Laboratory of Neurodegeneration: Kuznicki Laboratory
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 and using zebrafish, and mice as model organisms. Our major projects focus on the following:
1. Calcium homeostasis and calcium signaling
1.1. Role of STIM proteins in store-operated calcium entry in neurons
1.2. Functional and physical interaction between Alzheimer’s disease proteins and store-operated calcium entry machinery
1.3. Function of calmyrins in neuronal physiology and pathology
1.4. Dysregulation of calcium homeostasis in Alzheimer’s disease
2. Search for biomarkers and potential therapeutic targets in lymphocytes from Alzheimer’s disease patients
2.1. Calcium measurements
2.2. Analyses of the cell cycle and apoptosis
3. Role and regulation of β-catenin and transcription factors LEF1/TCF in mature neurons
4. Role of epigenetic mechanisms in regulation of calciumrelated genes involved in neurodegenerative diseases
5. Transgenic mice with dysregulated calcium homeostasis in neurons as a model of aged-induced neurodegeneration
1. Calcium homeostasis and calcium signaling
1.1. Role of STIM proteins in store-operated calcium entry in neurons (Joanna Gruszczyńska-Biegała)
The calcium sensors STIM1 and STIM2, located in the endoplasmic reticulum (ER), and calcium channel-forming protein ORAI1 are involved in store-operated calcium entry (SOCE).
The process relies on extracellular calcium influx through plasma membrane channels. In non-excitable cells, the STIM interaction with ORAI1 is a crucial element of SOCE, but its mechanism remains unclear in neurons. We previously showed that STIM1 is likely involved in thapsigargin-induced SOCE, whereas STIM2 is mostly active after the ethylene glycol tetraacetic acid-driven depletion of extracellular calcium (Gruszczynska-Biegala et al., PLoS One, 2011). The depletion of calcium from the ER by thansigargin increased the number of puncta-like colocalization of YFP-STIM1 and ORAI1 but not YFP-STIM2 and ORAI1. In contrast, a reduction of extracellular calcium levels triggered puncta formation for both YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1. As a next step, we focused on detecting complexes that contain endogenous STIM2 and ORAI1. Using a proximity ligation assay (PLA), we were able to visualize fluorescent dots that represent the site where two antibodies are bound: one against ORAI1 and another against STIM2. These dots identified the complexes between STIM2 and ORAI1. To confirm that the observed PLA dots represented authentic STIM2-ORAI1 complexes, we used different pairs of anti-STIM2 and anti-ORAI1 antibodies. The number of these complexes increased when intracellular and subsequently ER calcium concentrations decreased under the influence of BAPTA-AM or medium without calcium ions. These results were confirmed by co-immunoprecipitation of endogenous STIM2 and ORAI1 proteins. We also showed a strong correlation between the number of endogenous STIM2-ORAI1 complexes and calcium responses studied in the same neuronal cell. Our results indicated that STIM2 responds to changes in intracellular calcium levels and the small decrease in calcium levels in the ER in rat cortical neurons by interacting with ORAI1.
1.2. Functional and physical interaction between Alzheimer’s disease proteins and store-operated calcium entry machinery (Tomasz Węgierski, Kinga Gazda)
The genetic manipulation of proteins linked to Alzheimer’s disease (AD) results in disturbances in cellular calcium homeostasis. Specifically, alterations in the receptor-induced release of calcium from the ER and SOCE have been described.
These observations support the calcium hypothesis of the development of AD, but the precise mechanisms that underlie the dysregulation of calcium homeostasis in AD models are unclear. We aim to elucidate these mechanisms, particularly whether AD proteins exert a direct regulatory effect on key players of calcium homeostasis, such as the SOCE complex.
Using a split-ubiquitin system (i.e., a yeast genetic system) to search for interacting partners, we found a physical interaction between SOCE machinery and proteins crucially involved in the development of neurodegeneration. The interaction was confirmed using independent methodology, such as coimmunoprecipitation and co-immunolocalization assays. The functional relevance of this finding is being studied using overexpression systems and ablation of gene expression by RNA interference in various cell lines. We analyze the regulation of SOCE complexes using PLAs and calcium measurements with the help of the calcium indicator fura-2.
Fig. 1. DAB-staining of mice brain slices visualizing major component of Myelin Sheet – Myelin Basic Protein (MBP) in Hippocampus and Cortex (Author Łukasz Szewczyk).
1.3. Function of calmyrins in neuronal physiology and pathology (Katarzyna Dębowska; supervisor: Urszula Wojda)
Neuronal Ca2+ signaling regulates multiple cellular functions. Therefore, disturbances in Ca2+ signaling pathways can result in neuronal pathologies. We study the neuronal function of a novel family of Ca2+ signaling proteins called calmyrins (CaMy; also known as KIP or CIB proteins). We characterized the biochemical properties, localization, and protein ligands of CaMy1 and CaMy2 in the brain and showed that CaMy1 is involved in AD (BBA-Mol Cell Res, 2011; Arch Biochem Biophys, 2009; Calcium Binding Proteins, 2008; BBA-Mol Mech Diseases, 2006; Neuropathol Appl Neurobiol, 2005; Acta Biochim Pol, 2005).
