Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.

Uranium-Carbene-Imido Metalla-Allenes: Ancillary-Ligand-Controlled cis-/trans-Isomerisation and Assessment of trans Influence in the R2 C=U(IV) =NR' Unit (R=Ph2 PNSiMe3 ; R'=CPh3 ).

Uranium(IV)-carbene-imido complexes [U(BIPM(TMS) )(NCPh3 )(κ(2) -N,N'-BIPY)] (2; BIPM(TMS) =C(PPh2 NSiMe3 )2 ; BIPY=2,2-bipyridine) and [U(BIPM(TMS) )(NCPh3 )(DMAP)2 ] (3; DMAP=4-dimethylamino-pyridine) that contain unprecedented, discrete R2 C=U=NR' units are reported. These complexes complete the family of E=U=E (E=CR2 , NR, O) metalla-allenes with feasible first-row hetero-element combinations. Intriguingly, 2 and 3 contain cis- and trans-C=U=N units, respectively, representing rare examples of controllable cis/trans isomerisation in f-block chemistry. This work reveals a clear-cut example of the trans influence in a mid-valent uranium system, and thus a strong preference for the cis isomer, which is computed in a co-ligand-free truncated model-to isolate the electronic trans influence from steric contributions-to be more stable than the trans isomer by approximately 12 kJ mol(-1) with an isomerisation barrier of approximately 14 kJ mol(-1) .

Multimetallic cooperativity in uranium-mediated CO2 activation.

The metal-mediated redox transformation of CO2 in mild conditions is an area of great current interest. The role of cooperativity between a reduced metal center and a Lewis acid center in small-molecule activation is increasingly recognized, but has not so far been investigated for f-elements. Here we show that the presence of potassium at a U, K site supported by sterically demanding tris(tert-butoxy)siloxide ligands induces a large cooperative effect in the reduction of CO2. Specifically, the ion pair complex [K(18c6)][U(OSi(O(t)Bu)3)4], 1, promotes the selective reductive disproportionation of CO2 to yield CO and the mononuclear uranium(IV) carbonate complex [U(OSi(O(t)Bu)3)4(µ-κ(2):κ(1)-CO3)K2(18c6)], 4. In contrast, the heterobimetallic complex [U(OSi(O(t)Bu)3)4K], 2, promotes the potassium-assisted two-electron reductive cleavage of CO2, yielding CO and the U(V) terminal oxo complex [UO(OSi(O(t)Bu)3)4K], 3, thus providing a remarkable example of two-electron transfer in U(III) chemistry. DFT studies support the presence of a cooperative effect of the two metal centers in the transformation of CO2.

LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.

Parkinson's disease associated mutations in leucine rich repeat kinase 2 (LRRK2) impair mitochondrial function and increase the vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to oxidative stress. Since mitochondrial DNA (mtDNA) damage can compromise mitochondrial function, we examined whether LRRK2 mutations can induce damage to the mitochondrial genome. We found greater levels of mtDNA damage in iPSC-derived neural cells from patients carrying homozygous or heterozygous LRRK2 G2019S mutations, or at-risk individuals carrying the heterozygous LRRK2 R1441C mutation, than in cells from unrelated healthy subjects who do not carry LRRK2 mutations. After zinc finger nuclease-mediated repair of the LRRK2 G2019S mutation in iPSCs, mtDNA damage was no longer detected in differentiated neuroprogenitor and neural cells. Our results unambiguously link LRRK2 mutations to mtDNA damage and validate a new cellular phenotype that can be used for examining pathogenic mechanisms and screening therapeutic strategies.

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons.

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.

Synthesis, characterization, and reactivity of a uranium(VI) carbene imido oxo complex.

We report the uranium(VI) carbene imido oxo complex [U(BIPM(TMS))(NMes)(O)(DMAP)2] (5, BIPM(TMS) = C(PPh2 NSiMe3)2; Mes = 2,4,6-Me3C6H2; DMAP = 4-(dimethylamino)pyridine) which exhibits the unprecedented arrangement of three formal multiply bonded ligands to one metal center where the coordinated heteroatoms derive from different element groups. This complex was prepared by incorporation of carbene, imido, and then oxo groups at the uranium center by salt elimination, protonolysis, and two-electron oxidation, respectively. The oxo and imido groups adopt axial positions in a T-shaped motif with respect to the carbene, which is consistent with an inverse trans-influence. Complex 5 reacts with tert-butylisocyanate at the imido rather than carbene group to afford the uranyl(VI) carbene complex [U(BIPM(TMS))(O)2(DMAP)2] (6).

