Rosemary A. Fricker

The potential of high-resolution positron emission tomography to monitor striatal dopaminergic function in rat models of disease

The use of a recently commissioned small-diameter, high-resolution positron emission tomography (PET) to obtain a measure of specific binding of 3 carbon-11 labelled ligands in rat striatum is described. Using cerebellum as a reference tissue, compartmental modelling was used to obtain individual estimates of striatal binding potential (defined as the ratio of rate constants to and from the specifically bound compartment) for [11C]raclopride (D2 receptors), [11C]SCH 23390 (D1 receptors) and [11C]RTI-121 (dopamine transporter). The coefficients of variation in control, anaesthetized rats were of the order of 10%. Using two models of human disease, namely striatal injection of ibotenic acid to produce postsynaptic cell loss as in Huntingtons disease, and 6-hydroxydopamine injection into substantia nigra pars compacta to mimic dopaminergic terminal loss in Parkinsons disease, marked reductions in binding potential were observed for the corresponding pre- or postsynaptic markers. When the regions of interest are so small as to be of the order of the spatial resolution of the system, factor such as spill over and partial volume negate absolute quantification of tissue radioactivity. Nevertheless, the use of PET to monitor relative changes in dopaminergic integrity should be considered as a viable complement to established in vivo microdialysis and post mortem techniques.

A comparative study of preparation techniques for improving the viability of striatal grafts using vital stains, in vitro cultures, and in vivo grafts

Cell suspension grafts from embryonic striatal primordia placed into the adult rat striatum survive well and are able to alleviate a number of behavioral deficits caused by excitotoxic lesions to this structure. However, neither the anatomical connectivity between the graft and host nor the functional recovery elicited by the grafts is completely restored. One way in which the survival and function of embryonic striatal grafts may be enhanced is by the improvement of techniques for the preparation of the cell suspension prior to implantation, an issue that has been addressed only to a limited extent. We have evaluated a number of parameters during the preparation procedure, looking at the effects on cell survival over the first 24 h from preparation using vital dyes and the numbers of surviving neurons in vitro, after 4 days in culture, in addition to graft survival and function in vivo. Factors influencing cell survival include the type of trypsinization procedure and the age of donor tissues used for suspension preparation. The presence of DNase has no effect on cell viability but aids the dissociation of the tissue to form single cells. These results have important implications for the use of embryonic striatal grafts in animal models of Huntingtons disease, and in any future clinical application of this research.

Assessment of striatal graft viability in the rat in vivo using a small diameter PET scanner.

A small diameter positron emission tomography (PET) scanner has been used to monitor [11C]raclopride (D2 receptor) binding in vivo in either intact striatum, denervated striatum following an excitotoxic lesion with ibotenic acid, or lesioned and grafted striatum following implantation of cortical or striatal tissue grafts in rats. Binding of [11C]raclopride was localized in the intact striatum within 20 min of injection of the radioligand, and was much reduced within the lesioned striatum. Cortical grafts exhibited a similar low level of binding to the lesioned striatum, whereas striatal grafts showed specific binding at an intermediate level. The [11C]raclopride binding signal in vivo correlated well with the extent of surviving or grafted striatal tissue observed post morten by Nissl staining and acetylcholinesterase histochemistry. Thus, the distribution of dopamine receptors as seen in the PET scanner are consistent with post mortem anatomical observations of striatal, lesion and graft sizes, and suggest that PET can provide a useful tool for monitoring the viability of implanted striatal graft tissues in vivo.

Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression

During development a tightly controlled signaling cascade dictates the differentiation, maturation and survival of developing neurons. Understanding this signaling mechanism is important for developing therapies for neurodegenerative illnesses. In previous work we have sought to understand the complex signaling pathways responsible for the development of midbrain dopamine neurons using a proteomic approach. One protein we have identified as being expressed in developing midbrain tissue is the vitamin D receptor. Therefore we investigated the effect of the biologically active vitamin D3 metabolite, calcitriol, on primary fetal ventral mesencephalic cultures of dopamine neurons. We observed a dose responsive increase in numbers of rat primary dopamine neurons when calcitriol was added to culture media. Western blot data showed that calcitriol upregulated the expression of glial derived neurotrophic factor (GDNF). Blocking GDNF signaling could prevent calcitriol’s ability to increase numbers of dopamine neurons. An apoptosis assay and cell birth dating experiment revealed that calcitriol increases the number of dopamine neurons through neuroprotection and not increased differentiation. This could have implications for future neuroprotective PD therapies.

