Laboratory of Neurodegeneration: Kuznicki Laboratory
Current Projects
We are interested in the molecular mechanisms involved in neurodegeneration and memory formation, with a special emphasis on the role of calcium homeostasis and signaling. These processes are being studied at the genomic, proteomic, and cellular levels. Our major projects focus on:
1. Calcium homeostasis and calcium signaling:
1.1. Role of STIM proteins in store-operated calcium entry in neurons
1.2. Function of calmyrins in neuronal physiology and pathology
1.3. Dysregulation of calcium homeostasis in Alzheimer’s disease
2. Biomarkers and potential therapeutic targets in Alzheimer’s disease, based on:
2.1. Cell cycle analyses
2.2. Mutated p53
3. Role and regulation of β-catenin/Lef1 complex in mature neurons
1. Calcium homeostasis and calcium signaling
1.1. Role of STIM proteins in store-operated calcium entry in neurons (Joanna Gruszczyńska-Biegała, Aleksandra Szybińska, Tomasz Węgierski)
The interaction between Ca2+ sensors STIM1 and STIM2 and Ca2+ channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. To gain insights into the neuronal function of STIM proteins, we focused on identifying their interacting partners in the brain by undertaking both a proteomic approach, using mass spectrometry, and a yeast genetics approach, using a split-ubiquitin screening system. The identified hits are currently being analyzed (Danuta Korona, Joanna Gruszczyńska-Biegała, Tomasz Węgierski).
In parallel, we tested the hypothesis that the mechanism of SOCE in neuronal cells is based on STIM proteins and that alterations of SOCE may lead to pathology, such as Alzheimer’s disease. Our data indeed indicate that STIM1 and STIM2 proteins are involved in calcium homeostasis in neurons. Real-time PCR from cortical neurons proved that these cells contain significant amounts of Stim1 and Stim2 mRNA. In cultured cortical neurons that overexpress YFP-STIM1, YFP-STIM2, and ORAI1, we found that calcium depletion from the endoplasmic reticulum (ER) increased the number of STIM1/ORAI1 puncta much more than STIM2/ORAI1 puncta. In contrast, a reduction of extracellular calcium levels triggered puncta formation to a greater number for YFP-STIM2/ORAI1 than for YFP-STIM1/ORAI1. Our preliminary data indicate that the number of endogenous STIM2/ORAI1 complexes visualized by the Proximity Ligation Assay (Fig. 1) was enhanced by EGTA treatment. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. Our results indicate that STIM1is the main activator of TG-induced SOCE in neurons, whereas STIM2 regulates resting Ca2+ levels and activates constitutive calcium entry (Gruszczynska-Biegala et al., PLOS one, 2011).
Fig. 1. Analysis of the complexes formed from endogenous STIMs and ORAI1 using the Proximity Ligation Assay. Cortical neurons treated with 2 mM Ca2+ or 2 mM EGTA were incubated with mouse anti-STIM1 and rabbit anti-ORAI1 or goat anti-STIM2 and rabbit anti-ORAI1. The PLA signal, visualized as a fluorescent red dot, suggests the close proximity of STIM and ORAI1 antigens. Sample cell images with an overlay of fluorescence signal over phase contrast show that endogenous STIM and ORAI1 complexes are localized in the cells.
1.2. Function of calmyrins in neuronal physiology and pathology (Katarzyna Dębowska; supervisor: Urszula Wojda)
Fig. 2a. Cell cycle aberrations in lymphocytes from patients with Alzheimer’s disease. Schematic illustration of aberrant G1 phase cell cycle regulators identified in SAD lymphoblasts. The most significant change was detected in p21 protein levels.
Fig. 2b. Cell cycle aberrations in lymphocytes from patients with Alzheimer’s disease. Increased percentage of cells in G1 phase in lymphoblasts from SAD patients detected by flow cytometry upon labeling with propidium iodide.
Multiple neuronal functions rely on calcium signaling mediated by a group of Ca2+-binding proteins, such as calmodulin and calcineurin. We study the neuronal function of a novel family of Ca2+-signaling proteins called calmyrins (CaMy, known also as KIP or CIB proteins). We characterized the biochemical properties and localization of CaMy1 in the brain and found that CaMy1 is involved in Alzheimer’s disease (Calcium Binding Proteins 2008; BBA-Mol Mech Diseases 2006; Neuropathol Appl Neurobiol 2005; Acta Biochim Pol 2005).