The nature of the U=C double bond: pushing the stability of high-oxidation-state uranium carbenes to the limit.

Treatment of [K(BIPM(Mes)H)] (BIPM(Mes)={C(PPh2NMes)2}(2−); Mes=C6H2-2,4,6-Me3) with [UCl4(thf)3] (1 equiv) afforded [U(BIPM(Mes)H)(Cl)3(thf)] (1), which generated [U(BIPM(Mes))(Cl)2(thf)2] (2), following treatment with benzyl potassium. Attempts to oxidise 2 resulted in intractable mixtures, ligand scrambling to give [U(BIPM(Mes))2] or the formation of [U(BIPM(Mes)H)(O)2(Cl)(thf)] (3). The complex [U(BIPM(Dipp))(µ-Cl)4(Li)2(OEt2)(tmeda)] (4) (BIPM(Dipp)={C(PPh2NDipp)2}(2−); Dipp=C6H3-2,6-iPr2; tmeda=N,N,N',N'-tetramethylethylenediamine) was prepared from [Li2(BIPM(Dipp))(tmeda)] and [UCl4(thf)3] and, following reflux in toluene, could be isolated as [U(BIPM(Dipp))(Cl)2(thf)2] (5). Treatment of 4 with iodine (0.5 equiv) afforded [U(BIPM(Dipp))(Cl)2(µ-Cl)2(Li)(thf)2] (6). Complex 6 resists oxidation, and treating 4 or 5 with N-oxides gives [{U(BIPM(Dipp)H)(O)2- (µ-Cl)2Li(tmeda)] (7) and [{U(BIPM(Dipp)H)(O)2(µ-Cl)}2] (8). Treatment of 4 with tBuOLi (3 equiv) and I2 (1 equiv) gives [U(BIPM(Dipp))(OtBu)3(I)] (9), which represents an exceptionally rare example of a crystallographically authenticated uranium(VI)­carbon σ bond. Although 9 appears sterically saturated, it decomposes over time to give [U(BIPM(Dipp))(OtBu)3]. Complex 4 reacts with PhCOtBu and Ph2CO to form [U(BIPM(Dipp))(µ-Cl)4(Li)2(tmeda)(OCPhtBu)] (10) and [U(BIPM(Dipp))(Cl)(µ-Cl)2(Li)(tmeda)(OCPh2)] (11). In contrast, complex 5 does not react with PhCOtBu and Ph2CO, which we attribute to steric blocking. However, complexes 5 and 6 react with PhCHO to afford (DippNPPh2)2C=C(H)Ph (12). Complex 9 does not react with PhCOtBu, Ph2CO or PhCHO; this is attributed to steric blocking. Theoretical calculations have enabled a qualitative bracketing of the extent of covalency in early-metal carbenes as a function of metal, oxidation state and the number of phosphanyl substituents, revealing modest covalent contributions to U=C double bonds.

Transcript expression levels of full-length alpha-synuclein and its three alternatively spliced variants in Parkinson's disease brain regions and in a transgenic mouse model of alpha-synuclein overexpression.

Alternative splicing is a complex post-transcriptional process that can be regulated by cis-acting elements located within genomic non-coding regions. Recent studies have identified that polymorphic variations in non-coding regions of the α-synuclein gene (SNCA) locus are associated with an increased risk for developing Parkinson's disease (PD). The underlying mechanism(s) for this susceptibility may involve changes in α-synuclein mRNA expression and alternative splicing. As a first step towards understanding the biology of α-synuclein splice variants in PD, we characterized the levels of the full-length SNCA-140 mRNA transcript and SNCA-126, -112, and -98 alternatively spliced variants in different neuronal regions from PD patients or transgenic mice overexpressing human α-synuclein (ASO). In human post-mortem tissue, α-synuclein spliced transcripts were expressed in a region-specific manner in the cortex, substantia nigra, and cerebellum. We observed increased nigral SNCA-140 and SNCA-126 transcript levels in PD patients when compared to neurologically unaffected cases. Human α-synuclein splicing changes were also found to occur in a region-specific manner in ASO mice. Here, SNCA-126, -112, and -98 transcript levels did not increase proportionally with SNCA-140 levels, or parallel the region-specific mouse transcript ratios seen in wild-type (WT) littermates. While most transcripts were elevated in ASO mice when compared to WT mice, the most prominent increase was found in the ventral midbrain of 15-month-old ASO mice. These results demonstrate region-specific human α-synuclein transcript level abnormalities in PD patients and in a transgenic mouse model of α-synucleinopathy. This study is relevant to understanding the normal, adaptive, or pathological role(s) of α-synuclein splice variants.

Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.

Metal(oid)s content in High-Andean aquatic systems of the Atacama Desert, Chile: environmental assessment of extreme ecosystems.

The high-Andean mountain of northern Chile host numerous water systems that is in risk due to increased mining activities. Total and dissolved Cd, Cr, Cu, Hg, Ni, Pb, Se, and Zn in water, and Cd, Cu, Fe, Ni, Pb, Zn, As, Mo, Al, and V in sediments of 21 aquatic systems (rivers, saline lakes, salt flats), were studied. The presence of Pb, Cd, and As in waters and sediments could be explained, in part, by mining activities. Waters are not suitable for human consumption or irrigation due to high content of Cu and As and high pH that exceed Chilean water quality guideline values. The use of different background reference values influences noticeably the conclusion related to environmental quality of sediments, measured with different environmental indexes. The local geological background suggest that Cd, Mo, Pb, and As generate some degree of contamination, while the use of unpolluted systems as background suggest that all metals measured in sediments represent a low contamination risk. The use of background values of local unpolluted systems seems to be more realistic than geological formation or Upper Continental Crust reference values to assess the environmental condition. The ecological risk assessment suggests that Cd and As are threat for communities living in these aquatic environments. However, these systems support abundant wildlife, developing unique extreme ecosystems with great potential for non-consumptive use such as special interest tourism and conservation.

Loss of Lipid Carrier ApoE Exacerbates Brain Glial and Inflammatory Responses after Lysosomal GBA1 Inhibition.

Tightly regulated and highly adaptive lipid metabolic and transport pathways are critical to maintaining brain cellular lipid homeostasis and responding to lipid and inflammatory stress to preserve brain function and health. Deficits in the lipid handling genes APOE and GBA1 are the most significant genetic risk factors for Lewy body dementia and related dementia syndromes. Parkinson's disease patients who carry both APOE4 and GBA1 variants have accelerated cognitive decline compared to single variant carriers. To investigate functional interactions between brain ApoE and GBA1, in vivo GBA1 inhibition was tested in WT versus ApoE-deficient mice. The experiments demonstrated glycolipid stress caused by GBA1 inhibition in WT mice induced ApoE expression in several brain regions associated with movement and dementia disorders. The absence of ApoE in ApoE-KO mice amplified complement C1q elevations, reactive microgliosis and astrocytosis after glycolipid stress. Mechanistically, GBA1 inhibition triggered increases in cell surface and intracellular lipid transporters ABCA1 and NPC1, respectively. Interestingly, the absence of NPC1 in mice also triggered elevations of brain ApoE levels. These new data show that brain ApoE, GBA1 and NPC1 functions are interconnected in vivo, and that the removal or reduction of ApoE would likely be detrimental to brain function. These results provide important insights into brain ApoE adaptive responses to increased lipid loads.

Synthesis of a uranium(VI)-carbene: reductive formation of uranyl(V)-methanides, oxidative preparation of a [R2C═U═O]2+ analogue of the [O═U═O]2+ uranyl ion (R = Ph2PNSiMe3), and comparison of the nature of U(IV)═C, U(V)═C, and U(VI)═C double bonds.

We report attempts to prepare uranyl(VI)- and uranium(VI) carbenes utilizing deprotonation and oxidation strategies. Treatment of the uranyl(VI)-methanide complex [(BIPMH)UO(2)Cl(THF)] [1, BIPMH = HC(PPh(2)NSiMe(3))(2)] with benzyl-sodium did not afford a uranyl(VI)-carbene via deprotonation. Instead, one-electron reduction and isolation of di- and trinuclear [UO(2)(BIPMH)(µ-Cl)UO(µ-O){BIPMH}] (2) and [UO(µ-O)(BIPMH)(µ(3)-Cl){UO(µ-O)(BIPMH)}(2)] (3), respectively, with concomitant elimination of dibenzyl, was observed. Complexes 2 and 3 represent the first examples of organometallic uranyl(V), and 3 is notable for exhibiting rare cation-cation interactions between uranyl(VI) and uranyl(V) groups. In contrast, two-electron oxidation of the uranium(IV)-carbene [(BIPM)UCl(3)Li(THF)(2)] (4) by 4-morpholine N-oxide afforded the first uranium(VI)-carbene [(BIPM)UOCl(2)] (6). Complex 6 exhibits a trans-CUO linkage that represents a [R(2)C═U═O](2+) analogue of the uranyl ion. Notably, treatment of 4 with other oxidants such as Me(3)NO, C(5)H(5)NO, and TEMPO afforded 1 as the only isolable product. Computational studies of 4, the uranium(V)-carbene [(BIPM)UCl(2)I] (5), and 6 reveal polarized covalent U═C double bonds in each case whose nature is significantly affected by the oxidation state of uranium. Natural Bond Order analyses indicate that upon oxidation from uranium(IV) to (V) to (VI) the uranium contribution to the U═C σ-bond can increase from ca. 18 to 32% and within this component the orbital composition is dominated by 5f character. For the corresponding U═C π-components, the uranium contribution increases from ca. 18 to 26% but then decreases to ca. 24% and is again dominated by 5f contributions. The calculations suggest that as a function of increasing oxidation state of uranium the radial contraction of the valence 5f and 6d orbitals of uranium may outweigh the increased polarizing power of uranium in 6 compared to 5.