GABAergic Neurons from Mouse Embryonic Stem Cells Possess Functional Properties of Striatal Neurons In Vitro, and Develop into Striatal Neurons In Vivo in a Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is a neurodegenerative disease where GABAergic medium spiny neurons (MSNs) in the striatum degenerate. Embryonic stem cell-derived neural transplantation may provide an appropriate therapy for HD. Here we aimed to develop a suitable protocol to obtain a high percentage of functional GABAergic neurons from mouse embryonic stem cells (mESCs), and then tested their differentiation potential in vivo. The monolayer method was compared with the embryoid body and five stage method for its efficiency in generating GABAergic neurons from mESCs. All three methods yielded a similar percentage of GABAergic neurons from mESCs. Monolayer method-derived GABAergic neurons expressed the MSN marker dopamine- and cyclic AMP-regulated phosphoprotein (DARPP32). The pluripotent stem cell population could be eliminated in vitro by treating cells with puromycin and retinoic acid. Using patch-clamp recordings, the functional properties of GABAergic neurons derived from mESCs were compared to GABAergic neurons derived from primary lateral ganglionic eminence. Both types of neurons showed active membrane properties (voltage-gated Na+ and K+ currents, Na+-dependent action potentials, and spontaneous postsynaptic currents) and possessed functional glutamatergic receptors and transporters. mESC-derived neural progenitors were transplanted into a mouse model of HD. Grafted cells differentiated to mature neurons expressing glutamate decarboxylase, dopamine type 1 receptors, and DARPP32. Also, neural precursors and dividing populations were found in the grafts. In summary, mESCs are able to differentiate efficiently into functional GABAergic neurons using defined in vitro conditions, and these survive and differentiate following grafting to a mouse model of HD.

The Role of Vitamin D3 in the Development and Neuroprotection of Midbrain Dopamine Neurons

Vitamin D has long been synonymous with bone health. More recently, new health benefits are continually being associated with vitamin D, including a burgeoning field on neuroprotective properties. This has generated a huge explosion of interest in recent years in the potential for vitamin D to be used not only as a therapeutic in neurodegenerative disease, including Parkinsons disease, but also as biomarkers and for risk association. With an emphasis on Parkinsons disease, this chapter will discuss recent evidence supporting the assertion that vitamin D can be a useful therapeutic agent used as an intervention therapy to be combined with existing treatments; and the case for further development of novel treatments utilizing the potential of vitamin D. In addition, we present novel, previously unpublished evidence showing that in a unilateral model of Parkinsons disease, vitamin D can not only reduce the extent of denervation, but that this is also reflected in functional benefit to the animals. The potential of vitamin D is slowly being realized; in the future, it will be widely associated with far more than just bone health and may even contribute to an elusive treatment of neurodegenerative illness.

Nicotinamide promotes neuronal differentiation of mouse embryonic stem cells in vitro.

Factors controlling proliferation and differentiation are crucial in advancement of neural cell-based experimental neurodegenerative therapies. In this regard, nicotinamide has been shown to determine the fate of neural cells, enhance neuralization, and influence DNA repair and apoptosis. This study investigated whether the biologically active vitamin B3 metabolite, nicotinamide, could direct the differentiation of mouse embryonic stem cells, cultured as monolayers, into neurons at either early or late stages of development. Interestingly, we observed a dose-responsive increase in the percentage of neurons when nicotinamide was added at early stages to the cells undergoing differentiation (days 0–7). Nicotinamide (10 mM) had a significant effect on neuronal differentiation, increasing the &bgr;III-tubulin-positive neuronal population and concomitantly decreasing the total number of cells in culture, measured by quantification of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells. Nicotinamide added between days 7 and 14 had no effect on neuronal induction. High levels of nicotinamide (20 mM) induced cytotoxicity and cell death. Current work is focusing on elucidating the mechanism(s) mediating neural specification by nicotinamide – that is, induction of cell-cycle exit and/or selective apoptosis in non-neural populations. Preliminary data suggest a reduction in the proportion of proliferating cells in nicotinamide-treated cultures – that is, nicotinamide enhances cell-cycle exit, thereby promoting neuronal differentiation. Future work will focus on evaluating the effect of nicotinamide on the differentiation of midbrain dopamine neurons, towards a therapy for Parkinson’s disease.