Additionally, we identified the SCG10 protein (stathmin2) as a novel CaMy1 ligand in developing human brain. SCG10 is a microtubule-destabilizing factor that plays a role in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels.
In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca2+-dependent in vivo. CaMy1 interfered with SCG10 activity in a microtubule polymerization assay, and CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factorstimulated PC12 cells. Altogether, these data suggest that CaMy1, via SCG10, couples Ca2+ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain (Sobczak et al., BBA-Mol Cell Res 2011 Jan 5. [Epub ahead of print]). We further investigate the physiological significance of CaMy1/SCG10 interactions in developing neurons. Moreover, we pursued studies on CaMy2. CaMy2 transcript and protein were detected mainly in the hippocampus and cortex in the rat brain. Studies of rat primary hippocampal neurons showed that CaMy2 levels are controlled by NMDAR and Ca2+ and suggest a role for CaMy2 in the Ca2+ signaling that underlies NMDAR activation (Arch Biochem Biophys 2009). We cloned CaMy2 from the rat brain and demonstrated that CaMy2 binds Ca2+ and exhibits features of the Ca2+-sensor protein. We also identified new potential targets of CaMy2 in the rat brain by affinity chromatography followed by mass spectrometry and confirmed these interactions by several methods in vitro. The physiological significance of these interactions in neurons is currently under investigation.
1.3. Calcium homeostasis in Alzheimer’s disease (Aleksandra Szybińska, Anna Jaworska, and Tomasz Węgierski; collaboration: Honarnejad Kamran and Jochen Herms, Munich Center for Neurosciences)
Many studies showed that disturbed cellular calcium homeostasis is one of the features of Alzheimer’s disease (AD).
Calcium dyshomeostasis is an early event in AD pathogenesis that precedes other disease symptoms and can affect many cellular processes. Thus, finding a drug that is able to restore normal calcium signaling is important. In collaboration with Dr. Jochen Herms, we started a novel approach using highthroughput screening of chemical compounds based on intracellular calcium level measurements. The screen was conducted on several stably transfected HEK 293 cell lines that bear human mutated or wildtype PS1 and FRET-based fluorescent calcium sensor Yellow Cameleon 3.60. The created cell lines were subjected to treatment with a set of compounds. Changes in their fluorescence levels, reflecting the calcium response, were measured using an OPERA system. Preliminary data were presented at the Society for Neuroscience meeting in San Diego in 2010.
2. Search for biomarkers and potential therapeutic targets in lymphocytes from Alzheimer’s disease patients
Some molecular changes in AD can be observed not only in neurons, but also in peripheral cells, such as lymphocytes.
Because of difficulties in studying dynamic processes inpostmortem material, such peripheral cells have been used as a model to study the molecular mechanisms of AD.
Additionally, human lymphocytes have potential diagnostic value. In our studies, we use B-lymphocytes from AD patients immortalized with EB-virus.
2.1. Cell cycle analyses (Emilia Białopiotrowicz; supervisor: Urszula Wojda)
Mounting evidence indicates that the aberrant expression of cell cycle molecules in the brain contributes to the development of AD and causes neuronal death. We analyzed whether cell cycle alterations occur in lymphocytes from patients with sporadic and familial forms of AD with distinct PS1 mutations (SAD and FAD). The results of our experiments using real-time PCR arrays, immunoblotting, and flow cytometry demonstrated differences in the regulation of G1/S phases between SAD lymphocytes and cells from non-demented subjects, as well as between SAD and FAD cells. Compared with FAD lymphocytes, SAD lymphocytes showed differences in the expression profiles of 90 cell cycle genes, such as the genes encoding cyclin D and cyclin E, and a marked increase in the level of the p21 protein, which promotes G1-arrest. Accordingly, SAD but not FAD cells had a prolonged G1-phase. These data showed that SAD involves a prolongation of the G1 phase driven by the p21 pathway, which is not activated in FAD cells. Thus, the mechanism of SAD differs from FAD (Bialopiotrowicz et al., Neurobiol Aging 2010). We are continuing studies of the molecular and cellular aspects of cell cycle regulation in lymphocytes obtained from AD patients and investigate whether these cells may have diagnostic potential.
