Reduction in IGF1 mRNA in the Human Subependymal Zone During Aging.

The cell proliferation marker, Ki67 and the immature neuron marker, doublecortin are both expressed in the major human neurogenic niche, the subependymal zone (SEZ), but expression progressively decreases across the adult lifespan (PMID: 27932973). In contrast, transcript levels of several mitogens (transforming growth factor α, epidermal growth factor and fibroblast growth factor 2) do not decline with age in the human SEZ, suggesting that other growth factors may contribute to the reduced neurogenic potential. While insulin like growth factor 1 (IGF1) regulates neurogenesis throughout aging in the mouse brain, the extent to which IGF1 and IGF family members change with age and relate to adult neurogenesis markers in the human SEZ has not yet been determined. We used quantitative polymerase chain reaction to examine gene expression of seven IGF family members [IGF1, IGF1 receptor, insulin receptor and high-affinity IGF binding proteins (IGFBPs) 2, 3, 4 and 5] in the human SEZ across the adult lifespan (n=50, 21-103 years). We found that only IGF1 expression significantly decreased with increasing age. IGFBP2 and IGFBP4 expression positively correlated with Ki67 mRNA. IGF1 expression positively correlated with doublecortin mRNA, whereas IGFBP2 expression negatively correlated with doublecortin mRNA. Our results suggest IGF family members are local regulators of neurogenesis and indicate that the age-related reduction in IGF1 mRNA may limit new neuron production by restricting neuronal differentiation in the human SEZ.

Early-life decline in neurogenesis markers and age-related changes of TrkB splice variant expression in the human subependymal zone.

Neurogenesis in the subependymal zone (SEZ) declines across the human lifespan, and reduced local neurotrophic support is speculated to be a contributing factor. While tyrosine receptor kinase B (TrkB) signalling is critical for neuronal differentiation, maturation and survival, little is known about subependymal TrkB expression changes during postnatal human life. In this study, we used quantitative PCR and in situ hybridisation to determine expression of the cell proliferation marker Ki67, the immature neuron marker doublecortin (DCX) and both full-length (TrkB-TK+) and truncated TrkB receptors (TrkB-TK-) in the human SEZ from infancy to middle age (n = 26-35, 41 days to 43 years). We further measured TrkB-TK+ and TrkB-TK- mRNAs in the SEZ from young adulthood into ageing (n = 50, 21-103 years), and related their transcript levels to neurogenic and glial cell markers. Ki67, DCX and both TrkB splice variant mRNAs significantly decreased in the SEZ from infancy to middle age. In contrast, TrkB-TK- mRNA increased in the SEZ from young adulthood into ageing, whereas TrkB-TK+ mRNA remained stable. TrkB-TK- mRNA positively correlated with expression of neural precursor (glial fibrillary acidic protein delta and achaete-scute homolog 1) and glial cell markers (vimentin and pan glial fibrillary acidic protein). TrkB-TK+ mRNA positively correlated with expression of neuronal cell markers (DCX and tubulin beta 3 class III). Our results indicate that cells residing in the human SEZ maintain their responsiveness to neurotrophins; however, this capability may change across postnatal life. We suggest that TrkB splice variants may differentially influence neuronal and glial differentiation in the human SEZ.

Decline in Proliferation and Immature Neuron Markers in the Human Subependymal Zone during Aging: Relationship to EGF- and FGF-Related Transcripts.

Neuroblasts exist within the human subependymal zone (SEZ); however, it is debated to what extent neurogenesis changes during normal aging. It is also unknown how precursor proliferation may correlate with the generation of neuronal and glial cells or how expression of growth factors and receptors may change throughout the adult lifespan. We found evidence of dividing cells in the human SEZ (n D 50) in conjunction with a dramatic age-related decline (21-103 years) of mRNAs indicative of proliferating cells (Ki67) and immature neurons (doublecortin). Microglia mRNA (ionized calcium-binding adapter molecule 1) increased during aging, whereas transcript levels of stem/precursor cells (glial fibrillary acidic protein delta and achaete-scute homolog 1), astrocytes (vimentin and pan-glial fibrillary acidic protein), and oligodendrocytes (oligodendrocyte lineage transcription factor 2) remained stable. Epidermal growth factor receptor (EGFR) and fibroblast growth factor 2 (FGF2) mRNAs increased throughout adulthood, while transforming growth factor alpha (TGFα), EGF, Erb-B2 receptor tyrosine kinase 4 (ErbB4) and FGF receptor 1 (FGFR1) mRNAs were unchanged across adulthood. Cell proliferation mRNA positively correlated with FGFR1 transcripts. Immature neuron and oligodendrocyte marker expression positively correlated with TGFα and ErbB4 mRNAs, whilst astrocyte transcripts positively correlated with EGF, FGF2, and FGFR1 mRNAs. Microglia mRNA positively correlated with EGF and FGF2 expression. Our findings indicate that neurogenesis in the human SEZ continues well into adulthood, although proliferation and neuronal differentiation may decline across adulthood. We suggest that mRNA expression of EGF- and FGF-related family members do not become limited during aging and may modulate neuronal and glial fate determination in the SEZ throughout human life.

