Seminario boliogia molecular

The alterations of Ca2+/calmodulin/CaMKII/CaV1.2

signaling in experimental models of Alzheimer’s

disease and vascular dementia

Dongyu Mina,b,1 , Feng Guoa,1 , Shu Zhuc , Xiaoxue Xud , Xiaoyuan Maoa

, Yonggang Caoa , Xintong Lva , Q1 Qinghua Gaoa, Lei Wange, Tianbao

Chene, Chris Shawe, Liying Haoa, Jiqun Cai

Molecular Biology

III Semester

2013

Manuela Jiménez Obando

Juan Sebastián Marín Cárdenas

INTRODUCTION

• Alteration in the intracellular

Calcium is involved in Alzheimer

disease and vascular dementia.

• A lot of proteins are involved in this

pathological mecanism.

• The signaling

Ca2+/calmodulin/CaMKII/CaV1.2 is

really important in the Pathogenesis

of Alzheimer disease.

Proteins FuntionL-type calcium channel (LTCC) Survival and death transduction

medaited by calcium.

CaV1.2 channel Interfers in the effectively activate

cAMP response element-binding

(CREB)

CREB Mediates gene transcription.

Calmodulin (CaM) Basic neuronal functions

Calcium/calmodulin-dependent

protein kinase II (CaMKII)

Modulator of excitation-

transcription coupling in neurons -

synaptic plasticity

Brain-derived neurotrophic factor

(BDNF)

Development, differentiation,

maintenance and plasticity of brain

function

AB 1–42 Major component of amyloid

plaques, accumulates in neurons of

Alzheimer’s disease brains

INTRODUCTION

• Alzheimer’s disease was first identified on 1906 -

German physician, Dr. Alois Alzheimer.

• Abnormalities are deposits of the protein fragment 40–

43 amino acid peptide called b-amyloid (beta-pleated

sheet).

• Soluble beta-amyloid aggregates spontaneously into

fibrils that are indistinguishable, it is thought that

plaques result from raised b-amyloid levels.

• Strands of the protein tau when becomes hyper-

phosphorylated and this less efficient binding to

microtubules as well as evidence of nerve cell damage

and death in the brain.http://www.memorydr.com/alz.htm

INTRODUCTION

• Most common type of dementia-progressive deterioration of

thinking abilities severe enough to interfere with social,

occupational and intellectual functions.

• The term late-onset dementia refers to intellectual deterioration

which occurs after the age of 65 years (Vascular, Lewy bodies

(DLB), Mixed dementia, Parkinson’s disease, Frontotemporal lobar

degeneration, Creutzfeldt-Jakob)

• Accounts for an estimated 60 to 80 percent of dementia cases.

• Genetics factors (Prenilysin 1-2, Tau, Calmodulin,

Apolipoprotein-E-increase in the density of beta-amyloid

deposits)-non genetics Aetiology.http://year9diseases.wikispaces.com/Alzheimer's+disease

INTRODUCTION

• Vascular dementia is impaired the

judgment or ability to make plans is

more likely to be the initial symptom, as

opposed to the memory loss often

associated with the initial symptoms of

Alzheimer disease.

• Occurs because of brain injuries such

as microscopic bleeding and blood

vessel blockage. The location of the

brain injury determines how the

individual’s thinking and physical

functioning are affected.http://neuropsicologica.blogspot.com/2010/06/la-demencia-vascular-i.html

Ca2+/calmodulin/CaMKII/CaV1.2

There are important

interactions between Ca2+

and others proteins

mediateing some cellular

signaling that prevent the

cellular damage avoiding the

alterations presented in the

Alzheimer disease.

Proteins FuntionL-type calcium channel (LTCC) Survival and death transduction

medaited by calcium.

CaV1.2 channel Interfers in the effectively

activate cAMP response element-

binding (CREB)

CREB Mediates gene transcription.

Calmodulin (CaM) Basic neuronal functions

Calcium/calmodulin-dependent

protein kinase II (CaMKII)

Modulator of excitation-

transcription coupling in neurons

-synaptic plasticity

Brain-derived neurotrophic factor

(BDNF)

Development, differentiation,

maintenance and plasticity of

brain function

AB 1–42 Major component of amyloid

plaques, accumulates in neurons

of Alzheimer’s disease brains

Ca2+/calmodulin/CaMKII/CaV1.2

LTCC

Ca2+

CREBCaM

Ca2+

CaMKII

Nucleus

BDNF

Membrane

Development, differentiation, maintenance

and plasticity of brain function

P ser 33

General objective.

