Individual Projects

A01:  Activity-Dependent Regulation of Apoptosis in Developing Rodent Cerebral Cortex.
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luhmann-heiko

Heiko Luhmann Prof. Dr. rer. nat.
Institute of Physiology
University Medical Center of the JGU Mainz
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anne-sinning

Anne Sinning, Dr. rer. nat.
Institute of Physiology
University Medical Center of the JGU Mainz
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Programmed cell death (apoptosis) is an important physiological process during early development, ensuring a proper balance between excitatory and inhibitory neurons. In the brain, apoptosis is regulated by a number of extrinsic (e.g. neurotrophic factors, TNF-alpha) and intrinsic signals (Caspase-3 is of central relevance). We demonstrated that spontaneous synchronized network activity in developing neocortical networks plays a central role in the control of apoptosis in vitro and in vivo. Upon experimentally induced inflammation TNF-alpha is rapidly upregulated, spontaneous activity patterns become less synchronized and aCasp3-dependent apoptosis increases significantly within a few hours. When TNF-alpha is neutralized synchronized activity patterns are preserved and apoptosis is prevented.

In neurons undergoing apoptosis during the first postnatal week (Cajal-Retzius neurons), we identified a novel apoptosis pathway, which depends on NKCC1-mediated excitatory GABA action and p75NTR receptor activation. To obtain a detailed understanding of the events that will eventually trigger apoptosis in some cells while others are maintained, the challenge is to monitor individual cells over extended periods of time. Having established the technology for simultaneous recording of extracellular spikes from tens of neurons with multi-electrode arrays (MEA) and identification of active single neurons with calcium imaging we now can investigate activity-dependent apoptosis with single cell resolution followed by subsequent molecular analyses.

For the second funding period we propose to study (1) the mechanisms underlying remarkable region- and time-specific differences in the pattern of activity-dependent apoptosis in the developing mouse cerebral cortex. (2) The molecular mechanisms underlying activity-dependent apoptosis will be investigated in vitro at the single cell level by combining 120-channel MEA recordings, GCaMP6 calcium imaging and optogenetic single neuron stimulation. (3) We propose to study the development of neocortical cultures with different ratios of excitatory to inhibitory neurons, which spontaneous activity patterns these different cultures develop and which impact these induced changes in E-I imbalance have on apoptosis. These experiments will shed light on the molecular, cellular and network mechanisms underlying homeostatic regulation of physiological activity patterns and their influence on apoptosis in the developing cerebral cortex.

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A03:  EGFL7 & progranulin in neurogenesis: a notch above as a duo.5x800-white-divider

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Mirko HH Schmidt
, Prof. Dr. rer. nat.
Institute for Microscopic Anatomy and Neurobiology
University Medical Center of JGU Mainz
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Irmgard Tegeder, Prof. Dr. med.
Institute for Clinical Pharmacology
Goethe-University Frankfurt

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Neuronal homeostasis in the adult brain, i.e., the balance between the loss and gain of neurons, is sustained by a process termed adult neurogenesis. In the previous funding period the regulation of this cellular type of homeostasis by the non-canonical Notch ligand epidermal growth factor-like protein 7 (EGFL7) has been studied in the hippocampus and the subventricular zone by project A03 with a focus on neural stem cell (NSC) biology (Project-related publication PR1). In parallel, the influence of the secreted protein progranulin (PRGN) on neuronal repair subsequent to axonal injury has been analysed by project A09. In this context, PGRN was commonly identified by both projects as a novel ligand of Notch receptors (common publication PR2). Briefly, the loss-of-EGFL7 in young adult EGFL7-/- mice decreased neurogenesis but enhanced neuronal longevity. Deficiency of PGRN, however, increased neuronal death after injury and caused long-lasting and irreversible cognitive deficits. In our collaborative studies we discovered that both proteins directly interacted and converged on Notch receptor signalling, suggesting a mutual or additive interaction of EGFL7 and PGRN. In order to explore this finding in more detail, both projects have been fused in order to understand the role of the EGFL7-Notch-PGRN interface in the homeostatic mechanisms of adult neurogenesis, neuronal survival and repair.