Cell proliferation is reduced in the hippocampus in schizophrenia.

OBJECTIVE: The molecular and cellular basis of structural and functional abnormalities of the hippocampus found in schizophrenia is currently unclear. Postnatal neurogenesis contributes to hippocampal function in animal models and is correlated with hippocampal volume in primates. Reduced hippocampal cell proliferation has been previously reported in schizophrenia, which may contribute to hippocampal dysfunction. METHOD: We measured the cell proliferation marker, Ki67, in post-mortem hippocampal tissue from patients with schizophrenia (n = 10) and matched controls (n = 16). Ki67-labelled cells were counted within the dentate gyrus and hilus on sections taken from the anterior hippocampus. RESULTS: We replicated the finding of a significant reduction in Ki67+ cells/mm² in schizophrenia cases compared to controls (t24 = 2.1, p = 0.023). In our relatively small sample, we did not find a relationship between Ki67+ cells and age overall, or between Ki67 + cells and duration of illness or antipsychotic treatment in people with schizophrenia. CONCLUSION: Our results confirm that reduced hippocampal cell proliferation may be present in schizophrenia. Restoring hippocampal neurogenesis may be a potential therapeutic target for the treatment of hippocampal dysfunction in schizophrenia.

The effect of adolescent testosterone on hippocampal BDNF and TrkB mRNA expression: relationship with cell proliferation.

BACKGROUND: Testosterone attenuates postnatal hippocampal neurogenesis in adolescent male rhesus macaques through altering neuronal survival. While brain-derived neurotropic factor (BDNF)/ tyrosine kinase receptor B (TrkB) are critical in regulating neuronal survival, it is not known if the molecular mechanism underlying testosterone's action on postnatal neurogenesis involves changes in BDNF/TrkB levels. First, (1) we sought to localize the site of synthesis of the full length and truncated TrkB receptor in the neurogenic regions of the adolescent rhesus macaque hippocampus. Next, (2) we asked if gonadectomy or sex hormone replacement altered hippocampal BDNF and TrkB expression level in mammalian hippocampus (rhesus macaque and Sprague Dawley rat), and (3) if the relationship between BDNF/TrkB expression was altered depending on the sex steroid environment. RESULTS: We find that truncated TrkB mRNA+ cells are highly abundant in the proliferative subgranular zone (SGZ) of the primate hippocampus; in addition, there are scant and scattered full length TrkB mRNA+ cells in this region. Gonadectomy or sex steroid replacement did not alter BDNF or TrkB mRNA levels in young adult male rat or rhesus macaque hippocampus. In the monkey and rat, we find a positive correlation with cell proliferation and TrkB-TK+ mRNA expression, and this positive relationship was found only when sex steroids were present. CONCLUSIONS: We suggest that testosterone does not down-regulate neurogenesis at adolescence via overall changes in BDNF or TrkB expression. However, BDNF/TrkB mRNA appears to have a greater link to cell proliferation in the presence of circulating testosterone.

Schizophrenia and bipolar disorder show both common and distinct changes in cortical interneuron markers.

Schizophrenia and bipolar disorder are often viewed as distinct clinical disorders, however there is substantial overlap in their neuropathologies. While compromised cortical interneurons are implicated in both diseases, few studies have examined the relative contribution of the distinct interneuron populations to each psychotic disorder. We report reductions in somatostatin and vasoactive intestinal peptide mRNAs in prefrontal and orbitofrontal cortices in bipolar disorder (n=31) and schizophrenia (n=35) compared to controls (n=34) and increased calbindin mRNA in schizophrenia. We show, at the molecular level, shared deficits in interneuron markers in schizophrenia and bipolar disorder, and a unique interneuron marker increase in schizophrenia.

