Stimulation of the muscarinic receptor M4 regulates neural precursor cell proliferation and promotes adult hippocampal neurogenesis.

Cholinergic signaling plays a crucial role in the regulation of adult hippocampal neurogenesis; however, the mechanisms by which acetylcholine mediates neurogenic effects are not completely understood. Here, we report the expression of muscarinic acetylcholine receptor subtype M4 (M4 mAChR) on a subpopulation of neural precursor cells (NPCs) in the adult mouse hippocampus, and demonstrate that its pharmacological stimulation promotes their proliferation, thereby enhancing the production of new neurons in vivo. Using a targeted ablation approach, we also show that medial septum (MS) and the diagonal band of Broca (DBB) cholinergic neurons support both the survival and morphological maturation of adult-born neurons in the mouse hippocampus. Although the systemic administration of an M4-selective allosteric potentiator fails to fully rescue the MS/DBB cholinergic lesion-induced decrease in hippocampal neurogenesis, it further exacerbates the impairment in the morphological maturation of adult-born neurons. Collectively, these findings reveal stage-specific roles of M4 mAChRs in regulating adult hippocampal neurogenesis, uncoupling their positive role in enhancing the production of new neurons from the M4-induced inhibition of their morphological maturation, at least in the context of cholinergic signaling dysfunction.

The multifaceted role of platelets in mediating brain function.

Platelets, the small, anucleate blood cells that originate from megakaryocytes in the bone marrow, are typically associated with coagulation. However, it is now apparent that platelets are more multifaceted than originally thought, with their function extending beyond their traditional role in hemostasis to acting as important mediators of brain function. In this review, we outline the broad repertoire of platelet function in the central nervous system, focusing on the similarities between platelets and neurons. We also summarize the role that platelets play in the pathophysiology of various neurological diseases, with a particular focus on neuroinflammation and neurodegeneration. Finally, we highlight the exciting prospect of harnessing the unique features of the platelet proteome and extracellular vesicles, which are rich in neurotrophic, antioxidative, and antiinflammatory factors, for the development of novel neuroprotective and neuroregenerative interventions to treat various neurodegenerative and traumatic pathologies.

The relationship between adult hippocampal neurogenesis and cognitive impairment in Alzheimer's disease.

Neurogenesis persists throughout adulthood in the hippocampus and contributes to specific cognitive functions. In Alzheimer's disease (AD), the hippocampus is affected by pathology and functional impairment early in the disease. Human AD patients have reduced adult hippocampal neurogenesis (AHN) levels compared to age-matched healthy controls. Similarly, rodent AD models show a decrease in AHN before the onset of the classical hallmarks of AD pathology. Conversely, enhancement of AHN can protect against AD pathology and ameliorate memory deficits in both rodents and humans. Therefore, impaired AHN may be a contributing factor of AD-associated cognitive decline, rather than an effect of it. In this review we outline the regulation and function of AHN in healthy individuals, and highlight the relationship between AHN dysfunction and cognitive impairments in AD. The existence of AHN in humans and its relevance in AD patients will also be discussed, with an outlook toward future research directions. HIGHLIGHTS: Adult hippocampal neurogenesis occurs in the brains of mammals including humans. Adult hippocampal neurogenesis is reduced in Alzheimer's disease in humans and animal models.

p27kip1 Is Required for Functionally Relevant Adult Hippocampal Neurogenesis in Mice.