In the next funding period we will focus on studying how EGFL7 and PGRN govern the survival of adult-born neurons in the hippocampus and dorsal root ganglia (DRG). Therefore, I) EGFL7 and PGRN will be deleted from their predominant cellular sources using conditional knock-out (ko) approaches and the consequences for adult neurogenesis per se as well as the renewal functions in peripheral and central nervous system (PNS and CNS) models of axonal injury will be studied. Subsequently, II) the role of EGFL7 and PGRN in neuronal longevity will be determined because adult-born EGFL7-/- neurons display extended life spans in the hippocampus and the olfactory bulb, whereas PGRN-/- neurons die earlier. In particular, synaptic connectivity and plasticity of young adult-born neurons as well as their molecular fingerprints will be studied, e.g., in terms of NMDA receptor signalling or their epigenetic characteristics. Consequently, III) the combined effects of EGFL7 and PGRN deficiency will be defined in vivo. Behavioural paradigms of aversive and appetitive learning, memory acquisition and extinction for spatial, visual and social tasks will be applied. Interestingly, the influence of EGFL7 on adult neurogenesis is an age-dependent process and inverts in old animals; therefore, it will be determined whether EGFL7 and PGRN execute their functions in an additive or antagonistic manner using double mutant mice to define the role of both proteins in neural homeostasis during aging.soft-slit-separator

A05:  Deciphering the molecular adaptions underlying network homeostasis when facing the challenge of new neuron integration.

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Benedikt Berninger, Prof. Dr. rer. nat.
Institute for Physiological Chemistry
University Medical Center of JGU Mainz
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vijay-tiwari
Vijay Tiwari, PhD
Institute for Molecular Biology
(IMB)

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Integration of new neurons into a pre-existing neural circuit is a major challenge to network homeostasis. During the first funding period, we demonstrated that behaviourally relevant experience results in a remarkable remodelling of adult-born neuron connectivity in the hippocampus. We showed that exposure of adult mice to an enriched environment or physical exercise during a critical period after adult-born dentate granule cells’ (DGC) birth altered presynaptic input and the stability of this remodelling differed markedly between local and long-range inputs. Surprisingly, however, increased connectivity was countered by reduced excitatory input suggestive of homeostatic regulation of input strength. In the coming funding period we aim at unravelling the transcriptional and epigenetic changes occurring in adult-born DG neurons and their presynaptic partners when undergoing experience-dependent remodelling of connectivity, with the goal to decipher the molecular mechanisms underlying network homeostasis when challenged by the incorporation of new neurons.

In particular, we will establish a reductionist culture model of new neuron integration by adding immature hippocampal neurons to cultures of hippocampal neurons that have already established a robust neural network. First, we will use electrophysiological techniques (patch clamp, MEA) to assess the consequences of adding immature neurons to the network (work package 1, WP1)). We will then separate mature and immature neurons for transcriptome analyses using next generation sequencing to determine alterations in gene expression profiles caused by the addition of new neurons (WP2). We will pay particular attention to genes involved in establishing, maintaining and regulating synaptic function and neuronal excitability as potential executive components in the regulation of network homeostasis. Moreover, we will also put emphasis on transcriptional and epigenetic regulators among differential expressed genes to identify those genes, which might orchestrate a homeostatic response. Moving up in complexity we will then study the consequences of increased incorporation of new DGCs in the adult dentate gyrus in vivo. Towards this, we will employ patch-sequencing as an approach to determine the transcriptome of early and adult-born DGCs as well as their presynaptic partners at a single cell level. Early- and adult-born DGCs will be identified using retrovirus-mediated labelling, while presynaptic partners of adult-born neurons will be identified by rabies virus-mediated tracing of monosynaptic connections. Mice will be exposed to standard housing, enriched environment or voluntary exercise to cause specific alterations in network connectivity as previously shown. The goal is to identify genes that are differentially expressed in newly added DGCs versus earlier born DGCs under these different conditions (WP3) as well as genes that are regulated in presynaptic partners as a consequence of an altered integration of new DGCs (WP4). Among differential expressed genes again those with executive pre- or postsynaptic function as well as regulatory control (transcription factors and epigenetic regulators) will highlighted as potential candidates mediating network homeostasis, but also contributing to experience-dependent remodelling of connectivity. Finally, gain- and loss-of-function studies of selected candidate factors will serve to demonstrate a causal link between gene expression and altered connectivity (WP5). For those transcription factors and epigenetic regulators for which a functional involvement in network connectivity/homeostasis can be demonstrated, we will study genome-wide binding to obtain a global view of the genetic program underlying network remodelling and homeostasis.

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A06:  Gadd45a as a regulator of memory consolidation by post-transcriptional control mechanisms.

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Christof Niehrs, Prof. Dr. rer. nat.
Institute for Molecular Biology
(IMB)

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Beat Lutz, Prof. Dr. sc. nat.
Institute for Physiological Chemistry
University Medical Center of the JGU Mainz

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Growth Arrest and DNA Damage-inducible protein 45a (Gadd45a) is a stress response protein, which acts in a variety of molecular pathways, including cell-cycle, DNA repair and DNA demethylation. In the previous funding period, we established that Gadd45a is essential for the consolidation of hippocampus-dependent aversive memory in mouse, while numerous other behavioral domains (e.g., non-aversive memory, anxiety, and locomotion) were unaltered. Gadd45a deficiency also led to decreased hippocampal long-term potentiation (LTP), while hippocampal overexpression of Gadd45a led to increased memory consolidation and LTP. RNA-seq analyses of hippocampal tissue from mice at the memory consolidation phase revealed that Gadd45a regulates the stability of mRNAs encoding proteins, such as NMDA glutamate receptor subunits (Grin2a, Grin2b), which are centrally involved in synaptic plasticity and memory processes. This class of Gadd45a-regulated transcripts is much enriched in mRNAs containing very long (often >10 kb) non-canonical 3’UTRs. Our results raise the intriguing possibility that Gadd45a regulates RNA stability of a distinct class of mRNAs during the process of memory consolidation.

