Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted P<0.1). In depression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted P<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted P<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

Psychiatric brain collection in Macedonia: general lessons for scientific collaboration among countries of differing wealth.

Macedonia is a small country, and the current state has been independent for only 22 years. Medical research, which requires an extensive infrastructure, has been limited. We describe our experience in developing Macedonian research through a mutually beneficial collaboration between institutions in Macedonia and the United States.

Unaltered neuronal and glial counts in animal models of magnetic seizure therapy and electroconvulsive therapy.

Anatomical evidence of brain damage from electroconvulsive therapy (ECT) is lacking; but there are no modern stereological studies in primates documenting its safety. Magnetic seizure therapy (MST) is under development as a less invasive form of convulsive therapy, and there is only one prior report on its anatomical effects. We discerned no histological lesions in the brains of higher mammals subjected to electroconvulsive shock (ECS) or MST, under conditions that model closely those used in humans. We sought to extend these findings by determining whether these interventions affected the number of neurons or glia in the frontal cortex or hippocampus. Twenty-four animals received 6 weeks of ECS, MST, or anesthesia alone, 4 days per week. After perfusion fixation, numbers of neurons and glia in frontal cortex and hippocampus were determined by unbiased stereological methods. We found no effect of either intervention on volumes or total number or numerical density of neurons or glia in hippocampus, frontal cortex, or subregions of these structures. Induction of seizures in a rigorous model of human ECT and MST therapy does not cause a change in the number of neurons or glia in potentially vulnerable regions of brain. This study, while limited to young, healthy, adult subjects, provides further evidence that ECT and MST, when appropriately applied, do not cause structural damage to the brain.

Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression.

Impaired brain serotonin neurotransmission is a potential component of the diathesis of major depression. Tryptophan hydroxylase-2 (TPH2), is the rate limiting biosynthetic isoenzyme for serotonin that is preferentially expressed in the brain and a cause of impaired serotonin transmission. Here, we identify a novel TPH2 short isoform with truncated catalytic domain expressed in human brainstem, prefrontal cortex, hippocampus and amygdala. An exploratory study of 166 Caucasian subjects revealed association with major depression or suicide of a novel single nucleotide polymorphism (SNP) g.22879A>G located in exon 6 of this short isoform. This SNP and additional SNPs were discovered through a systematic characterization of the genetic architecture of the TPH2 gene for further genetic and functional investigations of its relationship to major depression and other psychopathology.

Postmortem mu-opioid receptor binding in suicide victims and controls.

BACKGROUND: Endogenous opiates may reinforce self-injurious behavior in animal and human subjects. Higher postmortem mu-opioid receptor binding is reported in some brain regions in young compared with older suicide victims. The present study compared opioid receptor binding kinetics in postmortem brains of young suicide victims and matched controls in two brain areas. METHODS: The density (Bmax) and affinity (K(D)) of the mu-opioid receptors were assayed postmortem using [3H] DAGO in prefrontal cortex (PFC) and pre-post central gyri (PPCG) in 9 suicide victims and 10 controls, matched for age and gender ratio. RESULTS: Binding indices did not differ between suicide victims and controls in either brain area and did not correlate with age. PFC Bmax was higher than PPCG Bmax (F=8.030; df=1,16; p=.012) for the combined sample. There was no brain region difference in K(D) between PFC and PPCG, but the interaction between K(D) and group was significant (F = 5.890; df = 1,16; p = .027). The K(D) in the suicide victims was lower than controls in the PFC and higher than controls in the PPCG. CONCLUSION: Our study demonstrated more mu-opioid receptors in PFC compared with PPCG binding regardless of suicide status. The region-dependent differences in binding affinity in suicide victims may reflect regionally different opiate transmission.

Abnormalities of myelination in schizophrenia detected in vivo with MRI, and post-mortem with analysis of oligodendrocyte proteins.

