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Alzheimer’s disease (AD) is a severe neurodegenerative disorder and the most common form of dementia, causing the loss of cognitive function. Our previous study has shown, using a doubly mutated mouse model of AD (APP/PS1), that the neural adhesion molecule L1 directly binds amyloid peptides and decreases plaque load and gliosis when injected as an adeno-associated virus construct (AAV-L1) into APP/PS1 mice. In this study, we microinjected AAV-L1, using a Hamilton syringe, directly into the 3-month-old APP/PS1 mouse hippocampus and waited for a year until significant neurodegeneration developed. We stereologically counted the principal neurons and parvalbumin-positive interneurons in the hippocampus, estimated the density of inhibitory synapses around principal cells, and compared the AAV-L1 injection models with control injections of green fluorescent protein (AAV-GFP) and the wild-type hippocampus. Our results show that there is a significant loss of granule cells in the dentate gyrus of the APP/PS1 mice, which was improved by AAV-L1 injection, compared with the AAV-GFP controls (p < 0.05). There is also a generalized loss of parvalbumin-positive interneurons in the hippocampus of APP/PS1 mice, which is ameliorated by AAV-L1 injection, compared with the AAV-GFP controls (p < 0.05). Additionally, AAV-L1 injection promotes the survival of inhibitory synapses around the principal cells compared with AAV-GFP controls in all three hippocampal subfields (p < 0.01). Our results indicate that L1 promotes neuronal survival and protects the synapses in an AD mouse model, which could have therapeutic implications.

The X-chromosome-linked cell adhesion molecule L1 (L1CAM), a glycoprotein mainly expressed by neurons in the central and peripheral nervous systems, has been implicated in many neural processes, including neuronal migration and survival, neuritogenesis, synapse formation, synaptic plasticity and regeneration. L1 consists of extracellular, transmembrane and cytoplasmic domains. Proteolytic cleavage of L1’s extracellular and transmembrane domains by different proteases generates several L1 fragments with different functions. We found that myelin basic protein (MBP) cleaves L1’s extracellular domain, leading to enhanced neuritogenesis and neuronal survival in vitro. To investigate in vivo the importance of the MBP-generated 70 kDa fragment (L1-70), we generated mice with an arginine to alanine substitution at position 687 (L1/687), thereby disrupting L1’s MBP cleavage site and obliterating L1-70. Young adult L1/687 males showed normal anxiety and circadian rhythm activities but enhanced locomotion, while females showed altered social interactions. Older L1/687 males were impaired in motor coordination. Furthermore, L1/687 male and female mice had a larger hippocampus, with more neurons in the dentate gyrus and more proliferating cells in the subgranular layer, while the thickness of the corpus callosum and the size of lateral ventricles were normal. In summary, subtle mutant morphological changes result in subtle behavioral changes.

Neuronal plasticity is a crucial mechanism for an adapting nervous system to change. It is shown to be regulated by perineuronal nets (PNNs), the condensed forms of the extracellular matrix (ECM) around neuronal bodies. By assessing the changes in the number, intensity, and structure of PNNs, the ultrastructure of the PNN mesh, and the expression of inhibitory and excitatory synaptic inputs on these neurons, we aimed to clarify the role of an ECM glycoprotein, tenascin-C (TnC), in the dorsal hippocampus. To enhance neuronal plasticity, TnC-deficient (TnC-/-) and wild-type (TnC+/+) young adult male mice were reared in an enriched environment (EE) for 8 weeks. Deletion of TnC in TnC-/- mice showed an ultrastructural reduction of the PNN mesh and an increased inhibitory input in the dentate gyrus (DG), and an increase in the number of PNNs with a rise in the inhibitory input in the CA2 region. EE induced an increased inhibitory input in the CA2, CA3, and DG regions; in DG, the change was also followed by an increased intensity of PNNs. No changes in PNNs or synaptic expression were found in the CA1 region. We conclude that the DG and CA2 regions emerged as focal points of alterations in PNNs and synaptogenesis with EE as mediated by TnC.

IntroductionThere is an increasing evidence supporting the hypothesis that traumatic experiences during early developmental periods might be associated with psychopathology later in life. Maternal deprivation (MD) in rodents has been proposed as an animal model for certain aspects of neuropsychiatric disorders.MethodsTo determine whether early-life stress leads to changes in GABAergic, inhibitory interneurons in the limbic system structures, specifically the amygdala and nucleus accumbens, 9-day-old Wistar rats were exposed to a 24 h MD. On postnatal day 60 (P60), the rats were sacrificed for morphometric analysis and their brains were compared to the control group.ResultsResults show that MD affect GABAergic interneurons, leading to the decrease in density and size of the calcium-binding proteins parvalbumin-, calbindin-, and calretinin-expressing interneurons in the amygdala and nucleus accumbens.DiscussionThis study indicates that early stress in life leads to changes in the number and morphology of the GABAergic, inhibitory interneurons in the amygdala and nucleus accumbens, most probably due to the loss of neurons during postnatal development and it further contributes to understanding the effects of maternal deprivation on brain development.

