55 research outputs found
Locomotor deficits in a mouse model of ALS are paralleled by loss of V1-interneuron connections onto fast motor neurons
Funding: This work was supported by the Lundbeck Foundation (I.A.), the Björklund foundation (I.A.), the A.P. Møller foundation (I.A.), the Novo Nordisk Laureate Program (O.K., NNF15OC0014186), The Lundbeck Foundation (O.K.), the Louis-Hansen foundation (R.M.R.) and The Faculty of Health and Medical Sciences (O.K.).ALS is characterized by progressive inability to execute movements. Motor neurons innervating fast-twitch muscle-fibers preferentially degenerate. The reason for this differential vulnerability and its consequences on motor output is not known. Here, we uncover that fast motor neurons receive stronger inhibitory synaptic inputs than slow motor neurons, and disease progression in the SOD1G93A mouse model leads to specific loss of inhibitory synapses onto fast motor neurons. Inhibitory V1 interneurons show similar innervation pattern and loss of synapses. Moreover, from postnatal day 63, there is a loss of V1 interneurons in the SOD1G93A mouse. The V1 interneuron degeneration appears before motor neuron death and is paralleled by the development of a specific locomotor deficit affecting speed and limb coordination. This distinct ALS-induced locomotor deficit is phenocopied in wild-type mice but not in SOD1G93A mice after appearing of the locomotor phenotype when V1 spinal interneurons are silenced. Our study identifies a potential source of non-autonomous motor neuronal vulnerability in ALS and links ALS-induced changes in locomotor phenotype to inhibitory V1-interneurons.Publisher PDFPeer reviewe
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
Modeling Motor Neuron Resilience in ALS Using Stem Cells
Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Toward this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of PHOX2A in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro-generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro that display a resilience similar to that seen in vivo.</p
Changing the Intrinsic Growth Capacity of motor and sensory neurons to promote axonal growth after injury
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
Directed midbrain and spinal cord neurogenesis from pluripotent stem cells to model development and disease in a dish
Induction of specific neuronal fates is restricted in time and space in the developing CNS through integration of extrinsic morphogen signals and intrinsic determinants. Morphogens impose regional characteristics on neural progenitors in a concentration-dependent fashion and establish distinct progenitor domains. Such domains are defined by unique expression patterns of fate determining transcription factors. These processes of neuronal fate specification can be recapitulated in vitro using pluripotent stem cells. In this review, we focus on the generation of dopamine neurons and motor neurons, which are induced at ventral positions of the neural tube through Sonic hedgehog (Shh) signaling, and defined at anteroposterior positions by fibroblast growth factor (Fgf) 8, Wnt1, and retinoic acid (RA). In vitro utilization of these morphogenic signals typically results in the generation of multiple neuronal cell types, which are defined at the intersection of these signals. If the purpose of in vitro neurogenesis is to generate one cell type only, further lineage restriction can be accomplished by forced expression of specific transcription factors in a permissive environment. Alternatively, cell-sorting strategies allow for selection of neuronal progenitors or mature neurons. However, modeling development, disease and prospective therapies in a dish could benefit from structured heterogeneity, where desired neurons are appropriately synaptically connected and thus better reflect the three-dimensional structure of that region. By modulating the extrinsic environment to direct sequential generation of neural progenitors within a domain, followed by self-organization and synaptic establishment, a reductionist model of that brain region could be created. Here we review recent advances in neuronal fate induction in vitro, with a focus on the interplay between cell intrinsic and extrinsic factors, and discuss the implications for studying development and disease in a dish
Neural circuit and synaptic dysfunctions in ALS-FTD pathology
Action selection is a capital feature of cognition that guides behavior in processes that range from motor patterns to executive functions. Here, the ongoing actions need to be monitored and adjusted in response to sensory stimuli to increase the chances of reaching the goal. As higher hierarchical processes, these functions rely on complex neural circuits, and connective loops found within the brain and the spinal cord. Successful execution of motor behaviors depends, first, on proper selection of actions, and second, on implementation of motor commands. Thus, pathological conditions crucially affecting the integrity and preservation of these circuits and their connectivity will heavily impact goal-oriented motor behaviors. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders known to share disease etiology and pathophysiology. New evidence in the field of ALS-FTD has shown degeneration of specific neural circuits and alterations in synaptic connectivity, contributing to neuronal degeneration, which leads to the impairment of motor commands and executive functions. This evidence is based on studies performed on animal models of disease, post-mortem tissue, and patient derived stem cells. In the present work, we review the existing evidence supporting pathological loss of connectivity and selective impairment of neural circuits in ALS and FTD, two diseases which share strong genetic causes and impairment in motor and executive functions
Neural circuit and synaptic dysfunctions in ALS-FTD pathology
Funding: This work was supported by the Lundbeck Foundation (IA) (Grant No. R346-2020-202) and the Department of Neuroscience, University of Copenhagen and School of Psychology and Neuroscience, University of St Andrews.Action selection is a capital feature of cognition that guides behavior in processes that range from motor patterns to executive functions. Here, the ongoing actions need to be monitored and adjusted in response to sensory stimuli to increase the chances of reaching the goal. As higher hierarchical processes, these functions rely on complex neural circuits, and connective loops found within the brain and the spinal cord. Successful execution of motor behaviors depends, first, on proper selection of actions, and second, on implementation of motor commands. Thus, pathological conditions crucially affecting the integrity and preservation of these circuits and their connectivity will heavily impact goal-oriented motor behaviors. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders known to share disease etiology and pathophysiology. New evidence in the field of ALS-FTD has shown degeneration of specific neural circuits and alterations in synaptic connectivity, contributing to neuronal degeneration, which leads to the impairment of motor commands and executive functions. This evidence is based on studies performed on animal models of disease, post-mortem tissue, and patient derived stem cells. In the present work, we review the existing evidence supporting pathological loss of connectivity and selective impairment of neural circuits in ALS and FTD, two diseases which share strong genetic causes and impairment in motor and executive functions.Publisher PDFPeer reviewe
Directed midbrain and spinal cord neurogenesis from pluripotent stem cells to model development and disease in a dish
Induction of specific neuronal fates is restricted in time and space in the developing CNS through integration of extrinsic morphogen signals and intrinsic determinants. Morphogens impose regional characteristics on neural progenitors and establish distinct progenitor domains. Such domains are defined by unique expression patterns of fate determining transcription factors. These processes of neuronal fate specification can be recapitulated in vitro using pluripotent stem cells. In this review, we focus on the generation of dopamine neurons and motor neurons, which are induced at ventral positions of the neural tube through Sonic hedgehog (Shh) signaling, and defined at anteroposterior positions by fibroblast growth factor (Fgf) 8, Wnt1, and retinoic acid (RA). In vitro utilization of these morphogenic signals typically results in the generation of multiple neuronal cell types, which are defined at the intersection of these signals. If the purpose of in vitro neurogenesis is to generate one cell type only, further lineage restriction can be accomplished by forced expression of specific transcription factors in a permissive environment. Alternatively, cell-sorting strategies allow for selection of neuronal progenitors or mature neurons. However, modeling development, disease and prospective therapies in a dish could benefit from structured heterogeneity, where desired neurons are appropriately synaptically connected and thus better reflect the three-dimensional structure of that region. By modulating the extrinsic environment to direct sequential generation of neural progenitors within a domain, followed by self-organization and synaptic establishment, a reductionist model of that brain region could be created. Here we review recent advances in neuronal fate induction in vitro, with a focus on the interplay between cell intrinsic and extrinsic factors, and discuss the implications for studying development and disease in a dish.</p
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