegeneration in AD

6. Conclusions

The characterization of intracellular signaling cascades and their interactions in further in vivo studies will shed new light on Ag-induced molecular pathways leading to selective neuronal loss in AD. Moreover, investigations

aimed at identifying

(I) the interactions

and (2) cascade

sequences of AB toxicity with other key factors of AD (e.g. tau-phosphotylation,

metabolic

994

T. Harkany et aL disturbances, Apo E allele heterogenity), (3) molecular mechanisms providing links between distinct neurotoxic pathways mediating Ag toxicity, and (4) excitoprotective neuroendocrine feedback regulation might outline Buittbl preventive or therapeutic strategies for AD. The authors hold the opinion that the summarized in vivo models and pharmacological approaches will contribute significantly to the ongoing attempts to decipher the cellular neurotoxic events elicited by Ap. The development

of modified

A/3 fragments,

conformational

peptide antagonists

or selective

pharmacological ligands will offer the necessary tools to help unravel fundamental processes of the neurotoxicity of Al3 and its contribution to the pathogenesis and progression of AD.

Acknowledgements

The authors wish to acknowledge the valuable contributions of Prof. B.E. Leonard and Dr. S. O’Mahony (University College, Galway. Republic of Ireland), Dr. I. AbraMin (Institute of Experimental Medicine, Hungarian Academy of Science-s, Budapest, Hungary) and Dr. G.I. De Jong (Dept. of Animal Physiology, University of Groningen, The Netherlands) to specific parts of the investigations described in this review. This work was supported by grants from The Hungarian Research Foundation (OTKA, #TO26451to C.N., #TO17751 and #TO22546 to B.P., #F023865 to T.H.), a joint short-term fellowship of NUFFTC (l%e Netherlands) and the Hungarian Ministv of Education (kf97114.538, to T.H.), The international SOROS Foundation (to T.H.) and a joint grant of The Netherfads

Science Founchtton (NW0 #048-01l-006, to

P.G.M.L., and OTKA #N26674, to. C.N.). Moreover, parts of the studies were supported by B&es Co. Ltd., Budapest, Hungary.

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Inquiries and reprint requests should be addressed to:

Dr. Tibor Harkany

Department of Animal Physiology University of Groningen Kerklaan 30, P.O. Box 14 9750 AA Haren, The Netherlands phone: +3 1 50 3632 353 fax: +3 1 50 3635 205 e-mail: [email protected]