The transcription factor Nurr1 is upregulated in amyotrophic … · receptor related 1 protein (Nurr1), also called NR4A2 (encoded by the gene NR4A2). Nurr1 is an orphan receptor

RESEARCH ARTICLE

The transcription factor Nurr1 is upregulated in amyotrophiclateral sclerosis patients and SOD1-G93A miceValeria Valsecchi123Dagger Marina Boido12 Francesca Montarolo24 Michela Guglielmotto12Simona Perga124 Serena Martire24 Santina Cutrupi5 Andrea Iannello5 Nadia Gionchiglia2Elena Signorino2 Andrea Calvo67 Giuseppe Fuda67 Adriano Chio 67 Antonio Bertolotto24 andAlessandro Vercelli12

ABSTRACTAmyotrophic lateral sclerosis (ALS) is a neurodegenerative diseasethat affects both lower and upper motor neurons (MNs) in the centralnervous system ALS etiology is highly multifactorial and multifariousand an effective treatment is still lacking Neuroinflammation is ahallmark of ALS and could be targeted to develop new therapeuticapproaches Interestingly the transcription factor Nurr1 has beendemonstrated to have an important role in the inflammatory processin several neurological disorders such as Parkinsonrsquos diseaseand multiple sclerosis In the present paper we demonstrate forthe first time that Nurr1 expression levels are upregulated in theperipheral blood of ALS patients Moreover we investigated Nurr1function in the SOD1-G93Amouse model of ALS Nurr1 was stronglyupregulated in the spinal cord during the asymptomatic and earlysymptomatic phases of the disease where it promoted theexpression of brain-derived neurotrophic factor mRNA and therepression of NFκB pro-inflammatory targets such as induciblenitric oxide synthase Therefore we hypothesize that Nurr1 isactivated in an early phase of the disease as a protectiveendogenous anti-inflammatory mechanism although not sufficientto reverse disease progression On the basis of these observationsNurr1 could represent a potential biomarker for ALS and a promisingtarget for future therapies

KEY WORDS ALS SOD1-G93A mice Motor neuron diseaseNeuroinflammation Nurr1

INTRODUCTIONAmyotrophic lateral sclerosis (ALS) is a neurodegenerative diseasethat affects both lower motor neurons (MNs) in the brainstemand spinal cord and upper MNs in the cerebral cortex (Pasinelli andBrown 2006) Loss of these neurons leads to muscle weaknessand paralysis and ultimately to death owing to respiratory failure2-5 years after diagnosis (Worms 2001) The incidence of the diseaseis about 1 to 2 individuals per 100000 per year with males beingaffected more frequently than females (Robberecht and Philips2013) Considering all cases 90 are classified as sporadic (sALS)and the remaining 10 as familial (fALS) with a Mendelian patternof inheritance In fALS mutations have been identified in genesencoding several proteins the copperzinc superoxide dismutase 1(SOD1) the transactive response DNA-binding protein (TARDBP)the fused in sarcomatranslocated in liposarcoma RNA-bindingprotein (FUSTLS) and the chromosome 9 open reading frame 72(C9orf72) Mutations in these genes account for two-thirds of allfALS cases and for approximately one-tenth of sALS cases althoughfor other incidences the etiology is unknown (Renton et al 2014Chia et al 2018) The clinical phenotype of fALS is usuallyindistinguishable from sALS and an effective therapy is still elusive

ALS etiology is multifarious and has been associated with manyfactors mitochondrial dysfunction and oxidative stress excitotoxicitydeficits in axonal architecturefunction and protein quality controlprotein misfolding and aggregation loss of neurotrophic factorsdysregulation of RNA metabolism and inflammation (Ferraiuoloet al 2011 Cozzolino et al 2012) In particular neuroinflammationis a common hallmark of neurodegenerative diseases such as ALSand Parkinsonrsquos disease (PD) Indeed in ALS a dramatic activationof microglia astrocytes and the complement system is reportedcontributing to neurodegeneration partly as a consequence of theupregulation of inflammatory genes (Haidet-Phillips et al 2011) Inaddition the role of the adaptive immune systemmodulates the balancebetween neuroprotection and neurotoxicity (Thonhoff et al 2018) Theeffects on the central nervous system (CNS) and peripheral blood alterover the course of the disease and the dysregulation of regulatory T cellsaffects the disease outcome (Henkel et al 2009) Hence asneuroinflammation makes a central contribution to ALS pathogenesisthe identification of novel therapeutic targets acting on inflammation isconsidered mandatory (Troost et al 1990 Zhao et al 2013)

In this scenario an interesting role is played by the nuclearreceptor related 1 protein (Nurr1) also called NR4A2 (encoded bythe gene NR4A2) Nurr1 is an orphan receptor belonging to thenuclear receptor subfamily 4 group A (NR4A) family which alsoincludes the nerve growth factor IB (NGFIB Nur77 NR4A1) andthe neuron-derived orphan receptor 1 (NOR-1 NR4A3) In theCNS Nurr1 has a well-established role in the development andmaintenance of midbrain dopaminergic (mDA) neurons

Handling Editor Steven J ClapcoteReceived 25 November 2019 Accepted 13 March 2020

1Department of Neuroscience Rita Levi Montalcini University of Turin via Cherasco15 10126 Turin Italy 2Neuroscience Institute Cavalieri Ottolenghi (NICO)University of Turin Regione Gonzole 10 10043 Orbassano Turin Italy3Department of Neuroscience Reproductive and Dentistry Sciences University ofNaples ldquoFederico IIrdquo via S Pansini 5 80131 Naples Italy 4Neurobiology UnitNeurology - CReSM (Regional Referring Center of Multiple Sclerosis) AOU SanLuigi Gonzaga Regione Gonzole 10 10043 Orbassano Turin Italy 5Departmentof Clinical and Biological Sciences University of Turin Regione Gonzole 10 10043Orbassano Turin Italy 6Department of Neuroscience Rita Levi MontalciniAmyotrophic Lateral Sclerosis Expert Center (CRESLA) University of Turin viaCherasco 15 10126 Turin Italy 7University Hospital Citta della Scienza e dellaSalute corso Bramante 88 10126 Turin ItalyThese authors contributed equally to this work

DaggerAuthor for correspondence (valsecchivyahoocom)

VV 0000-0002-0514-8579 SM 0000-0001-6889-888X AI 0000-0001-8940-1406 AB 0000-0002-7052-1907

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (httpscreativecommonsorglicensesby40) which permits unrestricted usedistribution and reproduction in any medium provided that the original work is properly attributed

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copy 2020 Published by The Company of Biologists Ltd | Disease Models amp Mechanisms (2020) 13 dmm043513 doi101242dmm043513

