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At one time, scientists believed that people were born with all the neurons they would ever have. While neurogenesis is quite limited compared to regeneration in other tissues, research in this area may lead to new treatments for disorders such as Alzheimer’s, stroke, and epilepsy.
How do scientists identify new neurons? A researcher can inject a compound called bromodeoxyuridine (BrdU) into the brain of an animal. While all cells will be exposed to BrdU, BrdU will only be incorporated into the DNA of newly generated cells that are in S phase. A technique called immunohistochemistry can be used to attach a fluorescent label to the incorporated BrdU, and a researcher can use fluorescent microscopy to visualize the presence of BrdU, and thus new neurons, in brain tissue.
Figure 1 shows fluorescently labeled new neurons in a rat hippocampus. Cells that are actively dividing have bromodoxyuridine (BrdU) incorporated into their DNA and are labeled in red. Cells that express glial fibrillary acidic protein (GFAP) are labeled in green. Astrocytes, but not neurons, express GFAP. Thus, cells that are labeled both red and green are actively dividing astrocytes, whereas cells labeled red only are actively dividing neurons.
Genetic control of hippocampal neurogenesis
Adult neurogenesis in the hippocampus is under complex genetic control. A recent comparative study of two inbred mouse strains using quantitative trait locus analysis has revealed that cell survival is most highly correlated with neurogenesis and identified candidate genes for further investigation.
Neurogenesis - the production of new neurons - is an ongoing process that persists in the adult brain of several species, including humans. It has been most intensively studied in the mouse in two discrete brain regions: the subventricular zone (SVZ) lining the lateral wall of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus  (Figure 1). These regions harbor relatively quiescent astrocyte-like stem cells, which divide and give rise to multipotential, rapidly dividing transit-amplifying cells that will eventually differentiate into neuroblasts. These later generate neuroblasts that are believed to have limited further mitotic potential [2, 3]. Neuroblasts from the SVZ and SGZ migrate and eventually mature into functional neurons within the olfactory bulb and dentate gyrus, respectively. Most recent evidence suggests that the stem cells in these regions can also give rise to astrocytes and oligodendrocytes of the glial lineage, indicating that in vivo, as in vitro, these cells are multipotent . A recent study by Kempermann et al.  in the Proceedings of the National Academy of Sciences of the USA sheds interesting new light on the genetic complexity of the regulation of neurogenesis.
Neurogenic zones in the adult mouse brain. Adult neurogenesis is best characterized in two zones in the adult mouse brain: the subventricular zone (SVZ) adjacent to the lateral ventricle (LV), where neurons are produced that subsequently migrate to the olfactory bulb via the rostral migratory stream (RMS) and the dentate gyrus (DG) of the hippocampus. The hippocampus (shown enlarged in the inset) consists of two interleaved layers of cells - the pyramidal cell layer (CA) and the dentate gyrus. Proliferating neural precursors and quiescent neural stem cells are found in a zone immediately adjacent to the dentate gyrus called the subgranular zone (SGZ).
Is Neurogenesis in the Hippocampus Linked to Depression?
On November 2 nd , the Neurotechnology Industry Organization revealed its top 10 neuroscience trends for 2007. While most of these trends had to do with technology, which makes sense because the company has a vested interest in neuroscience technology, trend #9 was that “new research continues to link neurogenesis to treatment of depression.” 
While some researchers would question the tenability of that statement, the NIO is correct in its assessment that this is one of the “hottest” topics in neuroscience today. The reasons for this are two-fold. The first reason is that neurogenesis is still a relatively new idea with broad-ranging implications. If the brain can create new functional cells in some regions of the brain, then one might speculate that it will eventually be able to create cells for any region in the brain. An understanding of the adult stem cells in the brain might bring us one step closer to that elusive fountain of youth. The next reason that the potential link between neurogenesis and depression is considered so important is that major depressive disorder is a widespread pathological disorder, affecting 16.2% of US adults at some point in their lifetime (Kesller et al., 2003). Novel models of understanding the disease could lead to improved pharmaceutical drugs capable of treating depression more acutely. The neurogenesis hypothesis is one model that has been suggested for helping to explain how unipolar depression forms and how it may be treated. This stated hypothesis is accepted to have two components: one, that deficiency of neurogenesis may be culpable for the onset of depression, and two, that current methods of treating depression may work in large part by fixing an abnormality in neurogenesis to the hippocampal region (Sapolsky, 2004). To test this neurogenesis hypothesis, a number of possible correlations between what we know about depressive disorder and what we know about neurogenesis need to be addressed. Also, a number of questions isolating the mechanisms behind neurogenesis and depression need to be answered. This paper will attempt to review the testing of the neurogenesis hypothesis.
