Evaluation of Proliferation of Neural Stem Cells In Vitro and In Vivo

Morte, Maria Inês; Carreira, Bruno P.; Machado, Vanessa; Carmo, Anália do; Nunes‐Correia, Isabel; Carvalho, Caetana M.; Araújo, Inês M.

doi:10.1002/9780470151808.sc02d14s24

Cited by 15 publications

(9 citation statements)

References 63 publications

(82 reference statements)

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“…Almost a decade ago, FNC of bromodeoxyuridine (BrdU) labeled mouse hippocampal nuclei was used to demonstrate the ability of therapeutics (e.g., imipramine) to double cell proliferation rates (Bilsland et al, 2006). However, BrdU labeling has its limitations and may be a better predictor of DNA repair, underestimating both the number of cells undergoing normal DNA replication based cell proliferation and cells with neurogenic potential (Zheng et al, 2011;Morte et al, 2013). Our work suggests that cell proliferation assays based on the analysis of populations of brain cell nuclei should be revisited as a powerful approach to examine neurodegenerative diseases and therapeutics.…”

Section: Cell-type-specific Analyses Of Disease Models Using Fnc and mentioning

confidence: 99%

Characterization of brain cell nuclei with decondensed chromatin

McKinney

Kandasamy

et al. 2014

Developmental Neurobiology

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Although multipotent cell types have enlarged nuclei with decondensed chromatin, this property has not been exploited to enhance the characterization of neural progenitor cell (NPC) populations in the brain. We found that mouse brain cell nuclei that expressed exceptionally high levels of the pan neuronal marker NeuN/FOX3 (NeuN-High) had decondensed chromatin relative to most NeuN-Low or NeuN-Neg (negative) nuclei. Purified NeuN-High nuclei expressed significantly higher levels of transcripts encoding markers of neurogenesis, neuroplasticity, and learning and memory (ARC, BDNF, ERG1, HOMER1, NFL/NEF1, SYT1), subunits of chromatin modifying machinery (SIRT1, HDAC1, HDAC2, HDAC11, KAT2B, KAT3A, KAT3B, KAT5, DMNT1, DNMT3A, Gadd45a, Gadd45b) and markers of NPC and cell cycle activity (BRN2, FOXG1, KLF4, c-MYC, OCT4, PCNA, SHH, SOX2) relative to neuronal NeuN-Low or to mostly non-neuronal NeuNNeg nuclei. NeuN-High nuclei expressed higher levels of HDAC1, 2, 4, and 5 proteins. The cortex, hippocampus, hypothalamus, thalamus, and nucleus accumbens contained high percentages of large decondensed NeuN-High nuclei, while the cerebellum, and pons contained very few. NeuN-High nuclei have the properties consistent with their being derived from extremely active neurons with elevated rates of chromatin modification and/or NPC-like cells with multilineage developmental potential. The further analysis of decondensed neural cell nuclei should provide novel insights into neurobiology and neurodegenerative disease. V C 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: [738][739][740][741][742][743][744][745][746][747][748][749][750][751][752][753][754][755][756] 2015

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Section: Cell-type-specific Analyses Of Disease Models Using Fnc and mentioning

confidence: 99%

Characterization of brain cell nuclei with decondensed chromatin

McKinney

Kandasamy

et al. 2014

Developmental Neurobiology

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“…Coronal sections from the hippocampal region were cryosectioned (30 μm thick, in 6-series) using a CryoStar NX50 cryostat (Thermo Fisher Scientific, Waltham, MA, USA) and stored in an antifreeze solution (30% ethylene glycol and 30% glycerol in phosphate buffer), at 4°C. Free-floating brain sections from one of the series were labeled against BrdU, DCX, EdU or EdU/NeuN (neuronal nuclei), as previously described (Morte et al, 2013; Machado et al, 2015). EdU labeling was performed using a commercially available kit (Click-iT ® EdU Alexa Fluor ® 488 HCS Assay, Thermo Fisher Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Calpastatin Overexpression Preserves Cognitive Function Following Seizures, While Maintaining Post-Injury Neurogenesis

Machado

Lourenço

Florindo

et al. 2017

Front. Mol. Neurosci.