The role that the immune system plays after injury of the peripheral nervous system is still not completely understood. Perforin, a natural killer cell- and T-lymphocyte-derived enzyme that mediates cytotoxicity, plays important roles in autoimmune diseases, infections and central nervous system trauma, such as spinal cord injury. To dissect the roles of this single component of the immune response to injury, we tested regeneration after femoral nerve injury in perforin-deficient (Pfp–/–) and wild-type control mice. Single frame motion analysis showed better motor recovery in Pfp–/– mice compared with control mice at 4 and 8 weeks after injury. Retrograde tracing of the motoneuron axons regrown into the motor nerve branch demonstrated more correctly projecting motoneurons in the spinal cord of Pfp–/– mice compared with wild-types. Myelination of regrown axons measured by g-ratio was more extensive in Pfp–/– than in wild-type mice in the motor branch of the femoral nerve. Pfp–/– mice displayed more cholinergic synaptic terminals around cell bodies of spinal motoneurons after injury than the injured wild-types. We histologically analyzed lymphocyte infiltration 10 days after surgery and found that in Pfp–/– mice the number of lymphocytes in the regenerating nerves was lower than in wild-types, suggesting a closed blood-nerve barrier in Pfp–/– mice. We conclude that perforin restricts motor recovery after femoral nerve injury owing to decreased survival of motoneurons and reduced myelination.

Early life stress negatively impacts brain development and affects structure and function of parvalbumin immunopositive (PV+) inhibitory neurons. Main regulators of PV+ interneurons activity and plasticity are perineuronal nets (PNNs), an extracellular matrix formation that enwraps PV+ interneurons mainly in the neocortex and hippocampus. To experimentally address the impact of early life stress on the PNNs and PV+ interneurons in the medial prefrontal cortex and dorsal hippocampus in rats, we employed a 24 h maternal deprivation protocol. We show that maternal deprivation in the medial prefrontal cortex of adult rats caused a decrease in density of overall PNNs and PNNs that enwrap PV+ interneurons in the rostral cingulate cortex. Furthermore, a staining intensity decrease of overall PNNs and PNN+/PV+ cells was found in the prelimbic cortex. Finally, a decrease in both intensity and density of overall PNNs and PNNs surrounding PV+ cells was observed in the infralimbic cortex, together with increase in the intensity of VGAT inhibitory puncta. Surprisingly, maternal deprivation did not cause any changes in the density of PV+ interneurons in the mPFC, neither had it affected PNNs and PV+ interneurons in the hippocampus. Taken together, our findings indicate that PNNs, specifically the ones enwrapping PV+ interneurons in the medial prefrontal cortex, are affected by early life stress.

Abstract Background Microglia are essential to maintain cell homeostasis in the healthy brain and are activated after brain injury. Upon activation, microglia polarize towards different phenotypes. The course of microglia activation is complex and depends on signals in the surrounding milieu. Recently, it has been suggested that microglia respond to ion currents, as a way of regulating their activity and function. Methods and results Under the hypothesis that HCN and KCNQ/Kv7 channels impact on microglia, we studied primary rat microglia in the presence or absence of specific pharmacological blockade or RNA silencing. Primary microglia expressed the subunits HCN1-4, Kv7.2, Kv7.3, and Kv7.5. The expression of HCN2, as well as Kv7.2 and Kv7.3, varied among different microglia phenotypes. The pharmacological blockade of HCN channels by ZD7288 resulted in cell depolarization with slowly rising intracellular calcium levels, leading to enhanced survival and reduced proliferation rates of resting microglia. Furthermore, ZD7288 treatment, as well as knockdown of HCN2 RNA by small interfering RNA, resulted in an attenuation of later microglia activation—both towards the anti- and pro-inflammatory phenotype. However, HCN channel inhibition enhanced the phagocytic capacity of IL4-stimulated microglia. Blockade of Kv7/KCNQ channel by XE-991 exclusively inhibited the migratory capacity of resting microglia. Conclusion These observations suggest that the HCN current contributes to various microglia functions and impacts on the course of microglia activation, while the Kv7/KCNQ channels affect microglia migration. Characterizing the role of HCN channels in microglial functioning may offer new therapeutic approaches for targeted modulation of neuroinflammation as a hallmark of various neurological disorders.