Rethinking schizophrenia in the context of normal neurodevelopment.

The schizophrenia brain is differentiated from the normal brain by subtle changes, with significant overlap in measures between normal and disease states. For the past 25 years, schizophrenia has increasingly been considered a neurodevelopmental disorder. This frame of reference challenges biological researchers to consider how pathological changes identified in adult brain tissue can be accounted for by aberrant developmental processes occurring during fetal, childhood, or adolescent periods. To place schizophrenia neuropathology in a neurodevelopmental context requires solid, scrutinized evidence of changes occurring during normal development of the human brain, particularly in the cortex; however, too often data on normative developmental change are selectively referenced. This paper focuses on the development of the prefrontal cortex and charts major molecular, cellular, and behavioral events on a similar time line. We first consider the time at which human cognitive abilities such as selective attention, working memory, and inhibitory control mature, emphasizing that attainment of full adult potential is a process requiring decades. We review the timing of neurogenesis, neuronal migration, white matter changes (myelination), and synapse development. We consider how molecular changes in neurotransmitter signaling pathways are altered throughout life and how they may be concomitant with cellular and cognitive changes. We end with a consideration of how the response to drugs of abuse changes with age. We conclude that the concepts around the timing of cortical neuronal migration, interneuron maturation, and synaptic regression in humans may need revision and include greater emphasis on the protracted and dynamic changes occurring in adolescence. Updating our current understanding of post-natal neurodevelopment should aid researchers in interpreting gray matter changes and derailed neurodevelopmental processes that could underlie emergence of psychosis.

Higher gamma-aminobutyric acid neuron density in the white matter of orbital frontal cortex in schizophrenia.

BACKGROUND: In the orbitofrontal cortex (OFC), reduced gray matter volume and reduced glutamic acid decarboxylase 67kDa isoform (GAD67) messenger (m)RNA are found in schizophrenia; however, how these alterations relate to developmental pathology of interneurons is unclear. The present study therefore aimed to determine if increased interstitial white matter neuron (IWMN) density exists in the OFC; whether gamma-aminobutyric acid (GABA)ergic neuron density in OFC white matter was altered; and how IWMN density may be related to an early-expressed inhibitory neuron marker, Dlx1, in OFC gray matter in schizophrenia. METHODS: IWMN densities were determined (38 schizophrenia and 38 control subjects) for neuronal nuclear antigen (NeuN+) and 65/67 kDa isoform of glutamic acid decarboxylase immunopositive (GAD65/67+) neurons. In situ hybridization was performed to determine Dlx1 and GAD67 mRNA expression in the OFC gray matter. RESULTS: NeuN and GAD65/67 immunopositive cell density was significantly increased in the superficial white matter in schizophrenia. Gray matter Dlx1 and GAD67 mRNA expression were reduced in schizophrenia. Dlx1 mRNA levels were negatively correlated with GAD65/67 IWMN density. CONCLUSIONS: Our study provides evidence that pathology of IWMNs in schizophrenia includes GABAergic interneurons and that increased IWMN density may be related to GABAergic deficits in the overlying gray matter. These findings provide evidence at the cellular level that the OFC is a site of pathology in schizophrenia and support the hypothesis that inappropriate migration of cortical inhibitory interneurons occurs in schizophrenia.

Developmental patterns of doublecortin expression and white matter neuron density in the postnatal primate prefrontal cortex and schizophrenia.

Postnatal neurogenesis occurs in the subventricular zone and dentate gyrus, and evidence suggests that new neurons may be present in additional regions of the mature primate brain, including the prefrontal cortex (PFC). Addition of new neurons to the PFC implies local generation of neurons or migration from areas such as the subventricular zone. We examined the putative contribution of new, migrating neurons to postnatal cortical development by determining the density of neurons in white matter subjacent to the cortex and measuring expression of doublecortin (DCX), a microtubule-associated protein involved in neuronal migration, in humans and rhesus macaques. We found a striking decline in DCX expression (human and macaque) and density of white matter neurons (humans) during infancy, consistent with the arrival of new neurons in the early postnatal cortex. Considering the expansion of the brain during this time, the decline in white matter neuron density does not necessarily indicate reduced total numbers of white matter neurons in early postnatal life. Furthermore, numerous cells in the white matter and deep grey matter were positive for the migration-associated glycoprotein polysialiated-neuronal cell adhesion molecule and GAD65/67, suggesting that immature migrating neurons in the adult may be GABAergic. We also examined DCX mRNA in the PFC of adult schizophrenia patients (n = 37) and matched controls (n = 37) and did not find any difference in DCX mRNA expression. However, we report a negative correlation between DCX mRNA expression and white matter neuron density in adult schizophrenia patients, in contrast to a positive correlation in human development where DCX mRNA and white matter neuron density are higher earlier in life. Accumulation of neurons in the white matter in schizophrenia would be congruent with a negative correlation between DCX mRNA and white matter neuron density and support the hypothesis of a migration deficit in schizophrenia.

