Development of an autism severity score for mice using Nlgn4 null mutants as a construct-valid model of heritable monogenic autism.

Autism is the short name of a complex and heterogeneous group of disorders (autism spectrum disorders, ASD) with several lead symptoms required for classification, including compromised social interaction, reduced verbal communication and stereotyped repetitive behaviors/restricted interests. The etiology of ASD is still unknown in most cases but monogenic heritable forms exist that have provided insights into ASD pathogenesis and have led to the notion of autism as a 'synapse disorder'. Among the most frequent monogenic causes of autism are loss-of-function mutations of the NLGN4X gene which encodes the synaptic cell adhesion protein neuroligin-4X (NLGN4X). We previously described autism-like behaviors in male Nlgn4 null mutant mice, including reduced social interaction and ultrasonic communication. Here, we extend the phenotypical characterization of Nlgn4 null mutant mice to both genders and add a series of additional autism-relevant behavioral readouts. We now report similar social interaction and ultrasonic communication deficits in females as in males. Furthermore, aggression, nest-building parameters, as well as self-grooming and circling as indicators of repetitive behaviors/stereotypies were explored in both genders. The construction of a gender-specific autism severity composite score for Nlgn4 mutant mice markedly diminishes population/sample heterogeneity typically obtained for single tests, resulting in p values of <0.00001 and a genotype predictability of 100% for male and of >83% for female mice. Taken together, these data underscore the similarity of phenotypical consequences of Nlgn4/NLGN4X loss-of-function in mouse and man, and emphasize the high relevance of Nlgn4 null mutant mice as an ASD model with both construct and face validity.

Common variants of the genes encoding erythropoietin and its receptor modulate cognitive performance in schizophrenia.

Erythropoietin (EPO) improves cognitive performance in clinical studies and rodent experiments. We hypothesized that an intrinsic role of EPO for cognition exists, with particular relevance in situations of cognitive decline, which is reflected by associations of EPO and EPO receptor (EPOR) genotypes with cognitive functions. To prove this hypothesis, schizophrenic patients (N > 1000) were genotyped for 5' upstream-located gene variants, EPO SNP rs1617640 (T/G) and EPORSTR(GA)(n). Associations of these variants were obtained for cognitive processing speed, fine motor skills and short-term memory readouts, with one particular combination of genotypes superior to all others (p < 0.0001). In an independent healthy control sample (N > 800), these associations were confirmed. A matching preclinical study with mice demonstrated cognitive processing speed and memory enhanced upon transgenic expression of constitutively active EPOR in pyramidal neurons of cortex and hippocampus. We thus predicted that the human genotypes associated with better cognition would reflect gain-of-function effects. Indeed, reporter gene assays and quantitative transcriptional analysis of peripheral blood mononuclear cells showed genotype-dependent EPO/EPOR expression differences. Together, these findings reveal a role of endogenous EPO/EPOR for cognition, at least in schizophrenic patients.

5-HT7R/G12 signaling regulates neuronal morphology and function in an age-dependent manner.

The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT(7) receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of Gα(12)-deficient mice reveals a critical role of G(12)-protein for 5-HT(7) receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT(7)R/G(12) signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT(7)/G(12) signaling are abolished. Moreover, inhibition of 5-HT(7) receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT(7) and Gα(12)-proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT(7) receptor and Gα(12)-protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.

Expression of constitutively active erythropoietin receptor in pyramidal neurons of cortex and hippocampus boosts higher cognitive functions in mice.

BACKGROUND: Erythropoietin (EPO) and its receptor (EPOR) are expressed in the developing brain and their transcription is upregulated in adult neurons and glia upon injury or neurodegeneration. We have shown neuroprotective effects and improved cognition in patients with neuropsychiatric diseases treated with EPO. However, the critical EPO targets in brain are unknown, and separation of direct and indirect effects has remained difficult, given the role of EPO in hematopoiesis and brain oxygen supply. RESULTS: Here we demonstrate that mice with transgenic expression of a constitutively active EPOR isoform (cEPOR) in pyramidal neurons of cortex and hippocampus exhibit enhancement of spatial learning, cognitive flexibility, social memory, and attentional capacities, accompanied by increased impulsivity. Superior cognitive performance is associated with augmented long-term potentiation of cEPOR expressing neurons in hippocampal slices. CONCLUSIONS: Active EPOR stimulates neuronal plasticity independent of any hematopoietic effects and in addition to its neuroprotective actions. This property of EPOR signaling should be exploited for defining novel strategies to therapeutically enhance cognitive performance in disease conditions.

