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GM1 ganglioside, nerve growth factor, and epidermal growth factor, enhance mesencephalic dopaminergic markers after a lesion with MPP+

Dalia, Asad Dawoud, Ph.D.

The Ohio State University, 1992

UMI 300 N. ZeebRd. Ann Arbor, MI 48106 GM1 GANGLIOSIDE, NERVE GROWTH FACTOR, AND

EPIDERMAL GROWTH FACTOR, ENHANCE MESENCEPHALIC

DOPAMINERGIC MARKERS AFTER A LESION WITH MPP+

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By

Asad D. Dalia, M.S, M.D. *****

The Ohio State University

1992

Dissertation Committee: ^ Approved by:

Maria Hadjiconstantinou-Neff /■ fve $rf-

Norton H. Neff Adviser

Allan J. Yates Department of Pharmacology TO MY FAMILY ACKNOWLEDGEMENTS

I express my sincere appreciation to my adviser, Dr. Maria Hadjiconstantinou-

Neff, for her guidance throughout this research project and my entire graduate education. I would also like to thank her for her patience, encouragement and friendship.

I am grateful to Dr. Norton H. Neff for his scientific input and friendship throughout this research project. I would also like to thank him for encouraging my interest in pharmacology.

I would like to thank Dr. Allan J. Yates for his friendship and critical evaluation of this document.

Thanks are also extended to my fellow pharmacology graduate students for their friendship and encouragement.

I am especially grateful to my parents who brought me to life.

To my wife Deanna Dalia for her continual love, support and encouragement throughout my educational endeavors. Special thanks to my little ones, Adam and

Julia, who let me sleep peacefully at night after some hard days at work. VITA

April 25, 1958 Born- Jerusalem, Palestine

1983 M.D., Timisoara Medical School Timisoara, Romania

1984-1985 Internship at Ramallah Hospital Ramallah, Palestine

1988 M.S., The Ohio State University Columbus, Ohio Department of Pathology College of Medicine

1988-1992 Graduate Research Associate Department of Pharmacology The Ohio State University College of Medicine Columbus, Ohio

PUBLICATIONS

Journal Articles

Hadjiconstantinou, M. Fitkin, J.G., Dalia, A. and Neff, N.H. Epidermal growth factor enhances striatal dopaminergic parameters in the l-methyl-4-phenyl-l,2,3,6- tetrahydropyridine-treated mouse. J Neurochem 57:479, 1991.

Dalia, A., Neff, N.H. and Hadjiconstantinou, M. GM1 ganglioside improves dopaminergic markers of rat mesencephalic cultures treated with MPP+. (submitted, J. Neurosci.). Abstracts

Dalia, A., Neff, N.H. and Hadjiconstantinou, M. GM1 ganglioside corrects the loss of DA uptake activity in embiyonic mesencephalic cultures treated with MPP+. Society for Neuroscience, 17:599, 1991.

Dalia, A , Neff, N.H. and Hadjiconstantinou, M. NGF corrects the loss of DA uptake activity in embryonic mesencephalic cultures treated with MPP+. Society for Neuroscience, 266:9, 1992.

Neff, N.H., Hadjiconstantnou, M., Dalia, A and Eaton, M.J. Neurotrophic factors and nigrostriatal dopaminergic neurons. Dopamine ’92, Italy.

FIELDS OF STUDY

Major Area: Pharmacology

Pharmacology Department of Pharmacology Faculty

Physiology Drs. J J. Curry III, J.A. Boulant, J.A. Lipsky, J.D. Wood

Biochemistry Drs. J.A Merola, K.E. Richardson

Neuropsychopharmacology Drs. M. Hadjiconstantinou-Neff, N.H. Neff

Radioisotopes Drs. D.R. Feller, L.A Malspeis, R.W. Brueggemeier TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iii

VITA ...... iv

LIST OF TABLES ...... x

LIST OF FIGURES ...... xii

LIST OF PLATES ...... xiii

LIST OF ABBREVIATION ...... xiv

INTRODUCTION ...... 1

A Pathophysiology of Parkinson’s disease...... 1

B. MPTP: a selective neurotoxin for nigrostriatal dopaminergic neurons...... 4

C. Neurotrophic factors ...... 7

D. GM1 ganglioside is a potential neurotrophic modulator for nigrostriatal dopaminergic neurons ...... 10

E. Nerve growth factor as a putative neurotrophic factor for injured nigrostriatal dopaminergic neurons ...... 13

F. Epidermal growth factor is a neurotrophic factor for dopaminergic neurons ...... 19

G. Specific aims and significance ...... 22 METHODS 25

I. Cell Culture T ech n iq u e...... 25

A. Materials ...... 25

1. Cell culture medium supplements ...... 25

2. Complete cell culture medium ...... 25

3. Dissociation solution ...... 25

B. Methods ...... 26

1. Coating Petri plates ...... 26

2. Preparation of mesencephalic cells ...... 26

3. Preparation of neuron rich mesencephalic culture ...... 28

4. Preparation of glia rich mesencephalic cultures ...... 29

II. Immunocytochemistry Techniques...... 29

A. Materials ...... 29

B. Methods ...... 30

1. Tyrosine hydroxylase immunocytochemistry...... 30

2. Glial fibrillary acidic protein immunocytochemistry...... 31

III. Dopamine Uptake ...... 32

A. Materials ...... 32

B. Methods ...... 32

IV. Measurement of dopamine and its metabolites ...... 33

A. Materials ...... 34

1. Chemicals ...... 34

vii 2. HPLC system ...... 34

3. Reagents ...... 34

4. Activated alumina ...... 34

5. Standards ...... 34

B. Methods ...... 34

C. Calculations ...... 35

V. Aromatic L-Amino Acid Decarboxylase Assay...... 36

A. Materials ...... 36

1. HPLC buffer ...... 36

2. Working buffer ...... 36

3. Incubation b u ffe r ...... 36

B. Standards ...... 37

C. Methods ...... 37

D. Calculations ...... 37

VI. MPP+, GM1, NGF, EGF, Cholera Toxin B subunit, and Anti-GMl Antibodies Treatments ...... 38

1. Neurotoxins; MFIP, MPP+ ...... 38

2. GM1 and trophic factors; NGF and EGF ...... 38

3. Cholera B subunit ...... 38

4. GM1 antibody ...... 38

B. Methods ...... 38

VII. Biochemical Analytical Techniques ...... 39

A. Protein assay ...... 39

viii 1. Reagents ...... 39

2. Standards ...... 39

3. Procedure ...... 39

B. Statistical Analysis ...... 40

RESULTS ...... 41

A. Primary Embryonic Mesencephalic Culture Conditions...... 41

B. Dopamine Uptake ...... 44

C. MPP+ Neurotoxicity...... 48

D. GM1 Effects ...... 52

E. NGF Effects...... 63

F. EGF Effects ...... 80

G. Combined Treatment of Neurotrophic Factors ...... 80

DISCUSSION ...... 84

A. MPP+ Neurotoxicity and Embryonic Mesencephalic Cultures .... 84

B. GM1 Improves Dopaminergic Markers of Mesencephalic Cultures Treated with MPP+ ...... 85

C. NGF Improves Dopaminergic Markers of Mesencephalic Cultures Treated with MPP+ ...... 91

D. EGF Improves Dopaminergic Markers of Mesencephalic Cultures Treated with MPP+ ...... 95

E. Conclusion ...... 97

LIST OF REFERENCES ...... 98

ix LIST OF TABLES

Table Page

1. Characteristics of DA uptake in embryonic mesencephalic cultures ... 47

2. DA uptake in glia rich mesencephalic culture ...... 49

3. MPTP or MPP+ decreases DA uptake in embryonic mesencephalic cu ltu res...... 50

4. GM1 enhances DA and DOPAC content of mesencephalic cultures treated with MPP+ ...... 53

5. GM1 enhances aromatic L-amino acid decarboxylase activity of mesencephalic cultures treated with MPP+ ...... 54

6. GM1 restores the number of TH-immunopositive cells in mesencephalic cultures treated with MPP+ ...... 55

7. GM1 does not prevent MPP+-induced reduction of DA uptake in embryonic mesencephalic cultures ...... 59

8. GM1 does not alter protein content in embryonic mesencephalic cultures ...... 61

9. GM1 restores the morphology of TH-immunopositive cells in mesencephalic cultures treated with MPP+ ...... 64

10. Cholera toxin B subunit or antibodies to GM1 prevent the GMl-induced increase of DA uptake in mesencephalic cultures treated with MPP+ . . 65

11. Enhanced DA uptake induced by GM1 in MPP+-treated mesencephalic cultures is not affected by prior treatment with Ara-C ...... 66

12. NGF 7S or NGF 2.5S enhances DA uptake in MPP+ -lesioned embryonic mesencephalic cultures ...... 67

x 13. NGF does not prevent MPP+-induced reduction of DA uptake in embryonic mesencephalic cultures ...... 72

14. NGF enhances DA and DOPAC content of mesencephalic cultures treated with MPP+ ...... 73

15. NGF enhances aromatic L-amino acid decarboxylase activity in mesencephalic cultures treated with MPP+ ...... 74

16. NGF does not alter protein content in embryonic mesencephalic cultures ...... 75

17. NGF restores the number of TH-immunopositive cells in mesencephalic cultures treated with MPP+ ...... 76

18. NGF restores the morphology of TH-immunopositive cells in mesencephalic cultures treated with MPP+ ...... 77

19. Treatment with Ara-C attenuates the NGF- induced increase of DA uptake in MPP+ treated mesencephalic cultures ...... 79

20. EGF enhances protein content in embryonic mesencephalic cultures ...... 81

21. EGF enhances DA uptake activity in embryonic mesencephalic cultures treated with MPP+ ...... 82

22. EGF does not prevent the MPP+-induced reduction of DA uptake in embryonic mesencephalic cultures ...... 83 LIST OF FIGURES

Figure Page

1. Dopaminergic pathways in the brain ...... 2

2. MPTP biotransformation ...... 6

3. Structure of GM1 ganglioside ...... 11

4. Structure of NGF ...... 16

5. Amino acid sequence of EGF ...... 20

6. Schematic representation of localization of ventral mesencephalon ... 27

7. DA-uptake as a function of cell density ...... 43

8. MPP+ concentration response curve ...... 51

10. GM1 time response study ...... 57

9. GM1 concentration response study...... 56

11. Discontinuation of GM1 treatment ...... 58

12. NGF dose response study ...... 68

13. NGF time response study ...... 69

14. Discontinuation of NGF treatment ...... 71 LIST OF PLATES

Plate Page

I. Development of mesencephalic cultures ...... 42

II. Glia rich mesencephalic cultures ...... 45

III. Neuron rich mesencephalic culture ...... 46

IV. GM1 induced recovery of TH-immunopositive cells ...... 62

V. NGF induced recovery of TH-immunopositive cells ...... 78

xiii ABBREVIATIONS

AAAD aromatic Damino acid decarboxylase Ab-GMl antibody to GM1 ganglioside Ara-C cytosine /9,D-arabino furanoside BDNF brain derived neurotrophic factor B CT B subunit of cholera toxin BSA bovine serum albumine BZT benztropine C-AMP cyclic adenosine monophosphate Cer ceramide CNS central nervous system DA 3,4-dihydroxyphenylethylamine DAB diaminobenzidine D.D double distilled DMI desipramine DOPA /3-3,4-dihydroxyphenylalanine DOPAC 3,4-dihydroxyphenylacetic acid DRG dorsal root ganglion E embryonic day, gestation day EAA excitatory amino acids EDTA ethyleneaminedisodium tetra acetic acid EGF epidermal growth factor FBS fetal bovine serum FGFb basic fibroblast growth factor Flux fluoxetine Gal galactose GalNAc N-acetylgalactosamine Glc glucose GFAP glial fibrillary acidic protein gP glycoprotein GS goat serum h c io 4 perchloric acid HPLC high performance liquid chromatography HS horse serum HVA homovanillylmandelic acid ICV intracerebral ventricle Ig immunoglobulin

xiv IGF-I insulin-like growth factor-I IS internal standard M molar MAO-A monoamine oxidase A MAO-B monoamine oxidase B MEM minimum essential medium MPDP l-methyl-2,3-dihydropyridinium MPP+ l-methyl-4-pyridinium MPTP l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine N normal NADH reduced nicotinamide adenine dinucleotide NAN N-acetylneuraminate NGF nerve growth factor NGFr nerve growth factor receptor NE norepinephrine NT-3 neurotrophin 3 NT-4 neurotrophin 4 NT-5 neurotrophin 5 ODS octyl docyl sulfate PAP peroxidase anti peroxidase complex PB phosphate buffer PBS phosphate buffered saline PC 12 phyochromocytoma cell line PD Parkinson disease PDGF platelet derived growth factor Pen/Strep penicillin/streptomycin Pmol picomole PNS peripheral nervous system Prot protein psi pounds per square inch rpm round per minute SEM standard error of mean TH tyrosine hydroxylase trk protooncogen containing tyrosine kinase [3H] DA tritiated dopamine 5-HT serotonin 6-OHDA 6-hydroxydopamine

XV INTRODUCTION

A. Pathophysiology of Parkinson’s Disease

Paralysis agitants type of Parkinson’s disease (PD) is a disorder of unknown etiology characterized clinically by: (1) a coarse rhythmic tremor of the hands, face and tongue, (2) akinesia, with loss of spontaneous movements, masked faces and poverty of voluntary movements and (3) intermittent or "cogwheel" rigidity [1].

Biochemically PD is associated with a dopamine (DA) deficiency in the nigrostriatal dopaminergic neurons, due to a loss of the DA containing cells in the substantia nigra pars compacta (Figure 1). Indeed, the most striking anatomic changes in

Parkinson’s disease are seen in the substantia nigra. Under gross examination the substantia nigra of Parkinson brains appears to be atrophic and pale [1], Under light microscopy there is a loss of most of the pigmented neurons, glial proliferation and phagocyte accumulation that contain the displaced pigment. Lewy bodies are the pathognomonic pathological finding of PD. They are spherical, eosinophilic, intracytoplasmic inclusions, composed of densely aggregated filaments, and are found in many of the residual neurons. Less dramatic, nonspecific degenerative changes are seen within the globus pallidus and, to a lesser extent, within the caudate nucleus and putamen [1,2].

