Chronic estrogen deficiency leads to molecular aberrations related to neurodegenerative changes in follitropin receptor knockout female mice

PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 2 7 8 - 6

Neuroscience Vol. 114, No. 2, pp. 493^506, 2002 K 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

www.neuroscience-ibro.com

CHRONIC ESTROGEN DEFICIENCY LEADS TO MOLECULAR ABERRATIONS RELATED TO NEURODEGENERATIVE CHANGES IN FOLLITROPIN RECEPTOR KNOCKOUT FEMALE MICE J. TAM,a N. DANILOVICH,b K. NILSSON,a M. R. SAIRAMb and D. MAYSINGERa a

Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Room 1314, McIntyre Medical Sciences Building, Montreal, QC, Canada H3G 1Y6 b

Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, QC, Canada

Abstract5The follitropin receptor knockout (FORKO) mouse undergoes ovarian failure, thereby providing an animal model to investigate the consequences of the depletion of circulating estrogen in females. The estrogen de¢ciency causes marked defects in the female reproductive system, obesity, and skeletal abnormalities. In light of estrogen’s known pleiotropic e¡ects in the nervous system, our study examined the e¡ects of genetically induced estrogen^testosterone imbalance on this system in female FORKO mice. Circulating concentrations of 17-L-estradiol (E2) in FORKO mice are signi¢cantly decreased (FORKO 3/3: 1.13 D 0.34 pg/ml; wild-type +/+: 17.6 D 3.5 pg/ml, P 6 0.0001, n = 32^41); in contrast, testosterone levels are increased (3/3: 37.7 D 2.3 pg/ml; wild-type +/+: 3.9 D 1.7 pg/ml, P 6 0.005, n = 25^33). The focus was on the activities of key enzymes in the central cholinergic and peripheral nervous systems, on dorsal root ganglia (DRGs) capacity for neurite outgrowth, and on the phosphorylation state of structural neuro¢lament (NF) proteins. Choline acetyltransferase activity was decreased in several central cholinergic structures (striatum 50 D 3%, hippocampus 24 D 2%, cortex 12 D 3%) and in DRGs (11 D 6%). Moreover, we observed aberrations in the enzymatic activities of mitogenactivated protein kinases (extracellular-regulated kinase and c-Jun N-terminal kinase) in the hippocampus, DRGs, and sciatic nerves. Hippocampal and sensory ganglia samples from FORKO mice contained hyper-phosphorylated NFs. Finally, explanted ganglia of FORKO mice displayed decreased neurite outgrowth (20^50%) under non-treated conditions and when treated with E2 (10 nM). Our results demonstrate that genetic depletion of circulating estrogen leads to biochemical and morphological changes in central and peripheral neurons, and underlie the importance of estrogen in the normal development and functioning of the nervous system. In particular, the ¢ndings suggest that an early and persisting absence of the steroid leads to neurodegenerative changes and identify several key enzymes that may contribute to the process. This model provides a system to explore the consequences of circulating estrogen deprivation and other hormonal imbalances in the nervous system. K 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: estrogen, follicle stimulating hormone, CNS, choline acetyltransferase, mitogen-activated protein kinase, neuro¢lament, dorsal root ganglia.

(Henderson, 1997), cardiac and vascular function (Matthews et al., 1989), and bone and mineral metabolism (Manolagas, 1998). The neurotrophic and neuroprotective properties of the steroid have been demonstrated in embryonic, neonatal, and adult systems (Beyer, 1999; Dubal and Wise, 2001; Wise et al., 2001a). Notably, in vitro studies show that estrogen promotes the survival of dorsal root ganglia (DRGs) deprived of nerve growth factor (NGF) (Patrone et al., 1999) and enhances neurite outgrowth from hypothalamic neurons (Ferreira and Caceres, 1991). The steroid also displays anti-in£ammatory e¡ects in primary rat microglia (Vegeto et al., 2001) and in the peripheral nervous system (PNS) (Dina et al., 2001). Estrogens can also act by decreasing the risk of neurologic disease and by lessening the adverse consequences of injury to the nervous system (reviewed in Garcia-Segura et al., 2001; Wise et al., 2001b). Finally, in in vivo studies in experimental models of axotomy (Tanzer and Jones, 1997), ischemia (Simpkins et al., 1997), Alzheimer’s disease (Henderson, 1997) and Parkinson’s disease (Dluzen et al., 1996) suggest that estrogens play a critical role in the CNS and PNS.

In the past several years, the understanding of estrogen’s actions outside of the reproductive system has increased considerably. Aside from its well known gonadal functions, estrogens play a role in cognition and memory

*Corresponding author. **Corresponding author. Tel.: +1-514-398-1264; fax: +1-514-3986690. E-mail addresses: [email protected] (M. R. Sairam), [email protected] (D. Maysinger). Abbreviations : ChAT, choline acetyltransferase ; DRG, dorsal root ganglion; E2, 17-L-estradiol ; EDTA, ethylenediaminetetraacetic acid ; ER, estrogen receptor; ERK, extracellular-regulated kinase; FORKO, follitropin receptor knockout; FSH-R, follicle stimulating hormone receptor; GAP-43, growth-associated protein 43; JNK, c-Jun N-terminal kinase ; MAPK, mitogen-activated protein kinase ; MEK, mitogen-activated protein kinase kinase ; NF, neuro¢laments ; NF-H, heavy neuro¢lament subunit; NF-L, light neuro¢lament subunit; NF-M, medium neuro¢lament subunit; NGF, nerve growth factor; PD98059, 2,2P-amino 3P-methoxy-phenyl-(oxanaphthalen-4-one); SN, sciatic nerve. 493

NSC 5719 15-8-02

494

J. Tam et al.

17-L-Estradiol (E2) binding to the classical nuclear receptors estrogen receptor (ER)-K and ER-L leads to their interaction with estrogen response elements on DNA and activates transcription of genes that promote cell survival (Cowley and Parker, 1999). E2 also exerts non-genomic e¡ects, possibly via the recently identi¢ed membrane-localized ER that couples to G-proteins and mobilizes calcium (Benten et al., 2001). In addition, E2 stimulates nitric oxide synthase activity (Pelligrino et al., 2000), inhibits free radical production (Moosmann and Behl, 1999), and activates signaling pathways for cyclic AMP (Zhou et al., 1996), phosphoinositide 3-kinase (Kelly and Levin, 2001), and the mitogen-activated protein kinases (MAPKs) (Watters et al., 1997). The MAPKs comprise a diverse family of serine/threonine protein kinases that regulate cell proliferation, differentiation, and survival in many cellular systems including neurons (Davis, 2000; Fukunaga and Miyamoto, 1998). MAPK family members include the extracellular-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 kinases. The MAPKs can act bene¢cially or detrimentally to the cell, and their role is dictated by the cellular context and environment. ERK, JNK, and p38 modulate neurite outgrowth from primary neuron cultures (Sjogreen et al., 2000) and neuronal cell lines (Hansen et al., 2000), as well as promote neuronal survival (Creedon et al., 1996; Xia et al., 1995) or death (Eilers et al., 2001). In addition, the MAPK family members participate in the phosphorylation of neuro¢lament (NF) proteins, the key structural proteins in central and peripheral axons (Li et al., 1999; Masaki et al., 2000; Pearson et al., 2001; Veeranna Amin et al., 1998). However, excesses in both MAPK activation and/or NF phosphorylation occur in various neurodegenerative disorders, including diabetic neuropathy (Fernyhough et al., 1999), Alzheimer’s disease (Hensley et al., 1999), and amyotrophic lateral sclerosis (Migheli et al., 1997). The ovary in females is the principal source of estrogen throughout reproductive life. This steroid is produced under the control of follicle stimulating hormone receptor (FSH-R) signaling. FSH-Rs expressed in ovarian granulosa in females and in testicular Sertoli cells in males interact with the pituitary glycoprotein hormone FSH to maintain reproductive capacity. The loss of FSH-R signaling in follitropin receptor knockout (FORKO) mice markedly a¡ects the reproductive axis of both sexes (Danilovich et al., 2000; Dierich et al., 1998). In FORKO females, ovarian underdevelopment leads to the loss of estrogen production as well as obesity and bone defects (Danilovich et al., 2000). Although the estrogen de¢cient condition manifests early in life, the ER signaling system appears to remain intact in target tissues. Thus, the FORKO mouse o¡ers new opportunities for investigating the physiological consequences of chronic estrogen depletion in females (Danilovich et al., 2000; Dierich et al., 1998). Among the hormonal imbalances that occur following genetic ablation of the FSHR, the virtual disappearance of estrogen from the circulation appears to have major functional consequences in females. Therefore we were interested in examining the

perturbations that might occur in the nervous system in FORKO females. The results reveal the occurrence of neurodegenerative changes in the CNS and PNS, identi¢ed at both the biochemical and functional levels. Thus, it may be possible to understand the early and long-term changes in the brain associated with hormonal imbalance in this genetically altered mouse model.

