Anti-Inflammatory and Neuroprotective Properties of Ladostigil and Its Primary Metabolites

Anti-inflammatory and neuroprotective properties of ladostigil and its primary metabolites

in vitro and in vivo

Thesis submitted for the degree of “Doctor of Philosophy”

Rony Panarsky

Submitted to the Senate of Hebrew University of Jerusalem

06/2012

This work was carried out under the supervision of: Prof. Marta Weinstock-Rosin

עבודה זו מוקדשת לאבי אלון ז"ל ולאמי ראיסה, שתזכה לשנים ארוכות, שתמיד

עודדו אותי להמשיך קדימה על אף מכשולים שבדרכי.

This work is dedicated to my beloved parents, Alon and Raisa, who always encouraged me to pursue my way despite all obstacles.

Acknowledgements:

I would like to express my deepest gratitude and appreciation to my mentor, Prof. Marta Weinstock – Rosin who’s personal and scientific wisdom, patience, optimism and humanity helped me to proceed successfully to my graduation and taught me to think critically and originally as a scientist and to improve my personal capabilities as a man.

I want to thank in addition to all my colleagues from the lab – Corina Beijar, Donna Appelbaum, Helena Shifrin, Efrat Groner, Yael Shoshani, Tehilla Billig, Dr. Lisandro Luques, Dr. Shai Shoham, Dr. Shiri Salomon and Dr. Yaarit Biala for providing me with their personal experience and knowledge.

Special thanks to Prof. Ioav Cabantchik who put his trust in me and encouraged me to continue believing in myself.

I want to thank Prof. Shlomo Rotshenker and Ms. Fanny Reichert for providing me with primary microglia cells during my work.

Thank you, my beloved wife Miri Gitik for all your love, patience and trust you placed in me as my partner and for scientific advice and knowledge you gave me as scientist.

Thank you to all my friends and the family for supporting me in this long journey.

Abstract

There is growing evidence that prolonged inflammation in the brain may promote and accelerate neurodegenerative processes leading to neuronal cell death and consequent deterioration in brain function. Inflammation, glial activation, selective cell death and abnormalities in oxidative metabolism are found in age related neurodegenerative diseases like Alzheimer's and

Parkinson disease.

The main source of inflammatory cytokines and oxidative and nitrative species in the brain are resident microglia, which make up about 10% of the brain parenchyma. Once activated, microglia produce a wide range of pro- and anti- inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10), as well as nitric oxide (NO) some of which can promote inflammation at the site of pathogen invasion or brain injury. While short-term inflammation has a beneficiary effect, prolonged and uncontrolled inflammatory response can have an undesirable impact on neuronal cells by promoting cell death and a decline in function of the entire brain region in which the chronic inflammation takes place.

In addition, it has been shown recently that chronic systemic inflammation may have a crucial impact on the risk of developing of Alzheimer's disease (AD) and other neurodegenerative diseases.

Ladostigil is a novel cholinesterase (ChE) and brain selective monoamine oxidase (MAO) inhibitor in vivo, designed as therapy for patients with AD co- morbid with depression. The three most abundant metabolites of ladostigil which are mostly responsible for inhibition ChE and MAO and have been identified in the circulation are R-MCPAI, R-CAI and R-HPAI. Treatment of aged female rats with ladostigil (1mg/kg) for one month increased the expression of genes that are associated with metabolism and oxidative processes in the hippocampus, which had been down-regulated as a result of aging. The same dose of ladostigil administered for 6 months to 16 month old male rats, prevented the increased expression of CD11b, an indication of microglial activation in the cortex and hippocampus and the development of memory deficits at 22 months of age. In order to understand how ladostigil produced this effect in the aging brain it was important to determine whether ladostigil and/or its metabolites acted primarily by preventing oxidative stress and thereby reducing microglial activation or whether ladostigil and its metabolites also had a direct action on activated microglia.

Our findings demonstrated that ladostigil and its three major metabolites, at concentrations of 10nM-1µM, decreased by 25-40% in primary cultures of mouse microglia the release of the LPS-induced NO into the culture medium.

The metabolites at concentrations of 10-100nM also downregulated the gene expression of IL-1β and TNF-α in LPS-treated microglial cells as demonstrated by RT-PCR. The same degree of inhibition (up to 25%) was achieved in gene expression of iNOS after 8 h of co treatment with LPS and 10nM of the compounds. All the compounds at concentrations of 10-100nM reduced the release of TNF-a protein in the microglia medium after 6 h LPS treatment to a similar degree. Western blot analysis of MAPK proteins revealed that the compounds reduced the phosphorylation of ERK1/2 and p38 by up to 35%, but had no significant effect on phosphorylation of Jnk1/2/3.

EMSA analysis demonstrated attenuation of NF-κB nuclear translocation by 20-

35%. These findings were supported by inhibition of IkBα degradation and intra nuclear staining of p65 component of NF-κB transcription factor. In all these measures ladostigil and R-MCPAI were generally more effective than the other metabolites.

The reduction of activated microglia in the brain after chronic treatment with ladostigil could also have been an indirect effect consequent to its prevention of age-related oxidative stress. We therefore also investigated the ability of ladostigil and its metabolites to protect SK-N-MC human neuroblastoma neuronal cells from the effects of H2O2. Measurement of the change in the mitochondrial potential by fluorescent analysis under oxidative stress conditions demonstrated the stabilization of the potential after 90 min of pretreatment of the cells with all the compounds at concentration of 10µM. All the compounds reduced activity of caspase 3/7 by up to 65%.

Our results obtained from in vivo experiments of acute inflammation show that when given orally, ladostigil (10mg/kg) significantly reduced the production of pro-inflammatory cytokines in LPS treated mice. Eight hours after ladostigil administration we demonstrate an 80% reduction of TNF-α and IL-1β protein levels in the cerebral cortex and 4 h after the administration, a 40% reduction of IL-6, TNF-α and IL-1β protein levels in the spleen. We also analyzed the response of splenocytes to anti-CD3 or LPS in terms of

IFN-γ release to the culture medium. Splenocytes were isolated from aged rats that have been treated for 6 months with ladostigil (1 and 8.5mg/kg/day) and compared to response of the cells obtained from aged and young untreated animals. We found that splenocytes from aged rats showed a higher reactivity to anti-CD3 than those of young rats. This was detected by a reduction in the ability of the splenocytes to produce IFN-γ cytokine after 48 h of activation with ex vivo. Moreover, our results point to a possible immunomodulatory effect of long term treatment with ladostigil on splenocytes in terms of IFN-γ production.

These data indicate that ladostigil and its major metabolites have neuroprotective and anti-inflammatory properties in vitro and in vivo in concentrations below and at those inhibiting ChE or MAO. Here we report for a first time that the mechanism of action underlying the anti-inflammatory properties of the compounds results from their attenuation of the activation of

MAPK pathway elements and consequent reduction of the nuclear translocation of NF-κB. The possible neuroprotective effect of ladostigil and the metabolites against oxidative stress may result from their ability to prevent the fall in mitochondrial potential and to reduce the activation of caspase 3/7 cascade.

Table of contents

Introduction 1 - 11

Methodology 12 - 24

Results 25 - 52

Discussion 53 – 63

References 64 - 68

List of abbreviations 69

Introduction

Inflammation, one of the most basic defensive processes in the body, is a response of the organism to invasion of pathogens, cancer cells, tissue damage or other traumatic events arising from endogenous or exogenous causes.

Originally intended for protection, this highly effective and tightly controlled chain of events is orchestrated by the immune system and usually culminates in complete or partial elimination of the origin of inflammation with subsequent termination of the inflammatory response.

During acute inflammation the majority of immune cells including macrophages, dendritic cells, natural killer (NK) cells and T-lymphocytes are recruited from the circulation to the site at which inflammation occurs. Within a very short time the site of inflammation becomes overloaded with variety of pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ etc.) the primary function of which is to recruit and to activate the cells of immune system, to produce edema in the inflammation site and to promote the maintenance of the inflammation required for combating the infection. These processes should be time and location restricted in order to preserve their efficacy and to prevent undesirable tissue damage, cell death or systemic overreaction.

Chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease or psoriasis, are all characterized by low grade prolonged inflammatory processes accompanied by ongoing pathophysiology of the involved organ, damage and consequent decline of its function. 2

Thus, finding a successful treatment of chronic inflammatory conditions and keeping these processes tightly controlled remains a main goal of modern pharmacological research.

It has previously been reported that inflammation, glial activation, selective cell death and abnormalities in oxidative metabolism are common in many age related neurodegenerative diseases like Alzheimer's and Parkinson disease (1,

2).

Alzheimer's disease (AD) is one of the most common forms of dementia affecting approximately 10% of the population over the age of 65 years.

Clinically AD is characterized by impairment in memory, visuospatial skills, complex cognition, language, emotion and personality.

In addition to the neuropathological hallmarks of the disease, namely neurofibrillary tangles and neuritic amyloid plaques, AD is characterized by a deficit in cholinergic neurotransmission, particularly affecting cholinergic neurons in the basal forebrain (3, 4). It has been reported previously that the reduction in activity of enzymes involved in degradation (acetylcholinesterase

(AChE)) or synthesis (choline acetyltransferase (ChAT)) of acetylcholine is the main finding in post mortem brains of AD and this cholinergic loss correlates well with cognitive impairment (5-7). Although the exact cause of AD remains elusive, mounting evidence continues to support the involvement of inflammation in its development (8). Furthermore, recent PET studies have reported inflammatory changes in one third of subjects with amnestic mild cognitive impairment (MCI) (9), a condition associated with slow progressive memory loss and considered as a prodromal phase of dementia. There is post 3

mortem evidence of decreased catalase activity in parietotemporal cortex and basal ganglia of AD brains suggesting the involvement, of oxidative stress in the progression of AD (10-13).

While it still remains unclear whether the inflammation is a cause or consequence of neurodegenerative pathology of AD the importance of inflammatory processes in the progression of the disease is generally accepted.

Chronic inflammatory processes that take place in the brain promote a hostile environment for neurons and supportive tissue and eventually may cause massive cell death accompanied by deterioration of brain function.

Microglial cells are the main source of inflammation in the brain. They make up about 10% of the brain cells. Since microglia originate from the same myelomonocytic lineage as macrophages they preserve their main features.

Residing in the brain, microglia continuously survey their microenvironment for the slightest changes and are designed to act immediately in case of pathogen invasion or brain trauma. Morphologically, microglial cells may be classified as ramified – a resting form, and amoeboid which is usually characterized as an activated state of the cells (Fig. 1). Once activated, microglia produce a wide range of pro-inflammatory cytokines, nitric oxide (NO) and growth factors in order to promote a suitable inflammatory environment to fight the potentially harmful agent. On switching from ramified to amoeboid form, microglial cells, like macrophages, acquire the property of phagocytosis which contributes to their ability to fight the infection or promote the elimination of damaged cells and tissues to ensure proper rehabilitation of the damaged region.