1 2 Dopaminergic Pathways in the Brain

Zono compoc'o Olfodory iubercl Median eminence \ lnterpedunculor nucleus Caudate nucleus j Arcuaie nucleus of Cemrai amyoaaioid nucleus nypothaiomus

Figure 1: Dopaminergic pathways in the brain, shown in a representative sagittal section of the brain of the rat. A8, A9 and A10 refer to identified groups of dopamine-containing neurons. A8 and A9 are in the substantia nigra and A10 is in the ventral tegmentum (modified from Ungerstedt, 1971), [3]. 3 Early in the research for the etiology of PD it was realized that the postmortem pathological and neurochemical findings alone were not enough to shed light into the pathogenic mechanism(s) of the disease [4]. The development of animals models for PD offered an alternative way for comparing neurochemical and pathological changes with the clinical state in humans. Primates, cats, rabbits, rodents, and other species have been used effectively for identifying the neurochemical events that accompany specific morphological changes and even clinical symptoms [4]. From this point of view studies with primates are of great advantage. Histological studies of brains of monkeys with various lesions of nigrostriatal projections have shown that fibers from the substantia nigra to the striatum are interrupted resulting in retrograde degeneration and eventual death of the nigral dopaminergic neurons [5]. This is evident by a reduction of the number of tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra and a significant decrease in the concentrations of DA in the striatum [6,7]. The loss of cell bodies and of DA content in the terminal fields parallel one another. In addition to DA, its metabolites 3,4-dihdroxyphenylacetic acid (DOPAC) and homovanillyl- mandelic acid (HVA), as well as its synthetic enzymes TH and aromatic L-amino acid decarboxylase (AAAD) are also decreased [8].

Over the years a number of different approaches have been used to lesion the nigrostriatal neurons, e.g. knife lesions, electrolytic lesions as well as selective neurotoxins as 6-hydroxy dopamine (6-OHDA) [9]. The discovery that l-methyl-4- phenyl-l,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans helped 4 the development of a new animal model of parkinsonism that has been extremely useful for primate studies [10,11,12,13]. Monkeys given MPTP systemically exhibit an extrapyramidal syndrome that resembles the human disease [14]. This syndrome is accompanied by a rather selective decrement of DA and its metabolites in striatum

[11,15,16,17]. Changes in brain norepinephrine (NE) and serotonin (5-HT) have been also reported [18,19,20]. The pars compacta of the substantia nigra is depigmented as a result of MPTP toxicity and there is a loss of DA neurons. This syndrome has been prevented by prior treatment of animals with the monoamine oxidase (MAO) inhibitors pargyline or deprenyl [8,21,22,23]. MPTP becomes neurotoxic only after its intracellular stepwise oxidation to l-methyl-4-pyridinium,

(MPP+) by MAO-B [21,22]. The cause of most cases of Parkinson’s disease is unknown, but the MPTP model suggests that some type of environmental toxicant might play a role.

B. MPTP: a Selective Neurotoxin for Dopaminergic Nigrostriatal Neurons

l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine, is a by-product of the illicit synthesis of a meperidine derivative that has been demonstrated to induce parkinsonism in both humans and experimental animals by destroying the melanin- containing dopaminergic neurons in the substantia nigra pars compacta [24]. While

MPTP neurotoxicity is directed selectively at the dopaminergic nigrostriatal tract, dopaminergic neurons in the limbic system, as well as central NE, and 5-HT neurons are apparently spared [11]. The toxicity of MPTP can be blocked by inhibition of 5 MAO-B (Figure 2) [8,12,23]. Therefore, it appears that MAO plays a central role in MPTP neurotoxicity. Because of its high lipophilicity, MPTP readily enters the brain, where it is preferentially oxidized to MPP+ by MAO-B present in glia and serotonergic neurons [24], The oxidative products MPDP+ and MPP+ then gain access to the brain’s extracellular space. MPP+ is then transported by the dopaminergic uptake system into the dopaminergic neurons [24], where via an uptake mechanism enters the mitochondria [25,26]. Within the mitochondria, MPP+ blocks electron flow from reduced nicotinamide adenine dinucleotide (NADH) to coenzyme

Q at or near the same site as do the mitochondrial toxins rotenone and piercidine

[27]. This alters the oxidative phosphorylation within the neurons. Since the melanin-containing dopaminergic neurons are highly dependent for their energy on the adenosine triphosphate that is generated through NADH-dependent oxidation, inhibition of NADH ultimately leads to neuronal death. Evidence also exists that

MPTP induces oxidative stress in the brain. For example, MPTP lowers glutathione levels in DA neurons [13]; glutathione or antioxidants protect dopaminergic neurons from MPTP neurotoxicity [13]; and finally there is increased MPTP toxicity in animals with vitamin E deficiency [28]. There is now accumulating evidence that free radicals can be generated during the biotransformation of MPTP to MPP+ [13].

Embryonic mesencephalic cultures are sensitive to the neurotoxic effects of

MPP+, and this model has been used successfully for studying the mechanism(s) of

MPP+ toxicity and the effects of various factors in preventing or restoring dopaminergic neuronal function after a MPP+ lesion [17,22,23,28]. 6 MPTP Biotransformation

a s t r o c y t e s

MPTP MPDP

SEROTONERGIC MPOP* MPP' catechqiaminergic NEURONS NEURONS iSuDsunua niqrai OA MPP

M A O B

HO M A O A m 0

HO HO

CH CH. CH, OA Oinvorosyonanvi |or otnar am mtii acaiaidarwaa MPTP MPDP* MPP MPP

MPTP MPOP MPP

OA

Figure 2: Hypothesis of MPTP biotransformation [29]. 7 Studies with primary cultures derived from embryonic midbrain have shown that: (1) MPTP is metabolized by the cultured cells and the metabolism can be partially antagonized by inhibition of MAO-B; (2) the metabolite formed diffuses readily into the culture medium; (3) increase in the amount of metabolite in the feeding medium parallels an increase in the toxicity of dopaminergic neurons; and

(4) reduction in the number of glial cells present in the culture suppresses the metabolism of MPTP [22].

C. Neurotrophic Factors

1. General concepts

Neurotrophic factors have been defined as "endogenous, freely diffusible proteins able to promote survival, growth and funcdon of developing or adult neurons". Very little is known about the function of neurotrophic factors in the adult or aging nervous system. Most of our knowledge about neurotrophic factors has been gained from studies using fetal or neonatal tissues and there is no doubt that neurotrophic factors play an important role during neuronal development. The number of known neurotrophic factors still remains relatively small but increasing.

Mechanisms and functions attributed to trophic factors have been studied thoroughly mainly for nerve growth factor (NGF) [30,31,32].

The central nervous system (CNS) in vertebrates derives from neuroepithelial cells contained within the embryonic neural tube. The developmental processes by which these neuroepithelial cells give rise to neurons and glia of the 8 mature CNS are characterized by a series of progressive stages: proliferation, differ­ entiation, migration, and selective neuronal survival. In the mouse, for instance, the major proliferative phase begins just after neuronal tube closure at embryonic day

10 (E10) and continues until E16 [30]. Studies examining the expression of neuronal and glial markers indicate that most neurons are differentiated by E16, while the differentiation of astrocytes and oligodendrocytes begins after E16. The same developmental sequences has been shown to be preserved when neuroepithelial cells and neurons are grown m vitro [33,34], suggesting that the regulation of these events is controlled either by endogenous factors and/or by other cell-autonomous programs of differentiation [30]. Trophic influences are considered to be involved in cell death or maintenance and participate in such functions as proliferation, hypertrophy, elongation and directional guidance, acquisition of functions and magnitude of functional activities. Trophic influences are defined as directed toward the qualitative regulation of the anabolic machinery of the cell, thus being equally responsible for decline and death (inadequate trophic supply), survival and maintenance (moderate supply), or overproduction (high supply) for growth or secretory activities [35]. The study of neurotrophic factors may lead to the development of a new, structurally oriented neuropharmacology. These neurotrophic factors may be useful in the treatment of neurodegenerative diseases which are associated with structural disintegration of selected neuronal system or brain areas

[36]. However, little information is available concerning the kinds of insults that growth factors may protect neurons from and the mechanism(s) involved. 9 Furthermore, it is unclear whether data obtained from animal studies are directly applicable to human central neurons [37].

2. Models for studying neurotrophic factors

Neuronal and glial progenitors can survive in vitro, even though this system allows only a limited number of cell divisions [38]. Most studies looking at the mechanism(s) of action of various trophic factors have been carried out on primary brain cultures. The major advantage of the culture systems is the ability of controlling the chemical and cellular environment in a way not possible in vivo.

Primary cultures however have disadvantages: 1) cell cultures are heterogenous and this may pose the problem of identifying the cellular origin of a given response to trophic factors. This disadvantage has been overcome in pure monolayer cultures of astrocytes and neurons to study these two cell populations separately [38,39] and 2) the projection fields are missing. Cocultures have been used successfully to overcome this problem. While cell culture is the most common system used for studying neurotrophic factor action and mechanism, it is obvious that the final answer to the questions requires both in vivo and in vitro studies [39]. 10 D. GM1 Ganglioside is a Potential Neurotrophic Factor Modulator for

Nigrostriatal Dopaminergic Neurons

1. Definition and distribution

Among the compounds that were recently shown to protect brain tissue from various types of injury, gangliosides are of special interest [30], Gangliosides, sialic acid-containing glycosphingolipids, are important constituents of all vertebrate cell membranes, but are particularly abundant in CNS where they are associated with nerve ending cell membranes [40,41]. The composition of the glycosphingolipids is cell type-specific and undergoes marked changes during development and transformation [40]. These glycosphingolipids have been implicated in various cell- surface phenomena, such as recognition of various bioeffectors, such as bacterial toxins and hormones [42,43], antigenic sites, ion binding and biotransduction of membrane mediated information. In the nervous system, gangliosides have been shown to modulate receptor function, neurotransmitter release [44,45,46], and promote cell proliferation and/or neurite formation and protein phosphorylation

[47,48,49].

2. Monosialoganglioside GM1

GM1 (II3NeuAc-GgOse4Cer) (Figure 3), is one of gangliosides present in

CNS. Numerous studies have shown that GM1 enhances neuronal survival and sprouting and facilitates the recoveiy of neurochemical, pharmacological and Structure of GM1 Ganglioside

Figure 3: Structure of ganglioside GM1. Abbreviations used: Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; NAN, N-acetylneuraminate; Cer, Ceramide. The types of linkages between the sugars-such as /31,4-are noted at the bonds joining them [52]. 12 behavioral impairments associated with a variety of lesions [50]. The effects of GM1

are not neurotransmitter neuron selective. The following is a summary of reported

effects of GM1 on lesioned DA nigrostriatal neurons in vivo. (1) GM1 stimulates

the regeneration of DA neurons in the CNS [51], (2) GM1 increases the survival of

dopaminergic neurons (3), GM1 favorably influences striatal energy metabolism,

blood flow, cyclic adenosine monophosphate (cAMP) levels, and phosphorylation

[53], (4) reverses the impairment in learning a complex spatial task seen in rats with

bilateral lesions of the caudate nucleus [41] and (5) facilitates the recovery of

dopaminergic neurochemical, morphological and behavioral markers in rodents

treated with MPTP [54,55,56,57,58,59]. The later studies were recently extended to

primates [60], In addition to facilitating the recovery of the dopaminergic system

after a lesion, GM1 also stimulates the recovery of serotonergic [61], noradrenergic

[62] and cholinergic [63,64,65,66] neurons after surgical or neurotoxin lesions.

Moreover, GM1 attenuates the neurotoxicity of glutamate and facilitates the recovery

of neurons after a kainate injury [67].

The basis of recovery is unknown. GM1 has apparently antineurotoxic

effects. But in addition it may have a neurotrophic-like effect by stimulating neuronal regrowth and protecting against secondary degenerative changes that follow neuronal injury, or both [49].

Gangliosides modify the physicochemical properties of the membranes where they are inserted, such as fluidity or thermotropic properties, and affect the activity of a number of membrane bound enzymes [68]. The morphological changes induced 13 by gangliosides depend on exogenous calcium and involve modest enhancement of

Ca2+ influx. Therefore, the increase of the amount of cell surface GM1 could be the

factor promoting Ca2+ influx and resultant differentiation [50]. In addition, it has

been hypothesized that gangliosides may prevent excitatory amino acid (EAA)

neurotoxicity by modulating the Ca2+ influx and preventing delayed neuronal death

[67,69].

Gangliosides have been shown to modulate neurotrophic factor receptor

affinity by facilitating the phosphorylation of receptor tyrosine kinases. There is

evidence now that gangliosides regulate receptor characteristics for epidermal growth

factor (EGF), platelet derived growth factor (PDGF) and insulin-like growth factor

I (IGF-I). It is possible that systemically administered GM1 might change the

number and/or the affinity of receptors for endogenous neurotrophic factors thus potentiating their effect.

E. Nerve Growth Factor as a Putative Neurotrophic Factor for Injured

Dopaminergic Nigrostriatal Neurons

Neurotrophic factors are involved in the development and maintenance of both the peripheral and central nervous system [70,71,72]. During brain development, the neurotrophic factors of the NGF-related gene family products, now termed "neurotrophins" [73], NGF, brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and neurotrophin-5 (NT-5) display distinct stage-specific and tissue specific patterns of expression [74]. The highest 14 level of NT-3 mRNA is found shortly after birth, whereas maximal expression of

BDNF and NGF mRNAs is observed 2 and 3 weeks postnatally, respectively [75].

The different neurotrophic factor mRNAs are transiently expressed in several brain regions during development with highest levels in the hippocampus [76,77,78].

However, the distribution of cells expressing BDNF, NGF, and NT-3 mRNA in adult rat hippocampus is unique for each factor [33,79]. Outside the hippocampus, BDNF mRNA has the most wide spread distribution and has been found in many brain regions including neocortex, piriform cortex, and amygdala [33,79,80]. From in vitro studies it is clear that all members of the neurotrophin family promote the survival and neurite outgrowth of neuronal crest-derived sensory neurons in the dorsal root ganglion (DRG) whereas BDNF and NT-3, but not NGF, support placodally derived sensory neurons of the nodose ganglion [73]. The functions of BDNF and NT-3 in

CNS are not yet known, but BDNF increases the survival in culture of retinal ganglia cells [81,82], basal forebrain cholinergic neurons [34], and ventral mesencephalic dopaminergic neurons [83,84]. The best characterized neurotrophic molecule is

NGF.