Moreover, we identified the SCG10 protein stathmin2 as a novel CaMy1 ligand in the human brain. SCG10 is a microtubuledestabilizing factor that plays a role in neuronal growth during brain development. Our study demonstrated that CaMy1, via SCG10, coupled Ca2+ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain (BBA-Mol Cell Res, 2011). More recently, we searched for the neuronal localization and function of another CaMy family member, CaMy2. We found that CaMy2 was preferentially expressed in neurons in the hippocampus and cortex. Endogenous CaMy2 was present in neurites and the Golgi apparatus and was found in the membranous fraction. Our search for CaMy2 protein ligands in neurons using affinity chromatography, mass spectrometry, and co-immunoprecipitation approaches revealed that CaMy2 interacted with key proteins involved in vesicular trafficking in vitro and in vivo, consistent with subcellular localization in neurons. Moreover, using RNA interference in primary hippocampal cultures, we demonstrated that CaMy2 affected the localization of early endosomes and endocytosis of neuronal surface receptors.
1.4. Calcium homeostasis in Alzheimer’s disease (Aleksandra Szybińska, Anna Jaworska, and Tomasz Węgierski; collaboration: Honarnejad Kamran and Jochen Herms, Munich Center for Neurosciences)
Calcium dyshomeostasis is an early event in the pathogenesis of AD that precedes other disease symptoms and can affect many cellular processes. Drugs with the ability to restore calcium homeostasis to values observed in healthy control cells could be applied as therapeutics in AD. In collaboration with Prof. Jochen Herms, we screened approximately 20,000 chemical compounds to determine their ability to influence intracellular calcium concentrations. The screen revealed over 300 compounds that decreased calcium levels. To address their putative mechanism of action, almost 160 of the best compounds were chosen for an enzyme-linked immunosorbent assay (ELISA) for γ-secretase activity, whose gain of function is believed to be a major factor in familial AD pathology. Using ELISA, we measured β-amyloid 1-42 levels in HEK 293 cells that overexpressed the wildtype or mutated presenilin 1 gene. Only a few compounds decreased β-amyloid 1-42 to control levels; thus, the majority of the compounds that influenced calcium signaling did not affect γ-secretase activity.
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 studying dynamic processes in postmortem 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.
2.1. Calcium measurements (Anna Jaworska)
Many studies have shown that disturbed cellular calcium homeostasis is one of the key features of AD. Calcium changes can be observed not only in neurons but also in peripheral cells, such as skin fibroblasts and lymphocytes. Lymphocytes, in contrast to other cell types, can be easily obtained and therefore have great diagnostic potential. Disturbed calcium handling was found by many research groups in immortalized human B-lymphocytes derived from patients with an inherited form of AD (i.e., familial AD), but observations of similar changes observed in cells derived from patients with the sporadic form of AD (SAD) are very limited. Mild cognitive impairment (MCI) is found to be a transitional stage between normal aging and dementia. It is often observed in individuals who develop AD later in life and therefore may be considered a risk factor for AD. To explore calcium homeostasis during the early stages of SAD and MCI, we investigated SOCE and inositol triphosphate receptor (IP3R)-mediated calcium release into the cytoplasm in unmodified B-lymphocytes from MCI subjects and SAD patients and compared them with non-demented subjects (NDS). Calcium levels in the endoplasmic reticulum were significantly similar in all three groups. However, we found that SAD and MCI cells were more prone to IP3R activation than NDS cells. Mild cognitive impairment cells exhibited an enhanced magnitude of calcium influx during SOCE, and MCI cells but not SAD cells were characterized by higher basal cellular calcium levels than NDS cells. In summary, perturbed calcium homeostasis was observed in peripheral cells from MCI and SAD patients, supporting the hypothesis that SAD is a systemic disease, and MCI is a risk factor for AD. Thus, lymphocytes obtained from MCI subjects may be promising in the early diagnosis of individuals who will eventually develop SAD (BBA MCR, 2013).
2.2. Analyses of the cell cycle and apoptosis (Emilia Białopiotrowicz and Katarzyna Sawicka; supervisor: Urszula Wojda)
According to the so-called cell cycle (CC) hypothesis, an important factor that contributes to the pathogenesis of AD is the failure to regulate the G1/S phases of the cell cycle and CC reentry in differentiated, postmitotic neurons. Recently, we and others detected CC alterations in lymphocytes from SAD patients (Bialopiotrowicz et al., Neurobiol Aging, 2011).
Our 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 is different from FAD. Moreover, disturbances of the CC in lymphocytes appear to have diagnostic value. Furthermore, we analyzed the effects of nine different PS1 mutations on CC regulation and Aβ production in immortalized lymphocytes from FAD patients and stably transfected human embryonic kidney cells. PS1 sustains the active site of γ-secretase, a membranous protein complex that cleaves transmembrane amyloid protein precursor (APP) to generate Aβ40 and Aβ42 peptides that in turn exert toxic effects in neurons. Mutations in PS1 that cause FAD increase the γ-secretase-mediated release of Aβ from APP. We found that both CC regulation and Aβ production differentiated PS1 mutations and that CC PS1 activity was mediated by p53/p21 signaling but not γ-secretase activity.
