Dr. Ralf Heinrich

Dept. of Cellular Neurobiology

Schwann-Schleiden Research Center

Julia-Lermontova-Weg 3

37077 Göttingen

Phone: +49(0)551 / 39177958
Fax: +49(0)551 / 39177952
E-mail: rheinri1@gwdg.de


Presence and function of erythropoietin (Epo) in invertebrate nervous systems

Erythropoietin (Epo) is a hematopoietic cytokine with multiple functions that are not exclusively related to vertebrate erythropoiesis. Epo is also expressed in the vertebrate nervous system where it serves important functions during neurodevelopment. Epo has been demonstrated to exert neuroprotective effects by interfering with apoptotic pathways and to promote the regeneration of damaged neurites in mammalian nervous systems. There is accumulating evidence that the functions of Epo/Epo-receptor in nervous tissues are independent from effects on the maturation of red blood cells. Particularly, the existence of endogenous splice variants and artificial derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis, suggested different Epo-responsive receptors on erythroid progenitor cells and cells of other tissues including the nervous system. While the classical Epo-receptor EpoR is present in erythrocyte progenitors and other tissues, non-hematopoietic tissues express additional types of Epo-receptors with different ligand profiles.

Insects and other invertebrates lack genes for Epo and EpoR. Nevertheless, similar to its neuroprotective and neuroregenerative functions in mammals, recombinant human Epo initiates beneficial mechanisms in grasshopper and beetle nervous systems. In vitro, Epo increases survival of primary cultured brain neurons in normoxic and hypoxic conditions and promotes the regeneration of neurites. Epo interferes with apoptotic mechanisms by activating AG490-sensitive Januskinase and STAT signaling. In vivo, Epo accelerates and improves axonal regeneration and reestablishment of sound source localization after crush injury of the tympanal nerve.

Locust and beetle brain neurons are also protected by  the non-erythropoietic  human Epo splice variant EV-3 and other non-erythropoietic Epo derivatives, suggesting that insect and mammalian neuroprotective Epo receptors share common structures that allow activation by the same non-erythropoietic agonists.

CRLF3 is highly conserved in eumetazoan species ranging from cnidarians to mammals. RNAi-mediated knockdown of CRLF3 expression abolishes Epo-mediated protection of hypoxia-challenged locust brain neuron in vitro. Locust hemolymph extracts protect both locust and beetle neurons against hypoxia-induced cell death.

Insect species like Locusta migratoria and Tribolium castaneum, in which recombinant human Epo mediates neuroprotection, express a single orthologue of the human orphan cytokine receptor-like factor 3 (CRLF3). CRLF3 belongs to the cytokine type 1 receptor family, which also includes the hematopoietic cytokine receptors EpoR, thrombopoietin receptor and GCSF receptor. Of this family only CRLF3 has orthologs in invertebrates and it is highly conserved from cnidarians to mammals including humans. Primary cultured insect neurons have been demonstrated to lose Epo-mediated protection in hypoxia following RNAi-mediated downregulation of CRLF3, indicating that this receptor represents the neuroprotective Epo-receptor in insects.
Its endogenous ligand (insects do not contain Epo) is contained in the circulating hemolymph since locust hemolymph extract protects both locust and beetle neurons from hypoxia-induced apoptosis through a CRLF3-dependentmechanism.

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Pro-apoptotic functions of insect acetylcholinesterase

Apoptosis contributes to the development of structured organs, enables the renewal of adult tissues, mediates the removal of compromised or malfunctioning cells and is critically involved in various degenerative diseases. Recent studies indicated that an early ancestor of all metazoans already possessed most components of the complex vertebrate-typical apoptotosis regulatory network. Consequently, the well investigated comparatively simple apoptotic networks of the model organisms C. elegans and D. melanogaster resulted from a secondary reduction of ancient complexity.  

Pizza of Death
Summary of apoptotic pathways in mammals, nematodes (C. elegans) and insects (D. melanogaster, lepidoptera (S. frugiperda and B. mori) and orthoptera (L. migratoria)). Experimental data from lepidopteran, coleopteran and orthopteran insects indicate the contribution of cytochrome c and acetylcholinesterase to insect apoptosis. Detailed description in Knorr et al. 2020)

We study apoptotic mechanisms in L. migratoria and T. castaneum, to (1) determine stimuli that promote apoptosis, (2) characterize the molecular mechanisms underlying apoptosis and (3) identify protective pathways that interfere with apoptotic cell death. Along these lines, we demonstrated that acetylcholinesterase activity promotes apoptosis of primary insect neurons.  Hence, acetylcholinesterase may have a similar role in apoptosome formation as it has previously been described for various mammalian cells types.

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Control of grasshopper sound production by the central complex

The central complex in the protocerebrum coordinates the type, intensity and timing of sound signals used for mate attraction, courtship and rivalry of acoustically communicating grasshoppers.

