Original Article

Low Levels of Nerve Growth Factor in Cerebrospinal Fluid of Children With Rett Syndrome Raija Lappalainen, MD;

Dan

Lindholm, PhD; Raili Riikonen,

PhD

ABSTRACT A role for neurotrophic factors has been postulated in some human neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. The known developmental effects of these substances suggested that, in some neurologic diseases

affecting children, neurotrophic factors might be inadequate. Using a sensitive, two-site enzyme-linked immunoassay, we examined the content of nerve growth factor in the cerebrospinal fluid of 11children with Rett syndrome and of 24 control patients with various neurologic diagnoses or suffering from other diseases. Nerve growth factor levels were significantly lower in the cerebrospinal fluid of the patients with Rett syndrome than in control patients. The lower level of cerebrospinal fluid nerve growth factor in Rett syndrome suggests that lack of nerve growth factor may be involved in the pathogenesis of this disease or reflect the underlying brain damage present. (J Child Neurol 1996;11:296-300).

Rett syndrome, a devastating disease of girls with unknown etiology, was first described by Andreas Rett in 19661 but received further attention only in the 1980s.1 At ages varying between 6 and 18 months, normal development of the child is arrested and regression ensues. The patient then loses her acquired skills, especially use of the hands and language abilities, and exhibits autistic traits. Hand stereotypies and so-called jerky-type ataxia are noted. Epilepsy and breathing irregularities are very common, in addition to growth retardation. To characterize the disease, it is divided into four stages (stages I through IV) according to the most distinct clinical problems: developmental slowing, the regression period, the pseudostationary period, and finally a stage of slow motor deterioration.3 The diagnosis of Rett syndrome is based on the clinical features, because no laboratory marker is available. A role for neurotrophic factors in this disease has been suggested previously, mainly because of the deceleration in

Received Jan 9, 1995. Received revised May 4 and August 8, for publication Sept 7, 1995.

1995. Accepted

From the Department of Child Neurology (Dr Lappalainen), Children’s Castle Hospital, Helsinki, Finland, the Department of Developmental Neuroscience (Dr Lindholm), Uppsala University, Biomedical Center, BOX 587, S-75123 Uppsala, Sweden, and the Department of Child Neurology (Dr Riikonen), University Central Hospital, Helsinki, Finland. Address correspondence to Dr Raija Lappalainen, Children’s Castle Hospital, Lastenlinnantie 2, 00250 Helsinki, Finland.

296

head

growth and

decreased

outgrowth

of neuronal

processes.4-7 Neurotrophic factors are important regulators of neuronal survival during the developmental period of natural cell death but also have a function in the adult nervous system.8 The first and best-known neurotrophic factor is a protein containing 118 amino acids, called (3-nerve growth factor. Brain-derived neurotrophic factor and neurotrophin3 are structurally similar to nerve growth factor, and together with nerve growth factor describe a neurotrophin gene family. However, the pattern of expression of these neurotrophins in the central nervous system and their actions may be quite distinct.’’ Nerve growth factor was first shown to act on sympathetic and sensory neurons, but later its trophic activity was demonstrated in the central nervous system, 10 where it acts especially on the cholinergic neurons of the basal forebrain.lul2 Nerve growth factor has been thought to be synthesized in the target cells and is then taken up by the nerve endings of nerve growth factorresponsive neurons and transported in a retrograde manner to the nerve cell body. These target cells are located, for example, in the cerebral cortex and the hippocampus, which are terminal regions of cholinergic neurons.1O,13 A role has been suggested for nerve growth factor in neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, and the possibility has been studied intensively. In Rett syndrome there may be some cholinergic neurochemical alterations similar to those in these

297

diseases&dquo; and because nerve growth factor is a trophic factor for basal forebrain cholinergic neurons, it is possible that

abnormalities in nerve growth factor might be responsible. The aim of our study was to examine (1) whether levels of nerve growth factor are low, corresponding with the involvement of basal forebrain cholinergic neurons in Rett syndrome, and (2) correlation of nerve growth factor levels in Rett syndrome with the other neuropediatric patients. We report here values for cerebrospinal fluid nerve growth factor in 11 girls with Rett syndrome and in neuropediatric patients of other types.

and then kept for 2 hours at room temperature in 1% bovine serum albumin dissolved in the carbonate buffer. After washing, 50 )JbL of

sample was added to each well, and the plates were incubated overnight at 4°C. The cerebrospinal fluid samples were diluted 1:1 with the sample buffer containing 2% bovine serum albumin and 0.2% polyethylene glycol pisoactylphenyl ether (Triton X-100). Each sample was then divided into two tubes, and 100 pg of the nerve growth factor standard was either the standard solution

added to

human and

stepwise

growth MATERIAL Eleven

patients

with Rett

syndrome

were

included in the

study.