The identified CC dysregulation linked with increased p53 and p21 protein levels distinguished the highly pathogenic PS1 P117R mutation and may contribute to the specific severity of the clinical progression of FAD associated with the mutation in the PS1 117 site (Fig 2). These findings suggest that impairment in the lymphocyte CC might play a pathogenic role in AD and may be relevant to the development of new diagnostic approaches and personalized therapeutic strategies (Bialopiotrowicz et al., J Alzheimers Dis, 2012).
Fig. 2. Schematic representation of the mechanism leading to G1 phase prolongation and simultaneous S phase shortening in human lymphocytes harboring PS1 P117R mutation. In cells bearing PS1 P117R the levels of p53, p21 and cyclin D1 are high. Increase of cyclin D1 protein level might additionally positively stabilize p21 protein by protecting it from proteasomal degradation and thus potentiate p21 elevation. Increased levels of p53, p21 and cyclin D1 result in prolongation of G1 phase and shortening of S phase observed in PS1 P117R cells. The CC changes may affect functions of B lymphocytes and in this way may contribute to the specific severity of the clinical progression of FAD associated with the mutation in the PS1 117 site.
3. Role and regulation of β-catenin and transcription factors LEF1/TCF in mature neurons (Katarzyna Misztal, Andrzej Nagalski, Łukasz Szewczyk, and Nikola Brożko; supervisor: Marta B. Wiśniewska)
β-catenin is a gene expression regulator in the canonical Wnt pathway that is involved in early brain patterning and neurogenesis. Growing evidence also implicates Wnt/β-catenin signaling in the proper functioning of the adult central nervous system. The aberrant regulation of β-catenin has been associated with psychotic and affective disorders (e.g., major depression, bipolar disorder, and schizophrenia) and neurodegenerative diseases (e.g., AD, Huntington’s disease [HD], and Parkinson’s disease). However, the physiological role of Wnt/β-catenin in the adult brain remains elusive. Pioneering research by our group demonstrated that β-catenin is constitutively and specifically present in the nuclei of thalamic neurons, independent of Wnt signaling activation, and associated with low levels of the proteins involved in β-catenin degradation (i.e., APC, AXIN1, and GSK3β; Misztal et al., J Biol Chem, 2011). Moreover, we demonstrated that β-catenin, together with LEF/TCF transcription factors, regulated the transcription of the Cacna1g gene that encodes Cav3.1 voltage-gated calcium channels, contributing to electrical signal propagation in thalamic neurons (Wisniewska et al., J Neurosci, 2010). Recently, we identified new β-catenin target genes in thalamic neurons by combining bioinformatics and experimental approaches: Gabra3 for the GABA receptor, Calb2 for the Ca2+-binding protein calretinin, and Kcna6 for the voltage-gated potassium channel; Wisniewska et al., BMC Genomics, 2012). Two other genes, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, but the binding of β-catenin to the regulatory sequences of these genes could not be confirmed. We conclude that β-catenin in the thalamus regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of β-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies. Additionally, we provided a comprehensive analysis of LEF1/TCF protein localization and the expression profile of their isoforms in cortical, thalamic, and midbrain regions in mice. The analysis of alternative splicing and promoter usage identified brain-specific TCF7L2 isoforms and revealed a developmentally coordinated transition in the composition of LEF1 and TCF7L2, suggesting that the role of these proteins in the adult brain might be different from their role in the embryonic brain.
4. Role of epigenetic mechanisms in the regulation of calcium-related genes involved in neurodegenerative diseases (Magdalena Czeredys; EraNet Russ project with Axel Methner, University of Mainz, Germany, and Elena Kaznacheyeva, St. Petersburg, Russia)
Calcium dyshomeostasis is an early event in the pathogenesis of neurodegenerative diseases. The literature suggests that epigenetic mechanisms, including DNA methylation and microRNA, can regulate gene expression in neurodegenerative diseases. Therefore, we focused on the expression of calciumrelated genes in AD and HD. We hypothesized that some epigenetic changes might affect the expression of components of calcium homeostasis and signaling pathways, thereby initiating or propagating the neurodegenerative processes of AD and HD.
To test this hypothesis, we analyzed mRNA levels in the brains of transgenic AD and HD mice using custom-made TaqMan Low Density Microarrays. Some genes whose expression was changed compared with control brains were further analyzed.
5. Transgenic mice with dysregulated calcium homeostasis in neurons as a model of age-induced neurodegeneration (Łukasz Majewski)
The vast majority of available animal models of AD are based on the β-amyloid/tau hypothesis. These mice overexpress one or more mutated proteins known to be responsible for the early onset of FAD. The FAD models, representing less than 5% of all human cases, appear to have little value for understanding the mechanisms of SAD. Our main research objective is to generate and characterize transgenic mouse models that have features characteristic of SAD. Transgenic mice with dysregulated Ca2+ homeostasis will be a suitable model for verifying the hypothesis that sustained increases in basal Ca2+ levels might be one of the early changes that lead to neurodegeneration.