Why do anti-inflammatory signals of bone marrow-derived stromal cells improve neurodegenerative conditions where anti-inflammatory drugs fail?

Neurodegenerative disorders share the final degenerative pathway, the inflammation-induced apoptosis and/or necrosis, irrespective of their etiology, be it of acute and chronic traumatic, vascular and idiopathic origin. Although disease-modifying strategies are an unmet need in these disorders, lately, (pre)clinical studies suggested favorable effects after an intervention with bone marrow-derived stromal cells (bm-SC). Recent interventions with intrathecal transplantation of these cells in preclinical rodent models improved the functional outcome and reduced the inflammation, but not anti-inflammatory drugs. The benefit of bm-SCs was demonstrated in rats with an acute (traumatic spinal cord injury, tSCI) and in mice with a chronic [amyotrophic lateral sclerosis (ALS)-like FUS 1-358 or SOD1-G93-A mutation] neurodegenerative process. Bm-SCs, were found to modify underlying disease processes, to reduce final clinical SCI-related outcome, and to slow down ALS-like clinical progression. After double-blind interventions with bm-SC transplantations, Vehicle (placebo), and (non)steroidal anti-inflammatory drugs (Methylprednisolone, Riluzole, Celecoxib), clinical, histological and histochemical findings, serum/spinal cytokines, markers for spinal microglial activation inclusive, evidenced the cell-to-cell action of bm-SCs in both otherwise healthy and immune-deficient tSCI-rats, as well as wild-type and FUS/SOD1-transgenic ALS-like mice. The multi-pathway hypothesis of the cell-to-cell action of bmSCs, presumably using extracellular vesicles (EVs) as carriers of messages in the form of RNAs, DNA, proteins, and lipids rather than influencing a single inflammatory pathway, could be justified by the reported differences of cytokines and other chemokines in the serum and spinal tissue. The mode of action of bm-SCs is hypothesized to be associated with its dedicated adjustment of the pro-apoptotic glycogen synthase kinase-3ß level towards an anti-apoptotic level whereas their multi-pathway hypothesis seems to be confirmed by the decreased levels of the pro-inflammatory interleukin (IL)-1ß and tumor necrosis factor (TNF) as well as the level of the marker of activated microglia, ionized calcium binding adapter (Iba)-1 level.

[Targeted inactivation of gamma-synuclein gene affects anxiety and exploratory behaviour of mice].

Gamma(gamma)-synuclein is a member of synuclein family of cytoplasmic and predominantly neuronal proteins found only in vertebrates. Gamma-synuclein is abundant in axons and presynaptic terminals of neurons localized in brain regions involved in emotions, learning and memory. However, the role of gamma-synuclein in these brain functions was not previously assessed. We have demonstrated for the first time that the loss of gamma-synuclein results in a significant increase in the level of orientation response in novel environment and decrease in the level of state anxiety.

Standardized human bone marrow-derived stem cells infusion improves survival and recovery in a rat model of spinal cord injury.

Spinal cord injury (SCI) is an incurable disorder with an unmet need of an effective treatment. Recently, autologous human bone marrow-derived stem cells have shown to promote functional improvement, due to their anti-inflammatory and regenerative/apocrine properties. In this study, the primary objective was to test whether a single intrathecal injection with a 100 µL suspension of 400,000 fresh human bone marrow-derived CD34+ and an equal number of CD105+ stem cells (Neuro-Cells (NC)), one day after balloon-compression of the spinal cord, improves motor function and reduces secondary damage in immunodeficient rats. During the first 5 weeks after this intervention, NC significantly improved locomotor recovery and induced less injury-associated adverse events compared to vehicle-treated rats. Histological analysis showed that NC reduced astrogliosis, and apoptosis early after administration (day 4), but not at a later stage (day 56) after SCI. Proteomic studies (at day 56) pointed to the release of paracrine factors and identified proteins involved in regenerative processes. As stem cells seem to reach their effects in acute lesions by mainly suppressing (secondary) inflammation, it is thus realistic to expect a lower magnitude of their eventual beneficial effect in T-cell deficient rats, a fact reinforcing the robustness of Neuro-Cells efficacy. Taken together, this study indicates that an intrathecal instillation of Neuro-Cells holds great promise as a neuro-regenerative intervention in a clinical setting with acute SCI patients.

