Project PNCDI 2 nr. 41-013 / 2007
EXPRESSION AND FUNCTION OF TIGHT JUNCTION PROTEINS – A STUDY
IN EXPERIMENTAL MODELS AND IN PATIENTS WITH DEMENTIA
(DEMENTJUNCTION)
Program: PARTNERSHIP IN PRIORITARY DOMAINS
The period covered by the project: 2007 – 2010
Project Budget: 1.045.359 lei (Project financed by CNMP)
Coordinator: “Victor Babes” National Institute of Pathology
Project Director: Dr. Bogdan Ovidiu POPESCU, CS I,
Biology Department, Molecular Medicine Laboratory, INCD Victor Babes
Tel.: 021-319.4528
e-mail: bogdan_ovidiu_popescu@yahoo.com ; bogdan.popescu@ivb.ro
Partners:
P1 – International Center of Biodynamics - Dr. Dana Cucu, CS II
P2 – Emergency University Hospital from Bucharest - Prof. Dr. Ovidiu Alexandru Bajenaru
Project Abstract
Key words:
Beta-amyloid, cholesterol, microangiopathy, tight junctions, blood-brain barrier
Dementias are among the most frequent diseases of aged population and are characterized by a
general cognitive and biological decline together with a decrease in quality of life of patients and their
families.
Current treatments for dementia are only symptomatic and all patients finally enter an inevitable
worsening course with premature death. Our project proposes a search for new scientific data, relevant for
the pathogenic mechanisms of dementias (both degenerative and vascular), which could establish new
targets for pharmacological treatment. The blood-brain barrier (BBB) is a unique structure of the human
body, with high selectivity and special regulation. The BBB suffer important alterations in both
Alzheimer’s disease and vascular dementia. Formed by protein complexes with partially unknown
functions, the tight junctions (TJs) hold the key of BBB integrity, which is important since the normal
function of the central nervous system depends on it. In this project we plan to explore the expression of
the TJs constitutive proteins within the brain tissue and to compare it with expression in other tissue types.
We also want to setup experimental models in order to study the function of these proteins in
physiological conditions. To reach our aims, we propose a partnership which guarantees access to patients
with dementia and to a brain bank, combined with basic neuroscience laboratory workup, targeting an
investigation of both morphological and functional properties of TJs proteins. These new scientific data
could generate an impact for the current pathogenic scenario of dementia and for the way we understand
passage of drugs to the central nervous system, at the level of BBB.
General and specific objectives of the project:
General objectives of the project are: the study of TJs proteins expression in brains of patients with
Alzheimer’s disease and vascular dementia, investigation of presence of TJs proteins or proteolysed
products in plasma and cerebrospinal fluid of patients with dementia, optimization of experimental models
for the study of TJs proteins function and expression regulation relevant for the BBB and for dementia
pathophysiology.
Specific objectives of the project are the following:
In 2007-2008:
1. Investigation of TJs proteins (occludin, claudins, ZO 1-3) expression in neurons. Through this aim we
target the quantification of neurons which express TJs proteins in cell cultures and in Alzheimer’s disease
and vascular dementia brains.
2. Development of cell culture systems for the study of TJs proteins. Through this aim we plan to select
and optimize cell lines which express endogenously TJs proteins. Moreover, we will also select cell lines
which can be transfected to artificially overexpress TJs proteins.
In 2009:
3. Evaluation of beta-amyloid effects on TJs. Through this aim we plan to investigate changes in
expression and function of TJs proteins induced by treatment with beta-amyloid peptide (A beta). A beta
is a small peptide composed of 39-43 amino acids, with high aggregation potential, which is the main
component of the senile plaques in Alzheimer’s disease brains. A beta is a result of a serial proteolysis of
the amyloid precursor protein (APP), an integral protein with an uncertain physiological function
(Popescu and Ankarcrona, 2004). Previous reports (e.g. Marco and Skaper, 2006) demonstrated that A
beta alters the TJs protein distribution in endothelial cells of brain capillaries. These data suggest a
dysfunction of BBB in Alzheimer’s disease. Our project will bring new informations about the A-beta
effects on TJs proteins expression and function in the selected cell lines (SH-SY5Y neuroblastoma, clones
of MDCK).
4. Investigation of vascular risk factors and dislipidemia in patients with dementia. Our purpose is to
study the modification of the transepithelial electric resistance following beta-amyloid exposure. We
would like to study also the incidence of hypercholesterolemia in patients with dementia.
In 2010:
5. Investigation of TJs proteins in biological material from patients with dementia and other pathologies.
Investigation of beta-amyloid effects on membrane fluidity; role of cholesterol. We will try to identify
occludin and claudins or proteolysed products in the plasma and cerebrospinal fluid of patients with
dementia as compared to patients with other diseases by molecular techniques. In this setting we will be
able to explore the expression and distribution of TJs proteins in the brain as compared to other tissue
types. Previous reports document the interaction of beta-amyloid and cholesterol in Alzheimer’s disease.
The effect of A beta on TJs proteins expression and function will be analyzed in cells with membranes
enriched or deprived of cholesterol.