Modification of cognitive performance in schizophrenia by complexin 2 gene polymorphisms.

CONTEXT: Schizophrenia is the collective term for a heterogeneous group of mental disorders with a still obscure biological basis. In particular, the specific contribution of risk or candidate gene variants to the complex schizophrenic phenotype is largely unknown. OBJECTIVE: To prepare the ground for a novel "phenomics" approach, a unique schizophrenia patient database was established by GRAS (Göttingen Research Association for Schizophrenia), designed to allow association of genetic information with quantifiable phenotypes.Because synaptic dysfunction plays a key role in schizophrenia, the complexin 2 gene (CPLX2) was examined in the first phenotype-based genetic association study (PGAS) of GRAS [corrected] DESIGN: Subsequent to a classic case-control approach, we analyzed the contribution of CPLX2 polymorphisms to discrete cognitive domains within the schizophrenic population. To gain mechanistic insight into how certain CPLX2 variants influence gene expression and function, peripheral blood mononuclear cells of patients, Cplx -null mutant mice, and transfected cells were investigated. SETTING: Coordinating research center (Max Planck Institute of Experimental Medicine) and 23 collaborating psychiatric centers all over Germany. PARTICIPANTS: One thousand seventy-one patients with schizophrenia (DSM-IV) examined by an invariant investigator team, resulting in the GRAS database with more than 3000 phenotypic data points per patient, and 1079 healthy control subjects of comparable ethnicity. Main Outcome Measure Cognitive performance including executive functioning, reasoning, and verbal learning/memory. RESULTS: Six single-nucleotide polymorphisms, distributed over the whole CPLX2 gene, were found to be highly associated with current cognition of schizophrenic subjects but only marginally with premorbid intelligence. Correspondingly, in Cplx2 -null mutant mice, prominent cognitive loss of function was obtained only in combination with a minor brain lesion applied during puberty, modeling a clinically relevant environmental risk ("second hit") for schizophrenia. In the human CPLX2 gene, 1 of the identified 6 cognition-relevant single-nucleotide polymorphisms, rs3822674 in the 3' untranslated region, was detected to influence microRNA-498 binding and gene expression. The same marker was associated with differential expression of CPLX2 in peripheral blood mononuclear cells. CONCLUSIONS: The PGAS allows identification of marker-associated clinical/biological traits. Current cognitive performance in schizophrenic patients is modified by CPLX2 variants modulating posttranscriptional gene expression.

Erythropoietin as neuroprotective and neuroregenerative treatment strategy: comprehensive overview of 12 years of preclinical and clinical research.

Erythropoietin (EPO), originally discovered as hematopoietic growth factor, has direct effects on cells of the nervous system that make it a highly attractive candidate drug for neuroprotection/neuroregeneration. Hardly any other compound has led to so much preclinical work in the field of translational neuroscience than EPO. Almost all of the >180 preclinical studies performed by many independent research groups from all over the world in the last 12 years have yielded positive results on EPO as a neuroprotective drug. The fact that EPO was approved for the treatment of anemia >20 years ago and found to be well tolerated and safe, facilitated the first steps of translation from preclinical findings to the clinic. On the other hand, the same fact, naturally associated with loss of patent protection, hindered to develop EPO as a highly promising therapeutic strategy for application in human brain disease. Therefore, only few clinical neuroprotection studies have been concluded, all with essentially positive and stimulating results, but no further development towards the clinic has occurred thus far. This article reviews the preclinical and clinical work on EPO for the indications neuroprotection/neuroregeneration and cognition, and hopefully will stimulate new endeavours promoting development of EPO for the treatment of human brain diseases.

Erythropoietin improves operant conditioning and stability of cognitive performance in mice.

