Crosstalk of copper and zinc in the pathogenesis of vascular dementia.

Copper and zinc are essential for normal brain functions. Both are localized in presynaptic vesicles and are secreted into synaptic clefts during neuronal excitation. Despite their significance, excesses of copper and zinc are neurotoxic. In particular, excess zinc after transient global ischemia plays a central role in the ischemia-induced neurodegeneration and pathogenesis of vascular type senile dementia. We previously found that sub-lethal concentrations of copper remarkably exacerbated zinc-induced neurotoxicity, and we investigated the molecular pathways of copper-enhanced zinc-induced neurotoxicity. The endoplasmic reticulum stress pathway, the stress-activated protein kinases/c-|Jun amino-terminal kinases pathway, and mitochondrial energy production failure were revealed to be involved in the neurodegenerative processes. Regarding the upstream factors of these pathways, we focused on copper-derived reactive oxygen species and the disruption of calcium homeostasis. Because excess copper and zinc may be present in the synaptic clefts during ischemia, it is possible that secreted copper and copper-induced reactive oxygen species may enhance zinc neurotoxicity and eventually contribute to the pathogenesis of vascular type senile dementia.

Copper as a Collaborative Partner of Zinc-Induced Neurotoxicity in the Pathogenesis of Vascular Dementia.

Copper is an essential trace element and possesses critical roles in various brain functions. A considerable amount of copper accumulates in the synapse and is secreted in neuronal firings in a manner similar to zinc. Synaptic copper and zinc modulate neuronal transmission and contribute to information processing. It has been established that excess zinc secreted during transient global ischemia plays central roles in ischemia-induced neuronal death and the pathogenesis of vascular dementia. We found that a low concentration of copper exacerbates zinc-induced neurotoxicity, and we have demonstrated the involvement of the endoplasmic reticulum (ER) stress pathway, the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) signaling pathway, and copper-induced reactive oxygen species (ROS) production. On the basis of our results and other studies, we discuss the collaborative roles of copper in zinc-induced neurotoxicity in the synapse and the contribution of copper to the pathogenesis of vascular dementia.

Neurometals in the Pathogenesis of Prion Diseases.

Prion diseases are progressive and transmissive neurodegenerative diseases. The conformational conversion of normal cellular prion protein (PrPC) into abnormal pathogenic prion protein (PrPSc) is critical for its infection and pathogenesis. PrPC possesses the ability to bind to various neurometals, including copper, zinc, iron, and manganese. Moreover, increasing evidence suggests that PrPC plays essential roles in the maintenance of homeostasis of these neurometals in the synapse. In addition, trace metals are critical determinants of the conformational change and toxicity of PrPC. Here, we review our studies and other new findings that inform the current understanding of the links between trace elements and physiological functions of PrPC and the neurotoxicity of PrPSc.

Carnosine as a Possible Drug for Zinc-Induced Neurotoxicity and Vascular Dementia.

Increasing evidence suggests that the metal homeostasis is involved in the pathogenesis of various neurodegenerative diseases including senile type of dementia such as Alzheimer's disease, dementia with Lewy bodies, and vascular dementia. In particular, synaptic Zn2+ is known to play critical roles in the pathogenesis of vascular dementia. In this article, we review the molecular pathways of Zn2+-induced neurotoxicity based on our and numerous other findings, and demonstrated the implications of the energy production pathway, the disruption of calcium homeostasis, the production of reactive oxygen species (ROS), the endoplasmic reticulum (ER)-stress pathway, and the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) pathway. Furthermore, we have searched for substances that protect neurons from Zn2+-induced neurotoxicity among various agricultural products and determined carnosine (ß-alanyl histidine) as a possible therapeutic agent for vascular dementia.

Amyloids: Regulators of Metal Homeostasis in the Synapse.

