Neurotrophin 3 (NT-3) is a member of the neurotrophin gene family which supports the survival of specific neurons. NT-3 was shown to prevent the death of adult central noradrenergic neurons in vivo, a neuronal population which is associated with the pathophysiology of major depression. We quantitated CSF levels of NT-3 in elderly patients with major depression (DE) and compared them to patients with Alzheimer's disease (AD), and mentally healthy control subjects (CTR). CSF levels of NT-3 were markedly and significantly elevated in the DE group, as compared to either the AD or the CTR group (P < 0.01, and P < 0.001, respectively). in terms of diagnostic accuracy, measurement of nt-3 levels in de resulted in 73.9% sensitivity, and 89.7% specificity. increased csf levels of nt-3 may indicate a disturbance of the central noradrenergic system in patients with de. nt-3 may constitute a biochemical candidate marker for clinical diagnosis and for the evaluation of therapeutic strategies in de.
Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) are members of the neurotrophin gene family which supports the survival, differentiation, maintenance, and repair of vertebrate neurons.1 NGF supports the cholinergic neurons of the basal forebrain system that are affected by neuronal loss in Alzheimer's disease (AD).2 BDNF supports cholinergic, dopaminergic and 5-hydroxytryptamine (5-HT) containing neurons.3, 4, 5 NT-3 was shown to prevent the death of adult central noradrenergic neurons in vivo,6 a neuronal population which is associated with the pathophysiology of major depression (DE).7 In addition, NT-3 reportedly promotes survival of ventral mesencephalic dopaminergic neurons,8 cerebellar granule neurons and Purkinje cells,9 and acts on sensory or sympathetic neurons of the dorsal root, nodose and sympathetic ganglia.10 Dysfunction of neurotrophic systems in the adult mammalian brain may be reflected by alterations of neurotrophin levels in the cerebrospinal fluid (CSF), and may thus be of diagnostic value. Therefore, we quantitated CSF levels of NT-3 in elderly patients with DE and compared them to mentally healthy control subjects (CTR), and to patients with Alzheimer's disease (AD). We show here that CSF levels of NT-3 are increased in patients with DE as compared to the AD and CTR groups.
Subjects and methods
Patients with major depression (DE) were diagnosed according to the ICD-10 (F32.0x/1x, F33.0x/1x) and DSM-III-R (296.20–22, 296.30–32) criteria. Diagnosis of probable AD was made according to criteria of the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's disease and Related Disorders Association (NINCDS–ADRDA).11 All patients were referred to the research ward from general practitioners, neurologists and psychiatrists for diagnostic purposes and screening for clinical trials. None of the patients was institutionalized. The group of healthy control subjects (CTR) consisted of patients who underwent lumbar puncture for orthopedic or neurologic diagnostic purposes and were shown to have normal CSF cell counts, total protein levels, and absence of signs of blood–brain barrier dysfunctions or cerebral IgG synthesis, as well as absence of psychiatric or central neurological disorders.
DE, AD and CTR patients were carefully examined and received a thorough clinical examination. Psychometric testing included the Mini Mental State (MMS),12 as a global screening instrument for dementia, and the Nurses’ Observation Scale for Geriatric Patients (NOSGER)13 as a functional measure of dementia severity. The patients with DE showed no cognitive disturbances in the clinical examinations and the Mini Mental State scores were within the normal range. Severity of depression was rated by using the Montgomery Asberg Depression Rating Scale (MADRS).14 Apolipoprotein (ApoE) genotyping, or, if DNA was not available, ApoE phenotyping was included in the laboratory screening in the DE and AD patients.
CSF was obtained for diagnostic purposes in the DE and AD patients in which no lumbar puncture had been previously done during the routine diagnostic work-up. Different CSF volumes were available for the analysis of the neutrophin proteins. This fact explains the different sample sizes for the individual measurements. All available CSF samples were used for the analyses.
NT-3 measurements. One hundred and twenty-five spinal fluid samples were examined. DE group: n = 23, 8 men, 15 women, mean age 70.5 ± 11.9 SD yrs, range 47–86 yrs, MMS score: mean 27.2 ± 2.5 SD. AD group: n = 39, 20 men, 19 women, mean age 67.2 ± 11.5 SD yrs, range 39–86 yrs, MMS score: mean 19.1 ± 5.3 SD. CTR group: n = 63, 35 men, 28 women, mean age 56.0 ± 15.0 SD yrs, range 28–84 yrs.
