Guo-Jen Huang

Mutations in α-Tubulin Cause Abnormal Neuronal Migration in Mice and Lissencephaly in Humans

Summary The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of α-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.

The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone

The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.

Stimulation of Neurogenesis in the Hippocampus of the Adult Rat by Fluoxetine Requires Rhythmic Change in Corticosterone

BACKGROUND Fluoxetine stimulates proliferation of progenitor cells in the dentate gyrus of the adult hippocampus. There are suggestions that this action may underlie the therapeutic effects of such drugs in depression. Glucocorticoids also regulate neurogenesis, and there are multiple interactions between serotonin and corticoids. Diurnal cortisol rhythms are dysregulated in depression. We explored the role of diurnal variations in corticosterone on the ability of fluoxetine to alter neurogenesis in the dentate gyrus. METHODS We manipulated plasma corticosterone by implanting corticosterone pellets or giving daily corticosterone injections to corticosterone-clamped adrenalectomized or intact rats that received fluoxetine or vehicle treatment. Proliferation of progenitor cells in the dentate gyrus was measured using BrdU or Ki-67. RESULTS Our results strongly suggest that a diurnal rhythm in corticosterone is necessary for fluoxetine to stimulate neurogenesis in the adult dentate gyrus in the male rat. Preliminary data suggest this may be related to the 5-HT1A receptor. CONCLUSIONS If altered neurogenesis in the dentate gyrus is part of the therapeutic response to antidepressants such as fluoxetine, the results we report suggest that concurrent manipulation of the HPA axis might improve sensitivity to selective serotonin reuptake inhibitors in some treatment-resistant patients.

Molecular Signatures of Major Depression

Summary Adversity, particularly in early life, can cause illness. Clues to the responsible mechanisms may lie with the discovery of molecular signatures of stress, some of which include alterations to an individual’s somatic genome. Here, using genome sequences from 11,670 women, we observed a highly significant association between a stress-related disease, major depression, and the amount of mtDNA (p = 9.00 × 10−42, odds ratio 1.33 [95% confidence interval [CI] = 1.29–1.37]) and telomere length (p = 2.84 × 10−14, odds ratio 0.85 [95% CI = 0.81–0.89]). While both telomere length and mtDNA amount were associated with adverse life events, conditional regression analyses showed the molecular changes were contingent on the depressed state. We tested this hypothesis with experiments in mice, demonstrating that stress causes both molecular changes, which are partly reversible and can be elicited by the administration of corticosterone. Together, these results demonstrate that changes in the amount of mtDNA and telomere length are consequences of stress and entering a depressed state. These findings identify increased amounts of mtDNA as a molecular marker of MD and have important implications for understanding how stress causes the disease.

Ablating Adult Neurogenesis in the Rat Has No Effect on Spatial Processing: Evidence from a Novel Pharmacogenetic Model

The function of adult neurogenesis in the rodent brain remains unclear. Ablation of adult born neurons has yielded conflicting results about emotional and cognitive impairments. One hypothesis is that adult neurogenesis in the hippocampus enables spatial pattern separation, allowing animals to distinguish between similar stimuli. We investigated whether spatial pattern separation and other putative hippocampal functions of adult neurogenesis were altered in a novel genetic model of neurogenesis ablation in the rat. In rats engineered to express thymidine kinase (TK) from a promoter of the rat glial fibrillary acidic protein (GFAP), ganciclovir treatment reduced new neurons by 98%. GFAP-TK rats showed no significant difference from controls in spatial pattern separation on the radial maze, spatial learning in the water maze, contextual or cued fear conditioning. Meta-analysis of all published studies found no significant effects for ablation of adult neurogenesis on spatial memory, cue conditioning or ethological measures of anxiety. An effect on contextual freezing was significant at a threshold of 5% (P = 0.04), but not at a threshold corrected for multiple testing. The meta-analysis revealed remarkably high levels of heterogeneity among studies of hippocampal function. The source of this heterogeneity remains unclear and poses a challenge for studies of the function of adult neurogenesis.

The role of 5-HT1A receptors in the proliferation and survival of progenitor cells in the dentate gyrus of the adult hippocampus and their regulation by corticoids

These experiments explore the role of 5-HT1A receptors in the regulation of cell proliferation in the dentate gyrus of the intact and adrenalectomized adult rat. Depleting 5-HT with p-chlorophenylalanine (300 mg/kg initially followed by 100 mg/kg/day) or stimulating 5-HT1A receptors with 8-OH-DPAT (1 mg/kg or 2 mg/kg, s.c. injections twice daily) for 14 days had no effect on cell proliferation as measured by Ki-67 or BrdU (5-bromo-3-deoxyuridine) immunocytochemistry in the dentate gyrus. However, combined treatment with p-chlorophenylalanine followed by 8-OH-DPAT significantly increased cell proliferation compared with p-chlorophenylalanine alone. Micro-injection of the 5-HT neurotoxin 5,7-dihydroxytryptamine into the fimbria-fornix (3.0 microg/side) and the cingulate bundle (1.8 microg/side) depleted hippocampal 5-HT locally but did not change cell proliferation 3 weeks after the surgery. However, 8-OH-DPAT (1 mg/kg, twice daily) stimulated cell proliferation in the dentate gyrus of hippocampal 5-HT-depleted rats compared with controls. These results suggest that 5-HT(1A) modulates cell proliferation in the hippocampus by a direct post-synaptic effect. Previous studies demonstrate that adrenalectomy increases hippocampal 5-HT1A receptor expression and binding, and thus we investigated whether the effect of adrenalectomy on cell proliferation and survival was dependent on the activity of the 5-HT1A receptors. In contrast to the null effect following twice-daily s.c. injection, 8-OH-DPAT (2.0 mg/kg/day) delivered by s.c. osmotic pumps increased proliferation in intact rats. The 5-HT1A antagonist WAY-100635 (1.5 mg/kg/day also delivered by osmotic pump) by itself did not alter cell proliferation, confirming that reduced serotonin activity does not change proliferation, but blocked the effect of 8-OH-DPAT. However, WAY-100635 could not block the stimulating action of adrenalectomy cell proliferation. 5-HT1A mRNA expression was not altered in the hippocampus by adrenalectomy. Thus, the effect of adrenalectomy on cell proliferation and survival is not 5-HT1A dependent, despite the interaction between 5-HT1A and corticosterone.