[Depressive-like state and sleep in laboratory mice].

In order to induce the state of anhedonia, a key symptom of depression, mice were subjected to a one-month stress procedure comprised of various stressors. Anhedonic state was defined by a reduction of preference for sucrose solution over tap water. Conventional cortical and neck-muscle electrodes were implanted to control and stressed animals under chloral-hydrate anesthesia. After a two-week recovery and habituation period, mice from chronically stressed group were re-subjected to five-day stress, and the anhedonic state was verified. As not all the stressed mice displayed a decrease in sucrose preference, animals were divided in two groups: stressed-non-anhedonic and stressed-anhedonic animals. Seven-day continuous polygraphic recording was carried out in animals from both stressed groups and the control group in recording chambers under conditions of 12/12-hour light/dark schedule. The anhedonic mice demonstrated a significant advanced shift in circadian distribution of paradoxical sleep and increased amount of paradoxical sleep during the light period. In the course of the dark period, the anhedonic group showed a slight but significant decrease in total amount of slow-wave sleep as compared to the non-anhedonic and control groups. The results suggest that the changes in sleep structure documented in the model of anhedonia are similar to those described for human depression.

Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition.

Cadherin-13 (CDH13), a unique glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules, has been identified as a risk gene for attention-deficit/hyperactivity disorder (ADHD) and various comorbid neurodevelopmental and psychiatric conditions, including depression, substance abuse, autism spectrum disorder and violent behavior, while the mechanism whereby CDH13 dysfunction influences pathogenesis of neuropsychiatric disorders remains elusive. Here we explored the potential role of CDH13 in the inhibitory modulation of brain activity by investigating synaptic function of GABAergic interneurons. Cellular and subcellular distribution of CDH13 was analyzed in the murine hippocampus and a mouse model with a targeted inactivation of Cdh13 was generated to evaluate how CDH13 modulates synaptic activity of hippocampal interneurons and behavioral domains related to psychopathologic (endo)phenotypes. We show that CDH13 expression in the cornu ammonis (CA) region of the hippocampus is confined to distinct classes of interneurons. Specifically, CDH13 is expressed by numerous parvalbumin and somatostatin-expressing interneurons located in the stratum oriens, where it localizes to both the soma and the presynaptic compartment. Cdh13(-/-) mice show an increase in basal inhibitory, but not excitatory, synaptic transmission in CA1 pyramidal neurons. Associated with these alterations in hippocampal function, Cdh13(-/-) mice display deficits in learning and memory. Taken together, our results indicate that CDH13 is a negative regulator of inhibitory synapses in the hippocampus, and provide insights into how CDH13 dysfunction may contribute to the excitatory/inhibitory imbalance observed in neurodevelopmental disorders, such as ADHD and autism.

Memory retrieval after contextual fear conditioning induces c-Fos and JunB expression in CA1 hippocampus.

Using specific polyclonal antisera against c-Fos, JunB, c-Jun and JunD, we tried to identify the candidate transcription factors of the immediate early gene family which may contribute to the molecular processes during contextual memory reconsolidation. For that purpose we analyzed the expression of these proteins in the hippocampus after contextual memory retrieval in a mouse model of fear conditioning. A single exposure to a foot shock of 0.8 mA was sufficient to induce robust contextual fear conditioning in C57BI/6N mice. In these mice context dependent memory retrieval evoked a marked induction of c-Fos and JunB, but not of c-Jun and JunD, in pyramidal CA1 neurons of the dorsal hippocampus. In contrast, mice exposed and re-exposed only to the context, without foot shock, did not show behavioral signs of contextual fear conditioning and exhibited significantly less expression of c-Fos and JunB in CA1 neurons. Mice which received a foot shock but were not re-exposed to the context revealed no immediate early gene induction. These results demonstrate that contextual memory retrieval is associated with de novo synthesis of specific members of the Fos/Jun transcription factor family. Therefore we suggest that these genes may contribute to plasticity and reconsolidation accompanying the retrieval process. The specific activation of CA1 neurons during the retrieval of contextual fear associations supports the postulated concept of a mnemonic role of this hippocampal subsector during the retrieval of contextual informations.