1. NGF sources and isolation:

NGF represents the prototype of target-derived, retrogradely transported neurotrophic molecules [85]. Typically, the trophic activities of such molecules are limited to subsets of peripheral and central neurons [86]. Trophic factors initiate their effect by binding to low affinity receptors. In the case of NGF this is followed 15 by uptake and retrograde transport to the cell body. The retrograde transport of exogenous NGF has been well documented in sympathetic and neuronal crest derived sensory neurons as well as for septal and basal cholinergic neurons [39]. Similarly, retrograde transport of endogenous NGF has been demonstrated [87], indicating that neurons do transport NGF under physiological conditions.

NGF has been isolated from submaxillaiy glands of the adult male mouse, snake venoms, guinea pig prostate gland, goldfish brain, rabbit prostate, bull semen, seminal plasma, and sarcoma tissue extract. The NGF protein can be isolated from the mouse submaxillary gland homogenate at neutral pH as a complex of three subunits (alpha, beta and gamma) that can be separated by ion-exchange chromatography. The stoichiometry of the complex is a2/372, and its molecular weight is approximately 13,000 dalton (Figure 4) [88]. Its sedimentation coefficient is 7S.

The 7S complex also contains one or two zinc ions [89]. The purified beta subunit is entirely responsible of the NGF biological activity [88]. The beta subunit is a dimmer of two identical chains held together by noncovalent forces. Each monomer is composed of 118 amine acids; the molecular weight of the dimmer is 26,518 and the PI is 9.3. There are both low and high affinity receptors for NGF (NGFr)

[90]. Low-affinity NGFrs have been shown to be associated with both neuronal and non-neuronal cells, while high-affinity NGFrs with neurons [91,92,93]. It appears that the high affinity NGFrs mediate the trophic activity of the factor [90]. Nerve growth factor receptor immunoreactivity has been found in neuronal cell bodies, axons and/or dendrites of the following brain areas: olfactory bulb, medial septum, islands NGF Structure

EZ] (EG

•fl DIMER CI\ (EG —_T_.

P dimer 2 7 subunits 2 a subunits (eacli chain M, 13.259) Mr - 26,000 Mr - 26,000 Nerve growlli facior Trypsinlike enzyme with arginine esteropeptidase activity

Figure 4: The active component of nerve growth factor is the beta subunit of a large prohormone. A. The prohormone is cleaved into the five subunits, which represent three types of peptides: 2 alpha, 1 beta, and 2 gamma. (Courtesy of D. Ishii). B. The amino acid sequence of a monomer of the beta subunit, which is the active growth-promoting component of the larger protein precursor. (From Angeletti and Bradshaw, in Patterson and Purves, 1982) [94]. 17 of Calleja, nuclei of diagonal band of Brocca, ventral pallidus, nucleus basalis of

Meynert, globus pallidus, entopeduncular nucleus, lateral hypothalamic area, medial amygdaloid nucleus, caudate-putamen complex, hippocampal formation, nuclei of the optic tract, substantia innominata, arcuate hypothalamic nucleus and cerebellum [95].

There is now accumulating evidence that the product of the tyrosine kinase, trk, family proto-oncogenes is necessary for the mediation of the trophic activity for the neurotrophin family of trophic factors. It appears that trkA [96,97], trkB [98,99], and trkC [100], are distinct, biologically related tyrosine kinase receptors that selectively bind a class of neurotrophins, although there is some degree of cross-talk

[87]. The current thinking is that all neurotrophins bind to the low affinity NGFr and that the presence of the specific trk product makes the neurons responsive to the particular neurotrophin. The high affinity NGFr-trk dimmer is believed to be responsible for mediating the biological activity of the neurotrophins. It is not known, whether cells transporting the individual neurotrophins contain mRNA and protein for their cognate trk receptor molecules and whether a single neuron might contain more than one trk. In addition, the question arises as to what role the low affinity NGFr plays in mediating transport and subsequent responsiveness of adult neurons to the various neurotrophins. It is known that BDNF, NT-3, NGF, NT-4 bind to the low affinity NGFr with comparable affinity [98,101,102] and that low affinity NGFr is itself retrogradely transported in peripheral and central NGF- responsive neurons [103]. 18 2. NGF mechanism(s) of action:

NGF is believed to play a role in maintenance of neuronal survival, promotion of neurite outgrowth, cell hypertrophy, induction of neurotransmitter synthesis enzymes, chemotropism, and cell differentiation [104]. There are multiple sites at which NGF can interact with its target cells: plasma membrane, cytoplasm and nucleus. One school of thinking is that NGF by binding to its receptors triggers a cascade of events that are mediated via second messenger systems. For example, cAMP has been shown to play a role for the NGF-induced regulation of neurite outgrowth, cell size, glycolipid content and neuropeptide synthesis [105,106,107,108].

Other investigators claim that the surface membrane receptors function only as carriers to facilitate the internalization of NGF and the primary site of action of

NGF are the cytoplasm and/or nucleus. A compromising approach is that each of the multiple sites of interactions plays a role in the NGF actions. Such a concept is consistent with the recent demonstration [109] that NGF-stimulated neurite outgrowth in pheochromocytoma cells (PC12) requires both transcription-dependent and independent pathways [39]. The fact that the biological effects exerted by this growth factor are numerous and qualitatively different support the notion that they are not elicited through the same molecular mechanism. Each distinct type of target cell may be genetically programmed to respond to NGF in a different fashion. Thus it is possible, for instance, that distinct molecular pathways are activated by NGF (1) in neurite outgrowth, (2) during chemotactic action on the growth cone [110], (3) in 19 the mitogenic effect on chromaffin cells [111], or (4) in the numerical increase of

most cells [112,113].

F. Epidermal Growth Factor is a Neurotrophic Factor for Dopaminergic

Neurons

Epidermal growth factor (Figure 5), like NGF originally found in the

submaxillaiy gland [114], has been shown to be a potent mitogen for many cell types

[115] and to have potential trophic action for neurons [116]. The above observations

raise the possibility that this molecule might be involved in the regulation of CNS

histogenesis [117]. During CNS histogenesis, an enormous diversity of different

neuronal cell types are generated from the multipotent neuroepithelial cells. Perhaps

mitogenic growth factors favor the production of particular classes of neurons or glia.

EGF has been identified in mammalian brain by immunohistological [118] and by

EGF mRNA in situ hybridization techniques [119]. Beginning at 15 days after birth,

EGF mRNA can be detected in the globus pallidus, endopeduncular nucleus, substantia nigra pars reticulata, ventral pallidum, and to a lesser extent in the stria terminalis and layers I-V of central cortex of rats [117]. Furthermore, EGF-like immunoreactivity is enriched in brain synaptosomes [120]. In cultures of brain cells,

EGF has been shown to promote neuronal survival and neurite outgrowth of dissociated neonatal rat brain cells in serum-free medium [121]. Receptors for EGF are present in the brain of various animal species [122,123,124,125] and appear to be located on both neuronal and glial cells [125,126]. Recently, a substrate for the 20 EGF Structure

NH,'

Figure 5: Amino add sequence of eoidennai growth factor. (After C.R. Savage. Jr., J.H. Hash, and S. Cohen. J. Bioi. Chem. 248:7669, 1973) [52], 21 tyrosine kinase activity of the EGF receptor has been found near the germinal zone

of developing brain [127]. Epidermal growth factor apparently stimulates DNA

synthesis [128] and cell division [129,130] in glial cells [131] or can increase glial enzymatic activities without stimulating DNA synthesis, as DNA synthesis is not necessaiy for EGF action [132]. EGF enhances survival and process outgrowth of cortical [131] and cerebellar [133] neurons in culture. There are reports that EGF moderately stimulates the uptake of DA in mesencephalic cultures and that this effect is dependent on the presence of glial cells [121]. Furthermore there are reports that EGF facilitates the recovery of lesioned dopaminergic neurons in vivo

[116], Thus in addition to its mitogenic and hormonal activities, EGF may act as a neurotrophic factor for selective neuronal populations. 22 G. Specific Aims and Significance

The aims of this project are:

I. To explore whether GM1 ganglioside can restore the function of embryonic

dopaminergic neurons in culture after a MPP+ lesion.

In vivo studies have shown that the depletion of DA, the rate of formation of DA, the size (but not the number) of TH-containing cells, and behavioral deficits induced by MPTP can be corrected by administering GM1 [134]. Reversal by GM1 is apparently not related to blockade of MAO B or DA reuptake. In addition, GM1 has been reported to enhance [3H]-DA uptake and survival of the dopaminergic neurons in culture in a time- and concentration-dependent manner [135]. These observations taken together, suggest that GM1 exerts some trophic influences on dopaminergic neurons during development and after injury. Some studies done in our laboratory and other laboratories are suggesting that GM1 might not be a trophic factor per se, but might modulate other trophic factors and causes neuronal trophic responses only in a proper environment(s). Many questions must be answered regarding the mechanism(s) of GM1 action in order to reach a reasonable conclusion about the therapeutic potential of this compound. Thus, the general aim of using

GM1 is to gain insight into the mechanism(s) of action of GM1. In particular:

(1) Does GM1 prevent or restore the function of dopaminergic neurons exposed

to MFP+? In other words, does it have an antineurotoxic and/or a

proneurotrophic action? 23 (2) Does GM1 rescue MPP+ injured neurons?

(3) Does GM1 exert its action directly on neurons or indirectly via glial

cells?

II. To explore whether NGF or EGF can restore function in cultures of

embiyonic dopaminergic neurons treated with MPP+.

NGF is a known neurotrophic factor for acetylcholinergic neurons in CNS and for sympathetic neurons in the peripheral nervous system (PNS) [136]. The current dogma is that NGF has no effect on central catecholaminergic neurons.

However, in vivo studies in this laboratory showed that NGF caused a remarkable recovery of dopaminergic neurons after a MPTP lesion of nigrostriatal neurons.

Therefore, it was critical to investigate the action of NGF on embryonic dopaminergic neurons in culture after lesioning with MPP+. It is unkown how NGF acts on this system and whether it exerts its actions directly on neurons or indirectly via other factors secreted by other cells (e.g. glia) present in the culture system.

Therefore, it was of interest to know:

(1) Whether NGF had any effect on dopaminergic neurons in culture.

(2) Whether NGF could prevent or restore the function of dopaminergic

neurons exposed to MPP+.

(3) Whether its actions were direct or indirect. 24 Similar questions were asked for EGF. EGF has been shown to have trophic activity for dopaminergic neurons in culture as well as other neuronal populations not responding to NGF. The major interest here was to see if EGF was able to restore dopaminergic function after a lesion with MPP+. METHODS

I. Cell Culture Techniques

A. Materials

1. Cell culture medium supplements

Fetal bovine serum (FBS) (Lot# 230-6140AJ), heat inactivated horse serum

(HS) (Lot# 230-6050AJ), Penicillin-Streptomycin, a stock solution of 10,000U/ml of penicillin G and 10,000 Mg/ml streptomycin, L-glutamine and minimum essential medium (MEM) (IX) were obtained from GIBCO, Grand Island, N.Y. D-Glucose, tiypsin (10X) (Lot# 79F-4628), cytosine /3-D-Arabino-Furanoside (Lot# 125F-7045) and poly-D-Lysine, mw > 300,000, were obtained from Sigma Chemical Co., St.

Louis, MO, USA.

2. Complete cell culture medium

MEM was supplemented with 5% FBS, 5% HS, 2 mM L-glutamine, 6 mg of glucose per ml, 100 U/ml penicillin and 100 Mg/ml streptomycin.

3. Dissociation solution

Phosphate buffered saline (PBS) pH 7.3, was prepared, with distilled water

25 26 without calcium and magnesium and supplemented with glucose and bovine serum

albumin (BSA) as follows: NaCl 0.14 M, KC1 2.7 mM, Na2HP 04 8 mM, KH2P0 4 1.5 ml, Glucose 5 mM, BSA 0.1 g/1.

B. Methods

1. Coating Petri plates

Thirty five mm culture dishes (Primaria) obtained from Fisher Scientific,

Pittsburgh, PA., were coated with poly-D-lysine 10 /xg/ml boric acid solution, pH 8.0, for at least 2 h at 4°C. Just prior to plating, dishes were rinsed gently with sterile distilled water 3 times and once with culture medium.

2. Preparation of mesencephalic cells

Timed pregnant Sprague-Dawley rats (Zivic Miller, PA, USA) 15 days in gestation were used for the establishment of mesencephalic cultures. The cultured embiyonic cells exhibit the normal characteristics of growing DA neurons including the presence of uptake and storage mechanisms for [3H]-DA, the presence of endogenous DA, the ability to release DA on depolarization and the accumulation of HVA due to the normal turnover [137]. The following method was used to dissect defined mesencephalic area including the substantia nigra (Figure 6): Rats were sacrificed by cervical dislocation and abdomen wiped with 70% ethanol and rinsed with sterile PBS to remove any remaining ethanol. Under sterile conditions, uterine horns were dissected out and placed into a large sterile petri dish containing a small 27 Schematic Representation of Ventral Mesencephalon

Figure 6: Schematic representation of localization of TH-immunopositive neurons in rat embryos E14. Mesencephalon (stippled area), rhombencephalon (striped area). Dotted line represents the cut made for initial gross dissection of piece containing mesencephalon and rhombencephalon. RI: rhombencephalic isthmus; CF: cervical flexure: PF: pontine flexure: MF: mesencephalic flexure [138]. 28 quantity of cold PBS. Embiyos were removed beginning at the distal end of a uterine

horn and amniotic sacs were peeled off. Heads were cut off and placed into a 35

mm petri dish containing 1.0 ml PBS. Under a dissecting microscope brains were

exposed by peeling off the skin and meninges using fine forceps taking care not to

damage the neuronal tissue, notably in the region of the pontine flexure, which is

particularly vulnerable due to its ventral position. The ventral two thirds of the

mesencephalon were pinched off and cleaned from the meninges and surrounding

tissue. Tissue pieces were collected in a 50 ml centrifuge tube containing 2 ml of ice

cold dissociation buffer. Tissues were dispersed mechanically with a fire polished

Pasteur pipette; tissue pieces were allowed to settle in the bottom of the tube at 4°C

and the supernatant containing suspended cells was collected. Five ml of dissociation

buffer was added to undissociated tissue and again triturated with a smaller caliber

Pasteur pipette. Supernatants were pooled, centrifuged for 5 min at 70 x g and pellet

resuspended in 10 ml completed growth medium. The number of viable cells was

counted as indicated below. One to one million and a half cells were plated per dish

in complete medium and incubated at 37°C in atmosphere of 5% C 02, 95% air and

100% relative humidity. Feeding medium was replaced with fresh medium every 3

days. Day 0 is considered as plating day. For immunocytochemistry, cells were

grown on precoated 22 x 22 mm glass coverslips and treated as described previously.