Expression of VGluT1 and VGAT mRNAs in human dorsolateral prefrontal cortex during development and in schizophrenia.

A balance between excitatory and inhibitory neurotransmission is important in normal brain function, and in schizophrenia a deficit in γ-aminobutyric acid (GABA)ergic inhibitory neurotransmission has been indicated by postmortem studies. We examined the ratio of excitatory to inhibitory vesicular neurotransmitter transporter mRNAs (VGluT1 to VGAT) and their ratio in the dorsolateral prefrontal cortex during normal human development and in people with schizophrenia and controls by quantitative RT-PCR. The ratio of VGluT1/VGAT increased gradually in development to reach a peak at school age (5-12 years), after which levels remained fairly constant into adulthood. The VGluT1 mRNA/VGAT mRNA ratio was unchanged in schizophrenia, as was the ratio of complexin 2 mRNA to complexin 1 mRNA (related to synaptic vesicle fusion in excitatory and inhibitory terminals, respectively). This suggests that the excitatory/inhibitory balance is attained prior to adolescence and is maintained across the rest of the life-span and also indicates that in schizophrenia this balance is not greatly disturbed.

Increased interstitial white matter neuron density in the dorsolateral prefrontal cortex of people with schizophrenia.

BACKGROUND: Interstitial white matter neurons (IWMNs) may reflect immature neurons that migrate tangentially to the neocortex from the ganglionic eminence to form cortical interneurons. Alterations of interneuron markers have been detected in gray matter of dorsolateral prefrontal cortex in schizophrenia, and IWMNs are also reported to be altered in schizophrenia. In this study, we considered whether a potential link exists between these two pathological findings. METHODS: From a cohort of 29 schizophrenia subjects and 37 control subjects, IWMN densities were determined in the dorsolateral prefrontal cortex by counting neuronal nuclear antigen (NeuN) and somatostatin (SST)-positive cells. Double-label immunofluorescence was carried out to determine the overlap between SST+/NeuN+ and SST+/neuropeptide Y + neurons. RESULTS: We found that density of NeuN + IWMNs in superficial white matter is significantly increased in schizophrenia subjects compared with control subjects. There was a significant negative correlation between SST mRNA expression in gray matter and NeuN + IWMN density. In schizophrenic patients with increased NeuN IWMN density, the density of SST-expressing neurons in white matter was also higher compared with control subjects. A subpopulation of SST immunopositive cells also show coexpression of neuropeptide Y. CONCLUSIONS: Our study confirmed previous results indicating that the density of NeuN + IWMNs is increased in superficial white matter in schizophrenia. We provide the first evidence that increased IWMN density correlates with a gray matter interneuron deficit, suggesting that migration of interneurons from white matter to the cortex may be deficient in some patients with schizophrenia, consistent with an interneuron deficit in schizophrenia.

Lack of change in markers of presynaptic terminal abundance alongside subtle reductions in markers of presynaptic terminal plasticity in prefrontal cortex of schizophrenia patients.