Their ages ranged from 1.8 to 17.4 years (mean, 9.0 years; standard deviation [SD], 5.7 years). Twenty-four children with other pediatric

neurologic diseases served as controls: control group 1 consisted of six patients with epilepsy and mental retardation; control group or

comprised six patients with epilepsy but without mental retardation ; and control group 3 consisted of 12 patients with no epilepsy 2

or

mental retardation. The Ethical Committee of the hospital approved the study. In

addition, informed consent for the cerebrospinal fluid examinations was obtained from the parents of all the patients. Control samples were obtained, with the permission of the parents, from patients in whom lumbar puncture was performed for some other reason. METHODS

cerebrospinal fluid samples were frozen immediately (in liquid nitrogen) and stored at -70°C. The content of nerve growth factor in the liquor samples was determined with a sensitive two-

All the

site ELISA as described in detail

elsewhere. 15,16 The human nerve

growth factor used as the standard was a kind gift from Genentech, San Francisco, CA. Briefly, 0.04 )Jbg of the monoclonal anti-nerve growth factor antibody, 27/21 (Boehringer Mannheim, Mannheim, Germany) was plated onto 96-microtiter plates (50 f-LUwell) in 50 mmol/L carbonate buffer and incubated overnight at 4°C. The plates were washed with the sample buffer [50 mmol/L tromethamine (Tris) HCI (pH 7.0), 150 mmol/L NaCI, and 5 mmol/L MgCl2] Table 1.

CSF

=

cerebrospinal fluid; NGF

=

Clinical and

nerve

Cerebrospinal Fluid

growth factor;

RS

=

Rett

one

tube

or a

control for any difference in recovery. The nerve growth factor standards were diluted

as a

mouse

to obtain

a

factor. After

concentration range of 1 to 250 pg nerve the plates were again incubated

washing,

overnight at 4°C with 0.1 U/mL of anti-¡3-nerve growth factor-(3galactosidase (Boehringer Mannheim). The plates were washed with 100 mmol/L phosphate buffer (pH 7.3) with 1 mmol/L MgCl 21 and room temperature in the presence of 0.2 mmol/L 4-methylbellifenyl-~3-galactosidase (Sigma, St. Louis,

then incubated for 2 hours at

MO). The increase in optical density was measured in nanometers using a Fluoroscan device. Nonspecific binding obtained in the absence of the first antibody was subtracted from the values for standards and samples. There was a linear range of values for human and mouse nerve growth factor up to the 200-pg standard. The reaction was specific for nerve growth factor because the antibody did not react with either recombinant brain-derived neurotrophic factor or recombinant neurotrophin-3. Statistical analyses between each control group and the Rett syndrome group were done using the nonparametric Mann-Whitney two-tailed two-sample test.

RESULTS Rett

Syndrome

Clinical features and values for cerebrospinal fluid nerve growth factor in 11 Rett syndrome patients are presented in Table 1. Mean cerebrospinal fluid nerve growth factor was 1.8 pg/mL (SD ± 2.6 pg/mL) (Figure 1). Altogether eight of the 11 patients had epilepsy. The two patients (6 and 4 years of age) with the highest values (6.7 and 6.6 pg/mL) also had epilepsy. Two girls had values of 3.0 and 2.7 pg/mL; their ages were 9 and 2 years, and the older patient had epilepsy. One

Nerve Growth Factor Data of 11 Patients With Rett

syndrome; EEG

=

electroencephalogram.

Syndrome

298

months to 5 years (mean, 2.7 years; SD, 1.7 years). The main clinical and cerebrospinal fluid nerve growth factor data are listed in Table 2. The highest value for cerebrospinal fluid nerve growth factor was 24.7 pg/mL, and only one patient had a value below the limit of detectability, the mean being 10.0 pg/mL (SD ± 8.1 pg/mL) (Figure 1). The cerebrospinal fluid nerve growth factor values of this control group and the Rett syndrome group were significantly different (P .024). In the second control group, all six patients had epilepsy, but they were not retarded. Their ages ranged from 6 months to 8 years (mean, 4.4 years; SD, 4.0 years). The data for this group are presented in Table 3. The highest value for cerebrospinal fluid nerve growth factor was 12.2 pg/mL, and the lowest, 2.5 pg/mL; the mean was 7.6 pg/mL (SD ± 3.1 pg/mL) (Figure 1). The cerebrospinal fluid nerve growth factor values for this control group were also significantly higher than the values for the Rett syndrome group (P .004). When we combined all 12 control patients with epilepsy (control groups 1 and 2) and compared them with the Rett syndrome group, the cerebrospinal fluid nerve growth factor values again differed significantly (P .0016), the values for cerebrospinal fluid nerve growth factor being lower in the Rett syndrome group. The third group consisted of 12 patients who had neither epilepsy nor mental retardation. They had a variety of =

