Department of Neurobiology, University of Osnabrück

Molecular mechanisms of neuronal development, aging and degeneration

(AG Roland Brandt)


Image "Research:CA1neuron.jpg"

Fig. 1: Expression of a fluorescent marker protein in an individual neuron from a brain slice. Note the small dendritic protrusions ("spines"), which represent the primary site of excitatory inputs (see also insert with an enlarged dendritic segment).

Neurons are one of the most extreme cell types in that they contain processes which can reach a meter or longer, contain more than 99% of the cellular volume, and provide the basis for innumerable synaptic input (Fig. 1). This requires the presence of a sophisticated molecular machinery in order to establish and maintain such a morphology. The cytoskeleton is the major intraneuronal structure that determines the shape of a neuron. From that it is not surprising that cytoskeletal mechanisms have an important role during the development of neurons and that abnormalities in the cytoskeletal organization are a hallmark of many neurodegenerative diseases. The group concentrates on the function of microtubules and their associated proteins, on neurofilaments and on the membrane cortex during neuronal development and neurodegeneration. In particular, a major part of the group concentrates on studying the involvement of the neuronal microtubule-associated protein tau during neurodegenerative processes in Alzheimer's disease and other tauopathies.



1. Cytoskeletal mechanisms of neurodegenerative diseases


The assembly of microtubules is regulated largely by microtubule-associated proteins (MAPs). From the neuronal MAPs, the tau proteins have attracted particular interest due to their potential role in neurodegenerative disorders ("tauopathies") including Alzheimer's disease (AD) (for reviews see Brandt et al., 2005; Bakota and Brandt, 2016).

In collaboration with the laboratory of Dr. Gloria Lee (Harvard Medical School, Boston, USA) we could show that tau not only binds to microtubules but interacts with neural plasma membrane components through its aminoterminal non-microtubule-binding projection domain (Brandt et al., 1995). Tau's interactions are critically affected by AD-relevant phosphorylation events suggesting that they are differentially influenced by signal transduction mechanisms during neuronal development and degeneration (Maas et al., 2000). In a collaborative approach with the Heidelberg-based proteomic company Cellzome and the ZMBE Münster we could show that tau interacts with the membrane component annexin A2 (AnxA2) and that this interaction is blocked by a tauopathy mutation (Gauthier-Kemper et al., 2011). The data suggest that impaired membrane binding, which critically involves annexins as membrane-cytoskeleton linkers, contributes to the pathological effects in tauopathies such as AD.

To understand the role of tau during the development of tauopathies we have created tau constructs in which we mutated disease-relevant phosphorylation sites to simulate a permanent, high stoichiometric tau phosphorylation characteristic for AD ("pseudohyperphosphorylated (PHP)-tau"; Eidenmüller et al., 2000). Using virus-mediated gene-transfer in human model neurons ("hNT neurons") and primary neurons we could show that PHP-tau exerts a neurotoxic effect which is closely associated with an induction of apoptosis (Fath et al., 2002). Furthermore, additional factors such as presenilin 1 and amyloid beta inversely modulate tau-dependent neurodegeneration at distinct steps (Leschik et al., 2007). The data indicate a "gain-of-toxic-function" of modified tau during AD and point towards a critical role of pathologic tau phosphorylation in disease progression. By targeted expression of tau in ex vivo cultures we could show that PHP-tau causes progressive neuronal degeneration in an authentic CNS environment (Shahani et al., 2006).