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(Zetterstroumlm et al 1997 Saucedo-Cardenas et al 1998) Given itscrucial functions altered Nurr1 expression has been implicated indopamine-associated brain disorders including PD Notably theexpression of Nurr1 in mDA neurons decreases in PD patients(Hering et al 2004) and single-nucleotide polymorphisms (SNPs)and mutations resulting in reduced expression of Nurr1 areassociated with familial and sporadic forms of PDInterestingly Nurr1 is a constitutively active transcription factor

binding its target genes as a monomer homodimer or heterodimerin association with retinoidX receptors (Aarnisalo et al 2002Mairaet al 1999 Wang et al 2003) In murine models of PD Nurr1 wasfound to have roles in both neuroprotection and immunomodulationIndeed Nurr1 has an anti-inflammatory role inhibiting expressionof the genes encoding pro-inflammatory components of the NFκBpathway in microglia and astrocytes (Saijo et al 2009) Specificallyknocking down Nurr1 in mice leads to an increased activation ofglial cells exposed to lipopolysaccharide (LPS) with subsequentproduction of higher levels of mRNAs encoding inflammatorycytokines and neurotoxic effector proteins such as tumor necrosisfactor α (TNF-α) inducible nitric oxide synthase (iNOS) andinterleukin 1β (IL-1β) responsible for inflammation-inducedneuronal death (Saijo et al 2009 McMorrow and Murphy 2011)In addition Nurr1 induces the expression of neurotrophic factorssuch as brain-derived neurotrophic factor (BDNF) (Barneda-Zahonero et al 2012) In addition to its role in the CNS Nurr1has an active role in PD as downregulated levels of gene expressionwere also found in peripheral blood obtained from PD patients withprogressive loss of mDA neurons (Le et al 2008 Liu et al 2012Montarolo et al 2016)Recently it has been shown that Nurr1 also controls the expression

of several nuclear-encodedmitochondrial genes involved in oxidativerespiration such as SOD1 Ts translation elongation factor (TSFM)and cytochrome c oxidase subunit 5B (COX5B) demonstrating animportant role in sustaining respiratory function (Kadkhodaei et al2013) Therefore we first analyzed Nurr1 gene expression levels inblood obtained from ALS patients in comparison with healthycontrols (HC) To better understand its role in ALS we theninvestigated the expression and function of Nurr1 in a murine modelof ALS the SOD1-G93A mouse

RESULTSNurr1 mRNA is upregulated in the peripheral blood of ALSpatientsGene expression analysis of Nurr1 was performed on wholeperipheral blood obtained from 43 ALS patients and 41 HC

subjects demographic and clinical characteristics are summarizedin Table 1 The ALS group comprising seven fALS and 36 sALSpatients showed higher Nurr1 mRNA levels compared with the HCgroup (Fig 1A) Furthermore there was a significant difference inage (Studentrsquos t-test P=0002 Table 1) but not in sex between ALSpatients and HC (Fisher exact test P=038 Table 1) To assesspotential bias related to age the correlation between age and geneexpression levels of Nurr1 was evaluated in HC subjects and nosignificant results were highlighted as previously reported byMontarolo et al (2016) (Pearson correlation coefficient r=013P=041) Also correlation analyses between Nurr1 gene expressionof ALS patients and age at the time of sampling (Pearson correlationcoefficient r=minus009 P=055) and age at disease onset (Pearsoncorrelation coefficient r=minus016 P=031) (Fig S1A) did not highlightsignificant results Similarly there are no significant differences inNurr1 expression between sexes in bothHC (Studentrsquos t-testP=053)and ALS patients (MannndashWhitney U-test P=032) (Fig S1B) Theinfluence on Nurr1 expression level of the pharmacological treatmentindicated in Table 1 was not assessed owing to the small sample sizeof each group Among fALS patients four carried mutations inC9Orf72 two in SOD1 and one in the TARDBP gene whereas threesALS patients carried mutations in C9Orf72 SOD1 and in the geneencoding optineurin (OPTN) We also reported Nurr1 expressionlevels for the different groups (Fig S1C)

Nurr1mRNAand protein are upregulated in the spinal cord ofSOD1-G93A mice in the asymptomatic and earlysymptomatic phases of the diseaseThe high levels of Nurr1 expression in samples of peripheral bloodfrom ALS patients suggested a potential role in ALS pathogenesisTo investigate the role of Nurr1 in ALS outcome we used atransgenic mouse model of ALS SOD1-G93A (TG) This modelhas a high copy number of the transgene and is one of the mostcommonly used in this area of research (Gurney et al 1994) Malemice develop first symptoms of the disease at approximately3 months of age and die about 4 weeks after disease onsetBehavioral tests such as rotarod and paw grip endurance (PaGE)were used to evaluate the appearance of the first motor deficitsand to divide the animals into three experimental groups(1) asymptomatic mice (2) early symptomatic mice and (3) latesymptomatic mice (Boido et al 2014) Age-matched wild-type(WT) male mice were used as controls Female animals wereexcluded from the study as significant differences were observedbetween male and female TG animals probably owing to anestrogen neuroprotective effect (Vercelli et al 2008)

Table 1 Demographic and clinical characteristics of the enrolled populations

HC (n=41) ALS (n=43) P-value

Age at time of sampling (years) mean (sd) 567 (1097) 646 (1203) 0002Sex female () 23 (561) 19 (442) 038dagger

Age at disease onset (years) mean (sd) ndash 6198 (1198) ndash

Patients treated with specific therapies n () ndash 3 (71) None14 (333) Riluzole22 (524) Riluzole+other drugs3 (71) Other drugsDagger

ndash

fALS patients with known mutations n () 7 (163) 4 C9Orf72 2 SOD1 1 TARDP43sALS patients with known mutations n () 36 (837) 1 C9Orf72 1 SOD1 1 OPTN

ALS amyotrophic lateral sclerosis HC healthy controlsStudentrsquos t-testdaggerFisher exact testDaggerOther drugs (L-acetilcarnitine tocopherol chininum sulphuricum baclofene gabapentin pregabalin pyridostigmine bromide escitalopram citaloprampalmitoylethanolamide)

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RESEARCH ARTICLE Disease Models amp Mechanisms (2020) 13 dmm043513 doi101242dmm043513

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First we measured Nurr1 mRNA levels in the whole peripheralblood obtained from early and late symptomatic TG animalsconsidered as a single group The analysis revealed no differencesin Nurr1 expression levels between TG and age-matched WTmice (Fig 1B) By contrast on evaluating Nurr1 expression levelsin the spinal cord of TG mice (Fig 2AB) we observed that Nurr1mRNA was significantly upregulated (up to 22-fold) in theasymptomatic phase of the disease compared with respectivecontrols In the early symptomatic phase of the disease Nurr1mRNA content was still higher in TG mice than in WT animals butto a slightly lesser extent (18-fold) in end-stage TG mice Nurr1mRNA levels were the same as for age-matched WT controls(Fig 2A)Nurr1 protein levels in nuclear extracts obtained from the

spinal cord of TG mice confirmed the results obtained withmRNA In particular Nurr1 protein was strongly upregulated by36- and 26-fold in the nuclei of asymptomatic and earlysymptomatic TG mice respectively compared with WT controls(Fig 2B) By contrast WT animals presented comparable levels ofNurr1 both at the mRNA and protein level in all groups analyzed(Fig 2AB)