We asked whether cell-cycle associated protein p27kip1 might be involved in the transition of precursor cells to postmitotic maturation in adult hippocampal neurogenesis. p27kip1 was expressed throughout the dentate gyrus with a strong nuclear expression in early postmitotic, calretinin-positive neurons and neuronally determined progenitor cells (type-3 and some type-2b), lower or absent expression in radial glia-like precursor cells (type-1) and type-2a cells and essentially no expression in granule cells. This suggested a transitory role in late proliferative and early postmitotic phases of neurogenesis. Inconsistent with a role limited to cell cycle arrest the acute stimuli, voluntary wheel running (RUN), environmental enrichment (ENR) and kainate-induced seizures increased p27kip1 expressing cells. Sequential short-term combination of RUN and ENR yielded more p27kip1 cells than either stimulus alone, indicating an additive effect. In vitro, p27kip1 was lowly expressed by proliferating precursor cells but increased upon differentiation. In p27kip1-/- mice neurogenesis was reduced in vivo, whereas the number of proliferating cells was increased. Accordingly, the microdissected dentate gyrus of p27kip1-/- mice generated more colonies in the neurosphere assay and an increased number of larger spheres with the differentiation potential unchanged. In p27kip1-/- monolayer cultures, proliferation was increased and cell cycle genes were upregulated. In the Morris water maze p27kip1-/- mice learned the task but were specifically impaired in the reversal phase explainable by the decrease in adult neurogenesis. We conclude that p27kip1 is involved in the decisive step around cell-cycle exit and plays an important role in activity-regulated and functionally relevant adult hippocampal neurogenesis. Stem Cells 2017;35:787-799.

Prominin-1 allows prospective isolation of neural stem cells from the adult murine hippocampus.

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However, despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone, no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo, including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting, only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation, self-renewal, and multipotentiality as assessed by a neurosphere assay. In addition, isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally, despite its expression by various stem and progenitor cells, Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless, a net decrease in hippocampal neurogenesis, by ∼30% was found in Prominin-1 knock-out mice, suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably, an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism, which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene.

Immature doublecortin-positive hippocampal neurons are important for learning but not for remembering.

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.

The Active Place Avoidance (APA) Test, an Effective, Versatile and Repeatable Spatial Learning Task for Mice.

Hippocampus-dependent spatial learning in rodents has been tested using a variety of methods. These include the Morris water maze (MWM), Y-maze, and novel object location (NOL) tasks. More recently, the active place avoidance (APA) task has been developed as an alternative to these more traditional approaches. In the APA task, mice must use spatial cues placed around a rotating arena to avoid a stationary shock zone. Due to the multiple parameters that can be adjusted, the APA task has been demonstrated to be a very versatile approach. It lends itself to being used longitudinally and repeatedly for the same cohort of mice. Here, we provide a detailed protocol to successfully conduct the APA task. We also highlight alternative APA approaches that can be used to examine different components of spatial learning. We describe the data collection and analysis processes. Critical steps during the APA task are discussed to increase the likelihood of successfully conducting the test. The APA task has several advantages over more traditional spatial navigation tests. It is appropriate to use with aged mice or those with disease phenotypes such as Alzheimer's disease. The complexity of the task can be easily altered, allowing a wide range of mouse strains to be tested. Further, the APA task is suitable for testing animals that have undergone surgery or experimental interventions that may have affected motor or neural function, such as stroke or traumatic brain injury.

Lrp8 knockout mice fed a selenium-replete diet display subtle deficits in their spatial learning and memory function.

Selenium is an essential trace element that is delivered to the brain by the selenium transport protein selenoprotein P (SEPP1), primarily by binding to its receptor low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2), at the blood-brain barrier. Selenium transport is required for several important brain functions, with transgenic deletion of either Sepp1 or Lrp8 resulting in severe neurological dysfunction and death in mice fed a selenium-deficient diet. Previous studies have reported that although feeding a standard chow diet can prevent these severe deficits, some motor coordination and cognitive dysfunction remain. Importantly, no single study has directly compared the motor and cognitive performance of the Sepp1 and Lrp8 knockout (KO) lines. Here, we report the results of a comprehensive parallel analysis of the motor and spatial learning and memory function of Sepp1 and Lrp8 knockout mice fed a standard mouse chow diet. Our results revealed that Sepp1 knockout mice raised on a selenium-replete diet displayed motor and cognitive function that was indistinguishable from their wild-type littermates. In contrast, we found that although Lrp8-knockout mice fed a selenium-replete diet had normal motor function, their spatial learning and memory showed subtle deficits. We also found that the deficit in baseline adult hippocampal neurogenesis exhibited by Lrp8-deficit mice could not be rescued by dietary selenium supplementation. Taken together, these findings further highlight the importance of selenium transport in maintaining healthy brain function. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Blood-brain crosstalk: the roles of neutrophils, platelets, and neutrophil extracellular traps in neuropathologies.

