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Antiparkinsonian carotid body (CB) cell therapy has been proven to be effective in rodent and nonhuman primate models of Parkinson’s disease (PD), exerting trophic protection and restoration of the dopaminergic nigrostriatal pathway. These neurotrophic actions are mediated through the release of high levels of glial-cell-line-derived neurotrophic factor (GDNF) by the CB transplant. Pilot clinical trials have also shown that CB autotransplantation can improve motor symptoms in PD patients, although its effectiveness is affected by the scarcity of the grafted tissue. Here, we analyzed the antiparkinsonian efficacy of in vitro-expanded CB dopaminergic glomus cells. Intrastriatal xenografts of rat CB neurospheres were shown to protect nigral neurons from degeneration in a chronic MPTP mouse PD model. In addition, grafts performed at the end of the neurotoxic treatment resulted in the repair of striatal dopaminergic terminals through axonal sprouting. Interestingly, both neuroprotective and reparative effects induced by in vitro-expanded CB cells were similar to those previously reported by the use of CB transplants. This action could be explained because stem-cell-derived CB neurospheres produce similar amounts of GDNF compared to native CB tissue. This study provides the first evidence that in vitro-expanded CB cells could be a clinical option for cell therapy in PD.

Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.

Brain aquaporins facilitate the movement of water between the four water compartments: blood, cerebrospinal fluid, interstitial fluid, and intracellular fluid. This work analyzes the expression of the four most abundant aquaporins (AQPs) (AQP1, AQP4, AQP9, and AQP11) in the brains of mice and discuss their contribution to hydrocephalus. We analyzed available data from single-cell RNA sequencing of the central nervous system of mice to describe the expression of aquaporins and compare their distribution with that based on qPCR, western blot, and immunohistochemistry assays. Expression of AQP1 in the apical cell membrane of choroid plexus epithelial cells and of AQP4 in ependymal cells, glia limitans, and astrocyte processes in the pericapillary end foot is consistent with the involvement of both proteins in cerebrospinal fluid homeostasis. The expression of both aquaporins compensates for experimentally induced hydrocephalus in the animals. Recent data demonstrate that hypoxia in aged animals alters AQP4 expression in the choroidal plexus and cortex, increasing the ventricle size and intraventricular pressure. Cerebral distensibility is reduced in parallel with a reduction in cerebrospinal fluid drainage and cognitive deterioration. We propose that aged mice chronically exposed to hypoxia represent an excellent experimental model for studying the pathophysiological characteristics of idiopathic normal pressure hydrocephalus and roles for AQPs in such disease.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sym pathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers un dergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic depen dency mediated through GDNF-family receptor-a2 (GFRa2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innerva tion for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-depen dent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innerva tion reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to mod erate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic efect in the treatment of GvHD. For this purpose, we studied the efect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without afecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efcacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia efect. This benefcial efect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer.

Aquaporin-4 (AQP4) is the principal water channel in the brain being expressed in astrocytes and ependymal cells. AQP4 plays an important role in cerebrospinal fluid (CSF) homeostasis, and alterations in its expression have been associated with hydrocephalus. AQP4 contributes to the development of hydrocephalus by hypoxia in aged mice, reproducing such principal characteristics of the disease. Here, we explore whether these alterations associated with the hydrocephalic state are permanent or can be reverted by reexposure to normoxia. Alterations such as ventriculomegaly, elevated intracranial pressure, and cognitive deficits were reversed, whereas deficits in CSF outflow and ventricular distensibility were not recovered, remaining impaired even one month after reestablishment of normoxia. Interestingly, in AQP4−/− mice, the impairment in CSF drainage and ventricular distensibility was completely reverted by re-normoxia, indicating that AQP4 has a structural role in the chronification of those alterations. Finally, we show that aged mice subjected to two hypoxic episodes experience permanent ventriculomegaly. These data reveal that repetitive hypoxic events in aged cerebral tissue promote the permanent alterations involved in hydrocephalic pathophysiology, which are dependent on AQP4 expression.