3. Preparation of neuron rich mesencephalic culture

The same technique was followed as in step B.2. On day 3, 1.25 juM of 29 cytosine £,D-arabino furanoside (Ara-C) was added to the cultures for 24 h. Then, plates were rinsed once with fresh culture medium to remove any remaining Ara-C, fresh completed medium was added and incubation continued under the above men­ tioned conditions.

4. Preparation of glia rich mesencephalic cultures

The same technique as in step B.2 was followed. On day 2 after plating plates were rinsed 3 times with sterile PBS at 4°C and then incubated in 0.1% trypsin solution at 37°C for 10 min. Cells were detached from the plates and collected in feeding medium and cell suspension centrifuged at 50 x g for 5 min. The resulted pellet was dispersed in growth medium consisting of MEM supplemented with 5% horse serum and 5% fetal calf serum and plated in 35 mm plates precoated with poly-D-lysine. Cells were fed eveiy 3 days with fresh supplemented medium.

II. Immunocytochemistiy techniques

A. Materials

The TH antibody used was a mouse monoclonal antibody (LNC 1) of IgGl class directed against TH purified from PC12 cells. This was a gift from Dr. G.

Kapatos (Wayne State Univ., Detroit, MI). Antibodies against glial fibrillary acidic protein (GFAP) IgGl, clone G-A-5,(Lot# G-3893), Sodium azide, BSA, and 3,3’- diaminobenzidine (DAB) (Lot# 51H3610), were obtained from Sigma Chemical Co. 30 St. Louis, MO. Goat anti-mouse (IgG + IgA + IgM) both (H + L) antibody (Lot#

65-6400), and a peroxidase anti peroxidase mouse monoclonal antibody (PAP) (Lot#

80-6520), were obtained from Zymed, San Francisco, CA. Goat serum (GS) was

obtained from GIBCO, Life Technologies, Inc., Grand Island, N.Y. Hydrogen peroxide 30% was obtained from Fisher, NJ. Paraformaldehyde was obtained from

Baker, Phillipsburg, NJ.

Buffers

Sodium phosphate buffer (0.1 M), pH 7.4, was prepared as follows: NaH2P0 4

(0.2 M) 95 ml/1, Na2HP 04 (0.2 M) 405 ml/1.

B. Methods

1. Tyrosine hydroxylase immunocytochemistiy

A method described by Beck et al. [139] was used for the immunocyto- chemical studies. Briefly, cultures were washed with PBS containing CaCl2,l mM,

MgS04, 1 mM, and glucose 5 mM, fixed with freshly made 4% paraformaldehyde in phosphate buffer for 30 min, washed again with PBS. Then they were incubated for

24 h with (LNC 1) (1:1000) anti TH monoclonal antibody, diluted in PBS containing

2% bovine serum albumin, 0.2% Triton X-100, 0.02% sodium azide, and 1% GS.

After washing, cultures were incubated for 2 h with anti- mouse monoclonal antibody

(1:100) dissolved in PBS containing 0.2% Triton X-100. Cultures were washed again and incubated for 2 h with mouse PAP (1:100). The peroxidase was visualized by 31 incubation with a solution of 1 mg/ml of DAB and 0.015% hydrogen peroxide in

PBS. For negative controls, the anti-TH monoclonal antibody was omitted. Whereas

for positive control, various dilutions of TH antibodies from 1:10 up to 1:1000 were

used. TH-immunopositive cells were studied using a bright field Zeiss microscope.

An image analyzer was used for the morphometries and the photography.

2. Glial fibrillary acidic protein immunocytochemistiy

GFAP was visualized immunocytochemically according to previous methods

[140,141]. Cultures were fixed with a mixture of 3 parts of acetone and 2 parts of

ethanol for 2 min at room temperature and then incubated for 12-24 h at 4°C with

mouse monoclonal antibody to GFAP (1:500) dissolved in 0.1 M sodium phosphate buffer, pH 7.4, 5% sucrose, 5% bovine serum albumin, 0.1% Triton X-100 and 1% normal goat serum. Cultures were washed 3 times, 5 min each, at room temperature

in PBS and then incubated with 0.2 ml goat anti-mouse IgG (1:200) for 2 h at 37°C.

Then they were washed 3 times in PBS at room temperature, incubated for 30 min with 0.2 ml mouse PAP (1:200) at 37°C and then washed again in PBS. The peroxidase was visualized by incubation with a solution of 1 mg/ml DAB and 0.015% hydrogen peroxide in PBS for 10 min. After rinsing 3 times with PBS, coverslips were mounted and GFAP immunopositive cells counted. Control cultures were incubated as above with the exception that the monoclonal antibodies for GFAP were omitted. 32 III. Dopamine uptake

A. Materials

Desipramine hydrochloride (DMI) was obtained from Merrell Dow Research

Center, Cincinnati, OH. 3,4-Dihydroxyphenylethylamine (DA) (Lot #39F-0287), pargyline (Lot# 94F-7702) and ascorbic acid (Lot# 18F-0523), were obtained from

Sigma, Sigma Chemical Co. St. Louis, MO. Benztropine (BZT) mesylate (STK#

86509) was obtained from Merck Sharp and Dohme Research Lab. West Point, PA.

Fluoxetine hydrochloride (Lot# 866-83F-176) was obtained from Eli Lilly and

Company, Indianapolis, IN. [3H]- DA was obtained from Dupont, Boston, MA.

ScintiVerse E was obtained from Fisher Scientific, Fiar Lawn, NJ.

B. Methods

Cells were rinsed 3 times with washing buffer (PBS supplemented with glucose

5 mM, CaCl2 1 mM, MgCl2 1 mM), at room temperature. Cells were preincubated at 37°C for 5 min with 800 /xl incubation buffer containing 0.1 mM pargyline, 0.1 mM ascorbic acid, 5 /xM DMI, and 1 /xM fluoxetine. [3H]-DA (37 Ci/mmol) was added to give a final concentration of 25 nM and the cultures were incubated at 37°C for

15 min. Uptake was stopped by removing the incubation buffer, immediately followed by 5 washes with ice cold washing buffer. Cells were then lysed and removed from the plates with 200 /xl ethanol/0.4 N perchloric acid solution, HC104,

(1:3). The cell homogenate was centrifuged at 50 x g for 10 min, then the cell 33 suspension was added to 10 ml ScintiVerse E to measure the radioactivity with a scintillation counter. Non specific DA uptake was determined in the presence of 5

»M BZT. To the pellet, 0.1 N NaOH was added and protein concentration was measured using the Lowiy method [142] as described below.

IV. Measurement of dopamine and its metabolites

A. Materials

1. Chemicals

3,4-dihydroxyphenylacetic acid (DOPAC) (Lot# 27-0214), DA, (Lot# 39F-

0287), 3,4-dihydroxyben2ylamine (IS) (Lot# 68F-3675), L-ascorbic acid (Lot# 18F-

0523), Trizma, (Lot# 129F-5610), chromatographic alumina, (Lot# 38F-0865), were obtained from Sigma Chemical Co. St. Louis, MO.

2. HPLC System

A reverse column (Pharmacia, spherisorb ODS-2,5 /xm, 4 x 100 mm) was used for catechol separation. The system was run with citric acetate buffer, pH 3.8. containing 4% methanol and octyl sodium sulphate 32 mg/1, at a pressure of about

1,500 psi. The buffer was prepared as follows: Citric acid, mw = 192.13, 2.4 g/1 was adjusted with sodium acetate, mw= 136.08, to pH 3.8 and degassed. Before use, octyl sodium sulfate 32 mg/1, and 4% methanol were added. 34 3. Reagents

Perchloric acid, 0.2 N: in 988 ml of double distilled water (d.d. H20) add 12

mis 70% HC104 and 10 mg sodium bisulfite. Tris/HCl,0.5 N: 60.55 g/1 Trizma base,

mw=121.1, adjust pH to 8.6 by adding HC1, 12 N dropwise.

4. Activated alumina

In a ratio 1:5 alumina is boiled in 2 N HC1 for 1 h. The acid is then aspirated

off, and alumina washed repeatedly with distilled water until the final pH is higher

than 3.5. The alumina is then activated in an oven for 2 h at 200-300°C and stored

in an anhydrous atmosphere.

5. Standards

Standards for DA, DOPAC and IS were prepared in 10 mM stock solutions in 0.2 N HC104 acid containing ascorbic acid, 1 mg/ml, and kept in aliquots in -20°C.

For the standard curves, serial dilutions of DA (60,30,15,7.5 and 3.75 pmol/40/il injection) as well as serial dilutions of DOPAC (120,60,30,15 and 7.5 pmol/40/il injection) were prepared in 0.2 N HC104 acid containing 1 mg/ml ascorbic acid.

To each sample and standard 10 pmol of IS was added.

B. Methods

Cells were rinsed 3x with ice-cold PBS supplemented with glucose 5 mM,

CaCl2 1 mM, MgCl2 1 mM. Cultures were scrapped in 400 fi\ 0.5 N HC104 35 supplemented with ascorbic acid 1 mg/ml, cells were harvested from the plates,

collected in Eppendorf tubes and homogenized by sonic disruption. Twenty /zl of the

homogenate were removed and stored for protein measurement. Twenty mg of

activated alumina was added into each tube. Tubes were filled with 0.5 N Tris/HCl,

handshaken for 15 min, making sure the alumina fell freely throughout the Tris/HCl

solution. Then alumina was allowed to precipitate and the supernatant was discarded

leaving the alumina intact. The alumina was washed twice with chilled d.d. H20 , 120

y\ of 0.5 N HC104 containing ascorbic acid was added to each tube and vortexed in

order to mix the alumina. Tubes were centrifuged at 18,000 rpm for 10 min.

Supernatant was transferred to HPLC vials, trying not to transfer any of the alumina,

and tubes were spun in a tabletop centrifuge to remove any air bubbles at 13,000

rpm for 5 min. Fortyn 1 of the supernatant was injected onto HPLC and catechols separated on a reverse phase column.

C. Calculations

The peaks for DA, DOPAC, and IS were measured from HPLC chromato­ graphs and then the ratios for DA/IS and DOPAC/IS for each sample as well for standards calculated. Using linear regression analysis, the slopes for DA and

DOPAC were determined from standards curves. In order to determine the concentrations (pmol/mg of prot) of DA and DOPAC in the samples, the following formula was applied: 36 peak ratio / standard slope pmol/mg prot = mg prot

V. Aromatic L- Amino Acid Decarboxylase Assay

A. Materials

1. HPLC buffer

The buffer was the same as for DA and DOPAC, but pH 4.0 and methanol

8% was used.

2. Working buffer

Fifty ml of phosphate buffer (14 ml of 0.2 M sodium phosphate monohydrate

+ 36 ml of 0.2 M sodium phosphate dibasic anhydrous) diluted in 150 ml d.d. H20 to a final concentration of 50 mM and pH 7.4.

3. Incubation buffer

Pargyline, 23 /xM, ascorbic acid, 170 /xM, ethyleneaminedisodium tetra acetic acid (EDTA) 100 /xM, mercaptoethanol, 1 mM, D-/3-3,4-dihydroxyphenylalanine (L-

DOPA) 500 /xM, pyridoxal 5’-phosphate, 10 /xM, were added to 200 ml of working buffer. D-DOPA was used for the estimation of blanks. 37 B. Standards

Standards were prepared in 0.2 N HC104 containing ascorbic acid, 1 mg/ml,

and serial DA dilutions, (600,300,150,75 and 37.5 pmol/40 /d injection). To each

sample and standard, 100 pmol of IS was added.

C. Methods

Cultures were washed 3 times in PBS supplemented with glucose 5 mM, CaCl2

1 mM, and MgCl2 1 mM. Cells were harvested in 130 /d sucrose 0.25 mM,

transferred in Eppendorf tubes and homogenized by sonication. Twenty #d of the

homogenate was added to 400 jd of incubation mixture and incubated at 37°C for 20 min. A portion of the remaining homogenate was used for protein determination.

Reaction was stopped by adding 80 jd of stop solution at room temperature. Stop solution was added to the standards as well. Twenty mg of activated alumina was added to each sample and standard in the presence of Tris/HCl, 1.5 ml. After 15 min of handshaking the supernatant was discarded, and the pellet was washed 2x with Tris/HCl and once with d.d. HaO. Finally, 120 /d of 0.2 N HC104 containing ascorbate, 1 mg/ml, was added to the tubes, which then centrifuged at 13,600 rpm for 5 min. From the supernatant, 20 /d were injected into an HPLC with electro­ chemical detection.

D. Calculations

The same procedure applied as in DA estimation in step IV-C. 38 VI. MPP+, GM1, NGF, EGF, Cholera toxin B subunit and anti GM1 antibody

treatment

A. Chemicals

MPTP and MPP+ were purchased from Research Biochemical, Natick, MA.

NGF 7S, NGF 2.5S, and EGF were purchased from Upstate Biotechnological Inc.,

Lake Placid, NY. Cholera toxin B subunit (choleragenoid) was purchased from List

Biological Laboratories, Campbell, CA. GM1 was a gift from FIDIA, Abano Terme,

Italy. Anti GM1 antibodies was a gift from Dr. Latov, Columbia University, NY.

B. Methods

MPTP or MPP+ dissolved in sterile saline was added in culture on day 4 for

24 h. Neurotoxins were removed by washing cultures 3 times with prewarmed growth medium. Trophic factors as well as GM1 were dissolved in growth medium and routinely, were added on days 5 and 8 in culture. Cholera toxin B subunit and anti

GM1 antibody were added at the same time with GM1 as mentioned in the result section. All studies were assessed on day 12 in culture, 7 days of treatment, except if otherwise indicated. 39 VII. Biochemical Analytical Techniques

A. Protein assay

1. Reagents

Reagent 1: 0.1 N NaOH

4% Na2C0 3

0.125 mg Copper Disodium EDTA [( Etylenedinitrilo)-tetraacetic Acid

Copper Disodium Salt]

Reagent 2: Phenol reagent: dilute 2 N Folin and Ciocalteau Phenol reagent in a

1:1 ratio with d.d. H20

2. Preparation of standards

BSA, 1 mg/ml d.d. H20 was prepared as a stock solution, working concentration was diluted 1:1 with 0.1 N NaOH. Serial BSA dilutions (5,10,15,20,25 fig) were used as standards.

