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Neurotoxicity ResearchNeurodegeneration,Neuroregeneration,Neurotrophic Action, andNeuroprotection ISSN 1029-8428Volume 20Number 2 Neurotox Res (2011)20:150-158DOI 10.1007/s12640-010-9230-y

Flavin-Containing Monooxygenase mRNALevels are Up-Regulated in ALS BrainAreas in SOD1-Mutant Mice

Stella Gagliardi, Paolo Ogliari, AnnalisaDavin, Manuel Corato, Emanuela Cova,Kenneth Abel, John R. Cashman, MauroCeroni & Cristina Cereda

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Flavin-Containing Monooxygenase mRNA Levelsare Up-Regulated in ALS Brain Areas in SOD1-Mutant Mice

Stella Gagliardi • Paolo Ogliari • Annalisa Davin • Manuel Corato •

Emanuela Cova • Kenneth Abel • John R. Cashman • Mauro Ceroni •

Cristina Cereda

Received: 31 May 2010 / Revised: 7 October 2010 / Accepted: 3 November 2010 / Published online: 17 November 2010

� Springer Science+Business Media, LLC 2010

Abstract Flavin-containing monooxygenases (FMOs)

are a family of microsomal enzymes involved in the oxy-

genation of a variety of nucleophilic heteroatom-containing

xenobiotics. Recent results have pointed to a relation

between Amyotrophic Lateral Sclerosis (ALS) and FMO

genes. ALS is an adult-onset, progressive, and fatal neu-

rodegenerative disease. We have compared FMO mRNA

expression in the control mouse strain C57BL/6J and in a

SOD1-mutated (G93A) ALS mouse model. Fmo expres-

sion was examined in total brain, and in subregions

including cerebellum, cerebral hemisphere, brainstem, and

spinal cord of control and SOD1-mutated mice. We have

also considered expression in male and female mice

because FMO regulation is gender-related. Real-Time

TaqMan PCR was used for FMO expression analysis.

Normalization was done using hypoxanthine–guanine

phosphoribosyl transferase (Hprt) as a control housekeep-

ing gene. Fmo genes, except Fmo3, were detectably

expressed in the central nervous system of both control and

ALS model mice. FMO expression was generally greater in

the ALS mouse model than in control mice, with the

highest increase in Fmo1 expression in spinal cord and

brainstem. In addition, we showed greater Fmo expression

in males than in female mice in the ALS model. The

expression of Fmo1 mRNA correlated with Sod1 mRNA

expression in pathologic brain areas. We hypothesize that

alteration of FMO gene expression is a consequence of the

pathological environment linked to oxidative stress related

to mutated SOD1.

Keywords FMO � Amyotrophic lateral sclerosis � qPCR �SOD1 � Exposure to toxins

Introduction

Amyotrophic lateral sclerosis (ALS) is an adult-onset,

progressive, and fatal neurodegenerative disease with

unknown pathogenesis due to a selective loss of motor

neurons in the brainstem and spinal cord. The majority

(i.e., 90%) of cases presents sporadic onset disease (i.e.,

SALS), while 10% are described as familial onset (i.e.,

FALS). Twenty percent of FALS are linked to mutations in

the gene encoding Cu–Zn superoxide dismutase (SOD1),

an enzyme converting superoxide anion into hydrogen

peroxide (Rosen et al. 1993). Familial and sporadic forms

of ALS present an overlapping clinical picture and disease

course suggesting a common pathogenesis that involves the

same cellular pathways and mechanisms such as oxidative

stress and protein aggregation (Cleveland and Rothstein

2001; Kruman et al. 1999).

It has been suggested that the onset of the sporadic ALS

(SALS) may be related to exposure to toxic environmental

factors (Steele and McGeer 2008). In 1954, epidemiological

studies showed that ALS was 100-fold more prevalent in the

Chamorro indigenous people of Guam Island (Mulder et al.

