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Physical exercise may reduce dementia risk in aging, but varying reports on its effectiveness make it challenging to ascribe what level of exercise will have significant longer-term effects on important functions such as hippocampal-based learning and memory. This study compared the effect of three different 6-month exercise regimens on hippocampal-dependent cognition in healthy, elderly individuals. Participants, aged 65-85 with no cognitive deficits, were randomly assigned to one of three exercise interventions (low (LIT), medium (MIT), and High intensity interval training (HIIT), respectively). Each participant attended 72 supervised exercise sessions over a 6-month period. A total of 151 participants completed all sessions. Cognitive testing for hippocampal performance occurred monthly, as did blood collection, and continued for up to 5 years following initiation of the study. Multimodal 7 Tesla MRI scans were taken at commencement, 6 and 12 months. After 6 months, only the HIIT group displayed significant improvement in hippocampal function, as measured by paired associative learning (PAL). MRI from the HIIT group showed abrogation of the age-dependent volumetric decrease within several cortical regions including the hippocampus and improved functional connectivity between multiple neural networks not seen in the other groups. HIIT-mediated changes in the circulating levels of brain-derived neurotrophic factor (BDNF) and cortisol correlated to improved hippocampal-dependent cognitive ability. These findings demonstrate that HIIT significantly improves and prolongs the hippocampal-dependent cognitive health of aged individuals. Importantly, improvement was retained for at least 5 years following initiation of HIIT, suggesting that the changes seen in hippocampal volume and connectivity underpin this long-term maintenance. Sustained improvement in hippocampal function to this extent confirms that such exercise-based interventions can provide significant protection against hippocampal cognitive decline in the aged population. The changes in specific blood factor levels also may provide useful biomarkers for choosing the optimal exercise regimen to promote cognitive improvement.

The hippocampus is a complex brain structure that plays an important role in various cognitive aspects such as memory, intelligence, executive function, and path integration. The volume of this highly plastic structure is identified as one of the most important biomarkers of specific neuropsychiatric and neurodegenerative diseases. It has also been extensively investigated in numerous aging studies. However, recent studies on aging show that the performance of conventional approaches in measuring the hippocampal volume is still far from satisfactory, especially in terms of delivering longitudinal measures from ultra-high field magnetic resonance images (MRIs), which can visualize more boundary details. The advancement of deep learning provides an alternative solution to measuring the hippocampal volume. In this work, we comprehensively compared a deep learning pipeline based on nnU-Net with several conventional approaches including Freesurfer, FSL and DARTEL, for automatically delivering hippocampal volumes: (1) Firstly, we evaluated the segmentation accuracy and precision on a public dataset through cross-validation. Results showed that the deep learning pipeline had the lowest mean ( L  = 1.5%, R  = 1.7%) and the lowest standard deviation ( L  = 5.2%, R  = 6.2%) in terms of volume percentage error. (2) Secondly, sub-millimeter MRIs of a group of healthy adults with test-retest 3T and 7T sessions were used to extensively assess the test-retest reliability. Results showed that the deep learning pipeline achieved very high intraclass correlation coefficients ( L  = 0.990, R  = 0.986 for 7T; L  = 0.985, R  = 0.983 for 3T) and very small volume percentage differences ( L  = 1.2%, R  = 0.9% for 7T; L  = 1.3%, R  = 1.3% for 3T). (3) Thirdly, a Bayesian linear mixed effect model was constructed with respect to the hippocampal volumes of two healthy adult datasets with longitudinal 7T scans and one disease-related longitudinal dataset. It was found that the deep learning pipeline detected both the subtle and disease-related changes over time with high sensitivity as well as the mild differences across subjects. Comparison results from the aforementioned three aspects showed that the deep learning pipeline significantly outperformed the conventional approaches by large margins. Results also showed that the deep learning pipeline can better accommodate longitudinal analysis purposes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lyu, Bartlett, Nasrallah and Tang.)

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., (© 2023. Springer Nature Limited.)