3. Procedure

Twentyfil of cell homogenate was solubilized in 80 fil of 0.1 N NaOH overnight. To 20 /xl of homogenate and the BSA standards 1 ml of reagent 1 was added and samples were incubated at room temperature for 30 min. One hundred fil of reagent 2 was added to each tube and incubated for 50 min. Tubes were 40 vortexed following each step. Absorbance was measured at 700 nm using a

spectrophotometer.

B. Statistical Analysis

Data were evaluated by analysis of variance followed by comparison of group differences with a Newman Keuls test RESULTS

A. Primary embryonic mesencephalic cultures

The ventral portion of midbrain of rat embryos contains both A8 and A9 as well as A10 nuclei that represent the DA containing cells of substantia nigra pars compacta and ventral tegmental area, respectively. Culturing this region of the embryonic brain results in a heterogenous culture consisting of at least two cell popultations; neurons, phase bright with several branching and a well-developed neuritic network within few days after plating; and glia, with large dark nuclei that forms a sheet underneath the neuronal population (Plate I). Moreover, in addition to dopaminergic neurons, a number of different neurons are present in this culture, such as gabaergic, noradrenergic and serotonergic, etc. These cultures grew very well in the presence of a coating substrate, poly-D-lysine, serum, and other nutrients present in the medium which enhance cell attachement, growth and maintenance.

Cell density has been reported [143] to be an important and critical variable for cell survival and DA uptake activity. In our cultures, increasing the number of cultured mesencephalic cells resulted in an increase of BZT-sensitive DA uptake (Figure 7).

Mesencephalic cells were seeded at different densities and cultures were evaluated

12 days later. The BZT-sensitive DA uptake per plate

41 42

Development of Mesencephalic Culture

Plate I: Phase-contrast photomicrograph of embryonic mesencephalic cultures; A) 3 h after plating; B) 12 d after plating. (X 250) DA-Uptake as a Function of Cell Density

2.0 t

UJ in +i c 1.5 • r H CD e a : rd in +-> □L 1.0 ID o> I i_ < D Q 4-> i—I D U 0.5 O E CL 0.0 0.05 0.10 0.50 0.75 1.50 2.00 106 cells/culture

Figure 7: Dopamine uptake by rat E15 mesencephalic cultures by increasing cell density. Mesencephalic cells were plated at various densities. DA uptake was assayed on day 12 in culture. Data presented are mean ± SEM. N = 10 plates per group from 3 different experiments.

u> 44 increased with the cell plating density and it was linear up to about 1-2X106 cells per plate. Therefore, this plating density was used for the remaining experiments.

Glia rich cultures were prepared as described in methods, and astrocytes were identified by GFAP immunohistochemistiy (Plate II). Astrocytes reached confluency by about 7 days after plating. Time to confluency depends on cell density at plating. There was a small number of cells bearing neurites, inidicating that the glial cell cultures were not pure and contained about 5-10% neurons.

Neuron rich cultures were isolated from mixed cultures treated with 1.25 fi.M Ara-C, on day 3 in culture for 24 h. The elimination of glia was evaluted by

GFAP immunostaining. The purity of neuronal cultures was more than 90%. The neuron rich cultures tended to clump and send long branches covering the whole area (Plate III). They were easily detached from the plate when kept for an extended period of time compared with the mixed cultures which could survive for longer periods of time.

B. Dopamine uptake

All DA uptake experiments were carried out using a saturation concentration of 25 nM [3H]-DA. DA uptake in mesencephalic cultures was temperature- and sodium-dependent and sensitive to BZT. Uptake of DA at 37°C was about 10-fold greater than that at 0°C (Table 1). The BZT-sensitive portion of

[3H]-DA uptake is a specific marker for dopaminergic cells and a reliable index of 45 GFAP Immunopositive Cells

Plate II: Bright-field photomicrograph of GFAP immunopositive cells of glia rich embryonic mesencephalic culture. (X 1000) 46 Neuron Rich Culture from Embryonic Mesencephalon

Plate HI: Phase-contrast photomicrograph of embryonic mesencephalic cultures; A) mixed culture; B) neuron rich culture. (X 100) 47

Table 1

Characteristics of DA Uptake in Embryonic Mesencephalic Cultures

DA Uptake (pmol/mg prot/15 min ± SEM)

Temperature Na (+) Na (-) BZT (5 nM)

37°C 3.49 ± 0.09 0.51 ± 0.12* 0.50 ± 0.02* 0°C 0.50 ± 0.04*

DA uptake in embryonic mesencephalic cultures was assayed on day 12 in culture. Incubation buffer for DA uptake was modified by omitting Na+ or by adding BZT for estimating non specific DA uptake. Incubation temperature also was modified from 37°C to 0°C. N=9-12 plates per group from 3 different experiments. * p< 0.05 compared with DA uptake activity measured in the presence of Na+ at 37°C. 48 the neurotransmitter in mesencephalic cell cultures (Table 1), [144,145,146].

Typically, BZT inhibited 70-80% of the total uptake.

A small activity of BZT-sensitive DA uptake was found in glia rich cultures

(Table 2). This counted for about 10 % of that found in mixed cultures. There are

no reports of the presence of DA uptake in glia. However, since the culture is

contaminated with neurons it is very difficult to say that the observed DA uptake is

due to glial cells or neurons or both. Elimination of glial cells did not significantly

change the DA uptake activity in neuron rich cultures when expressed per plate.

However, uptake activity was increased when expressed per mg prot, because of the

decrease of protein content/plate due to the elimination of glia.

The DA uptake was culture age dependent; it increased gradually starting on

day 4 in culture and reached a maximum by day 12-14 in culture and then started

declining.

C. MPP+ neurotoxicity

Both MPTP and its neurotoxic metabolite MPP+ decreased DA uptake in our culture system as it has been reported. Due to the fact that MPTP must be convert­ ed to MPP+ in glia, and our interest to study the effects of various trophic factors in neuronal cultures, a decision was made to use MPP+ for the lesioning of the cultures

(Table 3). MPP+ decreased DA uptake in a concentration-dependent manner.

MPP+ in concentrations from 3-10 /iM caused about 40-60% decrease of DA uptake when added for 24 h (Figure 8). 49

Table 2

DA Uptake in Glia Rich Mesencephalic Cultures

DA uptake (pimol/15 mini GFAP (+) prot per plate per mg prot % Mg

Mixed Culture 1.00 ± 0.13 2.25 ± 0.10 30 ± 5 441 ± 48 Glial Culture 0.12 ± 0.03 0.24 ± 0.06 88 ± 7 509 ± 18

Glial cultures were derived from mixed mesencephalic cultures on day 2 after plating as described in Methods. DA uptake, estimation and GFAP immunocytochemistry were performed on day 12 in culture. Data are presented as the mean ± SEM. N = 6-7 plates per group from 2 different experiments. 50

Table 3

MPTP or MPP+Decreases DA Uptake in Embryonic Mesencephalic Cultures

DA uptake (pmol/mg prot/15 min ± SEM)

Medium 2.85 ± 0.17 MPP+ (10 mM) 1.77 ± 0.06* MPTP (10 nM) 2.61 ± 0.04 MPTP (100 nM) 0.97 ± 0.06**

MPP+ or MPTP was added at day 4 in culture and 24 h later was removed by washing. DA uptake was assayed on day 12 in culture. N = 10-12 plates per group from 4 different experiments. * p < 0.05 compared with medium treated cultures. ** p<0.05 compared with medium and MPP+ (10 /iM) treated cultures. MPP+ Concentration-Response Curve

3.0 t LxJ CO +1 2.5- C •H a> e 2 . 0 - n3 in 1.5- 4 -' i O < L Q CL 1 . 0 - CD 0.5- O E CL 0 .0 - Vehicle 0.5 1.0 3.0 10 50 Mpp+ (/xM)

Figure 8: a ^ r d " 1™ d^fincuTtufe1^ T " 1 T ”" c0"Knlrati°"s <* MPP*. MPP* was

Ln 52 In addition, treatment with MPP+ decreased DA, DOPAC, AAAD and TH-

immunopositive cells by about 50% (Tables 4,5,6) without general toxic effects in the

culture as evidenced by nondetectable changes in the protein content/dish and in the

morphological appearance of cells with a phase contrast microscope. Therefore,

MPP+, 3 /xM, added on day 4 for 24 h was used for the remainder of our studies.

D. GM1 effects

Treating E15 mesencephalic cultures with the dopaminergic neurotoxin MPP+,

3 mM, for 24 h on day 4 in culture decreased all of dopaminergic markers evaluated

at later periods of time by 40-60%. GM1 treatment every three days enhanced DA

uptake in MPP+-lesioned cultures in a concentration and time- dependent manner,

(Figures 9, and 10). The response appeared maximal at a concentration of about 500

nM of GM1. Enhanced DA uptake was evident as early as 2 days after adding GMl

to the MPP+- lesioned cultures and reached a maximum about 7 days later, (Figure

9). When control mesencephalic cultures were incubated with GMl there was a small but significant increase of DA uptake when studied 7 days after initiating the

treatment, on day 12 in culture, but not at other earlier days. The response of the

MPP+-treated cultures to GMl was maintained as long as the compound was present in the medium. After removing GMl DA uptake decreased to values found for untreated MPP+-lesioned cultures (Figure 11). GMl did not prevent the MPP+- induced loss of DA uptake activity (Table 7). When GM l was added before or together with MPP+ for 24 h, there was no protection of DA uptake activity 53

Table 4

GMl Enhances DA and DOPAC Content of Mesencephalic Cultures Treated with MPP+

Conditions DA DOPAC (pmol/mg prot ±SEM)

Medium 6.8 ± 0.3 11.8 ± 0.5 GMl 9.5 ± 0.6* 9.2 ± 0.8 MPP+ 2.7 ± 0.3* 4.2 ± 0.5* MPP+ plus GM l 5.2 ±0.5** 9.5 ± 0.2**

Mesencephalic cultures were treated on day 4 in culture with MPP+, 3 ^M, for 24 h. On day 5 fresh medium or GMl, 500 nM, added where indicated. Fresh medium with GMl was added every 3 days. On day 12 in culture DA and DOPAC content were assayed. N = 10 plates per group from 2 different experiments. *p < 0.05 compared with medium treated cultures. **p<0.05 compared with MPP+ treated cultures. 54

Table 5

GM l Enhances Aromatic L-Amino Acid Decarboxylase Activity of Mesencephalic Cultures treated with MPP+

AAAD Activity Conditions (pmol/mg prot/20 min ± SEM)

Medium 3.9 ± 0.2 MPP+ 0.92 ± 0.08* GM l 4.4 ± 0.2* MPP+ plus GM l 1.6 ± 0.1**

Mesencephalic cultures were treated as described in methods. On day 12 in culture AAAD activity was assayed. N=5-10 plates per group from 3 different experiments. *p < 0.05 compared with medium treated cultures. **p<0.05 compared with MPP+ treated cultures. 55

Table 6

GMl Restores the Number of TH-Immunopositive Cells in Mesencephalic Cultures Treated with MPP+

Total TH-positive Percent of cells ± SEM Cells ± SEM Total Cells Conditions (x 104)

Medium 8.3 ± 0.1 819 ± 153 0.99 GMl 9.3 ± 0.3 1187 ± 92 1.27 MPP+ 8.7 ± 0.3 335 ± 35* 0.39 MPP+ plus GMl 8.2 ± 0.6 828 ± 51** 1.00

Mesencephalic cells were grown on coverslips (22 x 22 mm) and treated as described in methods. On day 12 in culture the cells were fixed and the presence of TH was evaluated by immunohistochemistiy. The number of cells on a coverslip was estimated by counting phase-bright cells in a superimposed counting frame and corrected for the size of the coverslip. N=4-6 slides per group from 4 different experiments. * p < 0.05 compared with medium treated cultures. * *p < 0.05 compared with MPP+ treated cultures. GMl Concentration-Response Study

UJ 00 +l c • H CD El ro in

4 -'' I O < (_ Q CL CD

Control 50 100 250 GMl Concentration (nM)

Figure 9: Dopamine uptake by rat E15 mesencephalic cultures treated with MPP+, and GMl ganglioside. After 4 days in culture MPP+, 3 nM, was added for 24 h. Then GMl, 500 nM, was added to the MPP+-treated cultures (solid bars) and to comparison normal cultures (open bars) and DA uptake assayed on day 12 in culture. Data presented as the mean ± SEM. N = 15 plates per group from 34 different experiments. * p<0.05 compared with control without GMl treatment. **p<0.05 compared with cultures treated with MPP+ alone. L r \ 0\ GMl Time-Response Study

UJ • Control + GMl * * on +i O Control c iH 0) ro in Q. □ MPP -M o Q CL cn e

i— i o £ CL 0.0 0 1 3 4 5 6 7 8 9 10 Days After Treatment Figure 10: Dopamine uptake by rat E15 mesencephalic cultures with time following treatment with MPP+, 3 /xM, or MPP+, 3 /xM, plus GMl, 500 nM. Cultures were treated with MPP+, 3 /xM, and GMl, 500 nM, added every three days and the incubation continued. Data are presented as the mean ± SEM for control, GMl treatment only, MPP+, and MPP+ treated with GMl. N = 12 plates per group from 3 different experiments. *p<0.05 compared with MPP+ alone. **p<0.05 compared with control.

Ui -4 Discontinuation of GMl Treatment 1 0 0

r— I O L_ oC o

c CD (U o L.

0 5 10 15 20 25 Days in Culture

Figure 11: Dopamine uptake by rat E15 mesencephalic cultures treated with MPP+ alone, MPP+ plus continuous treatment with GMl or MPP+ plus limited treatment with GMl. Cultures were treated with MPP+, 3 pM, on day 4 in culture for 24 h and then on day 5 MPP+ was removed and GMl, 500 nM, added to two groups. The two groups were maintained with GMl, 500 nM, until day 12 in culture. Then GMl was removed from one group (open circle) while GMl was maintained in the other (solid circles). A comparison group consisted of MPP+ treatment alone (solid squares). Data presented as the mean ± SEM. N=8 plates per group from 2-3 experiments. * p<0.05 compared with corresponding MPP+. ** p<0.05 compared with corresponding MPP+ plus GMl.

