These results provide evidence that a critical cell signaling mechanism is disrupted at the time of birth in individuals destined to develop schizophrenia as adults, such that neurotrophic factors fail to be recruited during biological stress invoked by birth hypoxia, potentially leading to dendritic atrophy and disruption of synaptogenesis in the fetus.
The downregulation of BDNF was specific to the fetus and was not observed in maternal samples obtained at the time of delivery. The specificity of this effect to the cord samples held regardless of whether the analysis of maternal samples was limited to those subjects who also had cord samples available. This pattern suggests that a fetus-specific vulnerability factor results in dysregulation of neurotrophic signaling in response to birth hypoxia. Notably, the downregulation of BDNF was specific to birth hypoxia – there were no differences in expression of BDNF in either cord or maternal samples in relation to maternal toxemia/SGA status or prematurity. Some degree of specificity of acute perinatal hypoxia is to be expected given that BDNF levels in cord samples from the time of delivery should index perinatal complications more directly than complications arising earlier in the course of pregnancy. It remains to be determined whether the latter may also be associated with failure to mount a fetal neuroprotective response via BDNF. Notably, however, a similar profile of specificity has been observed in the association of birth hypoxia, and not SGA status or prematurity, with gray matter reduction in schizophrenia patients and their siblings, and differentially so compared with normal controls (9
The factors responsible for the differential pattern of neurotrophin expression in cases versus controls as a function of hypoxia remain to be identified. We examined and ruled out the Val66Met polymorphism in the BDNF gene as moderating this effect, although it should be noted that the Met allele frequency observed in this predominantly African-American sample is much lower than has been seen in predominantly Caucasian samples from the US population (35
) but is comparable to other populations of African origin (see http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=6265
). Given that circulating BDNF may emanate from platelets, brain neurons, vascular endothelial cells, activated T cells, B cells and monocytes (36
), it is not possible to isolate the tissue source of the reduced BDNF levels observed in this study to brain neurons. Nevertheless, it is worth noting that postmortem studies have observed reduced mRNA expression of BDNF receptors TrkB and TrkC in the prefrontal cortex of schizophrenia patients (17
), and in animal models, a reduction in TrkB expression is associated with dendritic atrophy and decreased spine density in pyramidal neurons within kainic acid induced lesion sites (26
), while there are no reports examining its expression in platelets or vascular endothelial cells. Further, in patients with chronic schizophrenia, circulating BDNF levels are lower in serum but not whole blood, suggesting a deficit in neurotrophic factor release (38
). BDNF neuroprotection is regulated by the Ras-MAPK signaling pathway and depends on sustained activation of the cAMP response element-binding protein (CREB) and ERK 1/2 phosphorylation (18
). It is of interest in this regard that several putative susceptibility genes for schizophrenia are involved in Ras-MAPK and ERK signaling (39
) and along with BDNF and its receptors may thus be considered candidate genes for mediating the differential BDNF response to obstetric insults in individuals who develop schizophrenia as adults. Given that cord blood BNDF may reflect immune system function (45
), and considering that immune cell BDNF secretion is associated with white matter volume in multiple sclerosis (46
), it is also possible that an immunological mechanism is involved in the lower BDNF levels among cases exposed to hypoxia.
Overall, the rate of birth hypoxia was equivalent in the cases and controls in this study. However, in the overall cohort from which the cases and controls were drawn, exposure to birth hypoxia was associated with an increased risk for an early onset form of schizophrenia (2
). In the subset of cases and controls used in this study, the same degree of association between hypoxia and early-onset schizophrenia was present, but with the smaller sample size, at only a trend level of significance (p=0.08). A specific association between hypoxia-associated obstetric complications and an early onset of form of schizophrenia has also been replicated in an independent cohort from Finland (3
). In view of this pattern, and considering the differential BDNF response among cases versus controls, birth hypoxia may not increase the risk for developing schizophrenia in the absence of a particular biological vulnerability, such as failure to mount a neurotrophin protective response. However, the presence of both this biological vulnerability and birth hypoxia may contribute to an increased risk for a form of schizophrenia characterized by an early age at onset. While discussion of the potential mechanisms involved is necessarily speculative at this point, such a pattern could reflect a reduced degree of dendritic complexity and synaptic density arising at birth, which then interacts with subsequent regressive brain developmental processes during adolescence (e.g., synaptic pruning) in pushing individuals below some critical threshold of interneuronal connectivity associated with expression of psychosis (5
). If this interpretation is correct, it is possible that administration of neurotrophins (perhaps modified chemically to promote increased CNS penetration) during critical stages of brain development may have some protective benefits in individuals who are genetically susceptible to schizophrenia.
