Javier Villadiego

Carotid body autotransplantation in Parkinson disease: a clinical and positron emission tomography study.

Background: Carotid body (CB) glomus cells are highly dopaminergic and express the glial cell line derived neurotrophic factor. The intrastriatal grafting of CB cell aggregates exerts neurotrophic actions on nigrostriatal neurons in animal models of Parkinson disease (PD). Objective: We conducted a phase I–II clinical study to assess the feasibility, long term safety, clinical and neurochemical effects of intrastriatal CB autotransplantation in patients with PD. Methods: Thirteen patients with advanced PD underwent bilateral stereotactic implantation of CB cell aggregates into the striatum. They were assessed before surgery and up to 1–3 years after surgery according to CAPIT (Core Assessment Programme for Intracerebral Transplantation) and CAPSIT-PD (Core Assessment Programme for Surgical Interventional Therapies in Parkinson’s Disease) protocols. The primary outcome measure was the change in video blinded Unified Parkinson’s Disease Rating Scale III score in the off-medication state. Seven patients had 18F-dopa positron emission tomography scans before and 1 year after transplantation. Results: Clinical amelioration in the primary outcome measure was observed in 10 of 12 blindly analysed patients, which was maximal at 6–12 months after transplantation (5–74%). Overall, mean improvement at 6 months was 23%. In the long term (3 years), 3 of 6 patients still maintained improvement (15–48%). None of the patients developed off-period dyskinesias. The main predictive factors for motor improvement were the histological integrity of the CB and a milder disease severity. We observed a non-significant 5% increase in mean putaminal 18F-dopa uptake but there was an inverse relationship between clinical amelioration and annual decline in putaminal 18F-dopa uptake (r = −0.829; p = 0.042). Conclusions: CB autotransplantation may induce clinical effects in patients with advanced PD which seem partly related to the biological properties of the implanted glomus cells.

Selective glial cell line-derived neurotrophic factor production in adult dopaminergic carotid body cells in situ and after intrastriatal transplantation

Glial cell line-derived neurotrophic factor (GDNF) exerts a notable protective effect on dopaminergic neurons in rodent and primate models of Parkinsons disease (PD). The clinical applicability of this therapy is, however, hampered by the need of a durable and stable GDNF source allowing the safe and continuous delivery of the trophic factor into the brain parenchyma. Intrastriatal carotid body (CB) autografting is a neuroprotective therapy potentially useful in PD. It induces long-term recovery of parkinsonian animals through a trophic effect on nigrostriatal neurons and causes amelioration of symptoms in some PD patients. Moreover, the adult rodent CB has been shown to express GDNF. Here we show, using heterozygous GDNF/lacZ knock-out mice, that unexpectedly CB dopaminergic glomus, or type I, cells are the source of CB GDNF. Among the neural or paraneural cells tested, glomus cells are those that synthesize and release the highest amount of GDNF in the adult rodent (as measured by standard and in situ ELISA). Furthermore, GDNF expression by glomus cells is maintained after intrastriatal grafting and in CB of aged and parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals. Thus, glomus cells appear to be prototypical abundant sources of GDNF, ideally suited to be used as biological pumps for the endogenous delivery of trophic factors in PD and other neurodegenerative diseases.

Neuroprotection by Transgenic Expression of Glucose-6-Phosphate Dehydrogenase in Dopaminergic Nigrostriatal Neurons of Mice

Oxidative damage to dopaminergic nigrostriatal (DNS) neurons plays a central role in the pathogenesis of Parkinsons disease (PD). Glucose-6-phosphate dehydrogenase (G6PD) is a key cytoprotective enzyme that provides NADPH, the major source of the reducing equivalents of a cell. Mutations of this enzyme are the most common enzymopathies worldwide. We have studied in vivo the role of G6PD overexpressed specifically in the DNS pathway and show that the increase of G6PD activity in the soma and axon terminals of DNS neurons, separately from other neurons or glial cells, protects them from parkinsonism. Analysis of DNS neurons by histological, neurochemical, and functional methods showed that even a moderate increase of G6PD activity rendered transgenic mice more resistant than control littermates to the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The neuroprotective action of G6PD was also observed in aged animals despite that they had a greater susceptibility to MPTP. Therefore, overexpression of G6PD in dopaminergic neurons or pharmacological activation of the native enzyme should be considered as potential therapeutic strategies to PD.

Cellular properties and chemosensory responses of the human carotid body

•  The carotid body (CB) is a key chemoreceptor organ that mediates the hyperventilatory response to hypoxia, and contributes to the process of acclimatisation to chronic hypoxaemia. •  Knowledge of CB physiology at the cellular and molecular levels has advanced considerably in recent times thanks to studies on lower mammals; however, information on humans is practically absent. Here we describe the properties of human CB cells in slice preparations or after enzymatic dispersion. •  Besides glomus (type I) and glia‐like, sustentacular (type II) cells, adult human CBs contain nestin‐positive neural progenitor cells. The human CB also expresses high levels of glial cell line‐derived neurotrophic factor. These properties are maintained at an advanced age. •  Human glomus cells contain a relatively high density of voltage‐dependent Na+, Ca2+ and K+ channels. Membrane depolarisation with high extracellular K+ induces an increase of cytosolic [Ca2+] and quantal catecholamine release. •  Human glomus cells are responsive to hypoxia and hypoglycaemia, both of which induce an increase in cytosolic [Ca2+] and transmitter release. Chemosensory responses of glomus cells are also preserved at an advanced age. •  These findings on the cellular and molecular physiology of the CB provide novel perspectives for the systematic study of pathologies involving this organ in humans.