The general objective is to determinate the alterations of

Ca2+/calmodulin/CaMKII/CaV1.2 signaling in experimental

models of Alzheimer’s disease and vascular dementia

Evaluate the interacations between

this proteins and the Ca2+

concentrations in vascular and

Alzheimer dementia to dilucidate

the correct signaling process that

have not been well determinated.http://www.elmundo.es/elmundosalud/2008/02/06/neurociencia/1202303660.html

Materiales y metodos.

Etica: el estudio fue aprovado segun las

especificaciones propias del pais para las

investigaciones en el area de la salud.

Cultivo animal: se utilizaron ciertos ratones.

• Macho de nueve meses de edad APP/PS1-

Tg

• Tipo salvaje-jerbos. http://es.123rf.com/photo_9818004_un-raton-de-cosecha-trepar-a-traves-de-un-campo-de-trigo-antes-de-

tiempo-de-cosecha.html

Materiales y métodos.

Induccion de isquemia global: los ratones se anestesiaron

anteriormente, luego se indujo la isquemia utilizando pinzas arteriales, a

los 10 minutos, se retiraron las pinzas para reestablecer el flujo. El

raton de caracter control se hizo lo mismo pero son compresión

carotidea.

• Con esto se pretendio inducir la isquemia cerebral y así

evaluar los parámentros de la investigación con el modelo de

demencia vascular.


http://sosbiologiacelularytisular.blogspot.com/2010/09/biologia-celular-cuerpos-de-nissl.html

Tinción de Nissl: posterior a la aplicacion del test de Morris , se

extrageron todos los cerebros y fijados durante un dia, luego, se paso a

una solucion con sucrosa, se tomaron los fragmentos con el micrometro

y se aplico la tinsion de Nissl.

• Se realizó para evaluar la presencia de los cuerpos de Nissl que son

poliribosomas libres en el citoplasma principalmente en el soma y

que cuando hay alteraciones patológicas se encuentran disminuidos.


Cultivo primario y viabilidad celular

neuronal.• Células de un día de viejas del hipocampo fueron

sumergidas en una solución y luego trasladadas

a una caja Petri con suero bovino.

• 24 horas después el suero fue reemplazado por

suero neurobasal. Nueve días después fue de

nuevo reemplazado por el suero inicial y se le

sumo AB 1–42 a diferentes concentraciones (0,

116 1, 2, 4, 8, and 16 M).

• Se aplicó método MTT y se midió a 570nm.

http://www.google.com.co/imgres?q=mtt+method&um=1&hl=es-

419&sa=N&biw=1366&bih=667&tbm=isch&tbnid=8LdA2P_Xno3QNM:&imgrefurl=http://2010.igem.org/Team:Freiburg_Bioware/Filelist2&docid=mUBnj6GB6Em2BM&imgurl=http://2010.igem.or

g/wiki/images/c/ca/Freiburg10_MTT_method.png&w=543&h=458&ei=mt8_UaiBD5Ci8ASRh4D4BA&zoom=1&ved=1t:3588,r:0,s:0,i:77&iact=rc&dur=1398&page=1&tbnh=180&tbnw=213&start=

0&ndsp=17&tx=95&ty=118


Medición de Calcio intracelular.

Al noveno, las neuronas del control y pre-

tratadas con AB 1-42 (4uM) durante 24 h se

pusieron en un suero sin medio de crecimiento

por 30 min a 37 centígrados. La concentración

intracelular de Ca2 + se expresó como la

intensidad de fluorescencia por el software

(EZ-C1 3,70 128 FreeViewer NIKON) y la

intensidad media de cada neurona se calculó.

+CalcuioFluo-3/AM disuelto

en DMSO

Ester hidrolidazoFluorecencia


Western blot:

• Los tejidos del hipocampo de jerbos, ratones y los cultivos de neuronas tratadas 24 h con

AB 1-42 fueron utilizados.

• Los niveles totales de proteína se determinaron utilizando un kit de ensayo de proteína BCA

además Los anticuerpos primarios.

• Las membranas se incubaron con HRP(Peroxidasa de rábano picante)-conjugado con

anticuerpos secundarios durante 1 hora a temperatura ambiente.

• La inmunodetección se realizó con quimioluminiscencia seguido de seguro a película de

rayos X .