Conformational changes in amyloidogenic proteins, such as ß-amyloid protein, prion proteins, and α-synuclein, play a critical role in the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease, prion disease, and Lewy body disease. The disease-associated proteins possess several common characteristics, including the ability to form amyloid oligomers with ß-pleated sheet structure, as well as cytotoxicity, although they differ in amino acid sequence. Interestingly, these amyloidogenic proteins all possess the ability to bind trace metals, can regulate metal homeostasis, and are co-localized at the synapse, where metals are abundantly present. In this review, we discuss the physiological roles of these amyloidogenic proteins in metal homeostasis, and we propose hypothetical models of their pathogenetic role in the neurodegenerative process as the loss of normal metal regulatory functions of amyloidogenic proteins. Notably, these amyloidogenic proteins have the capacity to form Ca2+-permeable pores in membranes, suggestive of a toxic gain of function. Therefore, we focus on their potential role in the disruption of Ca2+ homeostasis in amyloid-associated neurodegenerative diseases.

Differentiation of 3D-shape-controlled mouse neural stem cell to neural tissues in closed agarose microchambers.

This paper describes three-dimensional (3D) tissue shape control of mouse neural stem cell (mNSC) micro tissues by using closed agarose microchambers for effective differentiation induction of neurons in vitro. Our agarose microchambers, made by micromolding, can be sealed with an agarose sheet to form the mNSC tissues along the shape of microchambers. We constructed lane-shaped mNSC tissues with different width (∼60-210 µm) and thickness (∼25-95 µm) dimensions and induced differentiation to neurons with differentiation medium. We found that in thick tissues (thickness: >60 µm), distribution of differentiated neurons was not uniform, whereas in thin tissues (thickness: ∼30 µm), differentiated neurons were uniformly distributed with high differentiation efficiency. Our system to construct in vitro 3D neural tissues having uniformly distributed neurons at high differentiation ratio, could become an effective tool for drug screening using 3D neural tissues and 3D mNSC tissues under differentiation induction.

Neural stem/progenitor cell-laden microfibers promote transplant survival in a mouse transected spinal cord injury model.

Previous studies have demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) into the lesioned spinal cord can promote functional recovery following incomplete spinal cord injury (SCI) in animal models. However, this strategy is insufficient following complete SCI because of the gap at the lesion epicenter. To obtain functional recovery in a mouse model of complete SCI, this study uses a novel collagen-based microfiber as a scaffold for engrafted NS/PCs. We hypothesized that the NS/PC-microfiber combination would facilitate lesion closure as well as transplant survival in the transected spinal cord. NS/PCs were seeded inside the novel microfibers, where they maintained their capacity to differentiate and proliferate. After transplantation, the stumps of the transected spinal cord were successfully bridged by the NS/PC-laden microfibers. Moreover, the transplanted cells migrated into the host spinal cord and differentiated into three neural lineages (astrocytes, neurons, and oligodendrocytes). However, the NS/PC-laden scaffold could not achieve a neural connection between the rostral end of the injury and the intact caudal area of the spinal cord, nor could it achieve recovery of motor function. To obtain optimal functional recovery, a microfiber design with a modified composition may be useful. Furthermore, combinatorial therapy with rehabilitation and/or medications should also be considered for practical success of biomaterial/cell transplantation-based approaches to regenerative medicine.

Metre-long cell-laden microfibres exhibit tissue morphologies and functions.

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

Effects of amyloid ß-peptides and gangliosides on mouse neural stem cells.

The interaction of amyloid ß-proteins (Aßs) with membrane lipids has been postulated as an early event in Aß fibril formation in Alzheimer's disease. We evaluated the effects of several putative bioactive Aßs and gangliosides on neural stem cells (NSCs) isolated from embryonic mouse brains or the subventricular zone of adult mouse brains. Incubation of the isolated NSCs with soluble Aß1-40 alone did not cause any change in the number of NSCs, but soluble Aß1-42 increased their number. Aggregated Aß1-40 and Aß1-42 increased the number of NSCs but soluble and aggregated Aß25-35 decreased the number. Soluble Aß1-40 and Aß1-42 did not affect the number of apoptotic cells but aggregated Aß1-40 and Aß1-42 did. When NSCs were treated with a combination of GM1 or GD3 and soluble Aß1-42, cell proliferation was enhanced, indicating that both GM1 and GD3 as well as Aßs are involved in promoting cell proliferation and survival of NSCs. These observations suggest the potential of beneficial effects of using gangliosides and Aßs for promoting NSC proliferation.