EXPERIMENTAL PROCEDURES

Generation of FSH-R 3/3 mice The generation and maintenance of FORKO mice has previously been described by Dierich et al. (Danilovich et al., 2000; Dierich et al., 1998). In brief, by deleting a portion of the promoter and the DNA segment in exon-1 that encodes the aminoterminal part of the FSH-R, the entire FSH-R signaling repertoire was ablated. Genotyping of the animals was performed after weaning by PCR of genomic DNA that allowed unequivocal identi¢cation of the +/+, +/3 and 3/3 mice (Danilovich et al., 2000). Animals All animal work was performed according to guidelines approved by the local Animal Research Committee of the Institute and all e¡orts were made to minimize animal su¡ering and to reduce the number of animals used. Female FORKO mice and corresponding littermates (aged 3^4 months) were used in the current investigation. They were fed ad libitum mouse standard diet and kept in a 12 h light/dark cycle. All animals were killed in the morning without regard to the stage of the reproductive cycle. For FORKO females this was of no consequence as none of them cycles (Danilovich et al., 2000). Steroid radioimmunoassays (RIAs) of plasma samples were performed as previously described (Danilovich et al., 2000). In brief, blood samples were obtained by cardiac puncture and collected in plastic centrifuge tubes containing EDTA. After centrifugation for 15 min at 2500Ug, the plasma was stored at 320‡C until use. RIAs of serum estrogen and testosterone levels were performed using Coat-A-Count kits (Diagnostic Products, Los Angeles, CA, USA) according to the manufacturer’s instructions. Materials The extracellular matrix material used was Matrigel obtained from Becton Dickinson USA. Cell culture media (RPMI 1640 not containing Phenol Red) was from Gibco-BRL (Life Technologies, Grand Island, NY, USA). Tissue culture plates were from Falcon (Becton Dickinson, Franklin Lakes, NJ, USA). E2 was purchased from Sigma-Aldrich (Oakville, ON, Canada). NGF was obtained from Cedar Lane Laboratories (Hornsby, ON, Canada), and PD98059 was purchased from Calbiochem (Hornsby, ON, Canada). Antibody to phosphorylated ERK (Promega, Madison, WI, USA) and non-phosphorylated ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used in a dilution of 1:5000 and 1:1000, respectively. Antibody to phosphorylated JNK (Promega, Madison, WI, USA) and non-phosphorylated JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied in a dilution of 1:5000 and 1:800, respectively. SMI31 monoclonal antibody to phosphorylated NF and SMI32 monoclonal antibody to non-phosphorylated NF were purchased from Sternberger Monoclonals (Baltimore, MD, USA) and used in a dilution of 1:800. Antibody to growthassociated protein 43 (GAP-43) was purchased from SigmaAldrich (Oakville, ON, Canada) and applied in a dilution of 1:1000. Secondary antibodies, anti-mouse IgG-conjugated horseradish peroxidase (HRP) and anti-rabbit IgG-conjugated HRP were purchased from Amersham.

NSC 5719 15-8-02

Neurodegenerative changes in FORKO mice

495

Choline acetyltransferase (ChAT) assay Wild-type and FORKO mice were decapitated, the skull was opened along the midline, and the parietal bones were moved aside hinging on the squamous bone thus allowing access to the entire brain (as described in Cuello and Carson, 1983), which was then immediately removed and placed on ice. Discrete brain areas (cortex, hippocampus and striatum) were dissected from the fresh, ice-chilled brain under an operating microscope. Details of the brain macrodissection procedures followed were as described by Glowinski and Iversen (1966). Microdissected brain regions were either homogenized immediately or stored at 380‡C until ChAT assays were performed. Tissues from the CNS were homogenized in Triton X-100 bu¡er, pH 7.4 (10 mM Na2 EDTA, 0.5% Triton X-100 in ddH2 O) on ice using a glass homogenizer (Kontes Glass, Duall 20 or Radnoti Glass, Monrovia, CA, USA). The volumes of homogenizing bu¡er were 300 Wl for cortex, 150 Wl for hippocampus and 75 Wl for striatum. DRGs (L3, L4, L5) were also dissected as described by Li (Li, 1998), and 25 Wl of homogenizing bu¡er was used for one DRG (two DRGs were usually lysed). The homogenized samples were centrifuged at 14 000 r.p.m. for 5 min at 4‡C, and the supernatant was collected. ChAT enzymatic activity was measured radioenzymatically as described by Fonnum (1975). Protein content was measured using Bio-Rad Protein Assay, based on the method developed by Bradford (1976) with bovine serum albumin as a standard. Gel electrophoresis and immunoblotting All dissected tissues (cortex, hippocampus, striatum, DRGs, sciatic nerves (SNs)) were lysed on ice in lysis bu¡er (50 mM Tris, 140 mM NaCl, 17 mM Nonidet P-40, 1.09 M glycerol, pH 8.0, 10 mM vanadate and one tablet ‘Complete Protease inhibitor’ Cocktail, Boehringer Mannheim, Montreal, QC, Canada). Cortical tissues were lysed in 200 Wl lysis bu¡er, hippocampal tissues in 100 Wl lysis bu¡er, and striatal tissues in 50 Wl lysis bu¡er. Dissected DRGs (L3, L4, L5) were lysed in 40 Wl lysis bu¡er and SNs were lysed in 50 Wl lysis bu¡er. DRG and SN tissues were sonicated on ice (3U5 s), and all tissues were centrifuged at 14 000 r.p.m. for 5 min at 4‡C. The supernatants were collected, and protein concentration was determined using Bio-Rad protein assay reagent. 6U sample bu¡er (1.5 M Tris^HCl, pH 6.8, 10% SDS, glycerol, Bromophenol Blue, 2-mercaptoethanol in ddH2 O) was added to the remaining lysates, and the samples were boiled for 4 min at 100‡C. Lysates were stored at 320‡C until analyzed by western blotting. Equivalent amounts of protein were loaded onto 12% SDS^ polyacrylamide gels and separated by electrophoresis (Bio-Rad, 100 V). Proteins were transferred to nitrocellulose membranes (Bio-Rad, 250 mA), which were then blocked with 0.1% bovine serum albumin in TBS+T (20 mM Tris^HCl pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 24 h. Separated proteins were visualized on gels by Coomassie staining. Primary antibodies (ERK-p, 1:5000; ERK, 1:1000; JNK-p, 1:5000; JNK, 1:1000; p38-p, 1:1000; p38, 1:1000; NF-p, 1:800; NF, 1:800; GAP-43, 1:2000) were applied to membranes for 24 or 48 h, followed by several washes with TBS+T. Membranes were incubated with HRP-conjugated secondary antibodies (goat antirabbit; sheep anti-mouse) for 1 h, followed by several washes with TBS+T. Immunoreactive bands were detected using an ECL detection system and Kodak Xomat-Blue ¢lm, and ¢lms were scanned at 600 d.p.i. with a Umax Astra 2000 scanner. Bands were quanti¢ed and analyzed with MCID-M5+5.1 image analysis software (Imaging Research) and SYSTAT 9 statistical software (SPSS). Di¡erences were considered signi¢cant where P 6 0.05. Outgrowth assay DRGs (L3, L4, L5) were dissected from wild-type and FORKO mice, and DRG explants were cultured in 7 Wl of Matrigel at 37‡C, 5% CO2 for 24 h in RPMI 1640 media free of Phenol Red and containing 1% penicillin/streptomycin. Neu-

Fig. 1. FSH-R knockout produces an imbalance in circulating steroid hormone levels. Blood samples from FORKO (3/3) and wild-type (+/+) female mice were collected by cardiac puncture, and RIAs for E2 and testosterone levels in plasma were performed as described in the Experimental procedures. The sensitivities of the assays were 1.4 pg/ml and 10 ng/dl, respectively. The columns represent mean serum steroid hormone levels D S.E.M. from three independent experiments (E2: n = 32^41; testosterone : n = 25^33). An asterisk (*) denotes signi¢cant change from wild-type mice (P 6 0.005).

rite outgrowth was followed for 7 days, and was visualized with an Olympus BX51 system microscope equipped with a DageMTI CCD300-RC camera coupled to a Powermate 8100 Pentium III computer (NEC) with 128 MB RAM. Images were stored in uncompressed tagged image ¢le format (TIFF) at a resolution of 600 d.p.i., and the total neuritic area was assessed using a method based on that of Bilsland et al. (1999). In brief, Adobe Photoshop 5.0 software (Adobe Systems) was used to isolate the total neurite portion of each image from the ganglionic portion. Relative optical density (ROD) of the neurite outgrowth (total neuritic area) was then assessed using MCIDM5+5.1 image analysis software (Imaging Research), statistical analysis of the measurements was performed with SYSTAT 9 software (SPSS). Pharmacological manipulations For western blotting experiments, DRG and SN tissues were incubated in Krebs^Ringer solution (125 mM NaCl, 3 mM KCl, 1.3 mM MgSO4 , 2.5 mM CaCl2 , 2.5 mM Na2 HPO4 , 26 mM NaHCO3 , 10 mM glucose, pH 7.4) D E2 (10 nM) for 30 min at 37‡C immediately following dissection. In the neurite outgrowth assays, E2 (10 nM), and/or PD98059 (50 WM), or NGF (50 ng/ ml) were added to the cultures. Statistical analysis Values are reported as mean D S.E.M. In all experiments, DRG preparations were kept paired, with one group acting as control and the other as the treated group. A ratio between the two groups was calculated and the statistical signi¢cance of any e¡ect was tested with two-way analysis of variance and post-hoc Bonferroni t-test. An experimental/control ratio signi¢cantly 6 1 thus re£ects inhibition by the experimental treatment, whereas ratios not di¡erent from or s 1 indicate no e¡ect or stimulation, respectively. Di¡erences were considered signi¢cant where P 6 0.05. In the outgrowth experiments, a ranking system for the control and treated groups was employed using the non-parametric Mann^Whitney U-test and the Bonferroni method. Di¡erences were considered signi¢cant where P 6 0.05.

