Quantitative proteomic profiling of the rat substantia nigra places glial fibrillary acidic protein at the hub of proteins dysregulated during aging: Implications for idiopathic Parkinson's disease.

There is a strong correlation between aging and onset of idiopathic Parkinson's disease, but little is known about whether cellular changes occur during normal aging that may explain this association. Here, proteomic and bioinformatic analysis was conducted on the substantia nigra (SN) of rats at four stages of life to identify and quantify protein changes throughout aging. This analysis revealed that proteins associated with cell adhesion, protein aggregation and oxidation-reduction are dysregulated as early as middle age in rats. Glial fibrillary acidic protein (GFAP) was identified as a network hub connecting the greatest number of proteins altered during aging. Furthermore, the isoform of GFAP expressed in the SN varied throughout life. However, the expression levels of the rate-limiting enzyme for dopamine production, tyrosine hydroxylase (TH), were maintained even in the oldest animals, despite a reduction in the number of dopamine neurons in the SN pars compact(SNc) as aging progressed. This age-related increase in TH expression per neuron would likely to increase the vulnerability of neurons, since increased dopamine production would be an additional source of oxidative stress. This, in turn, would place a high demand on support systems from local astrocytes, which themselves show protein changes that could affect their functionality. Taken together, this study highlights key processes that are altered with age in the rat SN, each of which converges upon GFAP. These findings offer insight into the relationship between aging and increased challenges to neuronal viability, and indicate an important role for glial cells in the aging process.

Parallelized Manipulation of Adherent Living Cells by Magnetic Nanoparticles-Mediated Forces.

The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising, since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following the passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.

Stathmin is enriched in the developing corticospinal tract.

Understanding the intra- and extracellular proteins involved in the development of the corticospinal tract (CST) may offer insights into how the pathway could be regenerated following traumatic spinal cord injury. Currently, however, little is known about the proteome of the developing corticospinal system. The present study, therefore, has used quantitative proteomics and bioinformatics to detail the protein profile of the rat CST during its formation in the spinal cord. This analysis identified increased expression of 65 proteins during the early ingrowth of corticospinal axons into the spinal cord, and 36 proteins at the period of heightened CST growth. A majority of these proteins were involved in cellular assembly and organization, with annotations being most highly associated with cytoskeletal organization, microtubule dynamics, neurite outgrowth, and the formation, polymerization and quantity of microtubules. In addition, 22 proteins were more highly expressed within the developing CST in comparison to other developing white matter tracts of the spinal cord of age-matched animals. Of these differentially expressed proteins, only one, stathmin 1 (a protein known to be involved in microtubule dynamics), was both highly enriched in the developing CST and relatively sparse in other developing descending and ascending spinal tracts. Immunohistochemical analyses of the developing rat spinal cord and fetal human brain stem confirmed the enriched pattern of stathmin expression along the developing CST, and in vitro growth assays of rat corticospinal neurons showed a reduced length of neurite processes in response to pharmacological perturbation of stathmin activity. Combined, these findings suggest that stathmin activity may modulate axonal growth during development of the corticospinal projection, and reinforces the notion that microtubule dynamics could play an important role in the generation and regeneration of the CST.

Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.

Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

The rat striatum responds to nigro-striatal degeneration via the increased expression of proteins associated with growth and regeneration of neuronal circuitry.

BACKGROUND: Idiopathic Parkinson's disease is marked by degeneration of dopamine neurons projecting from the substantia nigra to the striatum. Although proteins expressed by the target striatum can positively affect the viability and growth of dopaminergic neurons, very little is known about the molecular response of the striatum as nigro-striatal denervation progresses. Here, iTRAQ labelling and MALDI TOF/TOF mass spectrometry have been used to quantitatively compare the striatal proteome of rats before, during, and after 6-OHDA induced dopamine denervation. RESULTS: iTRAQ analysis revealed the differential expression of 50 proteins at 3 days, 26 proteins at 7 days, and 34 proteins at 14 days post-lesioning, compared to the unlesioned striatum. While the denervated striatum showed a reduced expression of proteins associated with the loss of dopaminergic input (e.g., TH and DARPP-32), there was an increased expression of proteins associated with regeneration and growth of neurites (e.g., GFAP). In particular, the expression of guanine deaminase (GDA, cypin) - a protein known to be involved in dendritic branching - was significantly increased in the striatum at 3, 7 and 14 days post-lesioning (a finding verified by immunohistochemistry). CONCLUSIONS: Together, these findings provide evidence to suggest that the response of the normal mammalian striatum to nigro-striatal denervation includes the increased expression of proteins that may have the capacity to facilitate repair and growth of neuronal circuitry.