AD and CTR patients were free of psychotropic medication. Patients with major depression were treated with various antidepressants: 11 were treated with tricyclics (TCA), two with a monoamine oxidase-A inhibitor (MAOI), one with a combination of TCA with MAOI, six with selective serotonin reuptake inhibitors (SSRI), and three were free of antidepressants at the time of lumbar puncture. It is currently unknown whether these treatments can influence CSF levels of neurotrophins. Informed consent was taken from each patient and their caregivers before the investigation. The study was approved by the local ethics committee. All procedures were in accordance with the 1983 revision of the 1975 Helsinki Declaration. Within one week of clinical work-up and dementia testing, CSF was obtained by lumbar puncture. CSF samples were frozen on dry ice immediately upon withdrawal at the bedside in 0.5-ml aliquots, and were stored at −85°C until biochemical analyses.
CSF levels of NT-3 were determined by using commercially available systems (Promega, Madison, WI, USA), and were performed according to the manufacturer's protocol. One hundred and twenty microlitres of undiluted CSF in carbonate buffer (pH 9.7) were added to 96-well immunoplates (Nunc Inc, Wiesbaden, Germany) at 4°C overnight. Anti-Human-NT-3 polyclonal antibodies (pAb) were used as capture Ab. Anti-NT-3 monoclonal Ab (mAb) were used as reporter Ab. After incubation with a species-specific Ab (anti-rat IgG) conjugated to horseradish peroxidase (HRP) as a tertiary reactant, and washing, the solution was incubated with the chromogenic substrate TMB. Absorbance was measured at 450 nm by using a microplate reader (Dynatech MR 700, Labsystems, Helsinki, Finland). Linear range 4.7–300 pg ml−1; cross-reaction with other neurotrophins at 10 ng ml−1 <3%; detection limit 6.0 pg ml−1. All CSF samples were assayed in duplicate determinations.
ApoE genotyping was by INNO-LiPA Apo E, Innogenetics, Belgium; ApoE phenotyping according to McDowell et al.15 The use of the ApoE phenotype synonymous with the ApoE genotype in the statistical analyses seemed to be appropriate, since ApoE genotyping compared with protein phenotyping showed conflicting results in less than 2%.16
Statistical analyses of data were performed using the Mann-Whitney U test as well as independent samples t-tests for group comparisons. Correlation analyses were performed by multiple regression using CSF levels of neurotrophins, as well as ApoE genotype (or phenotype, respectively), age, duration of the disease in AD, MMS, NOSGER and MADRS scores. Regression analysis was complemented with analysis of variance (ANOVA) by using SPSS for Windows (version 8.0). Statistical significance was assumed at P < 0.05.
CSF levels of NT-3 were significantly elevated in the DE group, as compared to both the AD and the CTR group (P < 0.01, and P < 0.001, Mann-Whitney U test). CSF levels of NT-3 were not different in the AD group, as compared to the CTR group (Figure 1). NT-3 concentrations in CSF of the DE group were 25.8 ± 4.3 pg ml−1 (mean ± SEM, range: 0.0–87.00, n = 23), compared to 14.0 ± 1.6 pg ml−1 in the AD-group (range: 0.0–41.0, n = 39), and 10.5 ± 1.6 pg ml−1 in the CTR group (range: 0.00–67.0, n = 63), respectively (Figure 1). To estimate the accuracy of CSF measurements of NT-3 in the diagnosis of DE, we calculated: (a) sensitivities; and (b) specificities, defined as follows: (i) true positives/(true positives + false negatives); and (ii) true negatives/(true negatives + false positives). To estimate the probability of disease, we calculated the predictive values of the tests. The positive predictive value (PPV) was defined as true positives/(true positives + false positives). The negative predictive value (NPV) was defined as true negatives/(true negatives + false negatives).
Diagnostic accuracy in DE: NT-3 measurement resulted in 73.9% sensitivity, 86.1% specificity, 50.0% PPV, 91.7% NPV using a cut-off value of ≥15.0 pg ml−1 (total n = 125, AD n = 39, DE n = 23, CTR n = 63) (Table 1).
Next, we grouped the DE patients (n = 23) according to treatment with substances that affect central noradrenergic neurotransmission (MAOI and TCA, n = 14) and those that selectively affect serotonergic neurotransmission (SSRI, n = 6). CSF levels of NT-3 were significantly lower in the MAOI/TCA group, as compared to the SSRI group (P < 0.05, independent samples t-test). NT-3 concentrations in CSF of the MAOI/TCA group were 19.7 ± 2.9 pg ml−1 (mean ± SEM, range: 0.0–35.0, n = 14), compared to 34.9 ± 8.5 pg ml−1 in the SSRI group (range: 14.0–63.0, n = 6), and 35.7 ± 26.3 pg ml−1 in the group with patients who were not treated with antidepressants at the time of lumbar puncture (range: 0.00–87.0, n = 3) (Figure 2).