Systemic inflammation, neurovascular dysfunction, and coagulopathy often occur concurrently in neuropathologies. Neutrophils and platelets have crucial synergistic roles in thromboinflammation and are increasingly suspected as effector cells contributing to the pathogenesis of neuroinflammatory diseases. In this review, we summarize the roles of platelet-neutrophil interactions in triggering complex pathophysiological events affecting the brain that may lead to the disruption of brain barriers, infiltration of toxic factors into the parenchyma, and amplification of neuroinflammation through the formation of neutrophil extracellular traps (NETs). We highlight the clinical significance of thromboinflammation in neurological disorders and examine the contributions of damage-associated molecular patterns (DAMPs) derived from platelets and neutrophils. These DAMPs originate from both infectious and non-infectious risk factors and contribute to the activation of inflammasomes during brain disorders. Finally, we identify knowledge gaps in the molecular mechanisms underlying neurodegenerative disease pathogenesis and emphasize the potential of interventions targeting platelets and neutrophils to treat neuroinflammatory diseases.

Selenium supplementation provides potent neuroprotection following cerebral ischemia in mice.

Despite progress in reperfusion therapy, functional recovery remains suboptimal in many stroke patients, with oxidative stress, inflammation, dysbiosis, and secondary neurodegeneration constituting the major hurdles to recovery. The essential trace element selenium is emerging as a promising therapeutic agent for stroke. However, although several rodent studies have shown that selenium can protect against cell loss following cerebral ischemia, no study has yet examined whether selenium can enhance long-term functional recovery. Moreover, published studies have typically reported a single mechanism of action underlying selenium-mediated stroke recovery. However, we propose that selenium is more likely to have multifaceted actions. Here, we show that selenomethionine confers a potent neuroprotective effect in a canonical filament-induced transient middle cerebral artery occlusion (tMCAO) mouse model. Post-tMCAO selenium treatment significantly reduces the cerebral infarct volume, oxidative stress, and ferroptosis and enhances post-tMCAO motor performance in the acute phase after stroke. Moreover, analysis of the gut microbiota reveals that acute selenium treatment reverses stroke-induced gut dysbiosis. Longer-term selenium supplementation activates intrinsic neuroprotective mechanisms, prevents secondary neurodegeneration, alleviates systemic inflammation, and diminishes gut microbe-derived circulating trimethylamine N-oxide. These findings demonstrate that selenium treatment even after cerebral ischemia has long-term and multifaceted neuroprotective effects, highlighting its clinical potential.

Platelet-derived exerkine CXCL4/platelet factor 4 rejuvenates hippocampal neurogenesis and restores cognitive function in aged mice.

The beneficial effects of physical activity on brain ageing are well recognised, with exerkines, factors that are secreted into the circulation in response to exercise, emerging as likely mediators of this response. However, the source and identity of these exerkines remain unclear. Here we provide evidence that an anti-geronic exerkine is secreted by platelets. We show that platelets are activated by exercise and are required for the exercise-induced increase in hippocampal precursor cell proliferation in aged mice. We also demonstrate that increasing the systemic levels of the platelet-derived exerkine CXCL4/platelet factor 4 (PF4) ameliorates age-related regenerative and cognitive impairments in a hippocampal neurogenesis-dependent manner. Together these findings highlight the role of platelets in mediating the rejuvenating effects of exercise during physiological brain ageing.