One of the fundamental assumptions of the neurogenesis hypothesis is that the hippocampal region in general is crucial to the development and treatment of depression. One attempt to find a correlation here is based on the idea that subjects who show depressive symptoms will show variance in the sizes of their hippocampal regions. Videbech and Ravnkilde (2004) conducted such a survey of studies which had measured hippocampal volume using MRI machines. They ensured that each study had identified which subjects qualified as having a major depressive disorder using a replicable method, and that each study had controlled for other variables such as age and drug abuse. They found that there was a significant effect size which correlated depression to a decrease in the volume of the hippocampus in both hemispheres of the brain. Since neurogenesis would be expected to increase the volume of the hippocampus, their results suggest that neurogenesis may have a role in depression. This paper will touch more on this point later. But while there is a correlation between the size of the hippocampus and depression, does the idea that the hippocampus causes or affects the symptoms of depression make intuitive sense? One of the commonly accepted symptoms of major affective disorder is cognitive impairment. Sweeny et al. (2000) tested groups of unipolar depressed subjects, bipolar subjects, and control subjects using computer programs designed to determine cognitive aptitude. Their study found that unipolar depressive patients predominantly showed deficiencies in episodic memory, but not much else. This is consistent with the known functions of the hippocampal regions in the brain (Ebmeimer et al, 2006). This link provides indirect evidence that the hippocampus plays at least somewhat of an important role in major depressive disorder. However, given how complicated the connections between known brain regions are known to be, it would be difficult to pinpoint exactly how much responsibility for depressive disorder can be placed on the hippocampal region of the brain.
However, just because the hippocampus plays a role in depression does not mean that neurogenesis in the hippocampus plays a functional role in the disease. Indeed, in past it has been somewhat unclear whether the newly generated neurons are even able to integrate themselves into the complicated neural networks of the hippocampus. Recently it has become more and more apparent that the new neurons are able to integrate themselves until existing neural networks in the hippocampus. Henriette van Praag et al. (2001) attempted to show this very fact—that stem cells can integrate and begin to act just as mature granule cells do in the dentate gyrus of the hippocampus. In order to do so, the researchers used a variety of staining techniques, including the use of green fluorescent protein (GFP) and 5-bromodeoxyuridine (BrdU). They then allowed their adult mice to live for a calculated period of time before they were killed: either a short amount of time to see how the cells are proliferating immediately after staining, or after a longer amount of time to see how the cells are integrating themselves into the neural network of the dentate gyrate. Based on the dendrite spine length of the stem cells, among other factors, they found that the cells were able to integrate themselves into the neural networks of the dentate gyrus. So it is possible for neurogenesis to lead to new functional neurons, which makes one wonder exactly what purpose, if any, these cells have in the brain.
The next piece of evidence surrounding the neurogenesis hypothesis is how it fits into the way that antidepressant treatments work. While the rationale that they work on serotonin reuptake and monoamine oxidase inhibitors is widely accepted, the fact that these drugs generally take two to three weeks to start working is not an obvious result. Some complex mechanisms of serotonin reuptake and monoamine oxidase inhibitors have been suggested to explain this phenomenon (Celada et al, 2004). However, the neurogenesis hypothesis could simplify our understanding of how the antidepressant drugs work. Indeed, it makes a certain amount of intuitive sense that the new neurons generated by the antidepressants would require a delay before they could begin to work themselves into the circuitry of the hippocampus. Some studies have found that new neurons may begin to be important for trace memory in the hippocampus after just 1-2 weeks (Shors et al, 2001). Zhao et al (2006) studied the development of newly formed granule cells in the dentrate gyrus of adult rats and mice. In order to do so, they injected GFP viruses into adult animals to indentify which cells had been newly formed at the time of injection. They then killed the animals at varying points of their development, isolated the cells that had been marked by the virus, and analyzed the stages of development that each of these cells had gone through. Based on how developed (ie, how long) the dendritic spines of the neurons were at the time of death, the researchers in the study attempt to draw conclusions about how they functioned in the animal’s brain at the time of death. They found that the dendritic spine had already started to grow by around 16 days in both adult rat and mice brains. These results, combined with the results of the previous study that new neurons help form trace memories after just 1-2 weeks, suggest that animals may be able to use the neurons to form new memories very soon after they appear in the dendrite gyrus. While abstracting results from animals to humans is tricky, the animal results suggest that the stem cells could begin to integrate themselves into the hippocampus around 2-3 weeks after they are formed, near the same time that the antidepressant drugs begin to work. Therefore, the neurogenesis hypothesis could help to simplify one of the not-so-well understood mechanisms of how andippressant drugs work, providing some indirect evidence for the neurogenesis hypothesis, since scientists have been taught to revere Occam’s razor.
The other piece of correlative evidence that supports the neurogenesis hypothesis is the relationship between stress and depression. There is a lot of data behind the theory that external stressors can lead to depression, and behind the idea that depressed subjects are less able to raise their cortisol levels when faced with a challenge (Miller et al, 2005). It has also been shown in double-blind studies that antiglucocorticoids can act as antidepressants if the patient is diagnosed as hypercortisolemic, which many depressed patients are (Wolkowitz et al, 1999). Additionally, excess of adrenal steroid and stress appear to damage neurons in the hippocampus, negatively affecting long-term potentation of neurons, cognition, and memory (Pavlides et al, 2002). Based on this relationship, scientists became curious as to whether or not stress inhibited neurogenesis of the hippocampus. Gould et al (1998) tested whether or not the growth of granule cells in the dendrite gyrus of adult primates would be affected by stressful experiences. In order to create a stressful experience, the researchers placed one male marmoset monkey in the cage of another male of the same species for an hour. The monkeys reacted to this by behaving subordinately: they remained still in one part of the cage in order to avoid a fight with the male that they had been placed with. This was considered a reasonable induction of stress in the “intruder” monkey. After the hour, the monkeys were removed from the cage and injected with BrdU, which acts as a marker of proliferating cells and their offspring. Two hours later, once the stem cells had a chance to form, the monkeys were killed, and sections of their brains were analyzed. The animals that were placed under the stress were then compared to control monkeys that had not been put in the stressful situation, and the monkeys that had been not inducted to the stress were found to have more stem cell proliferation. This result helps to illuminate the fact that stress is a potent inhibitor of neurogenesis, at least in non-human animals. However, since it would probably be unethical to force a human to undergo a very stressful experience in an experimental design, there is less evidence for this phenomenon in human subjects. If stress did not cause an inhibition of neurogenesis, that would be solid evidence against the neurogenesis hypothesis. As it is, the fact that stress might inhibit neurogenesis provides some more correlative evidence in support of it.