It is also possible that, rather than moderating the effect of birth hypoxia on cord BDNF levels, genetic factors associated with vulnerability to future schizophrenia cause both a higher rate of birth hypoxia and lower BDNF levels in fetal serum. This interpretation is somewhat less likely, however, given that presence of a family history of schizophrenia is not correlated with birth hypoxia independently of maternal health risk behaviors (48
One limitation of this study is that the majority of cases were diagnosed by chart review rather than by direct interview. The chart-review diagnoses were found to be reliable; independent evaluations of a random sample of medical records by different reviewers produced a high rate of diagnostic agreement (κ=.85, 93% simple agreement), and there was high agreement between the chart-review-based diagnoses and those based on the Structured Clinical Interview for DSM-IV diagnoses in the 15 cases who were interviewed directly (i.e., 92.8%).
Another limitation is that ascertainment of psychiatric morbidity relied on a recorded history of local psychiatric service utilization, which missed cases who were deceased or had not yet come to treatment at the time of follow-up, who had been treated at facilities that were no longer operating or did not cooperate with the chart reviews, or who, because of emigration or changes in social class, utilized psychiatric facilities other than those whose patient rolls were screened. Our use of a case-control (rather than a cohort analytic) design mitigates the potential biasing effects of incomplete ascertainment to the extent that the cases used in the study are representative of the overall pool of affected individuals in the birth cohort. One indication that the cases who were included in this study are likely to be representative is that they were found to be comparable in terms of demographics and obstetric history to the 121 probands with a treated history of schizophrenia or schizoaffective disorder whose charts were not available for review and were thus not included in the serological study. Of note, the control sample for this study included all remaining members of the cohort, not simply unaffected controls. This helps mitigate the concern that the observed differences between cases and controls could be attributable to atypical patterns of BDNF expression in the controls. While some cases of untreated or otherwise unascertained schizophrenia or affective psychosis could have been included as controls, the rate of ascertained morbidity for these diagnoses in this cohort was quite high and comparable to that in similar populations (2
). Further, the existence of any such misclassified controls would result in an under-estimation (rather than over-estimation) of the BDNF effect in the cases.
Although the samples of schizophrenia cases and controls were large and sufficient for detecting statistical differences between groups, the numbers of affective psychosis cases with and without birth hypoxia were relatively small, and small sample sizes could have mitigated against detecting effects in this group.
The samples were stored for 40+ years at −20° C. Protease inhibitors were not added to the samples, but serum contains a number of protease inhibitors such as alpha-1 antitrypsin so the amount of proteolytic cleavage while frozen is limited. Steroid protein measurements from NCPP samples are consistent with values from published studies of fresh samples collected at similar points in gestation (49
), and the measured values of BDNF from this study are comparable to those from other studies using fresh samples conducted by the Yolken lab. The fact that the samples for cases and controls were collected and stored in an identical fashion (blindly with respect to adult psychiatric outcome) makes it very unlikely that the results of this study could be due to storage artifacts.
Given the higher rate of schizophrenia among males in this cohort (2
), it could be argued that there is an under-ascertainment of female schizophrenia patients, such that the present results may generalize only to male schizophrenia patients. Arguing against this interpretation, however, is the fact that the hypoxia effect on risk for schizophrenia in this cohort was not
differential according to gender (2
), and the differential BDNF response to hypoxia in cases versus controls detected in this study was independent of gender.
Rates of schizophrenia are known to vary by factors such as urban residence, social class, and minority status, factors whose variability is greatly truncated in this cohort compared with the general population. Unfortunately, the truncated racial diversity did not permit us to examine race as a modifier of the differential BDNF response to birth hypoxia in cases versus controls. A race-specific effect seems unlikely, however, since hypoxia-related OCs have been found to be associated with schizophrenia in several Scandinavian countries whose populations are nearly entirely Caucasian (3
In conclusion, we found that individuals exposed to fetal hypoxia and who developed schizophrenia as adults showed a decreased level of BDNF in cord blood samples from the time of birth, findings that suggest dysregulation of neurotrophic signaling in the pathogenesis of schizophrenia and suggest novel molecular targets for preventive intervention.