Based on our data, we hypothesize that Gadd45a constitutes an important homeostatic factor which keeps the balance between neural stability and plasticity, when the brain has been challenged, e.g., by aversive encounters to the animal. Specifically our working hypothesis is that in neural learning and memory, Gadd45a acts as modulator of RNA post-transcriptional regulation of neural genes with extended 3’UTRs. In the next funding period, we aim to investigate the mechanisms underlying the regulation of mRNA stability in memory consolidation. Gadd45a is an RNA binding protein, and we therefore will investigate the physical interaction between Gadd45a and neuronal target RNAs and their consequences for target mRNA/protein localization within the neuron. The prediction is that Gadd45a directly binds to mRNAs which were found to be affected in stability in Gadd45a-deficient mice (e.g., Grin2a, Grin2b). First, RNA immunoprecipitations in basal state and under activated conditions will be carried out in extracts from neuronal cell lines and from hippocampi, followed by RNA-seq analysis. We will carry out a comprehensive bioinformatics analysis of the target RNAs identified. Second, using primary neuronal cultures, we aim at understanding what the consequences of 3’UTR length in Grin2a and Grin2b mRNAs are for the transport and localization of these mRNAs. We will finally test whether the localization of Grin2a and Grin2b mRNA and their encoded proteins are affected in Gadd45a mutants during memory consolidation.

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A11:  Interaction of homeostatic challenges in activity control for dopamine substantia nigra neurons by alpha-synuclein pathology, aging and cell loss.

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Jochen Roeper
, Prof. Dr. med.
Institute for Neurophysiology, Neuroscience Center
Goethe-University Frankfurt

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Dopamine (DA) substantia nigra (SN) neurons are particularly vulnerable and significant loss of these neurons is responsible for the clinical key symptoms of Parkinson Disease (PD). No treatment exists to prevent or even slow down the progressive loss of DA SN neurons in PD. While neurodegeneration of a DA SN neuron signifies the final collapse of its homeostatic defense against the onslaught of causal PD pathomechanisms such as the toxic aggregation of alpha-synuclein (aSYN), we propose that this fatal event is preceded by a yet mostly unknown cascade of adaptations in DA SN neuronal functions to ensure ongoing survival and functionality, even under increasing toxic loads. In addition, this homeostatic plasticity itself is likely to be impaired by aging processes and increasingly challenged by the need to compensate for more and more DA SN neurons being lost by progressive degeneration. Based on our progress in the first funding period in identifying selective but different electrophysiological adaptations of DA SN neurons to individual perturbations regarding (i) the PD key-pathomechanism of alpha-synuclein (aSYN) overexpression/aggregation (ii) aging itself and (iii) lesion-induced reduction of the DA SN population, we now are able to propose a refined follow-up research concept to study the interactions of these three factors for the homeostatic capacity of DA SN neurons in PD. We believe this project will improve our mechanistic understanding on how vulnerable and aging DA SN neurons functionally respond to a-synuclein pathology in the presence of ongoing DA neurodegeneration.

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B01:  Neuronal Proteostasis.
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Erin Schuman
, Prof. Ph.D.
Max Planck Institute for Brain Research

