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Brain Research 887 (2000) 309–315www.elsevier.com/ locate /bres

Research report

Immunohistochemical study on the distribution of Bcl-2 and Bax inthe central nervous system of the transgenic mice expressing a human

Cu/Zn SOD mutationa a a d,eChung-Min Shin , Yoon Hee Chung , Myeung Ju Kim , Dong Hoon Shin ,b f c a ,*Yong Sik Kim , Mark E. Gurney , Kwang Woo Lee , Choong Ik Cha

aDepartment of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South KoreabDepartment of Pharmacology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea

cDepartment of Neurology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South KoreadNeuroscience Research Institute, Medical Research Center, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea

eDepartment of Anatomy, Dankook University College of Medicine, Chonan, South KoreafCNS Diseases Research Unit Pharmacia Upjohn, Inc., Kalamazoo, MI 49001, USA

Accepted 19 September 2000

Abstract

In the present study, we performed immunohistochemical studies to investigate the changes of Bcl-2 and Bax in the central nervoussystem of the transgenic mice expressing a human Cu/Zn SOD mutation. In contrast to the controls, a high density of Bcl-2-IR astrocyteswere detected all around the gray matter of the spinal cord of the mutant transgenic mice. Bcl-2-IR astrocytes were also detected in thecerebellum and brainstem of transgenic mice. Specific immunoreactivity for Bax was seen in the spinal cord and brainstem of transgenicmice. Immunostaining for Bax was identified only in neurons and not in glial cells. Our present study demonstrated the distribution ofBcl-2 and Bax in detail using immunohistochemical methods through the central nervous system of the transgenic mice, for the first time. 2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Neuromuscular diseases

Keywords: Amyotrophic lateral sclerosis; Transgenic mice; Astrocyte; Bcl-2; Bax

1. Introduction possibly related to a toxic gain in function of mutant SOD1(mSOD1) [10].

Amyotrophic lateral sclerosis (ALS) is a neurodegenera- Several lines of evidence suggest that neuronal death intive disease characterized by progressive muscle weakness, ALS is apoptosis [9,18,21,22]. Apoptosis or programmedatrophy and spasticity, which ultimately leads to paralysis cell death is likely to be an important mechanism of celland death within 3–5 years [12]. Ninety percent of ALS loss in neurodegenerative diseases. Explosive researchcases are sporadic with no identifiable genetic or en- during the last several years has led to the identification ofvironmental risk factors. The remaining 10% shows famili- apoptosis-associated molecules, including Bcl-2, Bax, p53al inheritance and one fourth of these are associated with and interleukin 1b-converting enzyme. In vitro expressionmissense mutations in the antioxidant enzyme copper /zinc of mSOD1 can induce neuronal apoptosis and abnor-superoxide dismutase (SOD1). This neurodegeneration is malities in the production of free radicals [9,22]. Because

motor neuron survival depends on trophic factors, abnor-malities in neurotrophic support may result in apoptoticdeath of motor neurons in ALS by inducing a programmedcell death mechanism involving the generation of reactive*Corresponding author. Tel.: 182-2-740-8205; fax: 182-2-745-9528.

E-mail address: [email protected] (C.I. Cha). oxygen species. This hypothesis is supported indirectly by

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )03014-6

310 C.-M. Shin et al. / Brain Research 887 (2000) 309 –315

experiments showing oxidative damage in CNS tissues of tal conditions. We selected five slides in each area of theALS subjects and abnormalities in mRNA levels coding transgenic (n55) and control mice (n55), and counted allfor Bcl-2 and Bax in spinal motor neurons in ALS [21]. immunoreactive cells in each corresponding area of the

