Changing the intrinsic growth capacity of motor and sensory neurons to promote axonal growth after injury : the role of FGF2 in axonal regeneration /

Les lesions dels nervis perifèrics provoquen paràlisis, anestèsia i pèrdua del control autonòmic de la zona afectada. Després de lesió, la part distal dels axons queda desconectat del soma i degenera, provocant la denervació dels òrgans diana. La degeneració walleriana crea un microambient favorable per al creixement axonal, alhora que la neurona canvia a un fenotip proregeneratiu. Malauradament, la manca d'especificitat de la regeneració, en termes de creixement motor i sensorial i reinnervació, és un dels grans limitats de la recuperació. Els mecanismes moleculars mplicats en la regeneració axonal i després de lesió són complexes i les interaccions entre els axons i la glia, els factors tròfics, la matriu extracel·lular i els seus receptors són fonamentals. Per aquestes raons, hem caracteritzat un model qeu ens permet comparar sota les mateixes condicions, creixement neurític motor i sensorial. Hem posat a punt un model in vitro, basat en cultius organotípics de medul·la espinal i explants de ganglis de les arrels dorsals de rates postnatals de 7 dies, embeguts en una matriu de col·lagen. Afegitn difernets factors tròfics a la matriu, hem avaluat la fiabilitat de les preparacions de gangli i de medul·la espinal. A més a més, també hem posat a punt un co-cultiu amb cèl·lules de Schwann dissociades que mimetizen millor l'ambient permissiu del nervi perifèric. Amb aquest model, hem analitzat els efectes de diferents factors tròfics que potencialment podien afavorir la especificitat de la regeneració, i com aquests factors podien ser sobre-regulats de manera diferencial per branques de nervis motors i sensorials després de la lesió. Hem observat que l'FGF-2 (18 kDa) és el factor tròfic que exerceix un efecte més selectiu en el creixement de les motoneurones espinals, tant a nivell d'elongació com d'arborització de neurites. El mecanisme que provocaia aquest efecte sembla estar relacionat amb la capacitat de l'FGF-2 d'incrementar la interacció entre l'FGFR-1 i el PSA-NCAM. Les interaccions dels dos receptors són importants durant els estadis més primerencs de la neuritogènesis, mentres que la subunitat alfa7B de les integrines estaria més relacionada amb l'estabilització de les neurites. Amb l'objectiu d'explorar amb més detall la potencial habilitat de l'FGF-2 de promoure de manera selectiva la regeneració in vivo, hem produit un vector lentiviral (LV) que sobreexpressa FGF-2 i l'hem caracteritzat in vitro i in vivo. L'addició de cèl·lules de Schwann infectades amb el LV-FGF2 en la matriu de col·lagen que cobreix els explants de gangli o les medul·les espinals, incrementa el creixement de les neurites motores però no de les sensorials en comparació als co-cultius amb LV-GFP. Per tant, la sobreexpressió de l'FGF2 mitjançant el LV és tan eficaç com l'addició directe del factor en la matriu en la promoció selective de la regeneració motora. Quan el LV-FGF2 es va injectar directament al nervi ciàtic in vivo, vam corroborar que l'FGF2 se secretava a nivell de la lamina basal de les cèl·lules de Schwann. Els nivells de FGF-2 en els homogentats de nervi ciàtic una setmana després d'injectar 1μl LVFGF- 2 eren més alts que els dels nervis injectats amb vehicle o LV-GFP. Per tant, el vector LV pot ser utilitzat in vivo per tal de verificar les troballes in vitro i per investigar amb més detall la capacitate de l'FGF2 de promoure regeneració motora. En aquest treball també hem comparat la capacitat de la glia embolcalladora olfactiva i les cèl·lules de Schwann, en donar suport a la regeneració in vitro de neurites motores i sensorials. En els co-cultius de cèl·lules de Schwann i els explants de gangli i medul·les espinals, s'observava un increment de la regeneració motora, menters que la glia embolcalladora incrementava signifiativament el creixement neurític de les neurones sensorials. Per contra, quan la glia embolcalladora s'afegia al cultiu motor, s'observava una agregació d'aquestes cèl·lules. El comportament de la glia embolcalladora podria estar determinat pel manteniment de la citoarquitectura de les medul·les espinals, on trobem astròcits i cèl·lules Schwann endògenes. Les interaccions entre la cèl·lula de Schwnn, la glia olafctòria i els astròcits, a través del complexe FGFR1-FGF2-HSPG, poden provocar agregació cel·lular. De fet, els nivells elevats d'HSPG van detectar-se al costat de la barrera, i això pot explicar el paper complex d'aquestes neurones. Els nivells elevats d HSPG és van detecar a la zona de lesió , i això pot explicar el paper quimio-repelent dels agregats cel·lulars.Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. However, the lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. For these reasons, we characterized a model that allows us to compare under the same conditions motor and sensory neuron regeneration. We set up an in vitro model, based on organotypic cultures of spinal cord slices and dorsal root ganglia explants from P7 rats, embedded in a collagen matrix. By adding different neurotrophic factors in the collagen matrix, we evaluated the reliability of DRG and spinal cord preparations. Moreover, we also set up a co-culture with dissociated Schwann cells to further mimic the permissive environment of the peripheral nerve. Later, we screened in vitro the different capabilities of trophic factors with promising effect on specific reinnervation of target organs after peripheral nerve regeneration. Trophic factors which promoted in vitro neuritogenesis of sensory and motor neurons were up-regulated in Schwann cells obtained from axotomized sensory and motor branches respectively. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underling this effect in neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization. With the aim to further explore the potential capacity of FGF-2 to selectiveley promote motor regeneration in vivo, we produced a lentiviral (LV) vector to overexpress FGF-2 and we characterized it in vitro and in vivo. Addition of cultured Schwann cells infected with FGF-2 into a collagen matrix embedding spinal cords or DRG significantly increased motor neurite growth but not sensory outgrowth when compared to co-cultures with LV-GFP, thus demonstrating that the LV construct was as effective as direct addition of the trophic factor to selectively promote motor neuron growth. By injecting the LV construct direclty into the sciatic nerve in vivo, we corroborated the localization of the secreted FGF-2 in the basal lamina of Schwann cells. Levels of FGF-2 from homogenated sciatic nerves one week after injection of 1μl LV-FGF-2 were higher than from nerves injected with vehicle or LV-GFP. Therefore, the LV vector can be used in vivo to verify our in vitro results and further study the capacity of FGF-2 to enhance motor nerve regeneration. In the last part of our work, we compare the abilities of Olfactory Enshealting cells and Schwann cells in sustaining in vitro motor and sensory neuritogenesis. Co-culture of cells with DRG explants and spinal cord organotypic slices was set up. SCs were promoting motoneuron growth, whereas OEC were significantly increasing neurite outgrowth in DRGs. In contrast, when OEC were added into motoneuron culture, we saw cell clusters and motoneuron outgrowth inhibition. This behaviour of OEC could be due to the maintained cytoarchitecture of the spinal cord in vitro where astrocytes and endogenous Schwann cells were also present. Interactions of SC, OEC and astrocytes through FGFR1-FGF2-HSPG complex can cause cell clustering. In fact, high levels of HSPG were found into the boundary formations, and this can explain the chemorepellent role of the cluster on neurite outgrowth