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During synaptic plasticity, the covalent modifications of pre-existing proteins as well as new protein synthesis form the basis of the proteomic remodeling that underlies synaptic change. In order to identify these comprehensive changes one needs to address system-wide modifications in the newly synthesized proteome. In the previous funding period we addressed the issue of how the nascent proteome is modified to bring about the bi-directional responses that underlie homeostatic up- and down-scaling. To accomplish this, we induced homeostatic plasticity and then used BONCAT (biorthogonal non-canonical amino acid tagging) with click chemistry to tag and purify the newly synthesized proteins which we then identified using mass spectrometry. We achieved an unprecedented coverage of the nascent proteome- with 24 hrs of metabolic labeling we identified in excess of 5000 proteins. When we analyzed the size of the proteome between control, up-scaled (TTX-treated) and down-scaled (bicuculline-treated) neurons we find that the size of the total proteome (number of identified proteins) is unaltered. The extensive coverage of the nascent proteome allows us to see things that were simply not possible previously- we discovered ~300 proteins that were differentially regulated by homeostatic plasticity. These proteins are in important families, including ion channels (like Glutamate and GABA receptors), signaling molecules, and many molecules involved in the neurotransmitter transport and release. In many cases, opposite effects of up- or down-scaling can be observed on the new synthesis of a protein group. In the new funding period, we will examine how protein synthesis and degradation are coordinated.
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B02:  Mechanisms of synaptic vesicle recycling and of cAMP dependent synaptic homeostasis, studied by optogenetics & electron microscopy.
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Alexander Gottschalk, Dr. rer. nat., Prof.
Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry Goethe University
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Chemical synaptic transmission involves the release of neurotransmitter from synaptic vesicles (SVs), and the recycling of these vesicles for further use. Identity and characterization of proteins affecting these recycling processes are imperative to fully understand the mechanisms coupling exo- and endocytosis. They are also likely control points for mechanisms of synaptic homeostasis. By optogenetics, genetic screening, imaging, electrophysiology and time-resolved – ‘Flash-n-freeze’ – electron microscopy (at ms to s time scale, relative to channelrhodopsin-mediated optogenetic stimulation), we identify and characterize such proteins in Caenorhabditis elegans. Using optogenetic stimulation via photo-activated adenylyl cyclase (PAC) and electron microscopy, we furthermore uncovered a novel aspect of cAMP-dependent signalling at cholinergic synapses, which are altering their release probability, but also the acetylcholine content of their SVs in response to cAMP-induced neuropeptide release. This novel mechanism will be thoroughly analysed, particularly in the context of synaptic homeostasis, and to identify the intrinsic signals and pathways regulating this mechanism. Last, we will expand the optogenetics / EM approach to zebrafish, to assess whether a similar neuropeptide-mediated mechanism of synaptic homeostasis has been conserved also in vertebrates.
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B03:  Molecular mechanisms of homeostatic neuronal adaptations after denervation.
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thomas-deller
Thomas Deller
, Prof. Dr. med.
Institute for Clinical Neuroanatomy | Neuroscience Center
Goethe-University Frankfurt

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Neurons which are partially denervated after brain injury remodel their synaptic connections and eventually achieve a new stable state. Although this lesion-induced synaptic reorganization has long been described, its dynamics and the underlying molecular mechanisms are still insufficiently understood. During the first funding period we have focused on understanding the interdependency between denervation-induced functional and structural changes at the level of spines and synapses in organotypic entorhino-hippocampal slice cultures. After entorhinal denervation we found evidence for NMDAR-mediated heterosynaptic competition between homeostatically strengthened synapses and newly formed synapses, which retards the structural recovery of denervated neurons. TNFα and Synaptopodin (SP)/spine apparatus were identified as parts of the molecular and cellular machinery involved in synaptic strengthening after denervation. Preliminary data suggest that these molecules may act in concert. In addition, we found evidence that TNFα is not only involved in synapse remodelling but may also play a role in dendritic reorganization after denervation.

In the second funding period we will continue and extend our work on homeostatic mechanisms following denervation. Since we also found evidence for a homeostatic role of dendritic reorganization, we will extend our observations from the spine and synapse level to the level of dendritic trees. Using time-lapse imaging of denervated neurons in organotypic slice cultures, mouse genetics, electrophysiological recordings, pharmacology and viral transduction methods, we will focus on three aspects: (i) which molecules regulate homeostatic synaptic plasticity after denervation? In this first part we will follow-up on our preliminary observations that there may be a link between TNFα and SP/spine apparatus and will perform gain-of-function and loss-of-function experiments. The role of SP/spine apparatus in local protein synthesis will be investigated. (ii) Which molecules and/or conditions regulate denervation-induced dendritic plasticity? Following-up on our preliminary data on dendritic homeostasis after denervation we will investigate the role of TNFα signalling in dendritic remodelling. The role of neuronal activity for the maintenance of the dendritic arbour will be studied using viral transduction with channelrhodopsins and chronic stimulation of denervated neurons in a “slice culture disco”. The role of sprouting and reinnervation will be studied by co-culturing denervated slice cultures with a second entorhinal cortex (homologue sprouting) or a second hippocampus (heterologous sprouting). (iii) What is the relevance for brain repair? To demonstrate the in vivo relevance of our in vitro observation that NMDAR-mediated heterosynaptic competition delays spine density recovery after denervation, we will treat mice with entorhinal lesions with the NMDAR-antagonist Memantine and will study the reorganization of denervated granule cells. Using computer simulations of the network, we will simulate how interventions into the postlesional reorganization process could affect the firing properties of single neurons and the dentate gyrus network.

