Methods and products for biasing cellular development

Methods are described that bias cells, such as potent and multipotent stem cells, by transfection with a nucleic acid sequence, to differentiate to a desired end-stage cell or a cell having characteristics of a desired end-stage cell. In particular embodiments, human neural stem cells are transfected with vectors comprising genes in the homeobox family of transcription factor developmental control genes, and this results in a greater percentage of resultant transformed cells, or their progeny, differentiating into a desired end-stage cell or a cell having characteristics of a desired end-stage cell

Methods and products for biasing cellular development

Proper cellular functionand development depends on intrinsic signals and extracellular environmental cues. The interplay of these environmental influences and intracellular dynamics determine cellular ectivity including migration, differentiation and the manner in whihc it interacts with surrounding cells. An object of this invention invloves a way to mofigy cellular activity by gene insertion. Another object of this invention is the application of specially modified cells for therapeutic and research applications. Through gene insertion by means of a delivery system such as transfection we can create specialized cell types from various stem cells including adults these cells can be either be used to deliver proteins to a particular area in the body or develop into specialized cells. Examples include inner ear hair cells, cholinergic neurons or improved stem cells

Methods and products for biasing cellular development

Methods are described that bias cells, such as potent and multipotent stem cells, by transfection with a nucleic acid sequence, to differentiate to a desired end-stage cell or a cell having characteristics of a desired end-stage cell. In particular embodiments, human neural stem cells are transfected with vectors comprising genes in the homeobox family of transcription factor developmental control genes, and this results in a greater percentage of resultant transformed cells, or their progeny, differentiating into a desired end-stage cell or a cell having characteristics of a desired end-stage cell

A method of biasing implanted human neural stem cells away from differentiation into glial cells by (+)phenserine to modulate the concentration of soluble ßapp in tissue or csf

Disclosed herein are methods and materials for promoting neurogenesis of endogenous and transplanted stem cells. Specifically exemplified herein are methods that comprise transplanting neural stem cells in conjunction with a regimen of (+)phenserine treatment

β-Amyloid 1-42 Oligomers Impair Function of Human Embryonic Stem Cell-Derived Forebrain Cholinergic Neurons

Cognitive impairment in Alzheimer's disease (AD) patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs). Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ) peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES) cells with nerve growth factor (NGF) as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations) and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1–40 increased the number of functional neurons, whereas oligomeric Aβ1–42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1–40 and Aβ1–42 induced gliogenesis. These findings indicate that Aβ1–42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ

Positron emission tomography imaging and clinical progression in relation to molecular pathology in the first Pittsburgh Compound B positron emission tomography patient with Alzheimer’s disease

The accumulation of β-amyloid in the brain is an early event in Alzheimer’s disease. This study presents the first patient with Alzheimer’s disease who underwent positron emission tomography imaging with the amyloid tracer, Pittsburgh Compound B to visualize fibrillar β-amyloid in the brain. Here we relate the clinical progression, amyloid and functional brain positron emission tomography imaging with molecular neuropathological alterations at autopsy to gain new insight into the relationship between β-amyloid accumulation, inflammatory processes and the cholinergic neurotransmitter system in Alzheimer’s disease brain. The patient underwent positron emission tomography studies with 18F-fluorodeoxyglucose three times (at ages 53, 56 and 58 years) and twice with Pittsburgh Compound B (at ages 56 and 58 years), prior to death at 61 years of age. The patient showed a pronounced decline in cerebral glucose metabolism and cognition during disease progression, while Pittsburgh Compound B retention remained high and stable at follow-up. Neuropathological examination of the brain at autopsy confirmed the clinical diagnosis of pure Alzheimer’s disease. A comprehensive neuropathological investigation was performed in nine brain regions to measure the regional distribution of β-amyloid, neurofibrillary tangles and the levels of binding of 3H-nicotine and 125I-α-bungarotoxin to neuronal nicotinic acetylcholine receptor subtypes, 3H-L-deprenyl to activated astrocytes and 3H-PK11195 to microglia, as well as butyrylcholinesterase activity. Regional in vivo 11C-Pittsburgh Compound B-positron emission tomography retention positively correlated with 3H-Pittsburgh Compound B binding, total insoluble β-amyloid, and β-amyloid plaque distribution, but not with the number of neurofibrillary tangles measured at autopsy. There was a negative correlation between regional fibrillar β-amyloid and levels of 3H-nicotine binding. In addition, a positive correlation was found between regional 11C-Pittsburgh Compound B positron emission tomography retention and 3H-Pittsburgh Compound B binding with the number of glial fibrillary acidic protein immunoreactive cells, but not with 3H-L-deprenyl and 3H-PK-11195 binding. In summary, high 11C-Pittsburgh Compound B positron emission tomography retention significantly correlates with both fibrillar β-amyloid and losses of neuronal nicotinic acetylcholine receptor subtypes at autopsy, suggesting a closer involvement of β-amyloid pathology with neuronal nicotinic acetylcholine receptor subtypes than with inflammatory processes