As stated earlier in this paper, one of the more widely-accepted pathological symptoms of depressive disorder is that it decreases the volume of the hippocampus. However, the mechanisms that lead to this decrease in volume are not well understood. If they were found to be due to an inhibition of neurogenesis, perhaps from stress, this result would support the neurogenesis hypothesis. If the reasons for the decrease in volume were found to be from some other source, perhaps the death of existing hippocampal cells because of a lack of necessary nutrients, then it would provide support against the neurogenesis hypothesis (Sapolsky, 2004). Unfortunately, there is little data on the human central nervous system, in large part because it would be highly unethical to perform the stain and then brain slice technique that is used in animals, and in part because data on post-mortem tissue has thus far yielded inconclusive results (Feldmann et al, 2007). Despite the dearth of information, some scientists have attempted to make a few estimates about how widespread the neurogenesis is. Cameron and McKay (2001) posited that the amount of BrdU marker that had been used in previous studies was not ample, and only a portion of the stem cells were being found. Using a higher concentration of BrdU along with a thymide marker on adult rats, they found that over 9000 new stem cells were produced in the dentate gyrus of the hippocampus, for a total of over 250,000 a month. As Gould and Gross (2002) point out, this is out of a total of 1 to 2 million total neurons in the dentate gyrus of the adult rat, suggesting that the newly formed stem cells play a large role in the hippocampus. However, once again, it is difficult to extrapolate this data to humans, who could have entirely different amounts of cells in their dentate gyrus, and different amount of new cells formed each day. Even if hippocampal volume were shown to be lowered because of impairment in neurogenesis, this data would not prove a causal both parts of the neurogenesis hypothesis. Instead it must be shown that neurogenesis has an impact on the symptoms of depression (Feldmann et al, 2007), otherwise the data will remain simply correlative.
One study that attempted to get to the core of the neurogenesis hypothesis looked at the development of learned helplessness behavior in rats. Vollmayr et al. (2003) looked at whether or not the proliferation rate of new stem cells in the dentate gyrus would vary in animals that showed symptoms of learned helplessness versus those that did not. They subjected groups of rats to inescapable foot shocks, and decided based on their behavior whether they were displaying learned helplessness or not. The newly forming cells in the dentate gyrus were marked with BrdU, and the amounts of these cells were compared between those animals that had undergone learned helplessness and those that had not. In order to support the causal portion of the neurogenesis hypothesis, these results should have found that those animals that had experienced learned helplessness would have less cells, because the depression-like symptoms should have impaired their neurogenesis. Instead, the researchers found no significant difference between the two groups, and given that the sample size (200) of the study was so high, these results hurt the case for the neurogenesis hypothesis substantially. Additionally, there is some evidence that a decrease in neurogenesis will not necessarily lead to depressive symptoms, as it should based on the causal portion of the neurogenesis hypothesis (Malberg and Duman, 2003). These researchers also made their finding using a learned helplessness model in rats through foot shocks. This evidence seems to discount the possibility that there is a direct causal link between neurogenesis and depression. However, just as it was important to express caution over optimistic results for the neurogenesis hypothesis that relied on animal models earlier, it is important not to be too pessimistic about the hypothesis based solely on this animal data. Nevertheless, the results from the Vollmayr et al. and Malberg and Duman studies indicate that, as of now, we have no reason to suspect that there is a causal link between neurogenesis and depression.
The neurogenesis hypothesis has two parts. While the part about the causal link may have been refuted for now, the part about how current depression treatments work remains relevant. Researchers have found that many of the major antidepressant pharmaceutical agents stimulated neuron cell proliferation in the dentate gyrus of adult rats (Kodama et al., 2004). Additionally, the drugs in this study showed no effect on the number of cells in the subventrical zone or primary motor cortex, suggesting that they were isolated to areas where neurogenesis would take place. Electroconvulsive therapy is another method for treating depression that is widely held to be useful. Madsen et al. (2000) gave rats a series of either 1 or 10 electroconvulsive series, and stained their cells with BrdU marker at different points after the treatment. He found that the rats which had been given electroconvulsive therapy had higher levels of new stem cells in the dentate gyrus, and that these results showed themselves in a graded manner, so those rats that had undergone more electroconvulsive therapy showed a greater increase in neurogenesis. The only anti-depressive treatment that does not appear to yield an increase of new cells in the dentate gyrus is transcranial magnetic stimulation (Czeh et al., 2001). Although it is tempting to dismiss this results as non-significant because TMS is a new and relatively unproven method of treating depression, the careful scientist must review all the information at his or her disposal. Nevertheless, there appears to some sort of relationship between neurogenesis and the action of many of the methods for treating depression.
