Laboratory of Neurodegeneration

Head: Associate Professor: Postdoctoral fellows: PhD students: Office Manager: MSc Student:

Jacek Kuznicki, PhD, Professor Urszula Wojda, PhD Monika Klejman, PhD Marta Wisniewska, PhD Anna Skibinska-Kijek, PhD Joanna Gruszczynska, PhD Magdalena Blazejczyk, MSc (until March 2008, PhD defense Feb. 2008) Emilia Bialopiotrowicz, MSc Lukasz Bojarski, MSc Katarzyna Debowska, MSc Wojciech Michowski, MSc Katarzyna Misztal, MSc Adam Sobczak, MSc Aleksandra Szybinska, MSc Dominika Dubicka, MSc Mirosław Drab, Kamila Skieterska, Bozena Zebrowska

Collaborations:
Dr. Anna Filipek, Nencki Institute of Experimental Biology
Prof. Maria Barcikowska, MD, Institute of Experimental Medicine
Dr. Slawek Filipek and Krzysztof Jozwiak, Laboratory of Biomodelling, IIMCB
Dr. Malgorzata Mossakowska (IIMCB) and Dr. Katarzyna Broczek MD, Department of Clinical Geriatrics, Medical University of Warsaw
Dr. Jakub Golab, Medical University of Warsaw
Dr. Michael Kreutz, Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
Dr. Guido Tarone, Department of Biology, University of Turin

Current Projects:

We are interested in molecular mechanisms involved in learning and memory, as well as neurodegeneration; we study these processes at the genomic, proteomic and cellular levels. Our major projects are:
1. Search for bio-markers and potential therapeutic targets for Alzheimer’s disease (AD).
2. Analysis of proteins involved in Ca2+ homeostasis in neurons and non-neuronal cells.
3. Analysis of Ca2+-binding proteins calmyrin1 and calmyrin2 in neurons.
4. Regulation and role of β-catenin/Lef1 complex in mature neurons.
5. Characterization of biological functions of CHORD containing proteins in the nervous system.
6. Studies on the cyclin-dependent kinase 5 involvement in pathogenesis of Alzheimer’s disease.

1. Search for functional bio-markers and potential therapeutic targets of Alzheimer’s disease (Emilia Białopiotrowicz, Łukasz Bojarski, Mirosław Drab, Aleksandra Szybińska, Bożena KuŸniewska, Urszula Wojda, in collaboration with other laboratories)
In this area, several projects were carried out:
1.1. In cooperation with Prof. Maurizio Memo and Dr. Daniela Uberti (University of Brescia) the conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients was analyzed. Conformation of p53 protein was studied in cell lysates from our immortalized B lymphocytes from 13 sporadic AD (SAD) and 10 familial AD (FAD) patients and 12 control subjects by immunoprecipitation experiments. Cells from SAD and FAD patients specifically expressed an increased amount of conformationally altered p53 that makes them distinguishable from cells of age-matched non-AD subjects. This suggests a role for a dearrangement of protein controlling the cell cycle in AD pathogenesis (C. Lanni, et al., Mol Psych, 2008). Since p53 conformational tertiary structure is influenced by redox status of the cells, we also evaluated the oxidative profile of these patients. We found that among the markers of oxidative stress, hydroxytransnonenal-modified proteins were significantly increased in FAD patients. Furthermore it is interested to note that, besides increased levels of oxidative markers, the antioxidant defence mechanisms were compromised in these patients. These results supported and enhanced the first evidences of peripheral unfolded p53 associated with AD pathology and highlighted the identification of oxidative stress markers in peripheral, immortalised cells derived from FAD patients.
1.2. It has been suggested that the aberrant expression of cell cycle molecules in the brain contributes to the development of Alzheimer’s disease (AD) and causes neuronal death. The aim of the current study was to determine whether the alterations in cell cycle progression can be observed in lymphocytes from SAD and FAD patients. Immortalized B-lymphocytes from 17 SAD and 6 FAD patients (bearing distinct PS1 mutations) were studied in comparison to lymphocytes from 18 healthy individuals. 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 flowcytometry 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 Ca2+ homeostasis in neurons and non-neuronal cells (Łukasz Bojarski, Monika Klejman, Joanna Gruszczyńska-Biegała, Anna Skibińska-Kijek, in collaboration with partners from PROMEMORIA 6thFP 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. 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 amygdale (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 Ca2+ entry that are observed in AD (Bojarski et al., BBA MCR, 2008).

3. Analysis of Ca2+ - 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)
Ca2+-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 Ca2+-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 Ca2+-sensor properties, neuronal pattern of brain expression, and subcellular localization in the Golgi apparatus and dendrites. Moreover, we showed that CaMy2 expression in primary cultures of rat neurons is regulated by NMDA receptor activation and associated Ca2+ signaling (Blazejczyk et al., submitted). We have also identified several new potential targets of CaMy2 in rat brains and confirmed these interactions by several methods in vitro. Physiological significance of these interactions in primary neurons is currently under investigation.

