Torpor induces reversible tau hyperphosphorylation and accumulation in mice expressing human tau.

Tau protein hyperphosphorylation and aggregation are key pathological events in neurodegenerative tauopathies such as Alzheimer's disease. Interestingly, seasonal hibernators show extensive tau hyperphosphorylation during torpor, i.e., the hypothermic and hypometabolic state of hibernation, which is completely reversed during arousal. Torpor-associated mechanisms that reverse tau hyperphosphorylation may be of therapeutic relevance, however, it is currently not known to what extent they apply to human tau. Here we addressed this issue using daily torpor in wildtype mice that express mouse tau (mtau) and in mice that lack mtau expression and instead express human tau (htau). AT8, AT100 and Ser396 immunoblotting and immunohistochemistry were used to assess tau (hyper)phosphorylation at clinically relevant phosphorylation sites. We found that torpor robustly and reversibly increases the levels of phosphorylated tau in both mtau and htau mice. Immunohistochemistry revealed four brain areas that show prominent tau phosphorylation: the hippocampus, posterior parietal cortex, piriform cortex and cortical amygdala. Whereas wildtype mice primarily showed increased levels of diffusely organized hyperphosphorylated tau during torpor, htau mice contained clear somato-dendritic accumulations of AT8 reactivity resembling tau pre-tangles as observed in the Alzheimer brain. Interestingly, AT8-positive accumulations disappeared upon arousal, and tau phosphorylation levels at 24 h after arousal were lower than observed at baseline, suggesting a beneficial effect of torpor-arousal cycles on preexisting hyperphosphorylated tau. In conclusion, daily torpor in mice offers a quick and standardized method to study tau phosphorylation, accumulation and clearance in mouse models relevant for neurodegeneration, as well as opportunities to discover new targets for the treatment of human tauopathies.

Progression and recovery of Parkinsonism in a chronic progressive MPTP-induction model in the marmoset without persistent molecular and cellular damage.

Chronic exposure to low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in marmoset monkeys was used to model the prodromal stage of Parkinson's disease (PD), and to investigate mechanisms underlying disease progression and recovery. Marmosets were subcutaneously injected with MPTP for a period of 12weeks, 0.5mg/kg once per week, and clinical signs of Parkinsonism, motor- and non-motor behaviors were recorded before, during and after exposure. In addition, postmortem immunohistochemistry and proteomics analysis were performed. MPTP-induced parkinsonian clinical symptoms increased in severity during exposure, and recovered after MPTP administration was ended. Postmortem analyses, after the recovery period, revealed no alteration of the number and sizes of tyrosine hydroxylase (TH)-positive dopamine (DA) neurons in the substantia nigra. Also levels of TH in putamen and caudate nucleus were unaltered, no differences were observed in DA, serotonin or nor-adrenalin levels in the caudate nucleus, and proteomics analysis revealed no global changes in protein expression in these brain areas between treatment groups. Our findings indicate that parkinsonian symptoms can occur without detectable damage at the cellular or molecular level. Moreover, we show that parkinsonian symptoms may be reversible when diagnosed and treated early.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis.

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

The molluscan RING-finger protein L-TRIM is essential for neuronal outgrowth.

The tripartite motif proteins TRIM-2 and TRIM-3 have been put forward as putative organizers of neuronal outgrowth and structural plasticity. Here, we identified a molluscan orthologue of TRIM-2/3, named L-TRIM, which is up-regulated during in vitro neurite outgrowth of central neurons. In adult animals, L-Trim mRNA is ubiquitously expressed at low levels in the central nervous system and in peripheral tissues. Central nervous system expression of L-Trim mRNA is increased during postnatal brain development and during in vitro and in vivo neuronal regeneration. In vitro double-stranded RNA knock-down of L-Trim mRNA resulted in a >70% inhibition of neurite outgrowth. Together, our data establish a crucial role for L-TRIM in developmental neurite outgrowth and functional neuronal regeneration and indicate that TRIM-2/3 family members may have evolutionary conserved functions in neuronal differentiation.

LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis.

