Terapia celular y recuperación de lesiones cerebrales

En este seminario repasaremos diferentes tipos de células madre que se están aplicando en el tratamiento de la isquemia cerebral o traumatismos craneoencefálicos. Por otra parte, también mostraré resultados de nuestro laboratorio en donde tratamos de desarrollar una terapia celular para el tratamiento de encefalopatías epilépticas infantiles, como el Síndrome de West. Con ello pretendo dar una visión general de este prometedor e interesante campo biomédico.El cerebro humano es probablemente el órgano de mayor complejidad que se conoce. Por ello, su reparación es el reto más complicado y apasionante al que se enfrenta la Medicina Regenerativa. Las aproximaciones terapéuticas basadas en células han emergido con fuerza en las últimas décadas y suponen una gran esperanza. No obstante, para aplicarlas con éxito al sistema nervioso se requiere de una fuente adecuada de células madre que puedan mediar una mejora, ya sea mediante mecanismos de reemplazo celular, o por secreción de factores tróficos

Cryopreservation of GABAergic neuronal precursors for cell-based therapy

Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2 SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Postthaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies.Ministerio de Economía y Competitividad 09-07746 y 12-36853Junta de Andalucía CTS- 256

A 32-Channel Time-Multiplexed Artifact-Aware Neural Recording System

This paper presents a low-power, low-noise microsystem for the recording of neural local field potentials or intracranial electroencephalographic signals. It features 32 time-multiplexed channels at the electrode interface and offers the possibility to spatially delta encode data to take advantage of the large correlation of signals captured from nearby channels. The circuit also implements a mixed-signal voltage-triggered auto-ranging algorithm which allows to attenuate large interferers in digital domain while preserving neural information. This effectively increases the system dynamic range and avoids the onset of saturation. A prototype, fabricated in a standard 180 nm CMOS process, has been experimentally verified in-vitro with cellular cultures of primary cortical neurons from mice. The system shows an integrated input-referred noise in the 0.5–200 Hz band of 1.4 µVrms for a spot noise of about 85 nV / √Hz. The system draws 1.5 µW per channel from 1.2 V supply and obtains 71 dB + 26 dB dynamic range when the artifact-aware auto-ranging mechanism is enabled, without penalising other critical specifications such as crosstalk between channels or common-mode and power supply rejection ratios

Transplant of GABAergic Precursors Restores Hippocampal Inhibitory Function in a Mouse Model of Seizure Susceptibility

16 páginas, 8 figuras.-- Licencia Creative Commons Reconocimiento-No comercial.Defects in GABAergic function can cause epilepsy. In the last years, cell-based therapies have attempted to correct these defects with disparate success on animal models of epilepsy. Recently, we demonstrated that medial ganglionic eminence (MGE)-derived cells grafted into the neonatal normal brain migrate and differentiate into functional mature GABAergic interneurons. These cells are able to modulate the local level of GABA-mediated synaptic inhibition, which suggests their suitability for cell-based therapies. However, it is unclear whether they can integrate in the host circuitry and rescue the loss of inhibition in pathological conditions. Thus, as proof of principle, we grafted MGE-derived cells into a mouse model of seizure susceptibility caused by specific elimination of GABAergic interneuron subpopulations in the mouse hippocampus after injection of the neurotoxic saporin conjugated to substance P (SSP-Sap). This ablation was associated with significant decrease in inhibitory postsynaptic currents (IPSC) on CA1 pyramidal cells and increased seizure susceptibility induced by pentylenetetrazol (PTZ). Grafting of GFP+ MGE-derived cells in SSP-Sap-treated mice repopulates the hippocampal ablated zone with cells expressing molecular markers of mature interneurons. Interestingly, IPSC kinetics on CA1 pyramidal cells of ablated hippocampus significantly increased after transplantation, reaching levels similar to the normal mice. More importantly, this was associated with reduction in seizure severity and decrease in postseizure mortality induced by PTZ. Our data show that MGE-derived cells fulfill most of the requirements for an appropriate cell-based therapy, and indicate their suitability for neurological conditions where a modulation of synaptic inhibition is needed, such as epilepsy.This work was supported by grants from Spanish Ministry of Science and Innovation (SAF 07/61880 and FIS 07/0079), and the Regenerative Medicine Programme from CIPF. M.E.C. and I.Z. were recipients of Miguel Servet contract from Carlos III Institute (Spanish Ministry of Science and Innovation) and Ph.D. fellowship from Generalitat Valenciana, respectively.Peer reviewe