Fig. 1: Resting (A) and activated microglia (B). Arrows point on typical morphological changes of microglia after LPS activation

It is well known that microglia exhibit significant phenotypic changes during normal aging. Microglial cells from both aged humans and rodents show profoundly altered morphology, characterized by dystrophic processes, and abnormal clustering (14). These changes in morphology are accompanied by increased expression of activation markers such as MHCII (15), raised basal production of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 (16-18) and hyper responsiveness to inflammatory stimulation (19). 5

In addition, the recent findings clearly point to the existence of an additional state of microglia – the primed microglia. This phenotype has been found to be abundant even in the healthy aged brain and characterized by continuous low grade release of pro inflammatory cytokines, thereby promoting a chronic inflammatory environment (20). When exposed to an inflammatory trigger, this type of the microglia responds by exaggerated release of pro-inflammatory cytokines such as IL-1β. It has been shown that prolonged exposure of hippocampal cells to IL-1β promotes changes in the electrophysiological state of the cells, reduction of LTP and synaptic plasticity, eventually leading to spatial memory and learning impairments (21, 22).

Several lines of evidence suggest that adult microglia can also become chronically activated from a single stimulus in the absence of any predispositioning event or aging. For example, chronic microglial activation continues years after brief MPTP exposure in humans (23) and primates (24) through a process called reactive microgliosis.

Continuous and uncontrolled activity of microglia, producing low-grade inflammation and contributing to oxidative stress, could create undesirable conditions in their microenvironment leading to synaptic decline, stress and eventually apoptosis and cell death.

One of the hallmarks of apoptosis is a fall in the mitochondrial potential. During this process, the electrochemical gradient across the mitochondrial membrane collapses. The collapse is thought to occur through the formation of pores in the mitochondria by dimerized Bax or activated Bid, Bak, or Bad proteins (25-

27). Activation of these pro-apoptotic proteins is accompanied by the release of cytochrome c into the cytoplasm. Once released from damaged mitochondria 6

to the cytosol, cytochrome c interacts with cytosolic Apaf-1 protein which leads to formation of apoptosome (28). Apoptosome cleaves an inactive form of caspase 9, the cysteine aspartic acid protease proteolytic enzyme which plays a key role in promoting apoptosis by cleavage and activation of the cascade downstream of caspase 3 which in turn activates other caspases (caspase 6,7 and 9) to promote protein degradation within the cell and eventually the cell death (29). These events are tightly controlled and once triggered are irreversible.

Thus, one can conclude that keeping microglial activity under tight control should have a beneficial effect on the aging brain and may produce an attenuation of the neurodegenerative processes which cause AD.

In spite of fact that the most recent epidemiological studies reported that there was no notable effect of both non-steroidal anti-inflammatory drugs (NSAIDs) and steroids on the progression of AD once this occurs (30), earlier studies showed that NSAIDs may be able to prevent development of the disease but this needs to be further clarified.

The drugs most frequently employed for the treatment of AD are acetyl cholinesterase (AChE) inhibitors (31). One of these, donepezil, has been shown to reduce the cytotoxic effects of oxidative stress (32) and the release of NO and cytokines from activated microglia (33), but both these actions only occur at concentrations 10-100-fold higher than those inhibiting AChE (34). This lack of protective effect against neurodegenerative processes at relevant therapeutic doses could explain why chronic treatment with donepezil of patients with MCI did not reduce the proportion converting to AD (35, 36) and may even have accelerated its onset (37). 7

Ladostigil (6-(N- ethyl, N- methyl carbamyloxy)-N propargyl-1(R)-aminoindan hemitartrate, is an ChE and brain-selective monoamine oxidase (MAO) inhibitor in vivo (38) that was shown to protect cells against the toxic effects of oxidative and nitrative stress (39, 40). Based on two clinically approved drugs, rivastigmine - a potent AChE inhibitor and rasagiline – a MAO-B inhibitor, this compound has been designed to increase the therapeutic potential of the rivastigmine by providing the neuroprotective properties of rasagiline. It has been reported previously that the treatment of neuronal cells exposed to oxidative stress with rasagiline prevented the fall in mitochondrial potential and induced the activity of anti-apoptotic protein Bcl2 and antioxidant enzymes (39).

Rasagiline has been shown to have a neuroprotective effect in vivo. It accelerates the recovery of motor function and spatial memory after closed head injury in mice (41) and reduces the incidence of stroke and increases survival in stroke-prone spontaneously hypertensive rats (42).

Treatment for one month of aged female rats with ladostigil (1mg/kg) also increased the expression of genes that are associated with metabolism and oxidative processes in the hippocampus which had been down-regulated as a result of age (43). The same dose of ladostigil administered for 6 months to middle-aged male rats, prevented the increase in OX42 immunoreactivity, an indication of microglial activation in the cortex and hippocampus and the development of memory deficits (44).

Fig. 2: Chemical structure of ladostigil R-CPAI and its three most potent metabolites R-MCPAI, R-CAI and R-HPAI

Ladostigil is metabolized in rats (45) and humans (46) to three major primary active metabolites; R-MCPAI (6-(N-methyl carbamyloxy)-N-propargyl-1(R)- aminoindan) though de-ethylation by CYP 2C19 in the liver, R-CAI (6-(N-ethyl,

N-methyl carbamyloxy)-1(R)-aminoindan through de-propargylation by CYP

1A2 and R-HPAI (6-Hydroxy–N-propargyl-1(R)-aminoindan) through hydrolysis of the carbamate moiety by AChE, its target enzyme (Fig. 2).

R-MCPAI and R-CAI are about 30 and 12-fold respectively more potent inhibitors of ChE than ladostigil while R-HPAI accounts for all the MAO- inhibitory activity of the drug in vivo (47). R-CAI differs from all other compounds by lacking the propargyl group, a fact that may point to the relevance of this moiety to the pharmacological properties of the ladostigil. 9

It is not known whether the changes induced in the brain of aging rats result from actions of ladostigil itself, its metabolites or a combination of them. While the reduction in glial activation may be an indirect result of the effect of ladostigil on mitochondrial dysfunction and antioxidant enzymes, it is possible that the drug or its metabolites could also act directly on age-activated microglial cells and reduce the release of NO and pro-inflammatory cytokines.

Fig. 3: A scheme represents a general concept of TLR4 signal transduction by LPS

Brain located toll-like receptors (TLR) are thought to play a role in neurodegenerative processes and their stimulation by cytokines and pathogens leads to downstream activation of the transcription factor NF-κB (48).

It has been reported previously that microglia become activated by lipopolysaccharide (LPS) through TLR4 receptors which in turn activate downstream elements of MAPK pathway and eventually culminate in nuclear translocation of NF-κB which promotes gene transcription of wide range of pro- inflammatory cytokines and growth factors (49, 50) (Fig. 3).

A number of clinical studies have suggested an association between peripheral blood indicators of systemic inflammation and the subsequent development of

AD. The inflammatory proteins in plasma, notably C-reactive protein (CRP) and

IL-6 have been found to be elevated 5 years before the clinical onset of dementia, in a number of studies (51-53).

More recently, a prospective study suggested that cognitively intact individuals in the top tertile of peripheral blood mononuclear cell TNF-α or IL-1β production have three times the risk of developing AD than those in the lowest tertile (54). Another study has shown that high serum levels of TNF-α at baseline and increases in serum TNF-α associated with intermittent systemic infection are associated with a marked increase in the rate of cognitive decline in AD subjects over a 6-month period (55). Moreover, it has been reported that the risk of developing of AD also increases following the development of an infection in the absence of an obvious delirium. Thus, the presence of one or more infections over 5-year follow-up period increased the odds of developing of AD approximately 2-fold (56). 11

Given this data, we conclude that systemic inflammation may play an important role in initiation and progression of AD and modulation of inflammatory processes in older subjects may have a beneficial effect in preventing or attenuating neurodegenerative decline in brain function.

In this work we examined the possible immunomodulatory and neuroprotective properties of ladostigil and its primary metabolites on microglia in vitro and in animal models of acute inflammation and age related immunoreactivity in vivo.

The second goal was to evaluate the possible mechanism of action of the compounds by which they may produce their immunomodulatory effect.

Methodology

Reagents and cell cultures

Ladostigil (R-CPAI) hemitartrate; R-MCPAI HCl; R-CAI HCl and R-HPAI mesylate were provided by Avraham Pharmaceuticals. Bacterial LPS from

Salmonella typhosa was obtained from Sigma (St. Louis, MO, USA). Drug concentrations are expressed in terms of the salt of the particular compound.

Recombinant mouse interferon gamma (IFN-γ) was purchased from R&D

Systems (Minneapolis, MN, USA). Primary microglial cells were provided by

Prof. Rotshenker (Medical Neurobiology Department, Hebrew University) or prepared from brains of 1-3 day-old mice as previously reported (57). Brains were stripped of their meninges and enzymatically dissociated. The dissociated cells were plated on poly-L-lysine-coated flasks for one week and re-plated for two hours on bacteriological plates and non-adherent cells were removed by washing. The cells were cultured in Dulbecco's modified Eagle's medium

(DMEM; Biological Industries, Israel) containing 1g/L glucose, 10% fetal calf serum (FCS, Biological Industries), 10% of colony Stimulating Factor (CSF),

50µg/mL, 0.01% gentamicin and 1% L-glutamine (Biological Industries).

Human neuroblastoma SK-N-MC cell line was obtained from the ATCC Cell

Biology Collection (Manassas, VA, USA).

NO release assay

Microglial cells were co-treated with the compounds at concentrations ranging from 1nM to 1μM and LPS (10µg/mL), or IFN-γ (50ng/mL). NO released into the medium 24 h later was measured spectrophotometrically in the form of nitrites using Griess reagent (0.1% N-1-naphthyl ethylene diamine dihydrochloride and 1% sulphanilamide in 5% phosphoric acid). The supernatant (50µl) was mixed with an equal volume of Griess reagent in a 96- well plate and incubated at room temperature for 10 min. The concentration of nitrites was measured on a micro plate reader (Labsystems) at 540nm using serial dilutions of NaNO2 as a standard.

Cell viability assessment

Cell viability was assessed by either 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) test. Briefly, the cells were incubated with concentrations of ladostigil and its metabolites ranging from 1nM-1µM and with

MTT in final concentration of 0.5mg/mL in the medium for 30 min. The medium was then discarded and dimethyl sulphoxide (DMSO 100µl) was added to each well to dissolve the formazan dye. The plate was read at a wavelength of

580nm with background subtraction at 650nm.