Mice with targeted mutations of glucocorticoid and mineralocorticoid receptors: models for depression and anxiety?

Impaired corticosteroid receptor signaling is a key mechanism in the pathogenesis of stress-related psychiatric disorders such as depression and anxiety. Since in vivo expression and functional studies of corticosteroid receptors are not feasible in the human central nervous system, such analyses have to be done in animal models. Transgenic mice with mutations of corticosteroid receptors are promising tools, which allow us to investigate the role of these proteins in the pathogenesis of symptoms characteristic for depression and anxiety. This review summarizes the neuroendocrinological and behavioral findings that have been obtained in six different mouse strains with specific mutations that influence the expression or the function of the glucocorticoid or the mineralocorticoid receptor (MR). The analyses of these mice helped to define molecular concepts of how corticosteroid receptors regulate the activity of the hypothalamic-pituitary-adrenal (HPA) system. Furthermore, some of these mutant mice exhibited characteristic alterations in behavioral tests for anxiety and despair. However, so far, none of the mouse strains described here can be viewed as an animal model of a specific psychiatric disease defined by common diagnostic criteria. Using high throughput technologies for the identification of genes regulated by glucocorticoid receptor (GR) and MR in brain areas responsible for specific symptoms of stress-related disorders will yield potential new drug targets for the treatment of depression and anxiety.

Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-HTT-deficient mice.

The serotonin transporter gene (5-HTT/SLC6A4)-linked polymorphic region has been suggested to have a modulatory role in mediating effects of early-life stress exposure on psychopathology rendering carriers of the low-expression short (s)-variant more vulnerable to environmental adversity in later life. The underlying molecular mechanisms of this gene-by-environment interaction are not well understood, but epigenetic regulation including differential DNA methylation has been postulated to have a critical role. Recently, we used a maternal restraint stress paradigm of prenatal stress (PS) in 5-HTT-deficient mice and showed that the effects on behavior and gene expression were particularly marked in the hippocampus of female 5-Htt+/- offspring. Here, we examined to which extent these effects are mediated by differential methylation of DNA. For this purpose, we performed a genome-wide hippocampal DNA methylation screening using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip Mouse Promoter 1.0 R arrays. Using hippocampal DNA from the same mice as assessed before enabled us to correlate gene-specific DNA methylation, mRNA expression and behavior. We found that 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a subset of which showed overlap with the expression profiles of the corresponding transcripts. For example, a differentially methylated region in the gene encoding myelin basic protein (Mbp) was associated with its expression in a 5-Htt-, PS- and 5-Htt × PS-dependent manner. Subsequent fine-mapping of this Mbp locus linked the methylation status of two specific CpG sites to Mbp expression and anxiety-related behavior. In conclusion, hippocampal DNA methylation patterns and expression profiles of female prenatally stressed 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are promoter methylation-dependent, contribute to the behavioral effects of the 5-Htt genotype, PS exposure and their interaction.

Pregnancy or stress decrease complexity of CA3 pyramidal neurons in the hippocampus of adult female rats.

Pregnancy is a time of distinct neural, physiological and behavioral plasticity in the female. It is also a time when a growing number of women are vulnerable to stress and experience stress-related diseases, such as depression and anxiety. However, the impact of stress during gestation on the neurobiology of the mother has yet to be determined, particularly with regard to changes in the hippocampus; a brain area that plays an important role in stress-related diseases. Therefore, the aim of the present study was to understand how stress and reproductive state may alter dendritic morphology of CA1 and CA3 pyramidal neurons in the hippocampus. To do this, adult age-matched pregnant and virgin female Wistar rats were divided into two conditions: (1) control and (2) stress. Females in the stress condition were restrained for 1h/day for the last 2 weeks of gestation and at matched time-points in virgin females. Females were sacrificed the day after the last restraint session and brains were processed for Golgi impregnation. Dendritic length and number of branch points were quantified for apical and basal regions of CA1 and CA3 pyramidal neurons. Results show that regardless of reproductive state, stressed females had significantly shorter apical dendrites and fewer apical branch points in CA3 pyramidal cells. In addition, pregnant females, regardless of stress exposure, had less complex CA3 pyramidal neurons, as measured by Sholl analysis. No differences between conditions were seen in morphology of CA1 pyramidal neurons. This work shows that both repeated restraint stress and pregnancy affect dendritic morphology by decreasing complexity of CA3, but not CA1, neurons in the hippocampus.

