Transcript Expression of Estrogen Receptor α and β in the Brain and

Expression of Estrogen
Receptor α and β in the Brain
and Relationship with Behavior,
Cognition, Memory and Stress in
Health and Neurodegenerative
disease
*Radka MOLIKOVA , *Marcela BEZDICKOVA,
**Shan Shan HUANG, **He LI, ***Zdenka
MICHALIKOVA
* Dept. of General Anatomy, Faculty of Medicine, Palacky University Olomouc, Czech Rebublic Europe
** Division of Histology, Tongji Medical College, Huazhong University Science and Technology, Wuhan P.R.
of China
*** Medical Graphic Designer, Faculty of Medicine, Palacky University Olomouc, Czech Rebublic Europe
AKTUALIZOVÁNO: duben 2006
1
Introduction

ERα and ERβ are an important form of steroid
hormones with multiple targets, in the body and brain,
and exerting ubiquitous effects on behavior.

In the brain these receptors are regionally specific, but
both have widespread distributions, which are largely
non-overlapping .
[1, 7, 8]

In the CNS, estrogens are well known to cause a
importance for control of REPRODUCTION, including
SEXUAL BEHAVIOR but not only in the context of
reproduction, but also with reference to COGNITION
AND MEMORY .
[1, 4, 6]
2
Purpose

The physiological function of ERα and ERβ is under
intense study but certain results indicate that ERα and
ERβ have different or event opposite biological
actions .
[1]

Hoon´s preliminary studies show that hormone
receptors, estrogen receptors (ERs), and cAMP
response elements binding protein (CREB) are present
in the mitochondrial matrix of neurons and ER and
CREB could be regulated by novel signaling pathways
in the intact central nervous system and that their
functions in the mitochondria might be important in
neuronal survival .
[10]

On the basis of newly emerging complexities of
estrogen's mechanisms of action it is important :
[1, 7]
1.
To distinguish which pathways of ERα and ERβ are
involved in modifying which behavior, cognition and
memory.
2.
To study the mechanisms by which estrogens
mediate behavior, cognition and memory in which
parts of the brain during increasing
decreasing
3.
(siRNA)
(pEGFP-C2- ERα,β)
and
expression of ERαand ERβ.
To compare mediate mechanism and biological
function of ERαand ERβ in the brain and potential
relation to neurodegenerative diseases (PD, AD,
HD).
ERα and ERβ

ERα is localized on human chromosome 6, in
contrast to ERβ, which sits on chromosome 14.
ERα and ERβ thus represent two separate gene
products and share relationship .
[1, 6]

ERα is highly expressed in the classical estrogen
target tissues such as uterus, mammary gland,
bone and cardiovascular systems, whereas ERβ
is mainly expressed in non-classical tissues such
as prostate, ovary and urinary track .
[2]

Overall distribution of ERα and ERβ in different
tissues figure 1 .
[1]
Gustafsson A.J. 1999 Estrogen receptor β-a new dimension in estrogen mechanism of action. Journal of Endocrinology 163, 380.
Figure 1
[1]

ERα is widely expressed in the hypothalamus.
The distribution of ER αconcentrated in the band
of Broca, the medial mamillary nucleus, the
medial preoptic area, the paraventricular
nucleus,and the ventromedial nucleus .
[3]

ERα has been localized in the hippocampus,
with dorsal vs. ventral differences in the ERα
expression .
[5]

Sensitive binding study have also showed the
expression of estrogen receptors in cells of the
neocortex, where their accurate localization
and function is not so clear (resp.unknow),
but may involve mediation of estrogen action on
sexual behavior .
[4]
Neurodegenerative disease

Protein aggregation in the brain has been observed in
the brains of a big sort of diseases characterized of
progressive neurodegenetation, such as
Alzheimer Disease (AD)
Parkinson Disease (PD)
Huntington Disease (HD)
Creuzfeldt Jakob Disease (CJD) etc.

Abnormal protein aggregations are associated with
gene mutation, protein posttranslational aberrant
modification, chronical viral infection in the brain,
brain aging and environmental toxin as described as
follows.
ALZHEIMER DISEASE (AD)

AD is a progressive, degenerative disorders of
uncertain causes, although abnormal metabolism
and deposition of β-amyloid protein appears to be
closely linked to pathogenesis.

AD is the most common cause of dementia. Its
incidence rises from less than 1% per year to more
than 7% per year, and its prevalence from 3% to
almost 50%, between the ages of 65 and 85
years .
[13]

This translates into a prevalence of 10-20mil. cases
worldwide. Men and women are affected with equal
frequency, when adjusted for age .
[13,14]

AD is defined by characteristic histopathologic
features, especially neurofibrillary tangles and
neuritic (senile) plaques .
[13,14]

Examination of the brain will reveal variable cortical
atrophy with widening of the cerebral sulci that is
most pronounced in the frontal, temporal and
parietal lobe.

