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This study assesses the production potential, environmental impact, and economic viability of generating biohydrogen from biomethane obtained from the organic fraction of municipal solid waste (MSW) using steam methane reforming with carbon capture and storage (CCS). As the emissions are biogenic, CCS results in negative emissions. The methodology is based on a previously developed model, including techno-economic analysis based on the levelised cost of hydrogen (LCOH) and mobility (LCOM), and environmental assessment, focusing on production potential, cost estimates, and emissions impact. A case study is conducted to assess the feasibility of using this biohydrogen with negative emissions to decarbonize Madrid's public bus fleet. The findings reveal that Madrid's MSW could meet the entire hydrogen fuel demand if the fleet consisted of fuel-cell buses. However, given the high costs of replacing the entire fleet, a net-zero solution is proposed, combining 60% fuel-cell buses with existing natural gas-powered buses. In this configuration, the negative emissions from biohydrogen offset the fossil emissions from natural gas and 40% of biomethane is saved. The cost of the net-zero fleet ranges between 192.55 and 209.37 €/100 km, comparable with 100% natural gas fleet, which ranges between 176.19 and 217.69 €/100 km., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach., (Copyright © 2012 Elsevier Inc. All rights reserved.)

beta-chemokines are secreted factors that regulate diverse functions in the adult brain, such as neuro-immune responses and neurotransmission, but their function in the developing brain is largely unknown. We recently found that the orphan nuclear receptor, Nurr1, up regulates CCL2 and CCL7 in neural stem cells, suggesting a possible function of beta-chemokines in midbrain development. Here we report that two beta-chemokines, CCL2 and CCL7, and two of their receptors, CCR1 and CCR2, are expressed and developmentally regulated in the ventral midbrain (VM). Moreover, we found that the expression of CCL7 was down regulated in the Nurr1 knockout mice, linking CCL7 to dopamine (DA) neuron development. When the function of CCL2 and CCL7 was examined, we found that they selectively enhanced the differentiation of Nurr1+ precursors into DA neurons, but not their survival or progenitor proliferation in primary precursor cultures. Moreover, both CCL2 and CCL7 promoted neuritogenesis in midbrain DA neuron cultures. Thus, our results show for the first time a function of beta-chemokines in the developing brain and identify beta-chemokines as novel class of pro-differentiation factors for midbrain DA neurons. These data also suggest that beta-chemokines may become useful tools to enhance the differentiation of DA cell preparations for cell replacement therapy and drug discovery in Parkinson's disease (PD).

Wnt1 and -5a have been shown to modulate the proliferation and differentiation of midbrain dopaminergic (DA) neurons. However, it is not known whether other Wnts or which Frizzled (Fz) receptors are expressed in the developing midbrain. We found that 13 out of 19 Wnts, all 10 Fzs, and several intracellular Wnt signaling modulators, including Axin, FRAT, Naked, Par-1, and Ltap are developmentally regulated between embryonic days (E) 10.5 and 15.5. Next, we studied whether Fzs are differentially expressed in different cell types and examined neuronal-progenitor- or glial-enriched cultures and DA neurons isolated from TH-GFP reporter mice. We found that Fz8 is expressed at high levels in DA neurons at E11.5 and E13.5. Fz6 and -7 are the predominant transcripts in glial precursors, and Fz9, which is absent in DA neurons at E11.5, is the main receptor expressed in neuronal precursors. We therefore examined the function of Fz9 in DA cells and found that overexpression of Fz9 reduced Wnt5a- but not Wnt3a-induced hyperphosphorylation of Dishevelled. Thus, our results show that Fzs are developmentally regulated and differentially expressed in VM precursors, DA neurons, and glia. These findings suggest that Fz expression contributes to provide specificity to Wnt-mediated effects.

Glial cells have been classically described as supporting cells for neurons. Recently, additional roles during neural development have begun to emerge. Here, we report that ventral midbrain glia, including astrocytes and radial glia, are the source of signals required by neural precursors to acquire a dopaminergic phenotype. We found that ventral midbrain glia, but not cortical glia, secrete high levels of the glycolipoprotein Wnt-5a, express region-specific transcription factors such as Pax-2, En-1 and Otx-2 and increase the differentiation of cortical or ventral midbrain Nurr1 precursors into tyrosine hydroxylase-positive neurons. Moreover, blocking experiments using a Wnt-5a blocking antibody indicated that the effects of ventral midbrain glia on Nurr1-positive neural precursors are partially mediated by Wnt-5a. Thus, our results identify Wnt-5a as an important component of the dopaminergic inductive activity of the ventral midbrain glia.

Wnt7a has been reported to signal via the canonical pathway, but also in non-canonical pathways acting on the cytoskeleton. Since Wnt7a is expressed after neurulation, we set to investigate the effects of Wnt7a on brain regionalization. We engineered transgenic mouse embryos that, under control of the nestin second intron, overexpressed Wnt7a in neural stem/progenitor cells. Surprisingly, transgenic embryos failed to complete cranial neurulation due to reduced levels and an impaired distribution of actin microfilaments, beta-catenin, and N-cadherin at the neural tube adherens junctions. These transgenic embryos expressed high levels of Vangl2, an essential component of non-canonical Wnt signaling. In agreement with a disregulation of this pathway, aberrant spinal neurulation was detected in the transgenic embryos, revealing a novel function regulated by Wnts. Thus, our findings suggest that Wnt7a overexpression disrupts normal Wnt signaling in the neural tube, resulting in defective adherens junctions and neurulation.

