ATM phosphorylation of the actin-binding protein drebrin controls oxidation stress-resistance in mammalian neurons and C. elegans.

Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer's disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.

Investigation of hippocampal synaptic transmission and plasticity in mice deficient in the actin-binding protein Drebrin.

The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes.

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.

Defective actin dynamics in dendritic spines: cause or consequence of age-induced cognitive decline?

Ageing is a complex deteriorating process that coincides with changes in metabolism, replicative senescence, increased resistance to apoptosis, as well as progressive mitochondria dysfunction that lead to an increase production and accumulation of reactive oxygen species (ROS). Although controversy on the paradigm of the oxidative damage theory of ageing exists, persuasive studies in Caenorhabditis elegans and yeast have demonstrated that manipulation of ROS can modify the process of ageing and influences the damage of proteins, lipids and DNA. In neurons, ageing impacts on the intrinsic neuronal excitability, it decreases the size of neuronal soma and induces the loss of dendrites and dendritic spines. The actin cytoskeleton is an abundant and broadly expressed system that plays critical functions in many cellular processes ranging from cell motility to controlling cell shape and polarity. It is thus hardly surprising that the expression and the function of actin in neurons is crucial for the morphological changes that occur in the brain throughout life. We propose that alterations in actin filament dynamics in dendritic spines may be one of the key events contributing to the initial phases of ageing in the brain.

Phosphorylation of the actin binding protein Drebrin at S647 is regulated by neuronal activity and PTEN.

Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus--serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.

Induced tauopathy in a novel 3D-culture model mediates neurodegenerative processes: a real-time study on biochips.

Tauopathies including Alzheimer's disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer's disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner.Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer's disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy.Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates.

Impedance spectroscopy based measurement system for quantitative and label-free real-time monitoring of tauopathy in hippocampal slice cultures.

Alzheimer's disease (AD) and other tauopathies comprise death of cell bodies, synapses and neurites but there is surprising little knowledge of the temporal sequence and the causal relationships among these events. Here, we present a novel biosensoric approach to monitor retrograde neurite degeneration before cell death occurs. We induced tau hyperphosphorylation in organotypic hippocampal slice cultures (OHSC) and applied marker-independent real-time electrical impedance spectroscopy (EIS) for cellular real-time pathology monitoring. Using this approach, we were able to define two distinct phases of neurite degeneration, first a rapid swelling of axonal processes that manifests itself in relative impedance above control levels followed by a slower phase of collapse and subsequent fragmentation indicated by decreased relative impedance below control levels. Initial axon swelling is strictly dose-dependent and swelling intensity correlates with second phase impedance decrease implicating a causative link between both degenerative mechanisms. Moreover, suppressing tau hyperphosphorylation by kinase inhibition nearly prevented both phases of axon degeneration. Our findings demonstrate that the temporal sequence of tau-triggered neurite degeneration can be directly visualized by EIS-based, non-invasive and label-free monitoring. We therefore suggest this approach as a powerful extension of high content applications to study mechanisms of neurite degeneration and to exploit therapeutic options against AD and tau-related disorders.

A novel organotypic tauopathy model on a new microcavity chip for bioelectronic label-free and real time monitoring.

Herewith we developed a novel 3D in vitro Alzheimer's disease (AD) model, based on the human neuroblastoma cell line SH-SY5Y, which is well differentiated without the application of any agents. Furthermore AD-like pathological neurodegeneration can be induced by okadaic acid (OA) mediated hyperphosphorylation of the microtubule associated protein tau. Moreover, we established stable "rapid tauopathy cell lines" expressing additional EGFP-fused (enhanced green fluorescent protein) wildtype or a pathology-promoting mutant tau variant (P301L) by lentiviral transduction. For the sensitive and feasible quantitative detection of pathological effects on neuronal 3D-cultures by electrochemical impedance spectroscopy (EIS) we optimized and redesigned a microcavity array (MCA). The cellular contribution to impedance could be increased by the factor of 2.5 and the variance decreased by 40%. Using our optimized MCA and impedance measurement setup we were able to detect quantitatively an OA concentration- and time-dependent decrease of the impedance in 3D SH-SY5Y cultures. Moreover, we were able to detect and quantify distinct, AD-related effects triggered by tau-mutant (P301L) expression and hyperphosphorylation in our organotypic 3D-cultures with the help of impedance spectroscopy.