Sound production depends on the balance of fast and slow excitation and inhibition in central complex neuropils and various transmitters, modulators and intracellular signalling pathways that promote (ACh, proctolin, dopamine) or suppress (GABA, NO) sound production have been identified by pharmacological stimulation and confirmed by anatomical studies. Two of these signaling pathways have been associated with particular behavioral situations. Hearing and recognizing conspecific song activates cholinergic projections to the central complex leading to both nicotinic excitation of yet unknown targets and muscarinic excitation of columnar neurons. The latter is mediated by phospholipase C and adenylyl cyclase-initiated intracellular signaling pathways. Expression of muscarinic ACh receptors in the central complex is limited to a subset of columnar neurons with their cell bodies located in the pars intercerebralis, which are thought to contact pre-motor elements in the lateral accessory lobes. In contrast, a different set of pars intercerebralis neurons with columnar projections in the upper division of the central body and tangential neurons with cell bodies in the ventro-median protocerebrum contain the enzyme nitric oxide synthase and accumulate citrulline in situations that are unfavorable for sound production. Since liberation of nitric oxide in the central body inhibits sound production via soluble guanylyl cyclase activation and cyclic GMP production in the central body lower division, these citrulline-accumulating central complex neurons may translate inappropriate behavioral situations into nitric oxide-mediated suppression of sound production. By applying multiple antibodies directed against components of signaling pathways that contribute to the control of grasshopper sound production to the central complex and conducting physiological studies on pre-identified central complex neurons in primary cell culture, we are attempting to identify the points of convergence of different signals in order to trace the flow of information within the central complex.

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Modulation of female grasshoppers’ reproductive behaviour by nitric oxide and juvenile hormone

The sexual behaviour of female Ch. biguttulus grasshoppers changes with age, oocyte cycle and mating experience (for a description see Wirmer et al. 2010). Immediately after their imaginal molt, females reject all male mating attempts (“primary rejection”). Within a few days, females enter the state of “active copulatory readiness”, a state of high receptivity recognized by singing in response to male stridulation, orientation and active walking towards the partner. After mating, females assume the state of “secondary rejection” which lasts for several days and may end with another period of “copulatory readiness”.

Previous studies on various grasshopper species implicated two signalling systems in the control of female reproductive states, nitric oxide/cGMP signalling in the brain and juvenile hormone (JH) released from the corpora allata. Ch. biguttulus females that are injected with the nitric oxide synthase inhibitor aminoguanidine respond longer and more frequently to male calling songs (
Weinrich et al. 2008) while grasshopper females that lack JH remain in a rejective state and do not stridulate (Loher 1962).

We evaluate the effects of NO and juvenile hormone JH on reproduction related behaviors of female grasshoppers and particularly look for interactions in the brain and/or the corpora allata or for simultaneous regulation of both substances by upstream systems. We assess female responsiveness to male calling songs after diverse treatments that interfere with JH titers or NO production. Cellular sources and targets of NO as well as the distribution of neuroactive signals thought to control JH synthesis are investigated by immunocytochemistry. In contrast to previous beliefs that the corpora allata are regulated by the brain, we demonstrated the presence of neurons that project from the corpora allata to the protocerebrum, suggesting a more complex bi-directional information flow between brain and neurosecretory organs.

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Acoustic communication and social behavior of Drosophila melanogaster 

Drosophila melanogaster offers an arsenal of molecular genetic tools to identify the functions of individual genes and proteins, their interaction partners within cellular/molecular pathways and their impact on physiology and behavioral performance. Making use of this large variety of genetic methods to manipulate the formation and function of its nervous system, studies on Drosophila have increased our knowledge about nervous development, plasticity including learning and memory formation and the control of sexually dimorphic complex interactive behaviours like courtship and aggression.

Human neuro-developmental disorders such as autism spectrum disorders, schizophrenia, attention deficit hyperactivity disorders and Tourette syndrome are believed to result from interplay of multiple genetic risk factors with environmental stimuli. In many cases defects in synaptogenesis, synaptic maintenance and plasticity account for phenotypes that include deficits in social behavior, communication and cognitive functions. Drosophila’s behaviour, including its well-decribed social behavior, is increasingly used to study mechanisms underlying heritable human neuro-developmental disorders, pinpointing the contribution of genetic risk factors for these conditions. As one example, we expose neuroligin 2-deficient Drosophila to behavioural tests addressing their social interactions, space-dispersal, and behavioural switching and analysed their acoustic communication patterns. Neuroligins are a family of phylogenetically conserved postsynaptic adhesion molecules that code for postsynaptic cell adhesion molecules whose intracellular domains bind to synaptic scaffolding proteins while extracellular domains assemble with presynaptic Neurexins. Mutations in neuroligin genes have been identified as risk factors for the development of autism spectrum disorders (ASDs). We show that deletion of the dnl2 gene, coding for one of four Neuroligin isoforms, alters acoustic communication signals, affects the transition between different behaviours and impairs social interactions in Drosophila melanogaster. dnl2-deficient flies maintain larger distances to conspecifics and males perform less female-directed courtship and male-directed aggressive behaviours while the patterns of these behaviours and general locomotor activity resembled those of wild type controls. Since olfactory, visual and auditory perception were not altered in dnl2-deficient mutants, reduced social interactions seem to result from altered excitability in central nervous neuropils that initiate social behaviours. Our results demonstrate that neuroligins are phylogenetically conserved not only regarding their structure and direct function at the synapse but also their fine-tuning of synaptic function in brain circuits that regulate social behaviours dates back to common ancestors of humans and flies.

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Evolution and function of stick insects' "mushroom sensilla"

Some ground-dwelling stick insects contain sensilla of mushroom-like shapes (“mushroom-sensilla”) of yet unknown function.


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designed by A. Wirmer