David Petrik

Depression and Hippocampal Neurogenesis: A Road to Remission?

Adult-generated hippocampal neurons are required for mood control and antidepressant efficacy, raising hopes that someday we can harness the power of new neurons to treat mood disorders such as depression. However, conflicting findings from preclinical research—involving stress, depression, and neurogenesis—highlight the complexity of considering neurogenesis as a road to remission from depression. To reconcile differences in the literature, we introduce the “neurogenic interactome,” a platform from which to consider the diverse and dynamic factors regulating neurogenesis. We propose consideration of the varying perspectives—system, region, and local regulation of neurogenesis—offered by the interactome and exchange of ideas between the fields of learning and memory and mood disorder research to clarify the role of neurogenesis in the etiology and treatment of depression.

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo.

In vivo contribution of nestin- and GLAST-lineage cells to adult hippocampal neurogenesis.

Radial glia‐like cells (RGCs) are the hypothesized source of adult hippocampal neurogenesis. However, the current model of hippocampal neurogenesis does not fully incorporate the in vivo heterogeneity of RGCs. In order to better understand the contribution of different RGC subtypes to adult hippocampal neurogenesis, we employed widely used transgenic lines (Nestin‐CreERT2 and GLAST::CreERT2 mice) to explore how RGCs contribute to neurogenesis under basal conditions and after stimulation and depletion of neural progenitor cells. We first used these inducible fate‐tracking transgenic lines to define the similarities and differences in the contribution of nestin‐ and GLAST‐lineage cells to basal long‐term hippocampal neurogenesis. We then explored the ability of nestin‐ and GLAST‐lineage RGCs to contribute to neurogenesis after experimental manipulations that either ablate neurogenesis (i.c.v. application of the anti‐mitotic AraC, cytosine‐β‐D‐arabinofuranoside) or stimulate neurogenesis (wheel running). Interestingly, in both ablation and stimulation experiments, labeled RGCs in GLAST::CreERT2 mice appear to contribute to neurogenesis, whereas RGCs in Nestin‐CreERT2 mice do not. Finally, using NestinGFP reporter mice, we expanded on previous research by showing that not all RGCs in the adult dentate gyrus subgranular zone express nestin, and therefore RGCs are antigenically heterogeneous. These findings are important for the field, as they allow appropriately conservative interpretation of existing and future data that emerge from these inducible transgenic lines. These findings also raise important questions about the differences between transgenic driver lines, the heterogeneity of RGCs, and the potential differences in progenitor cell behavior between transgenic lines. As these findings highlight the possible differences in the contribution of cells to long‐term neurogenesis in vivo, they indicate that the current models of hippocampal neurogenesis should be modified to include RGC lineage heterogeneity.

Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule

Adult neurogenesis occurs throughout life in the mammalian hippocampus and is essential for memory and mood control. There is significant interest in identifying ways to promote neurogenesis and ensure maintenance of these hippocampal functions. Previous work with a synthetic small molecule, isoxazole 9 (Isx‐9), highlighted its neuronal‐differentiating properties in vitro. However, the ability of Isx‐9 to drive neurogenesis in vivo or improve hippocampal function was unknown. Here we show that Isx‐9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult‐generated dentate gyrus neurons. Isx‐9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx‐9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization. Molecular exploration of Isx‐9‐induced regulation of neurogenesis (via FACS and microarray of SGZ stem and progenitor cells) suggested the involvement of the myocyte‐enhancer family of proteins (Mef2). Indeed, transgenic‐mediated inducible knockout of all brain‐enriched Mef2 isoforms (Mef2a/c/d) specifically from neural stem cells and their progeny confirmed Mef2′s requirement for Isx‐9‐induced increase in hippocampal neurogenesis. Thus, Isx‐9 enhances hippocampal neurogenesis and memory in vivo, and its effects are reliant on Mef2, revealing a novel cell‐intrinsic molecular pathway regulating adult neurogenesis.—Petrik, D., Jiang, Y., Birnbaum, S. G., Powell, C. M., Kim, M.‐S., Hsieh, J., Eisch, A. J. Functional and mechanistic exploration of an adult neurogenesis‐promoting small molecule. FASEB J. 26, 3148–3162 (2012). www.fasebj.org