Schizophrenia unfolds during the late period of brain maturation, while myelination is still continuing. In the present study, we used MRI and T2 relaxation analysis to measure the myelin water fraction in schizophrenia. In schizophrenia (n=30) compared with healthy subjects (n=27), overall white matter showed 12% lower myelin water fraction (P=0.031), with the most prominent effects on the left genu of the corpus callosum (36% lower, P=0.002). The left anterior genu was affected in both first-episode (P=0.035) and chronic patients (P=0.011). In healthy subjects, myelin water fraction in total white matter and in frontal white matter increased with age, and with years of education, indicating ongoing maturation. In patients with schizophrenia, neither relation was statistically significant. Post-mortem studies of anterior frontal cortex demonstrated less immunoreactivity of two oligodendrocyte-associated proteins in schizophrenia (2',3'-cyclic nucleotide 3'-phosphodiesterase by 33%, P=0.05; myelin-associated glycoprotein by 27%, P=0.14). Impaired myelination in schizophrenia could contribute to abnormalities of neural connectivity and persistent functional impairment in the illness.

Altered immunoreactivity of complexin protein in prefrontal cortex in severe mental illness.

Recent imaging and postmortem studies suggest that impaired connectivity is involved in the pathophysiology of schizophrenia and major affective disorders. We investigated the presynaptic proteins complexin (Cx) I and Cx II in postmortem prefrontal cortex in schizophrenia (n = 13; six suicide, seven nonsuicide), major depression (n= 11, all suicide) and controls (n = 11) with an enzyme-linked immunoadsorbent assay (ELISA). Overall analysis indicated a significant difference between groups (F = 3.93, P = 0.007). Cx I (enriched in inhibitory terminals) was decreased 33% in schizophrenia (26% in schizophrenia/nonsuicide, 42% in schizophrenia/suicide) and 27% in major depression. Cx II (enriched in excitatory terminals) was not significantly different. Analysis of the ratio of Cx II/Cx I was carried out as an indication of the balance of excitatory to inhibitory terminals. A significant difference between groups (ANOVA, F = 6.42, P = 0.005) was observed. The mean value of Cx II/Cx I was significantly increased by 34% in schizophrenia (26% in schizophrenia/nonsuicide and 43% in schizophrenia/suicide) and by 32% in depression compared with control (Student-Newman-Keuls test, P = 0.05). Immunoreactivities of the two complexins were highly correlated in all groups. However, compared with controls and depression, samples from cases with schizophrenia appeared to have relatively less Cx I for similar amounts of Cx II. Immunocytochemical studies of rat frontal cortex after 3 weeks treatment with chlorpromazine, trifluoperazine or haloperidol revealed no differences in complexins, synaptophysin, SNAP-25, syntaxin or VAMP in comparison with animals treated with vehicle. Alterations of complexins may contribute to the molecular substrate for abnormalities of neural connectivity in severe mental disorders.

Extramedullary hematopoiesis within a frontoethmoidal encephalocele in a newborn with holoprosencephaly.

We present the first report of extramedullary hematopoiesis (EMH) in an encephalocele. The patient was a new-born with semilobar holoprosencephaly, a frontoethmoidal encephalocele, and a large subdural hematoma. The encephalocele appeared as a hemorrhagic mass, protruding from the forehead to cover the right eye, without involvement of the sinuses or nasopharynx. Computerized tomography and magnetic resonance imaging studies ruled out other forms of holoprosencephaly and confirmed the continuity of the brain with the extruded mass. Immunohistochemistry confirmed the presence of an atrophic epithelium covering the mass. Histologic examination of the encephalocele revealed EMH both within and adjacent to malformed cerebral cortex, with a tendency for the hematopoietic cells to line up in columns within malformed cerebral cortex. We propose that a single event during the fourth week of gestation could both interrupt closure of the neural tube, giving rise to the encephalocele, and impair migration of the neural crest, leading to holoprosencephaly secondary to failure of neural crest derivatives to induce basomedial telencephalic differentiation. EMH may have been induced from hematopoietic stem cells in the richly vascular meningeal component of the encephalocele, in response to anemia and hypoxia.

A serotonin transporter gene promoter polymorphism (5-HTTLPR) and prefrontal cortical binding in major depression and suicide.

