In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: Inducing effects of brain-derived neurotrophic factor

Neural stem cells proliferate in vitro and form neurospheres in the presence of epidermal growth factor (EGF), and are capable of differentiating into both neurons and glia when exposed to a substrate. We hypothesize that specific neurotrophic factors induce differentiation of stem cells from different central nervous system (CNS) regions into particular fates. We investigated differentiation of stem cells from the postnatal mouse hippocampus in culture using the following trophic factors (20 ng/mL): brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF). Without trophic factors, 32% of stem cells differentiated into neurons by 4 days in vitro (DIV), decreasing to 10% by 14 DIV. Addition of BDNF (starting at either day 0 or day 3) significantly increased neuron survival (31–43% by 14 DIV) and differentiation. Morphologically, many well-differentiated neurons resembled hippocampal pyramidal neurons. 5′-Bromodeoxyuridine labeling demonstrated that the pyramidal-like neurons originated from stem cells which had proliferated in EGF-containing cultures. However, similar application of NT-3 and GDNF did not exert such a differentiating effect. Addition of BDNF to stem cells from the postnatal cerebellum, midbrain, and striatum did not induce these neuronal phenotypes, though similar application to cortical stem cells yielded pyramidal-like neurons. Thus, BDNF supports survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons. The presence of pyramidal neurons in BDNF-treated hippocampal and cortical stem cell cultures, but not in striatal, cerebellar, and midbrain stem cell cultures, suggests that stem cells from different CNS regions differentiate into region-specific phenotypic neurons when stimulated with an appropriate neurotrophic factor. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 395–425, 1998     

Regulation of neurotrophin signaling in aging sensory and motoneurons

A hallmark of senescence is sensorimotor impairment, involving locomotion and postural control as well as fine-tuned movements. Sensory and motoneurons are not lost to any significant degree with advancing age, but do show characteristic changes in gene-expression pattern, morphology, and connectivity. This review covers recent experimental findings corroborating that alterations in trophic signaling may induce several of the phenotypic changes seen in primary sensory and motoneurons during aging. Furthermore, the data suggests that target failure, and/or breakdown of neuron-target interaction, is a critical event in the aging process of sensory and motoneurons.     

Green tea catechins potentiate the neuritogenic action of brain-derived neurotrophic factor: Role of 67-kDa laminin receptor and hydrogen peroxide

Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 μg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 μM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H₂O₂ in the potentiation. Consistently, exogenous sublethal concentrations of H₂O₂, added as a bolus dose (5 μM) or more effectively through a steady-state generation (1 μM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67LR and H₂O₂, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea.     

Whole mitochondrial DNA variations in hippocampal surgical specimens and blood samples with high-throughput sequencing: A case of mesial temporal lobe epilepsy with hippocampal sclerosis

Introduction

Hippocampal sclerosis is the most common lesion in patients with mesial temporal lobe epilepsy. Recently, there has been growing evidence on the involvement of mitochondria also in sporadic forms of epilepsy. In addition, it has been increasingly argued that mitochondrial dysfunction has an important role in epileptogenesis and seizure generation in temporal lobe epilepsy. Although mtDNA polymorphisms have been identified as potential risk factors for neurological diseases, the link between homoplasmy and heteroplasmy within tissues is not clear. We investigated whether mitochondrial DNA (mtDNA) polymorphisms are involved in a case report of a patient with mesial temporal lobe epilepsy-hippocampal sclerosis (MTLE-HS).

Design

We report the whole genome mtDNA deep sequencing results and clinical features of a 36-year-old woman with MTLE-HS. We used pyrosequencing technology to sequence a whole mitochondrial genome isolated from six different regions of her brain and blood. To assess the possible role of mitochondrial DNA variations in affected tissues, we compared all specimens from different regions of the hippocampus and blood.

Results

In total, 35 homoplasmic and 18 heteroplasmic variations have been detected in 6 different regions of the hippocampus and in blood samples. While the samples did not display any difference in homoplasmic variations, it has been shown that hippocampus regions contain more heteroplasmic variations than blood. The number of heteroplasmic variations was highest in the CA2 region of the brain and accumulated in ND2, ND4 and ND5 genes. Also, dentate and subiculum regions of the hippocampus had similar heteroplasmic variation profiles.

Discussion

We present a new rare example of parallel mutation at 16223 position. Our case suggests that defects in mitochondrial function might be underlying the pathogenesis of seizures in temporal lobe epilepsy.     

Activation of cultured astrocytes by amphotericin B: Stimulation of NO and cytokines production and changes in neurotrophic factors production

Amphotericin B (AmB) is a polyene antibiotic and reported to be one of a few reagents having therapeutic effects on prion diseases, such as the delay in the appearing of the clinical signs and the prolongation of the survival time. In prion diseases, glial cells have been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and neurotrophic factors. However, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotoxic and neurotrophic factors in microglia (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290–1296). In the present study, we examined the effects of AmB on cellular functions of rat cultured astrocytes. We found that AmB could activate astrocytes to produce nitric oxide via inducible nitric oxide synthase induction. AmB also induced mRNA expression of interleukin-1β and tumor necrosis factor-α, and productions of their proteins in astrocytes. Moreover, AmB changed levels of neurotrophic factor mRNAs and proteins. Among three neurotrophic factors examined here, neurotrophin-3 mRNA expression and its protein production in the cells were down-regulated by AmB stimulation. On the other hand, AmB significantly enhanced the amounts of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor proteins in the cells and the medium. These results suggest that AmB might show therapeutic effects on prion diseases by controlling the expression and production of such mediators in astrocytes.     

Matrix metalloproteinases and their endogenous inhibitors in neuronal physiology of the adult brain

More than 20 matrix metalloproteinases (MMPs) and four of their endogenous tissue inhibitors (TIMPs) act together to control tightly temporally restricted, focal proteolysis of extracellular matrix. In the neurons of the adult brain several components of the TIMP/MMP system are expressed and are responsive to changes in neuronal activity. Furthermore, functional studies, especially involving blocking of MMP activities, along with the identification of MMP substrates in the brain strongly suggest that this enzymatic system plays an important physiological role in adult brain neurons, possibly being pivotal for neuronal plasticity.