BACKGROUND: Executive functions, learning and attention are imperative facets of cognitive performance, affected in many neuropsychiatric disorders. Recently, we have shown that recombinant human erythropoietin improves cognitive functions in patients with chronic schizophrenia, and that it leads in healthy mice to enhanced hippocampal long-term potentiation, an electrophysiological correlate of learning and memory. To create an experimental basis for further mechanistic insight into erythropoietin-modulated cognitive processes, we employed the Five Choice Serial Reaction Time Task. This procedure allows the study of the effects of erythropoietin on discrete processes of learning and attention in a sequential fashion. RESULTS: Male mice were treated for 3 weeks with erythropoietin (5,000 IU/kg) versus placebo intraperitoneally every other day, beginning at postnatal day 28. After termination of treatment, mice were started on the Five Choice Serial Reaction Time Task, with daily training and testing extending to about 3 months.Overall, a significantly higher proportion of erythropoietin-treated mice finished the task, that is, reached the criteria of adequately reacting to a 1.0 sec flash light out of five arbitrarily appearing choices. During acquisition of this capability, that is, over almost all sequential training phases, learning readouts (magazine training, operant and discriminant learning, stability of performance) were superior in erythropoietin-treated versus control mice. CONCLUSION: Early erythropoietin treatment leads to lasting improvement of cognitive performance in healthy mice. This finding should be exploited in novel treatment strategies for brain diseases.

Aberrant function and structure of retinal ribbon synapses in the absence of complexin 3 and complexin 4.

Complexins regulate the speed and Ca(2+) sensitivity of SNARE-mediated synaptic vesicle fusion at conventional synapses. Two of the vertebrate complexins, Cplx3 and Cplx4, are specifically localized to retinal ribbon synapses. To test whether Cplx3 and Cplx4 contribute to the highly efficient transmitter release at ribbon synapses, we studied retina function and structure in Cplx3 and Cplx4 single- and double-knockout mice. Electroretinographic recordings from single and double mutants revealed a cooperative perturbing effect of Cplx3 and Cplx4 deletion on the b-wave amplitude, whereas most other detected effects in both plexiform synaptic layers were additive. Light and electron microscopic analyses uncovered a disorganized outer plexiform layer in the retinae of mice lacking Cplx3 and Cplx4, with a significant proportion of photoreceptor terminals containing spherical free-floating ribbons. These structural and functional aberrations were accompanied by behavioural deficits indicative of a vision deficit. Our results show that Cplx3 and Cplx4 are essential regulators of transmitter release at retinal ribbon synapses. Their loss leads to aberrant adjustment and fine-tuning of transmitter release at the photoreceptor ribbon synapse, alterations in transmission at bipolar cell terminals, changes in the temporal structure of synaptic processing in the inner plexiform layer of the retina and perturbed vision.

Erythropoietin enhances hippocampal long-term potentiation and memory.

BACKGROUND: Erythropoietin (EPO) improves cognition of human subjects in the clinical setting by as yet unknown mechanisms. We developed a mouse model of robust cognitive improvement by EPO to obtain the first clues of how EPO influences cognition, and how it may act on hippocampal neurons to modulate plasticity. RESULTS: We show here that a 3-week treatment of young mice with EPO enhances long-term potentiation (LTP), a cellular correlate of learning processes in the CA1 region of the hippocampus. This treatment concomitantly alters short-term synaptic plasticity and synaptic transmission, shifting the balance of excitatory and inhibitory activity. These effects are accompanied by an improvement of hippocampus dependent memory, persisting for 3 weeks after termination of EPO injections, and are independent of changes in hematocrit. Networks of EPO-treated primary hippocampal neurons develop lower overall spiking activity but enhanced bursting in discrete neuronal assemblies. At the level of developing single neurons, EPO treatment reduces the typical increase in excitatory synaptic transmission without changing the number of synaptic boutons, consistent with prolonged functional silencing of synapses. CONCLUSION: We conclude that EPO improves hippocampus dependent memory by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks. These mechanisms of action of EPO have to be further exploited for treating neuropsychiatric diseases.