10 A niversary of IIMCB

Additionally, cell cycle analysis was performed in transiently
and stably transfected HEK293 cells with wild type and
mutated PS1 constructs. The cell cycle was analyzed by
flow cytometry after staining of cells with propidium iodide.
Moreover, expression level of cell-cycle related proteins was
assessed by immunoblotting. The obtained data has shown
cell cycle disturbances in lymphocytes from AD patients.
Moreover, our results reveal a relationship between PS1
and the cell cycle regulation. Finally, this data indicates that
human lymphocytes sustain an easily accessible material
that can be used in studies on AD pathogenesis, and in
search for possible diagnostic markers and therapeutic
targets (Bialopiotrowicz et al., in preparation).
1.3. In collaboration with Dr. Jochen Herms (Ludwig
Maximilians University), we have also been analyzing
lymphocytes from patients with PS1 mutations showing
similar alterations in the calcium homeostasis to neurons
from transgenic animal models of familial AD. We are
performing cell-imaging screens for new potential
therapeutic targets for AD and also analyzing features of
calcium-related mechanisms of synapse formation and spine
morphology in hippocampal neurons from wild type and
PS1 mutant transgenic mice.
2. Analysis of proteins involved in Ca 2+ homeostasis in
neurons and non-neuronal cells (Łukasz Bojarski, Monika
Klejman, Joanna Gruszczyńska-Biegała, Anna Skibińska-
Kijek, in collaboration with partners from PROMEMORIA 6 th
FP of EU and from the Polish-German grant)
Store Operated Calcium Entry (SOCE) is well known in
non-excitable cells. It is based on the interaction of ER
calcium sensors STIM1 or STIM2 with the plasma membrane
calcium channel protein ORAI1. Although SOCE is ubiquitous
in non-excitable cells, it is also crucial for the nervous system.
Its alterations cause deregulation of calcium homeostasis
in the cell and may lead to pathology like Alzheimer’s and
Huntington’s disease.
Fig. 1. STIM1 forms puncta upon thapsigargin-induced ER depletion in
cortical neurons. Cortical neurons were co-transfected with ORAI1 and
YFP- STIM1 and treated with 3 µM thapsigargin for 15 min. Neurons were
analyzed using a confocal microscope and image represents 0.25 µM
thick confocal scan (author: Joanna Gruszczyńska-Biegała).
42 Annual Report 2008
We analyzed and compared the distribution of STIM1
and STIM2 in mice brains and in cultured cortical and
hippocampal neurons using various techniques. We showed
that the protein and mRNA levels of STIM1 and STIM2 vary
in different brain regions. Immunohistochemistry of brain
sections shows a distinct distribution of both proteins
mostly in the hippocampus, cerebellum and the amygdala
(Skibinska-Kijek et al., submitted). We also demonstrate that
STIM1 and STIM2 are present in cultured neurons and their
expression is accumulated mainly in the cell bodies.
Our data revealed that depletion of calcium stores
in cultured cortical neurons induces a change in the
localization of YFP-STIM1, YFP-STIM2 and ORAI1 from
disperse, in untreated, to puncta-like in thapsigargin treated
cells. We propose that, in neurons, just as in non-excitable
cells, the ORAI1 and STIM proteins are involved in store
operated calcium entry (Klejman et al., Neurochem Int 2008).
We also investigated the role of STIM proteins in presenilin
dependent alterations of capacitative Ca 2+ entry that are
observed in AD (Bojarski et al., BBA MCR, 2008).
3. Analysis of Ca 2+ - binding proteins calmyrin 1 and
calmyrin 2 in neurons (Magdalena Błażejczyk, Katarzyna
Dębowska, Adam Sobczak, under the supervision of Urszula
Wojda and in collaboration with the Laboratory of Molecular
and Cellular Neurobiology headed by Dr. Jacek Jaworski)
Ca 2+ -binding proteins in neurons regulate neuronal
development, plasticity, and neurodegeneration. They also
draw much attention due to implications in multiple brain
pathologies including Alzheimer’s disease. Our research
concentrated on a novel family of Ca 2+ -binding proteins
called calmyrins (CaMy, known also as KIP or CIB). In humans,
four genes encode calmyrin proteins (CaMy1 – CaMy4).
The aim of our studies is to elucidate functions of CaMy1
and CaMy2 in neurons by analysis of CaMy1 and CaMy2
localization, biochemical properties and protein ligands in
the brain.
We have previously demonstrated that CaMy1 is implicated
in Alzheimer’s disease and that it interacts specifically with
Alzheimer’s disease associated presenilin 2 (PS2) in vitro and
in vivo (Bernstein et al., Neuropathol Appl Neurobiol. 2005;
Blazejczyk et al., Biochim Biophys Acta. 2006). Our results
indicate, however, that the interaction of CaMy1 with PS2 in
neurons is limited and does not account for the involvement
of CaMy1 in Alzheimer’s disease. Therefore, we have
undertaken the search for other possible protein ligands of
CaMy1. Using several biochemical methods, we identified a
new potential target of CaMy1 in neurons and characterized
CaMy1 interaction with its novel protein ligand in vitro.
Currently, we are investigating the functional role of this
new CaMy1 interaction using cultured primary hippocampal
neurons (Sobczak et al., in preparation).
Moreover, we pursued studies on rat calmyrin 2 (CaMy2).
We cloned rat recombinant CaMy2 protein and obtained
polyclonal anti-CaMy2 antibodies. We demonstrated CaMy2
Ca 2+ -sensor properties, neuronal pattern of brain expression,
and subcellular localization in the Golgi apparatus and
dendrites. Moreover, we showed that CaMy2 expression