NSC 5719 15-8-02

496

J. Tam et al.

Fig. 2. ChAT enzymatic activity is reduced in the CNS of FORKO mice. ChAT enzymatic activity in the CNS (A) and PNS (B) of wild-type (+/+) and FORKO (3/3) female mice. ChAT enzymatic activity was measured as described in the Experimental procedures. (A) CNS : cortex, hippocampus, striatum, and (B) PNS: DRGs. The columns represent means D S.E.M. from three independent experiments (cortex, hippocampus: n = 9; striatum, DRG: n = 6). All measurements were performed in triplicate. An asterisk (*) denotes signi¢cant change from wild-type mice (P 6 0.05).

RESULTS

FORKO females exhibit an imbalance in circulating hormones At 3 months of age, E2 was almost non-existent in the circulation of FORKO mice as the levels were at the detection limit of our RIA (Fig. 1) (wild-type +/+: 17.6 D 3.5 pg/ml; FORKO 3/3: 1.13 D 0.34 pg/ml, P 6 0.0001, n = 32^41). Testosterone levels were markedly elevated as compared to wild-type mice (+/+: 3.9 D 1.7 pg/ml; 3/3: 37.7 D 2.3 pg/ml, P 6 0.005, n = 25^33). As the FORKO mice are acyclic, we did not make any distinction with respect to cycles in collecting samples from wild-type females. As previously reported, the hormonal imbalance (testosterone vs. estrogen) as well as regression of the urogenital tract were observed and concur with previously reported data (Danilovich et al., 2000). ChAT enzymatic activity is reduced in the CNS of FORKO mice ChAT is presently one of the most accurate markers for the cholinergic nervous system. Therefore we ¢rst assessed the enzymatic activity in the wild-type and genetically estrogen de¢cient female mice. Radioenzymatic assays showed decreased ChAT enzymatic activity in several CNS structures examined in FORKO (3/3) animals relative to wild-type (+/+) counterparts (Fig. 2). Reductions in ChAT activity were most signi¢cant in the

striatum (50%; +/+: 55.1 D 2.8 nmol Ach/mg protein/10 min; 3/3: 27.2 D 1.4 nmol Ach/mg protein/10 min, P 6 0.05, n = 6) and to a lesser extent in the hippocampus (24%; +/+: 47.3 D 1.9 nmol Ach/mg protein/10 min; 3/3: 38.52 D 0.9 nmol Ach/mg protein/10 min, P 6 0.05, n = 9) and cortex (12%; +/+: 41.9 D 0.98 nmol Ach/mg protein/10 min; 3/3: 34.0 D 1.2 nmol Ach/mg protein/10 min, P 6 0.05, n = 9). A trend in ChAT reduction was observed in the PNS cholinergic structures (DRG), but was not statistically signi¢cant (P s 0.05, n = 6). MAPKs in FORKO mice are di¡erentially phosphorylated In the CNS structures examined, the strongest signal of p42/p44 ERK phosphorylation as determined by densitometry of immunoreactive bands was found in the hippocampus (Fig. 3A), and it was not signi¢cantly different between the wild-type and FORKO mice (P s 0.05, n = 4). There was no signi¢cant di¡erence between the intensity of phosphorylated ERK in the cortex (P s 0.05, n = 4), whereas the intensities of immunoreactive bands were signi¢cantly lower in the striatum of FORKO mice (P 6 0.05, n = 4). Non-phosphorylated p42/p44 ERK was highly expressed and not signi¢cantly di¡erent between wild-type and FORKO animals in the examined structures (Fig. 3A, P s 0.05, n = 4). Phosphorylated JNK was at the detection limit in all structures (data not shown), although an antibody directed against JNK1 revealed strong expression of this kinase isoform (Fig. 3B). Densitometric analysis of immunoblots

NSC 5719 15-8-02

Neurodegenerative changes in FORKO mice

497

Fig. 3. MAPKs in FORKO mice are di¡erentially phosphorylated. (A) Expression and phosphorylation of p42/44 ERK in the cortex, hippocampus and striatum of wild-type (+/+) and FORKO (3/3) female mice under basal conditions as detected by western blotting. The blots shown are representative of four independent experiments in which n = 3 animals per group. (B) Expression of non-phosphorylated p46 JNK in identical preparations of the cortex, hippocampus and striatum of wildtype (+/+) and FORKO (3/3) mice under basal conditions. Phosphorylated JNK was not detected by western blotting. The blots presented are typical of four independent experiments in which n = 3 animals per group. (C) Expression of phosphorylated and non-phosphorylated p42/44 ERK in the DRG and SN of wild-type (+/+) and FORKO (3/3) female mice as detected by western blotting. The blots shown on the right are representative of three independent experiments in which n = 3 animals per group, and densitometric analysis of immunoreactive bands is presented on the left. Each column represents a ratio of the mean intensities of immunoreactive bands for phosphoryalted ERK1=2 versus non-phosphorylated ERK1=2 for each group. Error bars indicate group S.E.M. An asterisk (*) denotes signi¢cant change from wild-type conditions. Di¡erences were considered signi¢cant where P 6 0.05. (D) Expression of phosphorylated p46/54 JNK and of non-phosphorylated p46 JNK in identical preparations of the DRG and SNs. Densitometric analysis of phosphorylated JNKs is shown on the left. The blots shown on the right are representative of three independent experiments in which n = 3 animals per group, and densitometric analysis of immunoreactive bands is presented on the left. Each column represents a ratio of the mean intensities of immunoreactive bands for phosphorylated JNK1=2 versus non-phosphorylated JNK1 for each group. Error bars indicate group S.E.M. An asterisk (*) denotes signi¢cant change from wild-type conditions. Di¡erences were considered signi¢cant where P 6 0.05. (E) Phosphorylation of ERK1=2 in DRG from both FORKO (3/3) and wild-type (+/+) female mice is responsive to E2 treatment. The columns represent the relative ratios of the mean intensities of immunoreactive bands for phosphorylated versus non-phosphorylated ERK1=2 in non-treated and E2-treated DRG. In vitro E2 treatment (10 nM; 30 min) of DRG from wild-type mice produced an increase in p42/p44 ERK phosphorylation relative to non-treated DRG. E2 treatment also enhanced the phosphorylation of ERK1/2 in DRG from FORKO mice. Data represent group means D S.E.M. of three independent experiments in which n = 3 animals per group. An asterisk (*) denotes signi¢cant change from wild-type conditions or from non-treated conditions. Di¡erences were considered signi¢cant where P 6 0.05.

revealed no di¡erences in the expression levels of this kinase between FORKO and wild-type tissue (Fig. 3B, P s 0.05, n = 4). In the PNS, DRG tissues from FORKO mice contained greater amounts of constitutive p42/p44 ERK phosphorylation (Fig. 3C) (ROD: 0.99 D 0.10) relative to wild-type (ROD: 0.38 D 0.03) (1.5-fold increase, P 6 0.05, n = 3). Each column represents a ratio of the mean intensity of immunoreactive bands for phosphorylated ERKs versus non-phosphorylated ERKs for each group as determined by densitometry. The ratios of

phosphorylated and non-phosphorylated ERK1=2 phosphorylation in the SNs were not signi¢cantly di¡erent between FORKO and wild-type animals (P s 0.05, n = 3). Immunoblotting with an antibody directed against ERK1 (that partially recognizes ERK2 ) indicated that p42/44 ERK expression was not a¡ected in the DRG or SNs of FORKO mice (Fig. 3C, P s 0.05, n = 3). p46/54 JNK phosphorylation was elevated in DRG samples from FORKO mice (Fig. 3D) (ROD: 0.17 D 0.02) compared to wild-type (ROD: 0.07 D 0.02) (2.4-fold, P 6 0.05, n = 3). Each column represents a

NSC 5719 15-8-02

498

J. Tam et al.

Fig. 3 (Continued).