BACKGROUND: Reduced synaptic connectivity in frontal cortex may contribute to schizophrenia symptoms. While altered messenger RNA (mRNA) and protein expression of various synaptic genes have been found, discrepancies between studies mean a generalizable synaptic pathology has not been identified. METHODS: We determined if mRNAs encoding presynaptic proteins enriched in inhibitory (vesicular gamma-aminobutyric acid transporter [VGAT] and complexin 1) and/or excitatory (vesicular glutamate transporter 1 [VGluT1] and complexin 2) terminals are altered in the dorsolateral prefrontal cortex of subjects with schizophrenia (n = 37 patients, n = 37 control subjects). We also measured mRNA expression of markers associated with synaptic plasticity/neurite outgrowth (growth associated protein 43 [GAP43] and neuronal navigators [NAVs] 1 and 2) and mRNAs of other synaptic-associated proteins previously implicated in schizophrenia: dysbindin and vesicle-associated membrane protein 1 (VAMP1) mRNAs using quantitative polymerase chain reaction. RESULTS: No significant changes in complexin 1, VGAT, complexin 2, VGluT1, dysbindin, NAV2, or VAMP1 mRNA expression were found; however, expression of mRNAs associated with plasticity/cytoskeletal modification (GAP43 and NAV1) was reduced in schizophrenia. Although dysbindin mRNA did not differ in schizophrenia compared with control subjects, dysbindin mRNA positively correlated with GAP43 and NAV1 in schizophrenia but not in control subjects, suggesting low levels of dysbindin may be linked to reduced plasticity in the disease state. No relationships between three dysbindin genetic polymorphisms previously associated with dysbindin mRNA levels were found. CONCLUSIONS: A reduction in the plasticity of synaptic terminals supports the hypothesis that their reduced modifiability may contribute to neuropathology and working memory deficits in schizophrenia.

Expression of interneuron markers in the dorsolateral prefrontal cortex of the developing human and in schizophrenia.

OBJECTIVE: The onset of schizophrenia symptoms in late adolescence implies a neurodevelopmental trajectory for the disease. Indeed, the γ-aminobutyric acid (GABA) inhibitory system shows protracted development, and GABA-ergic deficits are widely replicated in postmortem schizophrenia studies. The authors examined expression of several interneuron markers across postnatal human development and in schizophrenia to assess whether protracted development of certain interneuron subpopulations may be associated with a particular vulnerability in schizophrenia. METHOD: RNA was extracted postmortem from dorsolateral prefrontal cortex of individuals from age 6 weeks to 49 years (N=68) and from a cohort of normal comparison subjects and schizophrenia patients (N=74, 37 pairs). Expression levels of parvalbumin, cholecystokinin, somatostatin, neuropeptide Y, calretinin, calbindin, and vasoactive intestinal peptide were measured by quantitative reverse transcription-polymerase chain reaction. Changes in calretinin protein levels were examined by Western blot. RESULTS: Interneuron marker genes followed one of three general expression profiles: either increasing (parvalbumin, cholecystokinin) or decreasing (somatostatin, calretinin, neuropeptide Y) in expression over postnatal life, with the most dramatic changes seen in the first few years before reaching a plateau; or increasing to peak expression in the toddler years before decreasing (calbindin, vasoactive intestinal peptide). mRNA expression of all genes, with the exception of calbindin (which increased), showed a reduction (8%-31%) in schizophrenia. Somatostatin showed the most dramatic reduction (31%) in schizophrenia. CONCLUSIONS: It appears that a heterogeneous population of interneurons is implicated in schizophrenia. Further studies are needed to determine whether specific interneuron subpopulations are altered or whether common or distinct upstream pathways are responsible for interneuron deficits in schizophrenia.

The effect of gonadectomy on prepulse inhibition and fear-potentiated startle in adolescent rhesus macaques.

Sex steroids, such as testosterone, can regulate brain development, cognition and modify psychiatric conditions. However, the role of adolescent testosterone in the emergence of cognitive deficits relevant to psychiatric illness has not been directly studied in primates. We examined whether removing testosterone during adolescence in rhesus macaques would affect prepulse inhibition (PPI) and fear-potentiated startle (FPS), which are translational tests of cognition affected in psychiatric disorders. Prepubertal macaques (30 months old) were castrated (n=6) or sham operated (n=6), and PPI and (FPS) were tested before the onset of puberty (34 months old) and after the pubertal surge in sex hormones 16 months later (50 months old). As expected there were no differences between the gonadectomized and intact groups' level of startle amplitude, PPI or (FPS) before puberty. After puberty, the intact group displayed substantially less PPI than the gonadectomized group, consistent with evidence that PPI is attenuated by endogenous increases in sex hormones. At the end of the study, testosterone among the intact monkeys was also correlated with tyrosine hydroxylase levels in the putamen, suggesting the attenuation of PPI by gonadal sex hormones may be influenced by subcortical dopamine. Thus, puberty involves significant increases in sex hormones, which in turn may modulate subcortical dopamine synthesis and affect cognitive functions impaired in psychiatric illnesses such as schizophrenia.

Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury.

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.