Juan José Toledo-Aral

LRRK2 G2019S mutation is the most common genetic cause of Parkinson's disease (PD) producing clinical manifestations similar to sporadic PD patients, hinting at the relevance of this mutation in the pathophysiological mechanisms of the disease. However, its role potentiating nigrostriatal neurodegeneration, particularly in response to mitochondrial dysfunction, remains unclear. Here, using histological, neurochemical and behavioral analyzes, we provide a complete characterization of a mouse model expressing the human LRRK2 G2019S mutation (hLRRK2*G2019S mice), in the context of aging and toxin‐induced parkinsonism. hLRRK2*G2019S young adult mice showed significant microglial activation and deficits in motor coordination, whereas aged transgenic displayed a hyperactive phenotype, despite the absence of evidence for nigrostriatal neurodegeneration in either young adult or aged animals. Furthermore, we assessed the vulnerability of LRRK2*G2019S transgenic mice to the chronic exposure to the mitochondrial complex I inhibitor 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Notably, no exacerbation of nigrostriatal neurodegeneration was observed in hLRRK2*G2019S mice, suggesting that this mutation does not increase sensitivity to mitochondrial stress. However, hLRRK2*G2019S MPTP‐treated mice present alterations, relative to WT counterparts, in the glial response induced by the neurotoxic treatment. These findings suggest that the G2019S mutation modulates the neuroimmune response but does not exacerbate nigrostriatal neurodegeneration in response to mitochondrial dysfunction. This highlights the mutation's complex role in PD pathophysiology. image

The development and survival of dopaminergic neurons are influenced by the fibroblast growth factor (FGF) pathway. Anosmin-1 (A1) is an extracellular matrix protein that acts as a major regulator of this signaling pathway, controlling FGF diffusion, and receptor interaction and shuttling. In particular, previous work showed that A1 overexpression results in more dopaminergic neurons in the olfactory bulb. Prompted by those intriguing results, in this study, we investigated the effects of A1 overexpression on different populations of catecholaminergic neurons in the central (CNS) and the peripheral nervous systems (PNS). We found that A1 overexpression increases the number of dopaminergic substantia nigra pars compacta (SNpc) neurons and alters the striosome/matrix organization of the striatum. Interestingly, these numerical and morphological changes in the nigrostriatal pathway of A1-mice did not confer an altered susceptibility to experimental MPTP-parkinsonism with respect to wild-type controls. Moreover, the study of the effects of A1 overexpression was extended to different dopaminergic tissues associated with the PNS, detecting a significant reduction in the number of dopaminergic chemosensitive carotid body glomus cells in A1-mice. Overall, our work shows that A1 regulates the development and survival of dopaminergic neurons in different nuclei of the mammalian nervous system.

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.

The development and survival of dopaminergic neurons are influenced by the fibroblast growth factor (FGF) pathway. Anosmin-1 (A1) is an extracellular matrix protein that acts as a major regulator of this signaling pathway, controlling FGF diffusion, and receptor interaction and shuttling. Furthermore, overexpression of A1 in vivo gives rise to higher number of dopaminergic neurons in the olfactory bulb. Here, using A1 overexpressing mice (A1-mice), we studied the effects of A1 on different populations of catecholaminergic neurons in the central (CNS) and the peripheral nervous systems (PNS). A1 overexpression increases the number of dopaminergic SNpc neurons and alters the striosome/matrix organization of the striatum. Interestingly, these numerical and morphological changes in the nigrostriatal pathway of A1-mice do not confer an altered susceptibility to experimental MPTP-parkinsonism with respect to wild type controls. Moreover, the study of the effects of A1 overexpression was extended to different dopaminergic tissues associated with the PNS, detecting a significant reduction in the number of dopaminergic chemosensitive carotid body glomus cells in A1-mice. Overall, these analyses confirm A1 as a principal regulator of the FGF pathway in the development and survival of dopaminergic neurons in different nuclei of the mammalian nervous system.

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.

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.

Background: The use of long‐term immunosuppressive treatments on neural transplantation has been controversial during the last decades. Although nowadays there is a consensus about the necessity of maintaining a permanent state of immunosuppression 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 discontinuous 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 striatal damage. Moreover, we reported that chronic administration of immunosuppressant 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 the final aim of using CB allografts as an option of cell therapy in PD patients.

Synaptic protein α-synuclein (α-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinson′s disease. Here, we report that α-SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also show that premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking α-SYN resembles the effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic α-SYN. Interestingly, NSC loss in α-SYN-deficient mice can be prevented by viral delivery of human α-SYN into their sustantia nigra or by treatment withl-DOPA, suggesting that α-SYN regulates dopamine availability to NSCs. Our data indicate that α-SYN, present in dopaminergic nerve terminals supplying the subependymal zone, acts as a niche component to sustain the neurogenic potential of adult NSCs and identify α-SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain.SIGNIFICANCE STATEMENTWe report an essential role for the protein α-synuclein present in dopaminergic nigral afferents in the regulation of adult neural stem cell maintenance, identifying the first synaptic regulator with an implication in stem cell niche biology. Although the exact role of α-synuclein in neural transmission is not completely clear, our results indicate that it is required for stemness and the preservation of neurogenic potential in concert with dopamine.

Neuroinflammation mediated by chronically activated microglia, largely caused by abnormal accumulation of misfolded α‐synuclein (αSyn) protein, is known to contribute to the pathophysiology of Parkinson's disease (PD). In this work, based on the immunomodulatory activities displayed by particular heat‐shock proteins (HSPs), we tested a novel vaccination strategy that used a combination of αSyn and Grp94 (HSPC4 or Gp96) chaperone and a murine PD model. We used two different procedures, first, the adoptive transfer of splenocytes from αSyn/Grp94‐immunized mice to recipient animals, and second, direct immunization with αSyn/Grp94, to study the effects in a chronic mouse MPTP‐model of parkinsonism. We found that both approaches promoted a distinct profile in the peripheral system—supported by humoral and cellular immunity—consisting of a Th1‐shifted αSyn‐specific response accompanied by an immune‐regulatory/Th2‐skewed general phenotype. Remarkably, this mixed profile sustained by αSyn/Grp94 immunization led to strong suppression of microglial activation in the substantia nigra and striatum, pointing to a newly described positive effect of anti‐αSyn Th1‐responses in the context of PD. This strategy is the first to target αSyn and report the suppression of PD‐associated microgliosis. Overall, we show that the αSyn/Grp94 combination supports a distinct and long‐lasting immune profile in the peripheral system, which has an impact at the CNS level by suppressing chronic microglial activation in an MPTP model of PD. Furthermore, our study demonstrates that reshaping peripheral immunity by vaccination with appropriate misfolding protein/HSP combinations could be highly beneficial as a treatment for neurodegenerative misfolding diseases.