One way to isolate the second part of the hypothesis is to attempt to discover whether the anti-depressive treatments depend upon neurogenesis in order to work. In a controversial study, Santarelli et al. (2003) attempted to do just this. First, they formed a rodent model of depression whose symptoms were alleviated by the anti-depressant. They then attempted to disrupt neurogenesis in the hippocampus by delivering low-dose x-radiation to the hippocampus while attempting to spare the brain and the most of the rest of the body. This x-radiation yielded an 85% decrease of BrdU-positive stained cells in the subgranular zone of the dentate gyrus. They found that when the x-rays regionally restricted the neurogenesis of the hippocampus, the anti-depressant effects on their rodent model by the anti-depressant drug (fluoxetine) were blocked. Although their study was well controlled for internally, it has still received some criticism. The main critique of the article has been that the research design used to model depression, whether the animal was willing to feed in a novel environment, does not actually test for depression but instead for anxiety (Sapolsky, 2004). While this criticism does not refute their result entirely, it does bring up some doubt. So while this study is one of the strongest pieces of evidence supporting the second part of the neurogenesis hypothesis, that neurogenesis is the reason that anti-depressive treatments work, it is not perfect, and further replication or a study of animals closer genetically to humans could yield more significant results.
This paper has reviewed our current understanding of the relationship between neurogenesis and depression. As stated earlier, the neurogenesis hypothesis has two components. The first, that there is a causal relationship between neurogenesis and depression, relies on a large amount of correlative and indirect evidence, such as the relationship between stress and neurogenesis, and the relationship between stress and depression. However, if there is to be a causal relationship, then depression must lead to a decrease in neurogenesis, which is not the case (Vollmayr et al., 2003), at least in rodents. However, the second component of the neurogenesis hypothesis, which is that anti-depressive treatments work via the stimulus of neurogenesis, remains a viable idea. Santarelli et al. (2003) in particular seemed to show that stimulating neurogenesis is the mechanism through which antidepressant treatments work, although the fact that TMS does not appear to work via neurogenesis is one knock against this theory. Further research, especially research that somehow could replicate these studies on humans, while remaining ethically sound, would be extremely advantageous to the study of the relationship between neurogenesis and depression. This topic is likely to remain “hot” given that neurogenesis is such a new idea. Indeed, the fact that we may be beginning to understand one of its potential functions is fascinating. Despite the obvious reasons to be excited about this research, we must strive to remain fundamentally sound in our scientific study of the neurogenesis hypothesis and its implications for the treatment of depression.
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Over the years, crucial extrinsic and intrinsic mechanisms regulating the acquisition of cell fate during neural development have been elucidated. Gradients of morphogens secreted by organizer centers instruct neural progenitor cells (NPCs) to activate the expression of transcription factor (TF) cascades that guide cells through every single step of the fate acquisition process. Genetic studies in vitro and in vivo, essentially based on the gain- and loss-of-function experiments, revealed that large arrays of TF cascades are indeed responsible for the specification of different neuronal subtypes.
This mechanistic knowledge was critical for the field of cell reprogramming to emerge. Indeed, the possibility to convert a cell type into another has been strictly dependent on seminal findings accumulated over the last 30 years in neurodevelopmental biology.
Back in the 1950s, it was not yet clear whether all cells belonging to the same organism contained the same set of genes. On this line, Weismann had suggested that genes whose function was no longer required might be lost or permanently inactivated in a specific cell type, seeding the concept that cell fate acquisition is an irreversible process being associated with loss of genetic material. This concept, well represented by the famous Waddington’s landscape (Waddington, 1957) was later challenged by Gurdon’s work. He performed pioneer experiments of somatic nuclei transfer in Xenopus oocytes during his Ph.D. studies, providing the first evidence for the preservation of genome integrity after cellular differentiation (Gurdon, 1962).
Up to date, it is consolidated the concept that epigenetic-mediated gene silencing, rather than gene loss, accompanies cell fate acquisition. This evidence opened a crack toward the plasticity of cell identity and the possibility of altering the fate of a differentiated cell.
In 1988, MyoD ectopic expression in mouse embryonic fibroblasts (MEFs) was revealed sufficient to convert them into muscle cells (Tapscott et al., 1988). Two decades later the breakthrough from the Yamanaka’s group showed that somatic cells can be reverted to a pluripotent state forcing the expression of the four factors Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006), that are mediating global chromatin remodeling allowing for the expression of the pluripotency gene machinery (Takahashi and Yamanaka, 2006 Boissart et al., 2012). First successful conversion of MEFs into functional induced neurons (iNs) was described a few years later through Ascl1, Brn2, and Myt1l misexpression (Vierbuchen et al., 2010). After this study, many others attempted to modify or enrich this TF combination to induce MEF differentiation toward specific neuronal subtypes localized to defined areas of the brain (reviewed in Masserdotti et al., 2016).
All these works highlighted the ability of accurately selected cocktail of TFs to alter the fate of fully differentiated cells and to obtain functional neuronal cells.
To define a cell reprogramming gene cocktail, the TFs to be tested in the screening are chosen for their capability to impose that specific neuronal fate (master regulator genes) or among genes enriched in the target cell population, but not necessarily with their functions already addressed. Very recently, unbiased screenings of TFs for neuronal conversion have been also performed with very informative results (Liu et al., 2018 Tsunemoto et al., 2018). Once the candidate TF list is selected, they are delivered in donor cells according to a “narrow down” or an one” strategy. Generally, TFs are delivered and expressed all simultaneously in the donor cells although they control different phases of the cell fate acquisition process. In other cases, genetic tricks (i.e., mix of constitutive promoter guided- and inducible promoter guided-TFs whose expression can be turned off at a defined time) are employed to allow sequential expression of TFs required in different phases of differentiation in a manner that tries to recapitulate the expression timing observed during in vivo development (Au et al., 2013 Colasante et al., 2015). Finally, in an even more sophisticated experimental setting, the endogenous loci of the desired TFs can be activated using the CRISPR/Cas9 system (Black et al., 2016 Liu et al., 2018). In all these cases, the final output of these studies can meet the initial expectations, but unpredicted results have not rarely been reported. In fact, in some instances, new features for the mechanisms of action of TFs have been emerging. In others, TFs whose role was not considered determining for a specific neuronal fate acquisition during in vivo development, have come out as pivotal in the neuronal specification during direct cell reprogramming. Even more surprisingly is the identification of TFs not related to neuronal development that are able to impose a neuronal identity when overexpressed in heterologous cells.