Figure 1. The bars show the highest and lowest cerebrospinal fluid nerve growth factor (CSF NGF) concentrations (pg/mL) in the Rett syndrome group (RS) and in three control groups (Contr 1 through Contr 3). The mean for cerebrospinal fluid nerve growth factor in each group (being in the Rett syndrome group 1.8 ± 2.6 pg/mL; in control group 1, 10.0 ± 8.1 pg/mL, in control group 2, 7.6 ± 3.1 pg/mL; and in control group 3, 5.2 ± 5.0 pg/mL) is marked by horizontal line. Characterizing each control group there are abbreviations: e epilepsy; + present; - absent. =

=

r

=

=

retardation;

=

=

patient (aged 16 years) had a detectable but low value for cerebrospinal fluid nerve growth factor, 0.4 pg/mL; she, too, had epilepsy. Taken together, four of the five patients having detectable cerebrospinal fluid nerve growth factor levels had epilepsy. In the remaining six patients with Rett syndrome, no cerebrospinal fluid nerve growth factor was detectable, and four of them had epilepsy. In these 11 patients with Rett syndrome, there was no linear correlation between the ages and the values for cerebrospinal fluid nerve growth factor.

diseases: cerebral infarct (one), arteriovenous malformation (one), juvenile rheumatoid arthritis (one), tic (one), and various symptoms with nonspecific diagnoses (eight). Their ages ranged from 1 month to 11 years (mean, 5.0 years; SD ± 3.7 years). The main clinical features, diagnoses, and cerebrospinal fluid nerve growth factor values for this control group are listed in Table 4. Mean of the value of cerebrospinal fluid nerve growth factor was 5.2 pg/mL (SD ± 5.0 pg/mL) (Figure 1). In their cerebrospinal fluid nerve growth factor values this group differed significantly from the Rett syndrome group (P .034), the values for the patients with Rett syndrome being lower. Mean of the cerebrospinal fluid nerve growth factor values of all 24 controls was 7.0 pg/mL (SD ± 5.7 pg/mL), and the values for cerebrospinal fluid nerve growth factor differed significantly when compared with the Rett syndrome

Controls

=

For ethical reasons, it is impossible to obtain cerebrospinal fluid specimens from healthy children. Our 24 controls were children with various pediatric or neuropediatric problems. The controls were divided into three groups. The first control group comprised six patients with epilepsy and mental retardation, their ages ranging from 9 Table 2.

CSF

=

Control

cerebrospinal fluid; NGF

=

nerve

Group

1 (Retardation and

growth factor;

MRI

=

Epilepsy):

group (P Clinical and

magnetic resonance imaging.

=

.002).

Cerebrospinal

Fluid Nerve Growth Factor Data

299

Table 3.

CSF

=

Control

cerebrospinal fluid; NGF

=

nerve

Group 2 (Epilepsy,

growth factor;

MRI

=

No Retardation): Clinical and

magnetic

resonance

imaging;

In the statistical analysis, there was not a linear correlation between the ages of the patients and their cerebrospinal fluid nerve growth factor values when all the control groups were taken together. Furthermore, the values for the male (12 patients) and female (12 patients) controls did not differ significantly from each other. DISCUSSION

The present results demonstrate that in the cerebrospinal fluid of children suffering from Rett syndrome, the level of nerve growth factor protein is lower than in controls. Previous knowledge of Rett syndrome favors a two-step process; a genetic age-dependent disorder as a basic pre-

disposing factor combined with impaired maturational, biosynthetic, and/or deficient production of some trophic factor affecting brain growth and causing general growth retardation.s The etiology of Rett syndrome is unknown, but the low cerebrospinal fluid nerve growth factor observed here suggests possible involvement of nerve growth factor in this syndrome. Neither the role nor the level of any neurotrophic factor has been determined previously in any of the human neuropediatric diseases. It has been shown earlier that nerve growth factor is elevated by inflammatory processes,1’ mediated in part by Table 4.

