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 in neurodegeneration; we study these processes at the genomic, proteomic and cellular levels. Our major projects are:
1. Identification of mutations in Alzheimer disease-related genes of Polish patients and functional analysis of these mutations
2. Search for bio-markers and potential therapeutic targets of Alzheimer disease
3. Studies on the cyclin-dependent kinase 5 involvement in pathogenesis of Alzheimer disease
4. Analysis of Ca2+-binding proteins under normal and pathological conditions in neurons
5. Analysis of proteins involved in Ca2+ homeostasis in neurons
6. Regulation and role of β-catenin/Lef1 complex in mature neurons
7. Characterization of biological functions of CHORD containing proteins in the nervous system

1. Identification of mutations in Alzheimer disease-related genes of Polish patients and functional analysis of these mutations (Aleksandra Szybinska in collaboration with the group of Maria Barcikowska and Cezary Zekanowski at the Medical Research Center, PAN)
More than 100 mutations linked to early-onset familiar Alzheimer disease (FAD) have been identified in presenilin proteins. Presenilin 1 and presenilin 2 are the catalytic components of the gamma-secretase enzymatic complex, which also comprises of nicastrin, Aph-1 and Pen-1 proteins. Gamma-secretase is responsible for intramembranous cleavage of amyloid precursor protein (APP) and some
other cellular substrates. Most FAD mutations in presenilins are located in their transmembrane domains, indicating that the intramembrane interactions play a crucial role in the stabilization and proper functioning of the enzyme. Presenilins interact with beta-catenin, calmyrin, and several other proteins. Despite extensive efforts, the structure and mechanism of presenilin activity remains unclear. To determine the spectrum of mutations in a group of Polish patients with clinically diagnosed early-onset Alzheimer disease, frontotemporal dementia and related dementias, we performed a screening for mutations in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), tau protein (MAPT), and prion protein (PRNP) genes. The total frequency of mutations in a group of familial AD patients was 21%. Screens in a group of 65 Polish patients with early onset AD identified three previously known mutations in PSEN1 gene: L153V, S170F, and L224H.
Clinical outcome of our living patient bearing S170F mutation strongly resembles the outcome of previously diagnosed patient with S170F mutation with atypical AD and Lewy bodies. We also identified novel mutations in PSEN1 (F226L, I213F, P117R) and in PSEN2 (Q228L). Lymphocytes of patients with identified mutations in presenilins have been immortalized and collected in the cell bank consisting of about 200 lymphoblast lines including those obtained during the Polish Centenarian Project. We examined the previously recognized pathogenic mutation in PSEN1 gene and novel, mutations identifi ed by us, on beta-amyloid production. Stable clones of human embryonic kidney HEK 293 Flp-In-239 cells with Swedish APP mutation KM670/671NL (obtained from Dr. Jessie Theuns, University of Antwerp), were stably transfected with constructs bearing the above-mentioned PSEN mutations or empty vector. Beta-amyloid 1-40 and 1-42 levels in serum-free culture media were estimated by ELISA. Cells with mutated presenilins produced higher amounts of beta-amyloid than control cells and the beta-amyloid 42/betaamyloid 40 ratio was significantly increased versus controls, indicating that novel mutations identified in Polish patients are likely responsible for FAD.

2. Search for functional bio-markers and potential therapeutic targets of Alzheimer disease (Emilia Bialopiotrowicz, Lukasz Bojarski, Miroslaw Drab, Aleksandra Szybinska, Urszula Wojda, Bozena Zebrowska in collaboration with other laboratories)
In this area, several projects were carried out: 2.1. In cooperation with Prof. Mauricio 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 9 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 Psychiatr, 2007; in press).
2.2. We have been analyzing FAD mutations in PS1 effects on basic cellular functions, such as cell cycle progression and apoptosis. These studies are performed using immortalized lymphocytes from patients with FAD PS1 mutants, from SAD patients and from the control groups. Effects of PS1 mutations are also being tested in HEK cells transfected with PS1 mutant constructs. We are searching for molecular mechanism(s) underlying changes observed in PS1 mutant cells in comparison to cells expressing wild type PS1.
2.3. 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.

3. Studies on cyclin-dependent kinase 5 involvement in pathogenesis of Alzheimer disease (Aleksandra Szybinska in collaboration with Aleksandra Wyslouch-Cieszynska 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 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 disease. It was shown recently that in AD patients, the brains expression and activation of cdk5 is upregulated. That upregulation results in MAP tau overphosphorylation together with that caused by GSK beta. Other consequences of cdk5 activity impairment regarding AD are poorly understood. In our studies, we compare, using the proteomics methods, protein expression and modifications in synaptosomes of transgenic mice, AD models bearing human mutated presenilin 1 and APP genes and p25 overexpressing animals, which are cdk5 hyperactivation models versus wild type animals to fi nd mechanisms of neurodegeneration processes in AD connected with cdk5 upregulation. Using different methods of samples of preparation and fractionation, we identified 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 disease.