4. Role and regulation of β-catenin in mature neurons (Katarzyna Misztal, Wojciech Michowski, Andrzej Nagalski, Marta Wiœniewska in collaboration with partners from PROMEMORIA 6th FP of EU)
β-catenin is involved in the regulation of proliferation and differentiation of neuronal precursor cells as an activator of the Lef1/Tcf transcription factor and component of the cadherin cell-adhesion complex. In mature neurons β-catenin participates in synaptogenesis and synaptic function in the cadherin complex. However, its transcriptional activity in mature neurons remain elusive. We are interested in the function of β-catenin in the adult brain, since new data suggests it might be involved in learning and memory formation, as well as in some brain pathology. We look for β-catenin/Lef1 target genes in mature neurons i) and explore the mechanism of stabilization of β-catenin in mature thalamic neurons ii).
i)Using Real Time PCR, immunohistochemistry and Western blot techniques we demonstrated that β-catenin and Lef1 are expressed at high levels in neurons of the adult thalamus, in contrast to other regions of the forebrain. Moreover, both proteins are present in the cell nuclei, implying their involvement in gene expression. To answer the question about the role of the Lef1/β-catenin complex in the adult thalamic neurons we investigated a possible involvement of the complex in regulating genes encoding proteins indispensable for thalamic functions. We hypothesized that the Lef1/β-catenin transcription complex enhances expression of Cacna1G encoding Cav 3.1 in mature thalamic neurons. We collected evidence in silico, in vitro and in vivo that corroborates with our assumption (Wisniewska et al., submitted).
ii) We established thalamic cultures and observed that 30%-40% of the cultured thalamic neurons contain nuclear β-catenin, in sharp contrast to cultures of cortical and hippocampal neurons that exhibit β-catenin only in membranes. This confirms an unusual characteristic of thalamic neurons regarding the subcellular distribution of β-catenin. We want to answer the question whether nuclear localization of β-catenin in thalamic cultures depends on extrinsic factors and which signaling pathway is engaged in this process (Misztal et al., in preparation).

5. Characterization of biological function of CHORD containing proteins in the nervous system (Wojciech Michowski, Anna Skibińska-Kijek, Kamila Skieterska in collaboration with Prof. Guido Tarone from University of Turin)
CHORD (Cys and His Rich Domain) domains contain a novel type of zinc fingers. In plants these domains are involved in defence against pathogens. In human genomes there are two genes encoding CHORD containing proteins, melusin and CHP-1. Melusin is present exclusively in cardiac and skeletal muscles. It protects the heart from the consequences of chronic aortic hypertension. The highest level of CHP-1 is found in the brain but it is also present in other tissues. The biological role of this protein remains unknown but it is believed that CHP-1 might be a chaperon responsible for maintaining proper cell function under stress conditions. CHP-1 gene is regulated by HSF-1 (Heat Shock Factor 1) and the protein interacts directly with the major cellular chaperon, HSP90 (Sbroggio et al., FEBS Lett, 2008). We characterize CHP-1 expression pattern in rodents brains under normal conditions and after insults causing stress. We are studying the CHP-1 chaperon activity, using an in vitro assay which is based on monitoring of an aggregation of a heat liable proteins, citrate synthase. We are interested in changes of subcellular distribution of CHP-1 under different stress conditions. We also investigated the susceptibility of cells with different CHP-1 levels (overexpression and RNAi) to stress induced apoptosis (Michowski et al., in preparation).

6. Studies on cyclin-dependent kinase 5 involvement in pathogenesis of Alzheimer’s disease (Aleksandra Szybińska in collaboration with Aleksandra Wysłouch-Cieszyńska and Prof. Michał Dadlez from laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics PAN)
Cyclin-dependent kinase 5 in complex with p35 protein has brain-specific activity and is known to play an important role in a variety of neuronal processes in both developing brains and adult brains. In an adult brain, cdk5 via its interactions with different synaptic, cytoskeletal and cellular adhesion proteins as well as NMDA receptors and calcium channels, is involved in synaptic plasticity, memory and learning processes impaired in Alzheimer’s disease. It was shown recently that in AD patients, the brain expression and activation of cdk5 is upregulated. That upregulation results in MAP tau overphosphorylation together with that caused by GSKβ kinase. Other consequences of cdk5 activity impairment regarding AD are poorly understood. Using the proteomics methods we analyse protein expression and modifications in synaptosomes of transgenic mice, AD models bearing human mutated presenilin 1 and APP genes. Using different methods of samples of preparation and fractionation, we identified almost over 1500 synaptic proteins. Preliminary statistical analysis of mass spectrometry data obtained from wild type and transgenic animals synaptosomes revealed a set of differential proteins, some of which are known to be dysregulated in Alzheimer’s disease but expression changes of some other proteins are being shown for the first time. Additionally, we have made an attempt to increase the efficiency of identification of membrane proteins which are known to be underrepresented in different proteomic analyses.

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).

Fig. 2. 21 days in vitro hippocampal culture from rat embryos E19; stimulated with NMDA; Duolink in situ Proximity Ligation Assay for Calmyrin 2 and NSF interaction (red dots); nucleus (blue) (author: Magdalena Błażejczyk).

Fig. 3. β-catenin accumulation in thalamic neurons – immunohistochemistry. The sections were labeled with β-catenin specific (in green) and neuronal marker NeuN specific antibodies (in red) (author: Marta Wiœniewska).

Fig. 4. β-catenin occupancy along Cacna1G promoter - chromatin immunoprecipitation (ChIP). Real Time PCR analysis of the ChIP products. The results are shown as fold enrichment above background (IgG) (author: Marta Wiœniewska).