ARTICLE IN PRESS

Ann Anat 190 (2008) 502—509

0940-9602/$ - sdoi:10.1016/j.

�CorrespondE-mail addr

1These autho

www.elsevier.de/aanat

RESEARCH ARTICLE

Immunohistochemical study on the distribution ofinsulin-like growth factor-binding protein 4 in thecentral nervous system of SOD1G93A transgenicmice as an in vivo model of amyotrophiclateral sclerosis

Dong Woo Kanga,1, Yoon Hee Chungb,1, Jae Chul Leea, Joon Seok Bangc,Daejin Kimb, Sung Su Kimb, Kyung Yong Kimb, Won Bok Leeb,Choong Ik Chaa,�

aDepartment of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu,Seoul 110-799, KoreabDepartment of Anatomy, College of Medicine, Chung-Ang University, 221 Heukseok-Dong, Dongjak-Gu,Seoul 156-756, KoreacDepartment of Pharmacology, College of Medicine, Chung-Ang University, 221 Heukseok-Dong, Dongjak-Gu,Seoul 156-756, Korea

Received 2 May 2008; accepted 8 August 2008

KEYWORDSAmyotrophic lateralsclerosis (ALS);SOD1G93A transgenicmice;Insulin-like growthfactor-bindingprotein 4 (IGFBP4);Cerebral cortex;Hippocampus;Cerebellum

ee front matter & 2008aanat.2008.08.001

ing author. Tel.: +82 2 7ess: cicha@snu.ac.kr (Crs contributed equally

SummaryIn the present study, we used the SOD1G93A mutant transgenic mice as an in vivomodel of amyotrophic lateral sclerosis (ALS) and performed immunohistochemicalstudies to investigate the changes of insulin-like growth factor-binding protein 4(IGFBP4) in the central nervous system. Decreased expression of IGFBP4 was obviousin the cerebral cortex, hippocampus, cerebellar cortex and inferior olive of SOD1G93A

transgenic mice. In the cerebral cortex, there was a significant decrease in IGFBP4immunoreactivity in the pyramidal cells. In the hippocampal formation, IGFBP4immunoreactivity was also decreased in the pyramidal cells of CA1–3 areas and thegranule cells of dentate gyrus. In the cerebellar cortex, IGFBP4 immunoreactivitywas prominent in the granular layer in wtSOD1 transgenic mice, compared to that inSOD1G93A transgenic mice. IGFBP4 immunoreactivity was decreased in the inferior

Elsevier GmbH. All rights reserved.

40 8205; fax: +82 2 745 9528..I. Cha).to this work.

ARTICLE IN PRESS

Decreased expression of IGFBP4 in ALS mice 503

olive of SOD1G93A transgenic mice. This study, showing decreased IGFBP4 in differentbrain regions of SOD1G93A transgenic mice, may provide clues to understanding thedifferential susceptibility of neural structures in ALS, suggesting a role of IGFBP4 inan abnormality of cognitive and/or motor function in ALS. The mechanisms andfunctional implications of these decreases require elucidation.& 2008 Elsevier GmbH. All rights reserved.

Introduction

Amyotrophic lateral sclerosis (ALS) is a progres-sive, fatal neurodegenerative disease, which ischaracterized by the death of upper and lowermotor neurons (Rowland and Shneider, 2001). Theetiologies of sporadic and familial ALS (FALS) sharecommon mechanisms, and the study of FALS casescan provide an understanding of sporadic cases(Al-Chalabi and Leigh, 2000). It has been documen-ted that 20–25% of FALS are linked to dominantlyinherited mutations in Cu/Zn superoxide dismutase(SOD1) (Al-Chalabi and Leigh, 2000). A variety ofstudies, including investigations of SOD1-mutantmice that develop motor neuron disease (Gurneyet al., 1994; Ripps et al., 1995), established thatmutant SOD1 causes motor neuron disease throughthe appearance of one or more toxic properties(Cleveland and Rothstein, 2001; Julien, 2001).Although the mechanisms whereby mutant SOD1causes selective motor neuron death are not yetknown, several hypotheses have been postulatedsuch as enhancement of protein nitrosylation bymutant SOD1; enhanced peroxidase activity; ex-posure of toxic copper at the active site; andaccumulation and aggregation of altered or abnor-mal proteins including SOD1 (Rowland and Shneider,2001).