Recently, several studies have shown that, aside from spinal cord and brain. Visual assessment and densitometricthe dramatic loss of motor neurons, gliosis is, as in human measurement using a NIH image program (Scion Image)ALS, a striking neuropathological feature of the spinal determined the density of immunoreactive cells.cord of an animal model of familial ALS (FALS) [2–4,11,12,24]. In the previous studies, we reported increasedexpression of NOS, nitrotyrosine and p53 in the astrocytes 3. Resultsin the brain of the mutant transgenic mice that are used asan animal model of FALS [2–4]. Troost et al. [26] In the spinal cord of the transgenic mice, immuno-reported Bcl-2 immunoreactivity in the astrocytes in the histochemistry showed intensely stained Bcl-2-immuno-brains of ALS patients. In the present study, we performed reactive (IR) glial cells with the appearance of astrocytesimmunohistochemical studies to investigate the changes of (Table 1), but no Bcl-2-IR glial cells were observed in theBcl-2 and Bax in the central nervous system of the spinal cord of the control mice (Fig. 1). The Bcl-2-IR glialtransgenic mice expressing a human SOD1 mutation. cells showing the morphology of astrocytes were con-

firmed as astrocytes by immunohistochemistry using an-tiserum directed against GFAP, which is the specific

2. Materials and methods marker for astrocytes (Fig. 2). Some GFAP-IR astrocyteswere observed in the spinal cord and brainstem of the

Five mutant transgenic and five control mice developed control mice, but these astrocytes did not show anyby Gurney et al. [10]were used for these experiments. immunoreactivity with the Bcl-2 antiserum. In contrast toThey were bred by The Jackson Laboratory (Bar Harbor, the controls, a high density of Bcl-2-IR astrocytes wereME) under the Strain designations B6SJL-TgN (SOD1- detected all around the gray matter of the spinal cord of theG93A) 1 Gur and B6SJL-TgN (SOD1) 2 Gur for mutant mutant transgenic mice. In the cervical, thoracic andtransgenic and control mice, respectively. All animals were lumbar segment, Bcl-2-IR astrocytes were distributedsacrificed at the age of 9 months, when clinical symptoms diffusely in the gray matter except in superficial layers.were manifested in the mutant transgenic mice. To avoid Bcl-2-IR astrocytes were also detected in the cerebellumsuffering during the experimental procedures, the authorsconformed to the Ethical Committee Guidelines for Lab-

Table 1oratory Animals.Distribution of Bcl-2 and Bax protein in the central nervous system of the

aThe mice were perfused transcardially with cold phos- transgenic micephate buffered saline (PBS, 0.02 M, pH 7.4), and then with

Area Subdivision Bcl-2 Baxice-cold 4% paraformaldehyde for 10 min at a flow rate of

Spinal cord Anterior horn 111 11150–60 ml /min. Brains were immediately removed andPosterior horn 11 1sliced into blocks 4–6 mm thick. Spinal cords were also

Cerebellum Molecular layer 1 2removed and sliced into cervical, thoracic and lumbarPurkinje cell layer 2 111segments of 3–10 mm in length. These blocks wereGranular layer 11 2

immersed in a cold fixative for 12 h and then cryoprotected Deep cerebellar nuclei 111 11in a series of cold sucrose solutions of increasing con-

Medulla Reticular formation 11 11centrations. Frozen sections were cut at 40 mm in theInferior olivary nucleus 11 2

coronal plane. Immunohistochemistry was performed in Hypoglossal nucleus 111 111accordance with the free floating method described earlier Medial vestibular nucleus 111 2

Spinal vestibular nucleus 11 2[2–4]. Polyclonal rabbit anti-rat /mouse Bcl-2 antibodyDorsal motor nucleus of vagus 111 111(PharmingenE, USA, Cat. No. 13456E, at a dilution of

1:1000), polyclonal rabbit anti-rat /mouse Bax antibody Pons Reticular formation 11 11

Trigeminal nucleus 11 11(PharmingenE, USA, Cat. No. 13686E at a dilution of 1:Facial nucleus 11 112000) and GFAP (SerotecE Cat. No. MCA 363A at a

Midbrain Periaqueductal gray matter 111 2dilution of 1:500) were used as primary antibodies.Reticular formation 11 2A sample of sections was reacted without primary

antiserum, and a different sample was exposed to primary Motor cortex 2 11

antiserum that had been preincubated for 24 h with Bcl-2, Hippocampus CA1 2 1Bax or GFAP. Sections from these samples did not exhibit CA2 2 1

CA3 2 111any of immunoreactivity described in this report. Sectionsafrom each transgenic and control mice were stained The density of Bcl-2 or Bax-immunoreactive cells was rated as 111,

together eliminating conflicts between different experimen- high; 11, moderate; 1, low; 2, no signal above background.