Ui 00 59

Table 7 GM1 does not Prevent MPP+-Induced Reduction of DA Uptake in Embryonic Mesencephalic Cultures DA Uptake Conditions (pmol/mg prot/15 min ± SEM) Contrast Culture Medium 2.79 ± 0.12 MPP+ 1.17 ± 0.04* Pre-Treatment GM1 2.90 ± 0.06 GM1 plus MPP+ 1.10 ± 0.06* Co-Treatment GM1 2.96 ± 0.05 GM1 plus MPP+ 1.09 ± 0.04*

Mesencephalic cultures were prepared as follows: Contrast Culture. On day 4 in culture, MPP+, 3 /xM, was added for 24 h, cultures washed and fresh medium added for the remainder of the study; Pre-Treatment. GM1, 500 nM, was added at plating and was present when, MPP+, 3 /zM, added on day 4 for 24 h. The cultures were washed and fresh medium added for the remainder of the study; Co-Treatment, on day 4 in culture both MPP+, 3 mM, and GM 1,500 nM, were added for 24 h, removed and fresh medium added for the remainder of the study. DA uptake was assessed on day 12 in culture. N=12 plates per group from three different experiments. *p<0.05 compared with culture medium alone or with GM1 treatment. 60

compared with cultures that were treated with MPP+ alone. GM1 treatment

elevated DA content in both control and MPP+ lesioned cultures. Only the lesioned

cultures had elevated DOPAC content (Table 4).

Treatment with GM1 increased AAAD activity in control cultures. Addition

of MPP+ decreased AAAD in the cultures and treatment with GM1 enhanced

enzyme activity (Table 5).

GM1 did not change the protein content in control or MPP+-treated cultures

(Table 8).

About 1% of the cells in our cultures displayed TH-like immunoreactivity. TH immunopositive cells appeared to be bipolar or multipolar with triangle or elongated somata (Plate IV).

Treatment with MPP+ decreased the number of TH-immunopositive cells by about 60% without significantly diminishing the total number of phase bright cells,

(Table 6). After MPP+, there were fewer TH-immunopositive cells. They were smaller, had fewer primary processes or no processes and the observed processes were shorter. Treatment of control cultures with GM1 did not change the number of TH-immunopositive cells, and they appeared similar to untreated control cells.

Addition of GM1 to the MPP+-treated cultures resulted in a 2-3 fold increase of TH- immunopositive cells to values found for control cultures and the cells appeared essentially normal (Plate IV). This is evident from quantitative image analysis of 61

Table 8

GM1 Does not Alter Protein Content in Embryonic Mesencephalic Cultures

fug protein /plate') Conditions Control MPP+

Medium 400 ± 41 443 ± 33 GM1 464 ± 45 477 ± 15

Mesencephalic cultures were assayed for protein content on day 12 in cultures. GM1, 500 nM, was added on day 5 and day 8, while MPP+, 3 /zM, was added on day 4 for 24 h. Values are mean ± SEM. N=20 plates per group from 5-6 different experiments. 62 GM1 Restores the Number and the Morphology of TH-immunopositive Cells in Embryonic Mesencephalic Cultures Treated with MPP+ Plate IV: Bright-field photomicrograph showing TH-immunopositive neurons in rat E15 ventral mesencephalic cultures after 12 days in culture. A) cells were maintained for 12 days in standard medium. B) after the fourth day in culture MPP+, 3 /iM, was added for 24 h. Hie cultures were then maintained until day 12 in standard medium. C) cultures were treated with MPP+ as in B, however GM1, 500 nM, was added to the cultures until assayed on day 12. D) control cultures were maintained from day 5 to day 12 in the presence of GM1, 500 nM. (X 400).

A B t

C D 63 soma perimeter, the number of primary and secondary processes and the length of the primary process (Table 9).

The specificity of response of the cultures to exogenous GM1, with either

GM1 antibody or the B subunit of cholera toxin, revealed that both treatments reduced DA uptake in control cultures and prevented the augmentation of transporter activity by added GM1. Both treatments also prevented the DA uptake augmentation that occurs following treatment of MPP+-lesioned cultures with GM1.

Neither treatment, however, altered DA uptake in MPP+-lesioned cultures without

GM1 (Table 10).

The uptake of DA in cultures treated with the anti- mitotic agent, Ara-C, did not alter the effect of GM1 when DA uptake was evaluated on day 12 in culture

(Table 11).

E. NGF effect

Both NGF 7S, the whole molecule, and NGF 2.5S, the active B subunit, were effective in restoring DA uptake activity in MPP+-treated cultures (Table 12).

NGF enhanced DA uptake in MPP+-lesioned cultures in a time- and concentration-dependent manner (Figure 12). The response appeared maximal at a concentration of about 500 ng/ml of NGF 2.5S. Enhanced DA uptake was evident as early as 1 day after adding NGF to the MPPMesioned cultures and reached a maximal about 7 days later (Figure 13). When control mesencephalic cultures were incubated with NGF there was no increase of DA uptake when studied 7 days Table 9

GM1 Restores the Morphology of TH-immunopositive Cells in Mesencephalic Cultures Treated with MPP+

Number of Primary Number of Primary Process Length Processes Secondary Pro­ Soma Perimeter (Mm ± SEM) (Mean ± SEM) cesses (fim ± SEM) (Mean ± SEM) Condition Medium 56 ± 3 164 ± 3 2.8 ± 0.17 5.8 ± 1 GM1 47 ± 4 180 ± 8 3.2 ± 0.5 5.8 ± 1 MPP+ 37 ± 1* 65 ± 5* 1.8 ± 0.14* 2.1 ± 0.2* MPP+ plus GM1 46 ± 1** 176 ± 5** 3.0 ± 0.2** 5.7 ± 0.4**

Mesencephalic cells were grown on coverslips (22 x 22 mm) and treated as described in methods. On day 12 in culture the cells were fixed and TH-containing cells visualized by immunostaining. Morphometric analysis was performed with an image analyzer (Magiscan). N=50 cells per group from 4 different experiments. * p<0.05 compared with Medium treated cultures. **p<0.05 compared with MPP+ treated cultures. 65

Table 10

Cholera Toxin B Subunit or Antibodies to GM1 Prevent the GMl-Induced Increase of DA Uptake in Mesencephalic Cultures Treated with MPP+

DA Uptake (pmol/mg prot/15 min ± SEM) Conditions Control MPP+

Medium 3.0 ± 0.09 1.7 ± 0.7 GM1 3.5 ± 0.2* 2.6 ± 0.2* Ab-GMl 2.3 ± 0.2 1.7 ± 0.2 CT-B 2.0 ± 0.2 1.7 ± 0.6 GM1 plus Ab-GMl 2.0 ± 0.2 1.5 ± 0.2 GM1 plus CT-B 2.4 ± 0.1 1.6 ± 0.2

Mesencephalic cultures were treated on day 4 in culture with MPP+, 3 /zM, for 24 h. On day 5 the medium was changed and the cultures treated for the remainder of the study as indicated: Medium alone; GM1, 500 nM; Antibody to GM1 diluted 1:100; Cholera toxin B subunit 5 Mg/ml; GM1,500 nM, plus antibody to GM1; GM1, 500 nM, plus cholera toxin B subunit. DA uptake was assayed on day 12 in culture. N = 12 plates per group from 3 different experiments. *p<0.05 compared with all Control or MPP+ groups, respectively. All MPP+ treated cultures were significantly different from Control cultures. 66

Table 11

Enhanced DA Uptake Induced by GM1 in MPP+-Treated Mesencephalic Cultures is not Affected by Prior Treatment with Ara-C

DA Uptake DA Uptake Protein (pmol/dish) (pmol/mg prot) (Mg/dish)

Mixed Cultures Medium 1.5 ± 0.1 4.06 ± 0.16 346 ± 20 MPP+ 0.72 ± 0.12* 2.11 ± 0.14* 331 ± 12 GM1 1.76 ± 0.05* 4.4 ± 0.2* 372 ± 12 MPP+ plus GM1 1.24 ± 0.05** 3.6 ± 0.4** 335 ± 13

Neuronal Cultures Medium 1.29 ± 0.07 8.6 ± 0.9 124 + 22 MPP+ 0.67 ± 0.09* 5.5 ± 1.0* 147 ± 15 GM1 1.39 ± 0.05 9.1 ± 1.3 157 ± 8 MPP+ plus GM1 1.82 ± 0.05** 10.9 ± 1.4** 153 ± 14

Mesencephalic cultures were treated with Ara-C, 1.25 nM, for 24 h on day 3 in culture. On day 4 in culture they were washed and MPP+, 3 /xM, added for 24 h. On day 5 in culture GM1, 500 nM, was added and maintained for the remainder of the study. DA uptake was assayed on day 12 in culture. Data are presented as the mean ± SEM. N= 3-4 plates per group from 3 different experiments. * p<0.05 compared with medium treated cultures. **p<0.05 compared with MPP+ treated cultures. 67

Table 12

NGF 7S or NGF 2.5S Enhances DA Uptake in MPP+- Lesioned Mesencephalic Cultures

DA uptake (pmol/mg prot/15 min ± SEM)

Medium 2.63 ± 0.17 MPP+ 1.21 ± 0.10* MPP+ + NGF 7S (lMg/ml) 2.03 ± 0.09** MPP+ + NGF 2.5S (ljug/ml) 2.16 ± 0.13**

Mesencephalic cultures were treated with MPP+, 3 juM, on day 4 in culture for 24 h. NGF 7S or NGF 2.5S was added on day 5 and day 8 in culture after MPP+, 3 juM, removal. DA uptake was assayed on day 12 in culture. Data presented are mean ± SEM. N = 15 plates per group from 3 separate experiments. * p<0.05 compared with medium treated cultures. ** p<0.05 compared with MPP+ treated cultures. NGF Dose-Response Study

LU OQ +i c iH in

-M o (_ CD CL

I—I o E o_

cont MPP+ 0.10 0.20 NGF [ /(g /m l ]

Figure 12: Dopamine uptake by rat E15 mesencephalic cultures treated with MPP+ and increasing concentrations of NGF. After 4 days in culture MPP+, 3 mM, was added for 24 h. Then NGF, 500 ng/ml, was added to one group of MPP+-treated cultures (stippled bars). An MPP+-treated culture (striped bar) was induced for comparison. DA uptake was assayed on day 12 in culture. N =15 plates per group from 3-4 different experiments. * p<0.05 compared with MPP+-treated cultures.

On OO NGF: Time Course Recovery of DA Uptake

bJ m +i c CD g 2.0 in cu Q. D I o L. o CL

Q"

01 2 3 4 5 6 7 8 9 10 Days after NGF Treatment

Figure 13: DA uptake by rat E15 mesencephalic cultures with time following MPP+ or after MPP+ plus NGF. Cultures were treated with MPP+, 3 /xM, as described in methods and NGF, 500 ng/'ml, added and the incubation continued. DA uptake was measured at various times after initiation of NGF treatment. Control (open squares), control treated with NGF, 500 ng/ml, (solid squares), MPP+, 3 /xM, (open circles), and MPP+, 3 /xM, treated with NGF, 500 ng/ml, (solid circles). Data are presented as mean ± SEM. N = 12 plates per group from 4-5 different experiments. * p<0.05 compared with MPP+ alone.

O'. 'O 70 later or at any other time in the culture. After removing NGF, DA uptake decreased to values seen for untreated MPP+-lesioned cultures (Figure 14). NGF did not prevent the MPP+-induced loss of DA uptake activity. When NGF was added before or together with MPP+ for 24 h, there was no protection of DA uptake activity compared with cultures that were treated with MPP+ alone (Table 13).

NGF treatment enhanced DA and DOPAC content and AAAD activity in

MPP+-lesioned cultures but had no effect on these markers in control unlesioned cultures (Tables 14 and 15).

NGF treatment had no significant effect on the content of protein in all situations studied (Table 16).

Treatment of control cultures with NGF did not change either the number or the morphology of TH immunopositive cells and they appeared similar to untreated control cells (Tables 17 and 18 and Plate V). Addition of NGF to the MPP+-treated cultures resulted in an increase of the number of TH-immunopositive cells to values found for control cultures and the cells appeared essentially normal. This is evident from quantitative image analysis of soma perimeter, the number of primary and secondary processes and the length of primary processes (Tables 17,18).

The response to NGF appears to be dependent on the presence of glial cells in the culture (Table 19). Discontinuation of NGF Treatment 100

r—H o (_ 4 -» C

0 5 10 15 20 25 Days in Culture

Figure 14: DA uptake by rat E15 mesencephalic cultures treated with MPP+ alone, MPP+ plus continuous treatment with NGF or MPP+ plus limited treatment with NGF. Cultures were treated with MPP+, 3 mM, on day 4 in culture for 24 h and then on day 5 MPP+ was removed and NGF, 500 ng/ml, added to two groups. The two groups were maintained with NGF, 500 ng/ml, until day 12 in culture. Then NGF was removed from one group (solid tirangle) while NGF, 500 ng/ml, was maintained in the other (solid circles). A comparison group consisted of MPP+ treatment alone (solid squares). Data are presented as the mean ± SEM. N=8 plates per group from 3 different experiments. * p<0.05 compared with MPP+ alone. 72

Table 13

NGF does not Prevent MPP+-Induced Reduction of DA Uptake in Embryonic Mesencephalic Cultures DA Uptake Conditions (pmol/mg prot/15 min ± SEM) Contrast Culture Medium 2.96 ± 0.12 MPP+ 1.02 ± 0.02* Pre-Treatment NGF 3.02 ± 0.07 NGF plus MPP+ 1.04 ± 0.04* Co-Treatment NGF 2.89 ± 0.05 NGF plus MPP+ 1.13 ± 0.04*

Mesencephalic cultures were prepared as follows: Contrast Culture, on day 4 in culture, MPP+, 3 nM, was added for 24 h, the cultures washed and fresh medium added for the remainder of the study; Pre-Treatment. NGF, 500 ng/ml, was added at plating and was present when, MPP+, 3 /liM, added on day 4 for 24 h, after washing fresh addition-free medium added for the remainder of the study; Co- Treatment. on day 4 in culture both MPP+, 3nM, and NGF, 500 ng/ml, were added for 24 h removed and fresh addition-free medium added for the remainder of the study. DA uptake was assessed on day 12 in culture. N = 12 plates per group from 3-4 different experiments. *p<0.05 compared with culture medium alone or with NGF treatment. 73

Table 14

NGF Enhances DA and DOPAC Content of Mesencephalic Cultures Treated with MPP+

DA DOPAC Conditions (pmol/mg prot ±SEM)

Medium 6.8 ± 1.0 10.7 ± 1.0 NGF 7.30 ± 0.6 8.6 ± 0.6 MPP+ 2.5 ± 0.1* 4.0 ± 0.7* MPP+ plus NGF 4.8 ± 0.4** 7.9 ± 1.9**

