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 BiologyIII Semester

2013

Manuela Jiménez ObandoJuan 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 Funtion L-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 Funtion L-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

dementiaEvaluate 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.

Materiales y métodos.

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.

Materiales y métodos.

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.org/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

Materiales y métodos.

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.

+Calcuio Fluo-3/AM disuelto en DMSO

Ester hidrolidazo

Fluorecencia

Materiales y métodos.

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.

Materiales y métodos.

Materiales y métodos.

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

RESULTS

NEURONA + Aβ 1–42

↓ VIABILIDAD CELULAR

(DEPENDIENDO DE LA DOSIS)

NEURONAS NORMALES

NEURONAS TRATADAS CON Aβ 1–42 (4μM)

↑ [Ca+2 ]i

RESULTS

RESULTADOS

WESTERN BLOTS

RESULTS

RESULTS

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, 382Genes, 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] “

DISCUSSION

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 phos- phorylation 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 memoryDeficits”

DISCUSSION

CONCLUSIONS

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.

CONCLUSIONS

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.

MAPS

MAPS

REFERENCES

• Hutton M ,Hardy J (1997) The presenilins and Alzheimer’s disease.Human Molecular Genetics 6: 1639–1646.