C.-M. Shin et al. / Brain Research 887 (2000) 309 –315 311

Fig. 2. Bcl-2-IR glial cells (A) showing the morphology of astrocytesFig. 1. Changes in Bcl-2-IR astrocytes in the spinal cord of SOD mutant were confirmed as astrocytes by immunohistochemistry in the adjacenttransgenic mice (A) compared with those of the control mice (B). In the sections using antiserum directed against GFAP (B), which is the specificspinal cord of the mutant transgenic mice, strong immunoreactivity for marker for astrocytes. Scale bar550 mm (A, B).Bcl-2 was observed, but no specific immunostaining was observed in thespinal cord of the control mice. Scale bar5100 mm (A, B).

nucleus, facial nucleus and pontine reticular formation. Inthe cerebellum, Bax-IR neurons were observed in the

and brainstem of transgenic mice (Table 1). In the Purkinje cell layer and cerebellar nuclei (Fig. 6). In thecerebellum, intensely stained Bcl-2-IR astrocytes were motor cortex, Bax-IR neurons in the pyramidal cell layerdetected in the fastigial, interposed and dentate nucleus showed intense staining (Fig. 7A). In the hippocampus,(Fig. 3). In the medulla, Bcl-2-IR astrocytes were observed Bax-IR neurons in the CA3 region showed intense stain-in the medullary reticular formation, inferior olivary ing, and the immunoreactivity was localized mainly in thenucleus, hypoglossal nucleus, medial vestibular nucleus, pyramidal cell layer (Fig. 7B). In contrast to the transgenicspinal vestibular nucleus and dorsal motor nucleus of the mice, no Bax-IR neurons were detected in the brainstemvagus. In the pons, Bcl-2-IR astrocytes were noted in the and spinal cord of the control mice.pontine reticular formation and trigeminal and facialnuclei. In the midbrain, Bcl-2-IR astrocytes were detectedin the periaqueductal gray matter (Fig. 4) and mesence- 4. Discussionphalic reticular formation. In contrast to transgenic mice,no Bcl-2-IR astrocytes were detected in the brainstem and In the present study, we reported increased Bcl-2spinal cord of control mice. expression in the astrocytes in the spinal cord and brain-

In the transgenic mice, specific immunoreactivity for stem of the transgenic mice (Table 1). In vitro studies haveBax was seen in the gray matter of the spinal cord (Table indicated that different types of cultured neurons and1) and especially in the anterior horn (Fig. 5). Immuno- differentiated PC12 cells transfected with mSOD1 [22] orstaining for Bax was identified only in neurons and not in neurons from primary cultures grown from transgenicglial cells. Bax-IR neurons were also detected in the mSOD mice [19] die, at least in part, by apoptosis. It hasbrainstem of transgenic mice (Table 1). In the medulla, been demonstrated that overexpression of the antiapoptoticthey were observed in the hypoglossal nucleus, dorsal protein Bcl-2 delays paralysis and death in transgenicmotor nucleus of the vagus and medullary reticular forma- mSOD mice [17]. It can be hypothesized that overexpres-tion. In the pons, their presence was noted in the trigeminal sion of Bcl-2, by increasing the availability of Bcl-2


Fig. 3. Bcl-2-IR astrocytes were detected in the intracerebellar nuclei of Fig. 4. Bcl-2-IR astrocytes were detected in the periaqueductal graythe mutant transgenic mice. Intensely stained Bcl-2-IR astrocytes were matter in the midbrain of the mutant transgenic mice (A, B). In the higherobserved in the dentate nucleus (A) and interposed nucleus (B) of the magnification (B), immunohistochemistry clearly showed the morphologymutant transgenic mice. Scale bar550 mm (A, B). of intensely stained Bcl-2-IR astrocytes. Scale bar5100 mm (A); 50 mm

(B).

molecules, enhances the capacity of neutralizing Baxthrough the formation of Bax:Bcl-2 heterodimers. Al- The Bcl-2 upregulation by astrocytes might have numer-though the cascade of molecular events underlying apop- ous functional consequences for the ALS brain. Astrocytestosis is quite complex and involves a large number of remove excitotoxins, produce extracellular matrix andproteins, both the above cited in vivo and in vitro adhesion molecules, restore the ionic milieu, and are anobservations strongly suggest that Bcl-2 plays a key role in essential component of the blood–brain barrier [6]. In themSOD1-mediated neurodegeneration. previous studies using the mutant transgenic mice, we