Upstream lipid and metabolic systems are potential causes of Alzheimer's disease, Parkinson's disease and dementias.

Brain health requires circuits, cells and molecular pathways to adapt when challenged and to promptly reset once the challenge has resolved. Neurodegeneration occurs when adaptability becomes confined, causing challenges to overwhelm neural circuitry. Studies of rare and common neurodegenerative diseases suggest that the accumulation of lipids can compromise circuit adaptability. Using microglia as an example, we review data that suggest increased lipid concentrations cause dysfunctional inflammatory responses to immune challenges, leading to Alzheimer's disease, Parkinson's disease and dementia. We highlight current approaches to treat lipid metabolic and clearance pathways and identify knowledge gaps towards restoring adaptive homeostasis in individuals who are at-risk of losing cognition.

Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.

The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/ß-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.

Fate mapping and lineage analyses demonstrate the production of a large number of striatal neuroblasts after transforming growth factor alpha and noggin striatal infusions into the dopamine-depleted striatum.

Infusion of transforming growth factor alpha (TGFalpha) into the adult dopamine (DA)-depleted striatum generates a local population of nestin(+)/proliferating cell nuclear antigen (PCNA)(+) newborn cells. The precise origin and fate of these new striatal cells are unknown, making it difficult to direct them for neural repair in Parkinson's disease. Experiments in rats using 5-bromo-2'-deoxyuridine (BrdU) to label neural progenitor cells showed that during TGFalpha infusion in the DA-depleted striatum, newborn striatal cells formed a homogeneous population of precursors, with the majority coexpressing nestin, Mash1, Olig2, and epidermal growth factor receptor, consistent with the phenotype of multipotent C cells. Upon TGFalpha pump withdrawal, the subventricular zone (SVZ) was repopulated by neuroblasts. Strikingly, during this period, numerous clusters of doublecortin(+)/polysialylated neuronal cell adhesion molecule(+) neuroblasts were also produced in the ipsilateral medial striatum. In parallel, striatal BrdU(+)/glial fibrillary acidic protein(+) astrocytes were generated, but no BrdU(+)/O4(+)/CNPase(+) oligodendrocytes were generated. Infusion of the neuralizing bone morphogenetic protein antagonist noggin after TGFalpha pump withdrawal increased the neuroblast-to-astrocyte ratio among new striatal cells by blocking glial differentiation but did not alter striatal neurogenesis. At no time or treatment condition were differentiated neurons generated, including DA neurons. Using 6-hydroxydopamine-lesioned nestin-CreER(T2)/R26R-YFP mice that allow genetic fate-mapping of SVZ nestin(+) cells, we show that TGFalpha-generated striatal cells originate from SVZ nestin(+) precursors that confirmed data from the rats on the phenotype and fate of striatal nestin(+)/PCNA(+) cells upon TGFalpha withdrawal. This work demonstrates that a large population of multipotent striatal C-like cells can be generated in the DA-depleted striatum that do not spontaneously differentiate into DA neurons.

The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals.