The progressive loss of neurons in the entire cortex
leads to slowly progressive mental deterioration.

Clinical symptoms include the loss of recent memory
(the most common early sign) and loss of long-term
memory. The disease is progressive and leads to
immobility in about ten years .
[14]
[9]
Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical School.
PARKINSON DISEASE (PD)

Parkinsonism occurs in all ethic groups, in the
United States and Western Europe it has a
prevalence of 1-2/1000 population, with an
approximately equal sex distribution. The disorder
becomes increasingly common with advancing age.

It is characterized by expressionless face (masklike),
tremor 4-6Hz („pill-rolling“), hypokinesia, rigidity,
and abnormal gait and posture .
[13,14]

It is important to compare HD with excessive
movement to PD with decreased movement .
[13]




Etiology (multiple)
Idiopathic (most common)
Postencephalitis
Autoimmune dysfunction
Trauma
Drugs (illicit drug MPTP).
Location
Substantia nigra and other brainstem centers,
cell loss in the globus pallidus and putamen and
the presence of Lewy Body, containing αsynuclein in the basal ganglia, brainstem, spinal
cord, and sympathetic ganglia.
Proces
Decreased dopamine in corpus striatum.
Treatment
Dopamine agonists.
HUNTINGTON DISEASE (HD)

HD is a hereditar disorders of the nervous system
characterized by the gradual onset and
subsequent of chorea and dementia. It occurs
throughout the world and in all ethnic group. Its
prevalence is about 5 per 100 000 population .
[13]

HD is an autosomal dominant disorder. HD is
caused by the increase in the length of a CAG
triplet repeat present in a gene located on
chromosome 4p16.3. The product of the mutated
gene is an abnormal protein - HUNTINGTINE .
[9]

The typical onset of HD appears usually in the 4th
decade (typically between 30 and 50 years of age),
juvenile and late-onset cases are rare.

First symptoms are motor or mental changes
(choreatic dyskinesias, later dystonic or rigid
symptoms, personality and behavioral changes,
affective disorders, progressive cognitive deficits and
later dementia) .
[9,13]

The pathophysiology of HD relates to degeneration
of GABA neurons (typical inhibitory neurons) in the
striatum. Decreasing functioning of inhibitory
neurons leads to increased movements .
[14]
[9]
Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical School.
Material and Methods-The Present State
Present
State
STEP 1
Detection of ER
Single Imunohistochemistry
RT-PCR
Immunoblot - Western Blot
STEP 2
Designe of Primer ERα, ERβ
(mus musculus and rattus norvegicus)
STEP 3
Isolation of mRNA from classical and non-classical
estrogen target tissues and RT PCR
STEP 4
Construction of vector pEGFP-C2- ERα, ERβ
STEP 5
Sequencing of pEGFP-C2- ERα, ERβ, amplification
STEP 6
Transfaction into cell line HEK 293
3V
MUS MUSCULUS Bregma - 1,70mm
Dilution 1:400
Paxinos, G., Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordination.
[11]
Single Imunohistochemistry
ER+
ER+
HYPOTHALAMIC AREA
VMHDM
3V
PeF
3V
VMHVL
DM
MCLH
LH
magn. 10x
magn. 20x
LH
3V
magn. 40x
magn. 40x
CEREBRAL CORTEX AREA
*
S1Tr/S1BF magn.10 x
S1Tr/S1BF magn.20 x
*
S1Tr/S1BF magn.40 x
S1Tr/S1BF magn.100 x
Expression of ER in the cerebral cortex area have been verify by RT PCR & Western Blot.
CEREBRAL CORTEX AREA - RT-PCR
Detection of ERα
+
+
Acknowledgements


I would like to thank my supervisor Prof. LI He, PhD., for his
expert advice and great help, Shen Shen Huang for her devoted
guidance relentless energy, enthusiasm, with which they guided me
through the entire duration of my study in the Tongji Medical
College.
Last but by no means the least; I must thank my collegues from
Division of Histology, Tongji Medical College for their
enormous tolerance and support.
This project was supported by scholarship cooperation Ministry of
Education Czech Republic and Ministry of Education P. R. of
China in the Tongji Medical College in the year 2005/06.
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…IN A PICTURE
ESTROGEN
RECEPTOR
www.komsta.net
www.breastcancer.org
www.few.vu.nl
A Estrogen receptor
B Estrogen
C Estrogen helper proteins
D nucleus
E DNA genetic material
www.ks.uiuc.edu
Cell with estrogen receptors, estrogen, and helper proteins.
Protocol for cloning eukaryotic DNA fragments in lambda phage
An example of DNA cloning using bacterial plasmids
Formation of a recombinant DNA molecule
Division of Histology
Tongji Medical College, P.R. of China
LABORATORY OF
Tongji Medical College
Department of Anatomy
Leipzig University, Germany
Molikova Radka
Bezdickova Marcela
Thank you
for your attention!