Target‐specific forebrain projections and appropriate synaptic inputs of hESC‐derived dopamine neurons grafted to the midbrain of parkinsonian rats

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non‐human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC‐derived progenitors were grafted into the midbrain of 6‐hydroxydopamine‐lesioned rats, and analyzed at 6, 18, and 24 weeks for a time‐course evaluation of specificity and extent of graft‐derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies‐based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft‐derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine‐induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC‐derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC‐derived DA neurons to the midbrain.     

An epilepsy‐associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model

ACTL6B is a component of the neuronal BRG1/brm‐associated factor (nBAF) complex, which is required for chromatin remodeling in postmitotic neurons. We recently reported biallelic pathogenic variants in ACTL6B in patients diagnosed with early infantile epileptic encephalopathy, subtype 76 (EIEE‐76), presenting with severe, global developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal central nervous system myelination. However, the pathophysiological mechanisms underlying their phenotype is unknown. Here, we investigate the molecular pathogenesis of ACTL6B p.(Val421_Cys425del) using in silico 3D protein modeling predictions and patient‐specific induced pluripotent stem cell‐derived neurons. We found neurons derived from EIEE‐76 patients showed impaired accumulation of ACTL6B compared to unaffected relatives, caused by reduced protein stability. Furthermore, EIEE‐76 patient‐derived neurons had dysregulated nBAF target gene expression, including genes important for neuronal development and disease. Multielectrode array system analysis unveiled elevated electrophysiological activity of EIEE‐76 patients‐derived neurons, consistent with the patient phenotype. Taken together, our findings validate a new model for EIEE‐76 and reveal how reduced ACTL6B expression affects neuronal function.     

Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity

A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported. When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression. In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8-14 days). SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum. The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation. This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions. When we grafted a cell suspension of 2 x 10(5), 2 x 10(4), or 2 x 10(3) cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells. In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo.     

Increased cell suspension concentration augments the survival rate of grafted tyrosine hydroxylase immunoreactive neurons

The poor survival rate (5-20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinson's disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/microl and transplanted into two 0.5 microl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl groups (p=0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.     

A comparative study of the neural stem cell niche in the adult hypothalamus of human,mouse, rat and gray mouse lemur (Microcebus murinus)

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non‐human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co‐expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.     

Zuckerkandl's organ improves long-term survival and function of neural stem cell derived dopaminergic neurons in Parkinsonian rats

Transplantation of neural stem cells (NSC) derived dopamine (DA) neurons has emerged as an alternative approach to fetal neural cell transplantation in Parkinson's disease (PD). However, similar to fetal neural cell, survival of these neurons following transplantation is also limited due to limited striatal reinnervation (graft with dense neuronal core), limited host-graft interaction, poor axonal outgrowth, lack of continuous neurotrophic factors supply and principally an absence of cell adhesion molecules mediated appropriate developmental cues. In the present study, an attempt has been made to increase survival and function of NSC derived DA neurons, by co-grafting with Zuckerkandl's organ (a paraneural organ that expresses neurotrophic factors as well as cell adhesion molecules); to provide continuous NTF support and developmental cues to transplanted DA neurons in the rat model of PD. 24 weeks post transplantation, a significant number of surviving functional NSC derived DA neurons were observed in the co-transplanted group as evident by an increase in the number of tyrosine hydroxylase immunoreactive (TH-IR) neurons, TH-IR fiber density, TH-mRNA expression and TH-protein level at the transplantation site (striatum). Significant behavioral recovery (amphetamine induced stereotypy and locomotor activity) and neurochemical recovery (DA-D2 receptor binding and DA and DOPAC levels at the transplant site) were also observed in the NSC+ZKO co-transplanted group as compared to the NSC or ZKO alone transplanted group. In vivo results were further substantiated by in vitro studies, which suggest that ZKO increases the NSC derived DA neuronal survival, differentiation, DA release and neurite outgrowth as well as protects against 6-OHDA toxicity in co-culture condition. The present study suggests that long-term and continuous NTF support provided by ZKO to the transplanted NSC derived DA neurons, helped in their better survival, axonal arborization and integration with host cells, leading to long-term functional restoration in the rat model of PD.     