Given that the striking pathological feature inALS is motor neuron loss, many neurotrophicfactors have been found to promote motor neuronsurvival in cell-culture models (Ekestern, 2004).However, few have been found to be of benefit inthe SOD1 mutant mice and all clinical trials usingsuch agents in ALS to date have been negative(Goodall and Morrison, 2006). Evidence that in-sulin-like growth factor I (IGF-I) rescues motorneurons both in in vivo and in vitro studies has ledto therapeutic trials of human recombinant IGF-I inALS. However, systemic delivery of human recom-binant IGF-I in these trials did not lead to beneficialclinical effects in ALS patients, raising the possibi-lity that IGF-binding proteins (IGFBPs) inactivateIGF-I by forming inactive complexes (Wilczak andDe Keyser, 2005).

IGFBPs can either inhibit or enhance presentationof IGF-I to their receptors. Insulin-like growth

factor-binding protein 4 (IGFBP4) is one of themajor IGFBPs expressed in the central nervoussystem, and most data indicate an inhibitory role ofIGFBP4 on IGF-I effects both in vivo and in vitro(Mazerbourg et al., 2004). Recent results haveshown that tumors treated with the IGFBP4 genehad higher expression of Bax, lower expression ofBcl-2 and IGF-I receptor (Durai et al., 2007), whichwas reported to be involved in the pathogenesis ofALS in our previous studies (Cho et al., 1999; Shin etal., 2000; Chung et al., 2003). In particular, wedemonstrated that IGF-I receptor immunoreactivitywas increased in the CNS of SOD1G93A transgenicmice (Chung et al., 2003). In ALS patients, serumconcentrations of IGF-I were significantly reduced(Torres-Aleman et al., 1998), and IGF-I expressionin spinal cords was normal (Kerkhoff et al., 1994).Spinal cords of ALS patients have more [125I]IGF-I-binding sites than do spinal cords without thisdisease (Dore et al., 1996). Although the IGF-Isignaling system is complex and regulated by IGF-Ireceptors and IGFBPs in the pathogenesis of ALS,relatively little information is available on expres-sional changes in these IGFBPs. Therefore, we usedthe SOD1G93A mutant transgenic mice as an in vivomodel of ALS and performed immunohistochemicalstudies to investigate the changes of IGFBP4 in thecentral nervous system.

Materials and methods

Twelve SOD1G93A transgenic and 10 wild-type(wt)SOD1 transgenic mice developed by Gurney etal. (1994) were used for these experiments. Theywere bred by The Jackson Laboratory (Bar Harbor,ME) under the strain designations B6SJL-TgN(SOD1G93A) 1Gur and B6SJL-TgN (SOD1) 2Gur formutant transgenic and wtSOD1 transgenic mice,respectively. B6SJL-TgN (SOD1) 2Gur strain carriesthe normal allele of the human SOD1 gene, and ithas been reported that the SOD1 protein level isthe same as in the transgenic strain carrying theSOD1G93A transgene. This strain serves as a controlfor the B6SJL-TgN (SOD1G93A) 1Gur. Animals weresacrificed at the age of 8 (presymptomatic) and 18weeks (symptomatic). Clinical symptoms weremanifested in 18-week mutant transgenic mice.

ARTICLE IN PRESS

D.W. Kang et al.504

The animals used in this experiment were treatedaccording to the ‘Principles of Laboratory AnimalCare’ (NIH publication no. 86-23).