Adhesion molecules play major roles in cell proliferation, migration, survival, neurite outgrowth and synapse formation during nervous system development and in adulthood. The neural cell adhesion molecule L1 contributes to these functions during development and in synapse formation and synaptic plasticity after trauma in adulthood. Mutations of L1 in humans result in L1 syndrome, which is associated with mild-to-severe brain malformations and mental disabilities. Furthermore, mutations in the extracellular domain were shown to cause a severe phenotype more often than mutations in the intracellular domain. To explore the outcome of a mutation in the extracellular domain, we generated mice with disruption of the dibasic sequences RK and KR that localize to position 858RKHSKR863 in the third fibronectin type III domain of murine L1. These mice exhibit alterations in exploratory behavior and enhanced marble burying activity. Mutant mice display higher numbers of caspase 3-positive neurons, a reduced number of principle neurons in the hippocampus, and an enhanced number of glial cells. Experiments suggest that disruption of the dibasic sequence in L1 results in subtle impairments in brain structure and functions leading to obsessive-like behavior in males and reduced anxiety in females.

Early life stress has profound effects on the development of the central nervous system. We exposed 9-day-old rat pups to a 24 h maternal deprivation (MD) and sacrificed them as young adults (60-day-old), with the aim to study the effects of early stress on forebrain circuitry. We estimated numbers of various immunohistochemically defined interneuron subpopulations in several neocortical regions and in the hippocampus. MD rats showed reduced numbers of parvalbumin-expressing interneurons in the CA1 region of the hippocampus and in the prefrontal cortex, compared with controls. Numbers of reelin-expressing and calretinin-expressing interneurons were also reduced in the CA1 and CA3 hippocampal areas, but unaltered in the neocortex of MD rats. The number of calbinin-expressing interneurons in the neocortex was similar in the MD rats compared with controls. We analyzed cell death in 15-day-old rats after MD and found no difference compared to control rats. Thus, our results more likely reflect the downregulation of markers than the actual loss of interneurons. To investigate synaptic activity in the hippocampus we immunostained for glutamatergic and inhibitory vesicular transporters. The number of inhibitory synapses was decreased in the CA1 and CA3 regions of the hippocampus in MD rats, with the normal number of excitatory synapses. Our results indicate complex, cell type-specific, and region-specific alterations in the inhibitory circuitry induced by maternal deprivation. Such alterations may underlie symptoms of MD at the behavioral level and possibly contribute to mechanisms by which early life stress causes neuropsychiatric disorders, such as schizophrenia.

The close homolog of L1 (CHL1) is a cell adhesion molecule involved in regulation of neuronal differentiation and survival, neurite outgrowth and axon guidance during development. In the mature nervous system, CHL1 regulates synaptic activity and plasticity. The aim of the present study was to evaluate the influence of CHL1 on peripheral nerve regeneration after trauma. Using the established model of mouse femoral nerve regeneration, CHL1 knock-out mice were investigated in comparison to the wild type littermates. First, non-injured mice of both genotypes were compared regarding the synaptic phenotypes in the corresponding spinal cord segment. While no differences in phenotypes were detectable in the femoral nerve, corresponding segments in the spinal cord were observed to differ in that inhibitory perisomatic innervation of motor neurons was increased in CHL1-deficient mice, and numbers of perisomatic cholinergic synapses on motor neuronal somata were reduced. Regarding the femoral nerve after injury, CHL1-deficient mice demonstrated preferential motor axon regrowth into the saphenous vs. quadriceps branch after nerve transection upstream of the nerve bifurcation by 8 weeks after transection, indicating decreased preferential motor re-innervation. Furthermore, in injured wild-type mice, enhanced CHL1 expression was observed in regenerating axons in the proximal nerve stump upstream of the bifurcation at days 1, 3, 5, 7 and 14, and in the distal stump at days 7 and 14 after injury, when compared to non-injured mice. Injury-related upregulation of CHL1 expression was more pronounced in axons than in Schwann cells. Despite a more pronounced capacity for preferential motor axon regrowth in wild-type vs. mutant mice, only a tendency for difference in recovery of motor functions was observed between genotypes, without statistical significance Taken together, these results indicate that CHL1 is involved in peripheral nerve regeneration, because it guides regrowing axons into the appropriate nerve branch and regulates synaptic coverage in the spinal cord.

