Tingyu Qu

Human neural stem cells improve cognitive function of aged brain.

The capability for in vitro expansion of human neural stem cells (HNSCs) provides a well characterized and unlimited source alternative to using primary fetal tissue for neuronal replacement therapies. The HNSCs, injected into the lateral ventricle of 24-month-old rats after in vitro expansion, displayed extensive and positional incorporation into the aged host brain with improvement of cognitive score assessed by the Morris water maze after 4 weeks of the transplantation. Our results demonstrate that the aged brain is capable of providing the necessary environment for HNSCs to retain their pluripotent status and suggest the potential for neuroreplacement therapies in age-associated neurodegenerative disease.

Putative role of neuronal 5-lipoxygenase in an aging brain

Aging is associated with increased incidence and/or severity of neurodegenerative pathologies. Oxygen‐mediated events are being considered as possible mechanisms responsible for the increasing neuronal vulnerability. Lipoxygenases are enzymes that, as cyclooxygenases (COX), can insert oxygen into the molecule of arachidonic acid and thereby synthesize inflammatory eicosanoids: leukotrienes [due to 5‐lipoxygenase (5‐LOX) activity] and prostaglandins (via COX activity). It appears that 5‐LOX is expressed in central nervous system neurons and may participate in neurodegeneration. 5‐LOX‐triggered cell death may be initiated by the enzymatic activity of 5‐LOX but could also occur via the nonenzymatic actions of the 5‐LOX protein; new data point to the possibility that 5‐LOX protein exerts actions such as interaction with tyrosine kinase receptors, cytoskeletal proteins, and the nucleus. The expression of neuronal 5‐LOX is susceptible to hormonal regulation, presumably due to the presence of hormone‐responsive elements in the structure of the 5‐LOX gene promoter. The expression of the 5‐LOX gene and the activity of the 5‐LOX pathway are increased in elderly subjects. One possible mechanism of such 5‐LOX up‐regulation implies the contribution of aging‐associated hormonal changes: relative melatonin deficiency and/or hyper‐glucocorticoidemia. Thus, the 5‐LOX pathway could become a promising target of neuroprotective therapies for the aging brain.—Manev, H., Uz, T., Sugaya, K., Qu, T. Putative role of neuronal 5‐lipoxygenase in an aging brain. FASEB J. 14, 1464–1469 (2000)

Survival and plasticity of basal forebrain cholinergic systems in mice transgenic for presenilin-1 and amyloid precursor protein mutant genes.

The basalo-cortical cholinergic system was characterized in mice expressing mutant human genes for presenilin-1 (PS1), amyloid precursor protein (APP), and combined PS/APP. Dual immunocytochemistry for ChAT and Aβ revealed swollen cholinergic processes within cortical plaques in both APP and PS/APP brains by 12 months, suggesting aberrant sprouting or redistribution of cholinergic processes in response to amyloid deposition. At 8 months, cortical and subcortical ChAT activity was normal (PS/APP) or elevated (PS, APP frontal cortex), while cholinergic cell counts (nBM/SI) and receptor binding were unchanged. ChAT mRNA was up-regulated in the nBM/SI of all three transgenic lines at 8 months. The data indicate that the basal forebrain cholinergic system does not degenerate in mice expressing AD-related transgenes, even in mice with extreme amyloid load. The PS1 or APP transgene appears to enhance the cholinergic phenotype in younger mice, but this involves aberrant sprouting and redistribution of cortical cholinergic processes.

Inflammatory 5-LOX mRNA and protein are increased in brain of aging rats.

5-Lipoxygenase (5-LOX) is the key enzyme in the synthesis of leukotrienes, inflammatory mediators of arachidonic acid. 5-LOX is also expressed in neurons (in particular in the hippocampus and the cerebellum), and it seems to be capable of promoting neurodegeneration. Recently, we observed greater 5-LOX mRNA content in the hippocampus of older (24 months) than younger (2 months) rats. In this study, we measured in the hippocampus and the cerebellum of younger and older male F344 rats the contents of: 5-LOX mRNA, FLAP (5-LOX activating protein) mRNA, and 5-LOX protein. By using a quantitative reverse transcription/polymerase chain reaction (PCR) (RT-PCR) with internal standards we found that 5-LOX but not FLAP mRNA content is greater (both in hippocampus and cerebellum) of older than younger rats. By using quantitative Western immunoblotting, we found a greater content of 5-LOX protein in the hippocampus and the cerebellum of older rats; we also established that the membrane/cytosol 5-LOX content ratio is larger in the brains of older than younger rats (statistically significant in the cerebellum). The latter can be considered an indication of 5-LOX translocation/activation during aging. Together these results suggest that aging increases both neuronal 5-LOX expression and protein translocation, and indicate that the 5-LOX system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Abstract Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.