The mice were perfused transcardially with coldphosphate-buffered saline (0.02M, pH 7.4), andthen with ice-cold 4% paraformaldehyde for 10minat a flow rate of 5–6ml/min. Brains were immedi-ately removed and sliced into 4–6-mm-thick blocks.Spinal cords were also removed and sliced intocervical, thoracic and lumbar segments of 3–10mmin length. These blocks were immersed in a coldfixative for 12 h and then cryoprotected in a seriesof cold sucrose solutions of increasing concentra-tion. Frozen sections were cut at 40 mm in thecoronal plane. Immunohistochemistry was per-formed in accordance with the free-floating meth-od described earlier (Chung et al., 2003). Goatpolyclonal IGFBP4 antibody (sc-6005, Santa CruzBiotechnology, Inc.) was used as primary antibody.

Table 1. Changes in mean densities of IGFBP4 immu-noreactivity in the central nervous system of SOD1G93A

transgenic mice

Area wtSOD1 SOD1G93A

Cerebral cortexSomatomotor area 89.274.7 61.276.7*Cingulate area 92.573.9 58.974.8*Insular area 89.775.1 57.675.8*Somatosensory area 86.873.5 61.375.7*Auditory area 84.376.2 55.374.9*Visual area 81.675.2 53.874.2*Entorhinal area 83.976.9 54.376.0*

HippocampusCA1 regionStratum oriens 49.373.3 51.073.8Pyramidal cell layer 81.777.1 58.874.7*Stratum radiatum 46.172.3 49.274.1

CA3 regionStratum oriens 46.272.3 47.673.2Pyramidal cell layer 85.475.6 61.475.0*Stratum radiatum 43.573.3 44.873.9

Dentate gyrusGranule cell layer 79.374.7 61.075.8*Polymorphic layer 72.175.9 55.875.1*

Cerebellar cortexMolecular layer 53.973.0 51.172.9Purkinje cell layer 39.676.1 45.275.2Granular layer 104.574.9 61.172.9*

Deep cerebellar nuclei 62.877.2 55.574.6Inferior olivary nuclei 126.072.2 79.574.1*

Mean density is the sum of the gray values of all the pixels in theselection that was divided by the number of pixels within theselection. Values are presented as the mean7standard devia-tion. The Mann–Whitney U-test was performed (*Po0.05).

To observe the stained cells, a microscope (LeicaDM4500B; Leica Microsystems, Germany) with acomputer-driven digital camera (DFC320; LeicaMicrosystems) was used.

To demonstrate the specificity of the antibodiesused in this study, a sample of sections was reactedwithout primary antiserum, and a different samplewas exposed to a primary antiserum that had beenpreincubated with blocking peptide (sc-6005P). Nosections from these samples exhibited any of theimmunoreactivity described in this report. Sectionsfrom each SOD1G93A transgenic and wtSOD1 trans-genic mice were stained together, eliminatingconflicts between different experimental condi-tions. We selected five slides in each area of theSOD1G93A transgenic (n ¼ 12) and wtSOD1 trans-genic mice (n ¼ 10). Visual assessment and densi-tometric measurement using Leica QWin Plus (LeicaMicrosystems Imaging Solutions, UK) determinedthe staining intensities (Table 1). The Mann–Whit-ney U-test was performed (*Po0.05).

Results

The brain and spinal cord sections from wtSOD1transgenic and presymptomatic SOD1G93A trans-genic mice exhibited very low immunoreactivitiesat the ages of 8 weeks, and the staining intensitydid not vary. In symptomatic SOD1G93A transgenicmice, decreased expression of IGFBP4 was obviousin the cerebral cortex, hippocampus, cerebellarcortex and inferior olive (Table 1). In the cerebralcortex of wtSOD1 transgenic mice, heavily stainedneurons were seen in layers II–VI in most corticalregions including the parietal association area(Figure 1A), somatomotor area, cingulated area,insular area, somatosensory area, auditory area,visual area and entorhinal area. High magnificationof IGFBP4-immunoreactive cells illustrated typicalmorphology of pyramidal neurons in layer V of theparietal association area (Figure 1B). In SOD1G93A

transgenic mice, there was a significant decrease inIGFBP4 immunoreactivity in the cell bodies andapical dendrites of the pyramidal cells in the samecortical regions (Figures 1C and D).