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In the adult mammalian brain, new neurons continue to be produced throughout life in two main regions in the brain, the subgranular zone (SGZ) in the hippocampus and the subventricular zone in the walls of the lateral ventricles. Neural stem cells (NSCs) proliferate in these niches, and migrate as neuroblasts, to further differentiate in locations where new neurons are needed, either in normal or pathological conditions. However, the endogenous attempt of brain repair is not very efficient. Calpains are proteases known to be involved in neuronal damage and in cell proliferation, migration and differentiation of several cell types, though their effects on neurogenesis are not well known. Previous work by our group has shown that the absence of calpastatin (CAST), the endogenous inhibitor of calpains, impairs early stages of neurogenesis. Since the hippocampus is highly associated with learning and memory, we aimed to evaluate whether calpain inhibition would help improve cognitive recovery after lesion and efficiency of post-injury neurogenesis in this region. For that purpose, we used the kainic acid (KA) model of seizure-induced hippocampal lesion and mice overexpressing CAST. Selected cognitive tests were performed on the 3rd and 8th week after KA-induced lesion, and cell proliferation, migration and differentiation in the dentate gyrus (DG) of the hippocampus of adult mice were analyzed using specific markers. Cognitive recovery was evaluated by testing the animals for recognition, spatial and associative learning and memory. Cognitive function was preserved by CAST overexpression following seizures, while modulation of post-injury neurogenesis was similar to wild type (WT) mice. Calpain inhibition could still be potentially able to prevent the impairment in the formation of new neurons, given that the levels of calpain activity could be reduced under a certain threshold and other harmful effects from the pathological environment could also be controlled.

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“…NSC were isolated from the SVZ of P0-3 Cast +/+ and Cast −/− mice and maintained in culture, as previously described (Morte et al, 2013 ). Dissociated NSC ( n = 3) were plated on coverslips coated with 0.1 mg/ml poly-L-lysine (Sigma Aldrich, St Louis, MO, USA) until 60–70% confluency was reached, and then treated with calpeptin 25 μM (Tocris Bioscience, Bristol, UK) for 6 h (untreated cells were used as controls).…”

Section: Methodsmentioning

confidence: 99%

Involvement of calpains in adult neurogenesis: implications for stroke

Machado

Morte

Carreira

et al. 2015

Front. Cell. Neurosci.

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Calpains are ubiquitous proteases involved in cell proliferation, adhesion and motility. In the brain, calpains have been associated with neuronal damage in both acute and neurodegenerative disorders, but their physiological function in the nervous system remains elusive. During brain ischemia, there is a large increase in the levels of intracellular calcium, leading to the activation of calpains. Inhibition of these proteases has been shown to reduce neuronal death in a variety of stroke models. On the other hand, after stroke, neural stem cells (NSC) increase their proliferation and newly formed neuroblasts migrate towards the site of injury. However, the process of forming new neurons after injury is not efficient and finding ways to improve it may help with recovery after lesion. Understanding the role of calpains in the process of neurogenesis may therefore open a new window for the treatment of stroke. We investigated the involvement of calpains in NSC proliferation and neuroblast migration in two highly neurogenic regions in the mouse brain, the dentate gyrus (DG) and the subventricular zone (SVZ). We used mice that lack calpastatin, the endogenous calpain inhibitor, and calpains were also modulated directly, using calpeptin, a pharmacological calpain inhibitor. Calpastatin deletion impaired both NSC proliferation and neuroblast migration. Calpain inhibition increased NSC proliferation, migration speed and migration distance in cells from the SVZ. Overall, our work suggests that calpains are important for neurogenesis and encourages further research on their neurogenic role. Prospective therapies targeting calpain activity may improve the formation of new neurons following stroke, in addition to affording neuroprotection.

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Evaluation of Proliferation of Neural Stem Cells In Vitro and In Vivo

Cited by 15 publications

References 63 publications

Characterization of brain cell nuclei with decondensed chromatin

Characterization of brain cell nuclei with decondensed chromatin

Calpastatin Overexpression Preserves Cognitive Function Following Seizures, While Maintaining Post-Injury Neurogenesis

Involvement of calpains in adult neurogenesis: implications for stroke

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