BACKGROUND: Major depression and suicide are associated with fewer serotonin transporter (5-HTT) sites. The 5'-flanking promoter region of the 5-HTT gene has a biallelic insertion/deletion (5-HTTLPR). We assayed prefrontal cortical (PFC) 5-HTT binding in major depression and suicide and examine the relationship to the 5-HTTLPR allele. METHODS: Postmortem brain samples from 220 individuals were genotyped for the 5-HTTLPR polymorphism. Binding of 5-HTT was assayed by quantitative autoradiography in the PFC of a subset of subjects (n = 159). Clinical information, including DSM-III-R Axis I diagnoses, was obtained by psychological autopsy and medical chart review. RESULTS: Binding to 5-HTT was lower in the ventral PFC of suicides compared with nonsuicides and was lower throughout the PFC of subjects with a history of major depression. The 5-HTTLPR genotype was associated with major depression but not with suicide or 5-HTT binding. CONCLUSIONS: A diffuse reduction of 5-HTT binding in the PFC of individuals with major depression may reflect a widespread impairment of serotonergic function consistent with the range of psychopathologic features in major depression. The localized reduction in 5-HTT binding in the ventral PFC of suicides may reflect reduced serotonin input to that brain region, underlying the predisposition to act on suicidal thoughts. The 5-HTTLPR genotype was not related to the level of 5-HTT binding and does not explain why 5-HTT binding is lower in major depression or suicide. Arch Gen Psychiatry. 2000;57:729-738

Structural abnormalities of subicular dendrites in subjects with schizophrenia and mood disorders: preliminary findings.

BACKGROUND: Postmortem studies of the subiculum from subjects with schizophrenia have detected smaller pyramidal cell bodies and diminished immunoreactivity for the dendritic protein, microtubule-associated protein 2. While these findings suggest that subicular pyramidal cell dendrites may be structurally altered in subjects with schizophrenia, this possibility had not been tested directly. METHODS: Rapid Golgi impregnation of archival brain specimens was used to compare the morphologic characteristics of subicular dendrites in subjects with schizophrenia (n = 13) and mood disorders (n = 6) with subjects without psychiatric disease (n = 8). The specimens were processed and analyzed by physicians blind to diagnosis. The extent of dendritic trees in the subiculum and fusiform gyrus was examined by Sholl analysis. Spine density on apical dendrites of subicular pyramidal cells was determined at a fixed distance from the cell body. RESULTS: Spine density and arborization of subicular apical dendrites were significantly related to diagnostic group. Spine density was significantly lower in the schizophrenia and mood disorder groups than in the nonpsychiatric group. Among the mood disorder cases, diminished spine density was apparently related to a strong family history of major psychiatric diseases. There were no significant effects of diagnostic group on Sholl analysis of nonapical subicular dendrites nor on Sholl analysis of dendrites of neocortical pyramidal cells in the fusiform gyrus. CONCLUSIONS: We have observed an association between schizophrenia and major mood disorders and structural abnormalities of subicular apical dendrites. Further studies are needed to test this association in a larger sample and to evaluate the potential role of family history and of confounding factors, such as medications and chronic institutionalization.

Local activation of the complement system in endoneurial microvessels of diabetic neuropathy.

Quantitative immunocytochemical analysis of complement proteins (CP) was performed on sural nerve biopsies from 15 patients with diabetic neuropathy (DN) and 18 nondiabetic patients with other forms of chronic neuropathy (ON). The mean age of the patients and the pathological severity of the neuropathy were similar in both groups. The percentage of patients that expressed strongly immunoreactive CP in the walls of endoneurial microvessels was significantly greater in DN than in ON for all proteins tested. C3d neoantigen was expressed in 100% of DN cases compared with 17% of ON; and membrane attack complex (MAC), C5b-9 neoantigen, in 93% of DN and 17% of ON. In the cases with DN, 81% of endoneurial microvessels, as identified by the endothelial marker, Ulex europaeus, contained C5b-9 neoantigen deposits, compared with 22% in those of ON, and the staining in DN was significantly more intense. Expression of the neoantigens of C3d and C5b-9 in nerve implies local activation of the complement system. In DN, activation of the complement pathway and formation of the MAC could injure blood vessels and adversely affect the circulation in the endoneurium.