Isolation and Culture of Adult Hippocampal Precursor Cells as Free-Floating Neurospheres.

The neurosphere assay is the most widely used in vitro tool to determine the proliferative and differentiation potential of adult neural precursor cells in rodents. Although originally developed for, and predominantly applied to, the growth of embryonic and adult subventricular zone-derived stem cells, hippocampal neurospheres are now routinely cultured by many laboratories. As hippocampal neurospheres are fewer in number, on average smaller in size, and more slowly growing than their ventricular counterparts, the methodology traditionally used to isolate and culture neurospheres from the subventricular zone is not optimal for hippocampal neurosphere growth. Here, we provide a detailed description of an optimized protocol for the microdissection, dissociation, and neurosphere generation from adult hippocampal dentate gyrus tissue. We also outline the protocols required to perform downstream passaging, differentiation, and immunohistological determination of the multipotentiality of hippocampal neurospheres.

Protocol for three alternative paradigms to test spatial learning and memory in mice.

Here, we describe three alternative paradigms to overcome the limitations of the most widely used spatial learning paradigm for rodents: the Morris water maze. We outline the preparation of behavioral testing rooms and mouse handling/habituation prior to testing. We then detail three spatial learning and memory tasks: the Barnes maze, active place avoidance, and novel object location tasks. These tests have been successfully used across multiple ages (from 2 to 24 months) in both wild-type and transgenic animals. For complete details on the use and execution of this protocol, please refer to Leiter et al. (2022).

Does a rare mutation in PTPRA contribute to the development of Parkinson's disease in an Australian multi-incident family?

The genetic study of multi-incident families is a powerful tool to investigate genetic contributions to the development of Parkinson's disease. In this study, we identified the rare PTPRA p.R223W variant as one of three putative genetic factors potentially contributing to disease in an Australian family with incomplete penetrance. Whole exome sequencing identified these mutations in three affected cousins. The rare PTPRA missense variant was predicted to be damaging and was absent from 3,842 alleles from PD cases. Overexpression of the wild-type RPTPα and R223W mutant in HEK293T cells identified that the R223W mutation did not impair RPTPα expression levels or alter its trafficking to the plasma membrane. The R223W mutation did alter proteolytic processing of RPTPα, resulting in the accumulation of a cleavage product. The mutation also resulted in decreased activation of Src family kinases. The functional consequences of this variant, either alone or in concert with the other identified genetic variants, highlights that even minor changes in normal cellular function may increase the risk of developing PD.

Selenium mediates exercise-induced adult neurogenesis and reverses learning deficits induced by hippocampal injury and aging.

Although the neurogenesis-enhancing effects of exercise have been extensively studied, the molecular mechanisms underlying this response remain unclear. Here, we propose that this is mediated by the exercise-induced systemic release of the antioxidant selenium transport protein, selenoprotein P (SEPP1). Using knockout mouse models, we confirmed that SEPP1 and its receptor low-density lipoprotein receptor-related protein 8 (LRP8) are required for the exercise-induced increase in adult hippocampal neurogenesis. In vivo selenium infusion increased hippocampal neural precursor cell (NPC) proliferation and adult neurogenesis. Mimicking the effect of exercise through dietary selenium supplementation restored neurogenesis and reversed the cognitive decline associated with aging and hippocampal injury, suggesting potential therapeutic relevance. These results provide a molecular mechanism linking exercise-induced changes in the systemic environment to the activation of quiescent hippocampal NPCs and their subsequent recruitment into the neurogenic trajectory.

Endogenous interferon gamma directly regulates neural precursors in the non-inflammatory brain.