4. Analysis of Ca2+-binding proteins under normal and pathological conditions in neurons (Magdalena Blazejczyk, Katarzyna Debowska, Bozena Zebrowska, Adam Sobczak, under the supervision of Urszula Wojda and in collaboration with other laboratories)
Ca2+-binding proteins in neurons regulate neuronal development, plasticity, and neurodegeneration and draw much attention due to implications in multiple brain pathologies including Alzheimer disease. Genomic databases indicated the existence of a novel family of Ca2+-binding proteins called calmyrins (CaMy, known also as KIP or CIB). Calmyrins are evolutionarily conserved from Nematoda to humans. In humans, four genes encode calmyrin proteins (CaMy1 – CaMy4) but until now the only member of the CaMy family that has been analyzed is CaMy1. We have previously demonstrated that CaMy1 is implicated in Alzheimer disease and that it interacts specifically with Alzheimer disease associated presenilin 2 (PS2) in vitro and in vivo (Bernstein et al, Neuropathol Appl Neurobiol. 2005, 31(3):314-24; Blazejczyk et al, Biochim Biophys Acta. 2006;1762(1):66-72). Our results indicate, however, that the interaction of CaMy1with PS2 in neurons is limited and does not account for the involvement of CaMy1 in Alzheimer disease. Therefore, we have undertaken the search for other possible binding protein partners of CaMy1 and using several biochemical methods, we identified a new potential target of CaMy1. Currently, we characterize CaMy1 interaction with its novel protein ligand. Moreover, we pursued fi rst 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 Golgi apparatus and dendrites. Moreover, regulation of CaMy2 expression has been studied in primary cultures of rat neurons. We have also searched for protein ligands of CaMy2 in rat brain and identified several new potential targets of CaMy2. These interactions were confirmed by several in vitro methods, and their physiological significance has become the aim of further studies. In addition, we are analyzing the changes in CaMy1 and CaMy2 biochemical properties, protein ligands binding and in involvement in neuronal functions as a result of neurodegenerative processes.

5. Analysis of proteins involved in Ca2+ homeostasis in neurons (Lukasz Bojarski, Monika Klejman, Joanna Gruszczynska, Anna Skibinska-Kijek, in collaboration with partners from PROMEMORIA 6th FP of EU and from the Polish-German grant)
We study STIM and Orai that are involved in the process of store operated Ca2+ entry. STIM is localized in the ER membrane where it serves as a Ca2+ sensor. Upon ER Ca2+ depletion STIM redistributes into punctuate structures, moves closer to the plasma membrane and activates Orai channels that refi ll Ca2+ stores. In our study we focus on analysis of STIM and Orai proteins in the brain: cellular localization and function. Using immunocytochemistry and other immunofluorescent methods, as well as biochemical analyses of the brain protein extracts, we describe expression pattern of STIM1 and STIM2 in mouse brain. We have developed rabbit polyclonal antibodies recognizing Orai1 protein and are currently optimizing conditions for application in immunohistochemical staining and immunoblotting.

6. Role and regulation of β-catenin in mature neurons (Monika Klejman, Katarzyna Misztal, Anna Skibinska-Kijek, Marta Wisniewska in collaboration with partners from PROMEMORIA 6th FP of EU)
β-catenin plays a crucial role in cell proliferation and development, and is a component of the adherens junctions. In addition to the membrane localized protein there is also a cytosolic pool of β-catenin, which is controlled by phosphorylation and subsequent ubiquitination and degradation. After wnt signaling activation, β-catenin phosphorylation is inhibited, the protein translocates to the nucleus and activates gene transcription as a cofactor of Lef1/Tcf4 transcription factor. We are interested in the function of β-catenin in the adult brain, since new data suggest it might be involved in learning and memory formation, as well as in some brain pathology. β-catenin and Lef1 expression levels were analyzed in the forebrain of adult mice and rats using immunocytochemical staining and immunofluorescent methods, as well as biochemical analysis of the brain protein extracts. Currently, we are exploring the mechanism of constant stabilization of β-catenin in mature thalamic neurons both in vivo and in vitro and are looking for β-catenin/Lef1 target genes in mature neurons.

7. Characterization of biological function of CHORD containing proteins in the nervous system (Wojciech Michowski, Anna Skibinska-Kijek, Kamila Skieterska in collaboration with Guido Tarone from University of Turin)
Two genes for CHORD containing proteins are present in the mammalian genome, melusin and chp-1. Melusin is a protein expressed in heart and skeletal muscles. It specifically senses mechanical stress induced by chronic aortic hypertension, mediates development of adaptive cardiac hypertrophy and protects cardiac muscle from consequences of pressure overload. We have identified melusin as a novel protein target of the S100 Ca2+-signal-sensing proteins. Chp-1 is an ubiquitously expressed protein which functions under stress conditions. It exhibits chaperoning activity and its mutants show mitotic aberrations. Since high level of chp-1 is observed in neuronal tissue, we have decided to explore its function in neurons. We are currently focused on elucidating the mechanisms involved in stress dependent nuclear accumulation of the CHP-1 protein. Using point and deletion mutants we characterize structural elements of CHP-1 that regulate cellular localization of the protein. By means of mass spectrometry we are trying to identify posttranslational modifications occurring in CHP-1 after exposure of cells to stress conditions. We are also determining the chaperoning activity of CHP-1 applying in vitro assays on purified proteins. These studies are complemented by looking at changes of CHP-1 expression pattern in brains of animal models of neurological diseases.

Figure 1.
Primary rat hippocampal neuron transfected with CaMy2-EGFP.