Experimental design
The first stage of the project plans the study of different claudin isoforms expression in
Alzheimer’s disease and vascular dementia brains. Access to the precious human brain material is granted
by our cooperation with Karolinska Institute Brain Bank, Stockholm, Sweden (Assoc. Prof. Nenad
Bogdanovic, written agreement). As techniques, we will use immunohistochemistry with anti-claudin sera
and quantification of claudin expressing neurons and glial cells in human brains of demented patients
compared to aged controls. Quantification will be performed with the optical dissector method (West et
al., 1991). These techniques are familiar to our research group, being used in a recent study (Romanitan et
al., 2007).
In the first stage we will also purchase transgenic mice which express mutant
APP or/and presenilin, in order to be able to analyze the results later, considering that for development of
beta-amyloid pathology these mice have to be aged.
In the second stage we will select appropriate cell lines to study the function of TJs proteins. As
cellular model the kidney MDCK cells will be used. These cells are extensively used in analyses of
cellular processes like transepithelial transport, modulation of epithelial barrier and regulation of
intercellular junction functions. TJs protein expression is different in different MDCK clones (I and II).
The two clones are characterized by different transepithelial resistance (TR) and different TJs protein
expression levels. Furuse & al. (2001) demonstrated that epithelia with higher TR (MDCK I) do not ionic
selectivity is also various in different epithelia due to diverse combined expression of claudin isoforms.
Physiological or pathological alterations of claudins can impact on the epithelial barrier properties,
modifying transport or morphological characteristics of the tissue. Understanding the function and the
selectivity properties of the TJs could lead to therapeutical applications for controlling the transport of the
drugs at the BBB level.
Occludin is a protein localized within the junctional complex as well and occludin overexpression
induces a rise in TR in epithelial cells (Feldman et al., 2005). However, complete lack of occludin
expression obtained by homologous recombination did not alter the polarization of epithelia or the
formation of TJs. From this proof it was concluded that occludin is not determinant for TJs formation and
it was predicted that the function of occludin is various and more complex.
In ICB the claudin 2-expressing MDCK I model is used on regular basis. We plan to test other
MDCK clones as well (II, C7, and C11) first by immunofluorescence (or real-time PCR) to study
localization/colocalization of different types of claudins and occludin. Moreover we target to study TJs
proteins by transfection in MDCK cell lines of wild type or truncated proteins.
When epithelial cells are grown on semipermeable filters, they develop polarization with apical
and basal membranes similar to their provenience epithelia. To study the function of TJs proteins we will
involve techniques which are currently used in ICB, among which the technique of impedance and noise
analysis spectroscopy (TINS), recently implemented. With TINS cellular and paracellular transport can be
measured and transmembrane pore kinetics can be studied. As a standard work protocol for TR
modulation variation of extracellular calcium concentration might be used.
The ICB work team has a vast experience with the use of these techniques on epithelial kidney
cells. Moreover, even though cation-selective transport properties were reported for claudin 2 by indirect
methods (bionic permeability), the electrophysiological methods (like noise analysis) can bring new and
detailed informations about pore function of these protein family. The paracellular permeability will be
studied by the measurement of transport between the apical to the basolateral of specific fluorochromes
(FITC-dextran, sodium –fluorescein, etc.) and by spectrophotometry. Variations of paracellular
permeability are to be done by changes in extracellular calcium concentration.
In the second stage of the project we also plan to study occludin, claudins and ZO 1-3 expression
in a stabilized cell line with neuronal phenotype that INCDVB laboratory currently uses (SH-SY5Y
neuroblastoma). This line is well characterized and is frequently used as a neuronal model, and the
INCDVB team used it in different previous studies (Popescu et al, 2001; Popescu et al., 2004). For this
purpose we will use s techniques the Western blot, imunocytofluorescence and possibly real-time PCR.
In the third stage of the project we will investigate the changes of above-mentioned parameters
(TR, paracellular permeability and pore kinetics) in the presence of the beta-amyloid peptide. In previous
studies morphological alterations of the BBB in the presence of A beta were reported but no consistent
data were produced about the functional changes of permeability or TR. This new experimental approach
will be enabled by the techniques that will be used in our project. Furthermore, these functional results
will be correlated with possible changes of protein expression caused by A beta treatment of cell cultures.
In the forth stage of the project a clinical retrospective study will be carried out in order to
establish the incidence of vascular risk factors and especially hypercholesterolemia in patients diagnosed
with dementia in the Department of Neurology, University Hospital Bucharest, in order to orientate and
prepare the collection of biological samples in the last stage of the project.
The fifth stage will be devoted to analyses of TJs expression and function by beta-amyloid peptide
in cell membranes with modified cholesterol composition. Modulation of membrane cholesterol will be
performed by a specific protocol, namely treatment with the sugar methyl-beta-cyclodextrine, which
extract the cholesterol from the membranes when is applied in the cell culture medium. In contrast, when
this compound is used in the presence of cholesterol has an effect of cholesterol enrichment on the cell
membranes. Previous reports show that cholesterol depletion is associated with cytoskeleton
reorganization and consecutive decrease of TR. We plan to investigate the effect of A beta in relation to
cholesterol composition modulation in membranes and to changes of TR, paracellular permeability and
expression of occludin and claudins. Together with changes of membrane composition in cholesterol
changes of membrane fluidity take place. Investigation of this parameter will be done in ICB by
fluorescence microscopy with total reflection, a method which is used on regular basis in our
laboratory. We will use this technique to detect differences for the presence as compared to absence of
cholesterol, which in high concentrations induces a rise in membrane rigidity. How beta-amyloid can
modulate the membrane fluidity and whether these effects are dependent on the membrane lipid
composition are largely unknown processes.