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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We have shown that the improvement in hippocampal-based learning in aged mice following physical exercise observed is dependent on neurogenesis in the dentate gyrus (DG) and is regulated by changes in growth hormone levels. The changes in neurocircuitry, however, which may underlie this improvement, remain unclear. Using in vivo multimodal magnetic resonance imaging to track changes in aged mice exposed to exercise, we show the improved spatial learning is due to enhanced DG connectivity, particularly the strengthening of the DG-Cornu Ammonis 3 and the DG-medial entorhinal cortex connections in the dorsal hippocampus. Moreover, we provide evidence that these changes in circuitry are dependent on neurogenesis since they were abrogated by ablation of newborn neurons following exercise. These findings identify the specific changes in hippocampal circuitry that underlie the cognitive improvements resulting from physical activity and show that they are dependent on the activation of neurogenesis in aged animals., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Hippocampal function is critical for spatial and contextual learning, and its decline with age contributes to cognitive impairment. Exercise can improve hippocampal function, however, the amount of exercise and mechanisms mediating improvement remain largely unknown. Here, we show exercise reverses learning deficits in aged (24 months) female mice but only when it occurs for a specific duration, with longer or shorter periods proving ineffective. A spike in the levels of growth hormone (GH) and a corresponding increase in neurogenesis during this sweet spot mediate this effect because blocking GH receptor with a competitive antagonist or depleting newborn neurons abrogates the exercise-induced cognitive improvement. Moreover, raising GH levels with GH-releasing hormone agonist improved cognition in nonrunners. We show that GH stimulates neural precursors directly, indicating the link between raised GH and neurogenesis is the basis for the substantially improved learning in aged animals., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Motor neuron disease (MND) comprises a group of fatal neurodegenerative diseases with no effective cure. As progressive motor neuron cell death is one of pathological characteristics of MND, molecules which protect these cells are attractive therapeutic targets. Accumulating evidence indicates that EphA4 activation is involved in MND pathogenesis, and inhibition of EphA4 improves functional outcomes. However, the underlying mechanism of EphA4's function in MND is unclear. In this review, we first present results to demonstrate that EphA4 signalling acts directly on motor neurons to cause cell death. We then review the three most likely mechanisms underlying this effect.

Identifying dynamic changes in biomarkers and clinical profiles is essential for understanding the progression of Alzheimer's disease (AD). The relevant studies have primarily relied on patients with autosomal dominant AD; however, relevant studies in sporadic AD are poorly understood. Here, we analyzed longitudinal data from 665 participants (mean follow-up 4.90 ± 2.83 years). By aligning normal cognition (CN) baseline with a clinical diagnosis of mild cognitive impairment (MCI) or AD, we studied the progression of AD using a linear mixed model to estimate the clinical and biomarker changes from stable CN to MCI to AD. The results showed that the trajectory of hippocampal volume and fluorodeoxyglucose (FDG) was consistent with the clinical measures in that they did not follow a hypothetical sigmoid curve but rather showed a slow change in the initial stage and accelerated changes in the later stage from MCI conversion to AD. Dramatic hippocampal atrophy and the ADAS13 increase were, respectively, 2.5 and 1 years earlier than the MCI onset. Besides, cognitively normal people with elevated and normal amyloid showed no significant differences in clinical measures, hippocampal volume, or FDG. These results reveal that pre-MCI to pre-AD may be a better time window for future clinical trial design., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microglia, the resident immune cells of the CNS, have emerged as key regulators of neural precursor cell activity in the adult brain. However, the microglia-derived factors that mediate these effects remain largely unknown. In the present study, we investigated a role for microglial brain-derived neurotrophic factor (BDNF), a neurotrophic factor with well known effects on neuronal survival and plasticity. Surprisingly, we found that selective genetic ablation of BDNF from microglia increased the production of newborn neurons under both physiological and inflammatory conditions (e.g., LPS-induced infection and traumatic brain injury). Genetic ablation of BDNF from microglia otherwise also interfered with self-renewal/proliferation, reducing their overall density. In conclusion, we identify microglial BDNF as an important factor regulating microglia population dynamics and states, which in turn influences neurogenesis under both homeostatic and pathologic conditions. SIGNIFICANCE STATEMENT (1) Microglial BDNF contributes to self-renewal and density of microglia in the brain. (2) Selective ablation of BDNF in microglia stimulates neural precursor proliferation. (3) Loss of microglial BDNF augments working memory following traumatic brain injury. (4) Benefits of repopulating microglia on brain injury are not mediated via microglial BDNF., (Copyright © 2021 the authors.)

