Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation.

The existence of endogenous neural progenitors in the nigrostriatal system could represent a powerful tool for restorative therapies in Parkinson's disease. Sox-2 is a transcription factor expressed in pluripotent and adult stem cells, including neural progenitors. In the adult brain Sox-2 is expressed in the neurogenic niches. There is also widespread expression of Sox-2 in other brain regions, although the neurogenic potential outside the niches is uncertain. Here, we analyzed the presence of Sox-2+ cells in the adult primate (Macaca fascicularis) brain in naïve animals (N = 3) and in animals exposed to systemic administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine to render them parkinsonian (N = 8). Animals received bromodeoxyuridine (100 mg/kg once a day during five consecutive days) to label proliferating cells and their progeny. Using confocal and electron microscopy we analyzed the Sox-2+ cell population in the nigrostriatal system and investigated changes in the number, proliferation and neurogenic potential of Sox-2+ cells, in control conditions and at two time points after MPTP administration. We found Sox-2+ cells with self-renewal capacity in both the striatum and the substantia nigra. Importantly, only in the striatum Sox-2+ was expressed in some calretinin+ neurons. MPTP administration led to an increase in the proliferation of striatal Sox-2+ cells and to an acute, concomitant decrease in the percentage of Sox-2+/calretinin+ neurons, which recovered by 18 months. Given their potential capacity to differentiate into neurons and their responsiveness to dopamine neurotoxic insults, striatal Sox-2+ cells represent good candidates to harness endogenous repair mechanisms for regenerative approaches in Parkinson's disease

Correction: Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation.

[This corrects the article on p. e66377 in vol. 8.]

Cell renewal and differentiation dynamics of progenitors in the striatum.

<p>Schematic representation of hypothetic dynamics of endogenous striatal progenitors and the effects of MPTP. <b>A.</b> For reference we represented on top, the results of fate mapping of Sox-2⁺ cells in the murine dentate gyrus showing that non-radial Sox-2⁺ progenitors have self-renew potential and give rise to both neurons and astrocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066377#pone.0066377-Muotri1" target="_blank">[26]</a>. <b>B, C.</b> Similarly, we propose than in the normal situation, Sox-2⁺ striatal progenitors (Sox-2⁺/GFAP^−/CR^–) divide to generate new Sox-2⁺ progenitors. Moreover, some Sox-2⁺ progenitors give rise to astrocytes and (few) Sox-2⁺/CR⁺ neurons that after several maturation steps could switch off Sox-2 expression and give rise to different mature phenotypes (including TH⁺). Following MPTP administration the number of Sox-2⁺ progenitors increases while the number of CR⁺ cells that express Sox-2 is decreased. Possible mechanisms are A) MPTP favours gliogenesis from Sox2 progenitors at the expense of neurogenesis. B) MPTP (or lack of dopamine) accelerates maturation of Sox-2⁺/CR⁺ cells. C) Sox-2⁺/CR⁺ cells are susceptible to MPTP toxicity. Abbreviations: 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine: MPTP; calretinin: CR; tyrosine hydroxylase: TH.</p

CAV-2-Mediated GFP and LRRK2G2019S Expression in the Macaca fascicularis Brain

Parkinson’s disease is characterized by motor and nonmotor symptoms that gradually appear as a consequence of the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Currently, no treatment can slow Parkinson’s disease progression. Inasmuch, there is a need to develop animal models that can be used to understand the pathophysiological mechanisms underlying dopaminergic neuron death. The initial goal of this study was to determine if canine adenovirus type 2 (CAV-2) vectors are effective gene transfer tools in the monkey brain. A second objective was to explore the possibility of developing a large nonhuman primate that expresses one of the most common genetic mutations causing Parkinson’s disease. Our studies demonstrate the neuronal tropism, retrograde transport, biodistribution, and efficacy of CAV-2 vectors expressing GFP and leucine-rich repeat kinase 2 (LRRK2G2019S) in the Macaca fascicularis brain. Our data also suggest that following optimization CAV-2-mediated LRRK2G2019S expression could help us model the neurodegenerative processes of this genetic subtype of Parkinson’s disease in monkeys

MPTP effect on the nigrostriatal system.

<p>Midbrain sections at the level of the 3rd nerve from a control (<b>A</b>), a MPTP short-term (<b>B</b>) and a MPTP long-term treated monkey (<b>C</b>) immunostained for TH. Note the severe reduction of TH immunoreactivity in the SNpc of the lesioned monkeys. Scale bar = 1 cm. <b>D.</b> Stereological quantification of TH⁺ neurons in the SNpc. Data represent median and quartiles, N = 3, *p≤0.05. Images of precommisural striatum sections from a control monkey (<b>E</b>), a MPTP short-term monkey (<b>F</b>) and a MPTP long-term monkey (<b>G</b>) immunostained for TH. Note the loss of TH⁺ fibers in the lesioned monkeys. Scale bar = 1 cm. Representative striatal sections of a control <b>(e’)</b> a MPTP short-term monkey (<b>f’</b>) and a MPTP long-term monkey (<b>g’</b>), at higher magnification showing the intrinsic TH⁺ neurons (arrows). Scale bar = 300 µm. <b>H.</b> Quantification of striatal TH⁺ neurons. There was a significant increase in the density of TH⁺ neurons after MPTP administration in both groups. Data represent median and quartiles, N = 3, *p≤0.05. <b>I.</b> Western blot to assess striatal TH levels showing a reduction in the MPTP-short term group and a partial recovery in the MPTP-long term group with respect to controls. Data represent median and quartiles, N = 3, *p≤0.05. Abbreviations: 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine: MPTP; tyrosine hydroxylase: TH; substantia nigra pars compacta: SNpc.</p

Ultra-structural analysis of Sox-2 expression.