1954). Guam ALS is clinically and pathologically identical

to classic ALS, and different environmental factors were

S. Gagliardi (&) � P. Ogliari � A. Davin � M. Corato � E. Cova

� M. Ceroni � C. Cereda

Lab of Experimental Neurobiology, IRCCS National

Neurological Institute ‘‘C. Mondino’’, Via Mondino, 2,

27100 Pavia, Italy

e-mail: stella.gagliardi@mondino.it

S. Gagliardi � M. Corato

Department of Neurological Sciences, University of Pavia,

Pavia, Italy

K. Abel � J. R. Cashman

Human BioMolecular Research Institute, San Diego, CA, USA

123

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DOI 10.1007/s12640-010-9230-y

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hypothesized to be involved. The most frequently cited

hypothesis of the environmental cause of Guam syndrome

has been the exposure to toxins from the Cycas micronesica

plant (Steele and McGeer 2008).

Environmental hypothesis has been supported also by an

association between the risk of developing SALS and the

exposure to heavy metals, industrial solvents, and pesti-

cides, especially organophosphates (Johnson and Atchison

2009; Weisskopf and Ascherio 2009). In particular, studies

have suggested associations between occupational expo-

sure to organophosphates related to playing professional

soccer and farming, and the risk of developing ALS (Li and

Sung 2003; Abel 2007).

Variations of genetic background involving the single

nucleotide polymorphisms (SNPs) of genes related to

detoxication pathways may also have a role in SALS. Data

from different populations have shown associations between

some SNPs belonging to the paraoxonase genes (PON 1, 2,

3) and SALS (Slowik et al. 2006; Mahoney et al. 2006;

Saeed et al. 2006; Ricci et al. 2010). PON genes encode

high-density lipoprotein-associated enzymes that play a role

in the detoxication of a large number of organophosphorus

compounds (Costa et al. 2003). Other reports have suggested

a role of Flavin-containing monooxygenase (FMO) genes in

ALS, underling a correlation between polymorphisms

located in the 30untranslated region of the FMO1 gene and

SALS (Malaspina et al. 2001; Cereda et al. 2006).

FMOs constitute a family of enzymes located in the

endoplasmic reticulum (ER) that catalyze the oxygenation

of a variety of endogenous and exogenous nucleophilic

compounds including organophosphates and pesticides

(Hines et al. 1994; Elfarra 1995; Cashman 1995) in

detoxication processes (Venkatesh et al. 1992; 1991; Sid-

dens et al. 2008; Leoni et al. 2008).

FMO enzymes are primarily expressed in tissues that

carry out detoxication processes (e.g., liver, kidney, and

lung) and they are also present in mammalian brain (Zhang

and Cashman 2006; Janmohamed et al. 2004), although

their role in the nervous system is not clear. Nine FMO

genes located in two clusters on chromosome 1 are present

in mouse. The first cluster contains five genes named

Fmo1, 2, 3, 4, and 6; the second comprises the Fmo9, 12,

and 13 genes. The Fmo5 gene is located on chromosome 3

(Cashman 1995).

The most highly expressed Fmo genes in mouse brain

are reported to be Fmo5 and Fmo1, while Fmo2 and Fmo4

are transcribed at relatively lower levels and Fmo3 mRNA

was not detectable (Janmohamed et al. 2004). Experi-

mental studies with ALS model mice carrying mutated

SOD1 have shown modified cellular metabolism and

development of multiple pathogenic cellular processes

similar to sporadic human ALS, including oxidative stress

and protein aggregation (Shaw 2005) especially in the

brainstem and spinal cord (Mahoney et al. 2006; Garbuz-

ova-Davis et al. 2007; Watanabe et al. 2001). Moreover,

Liu et al. (1998) showed that ALS model mice transgenic

for mutant SOD1-G93A showed increased levels of free

radical-derived products from hydrogen peroxide in spinal

cord. Instead, in brain, oxygen radical content in both

SOD1-G93A and non-transfected mice did not show sta-

tistically significant differences, suggesting that the ROS

increase in spinal cord was specific. Increased oxidative

stress is well documented in brain and spinal cord of

sporadic and familial ALS patients and seems to be both a

hallmark of the pathology and the SOD1 mRNA increase

(Liu 1996; Gagliardi et al. 2010).