[Molecular changes in inbred mice with individual vulnerability vs resilience to stress-induced depressive-like state].

The C57BL/6 mice were subjected to a chronic combined stress which resulted in the induction of a depressive-like state. The occurrence of a depressive-like state was defined by a decrease in sensitivity to the reward determined by the diminished preference of sweetened solutions over regular drinking water. Such decrease is generally considered as a sign of an unhedonic-like state: one of the key features of clinical depression. Applied here, the paradigm in mice allows unhedonia induction in a subpopulation of stressed animals (54% in the current study); remaining mice are regarded as resilient to stress-induced hedonic deficit. The resilient subgroup is taken, therefore, as a "functional control" for those effects of stress that are not accompanied by development of the stress-induced depressive-like state in mice. The analysis of the mRNA extracted from the hippocampi of stress-subjected and home-cage control mice enabled the assessment of gene expression level of over 13 000 genes. This study showed that unhedonic mice are characterized by an up-regulation of 278 and down-regulation of 174 genes related mostly to the CNS development and functions, inter-cellular interactions and signalling, neurological disorders, apoptosis and behavioural regulation. Resilient animals demonstrated up-regulation of 924 and down-regulation of only 29 genes that control formation of cell assemblies, molecular transport, CNS functioning, neurological disorders and various biochemical reactions. Thus, gene expression profiles in the hippocampus of susceptible vs resilient to stress-induced unhedonia inbred subgroups of animals are strictly distinct in both quantity and quality.

Stress-induced hyperlocomotion as a confounding factor in anxiety and depression models in mice.

Chronic stress is broadly used to model anxiety and depression. However, in chronic stress models, anxiety- and depression-like behaviors might be masked by unspecific effects of stress. We tested whether chronic stress in mice can induce unspecific changes in locomotion, and whether these changes interfere with the measurement of anxiety and forced-swimming behaviors. Also, we studied these latter behaviors in relation to the duration of stress, the lighting conditions during testing, and after the injection of diazepam. We employed a 4-week chronic stress paradigm, adopted from a model of stress-induced anhedonia and a 1-week subchronic stress, both consisting of rat exposure, restraint stress and tail suspension. Chronically stressed mice, tested under bright and moderate illumination, exhibited 'anxiolytic-like' behavior along with prolonged swimming and hyperactivity. These behaviors were not detectable under weak illumination or after the injection of diazepam (0.25 mg/kg). Instead, normal locomotion, increased anxiety and inhibited swimming were revealed under these conditions. Thus, chronic stress can induce hyperlocomotion in mice, which is triggered by acute stressors such as light, and interferes with the evaluation of anxiety and forced swimming. One week of stress did not change locomotion and forced swimming, and increased anxiety irrespective of illumination applied during testing. Our data can possibly explain previously reported contradictions in the behavioral testing of mice with chronic stress models of anxiety and depression.

Intrahippocampal administration of an antibody against the HNK-1 carbohydrate impairs memory consolidation in an inhibitory learning task in mice.

Many cell adhesion molecules express the HNK-1 carbohydrate involved in formation and functioning of synapses. To assess its role in learning, we injected the monoclonal HNK-1 antibody or nonimmune IgG into the hippocampus of C57BL/6J mice 1 h after training in a step-down avoidance task. In animals treated with the HNK-1 antibody, latencies of step down in a recall session 48 h after injection did not change compared to training values and were significantly shorter versus IgG-treated controls, which acquired the task normally. Similar differences between the two treatments were also observed after a stronger training protocol in a step-down avoidance paradigm. The HNK-1 antibody was effective only when injected 1 h, but not 48 h after training, thus affecting memory consolidation but not memory recall itself. The HNK-1 antibody impaired memory also in tenascin-R knock-out mice, indicating that extracellular matrix molecule tenascin-R, one of the carriers of the HNK-1epitope in the hippocampus, does not mediate the function of the HNK-1 carbohydrate in this task. Our observations show that the HNK-1 carbohydrate is critically involved in memory consolidation in hippocampus-dependent learning in mammals.