<p><b>A.</b> Sox-2 immuno-gold label is observed in the nucleus (arrowheads) in ependymal cells and astrocytes in ultra-thin sections of the SVZ. Boxed areas show an ependymal cell (E) and an astrocyte (As). Ependymal layer (EL), gap layer (HL), astrocyte ribbon layer (R). Scale bar = 20 um; Scale bar in boxed areas = 1 um. <b>a1.</b> Magnification shows a Sox-2⁺ astrocyte and lack of immunoreactivity in an oligodendrocyte (O) and a microglial cell (Mi). Astrocytes are recognized by the presence of dense bundles of intermediate filaments (arrows). Scale bar = 2 um. <b>B.</b> Sox-2 immuno-gold staining in a group of neuroblasts (type A cells). Scale bar = 20 um. <b>b1.</b> Magnification of three neuroblasts showing the nuclear staining. Neuroblasts form chain-like structures with characteristic intercellular spaces (arrows). Scale bar = 2 um. <b>C.</b> Light microscopy image of a semi-thin section showing the distribution and characterization of cells expressing Sox-2 in the dentate gyrus. Sox-2⁺ cells are visualized by the nuclear dark brown DAB precipitate. The section was counterstained with toluidine blue. The line marks the boundary between the granular layer (GrL) and the hilus (H) where most labeled cells were located. Scale bar = 100 um. <b>c1</b>) Neurons in the granular layer were not Sox-2⁺. Scale bar = 10 um; Insert = 1 um. <b>D.</b> Microphotograph of a striatal field of a control monkey showing a Sox-2⁺ astrocyte and unlabeled neuron (Neu) and microglial cells (Mi). Scale bar = 5 um. <b>d1.</b> Boxed area of the astrocyte (As) nucleus shows the gold particles (arrowheads). Scale bar = 1 um. <b>E.</b> A representative Sox-2⁺ neuron in the striatum of a control monkey. Scale bar = 2 um. <b>e1.</b> Boxed area shows deposition of gold particles over the nucleus (arrowheads) close to a synapse (arrow), which identifies this cell as a neuron. Scale bar = 1 um. <b>F.</b> Sox-2⁺ astrocyte in the substantia nigra pars compacta. Scale bar = 10 µm. <b>f1.</b> Boxed area shows a dense bundle of intermediate filaments (arrows) that characterizes astrocytes. Arrowheads indicate gold particles in the nucleus. Scale bar = 1 µm. Abbreviations: subventricular zone: (SVZ); 3′, 3′-diaminobenzidine: (DAB).</p

Expression of Sox-2 in neurogenic niches and the nigrostriatal system in the adult primate brain.

<p><b>A.</b> Schematic outline of the anatomical borders for the neurogenic niches as defined for the study. <b>B.</b> Confocal images of Sox-2⁺ cells in the neurogenic niches and nigrostriatal regions. Sox-2⁺ cells were evenly distributed throughout the brain parenchyma including the striatum and the SNpc. <b>C.</b> Stereological estimation of Sox-2⁺ cells revealed that the density of Sox-2⁺ cells was higher in the SNpc than in the striatum and lower in both regions than in the neurogenic niches (data shown for SGZ). Data represent median and quartiles, N = 3, *p≤0.05. Scale bar = 20 µm. Abbreviations: Substantia nigra pars compacta: SNpc; subgranular zone: SGZ.</p

List of the secondary antibodies used in the present study.

<p>List of the secondary antibodies used in the present study.</p

Characterization of Sox-2⁺ cells in the neurogenic niches.

<p>Confocal images showing Sox-2⁺ cells in the neurogenic niches. Representation of Sox-2⁺ cells in the SVZ that incorporate BrdU and Sox-2⁺ cells in the dentate gyrus of the hippocampus that co-express Ki67. Examples of Sox-2⁺/DCX⁺ in the SVZ and Sox-2⁺/GFAP⁺ in the SGL of the dentate gyrus. There were no differences between the control and MPTP treated animals. Scale bar = 50 µm. Abbreviations: subventricular zone: SVZ; doublecortin: DCX; subgranular layer: SGL; 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine: MPTP.</p

Phenotype of Sox-2 striatal cells in naïve adult primates.

<p><b>A.</b> Schematic drawing of the distribution of Sox-2⁺/CR⁺ neurons in control animals. Note that they are located close to the dorsolateral border of the striatum; the inmunofluorescence images show the co-localization (yellow arrowhead) of Sox-2 (red) and CR (green). An orthogonal reconstruction is shown in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066377#pone.0066377.s003" target="_blank">Fig. S3A</a>. <b>B.</b> TH⁺ striatal cells never co-localized with BrdU and the vast majority of CR⁺ striatal cells were BrdU^–. Scale bar = 20 µm. <b>C.</b> Detailed analysis of BrdU incorporation in the striatum showed that there were some Sox-2⁺/GFAP⁺/BrdU⁺ cells (yellow arrowhead) but the majority were negative for this glial marker (white arrowhead). <b>D.</b> Orthogonal confocal reconstruction of a z-stack showing co-localization of Sox-2 (red), BrdU (green) and GFAP (blue). Scale bar = 20 µm. Abbreviations: calretinin: CR; glial fibrillary acidic protein: GFAP; tyrosine hydroxylase: TH.</p