Mesencephalic cultures were treated on day 4 in culture with MPP+, 3 fiM, for 24 h. On day 5 fresh medium or NGF, 500 ng/ml, added when indicated. Fresh medium with NGF, 500 nM, was added every 3 days. On day 12 in culture DA and DOPAC content were assayed. N = 10 plates per group from 3 different experiments. *p<0.05 compared with medium treated cultures. * *p < 0.05 compared with MPP+ treated cultures. 74

Table 15

NGF Enhances Aromatic L-Amino Acid Decarboxylase Activity of Mesencephalic Cultures treated with MPP+

AAAD Activity Conditions (pmol/mg prot/20 min ± SEM)

Medium 3.9 ± 0.2 MPP+ 0.92 ± 0.08* NGF 3.5 ± 0.4 MPP+ plus NGF 1.3 ± 0.1**

Mesencephalic cultures were treated as described in Table 14. On day 12 in culture AAAD activity was assayed. N=5-10 plates per group from 2-3 different experiments. *p<0.05 compared with medium treated cultures. **p<0.05 compared with MPP+ treated cultures. 75

Table 16

NGF Does not Alter Protein Content in Embryonic Mesencephalic Cultures

Conditions (ug protein/platel Control MPP+

Medium 400 ± 41 443 ± 33 NGF 445 ± 10 478 ± 18

Mesencephalic cultures were treated as in Table 14 and assayed for protein content on day 12 in cultures. Values are mean ± SEM. N=20 plates per group from 5 different experiments. 76

Table 17 NGF Restores the Number of TH-immunopositive Cells in Mesencephalic Cultures Treated with MPP+

Total TH-positive Percent of cells ± SEM Cells ± SEM Total Cells Conditions (x 104) Medium 8.3 ± 0.1 819 ± 153 0.99 NGF 9.1 ± 0.3 1147 ± 82 1.25 MPP+ 8.7 ± 0.3 335 ± 35* 0.39 MPP+ plus NGF 7.9 ± 0.4 711 ± 64** 1.08

Mesencephalic cells were grown on coverslips (22 x 22 mm) and treated as described in Table 14. On day 12 in culture the cells were fixed and the presence of TH was evaluated by immunohistochemistry. The number of cells on a coverslip was estimated by counting phase-bright cells in a superimposed counting frame and corrected for the size of the coverslip. N=4-6 slides per group from 5 different experiments. * p < 0.05 compared with medium treated cultures. **p<0.05 compared with MPP+ treated cultures. Table 18 NGF Restores the Morphology of TH-immunopositive Cells in Mesencephalic Cultures Treated with MPP+

Number of Primary Number of Primary Process Processes Secondary Soma Perimeter Length (Mean ± SEM) Processes Condition (/im ± SEM) (Mm ± SEM) (Mean ± SEM) Medium 56 ± 3 164 ± 3 2.8 ± 0.17 5.8 ± 1 NGF 47 ± 1 178 ± 18 3.0 ± 0.10 4.9 ± 0.2 MPP+ 37 ± 1* 65 ± 5* 1.8 ± 0.14* 2.1 ± 0.2* MPP+ plus NGF 46 ± 2** 191 ± 4** 3.3 ± 0.2** 3.5 ±0.3**

Mesencephalic cells were grown on coverslips (22 x 22 mm) and treated as described in methods. On day 12 in culture the cells were fixed and TH-containing cells were visualized by immunostaining. Morphometric analysis was performed with an image analyzer (Magiscan). N=50 cells from 4 different experiments. *p<0.05 compared with medium treated cultures. **p<0.05 compared with MPP+ treated cultures. 78 NGF Restores the Number and the Morphology of TH-immunopositive in Embryonic Mesencephalic Cultures Treated with MPP+

Plate V: Bright-held photomicrograph showing TH-immunopositive neurons in rat E15 ventral mesencephalic cultures after 12 days in cultures. A) cells were maintained for 12 days in standard medium. B) after the fourth day in culture MPP+, 3 was added for 24 h. The cultures were then maintained until day 12 in standard medium. C) cultures were treated with MPP+, 3 fiM, as in B, however NGF, 500 ng/ml, was added to the cultures until assayed on day 12. D) control cultures were maintained from day 5 to day 12 in the presence of NGF, 500 ng/ml. (X-400). 79

Table 19

Treatment with Ara-C Attenuates the NGF-Induced Increase of DA-Uptake in MPP+-Treated Mesencephalic Cultures

DA uptake DA uptake Protein (pmol/dish/15 min) (pmol/mg prot/15 min) (/xg/dish)

Mixed Cultures Medium 1.43 ± 0.08 2.72 ± 0.25 503 ± 16 MPP+ 0.71 ± 0.04* 1.30 ± 0.24* 510 ± 18 NGF 1.76 ± 0.05 2.96 ± 0.20 455 ± 21 MPP+ plus NGF 1.25 ± 0.05** 2.50 ± 0.27** 501 ± 7

Neuron Rich Cultures Medium 1.63 ± 0.18 11.5 ± 3.7 175 ± 27 MPP+ 0.69 ± 0.06* 3.8 ± 1.0* 165 ± 8 MPP+ plus NGF 0.76 ± 0.07* 3.76 ± 0.05* 194 ± 10

Mesencephalic cultures were treated with Ara-C, 1.25 mM, for 24 h on day 3 in culture. On day 4 in culture they were washed and MPP+, 3 fiM, added for 24 h. On day 5 in culture NGF, 500 nM, was added, and maintained for the remainder of the study. DA uptake was assayed on day 12 in culture. Data are presented as the mean ± SEM. N=3-4 plates per group from 4 different experiments. * p<0.05 compared with medium treated cultures. * *p < 0.05 compared with MPP+ treated cultures. 80 F. EGF effect

Treatment with EGF, 10 ng/ml, resulted in an increase of DA uptake in control unlesioned cultures. This effect was statistically significant when DA uptake activity was expressed as pmol/plate. Due to the fact that EGF increases protein content the EGF effect on DA uptake was small (about 15%) and not statistically significant when expressed as pmol/mg prot (Tables 20 and 21). Treatment with

EGF enhances DA uptake activity after a lesion with MPP+, an increase of about

163% over the MPP+-treated group (Table 21). EGF had no protective effect against an MPP+ lesion. When it was added prior or simultanously with MPP+ for

24 h did not prevent the MPP+-induced decrease of DA uptake, (Table 22).

E. Combined treatments

In a series of experiments we asked the question whether GM1 has a synergistic relationship with NGF or EGF in promoting DA markers in control or

MPP+-lesioned cultures. GM1 did not influence the effect on the NGF on DA uptake activity in control or MPP+-treated cultures ruling out the possibility of a synergistic interaction between the two compounds regarding the modulation of DA uptake. The same observations appear to be true for EGF (data not shown). 81

Table 20

EGF Enhances Protein Content in Embryonic Mesencephalic Cultures

Conditions (ug protein/plate) Control MPP+

Medium 400 ± 41 443 ± 33 EGF 523 ± 63* 542 ± 36*

Mesencephalic cultures were assayed for protein content on day 12 in cultures. EGF, 10 ng/ml, was added on day 5 and day 8 , while MPP+, 3/xM, was added on day 4 for 24 h. Values are mean ± SEM. N=15 plates per group from 3-4 different experiments. * p 0<05 compared to medium treated cultures. 82

Table 21

EGF Enhances DA Uptake Activity in Embryonic Mesencephalic Cultures Treated with MPP+

DA uptake Treatment (pmol/mg prot/ 15 min ± SEM)

Medium 2.90 ± 0.18 MPP+ 0.95 ± 0.14* EGF 3.30 ± 0.40 MPP+ + EGF 2.50 ± 0.26**

Mesencephalic cultures were treated with MPP+, 3 /xM, on day 4 in culture for 24 h. EGF, 10 ng/ml, was added on day 5 and day 8 in culture after MPP+ removal. DA uptake was assayed on day 12 in culture. Data presented are mean ± SEM. N = 9 plates per group from 3 separate experiments. * p<0.05 compared with medium treated cultures. ** p<0.05 compared with MPP+ treated cultures. 83

Table 22

EGF Does not Prevent the MPP+-Induced Reduction of DA Uptake in Embryonic Mesencephalic Cultures

DA-Uptake Conditions (pmol/mg prot/15 min ± SEM)

Contrast Culture Medium 2.63 ± 0.07 MPP+ 1.13 ± 0.05*

Pre-Treatment EGF 2.76 ± 0.07 EGF + MPP+ 1.05 ± 0.05*

Co-Treatment EGF 2.69 ± 0.03 EGF + MPP+ 1.04 ± 0.03*

Mesencephalic cultures were prepared as follows: Contrast Culture. On Day 4 in culture, MPP+, 3 /jlM, was added for 24 h, the cultures washed and fresh medium added to the remainder of the study; Pre-Treatment. EGF, 10 ng/ml, was added at plating and was present when, MPP+, 3 /xM, added on day 4 for 24 h, after washing fresh addition-free medium added for the remainder of the study; Co-Treatment, on day 4 in culture both MPP+, 3 /xM, and EGF, 10 ng/ml, were added for 24 h removed and fresh addition-free medium added for the remainder of the study. DA uptake was assesed on day 12 in culture. N = 10 plates per group from 3 different experiments. * p<0.05 compared with medium treated cultures. DISCUSSION

A. MPP+ Neurotoxicity and Embryonic Mesencephalic Cultures.

Embryonic mesencephalic cultures are a reliable system to study MPP+ neurotoxicity on dopaminergic neurons and functional recovery after treatment with neurotrophic factors [143,144]. Addition of MPP+ to mesencephalic cultures de­ creases DA and DOPAC content, AAAD activity and DA uptake activity and reduces the number of TH immunopositive cells without having a general toxic effect on the culture [23,147].

No spontaneous recovery of the neurochemical parameters was observed in our studies. Embryonic mesencephalic cultures grow well and can be maintained for up to 2-3 weeks in the presence of serum. The same cultures can grow in chemically defined media only for about 7-8 days. Under the conditions used about 30-40% of the cells are glia, as evidenced by GFAP immunostaining, and the remainder are neurons. Only 1% of the neuronal population displays TH immunoreactivity. Using mixed cultures was advantageous for our studies since it appears that glial cells are important for the mediation of the trophic effects of EGF and NGF on the lesioned dopaminergic neurons. Such an effect could have been missed if pure neuronal cultures had been used.

84 85 Addition of MPP+ in embryonic mesencephalic cultures decreased DA uptake in a concentration-dependent manner. This effect was permanent and lasted as long as the cultures were followed. This indicates that the decrease in DA uptake reflects a neurodegenerative process (loss of processes and/or cells) and not an acute pharmacological effect. This conclusion is further supported by more detailed studies showing that MPP lesioned dopaminergic mesencephalic neurons in culture when surveyed by a number of biochemical and morphological criteria. There was about

40-60% loss of DA uptake, DA and DOPAC content, AAAD activity and TH immu­ nopositive cells when cultures surveyed 7 days after MPP+ treatment. This obser­ vation is consistent with in vivo [148,149] and in vitro [23,147] studies where there is evidence for selective destruction of dopaminergic neurons by MPP+. From an analysis of the TH-containing cells in the cultures, the progression of events appears to be loss of TH immunopositive processes with shrinkage of the soma and eventual loss of the soma image. Loss of DA uptake activity is consistent with the loss of terminal processes and, therefore, it can be used as an indirect biochemical index to evaluate nerve terminal status.

B. GM1 Improves Dopaminergic Markers of Mesencephalic Cultures Treated

with MPP+.

GM1 ganglioside has been reported to promote the recovery of cholinergic

[63,65,66], adrenergic [150], and dopaminergic [58,151,152] neuronal functions in the

CNS following surgical and neurotoxin lesions. There are also reports of recovery in 86 the peripheral sympathetic neurons [153,154,155]. The mechanism(s) for recovery

is unclear. However, GM1 may have a regrowth-stimulating effect, a protective

action against retrograde degeneration following nerve terminal lesions,or both [49].

Gangliosides are found in high concentrations in the CNS, and they are located in

the outer surface of neuronal membranes, an observation suggesting that they might

have a receptor role [156], Indeed, GM1 ganglioside is a receptor for cholera toxin

[157]. Incubation of GM1 with neuronal membranes results in incorporation into

membranes [158]. Thus, it is possible that some of the exogenous GM1 enters

neuronal membranes and serves as a receptor for endogenous neurotrophic factor(s).

Following injury, endogenous neurotrophic factors are released and promote survival

of injured neurons [159]. It is possible that the exogenously administered GM1 might

modulate receptor function for these neurotrophic factors. For example, GM1

enhances the effect of NGF lesion peripheral sympathetic neurons as well as on

cholinergic neurons in the brain of lesioned or aged animals [62,159,160].

GM1 added to normal or lesioned cultures increased DA uptake in a

concentration-dependent manner. MPP+-lesioned cells show enhanced uptake as

early as 2 days after adding GM1 and uptake enhancement is maintained whereas

control cultures show enhanced uptake only on day 12 in culture. With the

concentration of DA used for studying DA uptake, the changes observed probably

reflect an increased number of transporter sites. Indeed, it has been reported that

GM1 treatment increases the Vmax but not the Km for DA uptake in embryonic mesencephalic cultures [161]. GM1 was able to enhance AAAD activity as well as 87 DA and DOPAC content in both control and MPP+-treated cultures. Apparently the effect of GM1 on the biochemistry of dopaminergic neurons in culture is extensive and not a selective effect on DA uptake.

GM1 does not protect dopaminergic neurons in vivo when administered before or together with MPTP [59] and it does not protect mesencephalic dopaminergic neurons from destruction by MPP+ in our cultures. The finding that adding GM1 after a lesion has been initiated in the mesencephalic culture facilitates the recovery of all neurochemical parameters evaluated is consistent with in vivo studies [59]. In vivo, early initiation of treatment is required for successful recovery as there appears to be a finite time after insult when GM1 is effective [59].

Moreover, GM1 treatment in vivo must be uninterrupted or recovery deteriorates.

Recovery of dopaminergic parameters also deteriorates in the mesencephalic cultures if GM1 is withdrawn.

