1. 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

2. Manuela Jiménez Obando
Juan Sebastián Marín Cárdenas
Molecular Biology
III Semester
2013

3. INTRODUCTION
• Alteration in the intracellular Proteins Funtion
Calcium is involved in Alzheimer L-type calcium channel (LTCC) Survival and death transduction
medaited by calcium.
disease and vascular dementia.
CaV1.2 channel Interfers in the effectively activate
cAMP response element-binding
(CREB)
• A lot of proteins are involved in this CREB Mediates gene transcription.
pathological mecanism. Calmodulin (CaM) Basic neuronal functions
Calcium/calmodulin-dependent Modulator of excitation-
protein kinase II (CaMKII) transcription coupling in neurons -
• The signaling synaptic plasticity
Ca2+/calmodulin/CaMKII/CaV1.2 is Brain-derived neurotrophic factor Development, differentiation,
(BDNF) maintenance and plasticity of brain
really important in the Pathogenesis function
of Alzheimer disease. AB 1–42 Major component of amyloid
plaques, accumulates in neurons of
Alzheimer’s disease brains

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

5. 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

6. 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
http://neuropsicologica.blogspot.com/2010/06/la-demencia-vascular-i.html
functioning are affected.

7. Ca2+/calmodulin/CaMKII/CaV1.2
Proteins Funtion
There are important L-type calcium channel (LTCC) Survival and death transduction
medaited by calcium.
interactions between Ca2+ CaV1.2 channel Interfers in the effectively
activate cAMP response element-
and others proteins binding (CREB)
mediateing some cellular CREB Mediates gene transcription.
Calmodulin (CaM) Basic neuronal functions
signaling that prevent the Calcium/calmodulin-dependent Modulator of excitation-
cellular damage avoiding the protein kinase II (CaMKII) transcription coupling in neurons
-synaptic plasticity
alterations presented in the Brain-derived neurotrophic factor
(BDNF)
Development, differentiation,
maintenance and plasticity of
Alzheimer disease. brain function
AB 1–42 Major component of amyloid
plaques, accumulates in neurons
of Alzheimer’s disease brains

8. Ca2+/calmodulin/CaMKII/CaV1.2
Ca2+ Development, differentiation, maintenance
and plasticity of brain function
LTCC
Membrane
Ca2+
CREB BDNF
CaM
Nucleus
P ser 33
CaMKII

9. 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

10. 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

11. 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.

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

13. 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).
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
• Se aplicó método MTT y se midió a 570nm.

14. 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.
Fluo-3/AM disuelto
Calcuio +
en DMSO
Fluorecencia
Ester hidrolidazo

15. 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.
Conejo anti-CREB, de
• Los niveles totales de proteína se determinaron utilizando un kit de ensayo de proteína BCA conejo anti-BDNF,
ratón anti Cav1.2, de
además Los anticuerpos primarios. conejo anti-p-
CaMKII, ratón anti-
CaM y de conejo
beta-Actina.
• 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).

16. Materiales y métodos.

17. Materiales y métodos.
Inmunofluoresencia
Ratones
• Ratones anestesiados y perfundidos intracardialmente
con paraformaldehído- anestesia.
• Secciones coronales del cerebro, después se incubaron Células propias
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.
Anticuerpos-análisis
• La tinción nuclear se consiguió con DAPI. Fueron
examinados utilizando microscopia de barrido laser, se
documento los resultados con softhware.

18. NEURONA + Aβ 1–42
↓ VIABILIDAD
CELULAR
(DEPENDIENDO
DE LA DOSIS)

19. NEURONAS NEURONAS TRATADAS
NORMALES CON Aβ 1–42 (4μM)
↑ [Ca+2 ]i

21. APP/PS1:
CaMKII,
Cav1.2
VD Gerbils
CaMKII,
Cav1.2

22. Who? What did they say? Agree Disagree
• [2] K.A. Bruggink, W. Jongbloed, E.A. “APP/PS1 mice revealed significant deficits
Biemans, R. Veerhuis, J.A. Claassen, H.B. in hippocampal basal synaptic transmission
Kuiperij, M.M. Verbeek, Amyloid- oligomer
detection by ELISA in cerebrospinal fluid and
(BST), long-term potentiation (LTP) and
brain tissue, Analytical Biochemistry 433 memory [15] and the expression of Aβ 1–42
(2012) 112–120. is increased in the brain [2].”
• [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.
A. Kamata, H. Sakagami, H. Tokumitsu, M. Sanda, “CaMKII, a kinase that is highly-
Y. Owada, K. Fukunaga, H. Kondo, Distinct concentrated in postsynaptic regions and is
developmental expression of two isoforms of activated by Ca2+/CaM, is thought to have
Ca2+/calmodulin- dependent protein kinase
kinases and their involvement in hippocampal
a key role in this alteration [8] “
dendritic formation, Neuroscience Letters 423
(2007) 143–148.

23. Who? What did they say? Agree Disagree
Y. Yamamoto, N. Shioda, F. Han, S. “Cerebral ischemia reduced p-CaMKII levels
Moriguchi, A. Nakajima, A. Yokosuka, Y. in the hippocampal CA1 region “
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.
D. Zhao, J.B. Watson, C.W. Xie, Amyloid beta “Inhibition of CaMKII-dependent protein
prevents activation of calcium/calmodulin- phosphorylation was found to be essential
dependent protein kinase II and AMPA for A-induced LTP and memory
receptor phos- phorylation during Deficits”
hippocampal long-term potentiation,
Journal of Neurophysiology 92 (2004)
2853–2858.

24. 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.

25. 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.

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