Efficient alignment of primary CNS neurites using structurally engineered surfaces and biochemical cues

Tissue engineering strategies for the central nervous system (CNS) have been largely hampered by the complexity of neural cell interactions and limited ability to control functional circuit formation. Although cultures of primary CNS neurons give key insight into an in vivo state, these cells are extremely sensitive to local micro-environments and are therefore often replaced with cell lines. Here we aimed to combine primary CNS neurons with surface nano- and micro-topography, and biochemical cues, to direct neurite outgrowth. Neurons were cultured on nano-fibers and micro-grooves either coated with poly-L-lysine and laminin (PLL–LN) or pre-seeded with naturally supporting astrocyte cells. Developing neurites extended parallel to PLL–LN coated topography, significantly more on micro-grooved than nano-fiber substrata. Astrocytes were found to direct neurite alignment to a greater extent compared to structured surface cues, highlighting the importance for biochemical signalling and cellular architecture. Equally neuron–neuron interactions strongly influenced neurite outgrowth. On micro-structured surfaces neurite orientation was regulated by contact guidance cues at the edges of grooves. All of our findings show that we can control the behaviour of primary CNS neurons in vitro using surface engineering approaches. This will allow us to establish neuronal circuitry, to model neurodegenerative diseases and advance regenerative medicine strategies.

The Influence of Nicotinamide on Health and Disease in the Central Nervous System

Nicotinamide, the amide form of vitamin B3 (niacin), has long been associated with neuronal development, survival, and function in the central nervous system (CNS), being implicated in both neuronal death and neuroprotection. Here, we summarise a body of research investigating the role of nicotinamide in neuronal health within the CNS, with a focus on studies that have shown a neuroprotective effect. Nicotinamide appears to play a role in protecting neurons from traumatic injury, ischaemia, and stroke, as well as being implicated in 3 key neurodegenerative conditions: Alzheimer’s, Parkinson’s, and Huntington’s diseases. A key factor is the bioavailability of nicotinamide, with low concentrations leading to neurological deficits and dementia and high levels potentially causing neurotoxicity. Finally, nicotinamide’s potential mechanisms of action are discussed, including the general maintenance of cellular energy levels and the more specific inhibition of molecules such as the nicotinamide adenine dinucleotide-dependent deacetylase, sirtuin 1 (SIRT1).

14 – Factors Important in the Survival of Dopamine Neurons in Intracerebral Grafts of Embryonic Substantia Nigra

Publisher Summary This chapter focuses on the factors important in the survival of dopamine neurons in intracerebral grafts of embryonic substantia nigra. A number of common human neurodegenerative conditions of the central nervous system (CNS) are currently under consideration as potential targets for neural transplantation therapy. These include Parkinsons disease (PD), Alzheimers disease (AD), Huntingtons chorea (HC), and amyotrophic lateral sclerosis (ALS). In each case, the pathology that lies at the core of the disease is relatively well described, but the etiology remains largely unknown. Consequently, present approaches to treatment focus on pharmacological approaches to reversing neurochemical pathology rather than on halting the degeneration or replacing lost neurons. This approach has proved to be most successful in PD, with the use of dopaminergic agents to replace the degenerating nigrostriatal system, the loss of which constitutes the pathological hallmark of this condition. The chapter focuses on PD and its treatment by intracerebral transplantation of embryonic nigral neurons, as this condition has been the one that has received the most attention and has now entered the first stages of clinical trials.