Total RNA extraction, cDNA production and Real-Time Polymerized Chain

Reaction (RT-PCR) analysis

Microglial cells were co-treated with the compounds at concentrations of 10 and 100nM and LPS (10µg/mL) in 96-well plates for 2, 8 or 12 h before being detached from the wells, centrifuged and collected for RNA extraction. Total

RNA was isolated from the cells using Total RNA mini kit (Genaid Biotech,

Sijhih, Taiwan). Total cDNA was synthesized from the isolated total RNA using

High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad,

CA, USA). Total RNA (1-2µg) was added to the reagent mix to a final volume of 20µl for each sample. The RT reaction was run for 10 min at 25°C, 2 h at

37˚C and 5 min at 85˚C. Real time RT-PCR reaction was performed using

TaqMan the probe-based detection approach, in which 6µl of TaqMan Master

Mix (Applied Biosystems) with appropriate TaqMan probes (TNF-α, IL-1β, iNOS) was added to 4µl of cDNA solution (final amount of cDNA – 10ng/well) to a final volume of 10µl of reaction in each well. The reaction was run for 40 min at StepOnePlus™ RT-PCR System (Applied Biosystems).

Enzyme-linked immunosorbent assay (ELISA)

Microglial cells were co-treated with LPS (10µg/mL) and the compounds (10nM or 100nM) for 6 h and TNF-α protein was detected using the sandwich ELISA method. The cells were incubated for 24 h in 48-well plates. Medium (50μl) was applied to each well which was coated with capture anti-mouse TNF-α antibody

(BioLegend, San Diego, CA, USA) in 96-wells plate. Dilutions of recombinant

TNF-α protein ranging from 7.8-500pg/mL were used for a standard curve. The 15

plate was incubated overnight at 4⁰C. After 3 washes with PBS/Tween20

(PBST) solution the cells were blocked with 1% BSA in PBS solution for 1 h followed by 3 additional washes. Anti-mouse TNF-α antibody was applied for detection for 1 h at room temperature followed by 30 min incubation with alkaline phosphatase. A chemiluminescence reaction was performed by adding a pNPP One Component Microwell Substrate reagent (Southern

Biotech, Birmingham, AL, USA) and color intensity was evaluated on a plate reader at 405nm.

Western blot analysis

In order to evaluate the possible role of the compounds on MAPK signaling we analyzed phosphorylation of three major components of this pathway; extracellular signal-regulated kinases ERK1/2, p38 and c-Jun N-terminal kinase (Jnk) 1, 2, 3 proteins which may be activated upstream to NF-κB and are involved in transduction and proliferation of the pro-inflammatory signal induced by LPS at the TLR4 receptor. Microglial cells were seeded 24 h prior to the experiment in 6-well plates at a density of 2 x 106 cells/well. The cells were pre-treated for 90 min with the compounds (10nM), stimulated with LPS

(10µg/mL) for 30 min in presence of the compounds and the degree of phosphorylation of the MAPK proteins was evaluated. The 30 min time point was found in preliminary experiments to give optimal results for phosphorylation by of MAPK by all components and IκBα protein (data not shown). The cells were homogenized with RIPA buffer (Sigma) which protein included a cocktail of proteinase and phosphatase inhibitors and DNAses

(Sigma). Total protein concentration was determined using bi-cinchoninic acid 16

(BCA) assay (Sigma) and 20µg of it was loaded on 12% SDS-polyacrylamide gel (Sigma) and run for 40 min. The proteins were transferred onto nitrocellulose membranes and blocked for 30 min with 5% milk solution at Tris- buffered saline Tween-20 (TBST). The membranes were incubated overnight at 4ºC with appropriate primary antibodies on a shaking platform. Mouse anti- mouse ERK2, rabbit anti-mouse phospho ERK1/2, mouse anti-mouse p38, rabbit anti-mouse phospho p38, mouse anti-mouse JNK1/2/3, goat anti-mouse phospho JNK1/2/3 and rabbit anti-mouse IκBα (Santa Cruz Biotechnology,

Santa Cruz, CA, USA) were used in 2mL of 5% milk solution. Mouse anti- mouse tubulin antibody (Sigma) was used as a housekeeping protein at a dilution 1:15000. Goat anti-rabbit Alexa Fluor 680-conjugated IgG and IRDye

800-conjugated goat-anti mouse IgG (dilution 1:10000) were added for 1 hour at room temperature before visualization and analysis by the Odyssey IR imaging system (LI-COR Biosciences).

Immunofluorescence staining

Microglial cells were seeded on sterile glass coverslips 24 h before they were exposed to the compounds for 90 min at a concentration of 10nM, followed by activation for 30 min with LPS (10µg/mL) in presence of the compounds. The coverslips were fixed in 4% paraformaldehyde for 30 min at room temperature

(24˚C) and then in cold methanol for 10 min at −20 °C. Fixed cells were blocked in 1% bovine serum albumin (BSA). The cells were incubated with mouse anti- p65 antibody (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA,

USA) at 4 °C overnight and Alexa Fluor-488 labeled goat anti-mouse IgG

(1:2000 dilution; Molecular Probes Inc., Eugene, OR, USA) at room 17

temperature for 1 h. Stained cells were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and examined on a fluorescent microscope (Nikon Eclipse TE2000-E). DAPI staining was used for nuclei contrasting marking. Images were analyzed by means of

Volocity Image Analysis Software (PerkinElmer, USA) and the co-localization of Alexa Fluor-488 signal for p65 (green) and DAPI nuclear staining (blue) was estimated and represents the amount intra-nuclear p65.

Electro-mobility shift assay (EMSA)

Microglial cells were seeded 24 h prior to the experiment in 6-well plates, pretreated for 90 min with 10nM of the compounds followed by 10µg/mL LPS plus

10nM of the compounds for 30 min. Nuclear proteins were isolated from approximately 1x107 microglia cells using Cayman Nuclear Extraction Kit

(Cayman Chemical Company, Ann Arbor, MI, USA). Total protein concentration was determined using bicinchoninic acid (BCA) assay (Sigma).

Double stranded NF-κB IRDye 700 labeled oligonucleotide (LI-COR

Biosciences, Nebraska, USA) corresponding to NF-κB specific consensus sequence was used for the binding reaction at concentration of 50nM per reaction. The double stranded sequence for NF-κB was 5' -- AGT TGA GGG

GAC TTT CCC AGG C -- 3' and 3' -- TCA ACT CCC CTG AAA GGG TCC G

-- 5' while the underlined nucleotides represent the NF-κB binding site. The unlabeled oligonucleotides with the similar sequence (cold probe) or labeled nonspecific sequence were used as controls for specificity of the binding reaction. The binding reaction was performed using Odyssey Infrared EMSA kit (LI-COR Biosciences) according to appropriate protocol and with 50µg of 18

total nuclear protein per each reaction. For control of binding specificity the non-specific probe was added to vials containing protein extract from LPS treated cells alone, 10 min before adding the specific probe. The reaction took place during 40 min in the dark at room temperature and 2μl of Orange loading dye X10 (LI-COR Biosciences) was added to each vial before loading to the gel. A 4% polyacrylamide TBE gel was prepared and pre-run at 70 mV for 40 min before the samples were loaded. Protein-DNA complexes (20μl) were resolved by electrophoresis for 1 h at 70mV in the dark. The gels were scanned at 700μm using LI-COR Odyssey Imaging system. Band densities were quantitated by densitometric analysis using ImageJ software.

JC1 staining

The viability of SK-N-MC cells treated with hydrogen peroxide (H2O2), a direct- acting oxidative stress-inducing agent, was detected using fluorescent dye JC-

1. Briefly, the cells were seeded 24 h before an experiment in 48 well plates.

The cells were pretreated the compounds at different concentrations (10, 1 or

0.1µM) for 90 min at 37ºC. The medium was discarded and changed for the fresh medium containing 2.5µM of JC1 for 20 min. The medium was changed to the fresh one containing H2O2 (500µM). Every 15 min the snapshots of the cells were taken at 485nm for monomeric form of dye and 550nm for J- aggregate form of JC-1 using fluorescent microscopy (Nikon Eclipse 2000T).

The fall of the mitochondrial potential (∆ψ), representing cytotoxicity, was determined by calculating the ratio between green (low ∆ψ) and red (high ∆ψ) intensities of fluorescence obtained at each time point. 19

Measurement of caspase 3/7 activity

SK-N-MC cells were seeded on 6 well plates at density of 106 cells/well and were incubated in DMEM/10%FCS/1% gentamycin sulfate/1% sodium glutamate for 24 h before the experiment. The cells were pretreated with the

10µM of the compounds for 90 min and then exposed to 1mM of H2O2 for 180 min with appropriate controls. The cells were immediately scraped, centrifuged and counted. Measurement of caspase 3/7 activity was performed using

Promega Caspase-Glo®3/7 Assay (Promega,USA) protocol. Briefly, the cells were counted and diluted to the density of 2x105cells/mL. 100µl of the solution

(2 x104 cells) was placed in triplicates in the 96 well F96 MicroWell Plates

(NUNC, Denmark) and 100µl of the assay mix was added to each well.

Luminescence signal was measured 30–60 min later using a MITHRAS

Luminometer equipment.

Effect of ladostigil in mice injected with LPS

Male Balb-C mice aged 7-8 weeks were obtained from Harlan and randomly divided into three groups, 6-8 animals at each group. Every group was treated under appropriate controlled conditions according to the guidelines of the

University Committee for Institutional Animal Care, based on those of the

National Institutes of Health, USA. Animals were given free access to food and water.

In the first group, ladostigil 1, 5 or 10mg/kg in a volume of 0.25mL was given orally by gavage to each animal 15 or 120 minutes before the mouse was injected intraperitoneally (i.p.) with 10mg/kg LPS saline solution. 21

In the second, LPS control group, each animal was given water by gavage 15 or 120 min before it was injected i.p. with 10mg/kg LPS in saline solution.

In the third untreated control group (sham group), each mouse was given water by gavage and 15 min or 120 min later it was injected i.p. with appropriate volume of saline according to its weight.

The effect of ladostigil on the basic production of cytokines in the spleen of naïve (LPS untreated) animals was evaluated in a separate experiment in which ladostigil 10mg/kg in a volume of 0.25mL was given orally by gavage to each animal while in control group each mouse was given water by gavage.

The animals were kept in cages for additional 4, 8 or 16 h before they were sacrificed by cervical dislocation.

Four hours after treatment with and without the LPS the spleens of each animal was removed and immediately frozen in liquid nitrogen. The brain of each animal was carefully removed from the skull and briefly rinsed in ice cold saline solution to wash out the excess blood. Using coronal sections the cortex was separated from the sub-cortex and both brain compartments were collected and frozen in liquid nitrogen.