• Todos los datos se analizaron por software Quantity One (BioRad).

Conejo anti-CREB, de

conejo anti-BDNF,

ratón anti Cav1.2, de

conejo anti-p-

CaMKII, ratón anti-

CaM y de conejo

beta-Actina.



Inmunofluoresencia

• Ratones anestesiados y perfundidos intracardialmente

con paraformaldehído- anestesia.

• Secciones coronales del cerebro, después se incubaron

noche en una mezcla de anticuerpos, ratón anti-Cav1.2 y

de conejo FITC y Cy3.

• Cortes se incubaron anticuerpos anti-ratón y anti-

conejo durante 2 ha temperatura ambiente.

• La tinción nuclear se consiguió con DAPI. Fueron

examinados utilizando microscopia de barrido laser, se

documento los resultados con softhware.

Ratones

Células propias

Anticuerpos-análisis

NEURONA + Aβ 1–42

↓ VIABILIDAD

CELULAR

(DEPENDIENDO

DE LA DOSIS)

NEURONAS

NORMALES

NEURONAS TRATADAS

CON Aβ 1–42 (4μM)

↑ [Ca+2 ]i

CaMKII,

Cav1.2

APP/PS1:

VD Gerbils

CaMKII,

Cav1.2

Who? What did they say? Agree Disagree

• [2] K.A. Bruggink, W. Jongbloed, E.A.

Biemans, R. Veerhuis, J.A. Claassen, H.B.

Kuiperij, M.M. Verbeek, Amyloid- oligomer

detection by ELISA in cerebrospinal fluid and

brain tissue, Analytical Biochemistry 433

(2012) 112–120.

• [15] R.S. Reiserer, F.E. Harrison, D.C.

Syverud, M.P. McDonald, Impaired spatial

learn- 381

ing in the APPSwe +

PSEN1DeltaE9 bigenic mouse model of

Alzheimer’s disease, 382

Genes, Brain and Behavior 6 (2007) 54–65.

“APP/PS1 mice revealed significant deficits

in hippocampal basal synaptic transmission

(BST), long-term potentiation (LTP) and

memory [15] and the expression of Aβ 1–42

is increased in the brain [2].”

A. Kamata, H. Sakagami, H. Tokumitsu, M. Sanda,

Y. Owada, K. Fukunaga, H. Kondo, Distinct

developmental expression of two isoforms of

Ca2+/calmodulin- dependent protein kinase

kinases and their involvement in hippocampal

dendritic formation, Neuroscience Letters 423

(2007) 143–148.

“CaMKII, a kinase that is highly-

concentrated in postsynaptic regions and is

activated by Ca2+/CaM, is thought to have

a key role in this alteration [8] “

Who? What did they say? Agree Disagree

Y. Yamamoto, N. Shioda, F. Han, S.

Moriguchi, A. Nakajima, A. Yokosuka, Y.

Mimaki, Y. Sashida, T. Yamakuni, Y. Ohizumi,

K. Fukunaga, Nobiletin improves brain

ischemia-induced learning and memory

deficits through stimulation of CaMKII and

CREB phosphorylation, Brain Research

1295 (2009) 218–229.

“Cerebral ischemia reduced p-CaMKII levels

in the hippocampal CA1 region “

D. Zhao, J.B. Watson, C.W. Xie, Amyloid beta

prevents activation of calcium/calmodulin-

dependent protein kinase II and AMPA

receptor phosphorylation during

hippocampal long-term potentiation,

Journal of Neurophysiology 92 (2004)

2853–2858.

“Inhibition of CaMKII-dependent protein

phosphorylation was found to be essential

for A-induced LTP and memory

Deficits”

The findings of the present study could be the beginning of new

therapeutic targets both in Alzehimer disease and vascular

dementia.

Biomolecular techniques like Western Blot, are essential in the

development of investigations like the one in this article, and if

we do a correct use of them, the advances will be bigger and

would continue contributing to the science.

This study can show us the important role of calcium in the real

homestasis in brain cells , an clear up the complex

phisiopathologic mecanism in the Alzheimer disease and more

clues of the vascuar dementia.

The importance of the inglish in the biomedical sciences is very

significant to know the most recently news and knowdge.

REFERENCES

• Hutton M ,Hardy J (1997) The presenilins and Alzheimer’s

disease.Human Molecular Genetics 6: 1639–1646.

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Seminario boliogia molecular