Nurr1 is able toactivateBdnfexpressionand to preventNFκBtarget gene activation in the asymptomatic phase of thedisease of SOD1-G93A miceWe investigated the possible role of Nurr1 by comparing TG andWT spinal cord samples As it has been demonstrated that thetranscription factor Nurr1 can directly activate its target genes suchas Bdnf (Barneda-Zahonero et al 2012) we performed a real-timePCR analysis for Bdnf Furthermore Nurr1 can dock on p65 theNFκB transactivating subunit blocking the activation of its pro-inflammatory genes such as iNos (Saijo et al 2009 De Mirandaet al 2015) Therefore to investigate whether Nurr1 was involvedin the NFκB pathway we measured iNos mRNA and performedchromatin immunoprecipitation assay following quantitative real-time PCR (ChIP-qPCR) on the iNos promoter using antibodiesagainst Nurr1 or p65Our results showed a significant increase in BdnfmRNA levels in

the asymptomatic phases of the disease (Fig 3A) In particular a

significant increase of 18-fold was observed in asymptomatic TGanimals compared with WT In early symptomatic mice Bdnf levelswere still higher than in respective controls but not significantly(15-fold) In a late phase of the disease Bdnf levels werecomparable with those of respective WT controls

By contrast we observed a downregulation of iNosmRNA in theasymptomatic phases of the disease (up to 40) as compared withage-matched WT controls this downregulation was not seen inearly symptomatic mice Furthermore in a late phase of the diseaseiNos mRNAwas strongly upregulated up to 43-fold in TG animalscompared with respective controls (Fig 3B)

To assess whether iNos modulation depended on directcompetition between Nurr1 and NFκB binding on its promoterwe performed ChIP-qPCR assay (Fig 3C) In asymptomaticanimals Nurr1 binding expressed as TG to WT ratio was 22-fold higher in TG compared with age-matched WT animalsInterestingly at this stage Nurr1 binding was 46-fold higher than

Fig 2 Nurr1 mRNA and protein were upregulated in the spinal cord ofasymptomatic and early symptomatic SOD1-G93A mice (A) mRNAexpression levels of Nurr1 in the spinal cord of asymptomatic (Asym n=11)early symptomatic (Early Symp n=5) and late symptomatic (Late Symp n=4)SOD1-G93A animals (TG black columns) compared with age-matched WTmice (white columns) The Gapdh gene was used as endogenous control(B) Representative western blot displaying the expression levels of Nurr1protein in nuclear extracts from spinal cord of Asym (n=8) Early Symp (n=5)and Late Symp (n=3) TG and WT animals The graph below the image reportsthe quantification of Nurr1 expressed as a ratio with the endogenous controllamin A Each column represents the meanplusmnsem Statistically significantdifferences among means were determined by two-way ANOVA followed byBonferroni test Plt005 TG versus respective WT

Fig 1 Whole peripheral blood gene expression levels of Nurr1 in ALSpatients and in SOD1-G93Amice (A) Comparison of gene expression levelsof Nurr1 in 43 ALS patients and 41 HC Nurr1 is upregulated in ALS patientscompared with HC (Studentrsquos t-test P=001) Relative expression wascalculated using the normalized comparative cycle threshold (Ct) method(2^-ΔCt) (B) Comparison of gene expression levels of Nurr1 in seven WT andeight TG mice No differences were detected for Nurr1 in TG mice comparedwith HC (Studentrsquos t-test P=069) Relative expression was calculated usingthe normalized comparative cycle threshold (Ct) method (2^-ΔCt)

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p65 binding at the iNos promoter Furthermore during diseaseprogression Nurr1 binding decreased and this was mirrored by aparallel increase in p65 binding Specifically in late symptomaticTG animals p65 binding was 28-fold higher than for WT and theTGWT ratio of p65 binding was 26-fold higher compared withNurr1 (Fig 3C) Finally we performed ChIP-qPCR assay on theiNos promoter using antibody against trimethylation at Lys4 ofhistone H3 (H3K4me3) and trimethylation at Lys27 of histone H3(H3K27me3) markers for active and repressive genes respectively(Kim et al 2013) Our results showed that H3K4me3 enrichmentincreased 42-fold compared with H3K27me3 suggesting that theiNos promoter is active in accordance with increased iNos mRNAlevels (Fig 3D)

Nurr1 protein is expressed in motor neurons and to a lesserextent in astrocytes of SOD1-G93A miceTo investigate in which CNS cell types Nurr1 was expressedrepresentative double immunofluorescence experiments wereperformed in spinal cord sections with antibodies raised against

three markers (1) the neurofilament H (SMI32) a specific markerfor MNs (2) the glial fibrillary acidic protein (GFAP) anintermediate filament protein expressed mainly by astrocytes inthe CNS and (3) CD68 protein which is highly expressed bymacrophages and activated microglia

For the first time we observed that Nurr1 is physiologicallyexpressed in the cytoplasmic compartment of SMI32-positive cellsof WT animals indicating Nurr1 presence in MNs (Fig 4 aPrime-aprimePrimethin arrows) Interestingly in asymptomatic and early symptomaticTG mice Nurr1 expression was mainly evident in the nuclearcompartment of MNs (Fig 4 bPrime-bprimePrimecPrime-cprimePrime thick arrows) inagreement with the upregulation in nuclear extract observed withwestern blot (WB) analysis By contrast in late symptomatic TGanimals Nurr1 immunostaining was still present only in MNcytoplasm (Fig 4 dPrime-dprimePrime thin arrows)

Furthermore as neuroinflammation is a characteristic of ALS (seeFigs S2 and S3 in agreement with Ilieva et al 2009 Boido et al2014 Anzilotti et al 2018) and because Nurr1 is reported to havean anti-inflammatory role in astrocytes and microglial cells (Saijo