Determination of malondialdehyde (MDA) content in mouse brain by

TBARS analysis

Both anterior hemispheres of the brain were removed from the mice after cervical dislocation, rinsed in 0.9% NaCl solution to wash the excess of blood and immediately frozen in liquid nitrogen for further experiments

The tissue was homogenized in 1.15% KCl buffer at ratio 1:100 (1mg of brain in 100µl buffer) and centrifuged (3000rpm) for 10 min at 4°C. 200µl of supernatant solution was mixed with 100µl SDS, 750µl 20% acetic acid and

750µl TBE. The mixtures were boiled in 97°C for 60 min. 1mL of pyridine:n- butanol solution was then added, the mixtures mixed well and centrifuged

(3000 rpm) for 10 min at 4°C. The upper phase was collected and read at

540nm. The results were standardized to total protein.

Total RNA extraction from mouse brain and RT-PCR reaction

Total RNA was isolated from the cells using Total RNA mini kit (Genaid Biotech,

Sijhih, Taiwan) from about 20mg of the tissue. Total cDNA was synthesized from the isolated total RNA using High Capacity cDNA Reverse Transcription

Kit (Applied Biosystems, Carlsbad, CA, USA) (see description above). Real time RT-PCR reaction was performed using TaqMan the probe-based detection approach, in which 6µl of TaqMan Master Mix (Applied Biosystems) with appropriate TaqMan probes (TNF-α, IL-1β, iNOS, IL-6, COX-2, Galectin-

3) was added to 4µl of cDNA solution (final amount of cDNA – 10ng/well) to a final volume of 10µl of reaction in each well. The reaction was run for 40 minutes at StepOnePlus™ RT-PCR System (Applied Biosystems). 22

Enzyme-linked immunosorbent assay (ELISA) for tissues homogenates

Tissue homogenates (spleen, brain) were prepared in PBS/proteases inhibitor buffer from freshly frozen tissues using homogenizer. Total protein was determined using BCA assay. The diluted homogenates were placed in previously coated 96-well plates with either TNF-α, IL-1β or IL-6 anti-mouse capture antibodies (BioLegend, USA) and incubated overnight in 4⁰C. After 3 washes with PBST solution the cells were blocked with 1% BSA in PBS solution for 1 h followed by 3 additional washes. Anti-mouse TNF-α, IL-1β or

IL-6 antibodies were applied for detection for 1h at room temperature followed by 30 min incubation with alkaline phosphatase. A chemiluminescence reaction was performed by adding a pNPP One Component Microwell

Substrate reagent (Southern Biotech, Birmingham, AL, USA) and color intensity was evaluated on a plate reader at 405 nm.

Chronic ladostigil administration in aged rats

Experiments were performed on 25 retired breeders of the Wistar strain obtained from Harlan (Jerusalem) at the age of 10 months and six 4 month old adult rats according to the guidelines of the University Committee for

Institutional Animal Care, based on those of the National Institutes of Health,

USA. The rats were housed two per cage at an ambient temperature of 21±1oC and a reversed 12 h light-dark cycle. When they reached the age of 16 months and weighed 600-750g, ladostigil was added to acidified drinking water of two thirds of them to provide a daily intake of 1 or 8.5mg/kg/day. This method of dosing was chosen to avoid the considerable stress of daily gavage. The remaining rats served as controls and drank acidified water (pH 3-4). 23

Splenocyte culturing and activation

After sacrifice the spleens were removed and put through a BD falcon cell strainer, 70mm in HBSS medium and centrifuged at 4°C 1200RPM for 6 min.

The cells were re-suspended in 5mL of ACK medium for 15 min to get rid of erythrocytes and centrifuged at 4°C 1200RPM for 6min. The cells were re- suspended in clone medium containing DMEM medium/10% FBS high clone/1% essential amino acids/ 1% Pen-strep nystatine/1% Na- Pyruvate/1%

Hepes/2mL of 0.002% β-mercaptoethanol and counted. The cell suspensions were brought to a final concentration of 4x106 cells/mL of clone medium. 96- well plates were coated with 0.5 and 0.1µg/mL of anti-rat anti-CD3 antibody overnight at 4°C before the cell seeding. Immediately before cell seeding the coating solution was aspirated and 250µl of cell suspension was added in each well (106 cells/well). The plates were incubated for 48 h then the medium was collected and immediately frozen for further examination.

Enzyme-linked immunosorbent assay (ELISA) for splenocyte

Medium (50μl) collected from splenocytes was applied to each well which was coated with capture anti-rat INF-γ antibody (Eli-Pair, ENCO, Israel) or IL-2

Antigenix America Inc., ENCO, Israel) antibody in 96-wells plate. Dilutions of recombinant IFN-γ ranging from 31.5-1000pg/mL or IL- 2 protein ranging from

1 – 0.016ng/mL were used for a standard curve. The plate was incubated for 3 h with biotin linked anti-rat detecting antibody followed by 30 min incubation with alkaline phosphatase. The IL-2 capturing antibody was applied in coating solution in 96-wells plate overnight. The plate was washed with PBST solution and blocked for 2 h with 1% BSA solution. The samples were applied overnight 24

in 4°C. The plate was washed 3 times with PBST solution and IL-2 detection antibody was applied for 2 h followed by 30 min incubation with alkaline phosphatase. A chemiluminescence reaction was performed by adding a pNPP

One Component Microwell Substrate reagent (Southern Biotech, Birmingham,

AL, USA) and color intensity was evaluated on a plate reader at 405nm.

Statistical analysis

All results are expressed as mean ± SEM from 4-8 independent in vitro experiments with 3-4 replicates in each and at least 2 independent in vivo experiments with 6 – 8 animals at each experimental group. Statistical analysis of data was performed by one-way ANOVA with Duncan's post hoc test or

Dunnett’s test with multiple comparisons of means where appropriate.

Differences were considered statistically significant if the p value was <0.05.

Results

Release of nitric oxide from LPS or IFN-γ activated microglia

Activation of microglia by IFN-γ increased NO release to the cell medium 10-15 fold. Neither ladostigil nor its metabolites (1nM-1µM) caused any significant reduction in the release of NO after activation of the microglial cells with IFN-γ

(Fig. 4A). Therefore, no further experiments were performed with IFN-γ.

Exposure of microglial cells to LPS resulted in a 4-8 fold increase in the release of NO which was reduced significantly by 15-20% by 1nM all the compounds

(Fig. 4B). NO release was further decreased at concentrations ranging from

10nM-1µM to a maximum of 35-40% (p<0.001) depending on the compound

(Fig. 4C). The effect of R-MCPAI was significantly greater than that of ladostigil at a concentration of 1µM, and that of R-HPAI, at a concentration of 100nM.

Viability of microglia was unaffected by LPS 10µg/mL alone or when given together with the compounds in concentrations of 1nM-1µM in the MTT assay

(data not shown).

A) 20 15 10 5

Nitrites [µM] Nitrites 0 Vehicle + + Ladostigil R-MCPAI R-CAI R-HPAI IFN B) 8

6 ** ** ** ** 4 2

Nitrites [µM] Nitrites 0 Vehicle + + Ladostigil R-MCPAI R-CAI R-HPAI LPS C) 100 * * * 80 # * 60 40 20

% of LPS alone LPS of % 0 Ladostigil R-MCPAI R-CAI R-HPAI 1nM 10nM 100nM 1µM

Fig. 4: Effect of ladostigil and its metabolites on the release of NO from

IFN-γ and LPS activated microglia

Microglial cells were co-treated with the compounds and 50ng/mL IFNγ (A) or

10µg/mL LPS (B and C) for 24 h. Nitric oxide concentrations in medium were measured using Griess reagent (see Methodology). Significantly different from

LPS alone treated; ** p<0.01; significantly different from value for R-MCPAI and R-CAI; # p<0.05; significantly different from value produced by 10nM-1μM;

* p<0.05; 27

LPS-induced pro-inflammatory gene expression in microglia cells

In preliminary experiments we found that the largest increase (35-fold) in TNF-

α gene expression compared to that in control microglia was seen 2 h after stimulation with LPS, (10µg/mL), while those of IL-1β (1000-fold increase) and iNOS (5-fold increase) were seen at 12 h and 8 h respectively.

Co-treatment of the microglial cells with LPS and 10nM of each of the compounds for 2 h resulted in a similar significant reduction of 25-30% in TNF-

α gene expression for all metabolites (p<0.01) (Fig. 5A). When the concentration of each compound was increased to 100nM the reduction in the expression of TNF-α was significantly lower than with 10nM. By contrast, the expression of IL-1β was reduced to a similar extent at concentrations of 10nM and 100nM by all compounds.

Ladostigil showed the highest activity, reducing the levels of IL-1β mRNA by

35% at each concentration compared to <25% for the other compounds (Fig.

5B). At a concentration of 10nM all the compounds caused a reduction of similar magnitude in the expression of iNOS after 8 h of co-treatment with LPS (Fig.

5C). This agrees well with their effect on NO release from LPS activated microglia (Fig. 4B). 28

A) TNF- C) 100 100 iNOS

  80   80

60 60

40 40

20 20

% of LPS alone LPS of %

% of LPS alone LPS of % 0 0 Ladostigil R-MCPAI R-CAI R-HPAI Ladostigil R-MCPAI R-CAI R-HPAI B) D) 100 IL-1 600 ## 500 * 80 ‡‡  ** ** ‡‡ ** 400 ** ** 60 ** ** pg/ml 300

40  200

20 TNF 100

% of LPS alone LPS of %

0 0 Ladostigil R-MCPAI R-CAI R-HPAI Vehicle + + Ladostigil R-MCPAI R-CAI R-HPAI LPS - + + + + + 10nM 100nM

Fig. 5: Effect of ladostigil and its metabolites on expression of TNF-α, IL-

1β and iNOS mRNA and TNF-α protein in LPS activated microglia

Microglial cells were co-treated with the compounds and 10µg/mL LPS for 2 h

(A), 12 h (B) and 8 h (C). The cells were harvested for total RNA isolation and

further RT-PCR analysis. Cell medium was collected after 6 h of co-treatment

for ELISA analysis of TNF-α protein (D) (see Methodology). Significantly

different from LPS alone treated; ** p<0.01; significantly different from the

remaining compounds; ‡‡ p<0.01; significantly different from R-CAI and R-

HPAI; ## p<0.01; significantly different from LPS alone treated; * p<0.05;

LPS-induced TNF-α release from activated microglia cells

To determine whether the decrease in gene expression of the cytokines by the compounds is translated into reduced amounts of the protein we measured

TNF-α in the medium of the microglial culture. Maximal increase in TNF-α protein was seen 6 h after addition of LPS to microglia but it was not possible to detect sufficient amounts of IL-1β protein from these cells. Ladostigil, R-

MCPAI caused a similar reduction in TNF-α of 40% at a concentration of 10nM, but R-HPAI and R-CAI were significantly less effective. Increasing the concentration to 100nM did not further increase the effect of any of the compounds (Fig. 5D).