Dietary Trace Elements and the Pathogenesis of Neurodegenerative Diseases.

Trace elements such as iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) are absorbed from food via the gastrointestinal tract, transported into the brain, and play central roles in normal brain functions. An excess of these trace elements often produces reactive oxygen species and damages the brain. Moreover, increasing evidence suggests that the dyshomeostasis of these metals is involved in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, prion diseases, and Lewy body diseases. The disease-related amyloidogenic proteins can regulate metal homeostasis at the synapses, and thus loss of the protective functions of these amyloidogenic proteins causes neurodegeneration. Meanwhile, metal-induced conformational changes of the amyloidogenic proteins contribute to enhancing their neurotoxicity. Moreover, excess Zn and Cu play central roles in the pathogenesis of vascular-type senile dementia. Here, we present an overview of the intake, absorption, and transport of four essential elements (Fe, Zn, Cu, Mn) and one non-essential element (aluminum: Al) in food and their connections with the pathogenesis of neurodegenerative diseases based on metal-protein, and metal-metal cross-talk.

Cell fiber-based 3D tissue array for drug response assay.

For the establishment of a reproducible and sensitive assay system for three-dimensional (3D) tissue-based drug screening, it is essential to develop 3D tissue arrays with uniform shapes and high cell numbers that prevent cell death in the center of the tissue. In recent years, 3D tissue arrays based on spheroids have attracted increased attention. However, they have only been used in specific tissues with hypoxic regions, such as cancer tissues, because nutrient deprivation and hypoxic regions are formed in the core as spheroids grow. Herein, we propose a method to array cell-encapsulated tube-like tissue (cell fiber (CF)) with diameters < 150 µm to prevent nutrient deprivation and hypoxia using a device that can fix the CFs, section them in uniform sizes, and transfer them to a 96-well plate. We fabricated the arrays of CF fragments from cell lines (GT1-7), cancer cells (HeLa), mouse neural stem cells (mNSCs) and differentiated mNSCs, and performed drug response assays. The array of CF fragments assessed the drug response differences among different cell types and drug responses specific to 3D tissues. The array of CF fragments may be used as a versatile drug screening system to detect drug sensitivities in various types of tissues.

Distribution of olfactory marker protein on a tissue section of vomeronasal organ measured by AFM.

Distribution of olfactory marker protein (OMP) on a tissue section of vomeronasal organ (VNO) was successfully measured by atomic force microscopy (AFM). Anti-OMP antibodies were covalently crosslinked with the tip of the AFM and were used as a probe to observe the distribution of OMP on a tissue section. First, force measurements were performed using a glass surface on which OMP was covalently immobilized to verify the success of tip modification. Clear differences of interaction forces were observed between a specific pair and the control experiments, indicating that the tip preparation succeeded. Next, distributions of OMP on the tissue section were observed by AFM and were compared with immunohistochemical observations. For large scale observation, a microbead was used as a probe in the AFM measurements. The results of the AFM measurements were well overlapped with that of immunohistochemistry, confirming the reliability of our method. A mapping of the AFM measurement with high resolution was also successfully obtained, which showed an advantage of the application of the AFM measurement in analysis of proteins on the tissue section.

Assembly and Connection of Micropatterned Single Neurons for Neuronal Network Formation.

Engineering of neuronal network geometry by micropatterning technology is a key future technology for creating artificial brains on a chip. However, engineering of network geometry at the single-cell-level with functional morphology (axon/dendrite) and connectivity (synapses) is still challenging. Here, we describe a method for controlling the axon and dendrite morphology of single primary-cultured neurons and assembling a neural circuit using mobile microplates. The microplates enabled morphological control of neurons by their shapes and bringing their ends into contact caused the formation of physical connections. Functional synapse formation at the connection was indicated by immunostaining of synapse-related proteins and intracellular Ca2+ imaging of neural activity. We believe that the method will be useful in engineering neural circuits with selected neurons and defined morphology.

Zinc, Carnosine, and Neurodegenerative Diseases.

Zinc (Zn) is abundantly present in the brain, and accumulates in the synaptic vesicles. Synaptic Zn is released with neuronal excitation, and plays essential roles in learning and memory. Increasing evidence suggests that the disruption of Zn homeostasis is involved in various neurodegenerative diseases including Alzheimer's disease, a vascular type of dementia, and prion diseases. Our and other numerous studies suggest that carnosine (ß-alanyl histidine) is protective against these neurodegenerative diseases. Carnosine is an endogenous dipeptide abundantly present in the skeletal muscles and in the brain, and has numerous beneficial effects such as antioxidant, metal chelating, anti-crosslinking, and anti-glycation activities. The complex of carnosine and Zn, termed polaprezinc, is widely used for Zn supplementation therapy and for the treatment of ulcers. Here, we review the link between Zn and these neurodegenerative diseases, and focus on the neuroprotective effects of carnosine. We also discuss the carnosine level in various foodstuffs and beneficial effects of dietary supplementation of carnosine.

Rod-Shaped Neural Units for Aligned 3D Neural Network Connection.

This paper proposes neural tissue units with aligned nerve fibers (called rod-shaped neural units) that connect neural networks with aligned neurons. To make the proposed units, 3D fiber-shaped neural tissues covered with a calcium alginate hydrogel layer are prepared with a microfluidic system and are cut in an accurate and reproducible manner. These units have aligned nerve fibers inside the hydrogel layer and connectable points on both ends. By connecting the units with a poly(dimethylsiloxane) guide, 3D neural tissues can be constructed and maintained for more than two weeks of culture. In addition, neural networks can be formed between the different neural units via synaptic connections. Experimental results indicate that the proposed rod-shaped neural units are effective tools for the construction of spatially complex connections with aligned nerve fibers in vitro.

Differentiation Induction of Mouse Neural Stem Cells in Hydrogel Tubular Microenvironments with Controlled Tube Dimensions.

In this paper, a tubular 3D microenvironment created in a calcium alginate hydrogel microtube with respect to the effect of scaffold dimensions on the differentiation of mouse neuronal stem cells (mNSCs) is evaluated. Five types of hydrogel microtubes with different core diameters (≈65-200 µm) and shell thicknesses (≈30-110 µm) are fabricated by using a double coaxial microfluidic device, and differentiation of encapsulated mNSCs is induced by changing the growth medium to the differentiation medium. The influence of the microtube geometries is examined by using quantitative real-time polymerase chain reaction and fluorescent immunocytochemistry. The analyses reveal that differences in microtube thickness within 30-110 µm affected the relative Tuj1 expression but do not affect the morphology of encapsulated mNSCs. The diameters of cores influence both the relative Tuj1 expression and morphology of the differentiated neurons. It is found that the tubular microenvironment with a core diameter of less than ≈100 µm contributes to forming highly viable and aligned neural tissue. The tubular microenvironment can provide an effective method for constructing microfiber-shaped neural tissues with geometrically controlled differentiation induction.

Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

Developmental changes of GABAergic synapses formed between primary cultured cortical neurons.

The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.

Brain-derived neurotrophic factor protects cultured rat hippocampal neurons from aluminum maltolate neurotoxicity.

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer's disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate.

Differences in development and cellular composition between neuronal cultures of rat accessory and main olfactory bulbs.

We previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.