BACKGROUND: Co-ordinated cell movement is a fundamental feature of developing embryos. Massive cell movements occur during vertebrate gastrulation and during the subsequent extension of the embryonic body axis. These are controlled by cell-cell signalling and a number of pathways have been implicated. Here we use long-term video microscopy in chicken embryos to visualize the migration routes and movement behaviour of mesoderm progenitor cells as they emerge from the primitive streak (PS) between HH stages 7 and 10. RESULTS: We observed distinct cell movement behaviours along the length of the streak and determined that this is position dependent with cells responding to environmental cues. The behaviour of cells was altered by exposing embryos or primitive streak explants to cell pellets expressing Wnt3a and Wnt5a, without affecting cell fates, thus implicating these ligands in the regulation of cell movement behaviour. Interestingly younger embryos were not responsive, suggesting that Wnt3a and Wnt5a are specifically involved in the generation of posterior mesoderm, consistent with existing mouse and zebrafish mutants. To investigate which downstream components are involved mutant forms of dishevelled (dsh) and prickle1 (pk1) were electroporated into the primitive streak. These had differential effects on the behaviour of mesoderm progenitors emerging from anterior or posterior regions of the streak, suggesting that multiple Wnt pathways are involved in controlling cell migration during extension of the body axis in amniote embryos. CONCLUSION: We suggest that the distinct behaviours of paraxial and lateral mesoderm precursors are regulated by the opposing actions of Wnt5a and Wnt3a as they leave the primitive streak in neurula stage embryos. Our data suggests that Wnt5a acts via prickle to cause migration of cells from the posterior streak. In the anterior streak, this is antagonised by Wnt3a to generate non-migratory medial mesoderm.

Recent advances in cell-based therapy for Parkinson disease.

In this review, the authors discuss recent advances in the field of cell therapy for Parkinson disease (PD). They compare and contrast recent clinical trials using fetal dopaminergic neurons. They attribute differences in cell preparation techniques, cell type specification, and immunosuppression as reasons for variable outcome and for some of the side effects observed in these clinical trials. To address ethical, practical, and technical issues related to the use of fetal cell sources, alternative sources of therapeutic dopaminergic neurons are being developed. The authors describe the progress in enrichment and purification strategies of stem cell-derived dopaminergic midbrain neurons. They conclude that recent advances in cell therapy for PD will create a viable long-term treatment option for synaptic repair for this debilitating disease.

Neuroblast protuberances in the subventricular zone of the regenerative MRL/MpJ mouse.

The MRL mouse is unique in its capacity for regenerative healing of wounds. This regenerative ability includes complete closure, with little scarring, of wounds to the ear pinna and repair of cardiac muscle, without fibrosis, following cryoinjury. Here, we examine whether neurogenic zones within the MRL brain show enhanced regenerative capacity. The largest neurogenic zone in the adult brain, the subventricular zone (SVZ), lies adjacent to the lateral wall of the lateral ventricle and is responsible for replacement of interneuron populations within the olfactory bulb. Initial gross observation of the anterior forebrain in MRL mice revealed enlarged lateral ventricles; however, little neurodegeneration was detected within the SVZ or surrounding tissues. Instead, increased proliferation within the SVZ was observed, based on incorporation of the thymidine analogue bromodeoxyuridine. Closer examination using electron microscopy revealed that a significant number of SVZ astrocytes interpolated within the ependyma and established contact with the ventricle. In addition, subependymal, protuberant nests of cells, consisting primarily of neuroblasts, were found along the anterior SVZ of MRL mice. Whole mounts of the lateral wall of the lateral ventricle stained for the neuroblast marker doublecortin revealed normal formation of chains of migratory neuroblasts along the entire wall and introduction of enhanced green fluorescent protein-tagged retrovirus into the lateral ventricles confirmed that newly generated neuroblasts were able to track into the olfactory bulb.

Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.

We report the first post-mortem analysis of two patients with Parkinson's disease who received fetal midbrain transplants as a cell suspension in the striatum, and in one case also in the substantia nigra. These patients had a favourable clinical evolution and positive 18F-fluorodopa PET scans and did not develop motor complications. The surviving transplanted dopamine neurons were positively identified with phenotypic markers of normal control human substantia nigra (n = 3), such as tyrosine hydroxylase, G-protein-coupled inward rectifying current potassium channel type 2 (Girk2) and calbindin. The grafts restored the cell type that provides specific dopaminergic innervation to the most affected striatal regions in the parkinsonian brain. Such transplants were able to densely reinnervate the host putamen with new dopamine fibres. The patients received only 6 months of standard immune suppression, yet by post-mortem analysis 3-4 years after surgery the transplants appeared only mildly immunogenic to the host brain, by analysis of microglial CD45 and CD68 markers. This study demonstrates that, using these methods, dopamine neuronal replacement cell therapy can be beneficial for patients with advanced disease, and that changing technical approaches could have a favourable impact on efficacy and adverse events following neural transplantation.