2.2. Mutated p53 (Aleksandra Szybińska; cooperation: Maurizio Memo and Daniela Uberti, University of Brescia)
Our collaborative studies revealed an increased level of conformationally altered p53 protein in immortalized B lymphocytes from patients with sporadic and familial AD compared with lymphocytes from healthy controls. Thus, the conformational p53 mutant may be used as a marker to discriminate between AD and non-AD individuals (C. Lanni et al., Mol Psych 2008). Because the p53 conformational tertiary structure is influenced by the redox status of cells, an evaluation of the oxidative profile of these patients was performed. We found that among the markers of oxidative stress, hydroxytransnonenal-modified proteins were significantly increased in FAD patients. Furthermore, in addition to increased levels of oxidative markers, the antioxidant defense mechanisms were compromised in these patients because of decreased enzyme levels, such as superoxide dismutase. We also measured p53-regulated CD44 gene expression levels in lymphocytes from AD patients and healthy controls. Alzheimer’s disease lymphocytes showed significantly higher CD44 levels compared with controls, with increased misfolded p53 levels.
This finding may suggest that interactions between these two proteins can influence the peripheral immunological response during disease development (Uberti et al., Neurodeg Dis 2010). These results support the evidence of an association between peripheral unfolded p53 and AD pathology and indicate that immortalized peripheral cells from AD patients are suitable models for the identification of disease markers.
3. Role and regulation of nuclear β-catenin in mature neurons (Katarzyna Misztal, Andrzej Nagalski, Mateusz Ambrożkiewicz, and Nikola Brożko; supervisor: Marta B. Wiśniewska)
β-catenin participates in two distinct functions in a cell, serving as (i) a component of cadherin-based adherens junctions, and (ii) a gene expression regulator in canonical Wnt signaling as a cofactor of LEF1/TCF transcription factors. In the developing brain, nuclear β-catenin activates genes involved in the proliferation and differentiation of neuronal precursor cells. Interestingly, aberrant regulation of the Wnt pathway in the adult brain has been associated with neurodegenerative diseases and mood disorders.
However, the issue of the physiological role and regulation of the Wnt/β-catenin pathway in mature neurons is far from
resolved.
We recently demonstrated that, specifically in thalamic neurons of the adult brain, β-catenin is constitutively nuclear.
By exploring the mechanism of this phenomena, we found that neither disruption of the thalamic environment nor inhibition of Wnt/Dishevelled signal transduction affects nuclear levels of β-catenin in these cells, suggesting the existence of a mechanism that operates downstream of the WNT receptor. We reported that the β-catenin degradation rate is lower in thalamic neurons than in cortical neurons, which appears to be a consequence of low level of the β-catenin degradation complex (APC/AXIN1/GSK3β). Thus, the nuclear localization of β-catenin in thalamic neurons appears to be a cell-autonomous and cell-intrinsic feature (Misztal, Wisniewska, Ambrozkiewicz, Kuznicki, under revision).
We also showed that β-catenin, together with LEF1 transcription factor, regulates the Cacna1g gene, encoding the Cav3.1 T-type calcium channel subunit that contributes to electrical signal propagation in thalamic neurons and is involved in epilepsy (Wisniewska et al., J Neurosci 2010). Our main goal is to further identify new β-catenin-LEF1/TCF target genes, specific for neurons, which may help understand the role of nuclear β-catenin in the adult brain.
We perform in silico analyses (e.g., screening of conserved noncoding sequences for transcription factor motifs) and analyses of spatially correlated gene expression in the brain.
These investigations are performed in collaboration with Dr. Michal Dąbrowski, Nencki Institute, Warsaw. We also use custom-designed PCR arrays to profile gene expression in the brain and adenovirus-transduced primary neurons (Fig. 3). To experimentally confirm the actual targets, we perform luciferase assays, footprinting, and chromatin immunoprecipitation.
Fig. 3. Localization of β-catenin in cortical neurons treated with LiCl and overexpressing LEF1/TCF7L2. Cortical primary cultures were labeled with β-catenin-specific mouse monoclonal antibody. (A) Control neurons. (B) Neurons treated with 10 mM LiCl. (C) Neurons transduced with LEF1 and TCF7L2 expressing adenoviruses and treated with 10 mM LiCl.