Fig 3 Nurr1 activated Bdnf expression and repressed iNos transcriptional activation by docking with NFκB on the iNos promoter (AB) mRNAexpression levels of Bdnf (A) and iNos (B) in the spinal cord of asymptomatic (Asym n=10) early symptomatic (Early Symp n=5) and late symptomatic (LateSymp n=3 for panel A and n=4 for panel B) SOD1-G93A animals (TG black columns) compared with age-matchedWTmice (white columns) TheGapdh genewas used as an endogenous control Each column represents the meanplusmnsem Statistically significant differences among means were determined by two-wayANOVA followed by Bonferroni test Plt005 TG versus respective WT (CD) ChIP analysis of Nurr1 and p65 (C) and H3K4me and H3K27me (D) on the iNospromoter in the spinal cord of Asym Early Symp and Late Symp TG and WT animals The binding activity of each transcription factor was calculated as thepercentage of total input of chromatin DNA and represented as the ratio between TG and age-matched WT animals Each column represents the meanplusmnsem(n=3) Statistically significant differences between means were determined by two-way ANOVA followed by Bonferroni test Plt005 Nurr1 versus p65 sectPlt005p65 versus Nurr1 Plt0005 H3K4me versus H3K27me

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et al 2009) we investigated its expression in GFAP- and CD68-positive cellsDouble immunofluorescence staining with GFAP and Nurr1

showed the Nurr1 immunosignal to be present in the nuclearcompartment of rare astrocyte cells at early stages of the disease

(Fig 5A aPrime-aprimePrime thick arrows) but it was absent in the latesymptomatic phase (Fig 5A bPrimebprimePrime)

By contrast double immunofluorescence staining with CD68 andNurr1 did not highlight Nurr1 expression in CD68-positive cells(Fig 5B cPrimecprimePrimedPrimedprimePrime)

Fig 4 Nurr1 protein is expressed in MNs of TGanimals Representative confocal images showing thedouble-labeling of Nurr1 (red) and SMI32 (green) inlumbar spinal cord of WT animals (a-aprimePrime) asymptomatic(Asym b-bprimePrime) early symptomatic (Early Symp c-cprimePrimee-eprimePrime) and late symptomatic (Late Symp d-dprimePrime) TGmiceNuclei are labeled with DAPI (blue) Thin arrows showmainly cytoplasmic localization of Nurr1 whereasthick arrows indicate the nuclear localization of Nurr1in MNs Scale bars 20 microm

Fig 5 Nurr1 protein is expressed in astrocytes of TGanimals (AB) Representative confocal images showing thedouble-labeling of Nurr1 (red) and GFAP (green A) or CD68(green B) in lumbar spinal cord of early symptomatic (EarlySymp a-aprimePrime c-cprimePrime) and late symptomatic (Late Symp b-bprimePrimed-dprimePrime) TG mice Nuclei are labeled with DAPI (blue) Thinarrows show mainly cytoplasmic localization of Nurr1whereas thick arrows indicate nuclear localization of Nurr1 inastrocytes In the inset in a rotations along the x- and y-axesshow the superposition of the two colors on the z-axis In bPrime(TG Late Symp) it is evident that the astrocytes are notlabeled but they surround a plausible Nurr1-positive MN(Nurr1 expression at the cytoplasmic level thin arrow) Bdemonstrates the absence of Nurr1-positive microglial cellsfurther confirming its expression in plausible MNs (thickarrows) Scale bars 20 microm (a-aprimePrime) and 30 microm (b-bprimePrime c-cprimePrimeand d-dprimePrime)

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DISCUSSIONIn this paper we investigated the expression of the transcriptionfactor Nurr1 in blood obtained from ALS patients and in the bloodspinal cord of a murine model of ALS the SOD1-G93A mouseNurr1 is known to have an important role in the maturation of mDAneurons (Kadkhodaei et al 2009) linking its deficiency mainly toPD In fact several human mutations in the gene encoding for Nurr1proteinNR4A2 are associated with late-onset familial PD (Le et al2003) and the SNPsrs35479735 (insertiondeletion of a G) seems tobe a significant risk factor for the development of PD (Liu et al2017 Ruiz-Saacutenchez et al 2017) Furthermore Nurr1 geneexpression is downregulated in the blood of PD patients withprogressive loss of DA neurons (Le et al 2008 Liu et al 2012Montarolo et al 2016) Moreover conditional ablation of Nurr1 inadult mature mDA neurons in mice resulted in the downregulationof several genes involved in oxidative respiration and particularlyof SOD1 (Kadkhodaei et al 2013) As SOD1 is a key enzyme inALS disease (Renton et al 2014 Chia et al 2018) theseobservations prompted us to explore the functional role of Nurr1 inALS pathologyIn the whole blood obtained from ALS patients we noted a

significant increase in mRNA levels of Nurr1 compared with HCNurr1 has a central role in immune homeostasis where it regulatesthe induction maintenance and suppressor functions of regulatory Tcells (Tregs) Specifically it represses aberrant Th1 inductionthrough transcriptional activation of the master transcription factorof Treg cells the forkhead transcription factor (Foxp3) and inhibitsproduction of the cytokines interferon γ (IFNγ) and interleukin-17(IL-17) (Sekiya et al 2011 2013) Nurr1 mRNA upregulation inthe blood of ALS patients suggested an additional important role forthis transcription factor in ALS in which immune cells are able toexert either a detrimental or a protective action on MN survival(Chiu et al 2008 Hooten et al 2015) Indeed there is convincingevidence to suggest that ALS is a systemic disorder characterized byperipheral immune alterations (McCauley and Baloh 2019)Autopsy of ALS patients showed modifications in the frequencyof circulating immune cell populations and in cytokine expression(Zhang et al 2005 McCombe and Henderson 2011 Murdocket al 2017) However the peripheral mechanism that cruciallycontributes to the ALS disease process and whether or not itrepresents a consequence or has a causative role is still unclearNurr1 upregulation could be driven by a response to tissue damagein the brain initially sensed by resident glial cells then amplified andpropagated by the peripheral immune cells or by an intrinsicallyaltered peripheral immune system in ALS patients Indeed Nurr1expression has been reported in dendritic cells (Saini et al 2016) Tcells (Sekiya et al 2011 Won and Hwang 2016) and inmacrophages (Bonta et al 2006)To shed light on the role of Nurr1 in ALS pathology we

investigated Nurr1 expression and its mechanism of action in theSOD1-G93A mouse one of the most commonly used ALS murinemodels Specifically we assessed Nurr1 expression levels in theblood of early and late symptomatic animals considering them aunique group to better represent ALS patient sampling Howeverthe blood of TG mice did not show a significant increase in Nurr1expression compared with age-matched WT animals thisobservation was in contrast to the increase seen in ALS patientsThis discrepancy is probably a consequence of the small sample sizeandor because the phases of the disease in human ALS and themurine model are not parallel Furthermore our mouse modelcarried a mutation in the SOD1 gene which represents only 7 ofour ALS patient cohort