To examine a potential role for Fmo in ALS, we char-

acterized Fmo gene expression levels in different brain

regions and in spinal cord in both control C57BL/6J mice

and in mutated SOD1-G93A ALS model mice. We also

compared Fmo brain expression in female and male mice

because evidence exists that regulation of human and mice

Fmo gene expression is gender-dependent (Falls et al.

1997; Coecke et al. 1998). Moreover, we also searched for

a correlation between FMO and SOD1 expression to

understand better the pathogenetic mechanisms related to

the environmental factors and gender.

Materials and Methods

Animals

A group of 12 SOD1-G93A transgenic mice (6 males and 6

females C57BL/6J mice carrying the human SOD1-G93A

mutation) and a control group of 20 C57BL/6J mice (8

males and 12 females) were used. Transgenic SOD1-G93A

mice originally obtained from Jackson Laboratories (Bar

Harbor, Maine, USA) and carrying about 20 copies of the

mutant human hSOD1 gene with a Gly93Ala substitution

(B6SJL-TgNSOD-1-SOD1G93A-1Gur) were bred and

maintained on a C57BL/6 genetic background at Harlan

Italy S.R.L., Bresso (MI), Italy. Transgenic mice were

identified by PCR and were killed at the end stage of the

disease characterized by the complete paralysis of the hind

limbs and the difficulty to right through their own effort

within 30 s after being placed on both sides (approximately

21 weeks for males and 23 weeks for females). All mice

were housed at a temperature of 21 ± 1�C with relative

humidity 55 ± 10% and 12 h of light. Food (standard

pellets) and water were supplied ad libitum. Procedures

involving animals and their care were conducted in con-

formity with institutional guidelines that are in compliance

with national (D.L. No. 116, G.U. Suppl 40, Feb 18, 1992,

Circolare No. 8, G.U., 14 luglio 1994) and international

laws and policies (EEC Council Directive 86/609, OJL

Neurotox Res (2011) 20:150–158 151

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358, 1 Dec 12, 1987; NIH Guide for the Care and use of

Laboratory Animals, U.S. National Research Council,

1996). Brains were isolated by surgical ablation and were

split into right and left halves. Also spinal cords were

isolated. The right brain sections were used to study the

expression of FMO mRNAs in total brain, and the left

sections were utilized to obtain distinct brain areas (cere-

bellum, cerebral hemisphere, and brainstem). All samples

were stored in 1 ml of Trizol� (Sigma-Aldrich, Milan,

Italy) at -80�C.

RNA Isolation

Samples were homogenized, and total RNA was isolated

by Trizol� reagent (Sigma-Aldrich, Milan, Italy) follow-

ing the manufacturer’s specifications and quantified by

spectrophotometric analysis (Nanodrop�, Celbio, Milan,

Italy).

Reverse Transcription

RNA (11 ll at 550 ng/ll) was reverse-transcribed using

High Capacity cDNA Archive Kit (Applera, Monza, Italy)

according to the manufacturer’s recommendations. The

cDNA samples were stored at -20�C.

Validation of q-PCR Conditions

We designed, optimized, and validated primer and probe

systems with a quantitative real-time PCR (q-PCR) tech-

nique, using RNA from mouse liver because FMO

expression in this tissue was well characterized. The same

protocols were used to study the FMO mRNA expression

in brain. In addition, we checked specificity of the primers

by melting curve analysis to identify the presence of pos-

sible primer dimers or non-specific products. An assay for

the housekeeping Hypoxanthine–guanine phosphoribosyl

transferase (Hprt) q-PCR technique gene was chosen as a

control because of its relatively low mRNA levels in brain

that are similar to those of FMO genes observed in other

species (Janmohamed et al. 2004; Shaw 2005). Hprt

expression was invariable in all brain areas and spinal cord

in G93A transgenic and no transgenic control female and

male mice (data not shown).