There was no correlation of CSF levels of NT-3 with ApoE genotype (or phenotype, respectively), age, MADRS, MMS, or NOSGER scores.
This is the first study to demonstrate increased levels of the neurotrophin NT-3 in spinal fluid of elderly patients with major depression. CSF measurement of NT-3 levels showed a promising diagnostic accuracy in terms of sensitivity (73.9%) as well as specificity (86.1%). Thus, the NT-3 test constitutes a candidate tool for the biochemical diagnosis of major depression in the elderly. Further experiments will show whether these findings may be expanded to depressive populations of younger age.
Our finding of elevated levels of NT-3 in the CSF of patients with DE might reflect a cerebral alteration of NT-3 production or utilization, since NT-3 is the major neurotrophic factor for central noradrenergic neurons.6 NT-3, but not related neurotrophins, prevents the degeneration of noradrenergic neurons of the locus coeruleus in a 6-hydroxydopamine lesion model.6 Alterations in adrenoreceptor density and function, as well as changes in adrenoreceptors associated with the pituitary-adrenal axis function, strongly implicate abnormal central noradrenergic transmission in DE (for a review see Leonard).17 This dysfunction might be related to the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines. Alternatively, impaired uptake or transport of target-derived neurotrophic factors, including NT-3, may contribute to abnormalities in central noradrenergic function.
This is the first study of CSF levels of NT-3 in DE. NT-3 was previously determined in the CSF of patients with a variety of neurological conditions including hydrocephalus, meningitis, encephalitis, ventriculitis, brain tumors, and multiple sclerosis, with values ranging from 4.8 to 55.9 pg ml−1 determined with the same method as used in this study.18 In the report of Gilmore et al18 NT-3 was not detectable in the CSF of patients with schizophrenia. Another study showed no difference of CSF levels of NT-3 in AD as compared to normal control subjects,19 which is in full agreement with our findings. However, recently, a possible association of a missense mutation (Gly[-63]Glu) of the neurotrophin-3 gene with AD was described in a Japanese population.20 The mutated type was more frequent among the AD patients than the controls (P = 0.011, odds ratio 1.63, 95% CI 1.11–2.38).
Treatment with antidepressants may affect NT-3 levels in the DE group: Smith et al21 showed that chronic treatment with antidepressants that selectively blocked neuronal noradrenaline uptake decreased the expression levels of NT-3 in the locus coeruleus in rats. In contrast, treatment with selective serotonin reuptake inhibitors did not alter NT-3 mRNA levels. These findings suggest that some effects of antidepressants on the function of the locus coeruleus involve NT-3 expression. In addition, these findings make it rather unlikely that the increased CSF levels of NT-3 in patients with DE that were observed in the present study, are due to the effect of treatment with antidepressants. In fact, CSF levels of NT-3 were markedly lower within the DE group in patients treated with substances that affect central noradrenergic neurotransmission (MAOI/TCA) than in patients treated with substances that selectively affect serotonergic neurotransmission (SSRI) as well as in patients who were not treated with antidepressants at the time of lumbar puncture. Thus, treatment with antidepressants that block neuronal noradrenaline uptake may reduce previously increased CSF levels of NT-3 in patients with DE. In contrast, in rat brain, chronic (21-day) administration of several different antidepressant drugs, including tranylcypromine, sertraline, desipramine, significantly increased BDNF mRNA and trkB mRNA in the hippocampus.22 These and other findings constituted the framework for a molecular hypothesis of depression, which postulated that the therapeutic action of antidepressant treatments occurs via intracellular mechanisms that decrease or increase, respectively, neurotrophic factors.23, 24 To date, neither measurements of cerebral protein or expression levels of neurotrophins nor of their specific receptors and their signaling have been reported of patients with major depression. Thus, the present results may stimulate further studies to elucidate the role of NT-3 and related neurotrophins in the pathophysiology of major depression.
In conclusion, CSF levels of NT-3 were markedly increased in elderly patients with DE, as compared to mentally healthy controls and patients with AD. Increased CSF levels of NT-3 may indicate a disturbance of the central noradrenergic system in patients with DE. NT-3 may constitute a biochemical candidate marker for clinical diagnosis and for the evaluation of therapeutic strategies in DE.
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This work was supported by Grants 3100–049397.96/1 (Schweizerischer Nationalfonds) (UO, CH, FM-S), and SFB505 (Deutsche Forschungsgemeinschaft) (UO). We thank F Jancu for technical support and K Kräuchi, PhD, for help in statistical analyses.
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Hock, C., Heese, K., Müller-Spahn, F. et al. Increased cerebrospinal fluid levels of neurotrophin 3 (NT-3) in elderly patients with major depression. Mol Psychiatry 5, 510–513 (2000) doi:10.1038/sj.mp.4000743
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