This predictive value of the direct cell reprogramming methodology can be likely explained by the fact that during this process selected TFs are forced to operate in donor cell populations that are very distant from the target neuronal cells. This is the case for the fibroblast-to-neuron conversion, as fibroblasts have a mesodermic origin in the embryo contrary to the ectoderm-derived neurons. According to this different ontogeny, fibroblasts present both divergent global gene expression profiles and chromatin states compared to neurons. In this “unfavorable environment,” some neuronal TFs unexpectedly revealed to have a pioneer function being able to “open up” the chromatin and activate genes that are silenced in donor cells. Conversely, in vivo, their function might be facilitated by other TFs expressed earlier in the transcriptional cascades or their function might be hidden by complex gene regulation networks. With its ability to directly challenge TFs, the direct neuronal reprogramming provides a unique experimental system where to better appreciate their role in a relatively simple in vitro assay with a clear phenotypic analysis outcome.
The authors thank members of the Borrell laboratory, P. Bayly, R. Toro, M. Götz and W. Huttner, for insightful discussions. The authors apologize to colleagues whose research was not cited owing to the broad scope and space limitations of this Review. The authors’ research was supported by a European Research Grant (CORTEXFOLDING-309633), a Spanish Ministry of Economy and Competitiveness Grant (SAF2015-69168-R), the European Union Seventh Framework Programme (FP7/2007-2013, under the project DESIRE-602531) and the Spanish State Research Agency through the ‘Severo Ochoa’ Programme for Centers of Excellence in Research and Development (reference SEV-2017-0723).
Nature Reviews Neuroscience thanks H. Kawasaki and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Neurogenesis welcomes submissions of the following article types: Brief Research Report, Case Report, Clinical Trial, Community Case Study, Correction, Data Report, Editorial, General Commentary, Hypothesis and Theory, Methods, Mini Review, Opinion, Original Research, Perspective, Review, Specialty Grand Challenge, Systematic Review and Technology and Code.
All manuscripts must be submitted directly to the section Neurogenesis, where they are peer-reviewed by the Associate and Review Editors of the specialty section.
17.24: Neurogenesis - Biology
It is critical that applicants follow the Research (R) Instructions in the SF424 (R&R) Application Guide, except where instructed to do otherwise (in this FOA or in a Notice from the NIH Guide for Grants and Contracts). Conformance to all requirements (both in the Application Guide and the FOA) is required and strictly enforced. Applicants must read and follow all application instructions in the Application Guide as well as any program-specific instructions noted in Section IV. When the program-specific instructions deviate from those in the Application Guide, follow the program-specific instructions. Applications that do not comply with these instructions may be delayed or not accepted for review.
The overall objective of this initiative is to identify both molecular and physiological targets that are potentially amenable to intervention strategies for the prevention, mitigation and treatment of Alzheimer's disease as well as other neurodegenerative diseases.
The number of persons with neurodegenerative diseases (NDs) such as Alzheimer’s, Parkinson’s and Huntington’s diseases (AD, PD, HD) is on the rise as well as that of other, rarer conditions such as Pick’s disease, Creutzfeld-Jacob’s disease (CJD), progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS). Finding effective cures or prevention strategies for neurodegenerative diseases is still elusive in spite of intense research efforts over the last few decades. The challenges to achieve this goal are many. A major one is the identification of common molecular, cellular or environmental causes for any single disease. For instance, while a number of conditions share several common features, subsets of patients, even within a single disease category, display distinct clinical and pathological features. Some conditions, such as HD, appear to have a direct genetic cause. In the case of other NDs, the majority of patients do not share any common genetic trait. Within the small subset of individuals with an associated genetic linkage, neuronal degeneration and death results from one or more distinct mutations causing aberrant protein folding, abnormalities in protein degradation, mitochondrial dysfunction, or compromised axonal transport processes. Some of these pathological manifestations have served as primary targets for therapeutic interventions. Success, however, has been limited to temporary symptom relief without any significant alteration of disease progression. Beyond an incomplete understanding of CNS physiology, a major impediment to the discovery and clinical development of therapeutics is our still rudimentary grasp of ND etiology, in large part due to a lack of animal models that can simulate the long course and symptomatology of these diseases and allow the assessment of therapeutic effectiveness. Loss of brain functional ability and neurodegeneration are common features of aging throughout the phylogenetic scale. However, most animal models of terminal neurological diseases currently used in research consist of introducing known causative genetic mutations that are linked to human NDs into rodent models (mice and, in a few cases, rats). Most of them do not fully recapitulate neuronal loss, supporting the idea that neuronal death is caused by a combination of genetic, cellular, and environmental factors. Some models even fail to display the phenotypic alterations associated with the modeled diseases, evidence that humans could be more vulnerable than mice to the same triggers of degeneration. Taken together, these observations suggest the existence, in some species, of compensatory mechanisms that protect neurons from neuronal degeneration. Understanding what these mechanisms are might help identify the preclinical defects that trigger pathogenesis and underlie the presymptomatic phase of the disease. Comparative studies of cellular/molecular pathways that affect aging in different genetic backgrounds can provide a solid foundation to discover critical factors that may explain differences in ND protection between humans and other animal species.