Concerning SOD1 mRNA analysis, we evaluated

mRNA levels using Sybr Green Real-Time PCR. Sybr-

Green primers were designed with identical annealing and

melting temperatures using Primer Express Software

(Applera, Monza, Italy). The primers were designed

spanning introns to avoid amplification of genomic DNA.

We checked specificity of the primers by melting curve

analysis.

Real-Time PCR Primers and Probes

FMO quantitative experiments were done using Taq-Man

Real-Time PCR, while SOD1 mRNA level evaluation was

tested by Sybr Green Real-Time PCR. Taq-Man primers

and probes (Eurofins MWG Operon, Ebersberg, Germany)

were designed with identical annealing and melting tem-

peratures using Primer Express Software (Applera, Monza,

Italy). Primers, probes, sequences, and amplification effi-

ciencies are listed in Table 1. Real-Time PCR was done on

an iCycler PCR Detection System (iQ5 vers.2.0, Bio-Rad,

Segrate, Italy) using iQ Supermix (Bio-Rad, Segrate, Italy).

Each sample was tested with PCR using three replicates

with 5 ll of cDNA at a concentration of 300 ng/ll, forward

and reverse primers at a final concentration of 500 nM, and

Taq-Man probe at a final concentration 200 nM, 12.5 ll of

iQ buffer reaction (100 mM KCl, 40 mM Tris–HCl, pH

8.4, 1.6 mM dNTPs, 50 U/ml Taq DNA polymerase,

6 mM MgCl2) in 15 ll total volume. The PCR protocol

started with denaturation at 95�C for 5 min followed by 40

cycles (95�C 9 1500 and 59�C 9 10). Concerning SOD1,

oligonucleotides were used at a concentration of 150 nM in

a total volume reaction of 15 ll. Control reactions included

a template from the cDNA synthesis reaction, ± the

enzyme reverse transcriptase (RT), as well as the no-tem-

plate controls (NTC). The qPCR reaction was 95�C for

5 min, 95�C for 15 s, and 55�C for 30 s for 40 cycles, with

a melting curve starting at 55�C and increasing 0.5�C for

each 30 s. All the experiments were done twice with three

replicates. The average of the Ct values from the three

replicates for each sample was reported, and a standard

deviation equal to or less than 0.15 was considered a valid

result. The relative mRNA levels were displayed as rq

values normalized to Hprt expression using the 2-DDCt

comparative method related with an internal standard

(Livak and Schmittgen 2001).

Statistical Analysis

Kruskal–Wallis and Mann–Whitney tests were used to

compare expression of FMO genes in total brain and in

different brain sub regions. The Kruskal–Wallis test was

used to compare different FMO genes, and the Mann–

Whitney test was used to compare the expression differ-

ences between genders. Statistical analyses were done with

GraphPad Prism version 3.0 (GraphPad Software Inc, San

Diego, California). A P-value of 0.05 or lower was con-

sidered statistically significant.

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Results

Fmo mRNA Levels in SOD1-G93A and Control Mouse

Total Brain

Fmo gene-specific expression was defined in total brain in

ALS model and control mice and also compared in distinct

central nervous system (CNS) areas (cerebellum, cerebral

hemisphere, brainstem, and spinal cord). All Fmo genes

were detectably expressed in the murine CNS with the

exception of Fmo3. In total brain, Fmo mRNAs showed

generally higher expression in the ALS model mice than in

controls. A significant increase in Fmo4 expression was

observed between ALS and control mice (P \ 0.01)

(Fig. 1).