Chronic and progressive Parkinson's disease MPTP model in adult and aged mice.

Despite the different animal models of Parkinsons disease developed during the last years, they still present limitations modelling the slow and progressive process of neurodegeneration. Here, we undertook a histological, neurochemical and behavioural analysis of a new chronic parkinsonian mouse model generated by the subcutaneous administration of low doses of MPTP (20 mg/kg, 3 times per week) for 3 months, using both young adult and aged mice. The MPTP‐induced nigrostriatal neurodegeneration was progressive and was accompanied by a decrease in striatal dopamine levels and motor impairment. We also demonstrated the characteristic neuroinflammatory changes (microglial activation and astrogliosis) associated with the neurodegenerative process. Aged animals showed both a faster time course of neurodegeneration and an altered neuroinflammatory response. The long‐term systemic application of low MPTP doses did not induce any increase in mortality in either young adult or aged mice and better resembles the slow evolution of the neurodegenerative process. This treatment could be useful to model different stages of Parkinsons disease, providing a better understanding of the pathophysiology of the disease and facilitating the testing of both protective and restorative treatments.

The neurogenic niche in the carotid body and its applicability to antiparkinsonian cell therapy

The carotid body (CB) is a neural crest-derived organ whose major function is to sense changes in arterial O2 tension to elicit hyperventilation during hypoxia. The CB is composed of clusters of neuron-like glomus, or type I, cells that are highly dopaminergic and contain large amounts of the glial cell line-derived neurotrophic factor (GDNF). Glomus cells are enveloped by glia-like sustentacular, or type II, cells. In chronic hypoxia the CB grows with increase in glomus cell number. This adaptive response depends on a collection of neural progenitors that can be isolated and induced to form clonal neurospheres in vitro. CB neurospheres contain numerous newly differentiated glomus cells, which maintain their functional properties and the ability to synthesize dopamine and GDNF. Intrastriatal CB transplants have been assayed in animal models of Parkinson’s disease (PD) to test whether they increase the striatal dopamine levels and/or exert a neuroprotective action on the nigrostriatal pathway. Two pilot safety studies performed on PD patients subjected to CB autotransplantation have suggested that a major limitation of this technique is the small size of the organ. This could, however, be overcome by the in vitro formation of new CB tissue derived from adult CB stem cells.

Neuroprotective and reparative effects of carotid body grafts in a chronic MPTP model of Parkinson's disease

Intrastriatal transplantation of dopaminergic carotid body (CB) cells ameliorates parkinsonism in animal models and, with less efficacy, in Parkinsons disease patients. CB-based cell therapy was initially proposed because of its high dopamine content. However, later studies suggested that its beneficial effect might be due to a trophic action exerted on nigrostriatal neurons. Compatible with this concept are the high levels of neurotrophic factors encountered in CB cells. To test experimentally this idea, unilateral striatal transplants were performed with a sham graft in the contralateral striatum, as a robust internal control. Thereafter, the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6, -tetrahydropyridine was injected during 3 months. CB grafts protected from degeneration ipsilateral nigral dopaminergic neurons projecting to the transplant in a dose-dependent manner regarding size and glial cell line-derived neurotrophic factor expression. Grafts performed at different times after the onset of the neurotoxic treatment demonstrated with histological and behavioral methods protection and repair of the nigrostriatal pathway by CB transplants. This study provides a mechanistic explanation for the action of CB transplants on parkinsonian models. It should also help to improve cell therapy approaches to Parkinsons disease.

Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections

X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images. This method, combined with immunofluorescence, makes it possible to unequivocally identify X-gal-labelled cells in tissue sections, emerging as a valuable tool in gene expression and cell tracing analysis.

AQP1 mediates water transport in the carotid body

In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O2 tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB.

Bag1-L Is a Phosphorylation-Dependent Coactivator of c-Jun during Neuronal Apoptosis

ABSTRACT In the nervous system, cell death by apoptosis plays a critical role during normal development and pathological neurodegeneration. Jun N-terminal kinases (JNKs) are essential regulators of neuronal apoptosis. The AP-1 transcription factor c-Jun is phosphorylated at multiple sites within its transactivation domain by the JNKs, and c-Jun phosphorylation is required for JNK-induced neurotoxicity. While the importance of c-Jun as a mediator of apoptotic JNK signaling in neurons is firmly established, the molecular mechanism underlying the requirement for c-Jun N-terminal phosphorylation is enigmatic. Here we identify the multifunctional protein Bag1-L as a coactivator of phosphorylated c-Jun. Bag1-L preferentially interacts with N-terminally phosphorylated c-Jun, and Bag1-L greatly augments transcriptional activation by phosphorylated c-Jun. Chromatin immunoprecipitation experiments revealed binding of Bag1-L to the promoters of proapoptotic AP-1 target genes, and overexpression of Bag1-L augmented cell death in primary neurons. Therefore, Bag1-L functions as a coactivator regulating neurotoxicity mediated by phosphorylated c-Jun.