Research on model organisms such as mice, roundworms, fruit flies, and yeast has dramatically advanced our understanding of aging over the past 50 years. Comparative biology research can provide new insights into cellular and molecular mechanisms of aging and disease susceptibility by leveraging natural variants produced over evolutionary time. Several examples of the power of comparative biology come from studies recently supported by NIA. For instance, comparisons of long-lived and short-lived animal species have shown a strong correlation between longevity and enhanced proteostasis, in both vertebrates and invertebrates. Whether this enhanced proteostasis translates into improved resistance to neurodegenerative diseases remains to be investigated. Other studies conducted in multiple species of rotifers, on the other hand, have made a unique contribution in exploring the effect on lifespan of a variety of caloric restriction regimens, small molecule inhibitors, and dietary supplements, and have probed pathways using RNAi. By capitalizing on a broad range of genetic backgrounds, comparative biology approaches are likely to provide valuable insights into evolutionarily conserved cellular/molecular pathways that affect aging-related neurodegenerative diseases and conditions.
Studies involving comparative approaches, both across a broad range of species and across closely related species or strains with different degrees of neurodegeneration and/or resilience to it, could identify components in the pathways that are conserved in function and therefore, potential targets for intervention. At the molecular level, several major pathways that could be involved in resilience to terminal neurodegenerative diseases have been identified. They include autophagy and glycolysis, as well as signaling through metalloproteases, neurotrophins, noradrenaline, ERK and PKCdelta further exploration of these pathways in different contexts is necessary. The fact that these pathways are highly conserved among vertebrates (and some even in invertebrates) makes them potentially informative targets for comparative biology approaches, as well as druggable targets for potential intervention into humans.
The long-term goal of this initiative is to identify novel interventional targets, conserved across a number of species, for the prevention, mitigation and treatment of neurodegenerative diseases. This FOA will support exploratory comparative biology research projects that exploit similarities and differences among different species (or strains within a species) in important molecular, cellular and physiological pathways which affect neurodegeneration and resilience to it. Applicants are invited to submit innovative applications using comparative approaches to explore cellular physiology pathways that might influence neurodegeneration such as adaptation to stress, macromolecular damage, proteostasis and stem cell function and regeneration. Proposed projects should include but not be limited to comparative studies of how different species respond to challenges and damage to these pathways and thereby resist or succumb to age-related neurodegeneration. Approaches can be across vertebrate or invertebrate species or across strains of a given species (i.e. recombinant inbred mouse strains) and will include in vivo research in animal models or in vitro research in cells.
This initiative utilizes the R21 funding mechanism for exploratory and early stage research with a focus on comparative biology that examines how different animal species respond to challenges and damage to cellular physiology pathways that might influence the onset of Alzheimer's and other neurodegenerative diseases as well as resilience to them. These studies may involve considerable risk but may lead to a breakthrough in a particular area or to the development of novel techniques, agents, methodologies, models, or applications that could have a major impact on the field of neurodegeneration and of Alzheimer's disease.
See Section VIII. Other Information for award authorities and regulations.
Grant: A support mechanism providing money, property, or both to an eligible entity to carry out an approved project or activity.
Structure and function of the Mind bomb E3 ligase in the context of Notch signal transduction
N-terminal region of Mind bomb1 recognizes two separate epitopes on Notch ligands.
An architecture resembling Cullin-E3 ligases is proposed for full-length Mind bomb.
Bipartite tail recognition mechanism suggests potential for creating oligomeric assemblies.
The Notch signaling pathway has a critical role in cell fate determination and tissue homeostasis in a variety of different lineages. In the context of normal Notch signaling, the Notch receptor of the ‘signal-receiving’ cell is activated in trans by a Notch ligand from a neighboring ‘signal-sending’ cell. Genetic studies in several model organisms have established that ubiquitination of the Notch ligand, and its regulated endocytosis, is essential for transmission of this activation signal. In mammals, this ubiquitination step is dependent on the protein Mind bomb 1 (Mib1), a large multi-domain RING-type E3 ligase, and its direct interaction with the intracellular tails of Notch ligand molecules. Here, we discuss our current understanding of Mind bomb structure and mechanism in the context of Notch signaling and beyond.
Mouse study finds link between gut bacteria and neurogenesis
IMAGE: This visual abstract depicts the findings of Möhle et al., which show the impact of prolonged antibiotic treatment on brain cell plasticity and cognitive function. They were able to rescue. view more
Credit: Möhle et al./Cell Reports 2016
Antibiotics strong enough to kill off gut bacteria can also stop the growth of new brain cells in the hippocampus, a section of the brain associated with memory, reports a study in mice published May 19 in Cell Reports. Researchers also uncovered a clue to why-- a type of white blood cell seems to act as a communicator between the brain, the immune system, and the gut.
"We found prolonged antibiotic treatment might impact brain function," says senior author Susanne Asu Wolf of the Max-Delbrueck-Center for Molecular Medicine in Berlin, Germany. "But probiotics and exercise can balance brain plasticity and should be considered as a real treatment option."