In-Depth NMR Investigation of the Magnetic Hardening in Co Thin Films Induced by the Interface with Molecular Layers

The hybridization of the surface orbitals of thin ferromagnetic layers with molecular orbitals represents a soft but efficient technology that is able to induce in ferromagnetic component radical modifications of the key magnetic parameters, such as magnetization, magnetic anisotropy, and others. These effects are investigated in 7 nm thick polycrystalline Co films interfaced with C-60 and Gaq(3) molecular layers by combining Co-59 Ferromagnetic nuclear resonance spectroscopy (FNR) and magneto-optic kerr effect (MOKE) techniques. It is demonstrated that the surface hybridization produces a significant magnetic hardening with respect to a reference Co/Al system and that the molecule-induced effects modify the magnetic properties of entire Co layer, propagating for several nm from the interface. The FNR spectroscopy also reveals a reconstruction of the magnetic environment at the cobalt surface, whose observation in polycrystalline films is especially intriguing. The results shed new and unexpected light on the interfacial physics in such systems, whose understanding necessitates further experimental and theoretical research

Nanoscale phase separation in manganites

We study the possibility of nanoscale phase separation in manganites in the framework of the double exchange model. The homogeneous canted state of this model is proved to be unstable toward the formation of small ferromagnetic droplets inside an antiferromagnetic insulating matrix. For the ferromagnetic polaronic state we analyze the quantum effects related to the tails of electronic wave function and a possibility of electron hopping in the antiferromagnetic background. We find that these effects lead to the formation of the threshold for the polaronic state.Comment: 10 pages, 2 figures, invited talk on the workshop on Strongly Correlated Electrons in New Materials (SCENM02), Loughborough (UK). submitted to Journal of Physics A: Mathematical and Genera