In the hippocampal formation, there were layer-specific alterations in the staining intensity ofIGFBP4-immunoreactive neurons (Figure 2). It wasnoted that the pyramidal cells in the CA1–3 regionwere strongly immunoreactive for IGFBP4 inwtSOD1 transgenic mice (Figures 2A, B and E). InSOD1G93A transgenic mice, IGFBP4 immunoreactiv-ity was significantly decreased in the pyramidal celllayers of the CA1–3 areas (Figures 2C, D and F). In

ARTICLE IN PRESS

Figure 1. Localizations of IGFBP4-immunoreactive neurons in the cerebral cortex of wtSOD1 transgenic (A and B) andSOD1G93A transgenic (C and D) mice. In wtSOD1 transgenic mice, intensely stained neurons were seen in layers II–VI inthe parietal association cortex (A). High magnification of IGFBP4-immunoreactive cells illustrated typical morphology ofpyramidal neurons in layer V (B). In SOD1G93A transgenic mice, there was a significant decrease in IGFBP4immunoreactivity in the cell bodies and apical dendrites of the pyramidal cells in the same cortical regions (C and D).Scale bar ¼ 100 mm (A and C) and 25 mm (B and D).

Decreased expression of IGFBP4 in ALS mice 505

contrast, there were no differences between thetwo groups in the stratum oriens and stratumradiatum. In the dentate gyrus, immunoreactivityfor IGFBP4 was also decreased in the granularcell layers and polymorphic layers (Figures 2Gand H).

In the cerebellar cortex, IGFBP4 immunoreactiv-ity was prominent in the granular layer in wtSOD1transgenic mice (Figures 3A and B), whereasIGFBP4-immunoreactive cells were strikingly de-creased in the same layer in SOD1G93A transgenicmice (Figures 3C and D). In the deep cerebellarnuclei (Figures 3E and F) of SOD1G93A transgenicmice, IGFBP4 immunoreactivity was slightly de-creased in the neuropil. Interestingly, IGFBP4immunoreactivity was prominent in the inferiorolive in wtSOD1 transgenic mice (Figures 4A and B).The staining intensity was decreased in this areaof SOD1G93A transgenic mice (Figures 4C and D).Unexpectedly, IGFBP4 immunoreactivity washardly observed in other areas of the brainstemand spinal cord of both wtSOD1 and SOD1G93A

transgenic mice.

Discussion

Tightly regulating the actions of IGFs are struc-turally distinct IGFBPs that can inhibit or promoteIGF effects by preventing receptor interaction or bytransporting IGF to target cells (Mazerbourg et al.,2004). More recent studies have provided addi-tional evidence for IGF-independent effects ofseveral binding proteins, although the function ofIGFBP4 in the CNS remains largely unknown (Chesiket al., 2004). Regulatory molecules of the IGFsystem are known to be differentially expressed inthe course of neurodegenerative diseases. In recentyears, several mouse models and ALS patients havebeen subjected to extensive analyses of theinvolvement of the IGF system in the spinal cord(Kaspar et al., 2003; Wilczak et al., 2003). Highconcentrations of IGFBP2, 5 and 6 in spinal motorneurons prevented binding of IGF-I to IGF-I recep-tors in ALS (Wilczak et al., 2003). However, thehuman form of ALS includes pathology elsewhere inthe central nervous system, and other IGFBPs maybe involved in the pathogenesis of ALS. In our