The precise mechanism by which Bcl-2 prevents cell reported that expression of NOS, nitrotyrosine and p53,death is uncertain, but several hypotheses involve its which are known to be associated with apoptosis, in-intracellular location at mitochondrial, endoplasmic re- creased in the astrocytes [2–4]. Troost et al. [26] reportedticulum, and nuclear membranes, which are also implicated Bcl-2 immunoreactivity in the astrocytes in the brains ofas sites of oxygen free radical generation [13]. Brain cells ALS patients. Therefore, an increase in astrocyte size,and neurons, in particular, are very vulnerable to oxidative activity, or survival via an upregulation of Bcl-2 might bestress because of their high phospholipid content, high beneficial by increasing or helping maintain production oflevels of respiration, relatively low levels of antioxidants, neurotrophic factors including nerve growth factor (NGF),and high content of catecholamines and oxidases. Ghadge basic fibroblast growth factor (bFGF), platelet-derivedet al. [9] reported that the Bcl-2, which is known to affect growth factor (PDGF), ciliary neurotrophic factor andfree radical generation and cell viability [14], protected insulin-like growth factor [23], and protecting or helpingcells from apoptosis induced by mSOD. In their studies maintain the integrity of the blood–brain barrier [6]. It can[9], reactive oxygen species (ROS) appeared to be in- be hypothesized that Bcl-2 expression in the astrocytesvolved in the mSOD-induced cell death. Therefore, the may partially underlie astrocytic resistance and protectionBcl-2 expression in the astrocytes might represent a of motor neurons in ALS.mechanism for counter balancing ROS-mediated damage In the present study we demonstrated an increasedin ALS. expression of Bax in the spinal cord and brainstem of


Fig. 5. In the transgenic mice (A), specific immunoreactivity for Bax wasseen in the gray matter of the spinal cord and especially in the anteriorhorn, compared with no specific immunostaining of the control mice (B).Immunostaining for Bax was identified only in neurons and not in glialcells. Scale bar5100 mm (A, B).

transgenic mSOD1 mice (Table 1). Our findings agreedwith the report of Ekegren et al. [7] and Vukosavic et al.[27]. Ekegren et al. [7] reported increased Bax protein inthe spinal cord of ALS patients. Vukosavic et al. [27]reported increased Bax immunoreactivity in mice with ahuman SOD1 mutation, but they performed immuno-histochemical staining only in the spinal cord. So ourpresent study demonstrated the distribution of Bax in detail

Fig. 6. In the cerebellum of the mutant transgenic mice, a high density offor the first time using immunohistochemical methods in Bax-IR neurons was observed in the Purkinje cell layer (A) and cerebellarthe whole brains of the transgenic mice. nuclei (C). In contrast to the transgenic mice, no Bax-IR neurons were

One of the most striking findings in the present study is detected in the Purkinje cell layer of the control mice (B). P, Purkinje celllayer. Scale bar550 mm (A, B, C).the increased Bax immunoreactivity in the CA3 region of

hippocampus. Waldemar et al. [28] reported results ofsingle photon emission computed tomography study of 14ALS patients showing significantly reduced cerebral blood One intriguing possibility to explain how the demise offlow in the hippocampus and frontal cortex. As noted motor neurons in transgenic mSOD1 mice may be favoredearlier, Gallassi et al. [8], David and Gillham [5], and by the Bcl-2 family member deregulation, and especiallyIwasaki et al. [15] have all reported memory deficits in Bax upregulation, is by affecting mitochondrial function.ALS patients, an observation that further suggests the This would be consistent with demonstration that mito-presence of an abnormality of hippocampal function in chondrial function is impaired in the spinal cord ofALS. Recently, Kakita et al. [16] have found eosinophilic transgenic mice [1]. In addition, a relationship betweenintranuclear inclusions in the hippocampal pyramidal Bax upregulation and alterations in mitochondrial functionneurons of a patient with ALS. would be compatible with the mitochondrial localization of


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