NSC 5719 15-8-02

Neurodegenerative changes in FORKO mice

499

Fig. 4. NFs in FORKO mice are abnormally phosphorylated. (A) Expression of phosphorylated NF-H and NF-M subunits in the cortex, hippocampus and striatum of wild-type (+/+) and FORKO (3/3) female mice under basal conditions as detected by western blotting. The blots shown are representative of four independent experiments in which n = 3 animals per group. (B) Expression of GAP-43 in identical preparations of the cortex, hippocampus and striatum of wild-type (+/+) and FORKO (3/3) mice as detected by western blotting. The blots presented are typical for four independent experiments in which n = 3 animals per group. (C) Expression of phosphorylation of NF-H and NF-M in the DRG and SN of wild-type (+/+) and FORKO (3/3) female mice under basal conditions as detected by western blotting. The blots shown on the right are representative of three independent experiments in which n = 3 animals per group, and densitometric analysis of immunoreactive bands is presented on the left. Each column represents a ratio of the mean intensities of immunoreactive bands for phosphorylated NFH=M versus non-phosphorylated NFH=M for each group. Error bars indicate group S.E.M. An asterisk (*) denotes signi¢cant change from wild-type conditions. Di¡erences were considered signi¢cant where P 6 0.05. (D) Expression of GAP-43 in identical preparations of the DRG as detected by western blotting. The blots shown are typical for three individual experiments in which n = 3 animals per group.

ratio of phosphorylated JNK1=2 versus non-phosphorylated JNK1 for each group as determined by densitometry of the mean intensity of immunoreactive bands. In contrast, there was no signi¢cant di¡erence in the signal intensity for phosphorylated JNK1=2 in the SNs between wild-type and FORKO mice (P s 0.05, n = 3). Similarly, an antibody recognizing JNK1 did not reveal a signi¢cant di¡erence in the intensity of p46 JNK immunoreactive bands in the PNS structures of FORKO and wild-

type mice (P s 0.05, n = 3). However, the expression of JNK1 was signi¢cantly lower in the SNs as compared to the DRGs in either FORKO or wild-type mice (Fig. 3B). The ratios of relative mean intensities of immunoreactive bands for phosphorylated versus non-phosphorylated ERK1=2 in non-treated and E2-treated DRG are shown (Fig. 3E). In vitro E2 treatment (10 nM; 30 min) of DRG from wild-type mice produced an increase in p42/p44 ERK phosphorylation relative to non-treated

NSC 5719 15-8-02

500

J. Tam et al.

Fig. 5. Neurite outgrowth from sensory ganglia of FORKO mice is impaired. (A^D) DRG explants from wild-type (+/+) and FORKO (3/3) female mice were cultured in Matrigel and RPMI 1640 media (not containing Phenol Red) for 7 days (37‡C, 5% CO2 ) in (A, B) the absence or (C, D) the presence of 10 nM E2. Magni¢cation presented here was obtained with a 10U objective, and images were stored in TIFF for quantitative analysis. Note that the E2-stimulated neurite outgrowth in DRG explants from wild-type mice extends beyond the image border. Scale bar = 500 Wm. (E) Densitometric analysis of total neuritic area was performed as described in the Experimental procedures. Images were inverted using Adobe Photoshop 5.0 software to highlight the neurite processes, but otherwise unaltered digitally. Each column represents the mean ROD of the total neuritic area for each group. Error bars indicate group S.E.M. The results are typical for three paired preparations. An asterisk (*) denotes signi¢cant di¡erence from the wild-type condition or from the non-treated condition. Di¡erences were considered signi¢cant where P 6 0.05.

NSC 5719 15-8-02

Neurodegenerative changes in FORKO mice

DRG (Fig. 3E) (ROD ^ no treatment: 0.38 D 0.03; E2: 0.55 D 0.07) (0.4-fold, P 6 0.05, n = 3). The aberrantly phosphorylated ERK in DRG from FORKO mice was also enhanced by the same in vitro E2 treatment (Fig. 3E) (ROD ^ no treatment: 0.99 D 0.10; E2: 1.28 D 0.15) (0.3fold, P 6 0.05, n = 3). However, due to the signi¢cant increase in constitutive ERK phosphorylation in DRG from FORKO mice, the ROD of phosphorylated ERK immunoreactive bands was signi¢cantly higher in the DRG from E2-treated FORKO mice (ROD: 1.28 D 0.15) relative to E2-treated wild-type mice (ROD: 0.55 D 0.07) (1.3-fold, P 6 0.05, n = 3). In vitro E2 treatment did not signi¢cantly stimulate phosphorylation of JNKs in either the DRG or SNs of FORKO or wild-type tissues compared to the respective non-treated conditions (P s 0.05, n = 3, data not shown). NFs in FORKO mice are abnormally phosphorylated NF expression and phosphorylation were examined in the cortex, hippocampus and striatum (Fig. 4A). Densitometric analysis of immunoreactive bands for the heavy NF subunit (NF-H) was performed to give ratios between the phosphorylated and non-phosphorylated forms. Constitutive phosphorylation of NF-H was greater (P 6 0.05, n = 4) in the hippocampus of FORKO mice relative to wild-type (Fig. 4A). No signi¢cant di¡erences were detected in cortical samples (P s 0.05, n = 4). However, in the striatum the intensity of phosphorylated NF-H immunoreactive bands consistently showed a trend towards a decrease in FORKO mice compared to those in wild-type (Fig. 4A, P s 0.05, n = 4). The expression of non-phosphorylated NFs was not signi¢cantly di¡erent between the two groups of mice in these structures (data not shown). Similarly, the expression of another structural protein, GAP-43 (Fig. 4B), was not di¡erent in the CNS of FORKO and wild-type mice (P s 0.05, n = 4). In the PNS, an antibody that recognizes NF-H and to a lesser extent medium NF subunit (NF-M) revealed extensive constitutive NF phosphorylation in the DRG of FORKO mice (Fig. 4C) (ROD: 0.78 D 0.04) relative to wild-type (ROD: 0.31 D 0.07) (1.5-fold, P 6 0.05, n = 3). In the SNs, no di¡erences in phosphorylated NF levels were detected (P s 0.05, n = 3). In contrast to the dramatic increase in NF phosphorylation in the DRG, the expression levels of NFs were not signi¢cantly changed (P s 0.05, n = 3, data not shown). Similarly, the expression of GAP-43 protein (Fig. 4D) was not signi¢cantly di¡erent in the PNS tissues of FORKO and wild-type mice (P s 0.05, n = 3). However, the intensities of the immunoreactive bands of GAP-43 in the SNs were much stronger than those in the DRG. Neurite outgrowth from sensory ganglia of FORKO mice is impaired Neurite outgrowth from paired groups of L3 and L4 DRGs from wild-type and FORKO mice was assessed during 7 days in culture in the presence and absence of E2 in the culture medium. The image analysis method

501

used to measure total neuritic area (a combination of measure of neurite length and density) is based on the method described by Bilsland et al. (1999). Within the ¢rst 48 h of culturing, no di¡erences were observed between the wild-type and FORKO explants, with a hardly noticeable outgrowth in either culture. However, during the course of 1 week the extent of the neurite outgrowth in FORKO animals was less prominent than in wild-type mice. The ¢nal quanti¢cation (Fig. 5E) shows the signi¢cant de¢cits in neurite outgrowth (P 6 0.05, n = 3) in FORKO DRG explants under non-treated and E2-treated conditions. After 7 days in culture without E2, the total neuritic area was reduced in FORKO preparations (Fig. 5B) (ROD = 7.19 D 0.42) relative to wild-type (Fig. 5A) (ROD = 8.51 D 1.11), approximately by 16% (Fig. 5E, P 6 0.05, n = 3). Both DRGs from FORKO and wildtype mice responded to the treatment with E2 (Fig. 5C^ E). Treatment with 10 nM E2 strongly stimulated outgrowth from wild-type DRG (Fig. 5C) (ROD = 11.19 D 0.66) compared to the non-treated wild-type samples (Fig. 5A) (ROD = 8.51 D 1.11); the increase in neurite outgrowth was 32% (Fig. 5E, P 6 0.05, n = 3). Outgrowth from E2-treated FORKO DRG (Fig. 5D) (ROD = 6.36 D 1.64) was smaller compared to the E2-treated wild-type DRGs (Fig. 5C) (ROD = 11.19 D 0.66). The decreased responsiveness of FORKO DRG to E2 treatment was revealed by a 43% reduction in neurite length and density (Fig. 5E, P 6 0.05, n = 3). Addition of PD98059, a MAPK kinase (MEK) inhibitor, in the culture medium partially abolished the E2-induced neurite outgrowth from both FORKO and wild-type DRG (P 6 0.05, n = 3, data not shown).