ARTICLE IN PRESS

Figure 2. Localization of IGFBP4-immunoreactive cells in the hippocampus of wtSOD1 transgenic (A, B, E and G) andSOD1G93A transgenic (C, D, F and H) mice. There were layer-specific alterations in the staining intensity of IGFBP4-immunoreactive neurons in the hippocampus (A and C). It was noted that the pyramidal cells in CA1–3 region werestrongly immunoreactive for IGFBP4 in wtSOD1 transgenic mice (A, B and E). In SOD1G93A transgenic mice, IGFBP4immunoreactivity was significantly decreased in the pyramidal cell layers of the CA1–3 areas (C, D and F). In thedentate gyrus, immunoreactivity for IGFBP4 was also decreased in the granular cell layers and polymorphic layers (Gand H). CA1–3, fields CA1–3 of Ammon’s horn; DG, dentate gyrus; G, granule cell layer; L, stratum lucidum; O, stratumoriens; P, pyramidal cell layer; Po, polymorphic layer; and R, stratum radiatum. Scale bar ¼ 100 mm (A and C) and 25 mm(B and D–H).

D.W. Kang et al.506

analyses extended to the whole brain, significantdecreases in IGFBP4 were found in the cerebralcortex, hippocampus, cerebellum and inferiorolive, suggesting that these alterations of IGFBP4may provide clues for understanding a role ofIGFBP4 in differential susceptibility of neuralstructures in ALS.

Recently, Miyakoshi et al. (2001a) have suggestedthat systemic administration of IGFBP-4 at pharma-cological doses increases bone formation para-meters in mice by increasing IGF bioavailabilityvia an IGFBP-4 protease-dependent mechanism.Two additional in vitro studies suggest that IGFBP-4enhances IGF-I growth stimulation of melanocytes(Edmondson et al., 1999) and augments the survivalof rat neuronal cells (Kummer et al., 1996). In

terms of an IGF-independent effect of IGFBP-4,there is so far one study suggesting that IGFBP-4can exert inhibitory effects of steroidogenesis ofhuman granulosa cells in vitro by an IGF-indepen-dent mechanism (Wright et al., 2002). In contrastto this study, Miyakoshi et al. (2001b) have shownthat IGFBP-4, unlike IGFBP-5, cannot induce anincrease in bone formation in IGF-I knockout mice.Overall, in vivo and in vitro findings stronglysupport an inhibitory effect of IGFBP-4 on IGFsactions. Therefore, decreased IGFBP4 in ALS mightlead to a decrease in the inhibitory effect on IGF-Iinteraction with the IGF-I receptor, which wouldcompensate for a decrement in the IGF-I level.However, it cannot be excluded that the possibleIGF potentiating effects and IGF-independent

ARTICLE IN PRESS

Figure 3. Localization of IGFBP4-immunoreactive cells in the cerebellum of wtSOD1 transgenic mice (A, B and E) andSOD1G93A transgenic mice (C, D and F). In the cerebellar cortex, IGFBP4 immunoreactivity was prominent in the granularlayer in wtSOD1 transgenic mice (A and B), whereas IGFBP4-immunoreactive cells were hardly detected in the samelayer of SOD1G93A transgenic mice (C and D). In the deep cerebellar nuclei of SOD1G93A transgenic mice, IGFBP4immunoreactivity was decreased in the neuropil (E and F). Gr, granular layer; M, molecular layer; Pu, Purkinje celllayer; and W, white matter. Scale bar ¼ 100 mm (A and C) and 25 mm (B and D–F).

Figure 4. IGFBP4 immunoreactivity in the inferior olivary nuclei of wtSOD1 transgenic mice (A and B) and SOD1G93A

transgenic mice (C and D). IGFBP4 immunoreactivity was prominent in the inferior olive in wtSOD1 transgenic mice (Aand B). The staining intensity was decreased in this area of SOD1G93A transgenic mice (C and D). Scale bar ¼ 100 mm (Aand C) and 25 mm (B and D).

Decreased expression of IGFBP4 in ALS mice 507

actions of IGFBP-4 would be decreased due to adecrement in IGFBP4, although it remains uncer-tain in the central nervous system.