Hippocampal atrophy and cognitive decline are common sequelae of many neurodegenerative disorders, including stroke. To determine whether cognitive decline can be ameliorated by exercise-induced neurogenesis, C57BL/6 mice in which a unilateral hippocampal injury had been induced by injecting the vasoconstrictor endothelin-1 into their right hippocampus, were run voluntarily for 21 days on a running-wheel. We found the severe deficits in spatial learning, as detected by active place-avoidance task, following injury were almost completely restored in animals that ran whereas those that did not run showed no improvement. We show the increase in neurogenesis found in both the injured and contralateral hippocampi following running was responsible for the restoration of learning since bilateral ablation of newborn doublecortin (DCX)-positive neurons abrogated the cognitive improvement, whereas unilateral ablations of DCX-positive neurons did not prevent recovery, demonstrating that elevated neurogenesis in either the damaged or intact hippocampus is sufficient to reverse hippocampal injury-induced deficits.

Cognitive dysfunction and reactive microglia are hallmarks of traumatic brain injury (TBI), yet whether these cells contribute to cognitive deficits and secondary inflammatory pathology remains poorly understood. Here, we show that removal of microglia from the mouse brain has little effect on the outcome of TBI, but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. The beneficial effects of these repopulating microglia are critically dependent on interleukin-6 (IL-6) trans-signaling via the soluble IL-6 receptor (IL-6R) and robustly support adult neurogenesis, specifically by augmenting the survival of newborn neurons that directly support cognitive function. We conclude that microglia in the mammalian brain can be manipulated to adopt a neuroprotective and pro-regenerative phenotype that can aid repair and alleviate the cognitive deficits arising from brain injury., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The hippocampal dentate gyrus (DG) is a major region of the adult rodent brain in which neurogenesis occurs throughout life. The EphA4 receptor, which regulates neurogenesis and boundary formation in the developing brain, is also expressed in the adult DG, but whether it regulates adult hippocampal neurogenesis is not known. Here, we show that, in the adult mouse brain, EphA4 inhibits hippocampal precursor cell proliferation but does not affect precursor differentiation or survival. Genetic deletion or pharmacological inhibition of EphA4 significantly increased hippocampal precursor proliferation in vivo and in vitro, by blocking EphA4 forward signaling. EphA4 was expressed by mature hippocampal DG neurons but not neural precursor cells, and an EphA4 antagonist, EphA4-Fc, did not activate clonal cultures of precursors until they were co-cultured with non-precursor cells, indicating an indirect effect of EphA4 on the regulation of precursor activity. Supplementation with d-serine blocked the increased precursor proliferation induced by EphA4 inhibition, whereas blocking the interaction between d-serine and N-methyl-d-aspartate receptors (NMDARs) promoted precursor activity, even at the clonal level. Collectively, these findings demonstrate that EphA4 indirectly regulates adult hippocampal precursor proliferation and thus plays a role in neurogenesis via d-serine-regulated NMDAR signaling., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The blood-brain barrier presents a major challenge for the delivery of therapeutic agents to the brain; however, it can be transiently opened by combining low intensity ultrasound with microbubble infusion. Studies evaluating this technology have largely been performed in rodents, including models of neurological conditions. However, despite promising outcomes in terms of drug delivery and the amelioration of neurological impairments, the potential for long-term adverse effects presents a major concern in the context of clinical applications. Methods: To fill this gap, we repeatedly treated 12-month-old wild-type mice with ultrasound, followed by a multimodal analysis for up to 18 months of age. Results: We found that spatial memory in these aged mice was not adversely affected as assessed in the active place avoidance test. Sholl analysis of Golgi impregnations in the dentate gyrus of the hippocampus did not reveal any changes to the neuronal cytoarchitecture. Long-term potentiation, a cellular correlate of memory, was still achievable, magnetic resonance spectroscopy revealed no major changes in metabolites, and diffusion tensor imaging revealed normal microstructure and tissue integrity in the hippocampus. More specifically, all measures of diffusion appeared to support a neuroprotective effect of ultrasound treatment on the brain. Conclusion: This multimodal analysis indicates that therapeutic ultrasound for blood-brain barrier opening is safe and potentially protective in the long-term, underscoring its validity as a potential treatment modality for diseases of the brain., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Amyotrophic lateral sclerosis (ALS) is an untreatable, progressive, neurodegenerative disease specifically affecting motor neurons. Recently, the tyrosine kinase receptor EphA4 was directly implicated in ALS disease progression. We report that a long-lived mutated form of the EphA4 antagonist EphA4-Fc (mutEphA4-Fc), which blocks EphA4 binding to its ligands and inhibits its function, significantly improved functional performance in SOD1 G93A ALS model mice, as assessed by rotarod and hind-limb grip strength tests. Further, heterozygous motor neuron-specific EphA4 gene deletion in SOD1 G93A mice promoted significant improvement in functional performance during the disease course and a delay in disease onset relative to control mice. Importantly, mice in the heterozygous deletion group showed significantly improved survival of motor neurons and architecture of endplates of neuromuscular junctions compared with control and homozygous EphA4-deletion groups. Our novel results show that EphA4 signalling directly regulates motor neuron survival and that mutEphA4-Fc is a promising therapeutic candidate to slow disease progression in ALS.