DISCUSSION

Results from these studies provide the ¢rst information concerning the e¡ects of genetic depletion of circulating estrogens on the CNS and PNS of female FORKO mice in an in vivo setting. The FORKO model of estrogen de¢ciency is characterized by defects of the reproductive system, adiposity and abnormalities of bone structure. Quite remarkably phenotypic changes in the non-reproductive tissues that we have previously examined occurred early in life and intensi¢ed with aging despite the presence of elevated levels of circulating testosterone (Fig. 1). Such persistence of typical estrogen de¢ciency features suggests that local conversion of testosterone to estrogen at peripheral sites (e.g. ovaries and adipocytes) is minimal and the presence of high circulating testosterone levels is unable to substitute for the de¢cient estrogen (Danilovich et al., 2000) (Sairam et al., unpublished observations). However, these aspects including the status of ERs expression and functional status of aromatase in discrete regions of the brain remain to be explored in FORKO mice. Previous studies that mainly employed mature ovariectomized female rodents to study the e¡ects of estrogen de¢ciency (Gibbs et al., 1994) concur with the data presented here (Kaufman et al., 1988). An attractive feature of

NSC 5719 15-8-02

502

J. Tam et al.

FORKO mice is that they can be used in developmental studies to follow the progression of neurodegenerative changes without resorting to surgical manipulations. An additional experimental advantage lies in the development of age-dependent changes in ovarian/estrogen status induced by haploinsu⁄ciency of the FSH-R (Danilovich et al., unpublished observations). In the current study, we identify biochemical (ChAT, MAPKs, NFs) and functional (neurite outgrowth) changes in 3-month-old female FORKO mice that are consistent with a neurodegenerative state. ChAT enzymatic activity, a marker of the functional status of cholinergic neurons (Oda, 1999), is signi¢cantly reduced in the CNS structures examined. This reduction points to a general decline in the function of the central cholinergic system that is implicated in several neurodegenerative conditions (e.g. aging, Alzheimer’s disease) (Gibbs et al., 1994; Gibbs, 1994, 1998; McMillan et al., 1996). The cholinergic impairment in FORKO mice would predict a decline in memory and/or cognitive functions, and behavioral studies to verify this hypothesis are currently underway in our laboratories. Selective e¡ects of genetic estrogen depletion on central cholinergic activity The most signi¢cant ChAT activity decreases were in the striatum with lesser e¡ects in the hippocampus and cortex (Fig. 2). The cholinergic input to the neocortex originates largely from the nucleus basalis magnocellularis. The hippocampus receives cholinergic projections from the medial septal neurons and the nucleus of the vertical limbs. In contrast, the striatum contains intrinsic cholinergic neurons that may be more sensitive to E2 de¢ciency, resulting in degenerative changes or even loss of cell bodies in this structure. Recent studies by Wang et al. (2001) showed morphological abnormalities in the brains of ER-L knockout mice in the CNS. In these mice, a marked hypocellularity was seen in the cortex and a signi¢cant proliferation of astroglia was observed in the limbic system. Degeneration of the neuronal cell bodies was particularly evident in the substantia nigra. In our studies, we did not assess the number of neurons and glia, but it is conceivable that these cells may be di¡erentially a¡ected in the examined structures in FORKO mice. Our ¢ndings suggest that estrogen loss in FORKO mice preferentially a¡ects speci¢c cholinergic regions of the brain rather than causing a uniform and non-speci¢c decline of ChAT enzymatic activity across the cholinergic system. Whether these changes intensify with aging to produce functional disability in the mutants is an interesting and important question that remains to be explored. Additional studies will determine whether signi¢cant ChAT decreases occur in other structures (cerebellum, brainstem) from older FORKO mice or if these structures are less susceptible to genetic estrogen depletion. ERK activation in the hippocampus in FORKO mice The tyrosine kinase and MAPK pathways mediate

multiple e¡ects of estrogen in the hippocampus (Bi et al., 2001). Estrogens also activate ERKs in the cerebral cortex (Singh et al., 2000). By employing di¡erent inhibitors, those authors showed that ICI-insensitive and E2-sensitive ERs may mediate E2-induced activation of ERK in the brain. The most striking ¢nding in the present study is the signi¢cant increase in p42/44 ERK phosphorylation in the hippocampus of FORKO mice. Several possible causes could lead to the abnormality in the hippocampus, including a disturbance in trophic factor and membrane bound ERs. Estrogen provides trophic support to cholinergic neurons (Gibbs, 1998; McMillan et al., 1996), and several studies demonstrate co-localization of ERs and ChAT (Blurton-Jones et al., 1999; Mufson et al., 1999). ERs also co-localize with NGF receptors in the CNS (Blurton-Jones et al., 1999; Miranda et al., 1993) and modulate NGF receptors in sensory ganglia (Sohrabji et al., 1994). Since our previous work shows that ER levels in selected tissues examined are not a¡ected in FORKO mice (Danilovich et al., 2000), the ¢ndings reported here suggest a possible defective neurotrophin receptor (e.g. TrkA/p75) function in the CNS of the knockout animals (J.T., D.M. and M.R.S., unpublished observations). However, it is also conceivable that in spite of the presence of normally expressed ERs, fast non-genomic signaling pathways activated by E2 may be impaired (Hall et al., 2001). Our results do not indicate whether the hippocampal ERK activation represents a defensive mechanism against unavailable circulating estrogen or if it results from defective TrkA/p75 signal transduction. Investigations similar to those by Toran-Allerand (1996, 1999) of the TrkA/p75/ERK signaling would enhance our understanding of the biochemical basis of neurodegeneration exacerbated by chronic estrogen de¢ciency in the FORKO model. Hyper-phosphorylation of MAPKs and NFs in sensory cell bodies of FORKO mice The sensory ganglia of FORKO mice contain increased levels of constitutive p42/p44 ERK phosphorylation. Our study also reveals dramatic hyper-phosphorylation of NF-H/NF-M in the DRG of FORKO mice. NF proteins that comprise the major structural proteins in the peripheral nerves are targets of both ERK kinases (Li et al., 1999), although other kinases that phosphorylate NFs (Grant et al., 2001; Sihag et al., 1999; Nakamura et al., 1999, 2000) could also be disregulated (Namgung and Xia, 2000; Veeranna Amin et al., 1998; Yang et al., 1997). Thus, the phosphorylation state of NFs in FORKO mice provides a partial measure of the downstream consequences of defective MAPK^NF signaling. Our ¢ndings suggest that genetic estrogen depletion in the circulation a¡ects the peripheral nerves at the structural level of NFs via up-regulation of the ERK signaling cascades. In addition, the large amount of accumulated phospho-NFs in the DRG points to a sustained increase in p42/p44 ERK activity rather than an acute increase that would allow cellular phosphatases to restore the balance between phosphorylated and non-

NSC 5719 15-8-02

Neurodegenerative changes in FORKO mice

phosphorylated NFs. Further studies are required to determine which phosphatases might be disregulated in FORKO mice. Similar to the DRG, extensive phosphorylation of NF-H/NF-M was detected in the hippocampus of FORKO mice. These results point towards abnormal NF phosphorylation as a common denominator of neurodegenerative changes in both the CNS and PNS. However, in addition to NFs, other structural proteins (e.g. actin) and the microtubule-associated protein tau are implicated in a number of neurodegenerative disorders (Choudhary et al., 2001; del Alonso et al., 2001; Garcia and Cleveland, 2001) and may also be a¡ected in FORKO mice. Testosterone prevents the hyper-phosphorylation of tau (Papasozomenos, 1997) that is abnormally phosphorylated in Alzheimer’s disease. Testosterone is a substrate for aromatase, the biosynthetic enzyme that converts the steroid into E2 (Simpson and Davis, 2001). Although aromatase expression is increased in the brain after injury (Azcoitia et al., 2001), it appears that locally expressed aromatase in FORKO mice cannot utilize the excess circulating testosterone and compensate for E2 de¢ciency. Furthermore, it does not appear that excess circulating testosterone (Fig. 1) can reduce the hyperphosphorylation of NFs in the hippocampus and DRG of FORKO mice (Fig. 4A, C). The aromatase activity remains to be determined in these tissues and other brain structures as well as in the PNS in wild-type and FORKO mice. Assessment of JNK activity in the PNS (Giasson and Mushynski, 1996, 1997) suggests that the 46- and 54-kDa isoforms do contribute signi¢cantly to the NF hyperphosphorylation in sensory ganglia of FORKO mice. This result is in agreement with the ¢ndings in sensory neuropathy that implicate increased JNK activity as contributing to the neurodegenerative changes (Fernyhough et al., 1999; Purves et al., 2001). Implications of NF hyper-phosphorylation The phosphorylation state of the three NF subunits (NF-H, NF-M, light NF subunit (NF-L)) in£uences their association with each other (Gotow and Tanaka, 1994; Yabe et al., 2001) and with other intermediate ¢lament proteins (Athlan et al., 1997; Beaulieu et al., 1999, 2000) to form stable, functional nerves. Abnormal NF phosphorylation in FORKO mice may lead to reduced axonal caliber (Ho¡man et al., 1987; Xu et al., 1996) and subsequent slowing of axonal transport (Marszalek et al., 1996; Yabe et al., 2001). We observed accumulated phospho-NFs in the peripheral neurons but not in the axons of the SNs of FORKO mice. However, since phosphorylated NFs are normally transported anterogradely from the DRG through the axon hillock into and along axons (Yabe et al., 2001), our ¢ndings suggest that axonal transport might be impaired in the peripheral nerves of FORKO mice. If this hypothesis is true, phospho-NF levels in the SNs should not reach those of the DRG over an extended time period. Further studies will show if the axonal structure,