By contrast in the spinal cord of TG mice our data revealed thatNurr1 was strongly upregulated in the early phases of the disease inTG mice indeed (1) it translocated into the nuclei of MNs and (to alesser extent) astrocytes (2) it induced neurotrophic factor Bdnfexpression and (3) it inhibited iNos expression by docking withNFκB on the iNos promoter These observations have beenmade using reverse transcription PCR (RT-PCR) WB analysisChIP-qPCR assay and immunofluorescence staining Concerningimmunofluorescence experiments the Nurr1 antibody is mostcommonly used in immunocytochemistry (Zhou et al 2010 Leeet al 2012 Alvarez-Castelao et al 2013) as on tissue samples ittends to give an undesired noisy background (Baron et al 2012Garcia-Perez et al 2013) despite the antigen retrieval passageNevertheless we are quite confident about the labeling specificityseen here as it reflects the molecular trend observed with RT-PCRand WB assays

For the first time our results highlight Nurr1 expression in spinalcord MNs Indeed Nurr1 was physiologically present in thecytoplasmic compartment of MNs from WT animals suggesting apotential role for this transcription factor in the survival of cholinergicneurons which are affected in this neurodegenerative disease Notablyvery early in disease progression (in asymptomatic and earlysymptomatic phases) Nurr1 was upregulated at the transcriptionallevel as suggested by RT-PCR analysis Following activation Nurr1was translocated to the nucleus as indicated by its increased levels innuclear extracts of TG mice Nurr1 was found mainly in MNs andrarely in astrocytes as indicated by immunofluorescence experimentsWithin the nuclei of asymptomatic animals Nurr1 was able to activateBdnf its target gene and to repress the transcription of iNos IndeedNurr1 upregulation in the asymptomatic phase of the disease wasaccompanied by an increase in Bdnf mRNA and a reduction of iNosmRNA as demonstrated by RT-PCR assay Repression of iNoswas probably the result of direct competition between Nurr1 andNFκB on the iNos promoter as indicated by ChIP-qPCR assay inagreement with the mechanism of action proposed by Sajio andcoworkers for PD (Saijo et al 2009) In the early symptomatic phasehowever even if Nurr1 levels in the nuclear compartment of TGmice were higher than those in age-matched WT it was not able toactivate Bdnf or repress iNos Finally in late symptomatic animalswhen Nurr1 was no longer upregulated at the transcriptional level andapparently absent from nuclear compartments we observed a strongbinding of NFκB to the iNos promoter and an enrichment ofH3K4me3 indicating that the gene was active in accordance with theobserved increase in mRNA levels

Therefore we hypothesized that Nurr1 is not directly involved inthe development of ALS pathology but instead is likely to act as anendogenous means to delay the pathogenetic mechanisms Inparticular Nurr1 could be activated before symptom onset as anendogenous neuroprotective mechanism with an anti-inflammatoryrole although not sufficient to reverse disease progressionPreventing the final downregulation of Nurr1 and exploiting itsability to activate Bdnf and to repress iNos in the late stages of thedisease could represent a useful therapeutic approach

Nurr1 has recently emerged as having a key role in the mediationof cell-type specific inflammatory responses in several diseasessuch as cancer immune alterations metabolic cardiovascular andneurological diseases (Rodriacuteguez-Calvo et al 2017 Safe et al2016) In particular Saijo and coworkers (Saijo et al 2009)demonstrated Nurr1 to have an important anti-inflammatory rolefollowingmicroglial activation induced byLPS injection limiting theproduction of neurotoxic mediators by glial cells and protecting DAneurons from inflammation Furthermore a neuroprotective effect of

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Nurr1was demonstrated in themultiple sclerosis (MS)murinemodelrepresented by experimental autoimmune encephalomyelitis (EAE)Indeed it has been demonstrated that a preventive treatment with theNurr1 activator isoxazolopyridinone 7e delays onset and reduces theincidence and severity of EAE reducing inflammation in the spinalcord of treated mice probably through an NFκB-dependentmechanism (Montarolo et al 2014)Interestingly Nurr1 is able to protect hippocampal neurons

within the CA1 field following kainic acid insult in mice (Volakakiset al 2010) and also emerged as a mediator of CREB-dependentneuroprotection in mouse embryonic stem cell derived neurons(Volakakis et al 2010) By contrast Nurr1 and NR4A familymembers in general have also been described as pro-inflammatoryfactors with controversial results in several disease models(Rodriacuteguez-Calvo et al 2017)

ConclusionsCollectively our results demonstrate for the first time that Nurr1mRNA is upregulated in blood samples of ALS patients thereforewe speculate that it could be considered a biomarker candidate forALS However further studies will be necessary to confirm thisaspect and to determine the specific role of Nurr1 in the peripheralimmune system during disease progressionIn addition we demonstrate in a mouse model of ALS that Nurr1

is activated in the early symptomatic phase of the disease probablyas a neuroprotective endogenous mechanism This observation is inagreement with the current hypothesis supporting an initialactivation of glial cells aimed at sustaining MN viability throughneurotrophic factors (IGF-1) anti-inflammatory interleukins (IL-4IL-10) and cytokine secretion (Chiu et al 2008 Henkel et al 2009Murdock et al 2015 Hooten et al 2015) Later on during diseaseprogression we observed the inactivation of Nurr1 it was no longerupregulated at the mRNA level was absent from the nuclearcompartment and was unable to dock with NFκB on the iNospromoter Therefore we speculate that Nurr1 might represent apromising target for ALS therapy as neuroinflammation is arelatively unexplored field that can modify the course of ALSdisease Future therapeutics aimed at augmenting the anti-inflammatory effect through Nurr1 activation could mitigate thetoxic environment modulate neuroinflammation and foster the MNrepair process having a positive effect on ALS treatment

MATERIALS AND METHODSEnrolled subjectsA total of 43 patients affected by ALS and 41 healthy controls (HC) wereenrolled in the current study ALS patients were followed and clinicallymonitored by neurologists of the ALS Expert Center (CRESLA) lsquoCittagravedella Scienza e della Salutersquo University Hospital Turin Most of the ALSpatients received disease-specific drugs at the time of blood sampling suchas riluzole alone or combined with symptomatic therapies HC recruitedfrom volunteers were asked to complete a health questionnaire to excludeany acute or chronic inflammatory and neurological disease All subjectsenrolled in the study were of Caucasian origin Demographic and clinicalfeatures of patients and HC are summarized in Table 1

This study was approved by Piedmont and San Luigi University HospitalEthical Committee (Ndeg802011 25 May 2011) and was conducted inaccordance with the ethical standards laid down in the 1964 Helsinkideclaration and its later amendments Written informed consent was obtainedfrom all individual participants included in the study at the time of blooddrawing

Animal care and useExperiments were performed on male transgenic mice B6SJL-TgN(SOD1G93A)1Gur overexpressing human SOD1 containing the