Estudio de la neurogénesis en un modelo animal de epilepsia infantil (Síndrome de West)

La neurogénesis es el proceso mediante el cual se forman las células del Sistema Nervioso Central. Hoy en día se sabe que este proceso, además de en la etapa embrionaria, se da también en el individuo adulto, gracias a la presencia de células madre en la Zona Subventricular (ZSV), Hipocampo (SGZ) y Bulbo Olfativo. En condiciones patológicas o tras una lesión se observa un aumento de la neurogénesis; así, por ejemplo, se ha observado un incremento en la neurogénesis tras sufrir un ataques epilépticos. En este proyecto estudiamos si la tasa de neurogénesis aumenta en ratones con Síndrome de West, una encefalopatía epiléptica infantil. Estos ratones, poseen una mutación recesiva ligada al sexo (cromosoma X) en el gen ARX(GCG)7, consistente en el adicionamiento de 7 alaninas, que recapitulan las características fenotípicas de la mutación en humanos, siendo esta la mas común que se encuentra en niños con Síndrome de West. En estos modelos existe una reducción de interneuronas y una inhibición de la migración desde su origen, la eminencia media ganglionar (EMG), lo que provoca una hiperactividad causada por falta de la inhibición mediada por las interneuronas y crisis epilépticas. Queremos comprobar si existe un efecto sobre los niveles de neurogénesis debido a las crisis y si este proceso se ve alterado o modificado. Para ello estamos realizando distintos ensayos inmunohistoquímicos con anticuerpos que se unen específicamente a Brdu; nucleótido sintético análogo a timidina inyectado anteriormente a estos ratones, con el que marcamos todas aquellas células que estén en división celular. Un primer análisis preliminar ha confirmado la presencia de un mayor número de células positivas para BrdU en la SGZ de ratones portadores de la mutación frente a los control. A partir de los resultados obtenidos se realizarán estudios estadísticos para ver si hay diferencias significativas en el aumento de neurogénesis y se realizarán otros marcajes para Doblecortina y Fos, que ayudarán a entender mejor las posibles alteraciones de la neurogénesis en este modelo. Comprender mejor la neurogénesis puede ayudar a desarrollar nuevas terapias conta el Síndrome de West

Transcranial static magnetic stimulation reduces seizures in a mouse model of Dravet syndrome

[Abstract] Dravet syndrome is a rare form of severe genetic epilepsy characterized by recurrent and long-lasting seizures. It appears around the first year of life, with a quick evolution toward an increase in the frequency of the seizures, accompanied by a delay in motor and cognitive development, and does not respond well to antiepileptic medication. Most patients carry a mutation in the gene SCN1A encoding the α subunit of the voltage-gated sodium channel Nav1.1, resulting in hyperexcitability of neural circuits and seizure onset. In this work, we applied transcranial static magnetic stimulation (tSMS), a non-invasive, safe, easy-to-use and affordable neuromodulatory tool that reduces neural excitability in a mouse model of Dravet syndrome. We demonstrate that tSMS dramatically reduced the number of crises. Furthermore, crises recorded in the presence of the tSMS were shorter and less intense than in the sham condition. Since tSMS has demonstrated its efficacy at reducing cortical excitability in humans without showing unwanted side effects, in an attempt to anticipate a possible use of tSMS for Dravet Syndrome patients, we performed a numerical simulation in which the magnetic field generated by the magnet was modeled to estimate the magnetic field intensity reached in the cerebral cortex, which could help to design stimulation strategies in these patients. Our results provide a proof of concept for nonpharmacological treatment of Dravet syndrome, which opens the door to the design of new protocols for treatment.Instituto de salud Carlos III; PI21/00151Xunta de Galicia; ED431C 2022/05 (CR)Ministerio de Ciencia e Innovacion (España); PID2019-108250RJ-10