Myelin staining of archival brain tissue.

To evaluate the feasibility of staining for myelin in archival materials, paraffin blocks were prepared from brain tissue that had been in formalin for intervals ranging from 7 months to over 53 years. Verhoeff and Luxol fast blue stains of the resulting sections yielded staining whose quality was unaffected by duration of fixation. Myelinated and unmyelinated areas were clearly distinguished, and the morphology of individual myelin sheaths was well-preserved. No changes to conventional protocols were required, but it was necessary carefully to monitor the progress of differentiation. With antigen retrieval, it was possible to display immunoreactivity for myelin basic protein. While this persisted even after prolonged fixation, fine detail was lost from the myelin sheaths, and there was staining of oligodendroglial cytoplasm and nuclei, which was not seen in recently fixed tissue. In contrast to this loss of detail in myelin sheaths, immunohistochemistry for glial fibrillary acidic protein displayed astrocytic morphology clearly, even in the oldest tissue. We conclude that archival, formalin-fixed material can be adequately examined for myelin loss and astrocytosis.

Synaptic and plasticity-associated proteins in anterior frontal cortex in severe mental illness.

Abnormalities of proteins involved in neurotransmission and neural plasticity at synapses are reported in schizophrenia, and may be markers of dysregulated neural connectivity in this illness. Studies of brain development and neural regeneration indicate a dynamic interplay between neural and oligodendroglial mechanisms in regulating synaptic plasticity and axonal sprouting. In the present study, markers of synapses (synaptophysin), plasticity (growth-associated protein-43) and oligodendrocytes (myelin basic protein) were investigated in anterior frontal cortex homogenates from individuals with schizophrenia and depression. Synaptophysin immunoreactivity was reduced in schizophrenics who died of natural causes relative to controls. Myelin basic protein immunoreactivity was decreased in both schizophrenics and depressed individuals who died by suicide. Overall, no changes were observed in growth-associated protein-43 immunoreactivity. However, a slight increase in immunoreactivity in depressed suicides relative to control was observed. These findings support the hypothesis that synaptic abnormalities are a substrate for disordered connectivity in severe mental illness, and suggest that synaptic-oligodendroglial interactions may contribute to the mechanism of dysregulation in certain cases.

Application of cognitive scales to medical records of schizophrenia inpatients.

The Scales of Cognitive Impairment Rated From Institutional Records (SCIRFIR), a battery based on commonly used dementia rating instruments, was tested on the records of 26 chronically institutionalized, elderly schizophrenia patients, for the purpose of retrospectively evaluating the long-term course of cognitive change in schizophrenia and relating it to available autopsy materials. The inter-rater reliability of the component scales was high (Intraclass Correlations = 0.78-0.96), the final item scores were comparable to ratings on living subjects, and Alzheimer-type neuropathological changes were associated with a markedly deteriorating course. The substantial potential of this method is discussed.

Senile degeneration and cognitive impairment in chronic schizophrenia.

OBJECTIVE: This study was an investigation of the role of Alzheimer-type senile degenerative abnormalities in the cognitive impairment of chronic schizophrenia. METHOD: The study group comprised 145 deceased elderly institutionalized psychiatric patients: 66 with schizophrenia, 26 with mood disorders, 36 with dementia, and 17 with other psychiatric diagnoses. The comparison group included 16 deceased elderly individuals without neurologic or psychiatric disease. Psychiatric diagnoses and cognitive status were established by standardized review of medical records. Neuritic senile plaques and neurofibrillary tangles were identified immunohistochemically and counted, by investigators blind to clinical information, in standardized regions of each brain. RESULTS: Of the subjects with schizophrenia, 68% had definite cognitive impairment, but only 8% satisfied neuropathological criteria for Alzheimer's disease. Among the schizophrenia subjects without Alzheimer's disease, definite cognitive impairment was associated with higher levels of plaques and tangles. The schizophrenia subjects without definite cognitive impairment had fewer plaques and tangles than the unimpaired nonpsychiatric subjects. CONCLUSIONS: Most cases of cognitive impairment in schizophrenia could not be attributed to Alzheimer's disease. An association of mild Alzheimer-type pathology with definite cognitive impairment was unique to schizophrenia. Enhanced sensitivity to the effects of aging on the brain may be a manifestation of diminished cognitive reserve in schizophrenia.

