Alex Mercer

Peptide hormone exendin-4 stimulates subventricular zone neurogenesis in the adult rodent brain and induces recovery in an animal model of parkinson's disease

We investigated the effects of exendin‐4 on neural stem/progenitor cells in the subventricular zone of the adult rodent brain and its functional effects in an animal model of Parkinsons disease. Our results showed expression of GLP‐1 receptor mRNA or protein in the subventricular zone and cultured neural stem/progenitor cells isolated from this region. In vitro, exendin‐4 increased the number of neural stem/progenitor cells, and the number of cells expressing the neuronal markers microtubule‐associated protein 2, β‐III‐tubulin, and neuron‐specific enolase. When exendin‐4 was given intraperitoneally to naïve rodents together with bromodeoxyuridine, a marker for DNA synthesis, both the number of bromodeoxyuridine‐positive cells and the number of neuronal precursor cells expressing doublecortin were increased. Exendin‐4 was tested in the 6‐hydroxydopamine model of Parkinsons disease to investigate its possible functional effects in an animal model with neuronal loss. After unilateral lesion and a 5‐week stabilization period, the rats were treated for 3 weeks with exendin‐4. We found a reduction of amphetamine‐induced rotations in animals receiving exendin‐4 that persisted for several weeks after drug administration had been terminated. Histological analysis showed that exendin‐4 significantly increased the number of both tyrosine hydroxylase‐ and vesicular monoamine transporter 2‐positive neurons in the substantia nigra. In conclusion, our results show that exendin‐4 is able to promote adult neurogenesis in vitro and in vivo, normalize dopamine imbalance, and increase the number of cells positive for markers of dopaminergic neurons in the substantia nigra in a model of Parkinsons disease.

PACAP promotes neural stem cell proliferation in adult mouse brain

In recent years, it has become evident that neural stem cells in the adult mammalian brain continuously generate new neurons, mainly in the hippocampus and olfactory bulb. Although different growth factors have been shown to stimulate neurogenesis in the adult brain, very little is known about the role of neuropeptides in this process. Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a neuropeptide with pleiotropic effects acting through three receptors to which it has high affinity, namely, PACAP receptor 1 (PAC1), vasoactive intestinal peptide (VIP) receptor 1, and VIP receptor 2. We show that PAC1 is expressed in the neurogenic regions of the adult mouse brain, namely the ventricular zone of the lateral ventricle and the hippocampal dentate gyrus. Cultured neural stem cells isolated from the lateral ventricle wall of adult mice express PAC1 and proliferate in vitro in response to two PAC1 agonists, PACAP and Maxadilan, but not VIP at physiologic concentrations, indicating PAC1 as a mediator of neural stem cell proliferation. Pharmacologic and biochemical characterization of PACAP‐induced neural stem cell proliferation revealed the protein kinase C pathway as the principal signaling pathway, whereas addition of epidermal growth factor synergistically enhanced the proliferating effect of PACAP. Further in vitro characterization of the effect of PACAP on neural stem cells showed PACAP capable of stimulating ex novo in vitro formation of multipotent neurospheres with the capacity to generate both neuronal and glial cells. Finally, intracerebroventricular infusion of PACAP increases cell proliferation in the ventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus. We conclude that PACAP, through PAC1, is a potent mediator of adult neural stem cell proliferation.

Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method

BackgroundNeural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression.ResultsTo address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability.ConclusionsWe demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation

BackgroundThe recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.ResultsWe used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.ConclusionExperimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.