Gly93Ala mutation (The Jackson Laboratory stock number 002726)these mice have high transgene copy number as reported in the datasheetThe colony was derived by breeding male transgenic (TG) mice with naive(B6xSJLJ)F1 females (WT) (Janvier SAS) Overall 35 WT and 35 TGmice housed under diurnal lighting conditions (12 h darknesslight) wereused All experimental procedures on live animals were carried out in strictaccordance with the European Communities Council Directive 86609EEC(24 November 1986) Italian Ministry of Health and University of Turininstitutional guidelines on animal welfare (law 11692 on care andprotection of living animals undergoing experimental or other scientificprocedures authorization number 172010-B 30 June 2010 and 3672016-PR) additionally an ad hoc Ethical Committee of the University of Turinapproved this study All efforts were made to minimize the number ofanimals used and their suffering Transgenic mice were identified by PCRaccording to The Jackson Laboratoryrsquos genotyping protocol

Genotyping miceDNA was extracted from the mouse tail as previously described (Sirabellaet al 2018) PCR was performed on the extracted DNA to evaluate thepresence of the human transgene superoxide dismutase 1 (hSOD1) genethese mice were referred to as TG Two primers were used hSOD1 fwd5prime-CATCAGCCCTAATCCATCTGA-3prime and hSOD1 rev 5prime-CGCGACTA-ACAATCAAAGTGA-3prime

Behavioral testsTo identify symptom onset and to follow disease progression TG miceunderwent specific behavioral tests rotarod and PaGE tests were performedby a trained blind observer as previously reported (Valsecchi et al 2013Boido et al 2014) The tests started from postnatal day 60 (P60) a fullyasymptomatic phase of the disease The first 2 weeks of tests wereconsidered as training for the animals The tests were performed twice aweek The body weight was also monitored during the whole period ofobservation Briefly for the rotarod test we measured the time animals couldremain on the rotating cylinder in a 7650 accelerating model of a rotarodapparatus (Ugo Basile Italy) Each animal was given three trials Thearbitrary cut-off time was 300 s and the accelerated speed went from 4 to32 rpm For PaGE tests the animal was placed on the wire lid of aconventional housing cage the lid was gently shaken to prompt the mouseto hold onto the grid before it was swiftly turned upside down Grip scorewas measured as the length of time that the mouse was able to hang on to thegrid The arbitrary cut-off time was 90 s

This G93A stock of mice has a high transgene number and shows thefirst symptoms of disease at approximately 3 months of age with a rapidprogression of the disease in 1 monthWe considered three groups of animals(1) asymptomaticmice between 2 and 3 months of age that do not display anymotor performance deficit (2) early symptomatic animals that displayeddecreased motor behavioral performance in two consecutive testing sessionsapproximately at 35 months of age and (3) late symptomatic mice of 4and 45 months of age with seriously compromised motor conditions aspreviously reported

RT-PCR analysisALS patientsPeripheral whole blood samples from ALS patients and HC were collectedinto Tempus Blood RNA Tubes (Thermo Fisher Scientific) and stored atminus80degC until use Total RNAwas automatically extracted using the MaxwellRSC Station and products (Promega) following the manufacturerrsquosinstructions and was reverse-transcribed at a final concentration of20 ngμl using the RT High Capacity Transcription Kit followingmanufacturerrsquos instructions (Life Technologies)

MiceMice were deeply anaesthetized with 3 isoflurane vaporized in O2N2O5050 and sacrificed The blood was rapidly collected in Tempus BloodRNATubes (Thermo Fisher Scientific) and stored at minus80degC until use TotalRNA was automatically extracted using the Maxwell RSC Station andproducts (Promega) following the manufacturerrsquos instructions and wasreverse-transcribed at a final concentration of 20 ngμl (Thermo Fisher

7


Disea

seModelsampMechan

isms

Scientific) using the RT High Capacity Transcription Kit followingmanufacturerrsquos instructions (Life Technologies) The spinal cord wasrapidly removed and immediately frozen on dry ice and stored at minus80degCuntil use Total RNA was extracted with Trizol following supplierrsquosinstructions (Life Technologies) and cDNA was synthesized using 2 microg oftotal RNA with the High Capacity Transcription Kit followingmanufacturerrsquos instruction (Life Technologies) as previously reported(Sisalli et al 2014 Formisano et al 2015)

Gene expression analysis was performed by RT-PCR using AppliedBiosystemsrsquo TaqMan gene expression products (Life Technologies)For HC and ALS patients primers from Applied Biosystemsrsquo TaqManAssay-on-demandTM gene expression products were used glyceraldehyde-3-phosphate dehydrogenase (GAPDH Hs99999905_m1) and Nurr1(Hs00428691_m1) (Life Technologies) as previously reported(Montarolo et al 2015) For mice Applied Biosystemsrsquo TaqMan geneexpression assay Nurr1 (TaqMan ID Mm00443060_m1) and Gapdh (IDMm99999915_g1) were used as previously reported (Valsecchi et al2015) Expression levels of target genes were calculated by the normalizedcomparative cycle threshold (Ct) method (2^minusΔΔCt) using GAPDH asreference gene and the Universal Human Reference RNA (Stratagene) ascalibrator for human samples For blood murine samples expression levelsof target genes were calculated by the normalized comparative cyclethreshold (Ct) method (2^minusΔCt) using Gapdh as reference gene

WB analysisNuclear and cytoplasmic extracts were obtained as previously described(Guglielmotto et al 2014 Piras et al 2017) Briefly samples (spinal cord)were first washed with cold phosphate-buffered saline (PBS) and thenhomogenated with a 28-gauge needle syringe in ice-cold lysis buffer(10 mM HEPES pH 79 10 mM KCl 15 mM MgCl2 01 mM EGTA02 mM PMSF 10 mM NaF 1 mM Na3VO4 05 gml apronitin 1 gmlleupeptin 1 gml pepstatin) Tissues were allowed to swell on ice for10 min vortexed and collected by centrifugation The supernatant wasdiscarded and the pellet dissolved in ice-cold buffer (20 mM HEPES pH79 400 mM NaCl 15 mM MgCl2 01 mM EGTA 02 mM PMSF10 mM NaF 1 mM Na3VO4 05 gml apronitin 1 gml leupeptin 1 gmlpepstatin) and incubated on ice for 30 min for high salt extraction Cellulardebris was removed by centrifugation and the supernatant fraction stored atminus80degC

Protein concentration was determined using the Bio-Rad protein assayTo detect the proteins of interest specific antibodies were used anti-Nurr1(mouse monoclonal antibody 1750 Santa Cruz Biotechnology) and anti-lamin A (rabbit polyclonal 11000 Swant) Immunoreaction was revealedusing anti-mouse and anti-rabbit immunoglobulin G conjugated toperoxidase 12000 (GE Healthcare) by the ECL reagent (GE Healthcare)The optical density of the bands was determined by Chemi Doc ImagingSystem (Bio-Rad) and normalized to the optical density of lamin A