Phosphorylation of MAPK proteins and degradation of IκBα

For these experiments we used a concentration of 10nM of all the compounds since RT-PCR analysis of the TNF-α and IL-1β genes and measurement of NO release did not reveal a significantly greater inhibitory effect at 100nM. None of the compounds had a detectible effect on phosphorylation of JNK proteins

(data not shown). 31

A) C)

B) D) 1.20 1.20 ‡ 1.00 ‡ 1.00 0.80 0.80 0.60 0.60 0.40 0.40

0.20 Ratio pp38/p38 0.20

pERK/ERK Ratio pERK/ERK 0.00 0.00 Ladostigil R-CAI LPS Ladostigil R-CAI LPS R-MCPAI R-HPAI Medium R-MCPAI R-HPAI Medium

Fig. 6: Effect of ladostigil and its metabolites on phosphorylation of ERK and p38 induced by LPS in microglia

Microglial cells were treated with compounds for 90 min and then co-treated with compounds and 10µg/mL LPS for 30 min. The cells were harvested for total protein isolation and further Western blot analysis (see Methodology).

Significantly different from the rest of the compounds; ‡ p<0.01;

However, all the compounds significantly reduced the phosphorylation of ERK

(shown by the ratio of the sum of phosphorylated ERK1 and ERK2 to un- 31

phosphorylated ERK2) (Fig. 6B). The effect of ladostigil was significantly greater than that of R-CAI, but did not differ from that of the other metabolites

(Fig. 6A and 6B).

Tubulin

IkBα 2.50 B) 2.00 ‡ 1.50

1.00

/Tubulin Ratio /Tubulin 0.50



IkB 0.00 Ladostigil R-CAI LPS R-MCPAI R-HPAI Medium

Fig. 7: Increase in the stability of IκBα in LPS stimulated microglia by ladostigil and its metabolites

Microglial cells were treated with compounds for 90 min and then co-treated with compounds and 10µM LPS for 30 min. The cells were harvested for total protein isolation and further Western blot analysis (see Methodology).

Significantly different from the rest of the compounds; ‡ p<0.01; 32

Ladostigil, R-MCPAI and R-HPAI almost completely prevented the phosphorylation of p38 induced by LPS resulting in ratios of phosphorylated p38/p38 protein that did not differ from those in untreated cells. By contrast, R-

CAI had no effect on the phosphorylation of p38 (Fig. 6C and 6D).

Since p38 and ERK have been postulated as possible modulators of NF-κB activity (58-60) we examined the effect of the compounds on degradation induced by LPS of IκBα, the NF-κB inhibitor.

The degradation of IκBα releases the two functional subunits of NF-κB - p50/p65 - and enables the transcription factor to enter the nucleus culminating in the transcription of wide range of pro-inflammatory elements. Analysis of the

IκBα degradation at 30 min of co-treatment of the compounds with LPS showed that ladostigil, R-MCPAI and R-HPAI almost completely prevented the reduction in IκBα degradation induced by LPS. Although R-CAI had a significant effect on IκBα levels this was considerably weaker than that of the other compounds (Fig. 7A and 7B).

Nuclear translocation of p65, an NF-κB functional unit

The finding in the previous experiment indicated that the compounds promote their anti-inflammatory effect by modulation of the nuclear translocation of p50/p65 subunits. This was further investigated by two methods; immunofluorescence and electro-mobility shift assay (EMSA). As anticipated, microglial cells exposed to LPS increased p65 nuclear translocation by 100% showing that even a relatively short exposure of the microglia to the pathogen 33

can produce a rapid and substantial response (Fig. 8A and B). While all the compounds significantly reduced p65 nuclear entry after LPS, ladostigil and R-

MCPAI were the most effective (Fig. 8B). These changes in p65 translocation seen in the immunofluorescence assay were generally confirmed by the EMSA analysis in which a 25-35% reduction was seen compared to LPS alone (Fig.

8C). Again, the effect of R-MCPAI was significantly greater than that of the other compounds (Fig. 8D).

Fig. 8: Inhibition by ladostigil and its metabolites of the p65 nuclear translocation

Microglial cells were treated with compounds for 90 min and then co-treated with drugs and 10µM LPS for 30 min. The cells were prepared for immunofluorescence staining (A and B) or harvested for nuclear protein isolation and further EMSA analysis (C and D) (see Methodology). Significantly different from the effect of the rest of the compounds; ‡ p<0.01; 35

Immunofluorescent staining of the mitochondrial potential in SK-N-MC cells

Based on the previous data with rasagiline (see Introduction section) we were interested to explore the ability of ladostigil and its metabolites to act as neuroprotective agents in SK-N-MC human neuroblastoma-derived cell line under oxidative stress conditions. This cell line has been chosen as a common model of neurons in vitro (61, 62). The experiments have been performed with

500µM H2O2 in SK-N-MC cells pretreated with three different concentrations of the compound for 90 min and loaded with JC-1 dye.

Hydrogen peroxide treatment caused a significant reduction in mitochondrial potential of the cells (Fig. 9A) while no significant change has been observed in the cells without the H2O2 treatment. Pretreatment with the metabolites as well as with ladostigil significantly reduced the green/red ratio, thereby lowered the oxidative stress of the cells, up to 30% only at their higher concentrations

(10 and 1µM) but not at lower concentrations (Fig. 9B). R-CAI which lacks the propargyl moiety, present in rasagiline and ladostigil, does not differ in its neuroprotective effect on the cells from the ladostigil or propargyl containing metabolites at a concentration of 10µM but it has no effect in lower concentrations.

Recently it has been found in our lab that ladostigil possess an ability to act as a scavenging agent and shown to be able to neutralize nitric oxide in the medium by up to 40%. This effect of ladostigil, however, was observed at minimal concentration of 100µM.

Fig. 9: Effect of ladostigil and its metabolites on mitochondrial membrane potential stability in H2O2 treated SK-N-MC cells

SK-N-MC cells were treated with compounds for 90 min, loaded with 2.5µM of

JC-1 and exposed to 500µM of H2O2 in presence of the compounds for 60 min for immunofluorescence analysis of mitochondrial potential changes (see

Methodology). Significantly different from H2O2 treated controls; * p<0.01; significantly different from 10µM and 1µM drug treated; ‡ p<0.01; 37

These results indicate a potential neuroprotective role of ladostigil and its metabolites arising from their ability to stabilize the mitochondrial potential in

H2O2 stressed cells. The compounds possess this activity only in relatively high concentrations and neither the propargyl nor the carbamate moieties are required for this activity.

Analysis of activity of caspase 3 and 7 in SK-N-MC cells

The analysis of mitochondrial potential showed that the compounds might induce their neuroprotective effect by stabilization of mitochondrial membrane.

This effect may be accompanied by prevention of the release of cytochrome c and further activation of caspase intrinsic pathway. We performed the analysis of cellular activity of caspase 3 and 7 in SK-N-MC cells under oxidative stress produced by 1mM of H2O2 pretreated with the compounds. The results show that pretreatment with 10µM of the compounds for 90 min can attenuate caspase 3 and 7 activity in the cells under oxidative stress which was implemented for three hours by H2O2.

All the compounds reduced the activity of the caspase proteins 3 and 7 by up to 65% compared to that in cells treated with H2O2 alone (Fig. 10). No significant difference in the activity was detected between the compounds (p<0.05). It appears that the overall outcome of these experiments is in good agreement with the results obtained from the experiments with mitochondrial potential measurements.

120

100

60 * * * * 40

Relative level of caspase 3/7 activity (%) activity 3/7 caspase of level Relative

0 Ladostigil R-MCPAI R-CAI R-HPAI Vehicle + + + + + +

H2O2 - + + + + +

Fig. 10: Effect of ladostigil and its metabolites on caspase 3 and 7 activity in H2O2 treated SK-N-MC cells

SK-N-MC cells were pretreated with 10µM compounds for 90 min and then exposed to 1mM of H2O2 for 3 h. The cells were detached and collected for further analysis of caspase 3/7 activity assay (see Methodology). Significantly different from H2O2 alone treated cells; * p<0.05;

Evaluation of the effect of ladostigil administration in the acute inflammation mouse model

Evaluation of pro-inflammatory gene expression in the cerebral cortex of LPS treated mice

Cortical regions of mouse brains were collected 4 h after i.p. injection of LPS

(10mg/kg) in order to analyze the pattern of pro inflammatory gene expression in the animals which have been given ladostigil (10mg/kg).

A) B) 2 200 # #

1 100

RFU 0 RFU 0 TNF-a IL-1b C) # D) 60 20

40 # 10 20

0 0 IL-6 iNOS Sham LPS treated.

Fig. 11: Effect of i.p. injection of LPS on the gene expression of pro- inflammatory cytokines in mouse brain (RFU – relative fluorescent units)

Male Balb-C mice aged 7-8 weeks were injected i.p. with 10mg/kg LPS in saline solution. Sham group was injected i.p. with saline. The animals were sacrificed

4 h later and the cortex was collected and frozen for further total RNA isolation and RT-PCR analysis (see Methodology). Significantly different from LPS untreated controls; # p<0.05; 41

200

160 TNF- 15min

TNF- 120min

120 IL-1 15min

IL-1 120min * 80 * * IL-6 15min IL-6 120min

40 iNOS 15min

iNOS 120min

Relative level of cytokine gene expression (%) expression gene cytokine of level Relative 0 Sham LPS alone LPS+Ladostigil

Fig. 12: Effect of ladostigil pretreatment (15 min or 120 min) on pro- inflammatory cytokines gene expression in the brain of LPS treated mice

Male Balb-C mice aged 7-8 weeks were orally given the solution of 10mg/kg of ladostigil followed by injection i.p. of 10mg/kg LPS in saline solution. Control groups were: mice given saline orally and injected i.p. with saline (sham group) and mice given saline orally and injected i.p with LPS (LPS alone group). The animals were sacrificed 4 h later and the cortex was collected and frozen for further total RNA isolation and RT-PCR analysis (see Methodology).

Significantly different from LPS alone treated mice; * p<0.01;

We focused on three cytokine genes (TNF-α, IL-1β and IL-6) as well as on iNOS. These genes were shown to undergo the substantial upregulation in innate inflammatory response within a short time after LPS injection.