Paclitaxel is effective for controlling astrocyte proliferation in vitro: Implications for generating ventral mesencephalic cultures enriched with dopamine neurons.

BACKGROUND: Primary embryonic ventral mesencephalic (VM) cultures are a high throughput tool for understanding and manipulating dopamine neurons, to study the mechanisms that trigger their degeneration during Parkinson's disease (PD), and to test new drugs aimed at treating the disease. Unfortunately, primary cell cultures are often quickly overwhelmed by dividing astrocytes which both obscure neuronal cells and distort the cellular composition that exists in vivo. NEW METHOD: To develop a new in vitro system whereby astrocyte division can be readily controlled while maintaining neuronal integrity, VM cultures were treated with different doses (1.75, 3.5, 7, 14 nM) of the anti-mitotic drug paclitaxel for up to seven days in vitro. The study subsequently sought to determine the importance of astrocytes in dopamine neuron survival when challenged with an exposure to the toxin 6-hydroxydopamine (6-OHDA). RESULTS: Optical density (O.D.) measures of GFAP expression and counts of ß-III tubulin and tyrosine hydroxylase positive neurons reveals that a low dose of 3.5 nM of paclitaxel significantly reduced the density of GFAP + astrocytes in primary VM cultures, while maintaining the viability of neurons and dopamine neurons. Interestingly, a reduction of GFAP + astrocytes within primary VM cultures did not reveal any statistically significant differences in the number of dopamine neurons surviving treatment with 6-OHDA. CONCLUSIONS: These findings detail a quick and simple method for stabilising astrocyte numbers in primary VM cultures, without affecting the viability of dopamine neurons, and suggest that astrocytes may not enhance the survival of dopamine neurons when challenged with the 6-OHDA toxin.

Magnetic Mechanoactivation of Wnt Signaling Augments Dopaminergic Differentiation of Neuronal Cells.

Wnt signaling is a key developmental pathway that regulates dopaminergic progenitor cell proliferation and differentiation during neuronal development. This makes Wnt signaling an important therapeutic target for neurodegenerative conditions such as Parkinson's disease. Wnt signaling can be modulated using peptides such as UM206, which bind to the Wnt receptor Frizzled. Previous work has demonstrated remote activation of the Wnt pathway through Frizzled using peptide-functionalized magnetic nanoparticles (MNPs) with magnetic field stimulation. Using this technology, Wnt signaling is remotely activated in the neuronal cell line SH-SY5Y, and the phenotypic response to stimulation is assessed. Results indicate ß-catenin translocalization and activation of TCF/LEF responsive transcription in response to MNP and magnetic fields, which result in dopaminergic marker expression when synergistically combined with differentiation factors retinoic acid and the phorbol ester phorbol 12-myristate 13-acetate. This approach is translated into ex vivo postnatal rat brain slices modeling the developing nigrostriatal pathway. Dopaminergic marker expression is maintained in MNP-labeled SH-SY5Y cells after injection and magnetic stimulation. These results demonstrate the translational value of remote control of signal transduction for controlling neuronal precursor cell behavior and highlight the potential applications for controlled cell differentiation as part of cell therapies for neurodegenerative disease.

Monoclonal antibody Py recognizes neurofilament heavy chain and is a selective marker for large diameter neurons in the brain.