Brain aquaporin 1 (AQP1) and AQP4 are involved in cerebrospinal fluid (CSF) homeostasis and might participate in the origin of hydrocephalus. Studies have shown alterations of perivascular AQP4 expression in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer’s disease (AD). Due to the overlapping of clinical signs between iNPH and certain neurological conditions, mainly AD, specific biomarkers might improve the diagnostic accuracy for iNPH. The goal of the present study was to analyze and quantify the presence of AQP1 and AQP4 in the CSF of patients with iNPH and AD to determine whether these proteins can be used as biomarkers of iNPH. We examined AQP1 and AQP4 protein levels in the CSF of 179 participants (88 women) classified into 5 groups: possible iNPH (81 participants), hydrocephalus associated with other neurological disorders (13 participants), AD (41 participants), non-AD dementia (32 participants) and healthy controls (12 participants). We recorded each participant’s demographic and clinical variables and indicated, when available in the clinical history, the record of cardiovascular and respiratory complications. An ELISA showed virtually no AQP content in the CSF. Information on the vascular risk factors (available for 61 patients) confirmed some type of vascular risk factor in 86% of the patients with possible iNPH and 58% of the patients with AD. In conclusion, the ELISA analysis showed insufficient sensitivity to detect the presence of AQP1 and AQP4 in CSF, ruling out the possible use of these proteins as biomarkers for diagnosing iNPH.

Las acuaporinas son una familia de pequeñas proteínas integrales de membrana (≈ 30 kDa) que se expresan ampliamente en el cuerpo. Tradicionalmente se las han definido como facilitadores del transporte pasivo de agua a través de la membrana plasmática de las células, expresándose de forma abundante en epitelios y endotelios implicados en el transporte de fluidos. En esta tesis se ha estudiado cómo las acuaporinas cerebrales están implicadas en la homeostasis del líquido cefalorraquídeo, y su contribución a la hidrocefalia asociada a envejecimiento e hipoxia, por ser el principal componente del fluido extracelular del sistema nervioso central, junto con el líquido intersticial, y jugar un papel fundamental en el mantenimiento del equilibrio iónico y osmótico necesarios para una correcta función glial y neuronal. La regulación de los fluidos en el sistema nervioso central es de suma importancia y está estrictamente controlada, de manera que, en condiciones fisiológicas, debe existir un balance entre la producción y evacuación del líquido cefalorraquídeo. De cualquier otra forma, se desencadena un proceso patológico que conduce al establecimiento de una condición hidrocefálica o de edema parenquimal. Los resultados que se revelan del estudio de esta tesis sugieren la participación tanto de AQP1 como AQP4 en la formación del líquido cefalorraquídeo y que pueden modificar la dinámica de evacuación mismo. El estudio de los cambios de expresión de las principales acuaporinas cerebrales asociados a hipoxia y envejecimiento ha permitido el desarrollo de un modelo murino para el estudio de la hidrocefalia idiopática normotensiva (o hidrocefalia crónica del adulto). Por último, las alteraciones en la capacidad de evacuación del líquido cefalorraquídeo y en la distensibilidad ventricular que se establecen como consecuencia del desarrollo de esta condición patológica fueron irreversibles a largo plazo, y no se restablecieron tras retornar a un estado de oxigenación fisiológico, a

Aquaporin 1 (AQP1), expressed in epithelial cells of the choroid plexus, and aquaporin 4 (AQP4) present in ependymal cells and glia limitants have been proposed to play a significant role in cerebrospinal fluid (CSF) production and homeostasis. However, the specific contribution of each water channel to these functions remains unknown, being a subject of debate during the last years. Here, we analyzed in detail how AQP1 and AQP4 participate in different aspects of the CSF homeostasis such as the load and drainage of ventricles, and further explored if these proteins play a role in the ventricular compliance. To do that, we carried out records of intraventricular pressure and CSF outflow, and evaluated ventricular volume by magnetic resonance imaging in AQP1−/−, AQP4−/−, double AQP1−/−-AQP4−/− knock out and wild type mice controls. The analysis performed clearly showed that both AQPs have a significant participation in the CSF production, and additionally revealed that the double AQP1-AQP4 mutation alters the CSF drainage and the ventricular compliance. The data reported here indicate a significant extra-choroidal CSF formation mediated by AQP4, supporting the idea of an important and constant CSF production/absorption process, sustained by efflux/influx of water between brain capillaries and interstitial fluid. Moreover, our results suggest the participation of AQPs in structural functions also related with CSF homeostasis such as the distensibility capacity of the ventricular system.