New neurons are continually generated from the resident populations of precursor cells in selective niches of the adult mammalian brain such as the hippocampal dentate gyrus and the olfactory bulb. However, whether such cells are present in the adult amygdala, and their neurogenic capacity, is not known. Using the neurosphere assay, we demonstrate that a small number of precursor cells, the majority of which express Achaete-scute complex homolog 1 (Ascl1), are present in the basolateral amygdala (BLA) of the adult mouse. Using neuron-specific Thy1-YFP transgenic mice, we show that YFP+ cells in BLA-derived neurospheres have a neuronal morphology, co-express the neuronal marker βIII-tubulin, and generate action potentials, confirming their neuronal phenotype. In vivo, we demonstrate the presence of newly generated BrdU-labeled cells in the adult BLA, and show that a proportion of these cells co-express the immature neuronal marker doublecortin (DCX). Furthermore, we reveal that a significant proportion of GFP+ neurons (~23%) in the BLA are newly generated (BrdU+) in DCX-GFP mice, and using whole-cell recordings in acute slices we demonstrate that the GFP+ cells display electrophysiological properties that are characteristic of interneurons. Using retrovirus-GFP labeling as well as the Ascl1 CreERT2 mouse line, we further confirm that the precursor cells within the BLA give rise to mature and functional interneurons that persist in the BLA for at least 8 weeks after their birth. Contextual fear conditioning has no effect on the number of neurospheres or BrdU-labeled cells in the BLA, but produces an increase in hippocampal cell proliferation. These results demonstrate that neurogenic precursor cells are present in the adult BLA, and generate functional interneurons, but also show that their activity is not regulated by an amygdala-dependent learning paradigm.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease characterised by the degeneration of motor neurons, which are responsible for voluntary movement. There remains limited understanding of disease aetiology, with median survival of ALS of three years and no effective treatment. Identifying genes that contribute to ALS susceptibility is an important step towards understanding aetiology. The vast majority of published human genetic studies, including for ALS, have used samples of European ancestry. The importance of trans-ethnic studies in human genetic studies is widely recognised, yet a dearth of studies of non-European ancestries remains. Here, we report analyses of novel whole-exome sequencing (WES) data from Chinese ALS and control individuals., Methods: WES data were generated for 610 ALS cases and 460 controls drawn from Chinese populations. We assessed evidence for an excess of rare damaging mutations at the gene level and the gene set level, considering only singleton variants filtered to have allele frequency less than 5 × 10 -5 in reference databases. To meta-analyse our results with a published study of European ancestry, we used a Cochran-Mantel-Haenszel test to compare gene-level variant counts in cases vs controls., Results: No gene passed the genome-wide significance threshold with ALS in Chinese samples alone. Combining rare variant counts in Chinese with those from the largest WES study of European ancestry resulted in three genes surpassing genome-wide significance: TBK1 (p = 8.3 × 10 -12 ), SOD1 (p = 8.9 × 10 -9 ) and NEK1 (p = 1.1 × 10 -9 ). In the Chinese data alone, SOD1 and NEK1 were nominally significantly associated with ALS (p = 0.04 and p = 7 × 10 -3 , respectively) and the case/control frequencies of rare coding variants in these genes were similar in Chinese and Europeans (SOD1: 1.5%/0.2% vs 0.9%/0.1%, NEK1 1.8%/0.4% vs 1.9%/0.8%). This was also true for TBK1 (1.2%/0.2% vs 1.4%/0.4%), but the association with ALS in Chinese was not significant (p = 0.14)., Conclusions: While SOD1 is already recognised as an ALS-associated gene in Chinese, we provide novel evidence for association of NEK1 with ALS in Chinese, reporting variants in these genes not previously found in Europeans.