Fmo mRNA Levels in Specific CNS Areas in SOD1-

G93A and Control Mice

ALS model mice showed a different profile of Fmo gene

expression in the CNS areas examined when compared

with control mice (Fig. 2, Table 2). The most significant

difference was seen for Fmo1 expression in spinal cord,

where Fmo1 mRNA was detected at levels 16-fold greater

in SOD1-G93A than in control mice (P \ 0.001) (Fig. 2).

As in spinal cord, Fmo1 showed relatively higher expres-

sion in brainstem of SOD1-G93A mice (P \ 0.05). No

significant differences were detected in Fmo2–5 gene

expression in these areas, although the Fmo2, 4, 5 genes

were up-regulated in spinal cord (P \ 0.05) (Fig. 2). In

cerebellum, we observed 3-fold greater relative expression

of Fmo4 (P \ 0.001), and 2-fold greater Fmo2 and Fmo5

expression (P \ 0.01 and P \ 0.05, respectively) in the

ALS model than in C57BL/6J mice. In the cerebral

Table 1 q-PCR primers and probes

Primer Gene Sequence Amplification efficiency (90–110%)

Primer forward Hprt1 F 50-TCC CAG CGT CGT GAT TAG C-30

Primer reverse Hprt1 R 50-CGG CAT AAT GAT TAG GTA TAC AAA ACA-30 99.7%

Probe Hprt1 50-6-FAM-TGA TGA ACC AGG TTA TGA CC-30

Primer forward Fmo1 F 50-CGT GGA GGC CAG CCA CT-30

Primer reverse Fmo1 R 50-CAC CCA TGC CCC TCC AG-30 97.5%

Probe Fmo1 50-6-FAM-CAA AAA AGG TGT TCC TCA GC-30

Primer forward Fmo2 F 50-CAC ATC CAG CCT CAC CTG C-30

Primer reverse Fmo2 R 50-GGC CTC GGA ACC TCT CAA TAC-30 98.1%

Probe Fmo2 50-6-FAM-ACT CAA GTC ATT CCC-30

Primer forward Fmo3 F 50-TGA TTT GTT CTG GGC ATC ACA-30

Primer reverse Fmo3 R 50-AAC GGT TCA GTC CTG GAA AGG-30 101.6%

Probe Fmo3 50-6-FAM-CCA TGT ACC AAA AGA C-30

Primer forward Fmo4 F 50-TGG TTT GCA CTG GGC AAT T-30

Primer reverse Fmo4 R 50-TGG ATT CCA GGA AAG GAC TCC-30 99.4%

Probe Fmo4 50-6-FAM - TGA GCC CAC ATT TAC CTC-30

Primer forward Fmo5 F 50-AGA CTA CAG TGT GCA GCG TGA AG-30

Primer reverse Fmo5 R 50-GCC ATT GGC CCG AGG TA-30 100.9%

Probe FMO5 50-6-FAM-AGC AGC CTG ATT TC-30

Primer forward Sod1 F 50-ACT TCG AGC AGA AGG CAA GC-30

Primer reverse Sod1 R 50-TTA GAG TGA GGA TTA AAA TGA GGT C-30 99.7%

PCR primer and probe sequences for the real-time q-PCR analysis of Hprt housekeeping gene, Fmo and Sod1 genes are listed, with the

corresponding assay amplification efficiencies (90–110%)

Fig. 1 Fmo mRNA levels in C57BL/6J and SOD1-G93A mouse total

brain. The mRNA levels (combined males and females) were

normalized to Hprt expression using the 2-DDCt comparative method

related with a calibrator (Livak and Schmittgen 2001). Comparing

total brain RNAs from C57BL/6J (black bar) and SOD1-G93A (graybar) mice showed a higher expression of Fmo in the ALS model. A

statistically significant difference was observed for Fmo4 (**,

P \ 0.01 SOD1-G93A versus C57BL/6J mice)

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hemisphere, Fmo2, Fmo4, and Fmo5 were also expressed

at significantly greater levels in SOD1-G93A compared to

C57BL/6J mice (P \ 0.05) (Fig. 2, Table 2).