CSF

=

Control

cerebrospinal fluid; NGF

=

Group 3 (No Retardation

nerve

growth factor.

or

Epilepsy):

CT

=

Cerebrospinal

Fluid Nerve Growth Factor Data

computed tomographic scan.

interleukin,~ in addition to histamine and basophilic cells.15,19 In adult patients with head injuries, the cerebrospinal fluid nerve growth factor level increases rapidly (1 to 2 days) after injury and then decreases again,20,21 and in patients with multiple sclerosis, the cerebrospinal fluid nerve growth factor is elevated during acute attacks .22 These aspects were considered when we chose our control patients. None of them had signs of acute central nervous system infection or multiple sclerosis or head injury. There are results from animal studies showing that seizure activity of various types stimulates nerve growth factor in the brain.23-25 In the girls with Rett syndrome, the cerebrospinal fluid nerve growth factor level was significantly lower than in the epileptic control patients with or without retardation, although most of the patients with Rett syndrome (eight of 11 patients) also had epilepsy. Our results show a trend for lower concentrations of cerebrospinal fluid nerve growth factor especially in the oldest Rett syndrome patients with epilepsy. Is there some, maybe age-dependent, factor in Rett syndrome that impairs the ability of the brain to respond to epileptic activity, which usually seems to elevate the cerebrospinal fluid nerve growth factor? In Rett syndrome, there is evidence for decreased choline acetyltransferase activity in many cortical and subcortical Clinical and

Cerebrospinal

Fluid Nerve Growth Factor Data

300

regions of brain, and loss of basal forebrain cholinergic neurons, too.l4 These neurons respond to nerve growth factor and to brain-derived neurotrophic factor.26 As far as we know, there are no studies of nerve growth factor messenger RNA or protein in the brains of patients with Rett syndrome. Activation of the

14. 15.

glutamate27 and muscarine28 receptors has been shown

to enhance

growth factor expression in the rat hippocampus. However, whether Rett syndrome is accompanied by changes in specific neurotransmitters or their receptors remains an interesting possibility to be explored further, too. In animal models, intraventricular nerve growth factor nerve

treatment has been shown to

promote the survival of sepafter lesions to the fimbria fornix.11,2s Nerve growth factor also has protective effects against ischemic, excitotoxic, and hypoglycemia-induced neuronal damage.10-31 In some clinical trials, nerve growth factor has been infused into the brains of the patients with Alzheimer’s and Parkinson’s diseases.8,33 Recently, nerve growth factor has been conjugated to an antibody of the transfen-in receptor, and so can cross the blood-brain barrier, which in future may provide a possibility for treating neurodegenerative disorders by intravenous injections We conclude that in children with Rett syndrome, the cerebrospinal fluid content of nerve growth factor was very low. The pathophysiologic role of nerve growth factor in Rett syndrome needs to be investigated further, as well as any possibility of alleviating the effects of the disease by giving nerve growth factor treatment. tal

13.

16.

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Lindholm D, Heumann R, Meyer M, Thoenen H: Interleukin-1 regulates synthesis of nerve growth factor in non-neural cells of rat sciatic nerve. Nature 1987;330:658-659.

19.

Bischoff SC, Dahinden CA: Effect of nerve growth factor on the release of inflammatory mediators by mature human basophils. Blood 1992;79:2662-2669.

20.

Longo FM, Selak I, Zoviekian J, et al: Neurotrophic activities in cerebrospinal fluid of head trauma patients. Exp Neurol 1984;

neurons

84:207-218. 21.

Patterson SL, Grady MS, Bothwell M: Nerve growth factor and a fibroblast growth factor-like neurotrophic activity in cerebrospinal fluid of brain injured human patients. Brain Res 1993;605:43-49.

22.

Laudiero LB, Aloe L, Levi-Montalcini R, et al: Multiple sclerosis patients express increased levels of beta-nerve growth factor in cerebrospinal fluid. Neurosci Lett 1992;147(1):9-12.

23.

Gall CM, Isackson PJ: Limbic seizures increase neuronal production of messenger RNA for nerve growth factor. Science

24.

Gall C, Murray K, Isackson PJ: Kainic-acid-induced seizures stimulate increased expression of nerve growth factor mRNA in rat hippocampus. Mol Brain Res 1991;9:113-123.

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Bengzon J, Söderström S, Kokaia Z, et al: Widespread increase of nerve growth factor protein in the rat forebrain after kindlinginduced seizures. Brain Res 1992;587:338-342. Alderson RF, Alterman AL, Barde Y-A, Lindsay RM: Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 1990;5:297-306. Zafra F, Castren E, Thoenen H, Lindholm D: Interplay between glu-

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Supported by a grant from Arvo and Lea Ylpp6 Foundation.

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