An impedimetric microelectrode-based array sensor for label-free detection of tau hyperphosphorylation in human cells.

Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.

Tau kinase inhibitors protect hippocampal synapses despite of insoluble tau accumulation.

A better understanding of the cellular and molecular pathomechanisms of Alzheimer's disease (AD) is a prerequisite for the development of efficient treatments. We have used a novel assay system based on virus-transduced organotypic hippocampal slice cultures that mimics important aspects of tau-driven AD pathology in a short time frame. Human tau P301L, when expressed in pyramidal neurons of hippocampal slice cultures, was increasingly phosphorylated at several disease-relevant epitopes, leading to progressive neuronal dystrophy and formation of RIPA-insoluble tau. AD-like tau hyperphosphorylation was reduced by the tau kinase inhibitors lithium and SRN-003-556, but RIPA-insoluble tau remained unaffected after treatment with any of these substances. Only SRN-003-556 was able to protect hippocampal neurons from synaptic damage that was presumably caused by a toxic soluble tau fraction. These data provide first mechanistic insights towards the functional benefits of SRN-003-556 that have been observed in vivo.

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

Proteasome inhibition arrests neurite outgrowth and causes "dying-back" degeneration in primary culture.

Proteasome inhibitors such as lactacystin were first isolated when assaying their ability to stimulate neurite outgrowth in neuronal-like cell lines; however, their effect on neurites in primary culture has been largely neglected. We report here that lactacystin causes immediate arrest of nerve growth factor (NGF)-stimulated neurite outgrowth in sympathetic and sensory explant cultures. This is followed by neurite degeneration that in sympathetic cultures has a distinctive "dying-back" morphology. Remarkably, this occurs even at concentrations below that required to induce neurite outgrowth in PC12 cells. Thus, lactacystin opposes rather than potentiates the effect of NGF on sympathetic neurite outgrowth and the role of the ubiquitin proteasome pathway in growth and long-term maintenance of axons and dendrites differs from that in neuritogenesis in neuronal-like cell lines. Retrograde degeneration caused by blocking of the ubiquitin proteasome pathway may mimic some aspects of gracile axonal dystrophy, a dying-back axonopathy in mice caused by ubiquitin hydrolase (Uch-l1) deficiency, and may be relevant to human neurodegenerative diseases involving ubiquitination or proteasome abnormalities.

Age-dependent synapse withdrawal at axotomised neuromuscular junctions in Wld(s) mutant and Ube4b/Nmnat transgenic mice.

Axons in Wld(S) mutant mice are protected from Wallerian degeneration by overexpression of a chimeric Ube4b/Nmnat (Wld) gene. Expression of Wld protein was independent of age in these mice. However we identified two distinct neuromuscular synaptic responses to axotomy. In young adult Wld(s) mice, axotomy induced progressive, asynchronous synapse withdrawal from motor endplates, strongly resembling neonatal synapse elimination. Thus, five days after axotomy, 50-90 % of endplates were still partially or fully occupied and expressed endplate potentials (EPPs). By 10 days, fewer than 20 % of endplates still showed evidence of synaptic activity. Recordings from partially occupied junctions indicated a progressive decrease in quantal content in inverse proportion to endplate occupancy. In Wld(s) mice aged > 7 months, axons were still protected from axotomy but synapses degenerated rapidly, in wild-type fashion: within three days less than 5 % of endplates contained vestiges of nerve terminals. The axotomy-induced synaptic withdrawal phenotype decayed with a time constant of approximately 30 days. Regenerated synapses in mature Wld(s) mice recapitulated the juvenile phenotype. Within 4-6 days of axotomy 30-50 % of regenerated nerve terminals still occupied motor endplates. Age-dependent synapse withdrawal was also seen in transgenic mice expressing the Wld gene. Co-expression of Wld protein and cyan fluorescent protein (CFP) in axons and neuromuscular synapses did not interfere with the protection from axotomy conferred by the Wld gene. Thus, Wld expression unmasks age-dependent, compartmentally organised programmes of synapse withdrawal and degeneration.