Serotonin Modulates the Suppressive Effects of Corticosterone on Proliferating Progenitor Cells in the Dentate Gyrus of the Hippocampus in the Adult Rat

This series of experiments explores the interaction between corticosterone and serotonin (5-HT) in the regulation of cell proliferation in the dentate gyrus of the adult rat. Intracerebroventricular 5,7-DHT (5,7-dihydroxytryptamine) (either 200 or 300 μg) resulted in highly significant depletion of 5-HT as measured by high performance liquid chromatography in the frontal cortex but had no effect on the number of proliferating cells in the dentate gyrus by measuring 5-bromo-2′-deoxyuridine (BrdU) and Ki-67 cytochemistry. Treatment with PCPA (p-chlorophenylalanine: a tryptophan hydroxylase inhibitor: 300 mg/kg initially followed by 100 mg/kg/day) resulted in reduced proliferation as measured by Ki-67 after 3 days treatment, but not by BrdU uptake, and not after 14 days treatment by either method. In addition, injection of corticosterone (10–40 mg/kg/day) for 8 days significantly reduced proliferation in the dentate gyrus, as expected, measured by both BrdU uptake and Ki-67 immunostaining. Adrenalectomized (ADX) rats with a replacement subcutaneous pellet of corticosterone showed reduced proliferation when given additional corticosterone (10 mg/kg/day for 8 days), but this was prevented by 5-HT depletion (i.c.v. 5,7-DHT). Finally, a dose–response study showed that progressive doses of corticosterone (0–40 mg/kg/day) in ADX rats resulted in diminished suppression of proliferation in 5-HT-depleted compared with 5-HT-intact rats. These results strongly suggest that 5-HT regulates the sensitivity of proliferating cells in the dentate gyrus to corticosterone

Neurogenomic evidence for a shared mechanism of the antidepressant effects of exercise and chronic fluoxetine in mice.

Several different interventions improve depressed mood, including medication and environmental factors such as regular physical exercise. The molecular pathways underlying these effects are still not fully understood. In this study, we sought to identify shared mechanisms underlying antidepressant interventions. We studied three groups of mice: mice treated with a widely used antidepressant drug – fluoxetine, mice engaged in voluntary exercise, and mice living in an enriched environment. The hippocampi of treated mice were investigated at the molecular and cellular levels. Mice treated with fluoxetine and mice who exercised daily showed, not only similar antidepressant behavior, but also similar changes in gene expression and hippocampal neurons. These changes were not observed in mice with environmental enrichment. An increase in neurogenesis and dendritic spine density was observed following four weeks of fluoxetine treatment and voluntary exercise. A weighted gene co-expression network analysis revealed four different modules of co-expressed genes that were correlated with the antidepressant effect. This network analysis enabled us to identify genes involved in the molecular pathways underlying the effects of fluoxetine and exercise. The existence of both neuronal and gene expression changes common to antidepressant drug and exercise suggests a shared mechanism underlying their effect. Further studies of these findings may be used to uncover the molecular mechanisms of depression, and to identify new avenues of therapy.

A genetic and functional relationship between T cells and cellular proliferation in the adult hippocampus.

A large correlation between variation in T cell subsets and hippocampal neurogenesis suggests that the immune system has an unexpectedly large influence on the brain.

Subtypes of major depression: latent class analysis in depressed Han Chinese women.

BACKGROUND Despite substantial research, uncertainty remains about the clinical and etiological heterogeneity of major depression (MD). Can meaningful and valid subtypes be identified and would they be stable cross-culturally? METHOD Symptoms at their lifetime worst depressive episode were assessed at structured psychiatric interview in 6008 women of Han Chinese descent, age ⩾ 30 years, with recurrent DSM-IV MD. Latent class analysis (LCA) was performed in Mplus. RESULTS; Using the nine DSM-IV MD symptomatic A criteria, the 14 disaggregated DSM-IV criteria and all independently assessed depressive symptoms (n = 27), the best LCA model identified respectively three, four and six classes. A severe and non-suicidal class was seen in all solutions, as was a mild/moderate subtype. An atypical class emerged once bidirectional neurovegetative symptoms were included. The non-suicidal class demonstrated low levels of worthlessness/guilt and hopelessness. Patterns of co-morbidity, family history, personality, environmental precipitants, recurrence and body mass index (BMI) differed meaningfully across subtypes, with the atypical class standing out as particularly distinct. CONCLUSIONS MD is a clinically complex syndrome with several detectable subtypes with distinct clinical and demographic correlates. Three subtypes were most consistently identified in our analyses: severe, atypical and non-suicidal. Severe and atypical MD have been identified in multiple prior studies in samples of European ethnicity. Our non-suicidal subtype, with low levels of guilt and hopelessness, may represent a pathoplastic variant reflecting Chinese cultural influences.