胚胎多巴胺神经元移植治疗帕金森病的实验研究

目的探讨胚胎多巴胺神经元移植对帕金森病大鼠的治疗作用。方法立体定向注射6-羟多巴胺建立帕金森病大鼠模型,随机分为对照组(n=12)和细胞移植组(n=12)。细胞移植组将荧光染料CM-DiI标记的大鼠胚胎多巴胺神经元立体定向注入帕金森病大鼠纹状体区,对照组于相同部位注入生理盐水。用阿扑吗啡诱导帕金森病大鼠旋转行为评估细胞移植的治疗作用。细胞移植8周后取大脑标本行冷冻切片,荧光显微镜下观察移植细胞在脑内存活情况。结果与对照组比较,移植多巴胺神经元能显著改善阿扑吗啡诱导帕金森病大鼠的异常旋转行为(P0.01)。移植8周后,仅少量多巴胺神经元存活。结论多巴胺神经元移植可短期内改善帕金森病大鼠的运动障碍,但长期疗效不佳,可能与移植多巴胺神经元长期存活率较低有关。     

Developmental expression of Neuregulin-3 in the rat central nervous system

Neuregulin-3 (Nrg3) is a member of the Nrg family of growth factors identified as risk factors for schizophrenia. There are three Nrgs expressed in the nervous system (Nrg1-3) and of these Nrg1 has been the best characterized. To set the groundwork for elucidating neural roles for Nrg3, we studied its expression in the rat brain at both the RNA and protein levels. Using an antibody developed against Nrg3, we observed a developmental increase of Nrg3 protein expression from embryonic stages to adulthood and determined that it carries O-linked carbohydrates. In cortical neuronal cultures, transfected Neuro2a cells, and brain tissue sections Nrg3 protein was localized to the soma, neurites, and to the Golgi apparatus, where it is prominently expressed. Nrg3 was detected in excitatory, GABAergic and parvalbumin-expressing inhibitory neurons while expression in glia was limited. Nrg3 mRNA and protein were widely expressed during both embryonic and postnatal ages. At E17, Nrg3 was detected within the cortical plate and ventricular zone suggesting possible roles in cell proliferation or migration. At postnatal ages, Nrg3 was abundantly expressed throughout the cerebral cortex and hippocampus. Multiple thalamic nuclei expressed Nrg3, while detection in the striatum was limited. In the cerebellum, Nrg3 was found in both Purkinje cells and granule neurons. In the rodent brain, Nrg3 is the most abundantly expressed of the Nrgs and its patterns of expression differ both temporally and spatially from that of Nrg1 and Nrg2. These findings suggest that Nrg3 plays roles that are distinct from the other Nrg family members.     

Long‐term,dynamic synaptic reorganization after GABAergic precursor cell transplantation into adult mouse spinal cord

Transplanting embryonic precursors of GABAergic neurons from the medial ganglionic eminence (MGE) into adult mouse spinal cord ameliorates mechanical and thermal hypersensitivity in peripheral nerve injury models of neuropathic pain. Although Fos and transneuronal tracing studies strongly suggest that integration of MGE‐derived neurons into host spinal cord circuits underlies recovery of function, the extent to which there is synaptic integration of the transplanted cells has not been established. Here, we used electron microscopic immunocytochemistry to assess directly integration of GFP‐expressing MGE‐derived neuronal precursors into dorsal horn circuitry in intact, adult mice with short‐ (5–6 weeks) or long‐term (4–6 months) transplants. We detected GFP with pre‐embedding avidin–biotin‐peroxidase and GABA with post‐embedding immunogold labeling. At short and long times post‐transplant, we found host‐derived synapses on GFP‐immunoreactive MGE cells bodies and dendrites. The proportion of dendrites with synaptic input increased from 50% to 80% by 6 months. In all mice, MGE‐derived terminals formed synapses with GFP‐negative (host) cell bodies and dendrites and, unexpectedly, with some GFP‐positive (i.e., MGE‐derived) dendrites, possibly reflecting autoapses or cross talk among transplanted neurons. We also observed axoaxonic appositions between MGE and host terminals. Immunogold labeling for GABA confirmed that the transplanted cells were GABAergic and that some transplanted cells received an inhibitory GABAergic input. We conclude that transplanted MGE neurons retain their GABAergic phenotype and integrate dynamically into host‐transplant synaptic circuits. Taken together with our previous electrophysiological analyses, we conclude that MGE cells are not GABA pumps, but alleviate pain and itch through synaptic release of GABA.     

Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 × 10⁵) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.     

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.     

Adult neurogenesis and gliogenesis in the dorsal and ventral canine hippocampus

Dentate gyrus (DG) of the mammalian hippocampus gives rise to new neurons and astrocytes all through adulthood. Canine hippocampus presents many similarities in fetal development, anatomy, and physiology with human hippocampus, establishing canines as excellent animal models for the study of adult neurogenesis. In the present study, BrdU-dated cells of the structurally and functionally dissociated dorsal (dDG) and ventral (vDG) adult canine DG were comparatively examined over a period of 30 days. Each part's neurogenic potential, radial glia-like neural stem cells (NSCs) proliferation and differentiation, migration, and maturation of their progenies were evaluated at 2, 5, 14, and 30 days post BrdU administration, with the use of selected markers (glial fibrillary acidic protein, doublecortin, calretinin and calbindin). Co-staining of BrdU+ cells with NeuN or S100B permitted the parallel study of the ongoing neurogenesis and gliogenesis. Our findings reveal the comparatively higher populations of residing granule cells, proliferating NSCs and BrdU+ neurons in the dDG, whereas newborn neurons of the vDG showed a prolonged differentiation, migration, and maturation. Newborn astrocytes were found all along the dorso-ventral axis, counting however for only 11% of newborn cell population. Comparative evaluation of adult canine and rat neurogenesis revealed significant differences in the distribution of resident and newborn granule cells along the dorso-ventral axis, division pattern of adult NSCs, maturation time plan of newborn neurons, and ongoing gliogenesis. Concluding, spatial and temporal features of adult canine neurogenesis are similar to that of other gyrencephalic species, including humans, and justify the comparative examination of adult neurogenesis across mammalian species.     

Primate embryonic stem cell-derived neuronal progenitors transplanted into ischemic brain.

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.     

Generation of graftable dopaminergic neuron progenitors from mouse ES cells by a combination of coculture and neurosphere methods

Parkinson's disease is characterized by a loss of midbrain dopamine (DA) neurons and is generally viewed as a potential target for stem cell therapy. Although several studies have reported the generation of postmitotic DA neurons from embryonic stem (ES) cells, it is unknown whether the proliferative progenitors of DA neurons can be isolated in vitro. To investigate this possibility, we have developed a combined approach in which ES cells are cocultured with PA6 stromal cells to expose them to stromal cell-derived inducing activity (SDIA) and are then cultured as neurospheres. Mouse ES cell colonies were detached from PA6 feeder cells after 8 days of SDIA treatment and then expanded as spheres for another 4 days in serum-free medium supplemented with fibroblast growth factor-2. The spheres exhibited neural stem cell characteristics and contained few DA neurons at this stage of culture. After being induced to differentiate on polyornithine/laminin-coated dishes for 7 days, these spheres generated DA neurons in vitro at a relatively low frequency. Intriguingly, addition of PA6 cell conditioned medium to the sphere culture medium significantly increased the percentage of DA neurons to 25-30% of the total number of neurons. Transplantation of conditioned medium-treated day 4 spheres, which contained DA neuron progenitors, into the mouse striatum resulted in the generation of a significant number of graft-derived DA neurons. These findings suggest that progenitors of DA neurons are generated and can proliferate in ES cell-derived neurospheres induced by serial SDIA and PA6 conditioned medium treatment.     