In sum, this project aims at understanding the role of homeostatic plasticity mechanisms in brain repair. Understanding these mechanisms may help to identify new therapeutic targets for protecting and/or restoring neurons in brain diseases associated with neuronal loss and axonal denervation.
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B04:  Molecular mechanisms of dendritic development and maintenance.
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Amparo Acker-Palmer
, Prof. PhD.
Institute of Cell Biology and Neuroscience
Goethe University

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A crucial element of neuronal networks and their homeostatic plasticity are the dendritic trees, where neurons receive and begin to integrate information. The transmission and integration of information is heavily influenced by the structure of the dendritic arborisation, whose morphogenesis and maintenance are therefore highly regulated. Our aim is to better understand the functions in dendritogenesis and during synaptic plasticity of a core of key proteins, namely the glutamatergic receptor interacting protein (GRIP) 1 and its binding partners. In the first funding period we identified a regulatory role for GRIP1 during microtubule based transport and suggested a crucial function for 14-3-3 proteins in controlling kinesin-1 motor attachment during dendritic arborisation. Recently, we showed that GRIP1 bridges a complex including the Reelin receptor ApoER2, ephrinB2 and AMPA receptors and regulates AMPA receptor new insertion into the dendritic membrane, thereby modulating synaptic plasticity. In addition, we also have uncovered an interaction between ephrinB2 and the VEGF receptor 2 (VEGFR2) in neurons. In the next funding period, we plan to expand on these findings and dissect the molecular mechanisms behind the roles of the ApoER2/ephrinB2/GRIP1/AMPA receptor complex, as well as the ephrinB2/VEGFR2 interaction, in dendritic formation and maintenance and synaptic plasticity. In particular, we will assess dendrite and spine morphogenesis in the young or adult hippocampus of mice lacking one or a combination of these signalling partners. We will additionally, examine the role of these interactions in key mechanisms of neural homeostasis such as dendritic arborisation after lesion or homeostatic forms of synaptic plasticity.
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B05:  Role of bioactive lipid signaling in homeostatic control of excitatory transmission.
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Johannes Vogt
, Dr. med.
Institute for Microscopic Anatomy and Neurobiology
University Medical Center of JGU Mainz

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We have provided evidence for a role of plasticity-related gene 1 (PRG-1, (Brauer et al., 2003) in the maintenance of synaptic homeostasis by controlling the functional set-point at the glutamatergic junction. This signaling pathway involves lysophosphatidic acid (LPA) acting via presynaptic LPA2 receptors and PRG-1 acting from the postsynaptic side (Trimbuch et al., 2009). During the previous funding period, we assessed PRG-1 and its potential interaction partners at the PSD (Diestler et al., 2014), and determined a molecular pathway by which PRG-1 provides homeostatic control of both structural and functional spine plasticity in a cell-autonomous fashion (Liu et al., 2016). The role of bioactive synaptic lipid signaling in the homeostatic control of glutamatergic transmission was shown to affect neurotrophin signaling in the hippocampus (Petzold et al., 2016) and, importantly, cortical information processing (Unichenko et al., 2016). Genetic alterations in bioactive synaptic lipid signaling both in mice and man revealed that resulting changes in the homeostasis of cortical information processing may play a role in psychiatric disorders (Vogt et al., 2016).

In the upcoming funding period, we will 1) address the role of PRG-1/CaM binding which appears to be dynamically regulated using in vitro electrophysiology on a cellular and network level (LTP), employing pharmacological as well as specific peptide blockers, and study behavioral effects by interfering with this binding also in vivo. 2) Further, we will analyze the role of the LPA-synthesizing enzyme ATX, present at perisynaptic lamellae of astrocytes, using cellular studies of ATX-activity and its inhibition, as well as in vitro electrophysiology and behavioral studies. 3) Finally, we will assess LPA-uptake and resulting Ca2+-transients in dendritic spines using single-spine 2P-imaging with a temporal resolution in the milliseconds range (30 ms). These data will allow us to assess the kinetics of synaptic phospholipids action at the glutamatergic synapse and to arrive at a quantitative mathematical model for synaptic phospholipid function (in collaboration with the group of T. Tchumatchenko). This work will provide a new view on the homoeostatic control of synaptic signaling by bioactive lipids.