Fmo Gender-Specific Expression in SOD1-G93A

and Control Mice

Fmo1 expression showed the greatest gender-specific dif-

ferences in ALS and control mice. In male ALS model

mice, Fmo1 mRNA was detected at the highest level in

spinal cord (P \ 0.01), at levels 4-fold greater compared to

C57BL/6J brainstem (P \ 0.05) (Fig. 3a). In contrast,

Fmo1 expression was significantly decreased in spinal cord

of female SOD1-G93A compared to C57BL/6J female

mice (P \ 0.05) (Fig. 3b).

Fmo2 showed significantly higher expression in male

spinal cord of C57BL/6J than in SOD1-G93A mice

(P \ 0.01) (Fig. 3c). In female SOD1-G93A mice, Fmo2

was more highly expressed in cerebellum and cerebral

hemisphere than in female C57BL/6J mice (P \ 0.01,

P \ 0.05, respectively) (Fig. 3d). In male mice, the data of

Fmo4 expression showed no statistically significant dif-

ferences between control and ALS mice (Fig. 3e), while in

female SOD1-G93A mice Fmo4 expression showed the

greatest difference in cerebellum compared with control

mice (P \ 0.05) (Fig. 3f). Fmo5 did not show statistically

significant differences in mRNA levels between control

and ALS mice in both males and females (Fig. 3g, h).

Sod1 mRNA Expression in Pathological Areas

and Gender in G93A Mice

Sod1 mRNA was found to be increased in pathological

areas such as spinal cord and brainstem compared to non-

pathological regions such as cerebellum and cerebral

hemisphere.

ALS model mice showed a different profile of Sod1 gene

expression between females and males in the CNS areas

examined when compared with control mice (Fig. 4). In all

nervous areas examined, the Sod1 mRNA level was greater

in males than in female mice. In particular, Sod1 showed

increased expression in males compared to female mice

although the difference was not statistically significant.

Fig. 2 Fmo mRNA levels in specific mouse nervous regions. In

SOD1-G93A mouse spinal cord, Fmo1 expression was observed to be

16 times higher than in C57BL/6J mice (***, P \ 0.001) and 3 times

in brain stem (*, P \ 0.05). Fmo2 and Fmo4 levels were significantly

different in all areas (cerebellum: Fmo2 **, P \ 0.01; Fmo4 ***,

P \ 0.001; cerebral hemisphere and spinal cord: Fmo2 and Fmo4 *,

P \ 0.05). Fmo5 showed statistically significant differences between

controls and ALS mice in all tested regions (*, P \ 0.05)

Table 2 Fold-expression differences in specific CNS areas in SOD1-

G93A and C57BL/6J mice

Fmo1 Fmo2 Fmo4 Fmo5

Spinal cord ?16 ?1 ?0.5 –

Brain stem ?3 – – –

Cerebellum – ?2 ?3 ?2

Cerebral hemisphere – ?1 ?1 ?1

Fold differences are presented as related increases (positive values in

the table, SOD1-G93A versus C57BL/6J mice) or decreases in ALS

(negative values, SOD1-G93A versus C57BL/6J mice)

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Discussion

In this study, we examined for the first time the expression

of FMO genes in specific subregions of the CNS in ALS

model and control mice. We also compared FMO mRNA

levels between male and female mice of the two groups.

Our data showed that FMO genes were up-regulated in

ALS CNS areas studied relative to control C57BL/6J mice.