CHALLENGE 6: Exposing the roots of mental disorders

Mental disorders have devastating and increasing impact in our societies. CSIC researchers face the challenge of determining the biological and social causes and consequences of these disorders, and of finding efficient therapies. To these aims, the collaborative effort of neuroscientists, neurologists, psychiatrists, psychologists and human and social scientists, the use and development of state-of-the-art technologies and the contact with patient associations and pharma industry are required.Peer reviewe

BRAIN & SPINAL CORD DAMAGE & REHABILITATION

Stroke and traumatic injury in brain or spinal cord are often life-threating conditions and major causes of death or permanent disability with high impact in the health care system. There are several stages of intervention to improve the neurological outcome. Acutely, fast interventions aiming to reestablish cerebral blood flow in ischemic stroke, to stop bleeding after brain hemorrhage, and to reduce edema after contusions are amongst mandatory actions. Current studies aim to develop accompanying strategies for brain cell protection based on enhancing endogenous protective mechanism, blocking cell death pathways, or through immunomodulation. After the acute phase, interventions are intended to promote recovery of function using rehabilitation with state-of-the-art technologies enabled by robotics. Other advanced strategies include cell, gene, and immune therapies, and brain function modulation with the aid of smart nanotechnologies. There is great expectation in the fast evolving novel approaches for improvement of neurological deficits in these unpredictable and devastating conditionPeer reviewe

GABAergic deficits in absence of LPA1 receptor, associated anxiety-like and coping behaviors, and amelioration by interneuron precursor transplants into the dorsal hippocampus

Defects in GABAergic function can cause anxiety- and depression-like behaviors among other neuropsychiatric disorders. Therapeutic strategies using the transplantation of GABAergic interneuron progenitors derived from the medial ganglionic eminence (MGE) into the adult hippocampus reversed the symptomatology in multiple rodent models of interneuron-related pathologies. In turn, the lysophosphatidic acid receptor LPA has been reported to be essential for hippocampal function. Converging evidence suggests that deficits in LPA receptor signaling represent a core feature underlying comparable hippocampal dysfunction and behaviors manifested in common neuropsychiatric conditions. Here, we first analyzed the GABAergic interneurons in the hippocampus of wild-type and maLPA-null mice, lacking the LPA receptor. Our data revealed a reduction in the number of neurons expressing GABA, calcium-binding proteins, and neuropeptides such as somatostatin and neuropeptide Y in the hippocampus of maLPA-null mice. Then, we used interneuron precursor transplants to test links between hippocampal GABAergic interneuron deficit, cell-based therapy, and LPA receptor-dependent psychiatric disease-like phenotypes. For this purpose, we transplanted MGE-derived interneuron precursors into the adult hippocampus of maLPA-null mice, to test their effects on GABAergic deficit and behavioral symptoms associated with the absence of the LPA receptor. Transplant studies in maLPA-null mice showed that grafted cells were able to restore the hippocampal host environment, decrease the anxiety-like behaviors and neutralize passive coping, with no abnormal effects on motor activity. Furthermore, grafted MGE-derived cells maintained their normal differentiation program. These findings reinforce the use of cell-based strategies for brain disorders and suggest that the LPA receptor represents a potential target for interneuron-related neuropsychiatric disorders.This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities, co-funded by the European Regional Development Fund (ERDF, EU), (PSI2017-82604R, to LJS; PSI2017-83408-P to CP; SAF09-07746, to MAD; PI16/01510, to GET) and Andalusian Regional Ministries of Economy, Knowledge, Business and University (SEJ-4515 -to LJS; SEJ1863 to CP) and of Health and Families (Nicolas Monardes Programme to GET)

CXCL12-mediated murine neural progenitor cell movement Requires PI3Kß activation

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.BLH received an FPI predoctoral fellowship (BES-2006-12965) from the Spanish Ministry of Science and Innovation. This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF 2011-27370), the RETICS Program (RD08/0075/0010, RD12/0009/0009; RIER), the Madrid regional government (S2010/BMD-2350; RAPHYME), and the European Union (FP7-integrated project Masterswitch 223404).Peer reviewe