Identifying gene expression profiles associated with neurogenesis and inflammation in the human subependymal zone from development through aging

The generation of new neurons within the mammalian forebrain continues throughout life within two main neurogenic niches, the subgranular zone (SGZ) of the hippocampal dentate gyrus, and the subependymal zone (SEZ) lining the lateral ventricles. Though the SEZ is the largest neurogenic niche in the adult human forebrain, our understanding of the mechanisms regulating neurogenesis from development through aging within this region remains limited. This is especially pertinent given that neurogenesis declines dramatically over the postnatal lifespan. Here, we performed transcriptomic profiling on the SEZ from human post-mortem tissue from eight different life-stages ranging from neonates (average age ~ 2 months old) to aged adults (average age ~ 86 years old). We identified transcripts with concomitant profiles across these decades of life and focused on three of the most distinct profiles, namely (1) genes whose expression declined sharply after birth, (2) genes whose expression increased steadily with age, and (3) genes whose expression increased sharply in old age in the SEZ. Critically, these profiles identified neuroinflammation as becoming more prevalent with advancing age within the SEZ and occurring with time courses, one gradual (starting in mid-life) and one sharper (starting in old age).

Continual reelin signaling by the prime neurogenic niche of the adult brain

During development reelin sets the pace of neocortical neurogenesis enabling in turn newborn neurons to migrate, but whether and, if so, how reelin signaling affects the adult neurogenic niches remains uncertain. We show that reelin signaling, resulting in Dab1 phosphorylation, occurs in the ependymal-subependymal zone (EZ/SEZ) of the lateral ventricles where, along with its associated rostral migratory stream (RMS), the highest density of functional ApoER2 accumulates. Mice deficient for reelin, ApoER2 or Dab1 exhibit enlarged ventricles and dysplastic RMS. Moreover, while the conditional ablation of Dab1 in neural progenitor cells (NPCs) enlarges the ventricles and impairs neuroblasts clearance from the SEZ, the transgenic misexpression of reelin in NPCs of reelin-deficient mice normalizes the ventricular lumen and the density of ependymal cilia, ameliorating in turn neuroblasts migration; consistently, intraventricular infusion of reelin reroutes neuroblasts. These results demonstrate that reelin signaling persists sustaining the germinal niche of the lateral ventricles and influencing neuroblasts migration in the adult brain.

A schizophrenia subgroup with elevated inflammation displays reduced microglia, increased peripheral immune cell and altered neurogenesis marker gene expression in the subependymal zone

Inflammation regulates neurogenesis, and the brains of patients with schizophrenia and bipolar disorder have reduced expression of neurogenesis markers in the subependymal zone (SEZ), the birthplace of inhibitory interneurons. Inflammation is associated with cortical interneuron deficits, but the relationship between inflammation and reduced neurogenesis in schizophrenia and bipolar disorder remains unexplored. Therefore, we investigated inflammation in the SEZ by defining those with low and high levels of inflammation using cluster analysis of IL6, IL6R, IL1R1 and SERPINA3 gene expression in 32 controls, 32 schizophrenia and 29 bipolar disorder cases. We then determined whether mRNAs for markers of glia, immune cells and neurogenesis varied with inflammation. A significantly greater proportion of schizophrenia (37%) and bipolar disorder cases (32%) were in high inflammation subgroups compared to controls (10%, p < 0.05). Across the high inflammation subgroups of psychiatric disorders, mRNAs of markers for phagocytic microglia were reduced (P2RY12, P2RY13), while mRNAs of markers for perivascular macrophages (CD163), pro-inflammatory macrophages (CD64), monocytes (CD14), natural killer cells (FCGR3A) and adhesion molecules (ICAM1) were increased. Specific to high inflammation schizophrenia, quiescent stem cell marker mRNA (GFAPD) was reduced, whereas neuronal progenitor (ASCL1) and immature neuron marker mRNAs (DCX) were decreased compared to low inflammation control and schizophrenia subgroups. Thus, a heightened state of inflammation may dampen microglial response and recruit peripheral immune cells in psychiatric disorders. The findings elucidate differential neurogenic responses to inflammation within psychiatric disorders and highlight that inflammation may impair neuronal differentiation in the SEZ in schizophrenia.

Distribution of Aldh1L1-CreERT2 Recombination in Astrocytes Versus Neural Stem Cells in the Neurogenic Niches of the Adult Mouse Brain

In the adult central nervous system, neural stem cells (NSCs) reside in two discrete niches: the subependymal zone (SEZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Here, NSCs represent a population of highly specialized astrocytes that are able to proliferate and give rise to neuronal and glial progeny. This process, termed adult neurogenesis, is extrinsically regulated by other niche cells such as non-stem cell astrocytes. Studying these non-stem cell niche astrocytes and their role during adult neuro- and gliogenesis has been hampered by the lack of genetic tools to discriminate between transcriptionally similar NSCs and niche astrocytes. Recently, Aldh1L1 has been shown to be a pan-astrocyte marker and that its promoter can be used to specifically target astrocytes using the Cre-loxP system. In this study we explored whether the recently described Aldh1L1-CreERT2 mouse line (Winchenbach et al., 2016) can serve to specifically target niche astrocytes without inducing recombination in NSCs in adult neurogenic niches. Using short- and long-term tamoxifen protocols we revealed high recombination efficiency and specificity in non-stem cell astrocytes and little to no recombination in NSCs of the adult DG. However, in the SEZ we observed recombination in ependymal cells, astrocytes, and NSCs, the latter giving rise to neuronal progeny of the rostral migratory stream and olfactory bulb. Thus, we recommend the here described Aldh1L1-CreERT2 mouse line for predominantly studying the functions of non-stem cell astrocytes in the DG under physiological and pathological conditions.