The major finding was the up-regulation of the Fmo1 gene

in spinal cord and brainstem, where Fmo1 mRNA levels

were 16- and 3-fold greater, respectively, in the ALS model

compared with control mice. The contrasting data reported

Fig. 3 Comparison of brain Fmo genes expression between male and

female mice. In SOD1-G93A male mice (a), Fmo1 expression was

observed to be 3-fold greater than in C57BL/6J male mice (**,

P \ 0.01), while it was lower in SOD1-G93A females compared to

normal female mice (*, P \ 0.05) (b). Fmo2 was more highly

expressed in cerebral hemisphere of C57BL/6J male mice than in

SOD1-G93A mice (**, P \ 0.01) (c). An opposite trend we observed

in females, where SOD1-G93A mice showed higher Fmo2 expression

in cerebral hemisphere (*, P \ 0.05) and cerebellum (**, P \ 0.01)

than in control females (d). Although Fmo4 and Fmo5 mRNA levels

did not show statistically significant differences between male mice

(e, g). In females, they were more highly expressed in all areas in

SOD1-G93A than in C57BL/6J mice but we have statistically

differences only in cerebellum Fmo4 gene expression (*, P \ 0.05)

(f, h)

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by Malaspina et al. (2001) documenting down-regulation

of FMO1 in human ALS spinal cord compared to healthy

controls may be explained by species-specific gene regu-

lation differences. Alterations in the CNS of FMO gene

expression in ALS model mice may be a consequence of

the pathological environment linked to effects of the

mutant SOD1. Experimental studies with mutant SOD1

animal models revealed modified cellular metabolism and

development of multiple pathogenic processes including

oxidative stress, oxidation of proteins, and protein aggre-

gation (Shaw 2005), particularly in brainstem and spinal

cord (Garbuzova-Davis et al. 2007; Watanabe et al. 2001;

Mahoney et al. 2006). In particular, free radical products

from hydrogen peroxide were found to be increased in

spinal cord of ALS model mice transfected with mutant

SOD1-G93A and in autoptic brain samples of the ALS

patients (Liu et al. 1998; Liu 1996). Moreover, altered

FMO expression likely impacts oxidative-redox systems,

as described for the yeast flavin-containing monooxygen-

ase (yFMO) that is vital to the response of yeast to

reductive stress (Suh and Robertus 2000a, b). Interestingly,

Fmo1 mRNA up-regulation is strongly associated with

ALS pathological regions because it is up-regulated only in

spinal cord and brainstem and because it is the only Fmo

gene showing increased expression in brainstem. It is

possible that in ALS the activation of the FMO detoxica-

tion system may be increased by reactive oxygen species

(ROS) that have been found to be present in high con-

centrations in spinal cord extracts prepared form SOD1-

G93A mice (Liu et al. 1998).

In SALS not involving SOD1 mutations, the patholog-

ical environmental may be due to exposure to toxic factors

that increase free radicals and activate the FMO system

(Suh et al. 1999; Suh and Robertus 2000a, b). Studies have

shown that metals including iron, copper, mercury, nickel,

and lead deplete protein-bound sulfhydryl groups, resulting

in ROS production as superoxide ions, hydrogen peroxide,

and hydroxyl radicals. Also, it has been reported that

exposure to mercury is selectively toxic to spinal cord and

motor neurons in mouse and in rats, increasing ROS in

nervous regions affected by the metal (Su et al. 1998;

Pamphlett and Patricia 1996; LeBel et al. 1990).

The connection between genetic background and the

ability of detoxication processes to respond to toxic agents

has been shown previously. Polymorphisms in PON genes

have been associated with SALS, although results from

different populations are not conclusive (Wills et al. 2009;

Ricci et al. 2010). Cereda et al. (2006) also showed a

significant association between SALS disease and a genetic

polymorphism within the FMO1 gene. These data highlight

a likely role of at least one detoxication pathway in ALS

and may help to understand the involvement of FMO

enzymes in this disease. Moreover, both FMO and PON

enzymes metabolize organophosphate compounds that are

commonly present in farming and gardening. Studies have

shown increased incidence of ALS in specific occupational

categories such as professional soccer and farming (Li and

Sung 2003; Abel 2007).

Finally, we studied FMO gender-regulated expression

because the literature has shown the importance of hor-

monal regulation of both human and mouse FMO genes

mediated by sex steroids (Falls et al. 1997; Coecke et al.