Dissemination:
PUBLICATIONS
1. B.O. Popescu, Still debating a cause and diagnostic criteria for Alzheimer’s disease, Journal of Cellular
and Molecular Medicine, 11:1225-6, 2007.
2. D.F. Muresanu, X.A. Alvarez, H. Moessler, M. Buia, A. Stan, D. Pintea, F. Moldovan, B.O. Popescu, A
pilot study to evaluate the effects of Cerebrolysin on cognition and qEEG in vascular dementia: Cognitive
improvement correlates with qEEG acceleration, Journal of Neurological Sciences, 267:112-119, 2008
3. Popescu BO, Toescu EC, Popescu LM, Bajenaru O, Muresanu DF, Schultzberg M, Bogdanovic N.,
Blood-brain barrier alterations in ageing and dementia, J Neurol Sci. 2009 Aug 15;283(1-2):99-106 (ISI,
impact factor = 2,2)
4. Popescu BO, Bajenaru O, Ene A, Mindruta I, Manga G, Muresanu DF, Romanian O, Evaluation of
cognition and mood in post-stroke mixed dementia patients – a pilot trial,
Journal of Neurology, 2008, 7:153-156 (CNCSIS B+)
5. Romanitan O, Popescu BO, Spulber S, Popescu LM, Bajenaru O, Winblad B, Bogdanovic N., Altered
expression of claudin family proteins in Alzheimer's disease and vascular dementia brains, Journal of
Cellular and Molecular Medicine – in press (ISI, impact factor 5,1)
SCIENTIFIC MEETINGS
1. B.O. Popescu, Pathological changes of the blood-brain barrier in dementias, Fifth International
Congress on Vascular Dementia, WFN, Budapest, Hungary, november 2007 (oral presentation)
2. B.O. Popescu, Molecular pathology of Alzheimer’s disease – new promises in therapy, The present and
immediate future of the cellular and molecular medicine Symposium, by Medical Science Department of
the Roumanian Academy, Bucharest, Roumania, november 2007 (oral presentation)
3. B.O. Popescu, The contribution of the blood-brain barrier to neurodegeneration in dementia, 40th
International Danube Symposium in conjunction with the 6th Congress of Romanian Society of
Neurology, Sinaia, Roumania, may 2008 (oral presentation)
4. B.O. Popescu, Mechanisms of cell death and neuroprotection in Alzheimer’s disease, 40th International
Danube Symposium in conjunction with the 6th Congress of Romanian Society of Neurology, Sinaia,
Roumania, may 2008 (oral presentation)
5. B.O. Popescu, Alzheimer’s disease treatment – symptomatic benefits and beyond, 40th International
Danube Symposium in conjunction with the 6th Congress of Romanian Society of Neurology, Sinaia,
Roumania, may 2008 (oral presentation)
6. B. Popescu, Calcium dysregulation and altered neuronal signaling in Alzheimer’s disease, Diaspora in
roumanian scientific research Conference, Bucharest, Roumania, september 2008 (oral presentation)
7. B.O. Popescu, The development and survival of the nervous system – a long battle for trophic factors,
Cony – 2nd World Congress on Controversies in Neurology, Athens, Greece, october 2008 (oral
presentation)
8. B.O. Popescu, The dementia of Parkinson’s disease is mainly due to cholinergic deficiency, Cony –
2nd World Congress on Controversies in Neurology, Athens, Greece, october 2008 (oral presentation)
9. B.O. Popescu, Presenilin-mediated signal transduction – relevance to Alzheimer’s disease
pathogenesis, Second Joint Congress of GCNN and SSNN, Vienna, Austria, march 2009 Athens, Greece,
october 2008 (oral presentation)
10. Romanitan MO, Winblad B, Bajenaru O, Popescu BO, Bogdanovic N, The role of occludin and
claudins in vascular dementia, 18th European Stroke Conference, Stockholm, Sweden, may 2009 – (ISI
abstract in Cerebrovascular Diseases, 2009; 27, suppl.6: 110) (poster)
11. B.O. Popescu, Is there any neuroregeneration in Alzheimer’s disease?, 3rd World Congress on
Controversies in Neurology (CONy), Prague, Czech Republic, october 2009 (oral presentation)
12. Popescu BO, Alterations of blood-brain barrier triggered by neurodegeneration, 6th International
Congress on Vascular Dementia, Barcelona, Spain, november 2009 (oral presentation)
13. B.O. Popescu, Neurobiology of vascular cognitive impairment, 6th International Congress on Vascular
Dementia, Barcelona, Spain, november 2009 (oral presentation)