Cell Tracking of Adult Neural Stem Cells From The Subependymal Zone In Vitro Reveals Cell Type-Specific Impacts On Cell Cycle Progression By The Extracellular Matrix Molecule Tenascin-C

Adult neurogenesis has been described in two canonical regions of the adult central nervous system (CNS) of rodents, the subgranular zone (SGZ) of the hippocampus and the subependymal zone (SEZ) of the lateral ventricles. The stem cell niche of the SEZ provides a privileged environment composed of a specialized extracellular matrix (ECM) that comprises the glycoproteins tenascin-C (Tnc) and laminin-1 (LN1). In the present study, we investigated the function of these ECM glycoproteins in the adult stem cell niche. Adult neural stem cells (aNSPCs) of the SEZ were prepared from wildtype (Tnc+/+) and Tnc knockout (Tnc-/-) mice and analyzed using molecular and cell biological approaches. A delayed maturation of aNSPCs in Tnc-/- tissue was reflected by a reduced capacity to form neurospheres in response to epidermal growth factor (EGF). In order to examine a potential influence of the ECM on cell proliferation, aNSPCs of both genotypes were studied by cell tracking using digital video microscopy. aNSPCs were cultivated on three different substrates, namely poly-D-Lysine (PDL) and PDL replenished with either LN1 or Tnc for up to six days in vitro. On each of the three substrates aNSPCs displayed lineage trees that could be investigated with regard to cell cycle length. The latter appeared reduced in Tnc-/- aNSPCs on PDL and LN1 substrates, less so on Tnc that seemed to compensate the absence of the ECM compound to some extent. Close inspection of the lineage trees revealed a subpopulation of late dividing aNSPCslate that engaged into cycling after a notable delay. aNSPCslate exhibited a clearly different morphology, with a larger cell body and conspicuous processes. aNSPCslate reiterated the reduction in cell cycle length on all substrates tested, which was not rescued on Tnc substrates. When the migratory activity of aNSPC-derived progeny was determined, Tnc-/- neuroblasts displayed significantly longer migration tracks. This was traced to an increased rate of migration episodes compared to the wildtype cells that rested for longer time periods. We conclude that Tnc intervenes in the proliferation of aNSPCs and modulates the motility of neuroblasts in the niche of the SEZ.

Proliferative Capacity of Adult Mouse Brain

We studied cell proliferation in the postnatal mouse brain between the ages of 2 and 30 months and identified four compartments with different densities of proliferating cells. The first identified compartment corresponds to the postnatal pallial neurogenic (PPN) zone in the telencephalon; the second to the subpallial postnatal neurogenic (SPPN) zone in the telencephalon; the third to the white matter bundles in the telencephalon; and the fourth to all brain parts outside of the other three compartments. We estimated that about 3.4 million new cells, including 0.8 million in the subgranular zone (SGZ) in the hippocampus, are produced in the PPN zone. About 21 million new cells, including 10 million in the subependymal zone (SEZ) in the lateral walls of the lateral ventricle and 2.7 million in the rostral migratory stream (RMS), are produced in the SPPN zone. The third and fourth compartments together produced about 31 million new cells. The analysis of cell proliferation in neurogenic zones shows that postnatal neurogenesis is the direct continuation of developmental neurogenesis in the telencephalon and that adult neurogenesis has characteristics of the late developmental process. As a developmental process, adult neurogenesis supports only compensatory regeneration, which is very inefficient.

Reduced Insulin-Like Growth Factor Family Member Expression Predicts Neurogenesis Marker Expression in the Subependymal Zone in Schizophrenia and Bipolar Disorder

The generation of inhibitory interneurons from neural stem cells in the subependymal zone is regulated by trophic factors. Reduced levels of trophic factors are associated with inhibitory interneuron dysfunction in the prefrontal cortex and hippocampus in psychiatric disorders, yet the extent to which altered trophic support may underpin deficits in inhibitory interneuron generation in the neurogenic niche remains unexplored in schizophrenia and bipolar disorder. We determined whether the expression of ligands, bioavailability-regulating binding proteins, and cognate receptors of 4 major trophic factor families (insulin-like growth factor [IGF], epidermal growth factor [EGF], fibroblast growth factor [FGF], and brain-derived neurotrophic factor [BDNF]) are changed in schizophrenia and bipolar disorder compared to controls. We used robust linear regression analyses to determine whether altered expression of trophic factor family members predicts neurogenesis marker expression across diagnostic groups. We found that IGF1 mRNA was decreased in schizophrenia and bipolar disorder compared with controls (P ≤ .006), whereas both IGF1 receptor (IGF1R) and IGF binding protein 2 (IGFBP2) mRNAs were reduced in schizophrenia compared with controls (P ≤ .02). EGF, FGF, and BDNF family member expression were all unchanged in both psychiatric disorders compared with controls. IGF1 expression positively predicted neuronal progenitor and immature neuron marker mRNAs (P ≤ .01). IGFBP2 expression positively predicted neural stem cell and neuronal progenitor marker mRNAs (P ≤ .001). These findings provide the first molecular evidence of decreased IGF1, IGF1R, and IGFBP2 mRNA expression in the subependymal zone in psychiatric disorders, which may potentially impact neurogenesis in schizophrenia and bipolar disorder.