ÁREA: NEUROCIENCIAS

III REUNIÓN ANUAL DEL ÁREA DE NEUROCIENCIAS DEL IIS-FJD

8 de junio del 2021

Grupos:Grupo de Neurología

Responsable: José M. Serratosa FernándezIP: Marina Sánchez GarcíaInvestigación: Básica y Clínica

Grupo de Psiquiatría y Salud MentalResponsable: Enrique Baca GarcíaInvestigación: Clínica

Grupo de Señalización Mitocondrial del CalcioResponsable: Jorgina Satrústegui Gil-DelgadoInvestigación: Básica

ÁREA: NEUROCIENCIAS

NOMBRE DEL GRUPO: Señalización Mitocondrial del CalcioPONENTE: Beatriz Pardo

DATOS DE CONTACTO: [email protected], [email protected],

[email protected]



8 de junio del 2021

mailto:[email protected]





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“Deficiencia en Aralar/AGC1: tratamiento con dieta

cetogénica y cuerpos cetónicos"



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Satrústegui, Pardo & del Arco,

Physiol. Rev. (2007)

AGC1/Aralar

AGC2/Citrin

Skeletal MuscleHeartBrain

LiverHeart

Aralar/AGC1/SlC25a12: Structure and activation by cytosolic Ca2+

Modified from Thangaratnarajah, C.,

Ruprecht, J. J., & Kunji, E. R. (2014)



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Aralar/Slc25a12 as the regulatory component of the

NADH malate-aspartate shuttle(MAS)



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Aralar-MAS enables glycolysis to flow regenerating cytosolic NAD+



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Aralar-MAS provides substrates to neuronal mitocondrial for respiration



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Aralar-immunogold labeling in neurons: neurons (93.1% ± 18.0%) glia (7 % ± 0.5%)

Pardo et al, JCBFM 2011

Brain Aralar-Slc25a12 is mainly/abundantly localized in neuronal mitochondria

Neuron

Oligodendrocites precursor

Oligodendrocite

Astrocyte

Brain pericyte

Bergmann glial cell

Endothelial cell (brain)

Macrophage

Microglia

Natural killer cell

B cell

Hepatocyte

Endothelial cell (liver)

Kupffer cell

Cardiac Muscle cell

Smooth Muscle cell

myofibroblast cell

fibroblast

leukocyte

endocardial cell

Endothelial cell (hearth)



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Expression of MAS components AGC (AGC1/Slc25a12; or

AGC2/Slc25a13), OGC (Slc25a11) and the enzymes (GOT1, GOT2,

MDH1, MDH2) in cells and tissues

Mitochondrial respiration is drastically reduced in Aralar-KO neurons

Llorente-Folch et al., J Neurosci. (2016) O

CR

(p

mo

l/m

in)

OC

R (

pm

ol/m

in)

Glutamate

Pyruvate recovers mitocondrial respiration in Aralar-KO neurons

Llorente-Folch et al., J Neurosci. (2016)



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Global Aralar – KO mice. ( Jalil et al., 2005;

Ramos et al., 2011)

Aralar +/+ Aralar -/-

• Short lifespan(PND20)

• Delayed development

• Hypomyelination

• Epilepsy, seizures

• Tremor, lack of motor

coordination

• Ataxic posture

• Decreased Aspartate

and NAA

Control

KO

(Juaristi et al., 2013)

Characteristics/Phenotype of Aralar-KO mice

Aralar/AGC1 deficiency: Early infantile epileptic encephalopathy 39


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Effects of Aralar-MAS deficiency on brain cells, brain tissue and whole body



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I. To identify metabolic pathways bypassing the bioenergetic déficit and thepathological phenotype in Aralar-KO mice (and humans)

II. To investigate the contribution of each celullar type in brain to the globalpathological phenotype

Aralar/AGC1 deficiency: Early infantile epileptic encephalopathy 39

Ketogenic diet (KD) : Partial recuperation- Voluntary movement- Psichomotor development- Epilepsy- Ventricle size- Myelination

Standard diet Ketogenic diet

CarbohidratesFatsProteins

60,5 % 4 %

18 %

6,3 %

79,2 %

8 %

Ketogenic diet (KD):

Reduction in carbohydrate intake in favor of fatty acid intake

Metabolismo mitocondrial >> Glucólisis

ATP mitocondrial >> ATP glicolítico

Activación KATP channels

Hiperpolarización celular

Excitabilidad neuronal

CUERPOS CETÓNICOS >>> GLUCOSA

Diet

0

20

40

60

80

100

Survived

Pregnant

Died

No Aralar-KO

Not pregnant

No litter

Aralar+/- dams on KD

% S

ucc

ee

d a

t d

ay

30

Ketogenic diet had negative effects on pregnant Aralar+/- mice

+/+ +/- -/-0

20

40

60

80

100

120

140 Standard DietKetogenic Diet

% O

ffsp

rin

g s

urv

iva

l (P

ND

20

)

Aralar

And affected the survival of the offspring

PND 5

Dieta

0

20

40

60

80

100

Survived

Pregnant

Died

No Aralar-KO

Not pregnant

No litter

Aralar+/- dams on KD

% S

ucc

ee

d a

t d

ay

30

Ketogenic diet had negative effects on pregnant Aralar+/- mice

Excess fatty acids induce liver ketogenesis Ketone bodies are an alternative energy source to carbohydrates in brain

Intraperitoneal injections of β-Hydroxybutyrate in Aralar-KO mice

Phenotype BIOCHEMICAL ANALYSIS

1) DOPAMINERGIC METABOLISM2) MYELINIZATION

βOHB in vivo does not modify the lifespan or the weight of Aralar-KO mice

WT KO0

20

40

60

80

100

120

140 SalineOHB

% O

ffsp

rin

g su

rviv

al (

PN

D 1

2)

Aralar deficiency alters striatal dopamine metabolism

Llorente-Folch et al., (J. Neurochem, 2013)

=

βOHB partially reverses the alteration of dopamine metabolism and signaling in the striatum of Aralar-KO mice

=

βOHB i.p.

DOPAC/DA HVA/DA DOPAC/HVA0

100

200

300

400

Rel

ativ

e le

vels

(%vs

Wild

typ

e) Wild typeWild type + OHB

Aralar-KOAralar-KO + OHB

Dopamine DOPAC HVA0

50

100

150

Rel

ativ

e le

vels

(%vs

Wild

typ

e)

*

ns

Wild typeWild type + OHB

Aralar-KOAralar-KO + OHB

βOHB partially reverses the alteration of dopamine metabolism and signaling in the striatum of Aralar-KO mice

Wild type0.0

0.5

1.0

1.5

2.0

**

VehicleOHB

VM

AT2

/ -A

ctin

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KO

Wild type0.0

0.5

1.0

1.5

2.0

*

VehicleOHB

DA

RP

P-3

2/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KO

βOHB i.p.

=

Aralar deficiency causes postnatal hypomyelination

Aralar+/+

Aralar+/-

Aralar-/-

Ramos et al., J. Biol. Chem, (2011) Jalil et al., J. Biol. Chem (2005)

Asp

NAAAcCoA

NAA

Asp AcCoAAcetate

Fatty acids

Myelin lipids

Neuron

Oligodendrocyte

Asp-NAT

ASPA

ACS

Pyr AcCoA

Citrate

CitrateGlu

Satrústegui et al., Physiol Rev, (2007)

Wild type0.0

0.5

1.0

1.5

2.0

***

Veh

MA

G/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KO Wild type0

1

2

3

*

Veh

MB

P/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KO

Aralar deficiency causes postnatal hypomyelination

βOHB recovers cortical myelination

βOHB i.p.

Wild type0

1

2

3

*

VehOHB

*

MB

P/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KOWild type0.0

0.5

1.0

1.5

2.0

**

***

VehOHB

MA

G/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KO

Wild type0

1

2

3

*

VehOHB

*

MB

P/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KOWild type0.0

0.5

1.0

1.5

2.0

**

***

VehOHB

MA

G/

-Act

in

(Rel

ativ

e le

velsvs

Co

ntr

ol)

Aralar-KO

Aspartate NAA0.0

0.5

1.0

1.5

2.0

2.5

*

***

*

Wild typeAralar-KOAralar-KO + OHB

Bra

in (

nm

ol/

mg

tiss

ue)

Brain

Wild type0

5

10

15

20

***

***

VehOHB

Asp

arta

te

(nm

ol/

mg

pro

tein

)

Neurons

Aralar-KO Wild type0

5

10

15

20

***

***

VehOHB

NA

A

(nm

ol/

mg

pro

tein

)

Neurons

Aralar-KO

βOHB i.p.

βOHB recovers cortical myelination

and the neuronal synthesis of aspartate and NAA in Aralar deficiency

βOHB bypasses Aralar deficiency

COLABORACIÓN

• Oferta/Demanda: y este es el punto importante. El objetivo es:

1. Exponer al resto de los investigadores aquellas habilidades o tareas de las que te puedes

considerar experto:

Modelos animales modificados genéticamente, función mitocondrial, imaging in

vivo por FRET..

2. Demandar ayuda al resto de investigadores en alguna tarea en la que quieras profundizar

y en la que no seas experto (técnicas, equipos….)



8 de junio del 2021



8 de junio del 2021

Francesc PalauTakeyori SahekiFerdinando PalmieriPaola BovolentaSebastián CerdánClaes WollheimMichael DuchenGyorgy SzabadkaiJim HurleyEduardo RialAranzazu SanchezOscar HerrerasMaría José Casarejos

Irene

Llorente-

Folch

Laura

Contreras Irene

Perez-

Liébana

ÁREA: NEUROCIENCIAS

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