1998). Coecke et al. (1998) demonstrated that the human

sex hormone 17b-estradiol caused a significant decrease in

FMO activity both in cultured male rat hepatocytes and

also in male rat liver.

In this study, we showed that Fmo1 gene expression in

spinal cord and in brainstem was up-regulated in male ALS

mice but down-regulated in female ALS mice. Interest-

ingly, Fmo1 gene expression correlated with Sod1 mRNA

with regard to gender and specificity of affected areas. We

showed that Sod1 expression is decreased in ALS female

mice relative to ALS males. In particular, both Fmo1 and

Sod1 expression were down-regulated in female relative to

male spinal cord.

It was previously shown that SOD1 concentration in the

CSF is decreased in female relative to male ALS patients

(Frutiger et al. 2008). The epidemiological data presented a

different onset ratio between males and females in ALS as a

function of the fecund age of the females: the ratio between

males and females was 2.5 in the younger female group and

1.4 in the older female group (Manjaly et al. 2010).

In SOD1-G93A female mice, Sod1 mRNA may be

down-regulated because sex hormones may have a pro-

tective function against oxidative stress damage (Behl et al.

1995). Choi et al. (2008) reported that 17b-estradiol

appeared to have a protective effect in ALS. Treatment

with 17b-estradiol of G93A ovariectomized female mice

did not delay the onset of disease, but did retard the

Fig. 4 Comparison of brain Sod1 gene expression between SOD1-

G93A male and female mice. Sod1 expression was observed to be

greater in SOD1-G93A male mice than in SOD1-G93A female mice

in cerebellum, cerebral hemisphere, spinal cord, and brain stem. The

mRNA levels were normalized to Hprt expression using the 2-DDCt

comparative method related with a calibrator (Livak and Schmittgen

2001)

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progression of ALS motor dysfunctions. Recently, it was

shown that SOD1 interacts with estrogen receptor a (ER-a)

and this complex enhances the binding of ER-a to ERE

(estrogen response element)-containing DNA, representing

a regulation point of the various pathways (Rao et al.

2008). ER-a and SOD1 were found to be associated with

regions of the progesterone receptor gene, involved in

conferring estrogen-responsive gene expression. We sug-

gest that in female ALS mice, sex hormones may play a

protective role to maintain down-regulation of the Sod1

and Fmo system, while in ALS male mice Sod1 and the

Fmo system were up-regulated and their activation may be

necessary in response to the ROS increase. The potential

involvement of sex hormones in ALS pathology is partic-

ularly interesting in light of previous genetic studies

showing a significant association between FMO1 30UTR

SNP frequencies in female SALS patients (Cereda et al.

2006). The different genetic arrangements, as differential

interaction with FMO1 gene promoter regions by sex

hormone-regulated transcription factors gene in males and

females (Cereda et al. 2006), may influence FMO1 mRNA

levels observed to be gender-dependent in ALS model.

Overall, our findings of altered Fmo gene expression

within specific ALS brain regions in the SOD1-G93A

mouse model provide additional support for a role for FMO

enzymes in cellular response to ALS neurodegeneration.

Although specific functions have not been defined for

mammalian FMO isozymes in the CNS, the known func-

tions of certain FMO enzymes in humans and other species

suggest a role for detoxication in mutant SOD1-mediated

ALS, likely in response to increasing oxidative stress in

susceptible motor neurons.

Future work will help to understand the role of FMO-

mediated detoxication and redox balance in the CNS of

sporadic and familial ALS patients.

Acknowledgments We thank Dr. Caterina Bendotti, Mario Negri

Farmacological Institute, Milano, Italy, for supplying SOD1-G93A

mice tissues and for her help in writing the materials and methods

paragraph. This study was supported by the Ministry of Scientific

Research and Ministry of Health (Ex art.56/2003-Neurodegenerative

diseases).

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