Interestingly, IGFBP4 immunoreactivity was de-creased in the pyramidal cells of CA1–3 areas andthe granule cells of dentate gyrus in ALS. Single

ARTICLE IN PRESS

D.W. Kang et al.508

photon emission computed tomography study of 14ALS patients showed significantly reduced cerebralblood flow in the hippocampus (Waldemar et al.,1992). Eosinophilic intranuclear inclusions wereobserved in the hippocampal pyramidal neurons ofan ALS patient (Kakita et al., 1997). Recently,subclinical impairment of cognitive functions wasreported in patients with ALS (Hanagasi et al.,2002; Paulus et al., 2002). These observationssuggest the presence of an abnormality of hippo-campal function in ALS, resulting in cognitiveimpairment. Our first demonstration of decreasedexpression of IGFBP4 in the hippocampus oftransgenic mice may reflect a compensatory re-sponse to decreased IGF-I in impaired hippocampalcircuitry in ALS.

Another striking finding in the present study isdecreased immunoreactivity for IGFBP4 in thegranular layer of the cerebellar cortex and inferiorolive although there were no morphometric differ-ences in this region. The cerebellum is involved inmotor learning and control, and IGF-I has beenclosely correlated with the function of olivocer-ebellar interactions (Sherrard and Bower, 2003).Magnetic resonance imaging (MRI) indicated globalatrophy in the cerebellum of SOD1G93A mice (Zanget al., 2004). In our previous studies, we demon-strated in vivo evidence of oxidative damage in thecerebellum in the pathogenesis of ALS (Cho et al.,1999). However, functional MRI studies of the brainof ALS patients showed extra activation in thecerebellum, suggesting that the extra activationareas in ALS patients may indicate functionalcompensation (Han and Ma, 2006). In this respect,it is tempting to speculate about IGF-independenteffects of IGFBP-4 or a compensatory response to arelative deficiency of cerebellar IGF-I in olivocer-ebellar interactions in ALS. Although a role ofIGFBP4 need to be elucidated in the pathogenesisof ALS cerebellum, our first demonstration ofdecreased expression of IGFBP4 in the cerebellummay represent a reactive process to oxidativedamage in ALS.

In conclusion, the present study showing de-creased IGFBP4 in different brain regions ofSODG93A transgenic mice might be useful for under-standing a role of IGFBP4 in the differentialsusceptibility of neural structures in ALS. Inparticular, IGFBP4 was decreased in the hippocam-pal regions and cerebellum, suggesting compensa-tory or reactive responses to an abnormality ofcognitive and/or motor function in ALS, respec-tively. However, the mechanisms underlying thedecreased immunoreactivity for IGFBP4, and thefunctional implications of these decreases, requireelucidation.

Acknowledgment

This work was supported by the Korea ResearchFoundation Grant funded by the Korean Govern-ment (MOEHRD, Basic Research Promotion Fund)(KRF-2007-531-E00058).

References

Al-Chalabi, A., Leigh, P.N., 2000. Recent advances inamyotrophic lateral sclerosis. Curr. Opin. Neurol. 13,397–405.

Chesik, D., Wilczak, N., De Keyser, J., 2004. Insulin-likegrowth factor binding protein-4 interacts with centro-somes and microtubules in primary astrocytes. Neu-roscience 125, 381–390.

Cho, K.J., Chung, Y.H., Shin, C., Shin, D.H., Kim, Y.S.,Gurney, M.E., Lee, K.W., Cha, C.I., 1999. Reactiveastrocytes express p53 in the spinal cord of transgenicmice expressing a human Cu/Zn SOD mutation.Neuroreport 10, 3939–3943.

Chung, Y.H., Joo, K.M., Shin, C.M., Lee, Y.J., Shin, D.H.,Lee, K.H., Cha, C.I., 2003. Immunohistochemicalstudy on the distribution of insulin-like growth factorI (IGF-I) receptor in the central nervous system ofSOD1G93A mutant transgenic mice. Brain Res. 994,253–259.