Studies on the role of the hippocampus in higher cognitive functions such as spatial learning and memory in rodents are reliant upon robust and objective behavioral tests. This protocol describes one such test-the active place avoidance (APA) task. This behavioral task involves the mouse continuously integrating visual cues to orientate itself within a rotating arena in order to actively avoid a shock zone, the location of which remains constant relative to the room. This protocol details the step-by-step procedures for a novel paradigm of the hippocampal-dependent APA task, measuring acquisition of spatial learning during a single 20-min trial (i.e., short-term memory), with spatial memory encoding and retrieval (i.e., long-term memory) assessed by trials conducted over consecutive days. Using the APA task, cognitive flexibility can be assessed using the reversal learning paradigm, as this increases the cognitive load required for efficient performance in the task. In addition to a detailed experimental protocol, this paper also describes the range of its possible applications, the expected key results, as well as the analytical methods to assess the data, and the pitfalls/troubleshooting measures. The protocol described herein is highly robust and produces replicable results, thus presenting an important paradigm that enables the assessment of subtle short-term changes in spatial learning and memory, such as those observed for many experimental interventions.

Activated neurons express immediate-early genes, such as Arc. Expression of Arc in the hippocampal granule cell layer, an area crucial for spatial learning and memory, is increased during acquisition of spatial learning; however, it is unclear whether this effect is related to the task-specific learning process or to nonspecific aspects of the testing procedure (e.g. exposure to the testing apparatus and exploration of the environment). Herein, we show that Arc-positive cells numbers are increased to the same extent in the granule cell layer after both acquisition of a single spatial learning event in the active place avoidance task and exploration of the testing environment, as compared to naïve (i.e. caged) mice. Repeated exposure the testing apparatus and environment did not reduce Arc expression. Furthermore, Arc expression did not correlate with performance in both adult and aged animals, suggesting that exploration of the testing environment, rather than the specific acquisition of the active place avoidance task, induces Arc expression in the dentate granule cell layer. These findings thus suggest that Arc is an experience-induced immediate-early gene.

Cross-ethnic genetic studies can leverage power from differences in disease epidemiology and population-specific genetic architecture. In particular, the differences in linkage disequilibrium and allele frequency patterns across ethnic groups may increase gene-mapping resolution. Here we use cross-ethnic genetic data in sporadic amyotrophic lateral sclerosis (ALS), an adult-onset, rapidly progressing neurodegenerative disease. We report analyses of novel genome-wide association study data of 1,234 ALS cases and 2,850 controls. We find a significant association of rs10463311 spanning GPX3-TNIP1 with ALS (p = 1.3 × 10 -8 ), with replication support from two independent Australian samples (combined 576 cases and 683 controls, p = 1.7 × 10 -3 ). Both GPX3 and TNIP1 interact with other known ALS genes (SOD1 and OPTN, respectively). In addition, GGNBP2 was identified using gene-based analysis and summary statistics-based Mendelian randomization analysis, although further replication is needed to confirm this result. Our results increase our understanding of genetic aetiology of ALS.Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease. Here, Wray and colleagues identify association of the GPX3-TNIP1 locus with ALS using cross-ethnic meta-analyses.

Eph receptors have emerged as targets for therapy in both neoplastic and non-neoplastic disease, however, particularly in non-neoplastic diseases, redundancy of function limits the effectiveness of targeting individual Eph proteins. We have shown previously that a soluble fusion protein, where the EphA4 ectodomain was fused to IgG Fc (EphA4 Fc), was an effective therapy in acute injuries and demonstrated that EphA4 Fc was a broad spectrum Eph/ephrin antagonist. However, a very short in vivo half-life effectively limited its therapeutic development. We report a unique glycoengineering approach to enhance the half-life of EphA4 Fc. Progressive deletion of three demonstrated N-linked sites in EphA4 progressively increased in vivo half-life such that the triple mutant protein showed dramatically improved pharmacokinetic characteristics. Importantly, protein stability, affinity for ephrin ligands and antagonism of cell expressed EphA4 was fully preserved, enabling it to be developed as a broad spectrum Eph/ephrin antagonist for use in both acute and chronic diseases.