Neurogenesis in the Developing and Adult Brain—Similarities and Key Differences

Adult neurogenesis in the mammalian brain is often viewed as a continuation of neurogenesis at earlier, developmental stages. Here, we will critically review the extent to which this is the case highlighting similarities as well as key differences. Although many transcriptional regulators are shared in neurogenesis at embryonic and adult stages, recent findings on the molecular mechanisms by which these neuronal fate determinants control fate acquisition and maintenance have revealed profound differences between development and adulthood. Importantly, adult neurogenesis occurs in a gliogenic environment, hence requiring adult-specific additional and unique mechanisms of neuronal fate specification and maintenance. Thus, a better understanding of the molecular logic for continuous adult neurogenesis provides important clues to develop strategies to manipulate endogenous stem cells for the purpose of repair.

Modulation by the BK accessory β4 subunit of phosphorylation-dependent changes in excitability of dentate gyrus granule neurons

Large‐conductance voltage‐ and calcium‐activated potassium (BK) channels are large‐conductance calcium‐ and voltage‐activated potassium channels critical for neuronal excitability. Some neurons express so called fast‐gated, type I BK channels. Other neurons express BK channels assembled with the accessory β4 subunit conferring slow gating of type II BK channels. However, it is not clear how protein phosphorylation modulates these two distinct BK channel types. Using β4‐knockout mice, we compared fast‐ or slow‐gated BK channels in response to changes in phosphorylation status of hippocampus dentate gyrus granule neurons. We utilized the selective PP2A/PP4 phosphatase inhibitor Fostriecin to study changes in action potential shape and firing properties of the neurons. In β4‐knockout neurons, Fostriecin increases BK current, speeds up BK channel activation and reduces action potential amplitudes. Fostriecin increases spiking during early components of an action potential train. In contrast, inhibition of BK channels through β4 in wild‐type neurons or by the BK channel inhibitor Paxilline opposes Fostriecin effects. Voltage clamp recordings of neurons reveal that Fostriecin increases both calcium and BK currents. However, Fostriecin does not activate BK α channels in transfected HEK293 cells lacking calcium channels. In summary, these results suggest that fast‐gating, type I BK channels lacking β4 can increase neuronal excitability in response to reduced phosphatase activity and activation of calcium channels. By opposing BK channel activation, the β4 subunit plays an important role in moderating firing frequency regardless of changes in phosphorylation status.

Inducible knockout of Mef2a, -c, and -d from nestin-expressing stem/progenitor cells and their progeny unexpectedly uncouples neurogenesis and dendritogenesis in vivo

Myocyte enhancer factor (Mef)‐2 transcription factors are implicated in activity‐dependent neuronal processes during development, but the role of MEF2 in neural stem/progenitor cells (NSPCs) in the adult brain is unknown. We used a transgenic mouse in which Mef2a, ‐c, and ‐d were inducibly deleted in adult nestin‐expressing NSPCs and their progeny. Recombined cells in the hippocampal granule cell layer were visualized and quantified by yellow fluorescent protein (YFP) expression. In control mice, postmitotic neurons expressed Mef2a, ‐c, and ‐d, whereas type 1 stem cells and proliferating progenitors did not Based on this expression, we hypothesized that Mef2a, ‐c, and ‐d deletion in adult nestin‐expressing NSPCs and their progeny would result in fewer mature neurons. Control mice revealed an increase in YFP+ neurons and dendrite formation over time. Contrary to our hypothesis, inducible Mef2 KO mice also displayed an increase in YFP+ neurons over time—but with significantly stunted dendrites—suggesting an uncoupling of neuron survival and dendritogenesis. We also found non‐cell‐autonomous effects after Mef2a, ‐c, and ‐d deletion. These in vivo findings indicate a surprising functional role for Mef2a, ‐c, and ‐d in cell‐ and non‐cell‐autonomous control of adult hippocampal neurogenesis that is distinct from its role during development.—Latchney, S. E., Jiang, Y., Petrik, D. P., Eisch, A. J., Hsieh, J. Inducible knockout of Mef2a, ‐c, and ‐d from nestin‐expressing stem/progenitor cells and their progeny unexpectedly uncouples neurogenesis and dendritogenesis in vivo. FASEB J. 29, 5059–5071 (2015). www.fasebj.org