503

transport and other functions are impaired in FORKO mice. A recent report describes up-regulation of NF-L expression associated with cellular plasticity in the CNS (Hashimoto et al., 2000) and in ovariectomy (Vaucher et al., 2001). However, overexpression of NF-L does not appear to a¡ect PNS features such as axonal caliber (Monteiro et al., 1990). Functional de¢ciency in peripheral nerves of FORKO mice The functional assessment of the sensory ganglia reveals a reduced capacity for neurite outgrowth in the DRG from FORKO mice. Constitutive neurite outgrowth in explanted ganglia exhibits a combination of decreased length as well as altered neuritic density relative to wild-type cultures (16% reduction, P 6 0.05, Fig. 4AB). Estrogen enhances neurite elongation in primary neuronal cultures (Diaz et al., 1992) partly by e¡ects on structural proteins (Ferreira and Caceres, 1991). Thus, the genetic estrogen depletion in FORKO mice may lead to reduced neurite outgrowth via e¡ects on the neuronal cytoskeleton. To our knowledge, no studies have addressed the e¡ects of estrogen de¢ciency on nerve structural proteins in an in vivo setting. GAP-43 is a phosphoprotein expressed almost exclusively in neurons and is involved in neurite extension in the CNS (Mahalik et al., 1992) and the sensory ganglia (Van der Zee et al., 1989). Thus, it is conceivable that GAP-43 in addition to NFs is a cytoskeletal component involved in the neurodegenerative changes that result from genetic estrogen de¢ciency. Our study however (Fig. 4B, D) shows no di¡erences in the expression of these key structural proteins in the structures examined in the CNS or PNS of FORKO mice compared to wild-type. The localization of GAP-43 and NFs and the ratios between phosphorylated and non-phosphorylated isoforms along the lengths of the peripheral nerves could be di¡erent, and this remains to be studied. Retarded neurite outgrowth in FORKO mice may be due to abnormalities in the expression or internalization of trophic factor receptors (trk and p75) that are modulated by estrogens (Lanlua et al., 2001; Zhang et al., 2000). Reduced responsiveness of explanted ganglia to estrogen Neurite extension in the DRG from FORKO mice exposed to E2 in vitro also exhibits reduced responsiveness to the treatment. The ERKs are implicated in the process of trophic factor-stimulated neurite outgrowth in mouse DRG (Sjogreen et al., 2000) as well as in neuronal cell lines (Perron and Bixby, 1999). Thus, the decreased neurite outgrowth (total neuritic area) suggests a defective signaling pathway involving MAPK phosphorylation in the neurite outgrowth process in FORKO mice. PD98059 is an inhibitor of MEK1, the direct upstream activating kinase of the ERKs (Alessi et al., 1995), and may also inhibit JNK1 activity (Salh et al., 2000). PD98059 and UO126 were previously used to delineate the e¡ects of constitutive versus growth factor-stimulated ERK activity on neurite outgrowth from sensory ganglia

NSC 5719 15-8-02

504

J. Tam et al.

(Singh et al., 2000; Singh, 2001; Sjogreen et al., 2000). Our data utilizing PD98059 treatment show a partial inhibition of neurite outgrowth induced by E2, supporting the earlier ¢ndings of Singh (2001). However, non-ERKmediated signal transduction is also essential for neurite outgrowth (Levkovitz et al., 2001) and whether or not these pathways are a¡ected in FORKO mice remains to be established.

CONCLUSION

We have provided the ¢rst evidence of several alterations in biochemical targets implicated in neurogenerative changes that result from genetic depletion of circulating estrogens in an in vivo setting. The FORKO mouse model of chronic estrogen de¢ciency could serve as a useful tool to further investigate biochemical, functional and morphological changes that may be involved

in neurodegenerative processes associated with a decline in estrogen functions (Dubal and Wise, 2001; Henderson, 1997). Based on the results of this study, we believe that this model of hypoestrogenicity is well suited for the investigation of the dynamics of biochemical markers and morphological variables in the course of the development and progression of CNS and PNS degeneration in aging. Although testosterone remains elevated in these mice, we believe that it is unlikely to have contributed to the changes, either directly or indirectly, because the androgen is apparently unable to sustain biochemical parameters or gene expression of certain markers during neural remodeling in the gonadectomized rat (Monks et al., 2001).

Acknowledgements0The authors acknowledge the ¢nancial support of CIHR Canada (D.M. and M.R.S.).

REFERENCES

Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T., Saltiel, A.R., 1995. PD 098059 is a speci¢c inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489^27494. Athlan, E.S., Sacher, M.G., Mushynski, W.E., 1997. Associations between intermediate ¢lament proteins expressed in cultured dorsal root ganglion neurons. J. Neurosci. Res. 47, 300^310. Azcoitia, I., Sierra, A., Veiga, S., Honda, S., Harada, N., Garcia-Segura, L.M., 2001. Brain aromatase is neuroprotective. J. Neurobiol. 47, 318^ 329. Beaulieu, J.M., Jacomy, H., Julien, J.P., 2000. Formation of intermediate ¢lament protein aggregates with disparate e¡ects in two transgenic mouse models lacking the neuro¢lament light subunit. J. Neurosci. 20, 5321^5328. Beaulieu, J.M., Robertson, J., Julien, J.P., 1999. Interactions between peripherin and neuro¢laments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits. Biochem. Cell Biol. 77, 41^45. Benten, W.P., Stephan, C., Lieberherr, M., Wunderlich, F., 2001. Estradiol signaling via sequestrable surface receptors. Endocrinology 142, 1669^ 1677. Beyer, C., 1999. Estrogen and the developing mammalian brain. Anat. Embryol. (Berl.) 199, 379^390. Bi, R., Foy, M.R., Vouimba, R.M., Thompson, R.F., Baudry, M., 2001. Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway. Proc. Natl. Acad. Sci. USA 98, 13391^13395. Bilsland, J., Rigby, M., Young, L., Harper, S., 1999. A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis. J. Neurosci. Methods 92, 75^85. Blurton-Jones, M.M., Roberts, J.A., Tuszynski, M.H., 1999. Estrogen receptor immunoreactivity in the adult primate brain: neuronal distribution and association with p75, trkA, and choline acetyltransferase. J. Comp. Neurol. 405, 529^542. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein^dye binding. Anal. Biochem. 72, 248^254. Choudhary, S., Joshi, K., Gill, K.D., 2001. Possible role of enhanced microtubule phosphorylation in dichlorous induced delayed neurotoxicity in rat. Brain Res. 897, 60^70. Cowley, S.M., Parker, M.G., 1999. A comparison of transcriptional activation by ER alpha and ER beta. J. Steroid Biochem. Mol. Biol. 69, 165^ 175. Creedon, D.J., Johnson, E.M., Lawrence, J.C., 1996. Mitogen-activated protein kinase-independent pathways mediate the e¡ects of nerve growth factor and cAMP on neuronal survival. J. Biol. Chem. 271, 20713^20718. Cuello, A.C., Carson, S., 1983. Microdissection of fresh rat brain tissue slices. In: Cuello, A.C. (Ed.), Brain Microdissection Techniques. Wiley, New York, pp. 37^125. Danilovich, N., Babu, P.S., Xing, W., Gerdes, M., Krishnamurthy, H., Sairam, M.R., 2000. Estrogen de¢ciency, obesity, and skeletal abnormalities in follicle-stimulating hormone receptor knockout (FORKO) female mice. Endocrinology 141, 4295^4308. Davis, R.J., 2000. Signal transduction by the JNK group of MAP kinases. Cell 103, 239^252. del Alonso, C.A., Zaidi, T., Novak, M., Barra, H.S., Grundke-Iqbal, I., Iqbal, K., 2001. Interaction of tau isoforms with Alzheimer’s disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein. J. Biol. Chem. 276, 37967^37973. Diaz, H., Lorenzo, A., Carrer, H.F., Caceres, A., 1992. Time lapse study of neurite growth in hypothalamic dissociated neurons in culture: sex di¡erences and estrogen e¡ects. J. Neurosci. Res. 33, 266^281. Dierich, A., Sairam, M.R., Monaco, L., Fimia, G.M., Gansmuller, A., LeMeur, M., Sassone-Corsi, P., 1998. Impairing follicle-stimulating hormone (FSH) signaling in vivo : targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc. Natl. Acad. Sci. USA 95, 13612^13617. Dina, O.A., Aley, K.O., Isenberg, W., Messing, R.O., Levine, J.D., 2001. Sex hormones regulate the contribution of PKCepsilon and PKA signalling in in£ammatory pain in the rat. Eur. J. Neurosci. 13, 2227^2233. Dluzen, D.E., McDermott, J.L., Liu, B., 1996. Estrogen as a neuroprotectant against MPTP-induced neurotoxicity in C57/B1 mice. Neurotoxicol. Teratol. 18, 603^606. Dubal, D.B., Wise, P.M., 2001. Neuroprotective e¡ects of estradiol in middle-aged female rats. Endocrinology 142, 43^48. Eilers, A., Whit¢eld, J., Shah, B., Spadoni, C., Desmond, H., Ham, J., 2001. Direct inhibition of c-Jun N-terminal kinase in sympathetic neurones prevents c-jun promoter activation and NGF withdrawal-induced death. J. Neurochem. 76, 1439^1454.