Aquaporin-4, present in ependymal cells, in glia limiting and abundantly in pericapillary astrocyte foot pro cesses, and aquaporin-1, expressed in choroid plexus epithelial cells, play an important role in cerebrospinal fluid production and may be involved in the pathophysiology of age-dependent hydrocephalus. The finding that brain aquaporins expression is regulated by low oxygen tension led us to investigate how hypoxia and elevated levels of cerebral aquaporins may result in an increase in cerebrospinal fluid production that could be associated with a hydrocephalic condition. Here we have explored, in young and aged mice exposed to hypoxia, whether aquaporin-4 and aquaporin-1 participate in the development of age-related hydrocephalus. Choroid plexus, striatum, cortex and ependymal tissue were analyzed separately both for mRNA and protein levels of aquaporins. Furthermore, parameters such as total ventricular volume, intraventricular pressure, cerebrospinal fluid outflow rate, ventricular compliance and cognitive function were studied in wild type, aquaporin-1 and aquaporin-4 knock-out animals subjected to hypoxia or normoxia. Our data demonstrate that hypoxia is involved in the development of age-related hydrocephalus by a process that depends on aquaporin-4 channels as a main route for cerebrospinal fluid movement. Significant increases in aquaporin-4 expression that occur over the course of animal aging, together with a reduced cerebrospinal fluid outflow rate and ventricular compliance, contribute to produce more severe hydrocephalus related to hypoxic events in aged mice, with a notable impairment in cognitive function. These results indicate that physiological events and/or pathological conditions presenting with cerebral hypoxia/ischemia contribute to the development of chronic adult hydrocephalus.

Background: The use of long-term immunosuppressive treatments on neural trans plantation has been controversial during the last decades. Although nowadays there is a consensus about the necessity of maintaining a permanent state of immunosup pression to preserve the survival of cerebral grafts, little is known about the effects that chronic immunosuppression produces both on the neurodegenerative process and on transplants function. Methods: Here, we establish a new immunosuppressive protocol, based on the dis continuous administration of CsA (15 mg/kg; s.c.) and prednisone (20 mg/kg; s.c.), to produce long-term immunosuppression in mice. Using this treatment, we analyse the effects that long-term immunosuppression induces in a chronic 1-methyl-4-phenyl-1, 2,3,6,-tetrahydropyridine (MPTP) model of parkinsonism and on the neuroprotective and neurorestorative anti-parkinsonian actions exerted by rat carotid body (CB) xenografts. Results: This protocol preserves the survival of rat CB xenotransplants maintaining the general wellness of the grafted mice. Although permanent immunosuppression does not prevent the MPTP-induced cell death of nigral neurons and the consequent degeneration of dopaminergic striatal innervation, allowing for its use as Parkinson’s disease (PD) model, it reduces the microglial activation and slightly declines the stri atal damage. Moreover, we reported that chronic administration of immunosuppres sant drugs does not alter the neuroprotective and restorative anti-parkinsonian actions of rat CB xenografts into parkinsonian mice. Conclusions: This new immunosuppressive protocol provides a new murine model to assay the long-term effects of cerebral xenografts and offer a pharmacological alternative to the commonly used genetic immunodeficient mice, allowing the use of genetically modified mice as hosts. In addition, it will permit the experimental analysis of the effects produced by human CB xenografts in the chronic PD murine model, with t

Los trasplantes intraestriatales de cuerpo carotídeo (CC) han mostrado un claro efecto beneficioso en modelos animales de parkinsonismo, ejerciendo una acción trófica, mediada por la liberación del factor neurotrófico derivado de la glía (GDNF), sobre las neuronas dopaminérgicas de la vía nigrostriatal. El autotrasplante de CC en pacientes parkinsonianos produce un cierto efecto beneficioso, aunque de menor cuantía que el observado en modelos experimentales y condicionado por la edad del tejido implantado. Resultados recientes del grupo de investigación revelan que la expresión de GDNF del implante de CC se ve afectada por la edad del tejido implantado, limitando la eficacia terapéutica de los trasplantes de CC procedentes de donantes viejos. Estos resultados aportan una explicación a la relación observada entre la eficacia terapéutica y la edad de los pacientes parkinsonianos autotrasplantados. Por tanto, era necesario establecer un protocolo de inmunosupresión que permitiera el estudio experimental de las acciones ejercidas por xenotrasplantes de CC en el modelo crónico de parkinsonismo murino. En esta tesis doctoral se ha establecido un protocolo de inmunosupresión que garantiza la supervivencia del xenotrasplante y permite mantener los ratones inmunosuprimidos por un periodo prolongado de tiempo en buenas condiciones de bienestar animal, y por lo tanto es compatible con nuestro modelo crónico de parkinsonismo. También se ha determinado que dicho tratamiento inmunosupresor reduce la neuroinflamación asociada al parkinsonismo, mejora la inervación dopaminérgica y no altera la actividad neurotrófica de los xenotrasplantes de CC. Por último, se ha analizado el efecto que produce el implante de CC sobre las células gliales, comprobando que estos trasplantes producen activación microglial y reclutamiento de oligodendrocitos en el estriado trasplantado. El protocolo de inmunosupresión desarrollado en esta tesis será fundamental para que el laboratorio pueda ensayar la