Cleveland, D.W., Rothstein, J.D., 2001. From Charcot toLou Gehrig: deciphering selective motor neuron deathin ALS. Nat. Rev. Neurosci. 2, 806–819.

Dore, S., Krieger, C., Kar, S., Quirion, R., 1996.Distribution and levels of insulin-like growth factor(IGF-I and IGF-II) and insulin receptor binding sites inthe spinal cords of amyotrophic lateral sclerosis (ALS)patients. Brain Res. Mol. Brain Res. 41, 128–133.

Durai, R., Yang, S.Y., Sales, K.M., Seifalian, A.M.,Goldspink, G., Winslet, M.C., 2007. Insulin-like growthfactor binding protein-4 gene therapy increasesapoptosis by altering Bcl-2 and Bax proteins anddecreases angiogenesis in colorectal cancer. Int. J.Oncol. 30, 883–888.

Edmondson, S.R., Russo, V.C., McFarlane, A.C., Wraight,C.J., Werther, G.A., 1999. Interactions betweengrowth hormone, insulinlike growth factor I, and basicfibroblast growth factor in melanocyte growth. J. Clin.Endocrinol. Metab. 84, 1638–1644.

Ekestern, E., 2004. Neurotrophic factors and amyo-trophic lateral sclerosis. Neurodegener. Dis. 1,88–100.

Goodall, E.F., Morrison, K.E., 2006. Amyotrophic lateralsclerosis (motor neuron disease): proposed mechan-isms and pathways to treatment. Expert Rev. Mol.Med. 8, 1–22.

Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C.,Polchow, C.Y., Alexander, D.D., Caliendo, J., Hentati,A., Kwon, Y.W., Deng, H.X., Chen, W., Zhai, P., Sufit,R.L., Siddique, T., 1994. Motor neuron degeneration in

ARTICLE IN PRESS

Decreased expression of IGFBP4 in ALS mice 509

mice that express a human Cu, Zn superoxidedismutase mutation. Science 264, 1772–1775.

Han, J., Ma, L., 2006. Functional magnetic resonanceimaging study of the brain in patients with amyo-trophic lateral sclerosis. Chin. Med. Sci. J. 21,228–233.

Hanagasi, H.A., Gurvit, I.H., Ermutlu, N., Kaptanoglu,G., Karamursel, S., Idrisoglu, H.A., Emre, M., Demir-alp, T., 2002. Cognitive impairment in amyotrophiclateral sclerosis: evidence from neuropsychologicalinvestigation and event-related potentials. Brain Res.Cogn. Brain Res. 14, 234–244.

Julien, J.P., 2001. Amyotrophic lateral sclerosis. unfold-ing the toxicity of the misfolded. Cell 104, 581–591.

Kakita, A., Oyanagi, K., Nagai, H., Takahashi, H., 1997.Eosinophilic intranuclear inclusions in the hippocam-pal pyramidal neurons of a patient with amyotrophiclateral sclerosis. Acta Neuropathol. (Berlin) 93,532–536.

Kaspar, B.K., Llado, J., Sherkat, N., Rothstein, J.D.,Gage, F.H., 2003. Retrograde viral delivery of IGF-1prolongs survival in a mouse ALS model. Science 301,839–842.

Kerkhoff, H., Hassan, S.M., Troost, D., Van Etten, R.W.,Veldman, H., Jennekens, F.G., 1994. Insulin-like andfibroblast growth factors in spinal cords, nerve rootsand skeletal muscle of human controls and patientswith amyotrophic lateral sclerosis. Acta Neuropathol.(Berlin) 87, 411–421.

Kummer, J.L., Zawada, W.M., Freed, C.R., Chernausek,S.D., Heidenreich, K.A., 1996. Insulin-like growthfactor binding proteins in fetal rat mesencephaliccultures: regulation by fibroblast growth factor andinsulin-like growth factor I. Endocrinology 137,3551–3556.