Stem cells are currently considered as alternative cell resources for restorative transplantation strategies in Parkinson's disease. However, the mechanisms that induce differentiation of a stem cell toward the dopaminergic phenotype are still partly unknown thus hampering the production of dopaminergic neurons from stem cells. In the past, FGF-20 has been found to promote the survival of ventral mesencephalic (VM) dopaminergic (DA) neurons in culture. We hereby provide evidence that FGF-20, a growth factor of the FGF family, is expressed in the adult and 6-OHDA-lesioned striatum and substantia nigra, but is not expressed by VM glia or DA neurons, suggesting that FGF-20 may work on DA neurons in a paracrine- or target-derived manner. We also found that co-culture of Nurr1-NSCs with Schwann cells overexpressing FGF-20 induced the acquisition of a neuronal morphology by the NSCs and the expression of tyrosine hydroxylase (TH) as assessed by immunocytochemistry, cell ELISA, and Western blot analysis. RT-PCR showed, that both, Schwann cells and Nurr1-NSCs (differentiated or not), expressed the FGF-1 receptor suggesting that both direct and indirect actions of FGF-20 are possible. We show that differentiated Nurr1 cells retained both neuronal morphology and TH expression after transplantation into the striatum of 6-OHDA-lesioned postnatal or adult rats, but that neuritogenesis was only observed after postnatal grafts. Thus, our results suggest that FGF-20 promotes the differentiation of Nurr1-NSCs into TH-positive neurons and that additional factors are required for the efficient differentiation of DA neurons in the adult brain.

We have investigated functional roles of EphA/ephrin-A signaling in the development and function of the nigrostriatal system by overexpressing a soluble, broad-range EphA receptor antagonist in the central nervous system of transgenic mice. Adult transgenic mice showed a 30-40% reduction in the total volume of the substantia nigra (SN) without detectable differences in the number of dopaminergic neurons. Using fluorogold retrograde tracing from the striatum, we detected a 40-50% reduction in the number of dopaminergic neurons that could be traced from this structure in transgenic mice, suggesting that, a lower proportion of these cells were able to reach the striatum after disruption of EphA/ephrin-A signaling. In spite of this, total dopamine content in the striatum of transgenic mice was comparable to wild type. Analysis of locomotor activity and its regulation by pharmacological treatments that stimulate dopaminergic transmission revealed an unexpected dissociation of the behavioral responses to amphetamine and cocaine. In particular, transgenic mice were relatively insensitive to amphetamine while retaining normal responsiveness to cocaine, which, to the best of our knowledge, represents the first report of a dissociation of the behavioral responses to these two psychostimulants. Together, these results reveal an unexpected role for EphA/ephrin-A signaling in the normal connectivity and function of midbrain dopaminergic neurons., (Copyright 2004 Elsevier Inc.)

Persephin (PSP) is a neurotrophic factor of the GDNF family that has been found to promote the survival of multiple populations of neurons. In the present study we have examined: (1) the mechanism of action and the function of PSP on nigrostriatal dopamine neurons and (2) the therapeutic potential of PSP, delivered by neural stem cells (NSCs) in a model of Parkinson's disease. Interestingly we found that the prenatal ventral mesencephalon and the newborn striatum express high levels of PSP mRNA. Moreover, midbrain dopamine neurons express its preferred receptor GFRalpha4, allowing a cis type of action of PSP on dopamine neurons. Primary culture studies showed that PSP is as potent and efficacious as GDNF at promoting both survival and neuritogenesis of midbrain dopamine neurons. To study the function and therapeutic potential of PSP in vivo we engineered NSCs to overexpress PSP. PSP-c17.2 cells were found to stably express PSP mRNA and protein for at least 3 months in vivo, to disperse within the striatum, and to give rise to neurons, astrocytes, and a large proportion of oligodendrocytes that integrated within white matter tracts in the striatum. Moreover, PSP-c17.2 cells enhanced dopamine-dependent behavioral parameters in unlesioned mice and prevented the loss of dopamine neurons and the behavioral impairment of mice receiving intrastriatal 6-OHDA injections. Thus, our findings are consistent with a direct action of PSP on developing and adult midbrain dopamine neurons and suggest that the delivery of PSP by NSCs may constitute a very useful strategy in the treatment of Parkinson's disease.

In the present study we have evaluated changes in nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3 (NT-3) mRNA expression induced by different glutamate receptor agonists injected into the neostriatum. Up-regulation of NGF expression was observed at 24 h after intrastriatal quinolinate injection, an N-methyl-D-aspartate receptor agonist, and this increase was maintained up to 7 days after lesion. NGF up-regulation was also apparent in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) treatment from 6 to 16 h postinjection. Instead, BDNF was up-regulated only at 6 h after kainate or AMPA excitotoxicity. Interestingly, NT-3 mRNA was down-regulated from 10 to 16 h following AMPA lesion, while 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid injection enhanced NT-3 mRNA levels at 10 h. Our results show a specific neurotrophin response induced by stimulation of each glutamate receptor. These activity-dependent changes might be involved in neuronal plasticity processes and may underlie the differential vulnerability of striatal neurons observed in neurodegenerative disorders.