Although a number of growth factors have been shown to be involved in neurogenesis, the role of inflammatory cytokines remains relatively unexplored in the normal brain. Here we investigated the effect of interferon gamma (IFNgamma) in the regulation of neural precursor (NP) activity in both the developing and the adult mouse brain. Exogenous IFNgamma inhibited neurosphere formation from the wild-type neonatal and adult subventricular zone (SVZ). More importantly, however, these effects were mirrored in vivo, with mutant mice lacking endogenous IFNgamma displaying enhanced neurogenesis, as demonstrated by an increase in proliferative bromodeoxyuridine-labeled cells in the SVZ and an increased percentage of newborn neurons in the olfactory bulb. Furthermore, NPs isolated from IFNgamma null mice exhibited an increase in self-renewal ability and in the capacity to produce differentiated neurons and oligodendrocytes. These effects resulted from the direct action of IFNgamma on the NPs, as determined by single-cell assays and the fact that nearly all the neurospheres were derived from cells positive for major histocompatibility complex class I antigen, a downstream marker of IFNgamma-mediated activation. Moreover, the inhibitory effect was ameliorated in the presence of SVZ-derived microglia, with their removal resulting in almost complete inhibition of NP proliferation. Interestingly, in contrast to the results obtained in the adult, exogenous IFNgamma treatment stimulated neurosphere formation from the embryonic brain, an effect that was mediated by sonic hedgehog. Together these findings provide the first direct evidence that IFNgamma acts as a regulator of the active NP pool in the non-inflammatory brain.

Apple Peel and Flesh Contain Pro-neurogenic Compounds.

As mammals evolved with exposure to particular diets, naturally abundant compounds may have become part of the set of environmental co-determinants that shaped brain structure and function. Here we investigated whether bioactive factors found in apples directly affect hippocampal neurogenesis in the adult mouse. We found that quercetin, the most abundant flavanol in apple peel, was anti-proliferative at high concentrations but pro-neurogenic at low concentrations. This was confirmed in vivo, with intraperitoneally delivered quercetin promoting survival and neuronal differentiation, without affecting proliferation. Using a bioassay-guided fractionation approach we also identified additional pro-neurogenic compounds in apple flesh that were not related to flavonoids. We found that 3,5-dihydroxybenzoic acid significantly increased neural precursor cell proliferation and neurogenesis. This work shows that both flavonoids and 3,5-dihydroxybenzoic acid are pro-neurogenic, not only by activating precursor cell proliferation but also by promoting cell-cycle exit, cellular survival, and neuronal differentiation.

ROS Dynamics Delineate Functional States of Hippocampal Neural Stem Cells and Link to Their Activity-Dependent Exit from Quiescence.

Cellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of intracellular reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we here show that in the hippocampus of adult mice, quiescent neural precursor cells (NPCs) maintain the highest ROS levels (hiROS). Classifying NPCs on the basis of cellular ROS content identified distinct functional states. Shifts in ROS content primed cells for a subsequent state transition, with lower ROS content marking proliferative activity and differentiation. Physical activity, a physiological activator of adult hippocampal neurogenesis, recruited hiROS NPCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results provide a metabolic classification of NPC functional states and describe a mechanism linking the modulation of cellular ROS by behavioral cues to the activation of adult NPCs.

Platelets in Neurodegenerative Conditions-Friend or Foe?

It is now apparent that platelet function is more diverse than originally thought, shifting the view of platelets from blood cells involved in hemostasis and wound healing to major contributors to numerous regulatory processes across different tissues. Given their intriguing ability to store, produce and release distinct subsets of bioactive molecules, including intercellular signaling molecules and neurotransmitters, platelets may play an important role in orchestrating healthy brain function. Conversely, a number of neurodegenerative conditions have recently been associated with platelet dysfunction, further highlighting the tissue-independent role of these cells. In this review we summarize the requirements for platelet-neural cell communication with a focus on neurodegenerative diseases, and discuss the therapeutic potential of healthy platelets and the proteins which they release to counteract these conditions.

Author Correction: Mast cells increase adult neural precursor proliferation and differentiation but this potential is not realized in vivo under physiological conditions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.