Early Postnatal In Vivo Gliogenesis From Nestin-Lineage Progenitors Requires Cdk5

The early postnatal period is a unique time of brain development, as diminishing amounts of neurogenesis coexist with waves of gliogenesis. Understanding the molecular regulation of early postnatal gliogenesis may provide clues to normal and pathological embryonic brain ontogeny, particularly in regards to the development of astrocytes and oligodendrocytes. Cyclin dependent kinase 5 (Cdk5) contributes to neuronal migration and cell cycle control during embryogenesis, and to the differentiation of neurons and oligodendrocytes during adulthood. However, Cdk5’s function in the postnatal period and within discrete progenitor lineages is unknown. Therefore, we selectively removed Cdk5 from nestin-expressing cells and their progeny by giving transgenic mice (nestin-CreERT2/R26R-YFP/CDK5flox/flox [iCdk5] and nestin-CreERT2/R26R-YFP/CDK5wt/wt [WT]) tamoxifen during postnatal (P) days P2-P 4 or P7-P 9, and quantified and phenotyped recombined (YFP+) cells at P14 and P21. When Cdk5 gene deletion was induced in nestin-expressing cells and their progeny during the wave of cortical and hippocampal gliogenesis (P2-P4), significantly fewer YFP+ cells were evident in the cortex, corpus callosum, and hippocampus. Phenotypic analysis revealed the cortical decrease was due to fewer YFP+ astrocytes and oligodendrocytes, with a slightly earlier influence seen in oligodendrocytes vs. astrocytes. This effect on cortical gliogenesis was accompanied by a decrease in YFP+ proliferative cells, but not increased cell death. The role of Cdk5 in gliogenesis appeared specific to the early postnatal period, as induction of recombination at a later postnatal period (P7-P9) resulted in no change YFP+ cell number in the cortex or hippocampus. Thus, glial cells that originate from nestin-expressing cells and their progeny require Cdk5 for proper development during the early postnatal period.

Chromatin Remodeling Factor Brg1 Supports the Early Maintenance and Late Responsiveness of Nestin‐Lineage Adult Neural Stem and Progenitor Cells

Insights from embryonic development suggest chromatin remodeling is important in adult neural stem cells (aNSCs) maintenance and self‐renewal, but this concept has not been fully explored in the adult brain. To assess the role of chromatin remodeling in adult neurogenesis, we inducibly deleted Brg1—the core subunit of SWI/SNF‐like Brg1/Brm‐associated factor chromatin remodeling complexes—in nestin‐expressing aNSCs and their progeny in vivo and in culture. This resulted in abnormal adult neurogenesis in the hippocampus, which initially reduced hippocampal aNSCs and progenitor maintenance, and later reduced its responsiveness to physiological stimulation. Mechanistically, deletion of Brg1 appeared to impair cell cycle progression, which is partially due to elevated p53 pathway and p21 expression. Knockdown of p53 rescued the neurosphere growth defects caused by Brg1 deletion. Our results show that epigenetic chromatin remodeling (via a Brg1 and p53/p21‐dependent process) determines the aNSCs and progenitor maintenance and responsiveness of neurogenesis. Stem Cells 2015;33:3655–3665

Epithelial Sodium Channel Regulates Adult Neural Stem Cell Proliferation in a Flow-Dependent Manner

One hallmark of adult neurogenesis is its adaptability to environmental influences. Here, we uncovered the epithelial sodium channel (ENaC) as a key regulator of adult neurogenesis as its deletion in neural stem cells (NSCs) and their progeny in the murine subependymal zone (SEZ) strongly impairs their proliferation and neurogenic output in the olfactory bulb. Importantly, alteration of fluid flow promotes proliferation of SEZ cells in an ENaC-dependent manner, eliciting sodium and calcium signals that regulate proliferation via calcium-release-activated channels and phosphorylation of ERK. Flow-induced calcium signals are restricted to NSCs in contact with the ventricular fluid, thereby providing a highly specific mechanism to regulate NSC behavior at this special interface with the cerebrospinal fluid. Thus, ENaC plays a central role in regulating adult neurogenesis, and among multiple modes of ENaC function, flow-induced changes in sodium signals are critical for NSC biology.