Neuronal nicotinic receptor subtypes in normal ageing, Alzheimer's disease and schizophrenia : Influences of neuropathological mechanisms as studied in human autopsy brain and transgenic mice

Neuronal nicotinic acetylcholine receptors (nAChRs) are transmitter-gated ion channel receptors which are widely distributed in the brain. They mediate the effects of several neurotransmitters including ACh, DA, 5-HT and NA and are important for many normal physiological functions in the brain and are also implicated in a number of CNS disorders, such as AD, PD, schizophrenia, Tourette's syndrome and familial epilepsy. The overall aim of this thesis was to characterise changes in various nAChR subtypes during normal ageing, Alzheimer's disease (AD) and schizophrenia in postmortem human brain, and to investigate the neuropathological influences of characteristic AD lesions, eg. beta-amyloid (A-beta) deposition on nAChRs in the brains of two different transgenic mice models related to AD. Selective losses of particularly alpha3-containing nAChRs, but also alpha-4-beta-2 nAChRs were observed in different human autopsy brain regions at normal ageing. A comparison of nAChR deficits in cortical regions of sporadic AD cases versus those in subjects with familial Swedish APP 670/671 encoded mutation AD (FAD APP 670/671) showed that mutation carriers had similar deficits in nAChRs. This indicated that pronounced disturbances in APP metabolism result in similar neurotransmitter deficits as in cases where probably the mismetabolism is less pronounced. Investigation of the relationship between nAChR loss and neuropathological features i.e. deposition of amyloid plaques and neurofibrillary tangles in the cerebral cortex of FAD APP 670/671 carriers revealed no strict correlation. Two recently developed transgenic mice models, the APPSWE mouse harboring the 670/671 Swedish double mutation and the APPSWE /PS1 mouse expressing both the 670/671 Swedish double mutation and a FAD human PSI mutation, were used to study whether there was a potential link between A-beta production and accumulation and deficits in nAChRs as observed in the AD brain. Behavioural studies in the APPSWE mice revealed impairment in locomotion at 3 months, and impaired performance in water T-maze spatial working memory tasks at 10 months. Significant increases in alpha-7 nAChRs were observed at 4 months in various brain regions of APPSWE mice, preceding disturbances in learning and memory and A-beta pathology. This upregulation persisted at 17-19 months. Increases in alpha-4-beta-2 nAChRs were observed at 18 months, when the APPSWE mice showed heavy A-beta pathology. No changes in muscarinic receptors were detected. The upregulation of nAChRs in these transgenic mice probably reflect compensatory mechanisms in response to A-beta burden. No alterations of the binding sites of nAChR subtypes were observed in APPSWE /PS 1 transgenic mice, despite elevated levels of A-beta 1 -40 and A-beta 1 -42. These results indicate different effects on nAChRs between the two transgenic mice models. Moreover, the discrepancy in nAChR changes in transgenic mice brain and the AD brain provide evidence that these transgenic animals are not representative models for the neurodegenerative processes occuring in the human brain. We investigated changes in nAChRs in schizophrenia brain, and found selective reductions in 0C7 nAChRs in the cingulate cortex. In addition, a selective increase in high-affinity alpha-4-beta-2 nAChRs was also detected in the same brain region, probably reflecting the heavy tobacco use among schizophrenia patients. The lack of a loss of alpha-4-beta-2 nAChRs in schizophrenia is opposite to the marked loss of this receptor subtype observed in cortical regions and hippocampus in AD. It appears that the alpha-7 nAChRs are more vulnerable in schizoprenia compared to AD patients. These collective findings suggest that different nAChR subtypes are affected in the pathological cascades for these two diseases. In conclusion, these studies show that selective changes in different nAChR subtypes occur in diverse brain regions during various physiological and neurodegenerative conditions. Deducing changes in various subtypes in disease states from the normal may hopefully give us a further understanding of the function(s) of these receptor subtypes in specific pathways of the brain. There is a future challenge whether compounds can be developed which are selective in producing improvement in cognition and behavioural and attentional deficits associated with several CNS disorders without significant side effects