This is a continuation project of the successful previous project B05 (Nitsch/Huai). Johannes Vogt will serve as a PI after the move of Robert Nitsch to another position. Johannes Vogt has been a contributing researcher during the first funding period, and is first, but also senior author on publications derived from this project. Jisen Huai had a non-permanent contract which ends beginning of next year.
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B09:  The role of the protein receptor-mediated endocytosis 8 (RME8) in neuronal homeostasis.
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albrecht-clement
Albrecht Clement
, Dr. rer. nat.
Institute of Pathobiochemistry
University Medical Center of the JGU Mainz

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christian-behl
Christian Behl
, Prof. Dr. rer. nat.
Institute of Pathobiochemistry
University Medical Center of the JGU Mainz

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In the course of the first funding period of this CRC, we uncovered RME8 (for receptor-mediated endocytosis 8; human ortholog: DNAJC13) as a component of the proteostasis network through a functional RNAi-based screen in C. elegans and identified it as a positive modulator of autophagy in mammalian systems. RME8 is a 2243 amino acid long, ubiquitously expressed cytosolic protein containing a DNAJ domain which is a conserved motif for the interaction with HSP70. It has been initially identified in C. elegans as a component of the endocytic machinery and it has now become apparent that RME8 is involved in post-endocytic transport processes by interacting with PI(3)P, the retromer and WASH protein complexes. In the second funding period within this CRC we will now focus on the analysis of the role of RME8 in neuronal homeostasis. The importance of RME8 in neuronal, particular dopaminergic neuron function is documented as mutant RME8 variants (e.g. N855S) cause familial forms of Parkinson’s disease with Lewy body pathology. The endosomal compartment represents a switchboard where membrane proteins and lipids are sorted towards different destinations within the pre- and the postsynaptic compartments. These sorting processes which are only partially understood are essential to maintain the pool of synaptic vesicles as well as the correct number of neurotransmitter receptors within dendritic spines. To understand the basic neuronal function of RME8, we will initially analyze the subcellular localization of RME8 in primary neurons and in the nervous tissue with high resolution fluorescent and electron microscopy. Further, we will investigate whether RME8 is involved in neuronal activity by analyzing primary neurons that overexpress wild-type or mutant RME8 or reduced RME8 levels. As there is evidence that RME8 and the retromer complex are present on the pre- as well as on the postsynapse we will investigate the role of RME8 in synaptic vesicle and neurotransmitter receptor recycling in primary cells as well as in C. elegans. In addition, as exosomes derive from endosomes and may contribute to the prion-like propagation of misfolded proteins such as -synuclein, we will analyze the role of REM8 in exosome formation, release, and endocytosis. Finally, we aim to generate a conditional knock-out mouse to analyze the role of RME8 in neuronal homeostasis in vivo. With this study we aim to deepen our current understanding of the endosome- and RME8-linked molecular processes contributing to maintain neuronal and synaptic homeostasis.
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B10:  Homeostatic regulation of mTOR dependent synaptic function.
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schweiger-susann
Susann Schweiger, Prof.Dr. med.
Institute for Humangenetics
University Medical Center of the JGU Mainz

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luhmann-heiko
Heiko Luhmann
, Prof. Dr. rer. nat.
Institute of Physiology
University Medical Center of the JGU Mainz

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The mTOR (mechanistic target of rapamycin) kinase is the most important regulator of local dendritic and perisynaptic protein translation in the brain. It has been shown to fundamentally influence AMPA receptor activity by shifting GluA1 to GluA2 balance and to control the synthesis of several synaptic proteins involved in synaptic function and plasticity. Both, loss and gain of mTOR activity lead to significant disturbances of brain homeostasis and neuronal function resulting in intellectual disability(ID), epilepsy and behavioralalterations. Genetic syndromes with mTOR dysfunction includetuberous sclerosis, fragile X and Down syndrome,Rett and Opitz BBB/G syndrome.

Preliminary experiments in a Tsc2+/- animal model, which presents a lack of mTOR inhibition, show that mTOR hyperactivity leads to a significant increase in miniature (m) EPSCamplitudes and frequencies and tomIPSC frequencies but not amplitudesin cortical cultures. This can be blocked by the mTOR inhibitor rapamycin. Furthermore, Ca2+ permeability of AMPA receptors is increased in these cultures, which also can be blocked by rapamycin. Network analysis revealed increased functional connectivity and a shift in E/I balance towards excitation in Tsc2+/- animals.

Based on the fundamental role that mTOR kinase plays in synaptic protein synthesis and our preliminary results, we hypothesize that mTOR dysfunction leads to a set point shiftand significantly influences neuronal homeostasis by altering cell excitability, E/I balance, synaptic connectivity and plasticity.

In the proposedproject we will use two different mouse models:on one hand, Tsc2+/- animals carry a heterozygous mutation in the mTOR inhibitor gene Tcs2thatleads to chronic hyperactivity of mTOR.On the other hand,Mid1-/y and Mid2-/y double knock-out mice carry mutations in mTOR activating genes andpresent with reduced mTOR activity. We will compare mTOR hypo- and hyperactivity and analysetheir effectson homeostatic pathways and synaptic functionin these mouse models. We will combine excitability measurements and electrophysiological analysis of E/I balance in-vitro and in-vivo with outcomes of cognitive behavior. Furthermorewe will use a protocol that we have established recently for synaptosome preparations to identify differences in protein translation in postsynaptic cortical compartments to define the proteomic pattern in synaptic compartments in animals with hypo-, normo- and hyperactive mTOR.