Almost 30 years ago, the monoclonal antibody Py was developed to detect pyramidal neurons in the CA3 region of the rat hippocampus. The utility of this antibody quickly expanded when several groups discovered that it could be used to identify very specific populations of neurons in the normal, developing, and diseased or injured central nervous system. Despite this body of literature, the identity of the antigen that the Py antibody recognizes remained elusive. Here, immunoprecipitation experiments from the adult rat cortex identified the Py antigen as neurofilament heavy chain (NF-H). Double immunolabeling of sections through the rat brain using Py and NF-H antibodies confirmed the identity of the Py antigen, and reveal that Py/NF-H+ neurons appear to share the feature of being particularly large in diameter. These include the neurons of the gigantocellular reticular formation, pyramidal neurons of layers II/III and V of the cortex, cerebellar Purkinje neurons as well as CA3 pyramidal neurons. Taken together, this finding gives clarity to past work using the monoclonal Py antibody, and immediately expands our understanding of the importance of NF-H in neural development, functioning, and disease.

Development of a stereotaxic device for low impact implantation of neural constructs or pieces of neural tissues into the mammalian brain.

Implanting pieces of tissue or scaffolding material into the mammalian central nervous system (CNS) is wrought with difficulties surrounding the size of tools needed to conduct such implants and the ability to maintain the orientation and integrity of the constructs during and after their transplantation. Here, novel technology has been developed that allows for the implantation of neural constructs or intact pieces of neural tissue into the CNS with low trauma. By "laying out" (instead of forcibly expelling) the implantable material from a thin walled glass capillary, this technology has the potential to enhance neural transplantation procedures by reducing trauma to the host brain during implantation and allowing for the implantation of engineered/dissected tissues or constructs in such a way that their orientation and integrity are maintained in the host. Such technology may be useful for treating various CNS disorders which require the reestablishment of point-to-point contacts (e.g., Parkinson's disease) across the adult CNS, an environment which is not normally permissive to axonal growth.

Transplanting intact donor tissue enhances dopamine cell survival and the predictability of motor improvements in a rat model of Parkinson's disease.

Primary cell transplantation is currently the gold standard for cell replacement in Parkinson's disease. However, the number of donors needed to treat a single patient is high, and the functional outcome is sometimes variable. The present work explores the possibility of enhancing the viability and/or functionality of small amounts of ventral mesencephalic (VM) donor tissue by reducing its perturbation during preparation and implantation. Briefly, unilaterally lesioned rats received either: (1) an intact piece of half an embryonic day 13 (E13) rat VM; (2) dissociated cells from half an E13 rat VM; or (3) no transplant. D-amphetamine- induced rotations revealed that animals receiving pieces of VM tissue or dissociated cells showed significant improvement in ipsilateral rotation 4 weeks post transplantation. By 6 weeks post transplantation, animals receiving pieces of VM tissue showed a trend for further improvement, while those receiving dissociated cells remained at their 4 week scores. Postmortem cell counts showed that the number of dopaminergic neurons in dissociated cell transplants was significantly lower than that surviving in transplants of intact tissue. When assessing the correlation between the number of dopamine cells in each transplant, and the improvement in rotation bias in experimental animals, it was shown that transplants of whole pieces of VM tissue offered greater predictability of graft function based on their dopamine cell content. Such results suggest that maintaining the integrity of VM tissue during implantation improves dopamine cell content, and that the dopamine cell content of whole tissue grafts offers a more predictable outcome of graft function in an animal model of Parkinson's disease.

Stem cell-derived dopamine neurons for brain repair in Parkinson's disease.

One of the prospects for a curative treatment for Parkinson's disease is to replace the lost dopaminergic neurons. Preclinical and clinical trials have demonstrated that dissected fetal dopaminergic neurons have the potential to markedly improve motor function in animal models and Parkinson's disease patients. However, this source of cells will never be sufficient to use as a widespread therapy. Over the last 20 years, scientists have been searching for other reliable sources of midbrain dopamine neurons, and stem cells appear to be strong candidates. This article reviews the potential of different types of stem cells, from embryonic to adult to induced pluripotent stem cells, to see how well the cells can be differentiated into fully functional dopamine neurons, which cells might be the best candidates and how much more research is required before stem cell technology might be translated to a clinical therapy for Parkinson's disease.

A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development.

Re-examining the ontogeny of substantia nigra dopamine neurons.