Chromatin immunoprecipitation assayThe chromatin immunoprecipitation assay and qPCR quantification wereperformed as previously described (Valsecchi et al 2013 Guida et al2017 2018 Iannello et al 2019) Specifically tissues were crosslinkedwith 1 formaldehyde in PBS for 10 min at 37degC The reaction was stoppedby adding glycine to a final concentration of 125 mM at room temperature(RT) Crosslinked spinal cords were washed three times in cold PBScontaining proteinase inhibitors and then collected in 1 ml cell lysis buffer(5 mMPIPES pH 8 85 mMKCl and 05NP-40) After 10 min incubationon ice nuclei were collected by centrifugation and lysed with 400 microl ofnuclei lysis buffer (50 mM Tris-HCl pH 8 10 mM EDTA and 1 SDS)The lysates were incubated on ice for 10 min and then sonicated 20 times for20 s at 30 amplitude with SonoPlus HD2070 sonicator (Bandelin) Asmall portion of sonicated chromatin (25 microl) was used to verify that theaverage size of DNA fragments was in the range 250ndash500 bp For eachimmunoprecipitation a 1 microg sample of sheared chromatin was diluted in IPbuffer (167 mM Tris-HCl pH 8 167 mM NaCl 12 mM EDTA 001SDS 11 Triton X-100) and incubated with 05 microg of antibodies againstNurr1 and NFkB p65 (cat numbers sc-990 and sc-372 respectively SantaCruz Biotechnology) histones H3K4me3 and H3K27me3 (cat numbers

39915 and 39155 respectively Active Motif ) in a BSA pretreated 96-welldish at 4degC overnight on an orbital shaker Samples with IgG antibody (catnumber sc-2027 Santa Cruz Biotechnology) were run in parallel as negativecontrols The following day 30 microl of 50 Protein A Sepharosetrade 4 FastFlow (GE Healthcare) slurry was added and incubated for 2 h at 4degC tocapture the immune complexes Proteins and DNA not specificallyassociated with the beads were removed by sequentially washing withlow-salt buffer (01 SDS 1 Triton X-100 2 mM EDTA 20 mMTris-HCl pH 8 and 150 mM NaCl) high-salt buffer (01 SDS 1 TritonX-100 2 mM EDTA 20 mM Tris-HCl pH 8 and 500 mM NaCl) LiClwashing buffer (025 M LiCl 1 deoxycholate sodium salt 1 mM EDTA10 mM Tris-HCl pH 8 and 1 NP-40) and twice with Tris-EDTA buffer(10 mM TrisHCl pH 8 1 mM EDTA) at 4degC for 5 min each wash Theimmunoprecipitated DNA-protein complexes were purified using 10Chelexreg 100 Resin (Bio-Rad) for 10 min at 95degC Proteins were digested byincubating each sample with 20 μg of proteinase K (Thermo FisherScientific) for 30 min at 55degC and then 10 min at 95degC to obtain proteinaseK inactivation thus achieving DNA purification

Quantification of ChIP-enriched DNA was performed by real-time PCRusing iTaq Universal SYBR Green Supermix (Bio-Rad) The enrichment oftarget sequence in the immunoprecipitated samples was normalized oninput samples (1 of total chromatin used per immunoprecipitation) andexpressed as the ratio between WT and TG binding enrichment on iNospromoter for each transcription factor CustomChIP primers were employediNos promoter forward 5prime-ATGCCATGTGTGAAAATTCC-3prime andreverse 5prime-TGGGCTAGCCTGGTCTACAG-3prime Samples were amplifiedsimultaneously in triplicate in one assay run

ImmunofluorescenceImmunostaining procedures on spinal cord sections were performed aspreviously described (Boido et al 2014 2018) Briefly animals (TG n=3WT n=3 for each phase analyzed) were deeply anesthetized by gaseousanesthesia (3 isoflurane vaporized in O2N2O 5050) to undergointracardiac perfusion with 4 paraformaldehyde (PFA) pH 74 Thelumbar spinal cords were removed and post-fixed in PFA for 2 h at 4degCSamples were transferred overnight into 30 sucrose in 01 M phosphatebuffer at 4degC for cryoprotection embedded in cryostat medium (Killik Bio-Optica) and cut on the cryostat (Microm HM 550) in serial transverse14 μm-thick sections Sections were kept in PBS at 4degC or mounted ontogelatin-coated slides to be processed for immunostaining Unspecificbinding sites were blocked for 30 min at room temperature with 2 TritonX-100 and 10 normal donkey serum (Sigma-Aldrich) in PBS (pH 74)Sections were then incubated in the same solution with the followingprimary antibodies at 4degC overnight 1100 polyclonal rabbit anti-Nurr1(cat number sc991 Santa Cruz Biotechnology) 11000 monoclonal mouseanti-neurofilament H non-phosphorylated (SMI 32R cat number14974402 Covance) 11000 monoclonal mouse anti-glial fibrillaryacidic protein (GFAP cat number ab190288 Abcam) 11000 polyclonalrabbit anti-IBA1 (cat number 019-19741 Wako Chemicals) 11000monoclonal rat anti-mouse CD68 (cat number MCA1957 Bio-Rad) Thenext day sections were washed in PBS and incubated in 1200 cyanine3-conjugated anti-rabbit Alexa Fluorreg 488 anti-mouse (1200 respectivelycat numbers 711-165-152 and 715-546-150 Jackson ImmunoResearch) oranti-rat (1200 cat number ab150153 Abcam) secondary antibodiesdepending on the primary antibodies used Sections were then examinedwith a Leica TCS SP5 confocal laser-scanning microscope lightPhotomicrographs were eventually manipulated with autocontrastenhancement by Photoshop CS2 software

Statistical analysisRegarding ALS patients continuous data are presented as medians andranges and discrete data are given as counts and percentages Chi-squaretests were performed to compare groups of categorical data Studentrsquos t-testor MannndashWhitney U-test were used to compare continuous data asappropriate The correlation between Nurr1 gene expression levels andclinical and demographical variables was assessed by Pearson correlationsand fitting linear models In particular we considered (Table 1) (1) sex andage at sampling for all the groups (2) the age at disease onset Data obtained

8


Disea

seModelsampMechan

isms

from mice were expressed as meanplusmnse (sem) Statistically significantdifferences among means andor ratios were determined by two-wayANOVA test followed by Bonferroni test Statistical significance wasconsidered at Plt005 All analyses were carried out using R version 302and Prism 5 software (GraphPad Software)

Competing interestsThe authors declare no competing or financial interests

Author contributionsFormal analysis VV Investigation VV MB FM MG SP SC AI NGES A Calvo GF A Chio Data curation VV MB FM MG SM Writing -original draft VV MB FM Writing - review amp editing VV MB AVSupervision AV Funding acquisition MB AB AV