In the suggested project we will gain insight into the influence of aberrations in mTORon synaptic function, excitability, E/I balance and connectivity.We also aim at the identification of protein networks that mechanistically link mTORdysregulation with synaptic function and behavioral outcomeas measurements of neuronal homeostasis.Sinceall mouse models used resemble human diseases, data obtained in this project will provide insight into mTOR dependent alterationsin mouse and man.
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C01:  Immune Cytokines in the Regulation of Neuronal Homeostasis.
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Jonathan Kipnis
, Prof. Dr. rer. nat.
Clinic for Neurology
University Medical Center of the JGU Mainz

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Frauke Zipp
, Prof. Dr. med.
Clinic for Neurology
University Medical Center of the JGU Mainz

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Based on our work on the CRC topic “molecular mechanisms of neuronal homeostasis during inflammatory processes in the CNS” during the first funding period, (Ellwardt et al, 2016), we are now focusing on direct roles of major T cell-derived cytokines in the central nervous system (CNS). The neuron is a highly specialized cell whose neurites function partially independent of the cell bodies. Axon tracts in the spinal cord are up to one meter long in humans and, therefore, homeostatic mechanisms for the maintenance and integrity of axons are likely to exist. While glial cells are regarded as the classical supporting cells for axons, there are clear indications, including our findings from the last funding period that immune cells and their derived cytokines might contribute to axonal homeostasis. We have become increasingly interested in the roles of major T cell-derived cytokines in the CNS, concentrating on the immunoregulatory cytokine IL-4. Most neural cells bear receptors for certain cytokines and important roles have been ascribed to them in CNS functions, such as synaptic scaling, regulation of microglia differentiation, and neuronal survival. We have identified a role for the T helper 2 (TH2) cytokine IL-4 in neuroprotection in the optic nerve and spinal cord (Walsh et al, 2015). Our work suggests that the CNS has an intrinsic capability to restore homeostasis via the production of IL-4. In this project we plan to i) explore the mechanisms through which IL-4 acts on neurons and axons in depth; ii) investigate the role of IL-4 in neuron and axon homeostasis under physiological conditions; and (iii) investigate the neuronal homeostasis in neuroinflammation via therapeutic application of IL-4. We hypothesize that cytokines are relevant for the homeostatic regulation of neuron maintenance and axon integrity and function. This project might lead to the development of new therapeutic strategies to restore homeostasis in traumatic or inflammatory CNS injury.
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C02:  Adaptive cellular mechanisms of functional reorganization and recovery after traumatic brain injury (TBI).
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thomas-mittmann
Thomas Mittmann
, Prof. Dr. rer. nat.
Institute of Physiology
University Medical Center of the JGU Mainz

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Traumatic brain injury (TBI) is a challenging issue for public health, and it is one of the leading causes of mortality and morbidity in industrialized countries. A better understanding of the adaptive mechanisms underlying neuronal dysfunction in the vicinity of the injury and in more distant areas is crucial for the development of new therapies aiming to improve the recovery of brain functions. In the first funding period we disclosed an early neuronal hyperactivity in the somatosensory cortex of mice exclusively in the contralateral cortical hemisphere indicating the presence of transhemispheric diaschisis already in the acute phase of 24 hours after induction of controlled cortical impact. Furthermore, these adaptive functional change was accompanied by an altered synaptic strength of the glutamateric vs. GABAergic neurotransmission as measured by the ratio of AMPA- vs. GABA-A-receptor dominated postsynaptic currents. On top, we observed bidirectional changes in the function of two specific subtypes of GABAergic interneurons, and we recorded an imbalance of phasic versus tonic inhibition in the contralateral hemisphere post TBI. We could also show that oligodendrocyte precursor cells (OPCs) expressing the proteoglycan NG2 in somatosensory cortex of mice modulate AMPA-receptor mediated currents and behavior. In the second funding period will use the same brain injury model in mice to characterize in more detail the role of GABAergic inhibition for the functional reorganization in the contralateral hemisphere in the early phase 24h after TBI. We will investigate also additional homeostatic mechanisms that can stabilize the overall cortical network function after the injury: this includes an electrophysiological and optogenetic approach to study the function of callosal fibers projecting to the contralateral hemisphere. In addition, we will perform behavioral experiments in mice to evaluate the recovery of function following TBI, and we will also test the role of the inflammatory factors TNF-alpha and IL1-beta for the observed network changes post TBI.
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C03:  Activity driven homeostatic regulation of connectivity to optimize stimulus representation.
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tatjana-tchumatchenko
Tatjana Tchumatchenko
, Dr. rer. nat.
Theory of neural dynamics
Max Planck Institute for Brain Research