Retinoic Acid And Nerve Growth Factor Induce Differential Regulation Of Nicotinic Acetylcholine Receptor Subunit Expression In Sn56 Cells

Retinoic acid (RA) and nerve growth factor (NGF) have multiple functions in the regulation of neuronal development. In the present study, we characterized the expression of different nicotinic acetylcholine receptor (nAChR) subtypes in the cholinergic SN56 cell line and investigated the roles of RA and NGF in the expression of choline acetyltransferase (ChAT) and different nAChR subtypes. The nAChR agonist [3H]epibatidine was bound to two sites, with apparent affinities of 13 and 380 pM. RT-PCR analysis revealed expression of α3, α4, α5, α7, β2, and β4 nAChR subunits. RA treatment induced morphological changes, and the mRNA level of ChAT was maximally elevated after 4 days of exposure. The density of [3H]epibatidine binding sites and the mRNA and protein level of the α3 and β2 nAChR subunits were also increased by RA-induced differentiation. RA down-regulated the mRNA and protein level of the α4 nAChR subunit, whereas no significant change was observed in the mRNA and protein level of the α7 nAChR subunit. NGF treatment increased the mRNA and protein level of the α3 and β2 nAChR subunits. No morphological effects of NGF were observed, and the mRNA level of ChAT and mRNA and protein level of the α4 and α7 nAChR subunits were not significantly altered. Validation was performed with real-time RT-PCR. The present results show that RA and NGF have different effects on the expression of ChAT and the morphology and the expression pattern of different nAChR subunits in cholinergic SN56 cells. © 2007 Wiley-Liss, Inc

Glial Asthenia and Functional Paralysis: A New Perspective on Neurodegeneration and Alzheimer's Disease.

Neuroglia are represented by several population of cells heterogeneous in structure and function that provide for the homeostasis of the brain and the spinal cord. Neuroglial cells are also central for neuroprotection and defence of the central nervous system against exo- and endogenous insults. At the early stages of neurodegenerative diseases including Alzheimer's disease neuroglial cells become asthenic and lose some of their homeostatic, neuroprotective, and defensive capabilities. Astroglial reactivity, for example, correlates with preservation of cognitive function in patients with mild cognitive impairment and prodromal Alzheimer's disease. Here, we overview the experimental data indicating glial paralysis in neurodegeneration and argue that loss of glial function is fundamental for defining the progression of neurodegenerative diseases

Modulation of human neural stem cell differentiation in Alzheimer (APP23) transgenic mice by phenserine

In a previous study, we found that human neural stem cells (HNSCs) exposed to high concentrations of secreted amyloid-precursor protein (sAPP) in vitro differentiated into mainly astrocytes, suggesting that pathological alterations in APP processing during neurodegenerative conditions such as Alzheimer's disease (AD) may prevent neuronal differentiation of HNSCs. Thus, successful neuroplacement therapy for AD may require regulating APP expression to favorable levels to enhance neuronal differentiation of HNSCs. Phenserine, a recently developed cholinesterase inhibitor (ChEI), has been reported to reduce APP levels in vitro and in vivo. In this study, we found reductions of APP and glial fibrillary acidic protein (GFAP) levels in the hippocampus of APP23 mice after 14 days treatment with (+)-phenserine (25 mg/kg) lacking ChEI activity. No significant change in APP gene expression was detected, suggesting that (+)-phenserine decreases APP levels and reactive astrocytes by posttranscription regulation. HNSCs transplanted into (+)-phenserine-treated APP23 mice followed by an additional 7 days of treatment with (+)-phenserine migrated and differentiated into neurons in the hippocampus and cortex after 6 weeks. Moreover, (+)-phenserine significantly increased neuronal differentiation of implanted HNSCs in hippocampal and cortical regions of APP23 mice and in the CA1 region of control mice. These results indicate that (+)-phenserine reduces APP protein in vivo and increases neuronal differentiation of HNSCs. Combination use of HNSC transplantation and treatment with drugs such as (+)-phenserine that modulate APP levels in the brain may be a useful tool for understanding mechanisms regulating stem cell migration and differentiation during neurodegenerative conditions in AD