Wolf first saw clues that the immune system could influence the health and growth of brain cells through research into T cells nearly 10 years ago. But there were few studies that found a link from the brain to the immune system and back to the gut.
In the new study, the researchers gave a group of mice enough antibiotics for them to become nearly free of intestinal microbes. Compared to untreated mice, the mice who lost their healthy gut bacteria performed worse in memory tests and showed a loss of neurogenesis (new brain cells) in a section of their hippocampus that typically produces new brain cells throughout an individual's lifetime. At the same time that the mice experienced memory and neurogenesis loss, the research team detected a lower level of white blood cells (specifically monocytes) marked with Ly6Chi in the brain, blood, and bone marrow. So researchers tested whether it was indeed the Ly6Chi monocytes behind the changes in neurogenesis and memory.
In another experiment, the research team compared untreated mice to mice that had healthy gut bacteria levels but low levels of Ly6Chi either due to genetics or due to treatment with antibodies that target Ly6Chi cells. In both cases, mice with low Ly6Chi levels showed the same memory and neurogenesis deficits as mice in the other experiment who had lost gut bacteria. Furthermore, if the researchers replaced the Ly6Chi levels in mice treated with antibiotics, then memory and neurogenesis improved.
"For us it was impressive to find these Ly6Chi cells that travel from the periphery to the brain, and if there's something wrong in the microbiome, Ly6Chi acts as a communicating cell," says Wolf.
Luckily, the adverse side effects of the antibiotics could be reversed. Mice who received probiotics or who exercised on a wheel after receiving antibiotics regained memory and neurogenesis. "The magnitude of the action of probiotics on Ly6Chi cells, neurogenesis, and cognition impressed me," she says.
But one result in the experiment raised more questions about the gut's bacteria and the link between Ly6Chi and the brain. While probiotics helped the mice regain memory, fecal transplants to restore a healthy gut bacteria did not have an effect.
"It was surprising that the normal fecal transplant recovered the broad gut bacteria, but did not recover neurogenesis," says Wolf. "This might be a hint towards direct effects of antibiotics on neurogenesis without using the detour through the gut. To decipher this we might treat germ free mice without gut flora with antibiotics and see what is different."
In the future, researchers also hope to see more clinical trials investigating whether probiotic treatments will improve symptoms in patients with neurodegenerative and psychiatric disorders."We could measure the outcome in mood, psychiatric symptoms, microbiome composition and immune cell function before and after probiotic treatment," says Wolf.
This project was funded by the German Research Council
Cell Reports, Möhle, Mattei, and Heimesaat et al.: "Ly6Chi Monocytes Provide a Link between Antibiotic-Induced Changes in Gut Microbiota and Adult Hippocampal Neurogenesis" http://www.cell.com/cell-reports/fulltext/S2211-1247(16)30518-6
Cell Reports (@CellReports), published by Cell Press, is a weekly open-access journal that publishes high-quality papers across the entire life sciences spectrum. The journal features reports, articles, and resources that provide new biological insights, are thought-provoking, and/or are examples of cutting-edge research. Visit: http://www. cell. com/ cell-reports. To receive Cell Press media alerts, contact [email protected]
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Cell Biology of Mammalian Neurogenesis
Figure 1. Mouse and human neocortex development.
In mouse, aRG cells are the major neural stem cells. They are highly elongated, with an apical process in contact with the ventricular surface and a basal process connected to the pial surface of the brain. In human, the bRG cells (orange) have lost connection to the apical surface and are localized basally, in the highly enlarged OSVZ. I.P.: Intermediate Progenitor.
Cortical development occurs by proliferation of neural stem cells and migration of newborn neurons over substantial distances. In mice, neuroepithelial cells differentiate into apical radial glial cells (aRG), which act as the stem cells of the developing neocortex and give rise to all neocortical neurons, most glial cells and to the adult stem cells. aRG cells are highly elongated, extending a basal process all the way to the pial surface of the developing brain and an apical process that remains in contact with the ventricular surface (Figure. 1). They undergo fascinating cell cycle-dependent nuclear oscillations, a process known as Interkinetic Nuclear Migration (INM). We previously described the mechanism for G2 apical nuclear migration, which involves a Cdk1-triggered recruitment of the dynein motor to the nuclear pore complex (Hu et al., 2013 Baffet et al., 2015). aRG cells can divide asymmetrically to produce one aRG cell and one intermediate progenitor that will divide to produce two neurons.
Although the ventricular zone (VZ), where aRG cells are located, is the major neurogenic region in mice, the situation is very different in humans where 85% of mitoses occur in the sub-ventricular zone (SVZ). As a consequence, this region is massively thicker and becomes subdivided into two morphologically different regions, the inner SVZ (ISVZ) and the outer SVZ (OSVZ). This is largely due to a second type of neural stem cells, called the basal Radial Glial (bRG) cells (also known as oRG cells) (Figure. 1). bRG cells derive from aRG cells and share several characteristics with these cells, such as the extension of a basal process, the expression of a similar set of transcription factors and, most importantly, the capacity to self-renew. bRG cells are very rare in the smooth mouse brain (lissencephalic) and very abundant in the folded human brain (gyrencephalic) and are thought to be fundamental players of the massive size increase of the human brain.