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The new project C03 (Tchumatchenko) addresses how a neural circuit can homeostatically target an optimal set point for spiking activity and connectivity such that stimulus representation and stimulus separation can be accomplished. We will address how this can be accomplished on the network level and the cellular level. First, we will identify a minimal set of features that control both computations. To this end, we will generalize the stimulus representation task and address individually the roles synaptic plasticity, stimulus types and single neurons play for stimulus representation. Second, we will explain how a specific PRG-1 mediated molecular mechanism of homeostatic plasticity is contributing to these computations. To achieve the second goal of relating a specific molecular mechanism to stimulus representation, we will consider the
PRG-1 protein, which belongs to the family of plasticity related genes (PRGs). PRG-1 operates in an activity dependent manner at excitatory synapses and its interaction pathway has been experimentally characterized by the project B5. We now know that the lack of this homeostatic modulatory feedback loop in PRG-1−/− mice (compared to WT-litters) results in seemingly unaltered activity levels but leads to somatosensory discrimination deficits in the behaviour in these mice. In this project, we will build a computational model of the PRG-1 mediated excitatory homeostatic synaptic plasticity and will study its role in stimulus representation at the network level. Our third goal is to understand the homeostatic synaptic plasticity time scales. To this end, we build a mechanistic model to study how they are established and incorporate the time scales of synaptic protein transport, turnover and degradation that take place during plasticity events in neuronal dendrites using the data obtained in the project B1.
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C04:  Homeostatic regulation of REM-SWS balance in sleep.
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gilles-laurent
Gilles Laurent
, D.V.M., Ph.D.
Max Planck Institute for Brain Research

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This project builds on recent results from this lab establishing the existence of mammalian type sleep activity (REM and slow wave sleep, including sharp-wave ripples) in reptiles (Shein-Idelson et al., 2016). Exploiting several advantageous features of sleep in this animal (e.g., very short sleep cycle, clock-like regularity) and some experimental advantages of reptilian brains (resistance to anoxia, brain survival ex vivo), we wish to examine some mechanistic aspects of sleep homeostatic regulation.

Is the alternation of REM and SWS (non-REM) under homeostatic regulation? This question can be addressed in vivo by disrupting selectively the REM and non-REM segments of sleep, using real-time monitoring of LFP power spectra (ratio of power in  bands) during sleep. We will examine whether the fraction of REM and non-REM vary in a predictable and compensatory manner when either sleep subtype is experimentally disturbed (shortened).

What aspects of brain activity are correlated with homeostatic regulation of REM / non-REM? The homeostatic control of sleep/wake balance is thought to be a function of energetic expenditure related to activity during wake (e.g., via adenosine). Are similar correlations found for the regulation of REM/non-REM cycling? REM and non-REM are characterized by very different firing activities (high and awake-state-like in REM; very low and bursty in non-REM). By quantifying the population statistics of firing during baseline and disrupted REM/non-REM, we will be able to determine if an energy-based regulatory mechanism could be involved in this homeostatic loop.

Mechanisms of REM-nonREM cycle generation. By taking advantage of the reptilian brain as an experimental system, we will try and dissect out some of the cellular and circuit mechanisms underlying this ancient brain rhythm.
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C05:  Homeostatic maintenance of neuronal function in a dynamic network.
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rumpel-simon
Simon Rumpel
, Prof. Dr. rer. nat.
Institute of Physiology
University Medical Center of the JGU Mainz

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yonatan-loewenstein
Yonatan Loewenstein
, Prof.
Departments of Neurobiology and Cognitive Science
The Hebrew University of Jerusalem

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Dynamic remodelling of connectivity is a fundamental feature of neocortical circuits. As the response properties of neurons depend on the network architecture, the extent to which the cortical network reconfigures determines the level of plasticity. However, at the same time the stability of cortical representations needs to be ensured over days and weeks. The aim of this new research proposal is to unravel homeostatic mechanisms that ensure functional stability of neuronal ensembles forming auditory representations in a dynamic cortical architecture. To that goal, we will combine in vivo imaging approaches in mice with theoretical modelling to elucidate the link between volatility at the connectivity level and functionality at the level of neuronal ensembles. Specifically, we will develop a model that relates structural volatility, induced by dynamic remodelling of synapses, to the (in)stability of network activity and use calcium imaging in vivo to test it. We will study theoretically and experimentally the impact of transient, pharmacogenetic manipulation of cellular excitability on network activity (calcium imaging) and connectivity (spine imaging). Taken together, our combined approach will address the fundamental question of how homeostasis contributes to the stable function of cortical circuits in vivo.
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Tom J. Kay – Science and Finance Manager