We report the characterization of a cDNA encoding a novel -RFamide neuropeptide precursor that is up-regulated during parasitation in the snail Lymnaea stagnalis. Processing of this precursor yields five structurally related neuropeptides, all but one ending with the C-terminal sequence -LFRFamide, as was confirmed by direct mass spectrometry of brain tissue. The LFRFamide gene is expressed in a small cluster of neurons in each buccal ganglion, three small clusters in each cerebral ganglion, and one cluster in each lateral lobe of the cerebral ganglia. Application of two of the LFRFamide peptides to neuroendocrine cells that control either growth and metabolism or reproduction induced similar hyperpolarizing K+-currents, and inhibited electrical activity. We conclude that up-regulation of inhibitory LFRFamide neuropeptides during parasitation probably reflects an evolutionary adaptation that allows endoparasites to suppress host metabolism and reproduction in order to fully exploit host energy recourses.

Peptidergic modulation of male sexual behavior in Lymnaea stagnalis: structural and functional characterization of -FVamide neuropeptides.

In the simultaneous hermaphrodite snail Lymnaea stagnalis, copulation as a male is controlled by neurons that send axons to the male copulatory organs via a single penis nerve. Using direct mass spectrometry of a penis nerve sample, we show that one of the molecular ions has a mass corresponding to GAPRFVamide, previously identified from the buccal ganglia, and named Lymnaea inhibitory peptide (LIP). The identity of this peptide is confirmed by partial peptide purification from the penis nerve, followed by post source decay mass spectrometry. We cloned the LIP-encoding cDNA, which predicts a prohormone that gives rise to five copies of LIP (now re-named LIP A), two other -FVamide peptides (LIPs B and C), and five structurally unrelated peptides. The LIP gene is expressed in neurons of the right cerebral ventral lobe that send their axons into the penis nerve. We show that the LIP A peptide is present in these neurons and in the penis nerve, and confirmed the presence of LIP B and C in the penis nerve by post source decay mass spectrometry. Finally, we demonstrate that LIP A, B and C inhibit the contractions of the penis retractor muscle, thereby implicating their role in male copulation behavior.

Functional implications of neurotransmitter expression during axonal regeneration: serotonin, but not peptides, auto-regulate axon growth of an identified central neuron.

We studied the regenerative properties of one of two electrically coupled molluscan neurons, the serotonergic cerebral giant cells (CGCs) of Lymnaea stagnalis, after axotomy. The CGCs play a crucial role in feeding behavior, and when both cells are disconnected from their target neurons, animals no longer feed. When one CGC was permanently disconnected from its targets and the other was reversibly damaged by a nerve crush, the latter one regenerated over a period of 2 weeks to reform functional synapses with specific target neurons. At the same time, recovery of the feeding behavior was observed. After the crush, neuropeptide gene expression in the CGC was downregulated to approximately 50%. Serotonin synthesis, on the other hand, remained unaffected, suggesting that serotonin might have an active role in regeneration. In primary neuron culture, CGCs failed to extend neurites in the presence of serotonin; in cells that extended neurites in the absence of serotonin, focally applied serotonin, but not neuropeptides, induced growth cone collapse. Using serotonin-sensitive sniffer cells, we show that CGC neurites and growth cones release serotonin in culture. Finally, both the spontaneous and stimulation-induced release of serotonin from CGCs in culture resulted in growth cone collapse responses that could be blocked by the serotonin receptor antagonist methysergide. Our data suggest that auto-released serotonin is inhibitory to CGC neurite outgrowth in vitro. During regeneration in vivo, serotonin release might fine-tune axon guidance and branching by inducing local collapse responses in extending neurites.

Synapse formation between central neurons requires postsynaptic expression of the MEN1 tumor suppressor gene.

Synapse formation is a crucial step in the development of neuronal circuits and requires precise coordination of presynaptic and postsynaptic activities. However, molecular mechanisms that control the formation of functionally mature synaptic contacts, in particular between central neurons, remain poorly understood. To identify genes that are involved in the formation of central synapses, we made use of molluscan neurons that in culture form synaptic contacts between their somata (soma-soma synapses) in the absence of neurite outgrowth. Using single-cell mRNA differential display, we have identified a molluscan homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene encoding the transcription factor menin as a gene that is upregulated during synapse formation. In vitro antisense knock-down of MEN1 mRNA blocks the formation of mature synapses between different types of identified central neurons. Moreover, immunocytochemistry and cell-specific knock-down of MEN1 mRNA show that postsynaptic but not presynaptic expression is required for synapses to form. Together, our data demonstrate that menin is a synaptogenic factor that is critically involved in a general postsynaptic mechanism of synapse formation between central neurons.