COX2 15min COX2 120min

120

100 * 80

Relative expression of COX-2 (%) COX-2 of expression Relative 0 Sham LPS alone LPS+Ladostigil

Fig. 13: Effect of ladostigil pretreatment (15 min or 120 min) on COX-2 gene expression in the brain of LPS treated mice

Significantly different from LPS alone treated mice; * p<0.05; 42

In preliminary experiments it was found that 4 h is the optimal time point for measurement of gene expression in the cortex. RT-PCR analysis revealed that i.p. injection of LPS produced a significant upregulation of all the genes at 4 h

(Fig. 11 A-D).

While IL-1β and IL-6 showed the highest level of upregulation (x200 and x80 respectively) of gene expression compared to controls, that of TNF-α and iNOS was relatively low (x2 and x6 respectively).

Surprisingly, it was found that pretreatment conditions of 15 min resulted in a significant up regulation in the gene expression of TNF-α and IL-1β and no effect in gene expression of IL-6 and iNOS in the mice which were treated with ladostigil (Fig. 12 solid column). On the other hand, RT-PCR analysis of COX-

2 gene expression - revealed a significant reduction of 20% in the mice treated with 10mg/kg of ladostigil 4 h after LPS injection (Fig. 13 solid column). These findings were supported by two independent experiments.

However, when ladostigil was given 2 h before the LPS injection there was significant reduction in expression of all genes producing a lowering in levels of

TNF-α expression by 25%, IL-1β by 20% and iNOS by 30%. Ladostigil had no effect on the gene expression of IL-6 (Fig. 12 cross-hatched column). No additional effect was observed on the expression of COX-2 after 2 h of pretreatment with ladostigil (Fig. 13 cross-hatched column).

Evaluation of microglial activation in the cerebral cortex of LPS treated mice

The demonstration of microglial activation in the brain following i.p. injection of

LPS was performed by analysis of gene expression of Galectin-3.

120

100

80 * 60

Relative expression of Galectin -3 (%) -3 Galectin of expression Relative 0 Sham LPS alone LPS+Ladostigil

Fig. 14: Effect of ladostigil pretreatment on Galectin-3 gene expression in the brain of LPS treated mice

Significantly different from LPS alone treated mice; * p<0.05; 44

This marker is highly specific for activated microglia and undergoes a rapid upregulation when microglia are exposed to an inflammatory trigger.

Treatment of mice with ladostigil (10mg/kg) reduced Galectin-3 gene expression to its basal level compared to LPS treated controls (Fig. 14). This effect was observed 4 h after LPS injection. These results were confirmed by two independent experiments.

Evaluation of pro-inflammatory cytokines release in the cerebral cortex of LPS treated mice

The finding that the ladostigil treatment upregulates pro-inflammatory genes in the mouse brain 4 h after LPS injection led us to determine the effect of its administration on pro inflammatory cytokine release in the cortex of the LPS challenged mice.

Screening of different time points to determine the optimal signal to noise ratio between LPS treated and untreated animals (data not shown) demonstrated that 8 h was the most appropriate for cytokine detection. At this time point TNF-

α release in LPS challenged animals increased 5 fold compared to untreated mice, and IL-1β – up to 4 fold.

ELISA analysis of the ladostigil pretreated mice showed that 8 h after LPS injection, pretreated mice expressed up to 50% less TNF-α (Fig. 15A) and up to 40% less of IL-1β protein (Fig. 15B). 45

These results clearly show that oral administration of ladostigil at a dose of

10mg/kg 15 min prior to LPS injection can attenuate its pro-inflammatory effect in the brain.

A) TNF 120 100 80 60 * 40 20 0 Sham LPS alone LPS+Ladostigil

B) IL-1 120 100 80 * 60 40

Relative ammount of the cytokines (%) cytokines the of ammount Relative 20 0 Sham LPS alone LPS+Ladostigil

Fig. 15: Effect of ladostigil pretreatment on pro-inflammatory cytokines release in the brain of LPS treated mice

Ladostigil 10mg/kg was administered orally to male Balb-C mice aged 7-8 weeks followed by an i.p. injection of 10mg/kg LPS in saline solution. Control groups were: mice given saline orally and injected i.p. with saline (sham group) and mice given saline orally and injected i.p with LPS (LPS alone group). The animals were sacrificed 8 h later and the cortex was collected and frozen for further total protein isolation and Western blot analysis (see Methodology).

Significantly different from LPS alone treated mice; * p<0.05; 46

Evaluation of pro-inflammatory cytokines release in the spleen of LPS treated mice as a model of acute inflammation

In order to elucidate the possible involvement of ladostigil and its metabolites in modulation of systemic inflammation we treated the mice with ladostigil (10 and 5mg/kg) 15 min prior the LPS injection (10mg/kg) and harvested the spleens 4 h later. This time point was chosen as optimal for measuring pro- inflammatory cytokines TNF-α, IL-1β and IL-6. A significant increase in cytokine release was observed, producing amounts of 17, 2000 and 45 fold respectively greater than untreated controls (Fig. 16). Oral administration of 10mg/kg of ladostigil significantly reduced all three cytokines - TNF-α by 42%, IL-1β by 25% and IL-6 by 54% (Fig. 16). These results were confirmed in two independent experiments.

ELISA analysis of TNF-α, IL-1β and IL-6 proteins in the spleen of ladostigil treated animals did not show any noticeable effect compared to untreated mice

(data not shown).

We also determined the effect of a lower dose of ladostigil on pro inflammatory cytokines in spleen. The mice were treated under the same conditions with ladostigil at a dose of 5mg/kg and the spleens were harvested 4 h later. ELISA analysis of TNF-α, IL-1β and IL-6 proteins did not show any significant change in cytokine release compared to animals given LPS alone (data not shown).

47 *

A) TNF 20

10 *

0 Sham LPS alone LPS+Ladostigil B) 3000 IL-1

2000 *

1000

0 Sham LPS alone LPS+Ladostigil C) IL-6 60 50 40

Ammount of the cytokines (pg/ml) cytokines the of Ammount 30 * 20 10 0 Sham LPS alone LPS+Ladostigil

Fig. 16: Effect of ladostigil pretreatment on pro-inflammatory cytokines release in spleen of LPS treated mice

Male Balb-C mice aged 7-8 weeks were orally given the solution of 10mg/kg of ladostigil followed by injection i.p. of 10mg/kg LPS in saline solution. Control groups were: mice given saline orally and injected i.p. with saline (sham group) and mice given saline orally and injected i.p with LPS (LPS alone group). The animals were sacrificed 8 h later and the spleens were collected and frozen for further total protein isolation and Western blot analysis (see Methodology).

Significantly different from LPS alone treated mice; * p<0.05; 48

Based on these data we conclude that oral administration of ladostigil at a dose of 10mg/kg 15 min prior to LPS injection can attenuate its pro-inflammatory effect in the brain. This effect was not seen at a lower dose of ladostigil.

Measurement of extent of oxidative damage in the brain of ladostigil treated mice after LPS challenge using TBARS assay

To evaluate whether ladostigil can reduce oxidative damage in the brain induced in mice by LPS we quantified malondialdehyde (MDA) concentrations in the anterior hemispheres of ladostigil treated (10mg/kg) mice, 16 h after LPS

(10mg/kg) injection using thiobarbituric acid reactive substances (TBARS) assay.

These substances are formed as byproducts of lipid peroxidation as a result of exposure of the tissues to highly reactive oxygen species (ROS). The time point was chosen as most appropriate for successive detection of TBARS in the brain based on the previous experiments performed in our lab. The treatment with

LPS for 16 h doubled the content of MDA in the brain tissues (Fig. 17).

Fifteen min pretreatment with ladostigil prior to LPS injection reduced the content of MDA in the brain by 74% from 3.82 µM to 2.42 µM. These results were confirmed by 3 independent tests taken from the same experiment.

Based on this data we can conclude that 15 min pretreatment with ladostigil may have a neuroprotective effect on the brain tissues against LPS induced oxidative stress. This effect was observed to be lasting up to 16 h after LPS injection. 49

3 *

MDA (µM/mg of total protein) total of (µM/mg MDA 0 Sham LPS alone LPS+Ladostigil

Fig. 17: Effect of ladostigil on content of MDA in the brains of LPS treated mice

Male Balb-C mice aged 7-8 weeks were orally given the solution of 10mg/kg of ladostigil followed by injection i.p. of 10mg/kg LPS in saline solution. Control groups were: mice given saline orally and injected i.p. with saline (sham group) and mice given saline orally and injected i.p with LPS (LPS alone group). The animals were sacrificed 16 h later and both anterior hemispheres were collected and frozen for further analysis of MDA (see Methodology). Significantly different from LPS alone treated mice; * p<0.05;

Measurement of IFN-γ release from splenocytes of ladostigil chronically treated rats ex vivo

In order to elucidate whether chronic administration of ladostigil may possess a systemic immunomodulatory effect in aged animals we analyzed the release of IFN-γ from anti-CD3 activated splenocytes ex vivo using ELISA. Young controls did not show any response to anti-CD3 activation at both concentrations of the antibody (Fig. 18 A and B) but there was significant increase in release of IFN-γ in aged untreated controls.

Treatment of the splenocytes with 0.5µg/mL of anti-CD3 produced 430ng/mL of IFN-γ while concentration of 0.1µg/mL, 370ng/mL of the cytokine.

Chronic treatment of the aged animals at each dose of ladostigil showed an immunomodulatory effect on splenocytes that were activated with the lower concentration of anti-CD3 (0.1µg/mL) but not with the higher concentration.

ELISA analysis showed that IFN-γ was reduced by 38% and 62% for ladostigil

1 and 8.5mg/kg/day respectively, when splenocytes were activated with anti-

CD3 (0.1µg/mL).

A) anti-CD3 (0.5 µg/ml) 800 600 400

ng/ml) 200

 0 B) 500 anti-CD3 (0.1 µg/ml) 400 300 * * 200

Ammount of IFN of Ammount 100 0 Young untreated Ladostigil 1mg/kg Aged untreated Ladostigil 8.5mg/kg

Fig. 18: Effect of ladostigil on release of IFN-γ from the splenocytes of the aged rats

Splenocytes were isolated from spleens of ladostigil long term treated Winstar rats (see Methodology). The cells were activated with anti-CD3 antibody (0.1 or

0.5 µg/ml). Cell medium was collected 48 h later and frozen for further analysis of IFN-γ protein by ELISA (see Methodology). Significantly different from LPS alone; * p<0.05;

These results indicate that chronic treatment of aged rats with ladostigil can produce a dose-related systemic immunomodulatory effect.

Activation of the splenocytes with different concentrations of LPS (10, 5 and

1mg/mL) did not produce any detectable levels of IFN-γ or IL-2. Furthermore, no IL-2 was detected in the medium of anti-CD3 activated splenocytes (data not shown).