Using stem cells and iPS cells to discover new treatments for Parkinson's disease

Fetal cell transplantation can improve the symptoms of Parkinson's disease (PD) patients for more than a decade. In some patients, alpha-synuclein aggregates and Lewy bodies have been observed in the transplanted neurons without functional significance. Recently stem cells have emerged as an ethically acceptable source of cells for transplantation but, importantly, the type of stem cell matters. While the lineage restriction of adult neural stem cells limits their clinical applicability for patients with PD, human pluripotent stem cells provide an opportunity to replace specific types of degenerating neurons. Now, cellular reprogramming technology can provide patient-specific neurons for neural transplantation and problems with cell fate specification and safety are resolving. Induced pluripotent stem (iPS) cell-derived neurons are also a unique tool for interpreting the genetic basis for an individual's risk of developing PD into clinically meaningful information. For example, clinical trials for neuroprotective molecules need to be tested in presymptomatic individuals when the neurons can still be protected. Patient-specific neural cells can also be used to identify an individual's responsiveness to drugs and to understand the mechanisms of the disease. Along these avenues of investigation, stem cells are enabling research for new treatments in PD.     

Murine maternal dietary restriction affects neural Humanin expression and cellular profile

To understand the cellular basis for the neurodevelopmental effects of intrauterine growth restriction (IUGR), we examined the global and regional expression of various cell types within murine (Mus musculus) fetal brain. Our model employed maternal calorie restriction to 50% daily food intake from gestation day 10–19, producing IUGR offspring. Offspring had smaller head sizes with larger head:body ratios indicating a head sparing IUGR effect. IUGR fetuses at embryonic day 19 (E19) had reduced nestin (progenitors), β-III tubulin (immature neurons), Glial fibrillary acidic protein (astrocytes), and O4 (oligodendrocytes) cell lineages via immunofluorescence quantification and a 30% reduction in cortical thickness. No difference was found in Bcl-2 or Bax (apoptosis) between controls and IUGR, though qualitatively, immunoreactivity of doublecortin (migration) and Ki67 (proliferation) was decreased. In the interest of examining a potential therapeutic peptide, we next investigated a novel pro-survival peptide, mouse Humanin (mHN). Ontogeny examination revealed highest mHN expression at E19, diminishing by postnatal day 15 (P15), and nearly absent in adult (3 months). Subanalysis by sex at E19 yielded higher mHN expression among males during fetal life, without significant difference between sexes postnatally. Furthermore, female IUGR mice at E19 had a greater increase in cortical mHN versus the male fetus over their respective controls. We conclude that maternal dietary restriction-associated IUGR interferes with neural progenitors differentiating into the various cellular components populating the cerebral cortex, and reduces cerebral cortical size. mHN expression is developmental stage and sex specific, with IUGR, particularly in the females, adaptively increasing its expression toward mediating a pro-survival approach against nutritional adversity.     

GDF15 promotes simultaneous astrocyte remodeling and tight junction strengthening at the blood–brain barrier

Perivascular astrocyte processes (PAP) surround cerebral endothelial cells (ECs) and modulate the strengthening of tight junctions to influence blood–brain barrier (BBB) permeability. Morphologically altered astrocytes may affect barrier properties and trigger the onset of brain pathologies. However, astrocyte-dependent mediators of these events remain poorly studied. Here, we show a pharmacologically driven elevated expression and release of growth/differentiation factor 15 (GDF15) in rat primary astrocytes and cerebral PAP. GDF15 has been shown to possess trophic properties for motor neurons, prompting us to hypothesize similar effects on astrocytes. Indeed, its increased expression and release occurred simultaneously to morphological changes of astrocytes in vitro and PAP, suggesting modulatory effects of GDF15 on these cells, but also neighboring EC. Administration of recombinant GDF15 was sufficient to promote astrocyte remodeling and enhance barrier properties between ECs in vitro, whereas its pharmacogenetic abrogation prevented these effects. We validated our findings in male high anxiety-related behavior rats, an animal model of depressive-like behavior, with shrunk PAP associated with reduced expression of the junctional protein claudin-5, which were both restored by a pharmacologically induced increase in GDF15 expression. Thus, we identified GDF15 as an astrocyte-derived trigger of astrocyte process remodeling linked to enhanced tight junction strengthening at the BBB.