FundingThis work was supported by Universita degli Studi di Torino (Ricerca Locale 20142015 2016-2017 grant to MB) and by Ministero dellrsquoIstruzione dellrsquoUniversita edella Ricerca (Dipartimenti di eccellenza 2018-2022 to the Department ofNeuroscience lsquoRita Levi Montalcinirsquo Universita degli Studi di Torino)

Supplementary informationSupplementary information available online athttpdmmbiologistsorglookupdoi101242dmm043513supplemental

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(Zetterstroumlm et al 1997 Saucedo-Cardenas et al 1998) Given itscrucial functions altered Nurr1 expression has been implicated indopamine-associated brain disorders including PD Notably theexpression of Nurr1 in mDA neurons decreases in PD patients(Hering et al 2004) and single-nucleotide polymorphisms (SNPs)and mutations resulting in reduced expression of Nurr1 areassociated with familial and sporadic forms of PDInterestingly Nurr1 is a constitutively active transcription factor

binding its target genes as a monomer homodimer or heterodimerin association with retinoidX receptors (Aarnisalo et al 2002Mairaet al 1999 Wang et al 2003) In murine models of PD Nurr1 wasfound to have roles in both neuroprotection and immunomodulationIndeed Nurr1 has an anti-inflammatory role inhibiting expressionof the genes encoding pro-inflammatory components of the NFκBpathway in microglia and astrocytes (Saijo et al 2009) Specificallyknocking down Nurr1 in mice leads to an increased activation ofglial cells exposed to lipopolysaccharide (LPS) with subsequentproduction of higher levels of mRNAs encoding inflammatorycytokines and neurotoxic effector proteins such as tumor necrosisfactor α (TNF-α) inducible nitric oxide synthase (iNOS) andinterleukin 1β (IL-1β) responsible for inflammation-inducedneuronal death (Saijo et al 2009 McMorrow and Murphy 2011)In addition Nurr1 induces the expression of neurotrophic factorssuch as brain-derived neurotrophic factor (BDNF) (Barneda-Zahonero et al 2012) In addition to its role in the CNS Nurr1has an active role in PD as downregulated levels of gene expressionwere also found in peripheral blood obtained from PD patients withprogressive loss of mDA neurons (Le et al 2008 Liu et al 2012Montarolo et al 2016)Recently it has been shown that Nurr1 also controls the expression

of several nuclear-encodedmitochondrial genes involved in oxidativerespiration such as SOD1 Ts translation elongation factor (TSFM)and cytochrome c oxidase subunit 5B (COX5B) demonstrating animportant role in sustaining respiratory function (Kadkhodaei et al2013) Therefore we first analyzed Nurr1 gene expression levels inblood obtained from ALS patients in comparison with healthycontrols (HC) To better understand its role in ALS we theninvestigated the expression and function of Nurr1 in a murine modelof ALS the SOD1-G93A mouse

RESULTSNurr1 mRNA is upregulated in the peripheral blood of ALSpatientsGene expression analysis of Nurr1 was performed on wholeperipheral blood obtained from 43 ALS patients and 41 HC

subjects demographic and clinical characteristics are summarizedin Table 1 The ALS group comprising seven fALS and 36 sALSpatients showed higher Nurr1 mRNA levels compared with the HCgroup (Fig 1A) Furthermore there was a significant difference inage (Studentrsquos t-test P=0002 Table 1) but not in sex between ALSpatients and HC (Fisher exact test P=038 Table 1) To assesspotential bias related to age the correlation between age and geneexpression levels of Nurr1 was evaluated in HC subjects and nosignificant results were highlighted as previously reported byMontarolo et al (2016) (Pearson correlation coefficient r=013P=041) Also correlation analyses between Nurr1 gene expressionof ALS patients and age at the time of sampling (Pearson correlationcoefficient r=minus009 P=055) and age at disease onset (Pearsoncorrelation coefficient r=minus016 P=031) (Fig S1A) did not highlightsignificant results Similarly there are no significant differences inNurr1 expression between sexes in bothHC (Studentrsquos t-testP=053)and ALS patients (MannndashWhitney U-test P=032) (Fig S1B) Theinfluence on Nurr1 expression level of the pharmacological treatmentindicated in Table 1 was not assessed owing to the small sample sizeof each group Among fALS patients four carried mutations inC9Orf72 two in SOD1 and one in the TARDBP gene whereas threesALS patients carried mutations in C9Orf72 SOD1 and in the geneencoding optineurin (OPTN) We also reported Nurr1 expressionlevels for the different groups (Fig S1C)

Nurr1mRNAand protein are upregulated in the spinal cord ofSOD1-G93A mice in the asymptomatic and earlysymptomatic phases of the diseaseThe high levels of Nurr1 expression in samples of peripheral bloodfrom ALS patients suggested a potential role in ALS pathogenesisTo investigate the role of Nurr1 in ALS outcome we used atransgenic mouse model of ALS SOD1-G93A (TG) This modelhas a high copy number of the transgene and is one of the mostcommonly used in this area of research (Gurney et al 1994) Malemice develop first symptoms of the disease at approximately3 months of age and die about 4 weeks after disease onsetBehavioral tests such as rotarod and paw grip endurance (PaGE)were used to evaluate the appearance of the first motor deficitsand to divide the animals into three experimental groups(1) asymptomatic mice (2) early symptomatic mice and (3) latesymptomatic mice (Boido et al 2014) Age-matched wild-type(WT) male mice were used as controls Female animals wereexcluded from the study as significant differences were observedbetween male and female TG animals probably owing to anestrogen neuroprotective effect (Vercelli et al 2008)

Table 1 Demographic and clinical characteristics of the enrolled populations

HC (n=41) ALS (n=43) P-value

Age at time of sampling (years) mean (sd) 567 (1097) 646 (1203) 0002Sex female () 23 (561) 19 (442) 038dagger

Age at disease onset (years) mean (sd) ndash 6198 (1198) ndash

Patients treated with specific therapies n () ndash 3 (71) None14 (333) Riluzole22 (524) Riluzole+other drugs3 (71) Other drugsDagger

ndash

fALS patients with known mutations n () 7 (163) 4 C9Orf72 2 SOD1 1 TARDP43sALS patients with known mutations n () 36 (837) 1 C9Orf72 1 SOD1 1 OPTN

ALS amyotrophic lateral sclerosis HC healthy controlsStudentrsquos t-testdaggerFisher exact testDaggerOther drugs (L-acetilcarnitine tocopherol chininum sulphuricum baclofene gabapentin pregabalin pyridostigmine bromide escitalopram citaloprampalmitoylethanolamide)

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Documents

The transcription factor Nurr1 is upregulated in amyotrophic … · receptor related 1 protein (Nurr1), also called NR4A2 (encoded by the gene NR4A2). Nurr1 is an orphan receptor