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.

Affecting over 1.5 million people across the world, Parkinson's disease is a progressive neurological condition characterized, in part, by the loss of dopaminergic neurons in the substantia nigra pars compacta. It affects 1.5% of the global population over 65 years of age. As life expectancy is increasing, over the next few years the number of patients with Parkinson´s disease will grow exponentially. To date, there are no available treatments that are capable of curing Parkinson´s disease, and the current goal of therapy, dopamine replacement strategies, is to reduce symptoms. After several years of disease progression, treatment is complicated by the onset of motor fluctuations and dyskinesias. This information reveals the great importance and social need of finding an effective therapeutic intervention for Parkinson´s disease. This exemplary new book reviews some of the most outstanding examples of new drugs currently in pharmaceutical development or new targets currently undergoing the validation process to try to reach the Parkinson´s drug market in the next few years as potential disease modifying drugs. Providing up to date and comprehensive coverage, this book will be essential reading for researchers working in academia and industry in any aspect of medicinal chemistry or drug discovery.

El proyecto que se desarrolla en esta Tesis Doctoral se ha centrado en el estudio de los mecanismos celulares y moleculares por los que el trasplante de cuerpo carotídeo (CC) produce recuperación en modelos experimentales de la enfermedad de Parkinson (EP), con especial interés en la expresión del factor neurotrófico GDNF (del inglés “glial cell line derived neurotrophic factor”; Lin y col., 1993) en dicho órgano. Este trabajo surge a partir de estudios previos realizados en el grupo de investigación, en los que se observó que el implante intraestrial de agregados celulares de CC produce una importante recuperación histológica y funcional en modelos animales de parkinsonismo (Espejo y col., 1998; Luquin y col., 1999; Toledo-Aral y col., 2003). A partir de estos resultados se ha propuesto el uso del tejido carotídeo en terapia celular en la EP (Arjona y col., 2003; Mínguez-Castellanos y col., 2007), así como en otras alteraciones del sistema nervioso central (Yu y col., 2005). El objetivo general de esta Tesis Doctoral fue profundizar en el conocimiento de la expresión de GDNF en el cuerpo carotídeo, así como en las consecuencias que dicha expresión pueda tener en el uso de este órgano en terapia celular. A continuación se detallas los principales objetivos específicos. 1. Estudiar la selectividad de la expresión de GDNF en el cuerpo carotídeo, en comparación con otros tejidos catecolaminérgicos usados en trasplantes realizados en enfermos de Parkinson. Determinar dentro del cuerpo carotídeo el tipo celular que expresa el GDNF. 2. Analizar la capacidad de las células del cuerpo carotideo para secretar GDNF y compararla con otros tipos celulares dopaminérgicos. 3. Examinar si la alta expresión de GDNG observada en el cuerpo carotídeo, se mantiene una vez trasplantado al estriado. Comprobar si el implante intraestriatal de cuerpo carotídeo induce la expresión de GDNF en el parénquima cerebral o en macrófagos activados. 4. Evaluar en el modelo experimental de ratón, si

Glial cell line-derived neurotrophic factor (GDNF) exerts a notable protective effect on dopaminergic neurons in rodent and primate models of Parkinson’s 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 insomePDpatients. Moreover, the adult rodentCBhas 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 ofGDNFin the adult rodent (as measured by standard and in situ ELISA). Furthermore,GDNFexpression 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.