Mazerbourg, S., Callebaut, I., Zapf, J., Mohan, S.,Overgaard, M., Monget, P., 2004. Up date on IGFBP-4: regulation of IGFBP-4 levels and functions, in vitroand in vivo. Growth Horm. IGF Res. 14, 71–84.

Miyakoshi, N., Qin, X., Kasukawa, Y., Richman, C.,Srivastava, A.K., Baylink, D.J., Mohan, S., 2001a.Systemic administration of insulin-like growth factor(IGF)-binding protein-4 (IGFBP-4) increases bone for-mation parameters in mice by increasing IGF bioavail-ability via an IGFBP-4 protease-dependentmechanism. Endocrinology 142, 2641–2648.

Miyakoshi, N., Richman, C., Kasukawa, Y., Linkhart, T.A.,Baylink, D.J., Mohan, S., 2001b. Evidence that IGF-

binding protein-5 functions as a growth factor. J. Clin.Invest. 107, 73–81.

Paulus, K.S., Magnano, I., Piras, M.R., Solinas, M.A.,Solinas, G., Sau, G.F., Aiello, I., 2002. Visual andauditory event-related potentials in sporadic amyo-trophic lateral sclerosis. Clin. Neurophysiol. 113,853–861.

Ripps, M.E., Huntley, G.W., Hof, P.R., Morrison, J.H.,Gordon, J.W., 1995. Transgenic mice expressing analtered murine superoxide dismutase gene provide ananimal model of amyotrophic lateral sclerosis. Proc.Natl. Acad. Sci. USA 92, 689–693.

Rowland, L.P., Shneider, N.A., 2001. Amyotrophic lateralsclerosis. N. Engl. J. Med. 344, 1688–1700.

Sherrard, R.M., Bower, A.J., 2003. IGF-1 induces neonatalclimbing-fibre plasticity in the mature rat cerebellum.Neuroreport 14, 1713–1716.

Shin, C.M., Chung, Y.H., Kim, M.J., Shin, D.H., Kim, Y.S.,Gurney, M.E., Lee, K.W., Cha, C.I., 2000. Immunohis-tochemical study on the distribution of Bcl-2 and Baxin the central nervous system of the transgenic miceexpressing a human Cu/Zn SOD mutation. Brain Res.887, 309–315.

Torres-Aleman, I., Barrios, V., Berciano, J., 1998. Theperipheral insulin-like growth factor system in amyo-trophic lateral sclerosis and in multiple sclerosis.Neurology 50, 772–776.

Waldemar, G., Vorstrup, S., Jensen, T.S., Johnsen, A.,Boysen, G., 1992. Focal reductions of cerebral bloodflow in amyotrophic lateral sclerosis: a [99mTc]-d,l-HMPAO SPECT study. J. Neurol. Sci. 107, 19–28.

Wilczak, N., De Keyser, J., 2005. Insulin-like growthfactor system in amyotrophic lateral sclerosis. Endocr.Dev. 9, 160–169.

Wilczak, N., de Vos, R.A., De Keyser, J., 2003. Freeinsulin-like growth factor (IGF)-I and IGF bindingproteins 2, 5, and 6 in spinal motor neurons inamyotrophic lateral sclerosis. Lancet 361, 1007–1011.

Wright, R.J., Holly, J.M., Galea, R., Brincat, M., Mason,H.D., 2002. Insulin-like growth factor IGF-independenteffects of IGF binding protein-4 on human granulosacell steroidogenesis. Biol. Reprod. 67, 776–781.

Zang, D.W., Yang, Q., Wang, H.X., Egan, G., Lopes, E.C.,Cheema, S.S., 2004. Magnetic resonance imagingreveals neuronal degeneration in the brainstem ofthe superoxide dismutase 1 transgenic mouse model ofamyotrophic lateral sclerosis. Eur. J. Neurosci. 20,1745–1751.