NSC 5719 15-8-02

Neurodegenerative changes in FORKO mice

505

Fernyhough, P., Gallagher, A., Averill, S.A., Priestley, J.V., Hounsom, L., Patel, J., Tomlinson, D.R., 1999. Aberrant neuro¢lament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 48, 881^889. Ferreira, A., Caceres, A., 1991. Estrogen-enhanced neurite growth: evidence for a selective induction of Tau and stable microtubules. J. Neurosci. 11, 392^400. Fonnum, F., 1975. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24, 407^409. Fukunaga, K., Miyamoto, E., 1998. Role of MAP kinase in neurons. Mol. Neurobiol. 16, 79^95. Garcia-Segura, L.M., Azcoitia, I., DonCarlos, L.L., 2001. Neuroprotection by estradiol. Prog. Neurobiol. 63, 29^60. Garcia, M.L., Cleveland, D.W., 2001. Going new places using an old MAP : tau, microtubules and human neurodegenerative disease. Curr. Opin. Cell. Biol. 13, 41^48. Giasson, B.I., Mushynski, W.E., 1996. Aberrant stress-induced phosphorylation of perikaryal neuro¢laments. J. Biol. Chem. 271, 30404^30409. Giasson, B.I., Mushynski, W.E., 1997. Study of proline-directed protein kinases involved in phosphorylation of the heavy neuro¢lament subunit. J. Neurosci. 17, 9466^9472. Gibbs, R.B., 1994. Estrogen and nerve growth factor-related systems in brain. E¡ects on basal forebrain cholinergic neurons and implications for learning and memory processes and aging. Ann. N.Y. Acad. Sci. 743, 165^196. Gibbs, R.B., 1998. Impairment of basal forebrain cholinergic neurons associated with aging and long-term loss of ovarian function. Exp. Neurol. 151, 289^302. Gibbs, R.B., Wu, D., Hersh, L.B., Pfa¡, D.W., 1994. E¡ects of estrogen replacement on the relative levels of choline acetyltransferase, trkA, and nerve growth factor messenger RNAs in the basal forebrain and hippocampal formation of adult rats. Exp. Neurol. 129, 70^80. Glowinski, J., Iversen, L.L., 1966. Regional studies of catecholamines in the rat brain. I. The disposition of [3 H]norepinephrine, [3 H]dopamine and [3 H]dopa in various regions of the brain. J. Neurochem. 13, 655^669. Gotow, T., Tanaka, J., 1994. Phosphorylation of neuro¢lament H subunit as related to arrangement of neuro¢laments. J. Neurosci. Res. 37, 691^ 713. Grant, P., Sharma, P., Pant, H.C., 2001. Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neuro¢lament metabolism. Eur. J. Biochem. 268, 1534^1546. Hall, J.M., Couse, J.F., Korach, K.S., 2001. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 276, 36869^ 36872. Hansen, T.O., Rehfeld, J.F., Nielsen, F.C., 2000. Cyclic AMP-induced neuronal di¡erentiation via activation of p38 mitogen-activated protein kinase. J. Neurochem. 75, 1870^1877. Hashimoto, R., Nakamura, Y., Komai, S., Kashiwagi, Y., Matsumoto, N., Shiosaka, S., Takeda, M., 2000. Phosphorylation of neuro¢lament-L during LTD. NeuroReport 11, 2739^2742. Henderson, V.W., 1997. The epidemiology of estrogen replacement therapy and Alzheimer’s disease. Neurology 48, S27^S35. Hensley, K., Floyd, R.A., Zheng, N.Y., Nael, R., Robinson, K.A., Nguyen, X., Pye, Q.N., Stewart, C.A., Geddes, J., Markesbery, W.R., Patel, E., Johnson, G.V., Bing, G., 1999. p38 kinase is activated in the Alzheimer’s disease brain. J. Neurochem. 72, 2053^2058. Ho¡man, P.N., Cleveland, D.W., Gri⁄n, J.W., Landes, P.W., Cowan, N.J., Price, D.L., 1987. Neuro¢lament gene expression : a major determinant of axonal caliber. Proc. Natl. Acad. Sci. USA 84, 3472^3476. Kaufman, H., Vadasz, C., Lajtha, A., 1988. E¡ects of estradiol and dexamethasone on choline acetyltransferase activity in various rat brain regions. Brain Res. 453, 389^392. Kelly, M.J., Levin, E.R., 2001. Rapid actions of plasma membrane estrogen receptors. Trends Endocrinol. Metab. 12, 152^156. Lanlua, P., Decorti, F., Gangula, P.R., Chung, K., Taglialatela, G., Yallampalli, C., 2001. Female steroid hormones modulate receptors for nerve growth factor in rat dorsal root ganglia. Biol. Reprod. 64, 331^338. Levkovitz, Y., O’Donovan, K.J., Baraban, J.M., 2001. Blockade of NGF-induced neurite outgrowth by a dominant-negative inhibitor of the egr family of transcription regulatory factors. J. Neurosci. 21, 45^52. Li, B.S., Veeranna Gu, J., Grant, P., Pant, H.C., 1999. Activation of mitogen-activated protein kinases (Erk1 and Erk2) cascade results in phosphorylation of NF-M tail domains in transfected NIH 3T3 cells. Eur. J. Biochem. 262, 211^217. Li, R., 1998. Culture methods for selective growth of normal rat and human Schwann cells. Methods Cell Biol. 57, 167^186. Mahalik, T.J., Carrier, A., Owens, G.P., Clayton, G., 1992. The expression of GAP43 mRNA during the late embryonic and early postnatal development of the CNS of the rat: an in situ hybridization study. Brain Res. Dev. Brain Res. 67, 75^83. Manolagas, S.C., 1998. Cellular and molecular mechanisms of osteoporosis. Aging (Milan) 10, 182^190. Marszalek, J.R., Williamson, T.L., Lee, M.K., Xu, Z., Ho¡man, P.N., Becher, M.W., Crawford, T.O., Cleveland, D.W., 1996. Neuro¢lament subunit NF-H modulates axonal diameter by selectively slowing neuro¢lament transport. J. Cell Biol. 135, 711^724. Masaki, R., Saito, T., Yamada, K., Ohtani-Kaneko, R., 2000. Accumulation of phosphorylated neuro¢laments and increase in apoptosis-speci¢c protein and phosphorylated c-Jun induced by proteasome inhibitors. J. Neurosci. Res. 62, 75^83. Matthews, K.A., Meilahn, E., Kuller, L.H., Kelsey, S.F., Caggiula, A.W., Wing, R.R., 1989. Menopause and risk factors for coronary heart disease. N. Engl. J. Med. 321, 641^646. McMillan, P.J., Singer, C.A., Dorsa, D.M., 1996. The e¡ects of ovariectomy and estrogen replacement on trkA and choline acetyltransferase mRNA expression in the basal forebrain of the adult female Sprague^Dawley rat. J. Neurosci. 16, 1860^1865. Migheli, A., Piva, R., Atzori, C., Troost, D., Schi¡er, D., 1997. c-Jun, JNK/SAPK kinases and transcription factor NF-kappa B are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis. J. Neuropathol. Exp. Neurol. 56, 1314^1322. Miranda, R.C., Sohrabji, F., Toran-Allerand, C.D., 1993. Neuronal colocalization of mRNAs for neurotrophins and their receptors in the developing central nervous system suggests a potential for autocrine interactions. Proc. Natl. Acad. Sci. USA 90, 6439^6443. Monks, D.A., Getsios, S., MacCalman, C.D., Watson, N.V., 2001. N-cadherin is regulated by gonadal steroids in the adult hippocampus. Proc. Natl. Acad. Sci. USA 98, 1312^1316. Monteiro, M.J., Ho¡man, P.N., Gearhart, J.D., Cleveland, D.W., 1990. Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neuro¢lament density in axons without a¡ecting caliber. J. Cell Biol. 111, 1543^1557. Moosmann, B., Behl, C., 1999. The antioxidant neuroprotective e¡ects of estrogens and phenolic compounds are independent from their estrogenic properties. Proc. Natl. Acad. Sci. USA 96, 8867^8872. Mufson, E.J., Cai, W.J., Ja¡ar, S., Chen, E., Stebbins, G., Sendera, T., Kordower, J.H., 1999. Estrogen receptor immunoreactivity within subregions of the rat forebrain: neuronal distribution and association with perikarya containing choline acetyltransferase. Brain Res. 849, 253^274. Nakamura, Y., Hashimoto, R., Kashiwagi, Y., Aimoto, S., Fukusho, E., Matsumoto, N., Kudo, T., Takeda, M., 2000. Major phosphorylation site (Ser55) of neuro¢lament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture. J. Neurochem. 74, 949^959. Nakamura, Y., Hashimoto, R., Kashiwagi, Y., Wada, Y., Sakoda, S., Miyamae, Y., Kudo, T., Takeda, M., 1999. Casein kinase II is responsible for phosphorylation of NF-L at Ser-473. FEBS Lett. 455, 83^86.