Neurotrophic actions of a novel molluscan epidermal growth factor.

The mammalian epidermal growth factor (EGF) is expressed in the developing and adult CNS, and it has been implicated in the control of cell proliferation, differentiation, and neurotrophic events. Despite extensive evolutionary conservation of the EGF motif in a range of different types of proteins, secreted EGF homologs with neurotrophic actions have not been reported in invertebrates. In this study, we present a novel member of the family of EGF-like growth factors, an EGF homolog from the mollusc Lymnaea stagnalis (L-EGF), and we demonstrate that this protein has neurotrophic activity. Purified L-EGF is a 43-residue peptide and retains the typical structural characteristics of the EGF motif. The L-EGF cDNA reveals a unique precursor organization. In contrast to the multidomain mammalian EGFs, it consists of only two domains, a signal peptide and a single EGF motif. Conspicuously, the L-EGF precursor lacks a transmembrane domain, setting it apart from all other members of the EGF-family. L-EGF mRNA is expressed throughout embryonic development, in the juvenile CNS, but not in the normal adult CNS. However, expression in the adult CNS is upregulated after injury, suggesting a role of L-EGF in repair functions. This notion is supported by the observation that L-EGF evokes neurite outgrowth in specific adult Lymnaea neurons in vitro, which could be inhibited by an EGF receptor tyrosine kinase inhibitor. In conclusion, our findings further substantiate the notion that the EGF family has an early phylogenetic origin, and our data support a neurotrophic role for L-EGF during development and injury repair.

Early evolutionary origin of the neurotrophin receptor family.

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

Towards understanding the role of insulin in the brain: lessons from insulin-related signaling systems in the invertebrate brain.

Insulin is a molecule that has played a key role in several of the most important landmarks in medical and biological research. It is one of the most extensively studied protein hormones, and its structure and function have been elucidated in many vertebrate species, ranging from man to hagfish and turkey. The structure, function as well as tissue of synthesis of vertebrate insulins are strictly conserved. The structural identification of insulin-related peptides from invertebrates has disrupted the picture of an evolutionary stable peptide hormone. Insulin-related peptides in molluscs and insects turned out to be a structurally diverse group encoded by large multi-gene families that are uniquely expressed in the brain and serve functions different from vertebrate insulin. In this review, we discuss invertebrate insulins in detail. We examine how these peptides relate to the model role that vertebrate insulin has played over the years; however, more importantly, we discuss several unique principles that can be learned from them. We show how diversity of these peptides is generated at the genetic level and how the structural diversity of the peptides is linked to the exclusive presence of a single type of neuronal insulin receptor-related receptor. We also discuss the fact that the invertebrate peptides, in addition to a hormonal role, may also act in a synaptic and/or nonsynaptic fashion as transmitters/neuromodulators on neurons in the brain. It can be expected that the use of well-defined neuronal preparations in invertebrates may lead to a further understanding of these novel functions and may act as guide preparations for a possible role of insulin and its relatives in the vertebrate brain.

Altered gene expression in the host brain caused by a trematode parasite: neuropeptide genes are preferentially affected during parasitosis.

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

Co-evolution of ligand-receptor pairs in the vasopressin/oxytocin superfamily of bioactive peptides.

In order to understand the molecular mechanisms that underlie the co-evolution of related yet functionally distinct peptide-receptor pairs, we study receptors for the vasopressin-related peptide Lys-conopressin in the mollusc Lymnaea stagnalis. In addition to a previously cloned Lys-conopressin receptor (LSCPR1), we have now identified a novel Lys-conopressin receptor subtype, named LSCPR2. The two receptors have a differential distribution in the reproductive organs and the brain, which suggests that they are involved in the control of distinct aspects of reproduction and mediate transmitter-like and/or modulatory effects of Lys-conopressin on different types of central neurons. In contrast to LSCPR1, LSCPR2 is maximally activated by both Lys-conopressin and Ile-conopressin, an oxytocin-like synthetic analog of Lys-conopressin. Together with a study of the phylogenetic relationships of Lys-conopressin receptors and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates. Based on our findings, we provide a theory of the molecular co-evolution of the functionally distinct ligand-receptor pairs of the vasopressin/oxytocin superfamily of bioactive peptides.