Living organisms are exposed to the geomagnetic field (GMF) throughout their lifespan. Elimination of the GMF, resulting in a hypogeomagnetic field (HMF), leads to central nervous system dysfunction and abnormal development in animals. However, the cellular mechanisms underlying these effects have not been identified so far. Here, we show that exposure to an HMF (<200 nT), produced by a magnetic field shielding chamber, promotes the proliferation of neural progenitor/stem cells (NPCs/NSCs) from C57BL/6 mice. Following seven-day HMF-exposure, the primary neurospheres (NSs) were significantly larger in size, and twice more NPCs/NSCs were harvested from neonatal NSs, when compared to the GMF controls. The self-renewal capacity and multipotency of the NSs were maintained, as HMF-exposed NSs were positive for NSC markers (Nestin and Sox2), and could differentiate into neurons and astrocyte/glial cells and be passaged continuously. In addition, adult mice exposed to the HMF for one month were observed to have a greater number of proliferative cells in the subventricular zone. These findings indicate that continuous HMF-exposure increases the proliferation of NPCs/NSCs, in vitro and in vivo. HMF-disturbed NPCs/NSCs production probably affects brain development and function, which provides a novel clue for elucidating the cellular mechanisms of the bio-HMF response.

Objective: Traumatic spinal cord injury (SCI) elicits immediate neural cell death, axonal damage, and disruption of the blood-spinal cord barrier, allowing circulating immune cells and blood proteins into the spinal parenchyma. The inflammatory response to SCI involves robust complement system activation, which contributes to secondary injury and impairs neurological recovery. This study aimed to determine whether intravenous immunoglobulin (IVIg), an FDA-approved treatment for inflammatory conditions, can scavenge complement activation products and improve recovery from contusive SCI., Methods: We used functional testing, noninvasive imaging, and detailed postmortem analysis to assess whether IVIg therapy is effective in a mouse model of severe contusive SCI., Results: IVIg therapy at doses of 0.5-2 g/kg improved the functional and histopathological outcomes from SCI, conferring protection against lesion enlargement, demyelination, central canal dilation, and axonal degeneration. The benefits of IVIg were detectable through noninvasive diffusion tensor imaging (DTI), with IVIg treatment counteracting the progressive SCI-induced increase in radial diffusivity (RD) in white matter. Diffusion indices significantly correlated with the functional performance of individual mice and accurately predicted the degree of myelin preservation. Further experiments revealed that IVIg therapy reduced the presence of complement activation products and phagocytically active macrophages at the lesion site, providing insight as to its mechanisms of action., Interpretation: Our findings highlight the potential of using IVIg as an immunomodulatory treatment for SCI, and the value of DTI to assess tissue damage and screen for the efficacy of candidate intervention strategies in preclinical models of SCI, both quantitatively and noninvasively.

Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation.

The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however, whether these stimuli regulate the same or different precursor populations remains unknown. Here, we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population, which comprises the majority of neurosphere-forming precursors, there are two distinct subpopulations of quiescent precursor cells, one directly activated by high-KCl depolarization, and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus, and show that the NE-responsive precursors are selectively regulated by GABA, whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally, based on RNAseq analysis by deep sequencing, we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors, which may give rise to neural progeny with different functional capacity., (Copyright © 2015 the authors 0270-6474/15/358132-13$15.00/0.)

Recent advances in next-generation sequencing technology allow high-throughput cDNA sequencing (RNA-Seq) to be widely applied in transcriptomic studies, in particular for detecting differentially expressed genes between groups. Many software packages have been developed for the identification of differentially expressed genes (DEGs) between treatment groups based on RNA-Seq data. However, there is a lack of consensus on how to approach an optimal study design and choice of suitable software for the analysis. In this comparative study we evaluate the performance of three of the most frequently used software tools: Cufflinks-Cuffdiff2, DESeq and edgeR. A number of important parameters of RNA-Seq technology were taken into consideration, including the number of replicates, sequencing depth, and balanced vs. unbalanced sequencing depth within and between groups. We benchmarked results relative to sets of DEGs identified through either quantitative RT-PCR or microarray. We observed that edgeR performs slightly better than DESeq and Cuffdiff2 in terms of the ability to uncover true positives. Overall, DESeq or taking the intersection of DEGs from two or more tools is recommended if the number of false positives is a major concern in the study. In other circumstances, edgeR is slightly preferable for differential expression analysis at the expense of potentially introducing more false positives.