Discussion

Microglial activation during aging may have prominent consequences for brain function and mental health. Continuous and uncontrolled release of pro- inflammatory cytokines and reactive oxygen species promote neuronal cell death and are considered to be the possible triggers of neurodegenerative diseases like AD.

AD is progressive neurodegenerative condition characterized by massive cell loss at frontal and hippocampal regions of the brain leads eventually to cognitive deterioration and dementia. Current treatment options are limited and focus on palliative care of the clinical symptoms rather than to prevent the disease progression. The main treatment approach based on inhibition of AChE has been shown to be effective in improving some aspects of cognitive function and slowing progression of impairment for up to 1 year.

Ladostigil was designed by Prof. Weinstock-Rosin in order to combine the

AChE inhibitory properties of rivastigmine with the presumed neuroprotection activities of MAO-B inhibitors, by introducing a carbamate moiety into the MAO-

B selective inhibitor, rasagiline. However, it was found that ladostigil itself lacks

MAO inhibitory activity and is only a relatively weak AChE inhibitor in vitro but not in vivo. This is because ladostigil undergoes de-methylation by cytochrome

2C19 in the liver to form R-MCPAI which is about 30 times more potent than the parent drug as an AChE inhibitor. MAO-A and B activity in vivo results from the removal by cholinesterase of the carbamate moiety to form R-HPAI ((6-

Hydroxy–N-propargyl-1(R)-aminoindan). 54

The current study evaluated the neuroprotective and anti-inflammatory activity of the parent drug in vivo and also together with that of its primary metabolites in vitro.

The main findings of this research are that the ladostigil and its primary metabolites have immunomodulatory activity in the primary microglia cells and also neuroprotective capabilities against oxidative stress in a neuroblastoma cell line. Moreover we showed that when given orally to mice with acute systemic inflammation induced with LPS, ladostigil showed the ability to attenuate the immune response in the spleen and the brain cortex of the animals accompanied with reduced activation of microglia and brain tissue oxidative damage. Chronic oral administration of ladostigil to aged rats also reduced the immunoreactivity of the splenocytes to anti-CD3 thereby decreasing IFN-γ release. These effects are produced by the compounds in concentrations of ladostigil and metabolites at and below those that inhibit ChE and MAO.

We used a culture of mouse primary microglia as a model of brain microglia.

Using either lipopolysaccharide (LPS) or IFN-γ as microglia activators we tried first to elucidate the ability of ladostigil and its main metabolites – R-MCPAI, R-

CAI and R-HPAI – to modulate the inflammatory reaction of activated microglia in terms of NO release by measuring the concentration of nitrites – the main stable product of nitric oxide in the medium.

LPS and IFN-γ activate microglia through different pathways. LPS activates the

MAPK pathway in direct and indirect manner by interacting with TLR4. IFN-γ produces its inflammatory effect by interacting with its own receptor on the 55

surface of microglia. Upon receptor binding the cytosol regulatory elements of the pathway – JAK-STAT – undergo rapid phosphorylation and localize into nucleus where they promote their effect.

Treatment of IFN-γ activated microglia with a wide range of concentrations of the compounds did not cause any noticeable change in the pattern of NO release. However all the compounds caused a significant reduction of NO in

LPS activated microglia at concentrations ranging from 1nM-1µM. This suggests that they may act via modulation of MAPK pathway but not of the JAK-

STAT pathway. Therefore, all further experiments focused on the immunomodulatory ability of the compounds in LPS activated microglia.

We evaluated the effect of the compounds on expression of pro-inflammatory genes TNF-α, IL-1β and iNOS since they have been reported to be the most important in promoting the inflammatory environment caused by the response of the microglia to LPS. Furthermore, these genes have been shown to be regulated by the same molecular pathway involving MAPK cascade and NF-

κB transcription factor (18, 63-65).

All the compounds were effective at a concentration of 10nM in reducing the expression iNOS and pro-inflammatory cytokines but increasing the concentration did not increase the effect of the compounds appreciably.

ELISA analysis of the TNF-α cytokine release provides us with correlative results with the findings of gene expression. These results clearly show that all the metabolites as well as ladostigil at a concentration of 10nM produce their immunomodulatory effect on LPS activated microglia within short time frame

(from 2 h to 12 h) after the activation. 56

In order to discover the possible mechanism of action of the compounds we concentrated on three main elements of MAPK pathway which are activated when LPS interacts with the TLR4 – ERK, p38 and Jnk phosphorylation.

Western blot analysis showed reduction of up to 25% in ERK1/2 phosphorylation while phosphorylation of p38 was restored to basal levels by all compounds except R-CAI which lacks the propargyl moiety.

These findings suggest that these two main elements of MAPK pathway are potentially involved in the immunomodulatory activity of the compounds. The fact that Jnk phosphorylation was unaffected by the compounds may explain the moderate degree of the reduction of the pro-inflammatory genes despite the observation of complete prevention of p38 phosphorylation. Together with the moderate effect of the compounds on phosphorylation of ERK element, one may suggest that the compounds have an immunomodulatory rather than an immunosuppressive activity on the immune response. It is preferable to reduce excessive release of these cytokines rather than to cause immunosuppression with its attendant potential detrimental effects.

The main downstream component of MAPK pathway is IκBα protein which binds two functional NF-κB subunits – p50/p65 and inhibits their activity.

Phosphorylated ERK and p38 cause disassociation of IκBα and its further degradation resulting in the release and nuclear translocation of heterodimer.

We found that ladostigil, R-MCPAI and R-HPAI stabilized IκBα proteins, while

R-CAI, again, was less potent compared to others. 57

These findings were supported by further experiments that showed a reduction in nuclear translocation of the p65 subunit by up to 35% by the compounds compared to LPS alone treated microglia.

Based on these results we suggest a mechanism of action for ladostigil and its metabolites, in which reduction in phosphorylation of elements of the MAPK pathway results in attenuation of NF-κB nuclear translocation in the LPS activated microglia. The fact that R-CAI was less effective in its ability to reduce the phosphorylation of ERK and p38 proteins suggests that propargyl moiety may provide an advantage for this effect. Despite this finding, R-CAI was shown to be equally effective in reducing the gene expression of TNF-α, iNOS and the release of TNF-α cytokine. This suggests that the reduction in the phosphorylation of the MAPK elements by this compound was sufficient to produce immunomodulatory effect.

In order to investigate the neuroprotective properties of ladostigil and its metabolites we used human neuroblastoma cell line SK-N-MC stressed with hydrogen peroxide - as an in vitro model of neuronal cells. This model reproduces the oxidative damage that occurs in the brain as a result of exposure to ROS produced by astroglial cells during inflammation.

We show that the compounds are able to stabilize the mitochondrial potential in concentrations ranging from 1 to 10µM. R-CAI proved to be less potent than the other compounds in stabilizing the mitochondrial potential which again indicates the possible importance of propargyl moiety in the execution of this activity. 58

Since the fall in mitochondrial potential is associated with the release of cytochrome c and caspase activation we analyzed the caspase 3 and 7 activity in the stressed cells. Caspase 3 was chosen as an important factor in the apoptosis cascade, while a precursor of caspase 7 is cleaved by caspase 3 being converted to the active form which executes the apoptosis.

The subsequent reduction of caspase activity in the stressed cells treated with

10µM of the compounds accords with the data from previous experiments with

JC-1 and supports our hypothesis that the compounds possess neuroprotective and anti-apoptotic capabilities against oxidative stress. These concentrations are somewhat higher than those required for their anti-inflammatory effect but not high enough to scavenge ROS in the medium. This was shown recently in our lab, when it was found that ladostigil must to be presented at a minimum concentration of 100µM in the medium in order to act as scavenging agent.

Taken together, we can conclude that the compounds may promote their protective effect by reducing cell damage induced by oxidative stress and by modulation of the inflammatory response of microglia.

The blood brain barrier (BBB) functions as a physical obstacle for peripheral immune cells and prevents the uncontrolled exposure of the brain to undesirable factors. However, recent data point to the existence of cross talk between the brain and the peripheral immune system (66, 67).

The question whether peripherally administrated LPS has the ability to penetrate the BBB and induce its effect directly on the brain cells still remains unclear. However, recent data with radioactive labeled LPS suggest that the penetration of the LPS into the brain is minimal and mostly localized to brain 59

endothelial cells (68). The alternative mechanisms by which peripheral administration of LPS may promote the immune reaction in the brain may be through vagal or other afferent nerve stimulation (69, 70), release of substances from periphery that can cross BBB (71), altering the permeability of BBB or enhancing the interactions between BBB and immune cells (72, 73).

Activation of the innate immune system by LPS given i.p. induces the release of a wide range of pro-inflammatory cytokines. This was proven by detection of the raised levels of TNF-α, IL-1β and IL-6 cytokines in mouse spleen 4 h after an injection of LPS. The reason for using the spleen is that it serves as a good immunological model since it contains virtually all types of the immune cells and reflects the general state of peripheral immune system. Our findings that the short term pretreatment with ladostigil reduced the release of pro-inflammatory cytokines from the spleen suggest that the compounds may possess systemic immunomodulatory activity. This activity may have an important impact on the immune processes which take place in the brain.

By means of LPS i.p. injection we produced substantial upregulation of the gene expression of IL-1β, IL-6 and iNOS in cerebral cortex. The expression of TNF-

α was moderately elevated, doubling its amount compared to LPS controls.

This elevation of expression in 4 h suggests that these two processes occur simultaneously and probably subsequently, beginning with activation of innate immune system followed by a brain inflammatory response. Alternatively, it could result from continuous cross talk between the peripheral and central immune systems. Such an interaction takes place shortly after LPS injection and may be mediated by inflammatory factors (e.g. cytokines and chemokines) which cross the BBB and may activate the local microglia cells. 61

In mice given ladostigil 15 min prior to LPS a paradoxical upregulation of TNF-

α, IL-1β and iNOS gene expression was detected. The only gene that showed a 20% decrease in its expression was COX-2 which is responsible for the production of cyclooxygenase, the enzyme synthesizing prostaglandins as a response to pro inflammatory stimuli.

We speculate that the elevation of certain genes and downregulation of others

(COX-2) may be crucial for maintaining the early immune response.

Alternatively this may have occurred because there was not enough time for ladostigil or its metabolites to reduce gene expression. This agrees with our observation of reduction in TNF-α and IL-1β gene expression levels 8 h after administration of ladostigil and is supported by another experiment in which ladostigil was given 2 h before LPS, and reduced expression of IL-1β, TNF-α and IL-6 and iNOS by more than 20%. Another possibility is that the cytokine release can be regulated at the level of translation rather than gene expression.

This possibility, however, was not investigated in this research.

Treatment with ladostigil decreased the expression of the Galectin-3 gene in the cerebral cortex 4 h after LPS injection, virtually restoring it to basal levels.