Exogenous GM1 inserts into cell membranes and it induces differentiation and neurite outgrowth [162,163,164,165]. Antibodies to GM1 or the presence of the

B subunit of cholera toxin retards GMl-induced neurite outgrowth [50,166], demonstrating the importance of GM1 for these responses. DA uptake activity was decreased in control and MPP+-lesioned cultures if antibody to GM1 or the B subunit of cholera toxin were added, implying that DA uptake activity and perhaps other parameters are dependent, either directly or indirectly, on GM1. The GM1 content of serum is low [167], thus the response to antibody or to the B subunit of cholera toxin in the control untreated cultures may be the consequence of their 88 with GM1 generated in the culture. Indeed embiyonic mesencephalic cultures can

survive in a defined medium and added GM1 promotes DA uptake activity [143].

Both GM1 antibody and the B subunit prevented the GMl-induced increase of DA uptake in control and MPP+-treated cultures, implying that a contaminant in the

GM1 preparation is not responsible for the enhanced dopaminergic parameters.

Treatment with GM1 restored the number of TH immunopositive cells and their morphology to near normal in cultures lesioned with MPP+ yet it had little

effect on control cultures not lesioned with MPP+. This may represent rescue by

GM1 of the MPP+-injured neurons from eventual destruction. Alternatively, it is possible that MPP+ reduces TH protein and thus neuron detectibility by immunostaining and GM1 may upregulate TH expression restoring neuron detectabi­ lity. This interpretation has been offered to explain the effect of NGF on the return of choline acetyltransferase immunopositive neurons following injury to cholinergic neurons [168]. The fact that DA uptake does not recover in the MPP+-treated cultures with time, up to 4 weeks (data not shown), suggests that dopaminergic neu­ rons are eventually lost. Therefore, we postulate that GM1 rescues moribund neurons resulting in the return of dopaminergic parameters along with TH- immunoreactivity.

Although the mechanism(s) for the recovery of function after injury is unknown there is evidence that GM1 may have antineurotoxic and proneurotrophic actions. Neuronal insults are associated with excessive prolonged Ca2+ influx which is detrimental for the neuronal survival [169]. In glutamate-induced neurotoxicity 89 GM1 prevents the translocation of protein kinase C in injured neurons and may thereby limit intracellular Ca2+ increase thus protecting the neuron from delayed death [170]. MPP+ increases glutamate in brain [171] and NMDA receptor antagonists can prevent MPP+ neurotoxicity [172]. Pretreatment and cotreatment with GM1 together with MPP+ or M FIP does not appear to prevent toxic effects on dopaminergic neurons in culture or jn vivo. However, early and continuous treatment with GM1 after MPTP or MPP+ lesion is required for a response [59].

The early administration of GM1 after an insult may rescue neurons delayed degeneration and death associated with elevated glutamate release. BDNF has been shown to prevent the MPP+ neurotoxicity in mesencephalic cultures by inducing gluthathione synthetase activity [173].

In addition to possible antineurotoxic properties, GM1 may have restorative effects by modulating endogenous neurotrophic factor activity. Gangliosides modulate tyrosine kinase activity of trophic factor receptors, such as EGF, IGF-I, and

PDGF [174]. A tyrosine kinase receptor appears important for BDNF activity [175].

EGF and BDNF promote recovery of mesencephalic neurons in culture

[145,176,177]. Trophic factors for dopaminergic neurons are present in the brain and added GM1 may facilitate their actions on the injured neurons by modulation of their synthesis and release and/or receptor characteristics. For example, GM1 has a synergistic effect with NGF on brain cholinergic neurons [178,179,180] and in peripheral catecholaminergic neurons [181]. However, our findings do not suggest 90 that such a synergism operates between NGF and GM1 for central dopaminergic neurons.

Elimination of glial cells did not alter the ability of GM1 to enhance DA uptake in cultures lesioned with MPP+ negating a role for astrocytes in this response.

This is in agreement with reports that embryonic mesencephalic neurons in a defined culture medium show enhanced DA uptake when GM1 is introduced [161].

Based on reports in the literature and from this study with MPP+ neurotrophic peptides and GM1, it is postulated that the following events might occur in the culture lesioned with MPP+ and treated with GM1. MPP+ is transported into the mesencephalic dopaminergic neurons and inhibit mitochondrial energy metabolism and all dependent biochemical events. Phenotypic expression is lost and the neurons begin degenerating. Added GM1 inserted into membranes limits excessive Ca2+ influx and facilitating the action of neurotrophic factors present in the cultures, hence rescuing the cells and stimulating phenotypic expression, regeneration and repair. Apparently the rescued dopaminergic neurons are dependent on added GM1 as they lose their ability to take up DA if GM1 is removed. This observation is consistent with in vivo studies where dopaminergic parameters were found to deteriorate in MPTP lesioned animals when GM1 treatment were terminated. 91 C. NGF Improves dopaminergic markers in embryonic mesencephalic

cultures treated with MPP+

NGF is a well recognized neurotrophic factor and promotes differentiation,

growth and survival of peripheral sensory and sympathetic neurons as well of central

cholinergic neurons [136]. The current dogma is that NGF has no trophic effects on

central catecholaminergic neurons [182]. Indeed exogenously administered NGF is

not transported into locus coeruleus or substantia nigra [183,184] and addition of

NGF to embryonic mesencephalic cultures has no effect on DA uptake activity [84].

However, there is evidence now that although NGF does not exert a trophic action

on intact dopaminergic neurons it might have a restorative effect after a lesion has

occured. For example Cuello et al. reported that administration of NGF to animals with cortical lesions increases DA content in in vivo dialysis studies [185].

Furthermore, it has been reported that NGF increased DA and HVA in the striatum

of MPTP-treated mice [186]. We have completed studies showing that NGF adminis­

tration facilitates the recovery of DA and DOPAC content, TH activity, and DA

uptake in the striatum of MPTP-treated mice [abstract data not published]. Interest­

ingly, the neurochemical recovery was accompanied by a restoration of the number

and morphology TH immunopositive cells in the substantia nigra pars compacta [data not published]. NGF had no effects on normal unlesioned dopaminergic neurons.

This is contrary to the reported effects of NGF on central cholinergic neurons where the factor enhances function in both lesioned and unlesioned animals [185]. It appears, thus, that NGF is able to facilitate the recovery of function of central 92 dopaminergic neurons only after a lesion has occured. The above observations are

in agreement with our studies with MPP+ lesioned cultures.

NGF added to lesioned cultures increased DA uptake in a concentration-

dependent manner. MPPMesioned cells show enhanced uptake as early as 1 day

after adding NGF and uptake enhancement is maintained throughout the treatment.

With the concentration of DA used for studying DA uptake, the changes observed probably reflect an increased number of transporter sites. NGF was able to enhance

AAAD activity as well as DA and DOPAC content in MPP+-treated cultures.

Apparently the effect of NGF on the biochemistry of dopaminergic neurons in culture is extensive and not a selective effect on DA uptake only. In agreement with previous reports NGF had no effect on unlesioned control embryonic mesencephalic cultures [84].

NGF did not protect mesencephalic dopaminergic neurons from destruction by MPP+ in our cultures. The finding that adding NGF after a lesion has been initiated in the mesencephalic culture facilitates the recovery of all neurochemical parameters evaluated is consistent with in vivo studies [186]. Treatment with NGF must be uninterrupted, and recovery of dopaminergic parameters deteriorates in the lesioned mesencephalic cultures if NGF is withdrawn.

Treatment with NGF restored the number of TH-immunopositive cells and their morphology to near normal in cultures lesioned with MPP+, yet it had no effect on control not lesioned mesencephalic cultures. This may represent rescue by NGF of the MPP+-injured neurons from eventual destruction. Alternatively, it is possible 93 that MPP+ reduces TH protein and thus neuron detectibility by immunostaining and

NGF may upregulate TH expression restoring neuron detectability. This interpreta­ tion has been offered to explain the effect of NGF on the return of choline acetyltransferase immunopositive neurons following injury to cholinergic neurons

[185]. The fact that DA uptake does not recover in the MPP+-treated cultures with time (up to 4 weeks, data not shown) suggests that dopaminergic neurons are eventually lost and NGF rescues them from eventual death. Our in vivo studies support this notion as well. MPTP decreases both TH-immonopositive and total cell number, as evidenced with cresyl violet staining, in the substania nigra pars compacta and NGF returns total cell number and TH-immunopositive cells to control levels when studied 15 days after the completion of the lesion. Interestingly, at the time of the lesion the total cell number is intact, indicating that lesioned neurons start dying after the completion of the treatment apparently during the two week period of our studies. Taken together one can speculate that under our experimental conditions MPTP or MPP+ causes a delayed and not acute neuronal death and early treatment with NGF rescues moribund neurons.

At this point of our studies the mode of action of NGF is not clear.

Elimination of glial cells from the cultures prevent the NGF enhancement of DA uptake in MPP+-lesioned cultures. This indicates that the action of NGF is not directly on dopaminergic neurons but indirect via glial cells. EGF and FGFb have been shown to increase dopaminergic markers indirectly acting on glial cells [121].

The vast majority of glial cells present in mesencephalic culture were astrocyte type 94 I which augments DA neurons survival [187]. Lesion with MPTP causes proliferation

of reactive glia in the striatum and substantia nigra [188,189,190,191]. Nerve growth

factor receptors, gp75 NGFr, trkA, and trkB are expressed in astrocytes type I and

expression is more pronounced after lesioning [192]. It is possible that MPP+

increases the expression of NGFrs on reactive glial cells, making them more

responsive to the exogenously administered NGF [193]. In response to NGF glial

cells might produce dopaminergic neuron selective soluble neurotrophic factors which

in turn might rescue moribund neurons and restore function. Alternatively NGF might stimulate glial cells to produce cell adhesion factors that could regulate neuronal survival. There is accumulating evidence that cytokines might play a role in neuronal injury and repair. Indeed astrocytes produce IL-1 [194] which mediates wound healing and neuronal growth and repair [195]. The action of IL-1 may be mediated by other secondary mediators such as 11^6 [196] which has been shown to promote the survival of mesencephalic catecholaminergic neurons from postnatal rats in cultures [197]. Whether NGF can induce the synthesis and release of cytokines after an MPP+ lesion remains to be tested. Finally another possibility might be that the NGF induced glial factors might rescue the neurons from delayed death by interfering with Ca2+ homeostasis and/or oxidative stress. 95 D. EGF Improves Dopaminergic Markers in Mesencephalic Cultures Treated

with MPP+

Epidermal growth factor is a potent polypeptide mitogen with potential

trophic action for neurons. It has been identified in mammalian brain by

immunohistological [117] and EGF mRNA in situ hybridization [120] techniques.

EGF apparently stimulates DNA synthesis [128,129] and cell division [129,130] in

glial cells, and it enhances survival and process outgrowth of cortical [131] and

cerebellar [133] neurons in culture. There are reports that EGF moderately

stimulates the uptake of DA in embiyonic mesencephalic cultures and that this effect

depends on the presence of glial cells [121]. The stimulatoiy action of EGF on dopaminergic neurons appears to be rather selective, as it has no effect on central cholinergic neurons [121]. Thus, in addition to its mitogenic and hormonal activities,

EGF may act as a neurotrophic factor for selective neuronal populations.

The possibility that EGF might enhance the function of DA nigrostriatal neurons after a lesion with the neurotoxin MPTP or MPP+. The results showed that

EGF partially enhances the uptake of DA in MPP+-lesioned cultures.

Treatment with EGF increased DA uptake in embryonic mesencephalic cultures, as it has been reported [121]. In addition EGF restored the MPP+-induced decrease in DA uptake activity. Because our studies with EGF were limited, we cannot say whether the factor had any effect on the survival of neurons or that it rescued them from the detrimental effects of the neurotoxin. However EGF did not prevent the MPP+ neurotoxicity. There was also an apparent increase in total pro­ 96 tein in the culture dishes following EGF treatment, which is probably related to the proliferation of glial cells in the cultures [121]. Glial cells appear to be important for the neurotrophic activity of EGF since eliminating them from the cultures prevents the effects of the factor on DA uptake activity [121]. EGF is capable of recovering dopaminergic function after a MPTP lesion in vivo. Indeed, we have shown that ICV administration of EGF to mice treated with MPTP was able to facili­ tate the recovery of DA and DOPAC content as well as the DA uptake in the striatum [116]. Taken together, our data suggest that EGF can enhance some dopaminergic parameters after a relatively selective lesion. EGF may have enhanced

DA synthesis and neuronal sprouting. The molecular basis for the EGF effect on dopaminergic neurons is unknown. EGF and its receptors have been identified in mammalian brain. Most studies support the notion that they are present on both neurons and glia [125,126]. When EGF binds to its receptor, it triggers a cascade of transmembrane signals, i.e., it results in increased polyphosphoinositide hydrolysis and elevated pH and free Ca2+ in the cytoplasm [198]. Whether these processes operate in concert to induce plastic responses by dopaminergic neurons is currently under investigation. EGF can regulate its own receptor [199] and induce the appearance of high affinity EGF binding sites [200]. High-affinity binding sites have been postulated to play a role in signal transduction [201]. Induction of high affinity sites might explain our finding that treatment of unlesioned animals and embryonic mesencephalic neurons in culture with EGF also resulted in an increase in striatal

DA function. However, injury alone has been reported to induce the synthesis of 97 EGF receptors on reactive astrocytes [202], implying that injured tissue becomes more sensitive to EGF. These results support the hypothesis that EGF induces neurotrophic activity for dopaminergic neurons.

5. Conclusions

Our studies so far have shown that GM1, NGF and EGF are able of facilitating the recovery of dopaminergic function in embryonic mesencephalic cultures after a lesion with MPP+. None of the compounds prevented the MPP+ neurotoxicity indicating that their action is rather proneurotrophic and not antineurotoxic. The proneurotrophic effect of the agents was pronounced in MPP+ lesioned cultures, suggesting that injury makes the neurons more responsive to trophic stimuli. GM1 and EGF, but not NGF, also enhanced DA uptake in control noninjured dopaminergic neurons. Further detailed studies with GM1 and NGF showed that both agents increased DA and DOPAC content, AAAD activity and DA uptake, the latter in a concentration- and time-dependent manner. Continuous administration of GM1 or NGF is required for maintaining the response. Both GM1 and NGF increased the number and improved the morphology of TH-immuno- positive cells in cultures lesioned with MPP+. Eliminating glia cells did not prevent the GM1 effect on control or lesioned cultures, suggesting that GM1 acts dirrectly on dopaminergic neurons. In contrast elimination of glial cells prevented the effect of NGF on lesioned cultures, suggesting that the NGF action is indirect and mediated via glial cells. REFERENCES

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