Norepinephrine regulates latent neural stem cell activity and adult hippocampal neurogenesis, and has an important role in modulating hippocampal functions such as learning, memory and mood. Adult hippocampal neurogenesis is a multi-stage process, spanning from the activation and proliferation of hippocampal stem cells, to their differentiation into neurons. However, the stage-specific effects of noradrenergic receptors in regulating adult hippocampal neurogenesis remain poorly understood. In this study, we used transgenic Nestin-GFP mice and neurosphere assays to show that modulation of α2- and β-adrenergic receptor activity directly affects Nestin-GFP/GFAP-positive precursor cell population albeit in an opposing fashion. While selective stimulation of α2-adrenergic receptors decreases precursor cell activation, proliferation and immature neuron number, stimulation of β-adrenergic receptors activates the quiescent precursor pool and enhances their proliferation in the adult hippocampus. Furthermore, our data indicate no major role for α1-adrenergic receptors, as we did not observe any change in either the activation and proliferation of hippocampal precursors following selective stimulation or blockade of α1-adrenergic receptors. Taken together, our data suggest that under physiological as well as under conditions that lead to enhanced norepinephrine release, the balance between α2- and β-adrenergic receptor activity regulates precursor cell activity and hippocampal neurogenesis.

Research has shown that the hypomagnetic field (HMF) can affect embryo development, cell proliferation, learning and memory, and in vitro tubulin assembly. In the present study, we aimed to elucidate the molecular mechanism by which the HMF exerts its effect, by comparing the transcriptome profiles of human neuroblastoma cells exposed to either the HMF or the geomagnetic field. A total of 2464 differentially expressed genes (DEGs) were identified, 216 of which were up-regulated and 2248 of which were down-regulated after exposure to the HMF. These DEGs were found to be significantly clustered into several key processes, namely macromolecule localization, protein transport, RNA processing, and brain function. Seventeen DEGs were verified by real-time quantitative PCR, and the expression levels of nine of these DEGs were measured every 6 h. Most notably, MAPK1 and CRY2, showed significant up- and down-regulation, respectively, during the first 6 h of HMF exposure, which suggests involvement of the MAPK pathway and cryptochrome in the early bio-HMF response. Our results provide insights into the molecular mechanisms underlying the observed biological effects of the HMF.

Although tau is mainly known as an axonal microtubule-associated protein, many studies indicate that it is not restricted to this subcellular compartment. Assessing tau's subcellular distribution, however, is not trivial as is evident from transgenic mouse studies. When human tau is over-expressed, it can be immunohistochemically localized to axons and the somatodendritic domain, modeling what is found in neurodegenerative diseases such as Alzheimer's disease. Yet, in wild-type mice, despite its abundance, tau is difficult to visualize even in the axon. It is even more challenging to detect this protein in the nucleus, where tau has been proposed to protect DNA from damage. To establish a framework for future studies into tau's nuclear functions, we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain. While depending on the fixation and permeabilization protocol, we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells, we failed to do so in N2a murine neuroblastoma cells. As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form. When either full-length or truncated human tau was expressed, both accumulated in the cytoplasm, but were also found in the nuclear fraction. Because subcellular fractionation methods have their limitations, we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination. Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.

New neurons are continuously generated from resident pools of neural stem and precursor cells (NSPCs) in the adult brain. There are multiple pathways through which adult neurogenesis is regulated, and here we review the role of the N-methyl-D-aspartate receptor (NMDAR) in regulating the proliferation of NSPCs in the adult hippocampus. Hippocampal-dependent learning tasks, enriched environments, running, and activity-dependent synaptic plasticity, all potently up-regulate hippocampal NSPC proliferation. We first consider the requirement of the NMDAR in activity-dependent synaptic plasticity, and the role the induction of synaptic plasticity has in regulating NSPCs and newborn neurons. We address how specific NMDAR agonists and antagonists modulate proliferation, both in vivo and in vitro, and then review the evidence supporting the hypothesis that NMDARs are present on NSPCs. We believe it is important to understand the mechanisms underlying the activation of adult neurogenesis, given the potential that endogenous stem cell populations have for repopulating the hippocampus with functional new neurons. In conditions such as age-related memory decline, neurodegeneration and psychiatric disease, mature neurons are lost or become defective; as such, stimulating adult neurogenesis may provide a therapeutic strategy to overcome these conditions.

Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS.

Blocking the action of inhibitory molecules at sites of central nervous system injury has been proposed as a strategy to promote axonal regeneration and functional recovery. We have previously shown that genetic deletion or competitive antagonism of EphA4 receptor activity promotes axonal regeneration and functional recovery in a mouse model of lateral hemisection spinal cord injury. Here we have assessed the effect of blocking EphA4 activation using the competitive antagonist EphA4-Fc in a rat model of thoracic contusive spinal cord injury. Using a ledged tapered balance beam and open-field testing, we observed significant improvements in recovery of locomotor function after EphA4-Fc treatment. Consistent with functional improvement, using high-resolution ex vivo magnetic resonance imaging at 16.4T, we found that rats treated with EphA4-Fc had a significantly increased cross-sectional area of the dorsal funiculus caudal to the injury epicenter compared with controls. Our findings indicate that EphA4-Fc promotes functional recovery following contusive spinal cord injury and provides further support for the therapeutic benefit of treatment with the competitive antagonist in acute cases of spinal cord injury.

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.

Brain cancer research has been hampered by a paucity of viable clinical tissue of sufficient quality and quantity for experimental research. This has driven researchers to rely heavily on long term cultured cells which no longer represent the cancers from which they were derived. Resection of brain tumors, particularly at the interface between normal and tumorigenic tissue, can be carried out using an ultrasonic surgical aspirator (CUSA) that deposits liquid (blood and irrigation fluid) and resected tissue into a sterile bottle for disposal. To determine the utility of CUSA-derived glioma tissue for experimental research, we collected 48 CUSA specimen bottles from glioma patients and analyzed both the solid tissue fragments and dissociated tumor cells suspended in the liquid waste fraction. We investigated if these fractions would be useful for analyzing tumor heterogeneity, using IHC and multi-parameter flow cytometry; we also assessed culture generation and orthotopic xenograft potential. Both cell sources proved to be an abundant, highly viable source of live tumor cells for cytometric analysis, animal studies and in-vitro studies. Our findings demonstrate that CUSA tissue represents an abundant viable source to conduct experimental research and to carry out diagnostic analyses by flow cytometry or other molecular diagnostic procedures.

While it is now well-established that resident populations of stem and progenitor cells drive neurogenesis in the adult brain, a growing body of evidence indicates that these new neurons play a pivotal role in spatial learning, memory, and mood regulation. As such, interest is gathering to develop strategies to harness the brain's endogenous reservoir of stem and progenitor cells, with the view that newborn neurons may help overcome the loss of neural and cognitive function that occurs during neurodegenerative disease and psychiatric illness. Here we review evidence for the presence of endogenous stem cell populations in the adult hippocampus, especially large pools of latent stem and precursor cells, and the ways in which these populations can be stimulated to produce new neurons. While the translation of this research from animal models to human application is still in its infancy, understanding in detail the cellular and molecular mechanisms that regulate endogenous neurogenesis, offers the potential to use this innate reservoir of precursors to produce neurons that may be able to mitigate against cognitive decline and mood disorders.

Small interfering RNAs (siRNAs) are a potentially powerful new class of pharmaceutical drugs for many disease. However, the delivery of unprotected siRNAs is ineffective due to their susceptibility to degradation by ubiquitous nucleases under physiological conditions. Layered double hydroxide nanoparticles (LDHs) have been found to be efficient carriers of anionic drugs and nucleic acids. Our previous research has shown that LDHs (with the Z-average particle size of approximately 110 nm) can mediate siRNA delivery in mammalian cells, resulting in gene silencing. However, short double-stranded nucleic acids are mostly adsorbed onto the external surface and not well protected by LDHs. In order to enhance the intercalation of siRNA into the LDH interlayer and the efficiency of subsequent siRNA delivery, we prepared smaller LDHs (with the Z-average particle size of approximately 45 nm) with an engineered non-aqueous method. We demonstrate here that dsDNA/siRNA is more effectively intercalated into these small LDH nanoparticles, more dsDNA/siRNA is transfected into HEK 293T cells, and more efficient silencing of the target gene is achieved using smaller LDHs. Thus, smaller LDH particles have greater potential as a delivery system for the application of RNA interference., (Copyright © 2012 Elsevier Inc. All rights reserved.)