Co-localized neuropeptides conopressin and ALA-PRO-GLY-TRP-NH2 have antagonistic effects on the vas deferens of Lymnaea.

We examined functional aspects of co-localization of neuropeptides involved in the regulation of male copulation behaviour in the simultaneous hermaphrodite snail Lymnaea stagnalis. The copulation behaviour is controlled by several types of peptidergic neurons that include a cluster of neurons in the anterior lobe of the right cerebral ganglion. All anterior lobe neurons express the gene encoding Ala-Pro-Gly-Trp-NH2 (APGWamide), and a subset of neurons also express the vasopressin-related conopressin gene. Immunocytochemical and peptide chemical experiments show that both APGWamide and conopressin are transported to the penis complex and the vas deferens via the penis nerve. Co-localization of the two peptides was also observed in some, but not all, axon bundles that run along the vas deferens. APGWamide and conopressin were structurally identified from the penis complex with vas deferens. Conopressin excites the vas deferens in vitro, whereas APGWamide inhibits the excitatory effects of conopressin, both in a dose-dependent fashion. We propose that the antagonistic effects of these peptides on the vas deferens underlie its peristalsis. Thus, these peptides play an important role in the control of ejaculation of semen during copulation.

A novel G protein-coupled receptor mediating both vasopressin- and oxytocin-like functions of Lys-conopressin in Lymnaea stagnalis.

We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.

Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior.

It has been suggested that the gene duplication that led to the formation of the vasopressin/oxytocin two-gene family occurred early during vertebrate evolution. However, the existence of both vasopressin- and oxytocin-related peptides in invertebrates suggests that this duplication may have occurred much earlier, although there is no evidence for the co-occurrence of vasopressin- and oxytocin-related peptides in the same invertebrate species. We report here that in Lymnaea only the vasopressin-related peptide Lys-conopressin, but not an oxytocin-related peptide, is present. Moreover, it is very likely that an oxytocin-like cDNA or gene is absent. The conopressin gene is expressed in neurons that control male sexual behavior, and its gene products are present in the penis nerve and the vas deferens. Conopressin induces muscular contractions of the vas deferens and inhibits central neurons that control female reproductive behavior. Thus, although structurally related to vasopressin, conopressin has functional and behavioral characteristics typical for oxytocin. Physiological and receptor binding data suggest that conopressin and [Ile8]-conopressin, a synthetic oxytocin-like analog of conopressin, are functionally equivalent in Lymnaea, and that the chemical nature of the amino acid residue at position 8 does not result in a functional difference. Therefore, we suggest that invertebrates contain only a single member of the vasopressin/oxytocin gene family and that the amino acid change that distinguishes vasopressin from oxytocin is functionally neutral in invertebrates.

Alternative splicing generates diversity of VD1/RPD2 alpha peptides in the central nervous system of Lymnaea stagnalis.

1. Two giant peptidergic neurons, VD1 and RPD2, of the visceral ganglion and right parietal ganglion of Lymnaea stagnalis, respectively, play an important role in the modulation of complex physiological and behavioral adjustments that occur as a result of changes in O2 availability. 2. By cDNA cloning, we have identified two types of VD1/RPD2 transcripts expressed in VD1 and RPD2. In addition, these transcripts are also expressed in other neurons. 3. Both transcripts encode distinct yet related VD1/RPD2 preprohormones that may be cleaved to yield distinct but overlapping sets of neuropeptides. 4. Using the polymerase chain reaction technique, we could show the existence of additional splice variants. 5. Analysis of the organization of the VD1/RPD2 gene indicates that the alpha peptide coding region is interrupted by a number of introns. 6. We concluded that the mRNA segment encoding the alpha peptide domain of the VD1/RPD2 preprohormones is alternatively spliced, thus generating different alpha peptides.

Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis.

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not clear. To investigate this issue, we cloned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressin, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapeptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalian copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.