Resistivity and 1/f Noise in Non-Metallic Phase Separated Manganites

A simple model is proposed to calculate resistivity, magnetoresistance, and noise spectrum in non-metallic phase-separated manganites containing small metallic droplets (magnetic polarons). The system is taken to be far from the percolation transition into a metallic state. It is assumed that the charge transfer occurs due to electron tunneling from one droplet to another through the insulating medium. As a result of this tunneling, the droplets acquire or lose extra electrons forming metastable two-electron and empty states. In the framework of this model, explicit expressions for dc conductivity and noise power of the system are derived. It is shown that the noise spectrum has 1/f form in the low-frequency range.Comment: 6 pages, 1 fugure include

Effective conductivity of 2D isotropic two-phase systems in magnetic field

Using the linear fractional transformation, connecting effective conductivities sigma_{e} of isotropic two-phase systems with and without magnetic field, explicit approximate expressions for sigma_{e} in a magnetic field are obtained. They allow to describe sigma_{e} of various inhomogeneous media at arbitrary phase concentrations x and magnetic fields. the x-dependence plots of sigma_e at some values of inhomogeneity and magnetic field are constructed. Their behaviour is qualitatively compatible with the existing experimental data. The obtained results are applicable for different two-phase systems (regular and nonregular as well as random), satisfying the symmetry and self-duality conditions, and admit a direct experimental checking.Comment: 9 pages, 2 figures, Latex2e, small corrections and new figure

Percolative conductivity and critical exponents in mixed-valent manganites

Recent experiments have shown that some colossal magnetoresistance (CMR) materials exhibit a percolation transition. The conductivity exponent varies substantially with or without an external magnetic field. This finding prompted us to carry out theoretical studies of percolation transition in CMR systems. We find that the percolation transition coincides with the magnetic transition and this causes a large effect of a magnetic field on the percolation transition. Using real-space-renormalization method and numerical calculations for two-dimensional (2D) and three-dimensional (3D) models, we obtain the conductivity exponent $t$ to be 5.3 (3D) and 3.3 (2D) without a magnetic field, and 1.7 (3D) and 1.4 (2D) with a magnetic field.Comment: 4 pages, 4 figures. To appear in Rapid Communications of Phys. Rev.

Synaptotagmin 13 is neuroprotective across motor neuron diseases

In amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), spinal and lower brainstem motor neurons degenerate, but some motor neuron subtypes are spared, including oculomotor neurons (OMNs). The mechanisms responsible for this selective degeneration are largely unknown, but the molecular signatures of resistant and vulnerable motor neurons are distinct and offer clues to neuronal resilience and susceptibility. Here, we demonstrate that healthy OMNs preferentially express Synaptotagmin 13 (SYT13) compared to spinal motor neurons. In end-stage ALS patients, SYT13 is enriched in both OMNs and the remaining relatively resilient spinal motor neurons compared to controls. Overexpression of SYT13 in ALS and SMA patient motor neurons in vitro improves their survival and increases axon lengths. Gene therapy with Syt13 prolongs the lifespan of ALS mice by 14% and SMA mice by 50% by preserving motor neurons and delaying muscle denervation. SYT13 decreases endoplasmic reticulum stress and apoptosis of motor neurons, both in vitro and in vivo. Thus, SYT13 is a resilience factor that can protect motor neurons and a candidate therapeutic target across motor neuron diseases

Continuum Double Exchange Model

We present a continuum model for doped manganites which consist of two species of quantum spin 1/2 fermions interacting with classical spin fields. The phase structure at zero temperature turns out to be considerably rich: antiferromagnetic insulator, antiferromagnetic two band conducting, canted two band conducting, canted one band conducting and ferromagnetic one band conducting phases are identified, all of them being stable against phase separation. There are also regions in the phase diagram where phase separation occurs.Comment: 14 pages, LaTeX2e file, two eps included figures. Published versio

``Flux'' state in double exchange model

We study the ground state properties of the double-exchange systems. The phase factor of the hopping matrix elements arises from $t_{2g}$ spin texture in two or more dimensions. A novel ``flux'' state is stabilized against the canted antiferromagnetic and spiral spin states. In a certain range of hole doping, the phase separation occurs between the ``flux'' state and antiferromagnetic states. Constructing a trial state which provides the rigorous upper bound on the ground state, we show that the metallic canted antiferromagnetic state is not stable in the double exchange model.Comment: REVTEX, 8 pages and 4 PS figure

Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3\u3b2 phosphorylation and \u3b2-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1G93A ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration