II JORNADAS INTERNACIONALES PROGRAMA DE INVESTIGACIÓN DE EXCELENCIA INTERDISCIPLINARIA EN

ENVEJECIMIENTO SALUDABLE (PIEI-ES)Talca, Noviembre, 2014

Enfermedades neurodegenerativas y

acumulación de hierro.

Dr. Miguel Arredondo Olguín

INTA, Universidad de Chile

IMPORTANCIA DEL HIERRO TRANSPORT

EDe

OXIGENOHb, Mb

TRANSFERENCIA

De e*Citocromos

PROTEINA ENZIMAS. Fe-

SCatalasa

Peroxidasa Hidrolasa

ALTA TOXICIDADFe2+ + H2O2 Fe3+ + OH° + OH-

Fe3+ + NADH Fe2+ + [NADH°+]

ALTA REACTIVIDADCAPACIDAD PARA CAPTAR e*

-OH-, -COOH, NH2, -SH

Esencialidad vs Toxicidad

Esencialidad: Anemia por deficiencia de hierroPoblaciones susceptibles

HIERRO, VIDA Y EVOLUCIÓN

Los seres vivos necesitan poco hierro

El hierro abunda en la biosfera

El hierro en atmósfera oxidante es poco soluble

A pesar de la abundancia de hierro en la biosfera su bio-disponibilidad es muy baja.

Los seres vivos han de ser capaces de “disolver” el hierro para poder asimilarlo.

Primera paradoja: “ESCASEZ EN PLENA ABUNDANCIA”

Segunda Paradoja:

“El nutriente más tóxico”: “El estrés oxidativo”

El Hierro es potencialmente tóxico en ambientes donde abunda el O2.

La concentración de hierro intracelular ha de estar controlada.

Origen de la toxicidad del Fe: la reacción de Fenton:

Fe (II) + H2O2 ----> Fe (III) + OH - + .OH

El hierro “libre” intracelular se relaciona directamente con el estrés oxidativo y sus consecuencias patológicas.

La toxicidad del hierro en ambientes aerobios se manifiesta en todos los seres vivos .

Los seres de vida aerobia disponen de mecanismos que atenúan efectos del estrés oxidativo.

¡Dos veces más potenteque la lejía !

IMPORTANCIA DEL HIERRO

HOMEOSTASIA DEL HIERRO

TRANSPORTE

DE OXIGENOHb, Mb

TRANSFERENCIA

DE e*Citocromos

PROTEINA ENZIMAS. Fe-

SCatalasa

Peroxidasa Hidrolasa

ALTA TOXICIDADFe2+ + H2O2 Fe3+ + OH° + OH-

Fe3+ + NADH Fe2+ + [NADH°+]

ALTA REACTIVIDADCAPACIDAD PARA CAPTAR e*

-OH-, -COOH, NH2, -SH

[Fe] [F

e]

Esencialidad vs Toxicidad

Balance coordinado entre captación, utilización y almacenamiento intracelular

Metabolismo del Hierro

Increased iron stores and inflammation induce hepcidin synthesis Suppression: hypoxia, anemia, increased and/or ineffective erythropoiesis in bone marrow. Hepcidin is induced under infection, decreasing the available host iron pool that is essential for

survival of invading pathogens.

MECANISMOS DE REGULACIÓN

Iron and hepcidin: a story of recycling and balanceClara Camaschella. Hematology 2013

Hepcidin

Absorción de

Fe hem

Circulación

LIP

Lumen

Apical

Basolateral

HemHpx

Hem

Fe(II)

HC

P1

Hem

FLV

CR

HO-1

Figure 2. Effect of age on body iron. All values for body iron are positive and indicate the amount of storage iron. Data are based on a convenience sample of 2057 specimens collected in NHANES III. Shaded areas represent the mean 1 SEM for each 5-year interval.

Figure 3. Cumulative frequency distributions of body iron calculated from the ratio of the serum transferrin receptor to serum ferritin. The clear area and positive values indicate storage iron, and the shaded area and negative values indicate tissue iron deficiency. Data are shown for pregnant Jamaican women aged 16-35 years, US women aged 20-45 years, and US men aged 20-65 years.

RNAm Ferritina

Ferritina

Fe+2

Fe+2

Fe libreInsulina

Internalización de Insulinay Acciones Biológicas

Transferrina

Transferrina

Glicación deproteínas

Estrés oxidativo

HiperinsulinemiaInsulino resistencia

RTf

-

+

+

Intracelular

Extracelular

+

+

Reactividad Vascular anormal Daño celular y tisular

¿Cuál es la relación entre metabolismo de Fe y Sindróme Metabólico y diabetes?

Hiperglicemia

GLICOLISIS

Glucosa

Fructosa-6P

Gliceraldehido 3-P

1,3 Difodfoglicerato

Piruvato

Formación de AGEs

Glicación de Proteínas AGEs

RAGE

Glicación de SOD y Catalasa (↑ H2O2)Glicación de Transferrina

Activación de NADPH oxidasa (↑ O2

.- ) Activación de Nf-

κB (↑ NOS, NO,

ONOO - ) Cadena Transportadora de electrones en la Mitocondria.

Fe y Diabetes(Arredondo et al., AJCN, 2007)

No solo altos niveles de Fe se han asociado a DM2

HEM Fe + CO2 + BiliverdinaHO-1

Polimorfismo en el promotor de la HO-1 de repeticiones

(GT) n

Pacientes DM2 portadores de repeticiones cortas

> niveles de Ferritina> actividad de HO-1

complicaciones por estrés oxidativo

Hb (g/dL) 14,0 ± 1,4 NS

Fe (mg/dL) 128,3 ± 52,8 < 0,002

FS (µg/L)& < 0,001

HO& < 0,001(nmoles bilirrubina/mg proteína/hr)

DM2 SM C

61 (35-107)#

# : Diferencia estadística entre Mujeres/grupos&: Promedio geométrico + rangoA: Diferencia estadística entre DM2 y C

0,7 (0,3-2,0)#

Parámetros hematológicos: niveles de Fe

RTf (mg/L)& 5,5 ± 2,0

14,2 ± 1,4

126,5 ± 44,5

52 (27-100)#

0,6 (0,2-1,7)#

6,3 ± 1,7

13,8 ± 1,5

108,2 ± 37,5

34 (15-76)#

0,3 (0,1-0,7)#

6,8 ± 2,7 < 0,001A

Controln=146

OBn=132

T2Dn=60

T2DOBn=106

Hemoglobin (g/dl) 15.7±1.3 16.2±1.2a 14.7±1.8c 15.3±1.6

Serum Ferritin (µg/L)1 56.5(33.7-90.9)

75.5c

(50.0-111.0)70.3b

(42.2-118.1)82.3c

(53.9-125.7)

Serum Fe (µg/dl) 107.5±37.4 100.2±35.1 124.2±88.2 107.9±50.7

Transferrin Saturation (%) 32.7±12.0 30.5±9.0 35.6±15.9 27.9±9.7a

Transferrin Receptor (µg/mL)1 2.8(1.2-6.4)

3.9(1.3-6.6)

3.6(1.8-7.4)

3.6(1.9-6.5)

TBI (mg/kg) 8.5±3.2 9.7±3.3a 9.1±3.7 9.6±2.7a

Hepcidin (ng/mL) 19.0±8.7 25.0±11.5a 23.4±10.6a 25.2±10.8b

RBP4 (µg/mL) 26.1±8.4 33.6±7.3a 31.7±9.6a 32.7±9.3a

hsCRP (µg/dl)1 0.8(0.2-4.4)

1.8(0.5-6.8)

1.9b

(0.4-7.8)2.0b

(0.4-9.0)HO-1 (nmole bilirubin/mg protein/h)1 2.6

(0.9-7.1)4.2a

(1.7-10.1)4.6c

(1.7-12.4)3.4

(0.4-9.0)TBARS (nmoles/mL)1 0.99

(0.4-2.4)1.4a

(0.7-2.7)1.7b

(1.0-3.1)2.1c

(1.2-3.5)

Iron Nutrition and oxidative stress parameters in studied subjects

TBI: Total body iron; RBP4: Retinol Binding protein 4; hsCRP: high-sensitivity C reactive protein; HO-1: heme oxygenase-1; TBARS: Thiobarbituric Acid Reactive Species.Values are mean ± SD; 1Values are geometric mean±(Range)One way ANOVA, post hoc Dunnett`s ap<0.05; bp<0.01; cp<0.001

OR without to adjust

CI p OR adjusted*

CI P

Ferritin Q1: <50 μg/L 1.000 1.000

Ferritin Q2: 50-100 μg/L 1.021 0.42-1.71 0.12 1.101 0.87-1.57 0.66

Ferritin Q3: 100-150 μg/L 1.302 0.45-1.91 0.09 1.133 0.66-1.83 0.08

Ferritin Q4: 150-200 μg/L 1.377 0.97-2.19 0.07 1.782 1.61-1.92 <0.01

TBARS 1.980 1.91-2.28 <0.05 2.250 1.89-3.25 <0.05

Table 3Risk of developing type 2 diabetes (OR) according to

ferritin quartiles and TBARS concentration.

* Adjusted to age, BMI and hsCRPOR were estimated through logistic regression

OR CI p ORAdjusted*

CI p

Hepcidin Q1 1.000 1.000

Hepcidin Q2 1.483 1.11-1.98 0.007 1.300 0.95-1.11 0.470

Hepcidin Q3 1.980 0.89-4.37 0.091 2.120 0.89-5.03 0.087

Hepcidin Q4 3.291 1.39-7.75 0.006 4.370 1.67-11.42 0.003

Table 4:Risk of developing type 2 diabetes (OR) according to

hepcidin expression quartiles

* Adjusted to age, BMI and hsCRPOR were estimated through logistic regression.

HO5` 3`

GTn

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

20

40

60

80

100

120

Nº

Ind

ivid

ual

s

GT Repetitions

Frecuencia alélica(%)

Hardy-Weinberg Equilibrium

S = < 27 (GT)n

M = 27-32 (GT)n

L = >32 (GT)n

Micro-polimorfismo

MICRO-POLIMORFISMO EN EL PROMOTER DEL GEN DE LA ENZIMA HEM OXIGENASA

S M LC 6.2 9.6 4.2MS 8.0 10.9 1.1OB 7.9 8.9 3.1DM 7.6 8.7 3.7DMOB

8.3 8.0 3.7

SM SL ML MM SS LL

C 7.8 2.3 0.8 5.5 1.3 2.7

MS 13.7 0.4 1.2 6.0 2.8 0.6

OB 12.3 1.1 1.9 2.8 2.1 2.0

DM 10.8 2.1 1.6 2.6 1.2 1.9

DMOB

7.5 1.8 1.3 0.7 0.7 0.8

GENOTIPO (%)

C 175MS 210OB 189DM 172DMOB 109

Total 855

C OB T2D T2DOB0

5

10

15

20 ****

***

He

pci

din

mR

NA

0

10

20

30

40

***

*** IL

-6 m

RN

A

C OB T2D T2DOB

0

50

100

150

200

250

******

TL

R-2

mR

NA

C OB T2D T2DOB

0

5

10

15

20

******

***

TN

F-α

mR

NA

C OB T2D T2DOB0

10

20

30**

******

TL

R-4

mR

NA

C OB T2D T2DOB

0

5

1030

40

50

60

**

*** ***

NF

-kB

mR

NA

C OB T2D T2DOB

A B

C D

E F

Figure 1: Relative abundance of genes related to inflammation in OB, T2DOB, T2D and Cn subjects.

A) Hepcidin; B) IL6; C) NF-B; D) TLR-2; E) TLR-4; F) TNF-.

Values are mean ± SEM. Data were analyzed using the Kruskal-Wallis test.

*p<0.05; **p<0.01; ***p<0.001.

Hierro y cerebro.Es importante para la función

neuronal.

Funciones:Es un componente esencial del citocromo

a, b y c oxidasa.Componente del complejo hierro – sulfuro

de la cadena oxidativa.Es un cofactor para la tirosina, triptófano

hidroxilasa, ribonucleótido reductasa, succinato deshidrogenasa y aconitasa.

Es esencial para síntesis de lípidos, colesterol y un rol en el sistema GABA.

Existen altas concentraciones en globo pálido, sustancia nigra, núcleo dentado y corteza motora.

Las neuronas lo almacenan como Ferritina de cadena liviana o pesadas.

La alteración de Ferritina (por inserción de adenosina) produce la “neuroferritinopatía”.

Existen múltiples vías de regulación para el metabolismo del hierro

Lista de enfermedades neurodegenerativas Enfermedad de AlzheimerDemencia con cuerpos de LewyDemencia frontotemporalDemencia mixta (multi-infarto y E. de

Alzheimer)Enfermedad de ParkinsonAtrofia MultisistémicaParálisis supranuclear progresivaDegeneración córticobasalEsclerosis lateral amiotrófica.Enfermedad de Creutzfeldt-Jacob.

Desordenes asociados con neurodegeneración y anormalidades en

la regulación del hierro que resultan depósitos de hierro en el cerebro.Desordenes asociados a depósitos de hierro

primarios con anormalidades genéticas en las vías metabólicas del hierro.

Neuro-degeneración asociada a la pantotenato kinasa (PKAN)

Hipo-prebetalipoproteinemia, acantosis y retinitis pigmentosa con degeneración del pallidal (HARP)

Neuro-degeneración con acumulación de hierro en cerebro (NBIA)

Neuro-ferritinopatía Aceruloplasminemia Hemocromatosis

Desordenes con cambios secundarios en las vias regulatorias del metabolismo de hierro

Enfermedad de Huntington Enfermedad de Parkinson Friedreich´s ataxia.

FIGURE 1 | Inflammation causes ROS/RNS production, mitochondrial dysfunction, and iron accumulation. Inflammation, oxidative damage, and mitochondrial dysfunction are common features of neurodegenerative diseases. A complex net of relationships connect these features, which through feedback mechanisms contribute to the evolvement of neuronal death.

InflammationNFB activationInfl cytokines

Dementia with Lewy bodies (DLB)

Fig. 1. Expression levels of several iron metabolism genes in patients with AD compared with controls.

The distribution of the expression levels of (A) TFRC (B) TFR2 (C) SLC40A1 (D) HAMP and (E) SLC11A2 of patients with AD and control subjects are represented by the box-plots. The quantification was performed by normalizing the sample to a pool of individuals and using HPRT1 as a housekeeping gene. Dots represent the mild outliers. The number of individuals analyzed is indicated in brackets. p-values were obtained by covariance analysis.

Fig. 2 Macrophages migrating into the brain release nitric oxide radicals (NO•), a process that involves the catalytic oxidation of ferrous iron. NO• is capable of diffusing pass the cellular membranes and into neurons where it can react with superoxide (O2•−) and promote formation of the highly reactive and toxic peroxynitrite (ONOO−)

Fig. 4 Extravasated macrophages phagocytose and degrade damaged neurons and subsequently die to terminate their function, which leads to the release of iron into the extracellular space of the CNS on a low molecular weight form

Fig. 5 The macrophages, like monocytes and microglia, are capable of secreting hepcidin into the brain extracellular space. Hepatic hepcidin is synthesized in response to inflammatory signals and secreted into blood plasma from where it can diffuse into the brain in areas with a compromised blood–brain barrier. The hepcidin is capable of binding and inhibiting ferroportin needed for export of iron from neurons, which may result in neuronal iron accumulation and increased the likelihood of neuronal damage via Fenton chemistry

Fig. 6 Potential pharmacological intervention points to inhibit the impact of migrating macrophages on their deposition of iron in the brain. a) Inhibition of monocytes migration into the brain via transfer through the

brain capillaries. b) Inhibition of the functioning of the brain macrophages for phagocytosis

and nitric oxide (NO•) release. c) Extracellular chelation of low molecular weight iron released from dying

macrophages. d) Intracellular chelation of iron in neurons subsequent to their uptake of

low molecular weight iron from the extracellular space

Fig. 6. A hypothetical scheme for the pharmacologic mechanisms of Huperzine A (HupA) in the treatment of Alzheimer’s disease. In addition to acting as an acetylcholinesterase inhibitor, Huperzine A (HupA) has the ability to inhibit transferring receptor 1 (TfR1) expression and then reduce transferrin-bound iron (TBI) uptake by the neurons or other brain cells which has TfR1 expression on the membrane. This will lead to progressive reduction in iron contents in the brain and thus protecting neurons and other brain cells from damage and apoptosis probably by inhibiting the iron-associated oxidative stress. “Reducing iron in the brain” is a novel pharmacologic mechanism of HupA in the treatment of Alzheimer’s disease. Abbreviations: HupA, Huperzine A; TBI, transferrin-bound iron; TfR1, transferring receptor 1.

REDUCING IRON IN THE BRAIN: A NOVEL PHARMACOLOGIC MECHANISM OF HUPERZINE A IN THE TREATMENT OF ALZHEIMER’S DISEASE. Xiao-Tian Huang. Neurobiology of Aging 35 (2014) 1045

Low-copper diet as a preventive strategy for Alzheimer’s disease

Rosanna Squitti , Mariacristina Siotto , Renato PolimantiNeurobiology of Aging

DOI: 10.1016/j.neurobiolaging.2014.02.031

Low-copper diet as a preventive strategy for Alzheimer’s disease

Rosanna Squitti , Mariacristina Siotto , Renato PolimantiNeurobiology of Aging

DOI: 10.1016/j.neurobiolaging.2014.02.031

Fig. 1. Biochemical basis of the theoretical model of copper toxicity in AD.

b-APP binds and reduces copper from Cu(II) to Cu(I), which modulatescopper-induced toxicity based on redox reactions through the production of H2O2,triggering chain reactions of oxidative stress and lipid peroxidation. A and metalsare packed together in plaques, and it has been postulated that A plaques disturbneuronal physiology, entrapping metals within the plaques, while cell-associatedcopper could be decreased. On this basis it could be assumed that decopper-ing agents, as for example zinc therapy, can reduce systemic Non-Cp copper andstop the feeding of noxious copper entering redox cycles with A, thus halting theprogression of A plaques and promoting their solubilization. Additionally, metalionophores can improve neuroregenerative processes, restoring the physiologicaluptake of metals in neurons, which would suffer because of the copper entrappedwithin the extracellular A plaques.

Copper subtype of Alzheimer’s disease (AD): Meta-analyses, geneticstudies and predictive value of non-ceruloplasmim copper in mildcognitive impairment conversion to full AD Rosanna Squitti, JTEMB, 2014

En resumen…….

1) Existe asociación entre el metabolismo del hierro y Enfermedades neurodegenerativas

2) Elemento común desencadenador: Eje Inflamación – Hepcidina

3) Elemento mediador: Estrés Oxidativo

4) Consecuencia final: Muerte Celular

Mónica Andrews, PhD (INTA)Valeria Candia, MSc (INTA)Dr. Manuel Olivares (INTA)

Dr. Néstor Soto (Hospital Arriarán)

Solange Le Blanc , PhDc (Suiza)Alejandra Espinoza, PhDcMarcela Fuentes, PhD (PUC)

“Muchas gracias por su atención”