Differentiation of human neural stem cells into retinal cells

We have previously reported that transplanted human neural stem cells (HNSCs) display extensive migration and positional incorporation into the aged rat brain, which is associated with an improvement in cognitive function. In the current study, to investigate whether HNSCs are capable of differentiating into retinal cells, we treated HNSCs with human transforming growth factor-&bgr;3 (TGF-&bgr;3) under a serum-free differentiation condition. After 5 days of differentiation in vitro we detected opsin-immunopositive cells in the culture treated with TGF-&bgr;3. We also transplanted TGF-&bgr;3-treated HNSCs into the rat vitreous cavity. The donor cells migrated and differentiated into opsin-positive cells in the host retinal cell layer. Here we show for the first time that TGF-&bgr;3-treated HNSCs differentiate into retinal cells.

5-Lipoxygenase and cyclooxygenase mRNA expression in rat hippocampus:early response to glutamate receptor activation by kainate

Recent research has identified in central nervous system neurons the expression of two enzymes from the inflammatory pathway of the metabolism of arachidonic acid, the 5-lipoxygenase (5LOX) and the cyclooxygenase-2 (COX2). Expression of both enzymes appears to be upregulated during aging; upregulated 5LOX/COX2 expression in neurons may be responsible for the increased neuronal vulnerability to degeneration. Involvement of the excitatory neurotransmitter glutamate in aging-associated neurodegeneration has also been suggested. Stimulation of glutamate receptors by kainic acid (kainate) has been shown independently to affect the brain expression of 5LOX or COX2. Using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to measure the contents of mRNAs we found 3h after kainate injection (intraperitoneally; 10 mg/kg) increased mRNA levels of 5LOX and COX2, but not that of COX1 in the hippocampus of rats. Pretreatment with the COX2 inhibitor NS-398 (9 mg/kg, 1h prior to kainate) inhibited the kainate-stimulated increase of 5LOX and COX2 mRNA levels. Our results indicate that hippocampal expression of both 5LOX and COX2 increases rather promptly when glutamate receptors are stimulated by kainate. The mechanism of how NS-398 inhibits this action of kainate should be further investigated.

Neuronal expression of arylalkylamine N-acetyltransferase (AANAT) mRNA in the rat brain

The role of arylalkylamine N-acetyltransferase (AANAT) in neuronal functioning has been suggested based on biochemical assays; only scarce evidence indicates neuronal expression of the mRNA encoding for this enzyme that catalyzes the conversion of serotonin into N-acetylserotonin. Using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay with internal standards, and an in-situ RT-PCR hybridization assay we found evidence for the expression of AANAT in the rat brain. In the localization studies, the most prominent AANAT mRNA signal was found in the granule neurons of the hippocampus, the olfactory bulb, and the cerebellum, and in the gray matter of the spinal cord. Diurnal differences in AANAT mRNA content were observed in the pineal gland but not in the hippocampus; the content of AANAT mRNA was lower both in the pineal gland and the hippocampus of old (24 months) compared with young (2 months) rats. These data are consistent with the hypothesis that AANAT may play a physiological role in mammalian central nervous system neurons. Further studies are warranted into the possible functional significance of neuronal expression of AANAT mRNA.

Membrane properties of neuron-like cells generated from adult human bone-marrow-derived mesenchymal stem cells.

Adult mesenchymal stem cells (MeSCs) isolated from human bone marrow are capable of generating neural stem cell (NSC)-like cells that can be subsequently differentiated into cells expressing molecular markers for neurons. Here we report that these neuron-like cells had functional properties similar to those of brain-derived neurons. Whole-cell patch-clamp recordings and calcium imaging experiments were performed on neuron-like cells differentiated from bone-marrow-derived NSC-like cells. The neuron-like cells were subjected to current pulses to determine if they were capable of generating depolarization-induced action potentials. We found that nearly all of the cells with neuron-like morphology exhibited active membrane properties in response to the depolarizing pulses. The most common response was a single spike-like event with an overshoot and brief afterhyperpolarization. Cells that did not generate overshooting spike-like events usually displayed rectifying current-voltage relationships. The prevalence of these active membrane properties in response to the depolarizing current pulses suggested that the human MeSCs (hMeSCs) were capable of converting to a neural lineage under defined culture conditions. The spike-like events were blocked by the voltage-gated sodium channel inhibitor lidocaine, but unaffected by another sodium channel inhibitor tetrodotoxin (TTX). In calcium imaging experiments, the neuron-like cells responded to potassium chloride depolarization and l-glutamate application with increases in the cytoplasmic calcium levels. Thus, the neuron-like cells appeared to express TTX-resistant voltage-gated sodium channels, voltage-gated calcium channels, and functional l-glutamate receptors. These results demonstrate that hMeSCs were capable of generating cells with characteristics typical of functional neurons that may prove useful for neuroreplacement therapies.

Genetically Engineered Human Mesenchymal Stem Cells Produce Met-Enkephalin at Augmented Higher Levels in Vitro:

We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patients bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.