NSC 5719 15-8-02

506

J. Tam et al.

Namgung, U., Xia, Z., 2000. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogenactivated protein kinase. J. Neurosci. 20, 6442^6451. Oda, Y., 1999. Choline acetyltransferase : the structure, distribution and pathologic changes in the central nervous system. Pathol. Int. 49, 921^937. Papasozomenos, S.C., 1997. The heat shock-induced hyperphosphorylation of tau is estrogen-independent and prevented by androgens: implications for Alzheimer disease. Proc. Natl. Acad. Sci. USA 94, 6612^6617. Patrone, C., Andersson, S., Korhonen, L., Lindholm, D., 1999. Estrogen receptor-dependent regulation of sensory neuron survival in developing dorsal root ganglion. Proc. Natl. Acad. Sci. USA 96, 10905^10910. Pearson, G., Robinson, F., Beers, G.T., Xu, B.E., Karandikar, M., Berman, K., Cobb, M.H., 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev. 22, 153^183. Pelligrino, D.A., Ye, S., Tan, F., Santizo, R.A., Feinstein, D.L., Wang, Q., 2000. Nitric-oxide-dependent pial arteriolar dilation in the female rat: e¡ects of chronic estrogen depletion and repletion. Biochem. Biophys. Res. Commun. 269, 165^171. Perron, J.C., Bixby, J.L., 1999. Distinct neurite outgrowth signaling pathways converge on ERK activation. Mol. Cell. Neurosci. 13, 362^378. Purves, T., Middlemas, A., Agthong, S., Jude, E.B., Boulton, A.J., Fernyhough, P., Tomlinson, D.R., 2001. A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB J. 15, 2508^2514. Salh, B.S., Martens, J., Hundal, R.S., Yoganathan, N., Charest, D., Mui, A., Gomez-Munoz, A., 2000. PD98059 attenuates hydrogen peroxideinduced cell death through inhibition of Jun N-terminal kinase in HT29 cells. Mol. Cell Biol. Res. Commun. 4, 158^165. Sihag, R.K., Ja¡e, H., Nixon, R.A., Rong, X., 1999. Serine-23 is a major protein kinase A phosphorylation site on the amino-terminal head domain of the middle molecular mass subunit of neuro¢lament proteins. J. Neurochem. 72, 491^499. Simpkins, J.W., Rajakumar, G., Zhang, Y.Q., Simpkins, C.E., Greenwald, D., Yu, C.J., Bodor, N., Day, A.L., 1997. Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. J. Neurosurg. 87, 724^730. Simpson, E.R., Davis, S.R., 2001. Minireview : aromatase and the regulation of estrogen biosynthesis^some new perspectives. Endocrinology 142, 4589^4594. Singh, M., 2001. Ovarian hormones elicit phosphorylation of Akt and extracellular-signal regulated kinase in explants of the cerebral cortex. Endocrine 14, 407^415. Singh, M., Setalo, G., Jr., Guan, X., Frail, D.E., Toran-Allerand, C.D., 2000. Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-alpha knock-out mice. J. Neurosci. 20, 1694^1700. Sjogreen, B., Wiklund, P., Ekstrom, P.A., 2000. Mitogen activated protein kinase inhibition by PD98059 blocks nerve growth factor stimulated axonal outgrowth from adult mouse dorsal root ganglia in vitro. Neuroscience 100, 407^416. Sohrabji, F., Miranda, R.C., Toran-Allerand, C.D., 1994. Estrogen di¡erentially regulates estrogen and nerve growth factor receptor mRNAs in adult sensory neurons. J. Neurosci. 14, 459^471. Tanzer, L., Jones, K.J., 1997. Gonadal steroid regulation of hamster facial nerve regeneration : e¡ects of dihydrotestosterone and estradiol. Exp. Neurol. 146, 258^264. Toran-Allerand, C.D., 1996. Mechanisms of estrogen action during neural development : mediation by interactions with the neurotrophins and their receptors ? J. Steroid Biochem. Mol. Biol. 56, 169^178. Toran-Allerand, C.D., Singh, M., Setalo, G., Jr., 1999. Novel mechanisms of estrogen action in the brain: new players in an old story. Front. Neuroendocrinol. 20, 97^121. Van der Zee, C.E., Nielander, H.B., Vos, J.P., Lopes, S., Verhaagen, J., Oestreicher, A.B., Schrama, L.H., Schotman, P., Gispen, W.H., 1989. Expression of growth-associated protein B-50 (GAP43) in dorsal root ganglia and sciatic nerve during regenerative sprouting. J. Neurosci. 9, 3505^3512. Vaucher, E., Pierret, P., Julien, J., Kuchel, G.A., 2001. Ovariectomy up-regulates neuronal neuro¢lament light chain mRNA expression with regional and temporal speci¢city. Neuroscience 103, 629^637. Veeranna Amin, N.D., Ahn, N.G., Ja¡e, H., Winters, C.A., Grant, P., Pant, H.C., 1998. Mitogen-activated protein kinases (Erk1, 2) phosphorylate Lys-Ser-Pro (KSP) repeats in neuro¢lament proteins NF-H and NF-M. J. Neurosci. 18, 4008^4021. Vegeto, E., Bonincontro, C., Pollio, G., Sala, A., Viappiani, S., Nardi, F., Brusadelli, A., Viviani, B., Ciana, P., Maggi, A., 2001. Estrogen prevents the lipopolysaccharide-induced in£ammatory response in microglia. J. Neurosci. 21, 1809^1818. Wang, L., Andersson, S., Warner, M., Gustafsson, J.A., 2001. Morphological abnormalities in the brains of estrogen receptor beta knockout mice. Proc. Natl. Acad. Sci. USA 98, 2792^2796. Watters, J.J., Campbell, J.S., Cunningham, M.J., Krebs, E.G., Dorsa, D.M., 1997. Rapid membrane e¡ects of steroids in neuroblastoma cells: e¡ects of estrogen on mitogen activated protein kinase signalling cascade and c-fos immediate early gene transcription. Endocrinology 138, 4030^4033. Wise, P.M., Dubal, D.B., Wilson, M.E., Rau, S.W., Bottner, M., 2001a. Minireview : neuroprotective e¡ects of estrogen^new insights into mechanisms of action. Endocrinology 142, 969^973. Wise, P.M., Dubal, D.B., Wilson, M.E., Rau, S.W., Liu, Y., 2001b. Estrogens : trophic and protective factors in the adult brain. Front. Neuroendocrinol. 22, 33^66. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., 1995. Opposing e¡ects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326^1331. Xu, Z., Marszalek, J.R., Lee, M.K., Wong, P.C., Folmer, J., Crawford, T.O., Hsieh, S.T., Gri⁄n, J.W., Cleveland, D.W., 1996. Subunit composition of neuro¢laments speci¢es axonal diameter. J. Cell. Biol. 133, 1061^1069. Yabe, J.T., Chylinski, T., Wang, F.S., Pimenta, A., Kattar, S.D., Linsley, M.D., Chan, W.K., Shea, T.B., 2001. Neuro¢laments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites. J. Neurosci. 21, 2195^2205. Yang, D.D., Kuan, C.Y., Whitmarsh, A.J., Rincon, M., Zheng, T.S., Davis, R.J., Rakic, P., Flavell, R.A., 1997. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 389, 865^870. Zhang, Y., Moheban, D.B., Conway, B.R., Bhattacharyya, A., Segal, R.A., 2000. Cell surface Trk receptors mediate NGF-induced survival while internalized receptors regulate NGF-induced di¡erentiation. J. Neurosci. 20, 5671^5678. Zhou, Y., Watters, J.J., Dorsa, D.M., 1996. Estrogen rapidly induces the phosphorylation of the cAMP response element binding protein in rat brain. Endocrinology 137, 2163^2166. (Accepted 19 February 2002)

NSC 5719 15-8-02