Archival, formalin-fixed tissue: its use in the study of Alzheimer's type changes.

Preparing for a retrospective study of senile degeneration in schizophrenia, we had occasion to explore the suitability of an old collection formalin-fixed brains and paraffin blocks for study by modern staining methods. Tissue that had been in formalin for 50 years was embedded in paraffin. Sections were then stained with thioflavine S and with immunoperoxidase stains using Alz 50 and antibodies to paired helical filaments, ubiquitin, and beta-amyloid. In all 4 cases that had originally (50 years earlier) received neuropathologic diagnoses of Alzheimer's disease, large numbers of neocortical senile plaques and neurofibrillary tangles were clearly demonstrated by thioflavine S stain and by immunohistochemistry for paired helical filaments, ubiquitin, and beta-amyloid. In each of 4 other cases, in which the original neuropathologic examination had not revealed Alzheimer's disease, no plaques or tangles were observed. Immunoreactivity with Alz 50 was completely absent after 50 years in formalin. Examination of additional cases of Alzheimer's disease revealed that Alz 50 immunoreactivity was well preserved after 10 years in formalin and completely absent after 30 years in formalin. Alzheimer's disease tissue stored in paraffin for 30 years was clearly stained by all modalities. We conclude that immunohistochemical identification of senile plaques and neurofibrillary tangles is practical even after decades of storage in formalin or paraffin. The applicability of techniques that did not exist when these specimens were collected indicates that the systematic, permanent retention of formalin-fixed material may yield unanticipated future benefits.

Postmortem studies of the hippocampal formation in schizophrenia.

Many postmortem studies report differences between the hippocampal formations of patients with schizophrenia and those of controls. These differences include volume changes, cell density changes, periventricular gliosis, senile degenerative changes, and abnormalities of neuronal size, position, or orientation. However, the findings are almost never common to all schizophrenia patients within a series. Furthermore, some well-designed studies are negative, and different positive reports are mutually contradictory. Some of the inconsistencies are methodological. The normal variation, over small distances, in the cytoarchitecture of the temporal allocortex creates particular difficulties when this region is studied with a limited number of sections, especially if the sample size is small. Other inconsistencies are probably the result of case selection. We review the methods and findings of some of these studies, stressing the dangers of eliminating (rather than evaluating) cases with definite neuropathologic changes. We conclude that the existing postmortem studies of temporal lobe morphology provide little conclusive evidence for the neural substrate of schizophrenia.

Effects of diet and development upon the uptake and distribution of cerebral iron.

In order to determine whether iron sequestered by the rat brain during the third week of postnatal life could be mobilized by subsequent dietary iron deficiency (ID), iron-59 (59Fe) was administered to rats at 2 weeks of age. The animals were placed on an ID or a control diet from age 4 through 8 weeks and killed by perfusion. Brain radioactivity was identical for both groups, and autoradiography revealed no differences in the distribution of radioactivity. Thus, neither the sequestration of cerebral iron acquired at age 2 weeks nor its subsequent redistribution was affected by ID. Since ID beginning after age 3 weeks reportedly produces a cerebral iron deficit that is in part reversible, an attempt was made to determine whether 59Fe administered after ID was preferentially delivered to any brain region. Rats were placed on an ID or a control diet from age 3 through 7 weeks and then injected with 59Fe, placed on a normal diet, and killed 2 weeks later. Thre was no difference between groups in amount or distribution of brain 59Fe, except in the choroid plexus, which was more radioactive in the ID rats than in the controls. This finding may represent a mechanism by which the choroid plexus buffers the brain against rapid rises in plasma iron content.