Since Galectin-3 is specific marker of microglial activation in the CNS, it suggests that ladostigil may have a direct effect on the activation state of microglia. This finding fits well with our in vitro data since it has been reported that Galectin-3 expression is regulated by MAPK pathway (74). Although we did not examine Galectin-3 gene expression in the in vitro model of the microglial activation one may suggest that alteration by the compounds of the activity of components of the MAPK pathway may explain their general action on activated microglia. Reduction of microglia activation might provide an 61

explanation for our findings that ladostigil can reduce oxidative stress, as indicated by the 75% reduction in brain malondialdehyde 16 h after LPS injection. Again, this may result by direct or indirect or both mechanisms we proposed above leading to prevention of excessive microglial activation.

Taken together with our in vitro neuroprotection data we suggest that the ladostigil acts by different mechanisms to protect the brain from the harmful impact of acute inflammation. It modulates the state of activation of microglia and prevents cell damage induced by oxidative stress by attenuation of the fall of their mitochondrial potential and activation of caspase cascade.

Thus, we can conclude that ladostigil may act indirectly by modulating the response of peripheral immune system and directly by attenuating of MAPK pathway activity leading to reduction in the response of the microglia to peripheral administration of LPS. However, we cannot negate the possibility that the decrease in microglia activation may arise solely from modulation of peripheral immune system with alteration of the activity of vagal or other afferent nerve pathways.

Recent epidemiological data suggest the existence of possible link between chronic systemic inflammation and the probability of development of neurodegenerative diseases in the future (75-77). This condition, characterized by prolonged immunoreactivity as a result of chronic pathological processes or aging, may provoke or exaggerate the variety of neurodegenerative illnesses, and AD among them, in the older population.

Our findings with aged rats that were treated chronically with ladostigil may point on the possible role of ladostigil in immunomodulation of low grade and 62

age related systemic inflammation. The fact that the prolonged treatment with low dose of the drug attenuated IFN-γ release induced by anti-CD3 in concentration of 0.5µM brought us to suggest that ladostigil may act as immunomodulatory agent not only in the case of acute but also that of chronic low grade inflammation which, by itself, may promote the outbreak of the neurodegenerative conditions or exacerbate the current illness. The finding that ladostigil treatment reduced the excessive release of IFN-γ in aged rats suggests that it may also act indirectly to modify continuous activation of microglia in the aged brain. Our in vitro data show that ladostigil is unable to prevent the consequences of microglia activation by IFN-γ.

It has been reported recently that donepezil, another AChE inhibitor being used as a treatment for mild to moderate stages of Alzheimer's disease, may possess immunomodulatory activity in the cultured microglia (33). This drug was shown to reduce the gene expression of TNF-α, IL-1β and iNOS in concentrations ranged from 5 to 20µM. Moreover, it was reported to reduce the NF-κB nuclear translocation at concentration of 10µM.

These findings clearly show that although this drug may have some immunomodulatory effect on LPS activated microglia this would only be achieved at concentrations that are much higher than that which inhibits AChE thereby inducing adverse effects due to increased cholinergic activity. This problem does not exist for ladostigil since the immunomodulatory effect of its major metabolites occurs at slightly lower concentrations (10nM-1µM) than those that inhibit AChE (IC50 0.9 µM). 63

Taken together, we can conclude that ladostigil and its metabolites may possess anti-inflammatory, immunomodulatory and neuroprotective capabilities which enable this compound to be a good candidate for the treatment of Alzheimer's disease.

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List of abbreviations:

AD – Alzheimer’s disease

AChE – acetylcholine esterase

ChE – choline esterase

MAO – monoamine oxidase

LPS – lipopolysaccharides

ROS - reactive oxygen species

NO – nitric oxide

IFN – interferon

TNF – tumor necrosis factor

IL – interleukin iNOS – inducible nitric oxide synthase

TLR4 – Toll-like receptor 4

MAPK – mitogen-activated protein kinase

ERK - extracellular-signal-regulated kinase

Jnk – c-Jun N-terminal kinase

NF-κB – nuclear factor kappaB

MDA – malondialdehyde

TBARS - thiobarbituric acid reactive substances

EMSA – elctromobility shift assay

WB – western blot

RT-PCR – real time PCR

ELISA - Enzyme-linked immunosorbent assay

COX – cyclooxygenase i.p. – intra peritoneal

תקציר:

עדויות הולכות ומצטברות מעידות על כך שתהליכים דלקתיים כרוניים במוח עשויים לגרום

לתמותה של תאים נוירונליים וירידה מתמשכת בפעילות קוגניטיבית. מצב זה עשוי להוביל

למחלות נוירודגנרטיביות כגון מחלות האלצהיימר ופרקינסון.

הגורם העיקרי לדלקת במוח הינו תאי מיקרוגליה השייכים לתאי תמך ומהווים כ – 11% מתאי

מוח. תפקידם העיקרי הינו הגנה על המוח מפני חדירת פתוגן והם מהווים מקור בלעדי לתגובה

דלקתית. כתוצאה מחשיפת תאי מיקרוגליה לגורם מזיק, התאים עוברים ממצב לא משופעל

למצב משופעל תוך כדי שינויים מורפולוגיים ותפקודיים. שינויים אלו מלווים בהפרשת גורמי

דלקת (TNF-α, IL-1β, IL-6) ומולקולות ריאקטיביות )RNS ,ROS( שתפקידם לנטרל את

הגורם המזיק ולהגן על רקמת המוח. בעוד שלתגובה דלקתית קצרת טווח יתרונות רבים

בסילוק הגורם הדלקתי, עירור מתמשך של מיקרוגליה עשוי להסב נזק בלתי הפיך לרקמות

המוח ולהוביל לנוירודגנרציה.

Ladostigil הינו חומר חדש שסונתז על בסיס שתי תרופות ידועות לטיפול במחלת האלצהיימר

)Rivastigmine( ובמחלת הפרקינסון )Rasagiline( במטרה לשלב את פעילות הפרמקולוגית

של שני החומרים. Ladostigil הינו מעכב חלש של כולין אסתראז )ChE( וחסר פעילות

המעכבת את המונואמין אוקסידאז (MAO( בתנאי in vitro. יחד עם זאת, כשניתן דרך הפה, ladostigil מתפרק על ידי מערכות אנזימטיות בגוף לשלושה מטבוליטים פעילים. R-MCPAI

ו-R-CAI האחראים עיכוב פעילות ה- ChE ו – R-HPAI האחראי לעיכוב הפעילות MAO

ברקמת המוח.

בעבודות קודמות נמצא כי טיפול כרוני של חולדות זקנות במינון נמוך של ladostigil ביטל את

הירידה בביטוי גנים הקשורים למטבוליזם ותהליכי חמצון, מנע עליה בביטויו של CD11b (סמן

לאקטיבציה של מיקרוגליה בהיפוקמפוס וקורטקס) ומנע ירידה בזיכרון של החיות.

מטרת עבודה זו הייתה להבין האם ladostigil והמטבוליטים שלו גורמים לאפקטים הללו דרך

פעילותם הישירה על תאי המיקרוגליה או באופן עקיף על ידי מניעת נזקי עקת חמצון לתאי

מוח ושפעול של מיקרוגליה כתוצאה מכך. ממצאים עיקריים של המחקר הנוכחי מראים ש- ladostigil והמטבוליטים הורידו את השפעול של מיקרוגליה באמצעות LPS. מצאנו שבטווח

רחב של ריכוזים )10nM-1µM( כל החומרים שהודגרו עם תאי מיקרוגליה המשופעלים

באמצעות LPS למשך 24 שעות הורידו שחרור NO למדיום של התאים עד 41%. טיפול בתאי

מיקרוגליה המשופעלים באמצעות החומרים בריכוז של 10nM גרם לירידה של עד 25%

בביטוי גנים פרו-דלקתיים ו iNOS וכן לירידה בשחרור של TNF-α מהתאים. פעילות אנטי-

דלקתית זו נובעת מיכולת החומרים לעכב את זירחון ושפעול של ERK וp38- וכתוצאה מכך

לעכב כניסתו של פקטור שעתוק NF-κB לגרעין. טיפול ב 10µM של החומרים שונים בתאי

נוירובלסטומה, המהווים מודל in vitro של נוירונים, שעברו עקת חמצון באמצעות מי חמצן,

הקטין את נפילת הפוטנציאל המיטוכונדריאלי ואת שפעול מסלול הקספזות 3 ו7- ברמה של

עד 65%.

ממצאים מניסויי ה – in vivo מראים כי טיפול חד פעמי ב – ladostigil בעכברים בהם

הושרתה תגובה דלקתית על יד הזרקה פריפרית של LPS גרם לירידה של עד 81% בביטוי

גנים פרו-דלקתיים בקורטקס 8 שעות לאחר הטיפול. בנוסף לכך, הטיפול גרם לעיכוב של עד

41% בשחרור של ציטוקינים פרו-דלקתיים בטחול של החיות שעברו טיפול עם ladostigil 4

שעות קודם לכך.

מניסויים של מתן כרוני של ladostigil לחולדות זקנות המהוות מודל לדלקת כרונית שקשורה

לגיל ניתן היה ללמוד כי הטיפול במינונים של 8.5mg/kg/day ו - 1mg/kg/day גרם לירידה

בריאקטיביות של תאי טחול בהשוואה לחיות זקנות שלא עברו טיפול. הטיפול גרם לירידה בכ

- 61% של שחרור IFN-γ מתאי טחול משופעלים על ידי נוגדן ל – CD3. הפעילויות האנטי

דלקתיות המתוארות של החומרים נצפו בטווח מינונים נמוך או שווה לאלו הדרושים לעיכוב

.in vitro ChE

תוצאות אלו מצביעות על יכולת החומרים לפעול באופן ישיר ולמתן את התגובה הדלקתית של

תאי המיקרוגליה בתגובה ל-LPS. החומרים נמצאו כבעלי יכולות נוירופרוטקטיביות ואנטי

אפופטוטית בתאים שעברו עקת חמצון. בעבודה זו הראנו לראשונה, שהשפעת החומרים

נובעת מיכולתם לעכב את מסלול ה- MAPK וטרנסלוקצית פקטור שעתוק NF-κB לגרעין

התא ועל ידי מניעת נפילת הפוטנציאל המיטוכונדריאלי ושפעול מסלול הקספזות.

עבודה זו נעשתה תחת הדרכתה של פרופ' מרתה ווינשטוק - רוזין

תכונות אנטי-דלקתיות ונוירופרוטקטיביות של ladostigil והמטבוליטים הטבעיים שלו בתאים ובחיות

חיבור לשם קבלת תואר דוקטור לפילוסופיה

מאת

רוני פנרסקי

הוגש לסנט האוניברסיטה הערית בירושלים

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