Recently, the need to detail the precise ontogeny of nigrostriatal dopamine neurons has grown significantly. It is now thought that the gestational day on which the majority of these neurons are born is important not only for maximizing the yield of primary cells for transplantation but also for extracting suitable dopamine neural precursors (as stem cells) for expansion in vitro. Historically, peak ontogeny of substantia nigra pars compacta (SNc) dopamine neurons in the rat has been considered to occur around embryonic day (E)14. However, such a concept is at odds with recent studies that reveal not only that substantial numbers of tyrosine hydroxylase-immunopositive cells reside in the ventral mesencephalic region of rats at E14 but that many of these cells have matured extensive axonal projections to the ventral forebrain. Here, then, the ontogeny of SNc neurons in rats commonly used as a source of donor tissue for experimental cell transplantation in animal models of Parkinson's disease has been re-examined. Using a combination of bromodeoxyuridine (BrdU) administration at E11, E12, E13 or E14 with immunocytochemical stainings for both BrdU and tyrosine hydroxylase after 4 weeks of postnatal development, this characterization reveals that the vast majority (perhaps 80%) of SNc dopamine neurons are probably born on E12 in Sprague-Dawley rats. Such findings are important in refining the use of embryonic tissues for primary cell transplantation and may provide more precise timing for identifying the cellular and molecular events that drive neural stem cells toward a dopaminergic phenotype during development.

Striatal neurons in striatal grafts are derived from both post-mitotic cells and dividing progenitors.

Transplants of embryonic striatal tissue are characteristically heterogeneous, containing patches (P-zones) of striatal medium spiny projection neurons. It is not yet known how this morphology develops, and whether the striatal neurons in the grafts are derived from post-mitotic neuroblasts in the embryonic brain or from striatal progenitors that continue to divide after transplantation. To address this question we labelled dividing cells in the transplants with bromodeoxyuridine (BrdU), either prior to or after transplantation into the adult lesioned rat striatum. Cells for transplantation were either pre-labelled in utero by intraperitoneal (i.p.) injections of BrdU, or post-labelled after transplantation by i.p. injections to the hosts. Either two or six months after transplantation the brains were processed using double immunohistochemical techniques to detect BrdU and calbindin-positive neurons in the transplants. In the transplants pre-labelled with BrdU, approximately 30% of calbindin-positive cells were heavily labelled with BrdU, suggesting these had undergone a final division prior to transplantation. In transplants where cells had been labelled post-transplantation, approximately 17% of calbindin cells were heavily BrdU labelled. These results suggest that whereas a proportion of striatal medium spiny neurons in the striatal grafts were post-mitotic at the time of transplantation, other striatal progenitor cells can continue to divide after transplantation, and then complete an appropriate neuronal maturation programme in the adult host brain environment.

Spatially and temporally restricted chemoattractive and chemorepulsive cues direct the formation of the nigro-striatal circuit.

Identifying cellular and molecular mechanisms that direct the formation of circuits during development is thought to be the key to reconstructing circuitry lost in adulthood to neurodegenerative disorders or common traumatic injuries. Here we have tested whether brain regions situated in and around the developing nigro-striatal pathway have particular chemoattractive or chemorepulsive effects on mesencephalic dopamine axons, and whether these effects are temporally restricted. Mesencephalic explants from embryonic day (E)12 rats were either cultured alone or with coexplants from the embryonic, postnatal or adult medial forebrain bundle region (MFB), striatum, cortex, brain stem or thalamus. Statistical analysis of axon growth responses revealed a potent chemoattraction to the early embryonic MFB (i.e. E12-15) that diminished (temporally) in concert with the emergence of chemoattraction to the striatum in the late embryonic period (i.e. E19+). Repulsive responses by dopaminergic axons were obvious in cocultures with embryonic brain stem and cortex, however, there was no effect by the thalamus. Such results suggest that the nigro-striatal circuit is formed via spatially and temporally distributed chemoattractive and chemorepulsive elements that: (i) orientate the circuit in a rostral direction (via brain stem repulsion); (ii) initiate outgrowth (via MFB attraction); (iii) prevent growth beyond the target region (via cortical repulsion); and (iv) facilitate target innervation (via striatal chemoattraction). Subsequent studies will focus on identifying genes responsible for these events so that their products may be exploited to increase the integration of neuronal transplants to the mature brain, or provide a means to (re)establish the nigro-striatal circuit in vivo.