Our group currently focuses on three main topics:
1/ Mechanisms of polarized trafficking in RG cells
In the highly elongated aRG and bRG cells, long-range polarized trafficking occurs to deliver critical molecules, including surface receptors or secreted factors. To investigate these processes, we have recently developed a method for high resolution subcellular live imaging within thick embryonic brain slices. This approach allows to resolve individual fast-moving structures, such as RAB6+ positive vesicles while they are transported in the apical or basal process (Figure 2).
/> Figure 2. Subcellular live imaging in RG cells in situ.
A. Experimental set-up for mouse embryonic brain in utero electroporation and brain slice preparation. B. Live imaging of GFP-RAB6 in mouse radial glial cell in situ.
Using this method, we have identified the organization of the microtubule cytoskeleton in RG cells (Coquand et al., 2020).We showed that, while microtubules in the apical process of aRG cells in uniformly oriented basally, microtubules in the basal process display a mixed polarity, reminiscent of the mammalian dendrite (Figure 3). We furthermore showed that this acentrosomal microtubule network is organized from CAMSAP+ varicosities of the basal process, an organization conserved in human bRG cells. We are currently initiating a project aimed at identifying the factors that control this acentrosomal bipolar microtubule network organization.
Figure 3. Microtubule organization in RG cells in situ.
Microtubules in the apical process have a unipolar basal orientation, while microtubules in the basal process are bipolar and are organized from varicosities.
Our second project aims at identifying the mechanism for post-Golgi apical trafficking in epithelial cells, using aRG cells as a model system (Brault et al, in preparation). Using our live imaging method, as well as a newly generated RAB6A/B double knock-out mouse, we have identified the core machinery that drives post-Golgi transport of the Crumbs apical determinant to the apical surface of mouse aRG cells. We show that impairment of this pathways leads to a delamination of aRG cells that now divide basally, adopting a bRG-like identity. We have now begun a novel project aimed at identifying the mechanisms for basal secretion in RG cells.
2/ Human neurogenesis
To image human bRG cells, we have established the generation of iPS cell-derived human cerebral organoids. We observe the appearance of bRG cells after 5 weeks of culture, which gradually increases in the following weeks. While organoids usually display mild amounts of bRG cells, our adapted protocol leads to the formation of a large OSVZ containing a very abundant bRG cell population (Figure 4A). In parallel, we have developed the culture and imaging of human fetal brain tissue, obtained through collaborations with two Parisian hospitals (Figure 4B). Because of the dynamic nature of the processes we investigate, we have developed methods for quantitative live imaging of human fetal tissue and cerebral organoids. Tissues are sliced and put into culture for 7 days. Plasmids encoding fluorescent reporters or knockdown constructs are delivered using retroviral infection and slices are live imaged using a CSU-W1 Spinning wide microscope for 24 to 48 hours (Figure 4C, D). Finally, we have developed a protocol to generate in vitro cultures of human RG cells, obtained from human fetal tissue or cerebral organoids. We have developed assays based on microfabrication techniques allowing to align them on micropatterns of adhesion, in order to manipulate their shape (Figure 4E, F).
Figure 4. Methods for the investigation of human bRG cells.
A. Human cerebral organoid stained for the RG cell marker Sox2, the intermediate progenitor marker Tbr2 and the neuronal marker NeuN. B. Human fetal brain slice stained for the RG cell marker Sox2 and the mitotic RG cell marker Phospho-Vimentin (P-Vim). C. Schematic representation of brain slice infection and in vitro RG cell culture from fetal brain tissue biopsy. D. Live imaging of migrating and dividing bRG cell in human fetal cortex. E. In vitro cultivated human RG cells. F. A RG cell aligned on an elongated fibronectin pattern.
We currently pursue two main projects that focus on human bRG cells. First, we investigate how fate decisions are made in these cells. We have developed a live-fix correlative microscopy method, that allows us to live image dividing bRG cells in situ, and to identify the fate of the daughter cells. This approach has allowed us to generate a map of bRG cell division modes. Remarkably, this map is highly conserved between human fetal tissue and cerebral organoids (Coquand et al, in preparation). We are now using this knowledge to identify the mechanisms controlling asymmetric cell division, whereby one bRG cells divides into one self-renewing bRG cell and one differentiating cell. In a second project, we are investigating the cytoskeletal-based mechanisms that drive the migration of bRG cells into the developing human neocortex.
3/ Brain Malformations
Alterations in RG cells can lead to a variety of cerebral malformations, which can have genetic or environmental causes (including viral infection). We developed an assay to infect brain slices with flaviviruses and demonstrated that the microcephaly-causing Zika virus (but not other closely-related flaviviruses) specifically infect RG cells and lead to apoptotic cell death (Brault et al., 2016).
In another project, our collaborators have identified mutations in the WDR81 gene that causes extreme microcephaly in newborn children. Using a newly generated WDR81 KO mouse model as well as patient-derived cells, we demonstrated that reduced brain size is specifically due to reduced proliferation rates of RG cells. We showed that WDR81 regulates endosomal trafficking of EGFR, and that loss of function leads to reduced MAP kinase pathway activation (Carpentieri et al., 2020).
Finally, we use patient-derived iPS cells and cerebral organoids to model how mutations can affect human brain development. In particular, we focus on mutations in the DHC1H1 gene, which codes for the dynein heavy chain. Strikingly, our collaborators have identified clusters of mutations that can lead either to cortical malformations, or to motor neuron degeneration (Spinal Muscular Atrophy). We investigate how these different types of mutations differentially affect dynein-based process in the developing cerebral tissue (Farcy et al, in preparation).