Most neurodegenerative diseases are directly or indirectly associated with changes in the dynamics of cytoskeletal components (Bakota and Brandt, 2010). A major part of our recent work concentrates on the development of novel “live cell imaging” approaches in combination with molecular modeling to analyze protein dynamics in neurons (Weissmann et al., 2009). We developed methods to quantitatively determine cytoskeletal dynamics from live cell imaging experiments (Gauthier and Brandt, 2010, Igaev et al., 2014). Using single molecular tracking of tau in living neurons, we could show that tau interacts with microtubules by a "kiss and hop" mechanism (Janning et al., 2014) and that tau modifications affect tau's microtubule interactions in a potentially disease-relevant manner (Niewidok et al., 2016). In addition, we develop and employ imaging techniques to visualize and evaluate neuronal morphology and individual dendritic spines using algorithm-based methods (Tackenberg and Brandt, 2009). We could show that amyloid beta and tau cooperate to induce dendritic simplification through dysregulation of microtubule dynamics (Golovyashkina et al., 2015) and provided evidence that stabilization of microtubules by subnanomolar concentrations of epothilone D reverses amyloid beta-induced spine loss (Penazzi et al., 2016).

As a second major effort, we focus on developing novel animal models for tau pathologies. In collaboration with the laboratory of Dr. Harald Hutter (Simon Fraser University, Burnaby, Canada), we developed a Caenorhabditis elegans model of tau hyperphosphorylation (Brandt et al., 2009). In addition, we developed a mouse model, which expresses disease-like modified tau (PHP-tau) in forebrain neurons (Hundelt et al., 2011).

Image "Research:Tau-schematic.jpg"

Fig. 2: Tau and the neurodegenerative triad in Alzheimer's disease (from: Bakota and Brandt (2016)).



2. Molecular mechanisms of neuronal development and aging


In a combined biochemical-immunological approach we have generated a panel of novel monoclonal antibodies against cytoskeletal and membrane cortex proteins from human neurons. Currently we utilize several of the new antibodies as efficient tools to analyze the role of individual cytoskeletal components during neuronal development.

One of the antibodies specifically detects the protein gravin, a high-molecular-weight scaffolding protein that associates with the membrane cortex of various cell types and which appears to be involved in the autoimmune disease Myasthenia gravis. We could show that gravin distributes in putative "signalling complexes" in selected regions of developing human neurons where it may have a role in localizing kinases to neuronal branching points (Piontek et al., 2003). Using virus-mediated gene transfer of function blocking gravin fragments we currently analyze the role of gravin during neuronal morphogenesis.

In a second project, we have utilized an antibody that specifically detects an O-glycosylated epitope in neurofilament M (NF-M). We could show that O-glycosylation of NF-M is inversely regulated to phosphorylation and is drastically reduced in an animal model of amyotrophic lateral sclerosis suggesting a disturbance of neurofilament O-glycosylation during disease progression (Lüdemann et al., 2005). The antibody provides a useful tool to determine disease- and age-related changes of cytoskeletal proteins. E.g., in collaboration with the group of Dr. Cheng-Xin Gong (New York State Institute for Basic Research in Developmental Disabilities, Staten Island, USA), we could show that O-glycosylation of NF-M is dysregulated in AD (Deng et al., 2007).

In a third project, we collaborated with Dr. Helmut Rosemeyer (Institute of Chemistry, University of Osnabrück) and the lab of Christian Kaltschmidt (University of Bielefeld) to identify new, biologically active "small molecules" and to determine their effect on neural stem cell differentiation. Here, we used some of the antibodies to monitor the effect of nucleoside-based compounds as potential candidate molecules on the differentiation of human NT2 cells as a model for neural stem cell differentiation (Raasch et al., 2015).



3. Identification of novel regulators of microtubule assembly


Microtubule assembly has to be temporarily and spatially precisely regulated in order to establish the characteristic cytoskeletal organization found in all eukaryotic cells. It is likely that mechanisms exist which regulate the pool of assembly-competent microtubule protein by binding to tubulin subunits. Using a biochemical screen to identify neuronal factors which may regulate microtubule assembly by binding to tubulin subunits we could identify an activity from neural cells, which specifically inhibits microtubule nucleation and was thus named MINUS (for "microtubule nucleation suppressor") (Fanara et al., 1999).

To further characterize MINUS, an effective purification scheme has been established to purify the factor from various sources including C. elegans, Drosophila and yeast (Shahani et al., 2006). Current research concentrates on sequencing the factor and analyzing its distribution and function in cells.



Selected References

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Doctoral Theses