The nervous system is a notable exception to the rule that the cell is the structural and functional unit of tissue systems and organs. The functional unit of the nervous system is the synapse, the contact between two nerve cells. As such, synapses are the foci of investigations of nervous system organization and function, as well as a potential readout for the progression of various disorders of the nervous system. In the past decade the development of antibodies specific to presynaptic terminals has enabled us to assess, at the optical, laser scanning microscopy level, these subcellular structures, and has provided a simple method for the quantification of various synapses. Indeed, excitatory (glutamatergic) and inhibitory synapses can be visualized using antibodies against the respective vesicular transporters, and choline-acetyl transferase (ChAT) immunoreactivity identifies cholinergic synapses throughout the central nervous system. Here we review the results of several studies in which these methods were used to estimate synaptic numbers as the structural equivalent of functional outcome measures in spinal cord and femoral nerve injuries, as well as in genetic mouse models of neurodegeneration, including Alzheimer’s disease (AD). The results implicate disease- and brain region-specific changes in specific types of synapses, which correlate well with the degree of functional deficit caused by the disease process. Additionally, results are reproducible between various studies and experimental paradigms, supporting the reliability of the method. To conclude, this quantitative approach enables fast and reliable estimation of the degree of the progression of neurodegenerative changes and can be used as a parameter of recovery in experimental models.

Evolutionary elaboration of tissues starts with changes in the genome and location of the stem cells. For example, GABAergic interneurons of the mammalian neocortex are generated in the ventral telencephalon and migrate tangentially to the neocortex, in contrast to the projection neurons originating in the ventricular/subventricular zone (VZ/SVZ) of the dorsal telencephalon. In human and nonhuman primates, evidence suggests that an additional subset of neocortical GABAergic interneurons is generated in the cortical VZ and a proliferative niche, the outer SVZ. The origin, magnitude, and significance of this species-specific difference are not known. We use a battery of assays applicable to the human, monkey, and mouse organotypic cultures and supravital tissue to identify neuronal progenitors in the cortical VZ/SVZ niche that produce a subset of GABAergic interneurons. Our findings suggest that these progenitors constitute an evolutionary novelty contributing to the elaboration of higher cognitive functions in primates.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the most common cause of elderly dementia. In an effort to contribute to the potential of molecular approaches to reduce degenerative processes we have tested the possibility that the neural adhesion molecule L1 ameliorates some characteristic cellular and molecular parameters associated with the disease in a mouse model of AD. Three-month-old mice overexpressing mutated forms of amyloid precursor protein and presenilin-1 under the control of a neuron-specific promoter received an injection of adeno-associated virus encoding the neuronal isoform of full-length L1 (AAV-L1) or, as negative control, green fluorescent protein (AAV-GFP) into the hippocampus and occipital cortex. Four months after virus injection, the mice were analyzed for histological and biochemical parameters of AD. AAV-L1 injection decreased the Aβ plaque load, levels of Aβ42, Aβ42/40 ratio and astrogliosis compared with AAV-GFP controls. AAV-L1 injected mice also had increased densities of inhibitory synaptic terminals on pyramidal cells in the hippocampus when compared with AAV-GFP controls. Numbers of microglial cells/macrophages were similar in both groups, but numbers of microglial cells/macrophages per plaque were increased in AAV-L1 injected mice. To probe for a molecular mechanism that may underlie these effects, we analyzed whether L1 would directly and specifically interact with Aβ. In a label-free binding assay, concentration dependent binding of the extracellular domain of L1, but not of the close homolog of L1 to Aβ40 and Aβ42 was seen, with the fibronectin type III homologous repeats 1–3 of L1 mediating this effect. Aggregation of Aβ42 in vitro was reduced in the presence of the extracellular domain of L1. The combined observations indicate that L1, when overexpressed in neurons and glia, reduces several histopathological hallmarks of AD in mice, possibly by reduction of Aβ aggregation. L1 thus appears to be a candidate molecule to ameliorate the pathology of AD, when applied in therapeutically viable treatment schemes.

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.

Oligodendrocytes are cells that myelinate axons, providing saltatory conduction of action potentials and proper function of the central nervous system. Myelination begins prenatally in the human, and the sequence of oligodendrocyte development and the onset of myelination are not thoroughly investigated. This knowledge is important to better understand human diseases, such as periventricular leukomalacia, one of the leading causes of motor deficit in premature babies, and demyelinating disorders such as multiple sclerosis (MS). In this review we discuss the spatial and temporal progression of oligodendrocyte lineage characterized by the expression of specific markers and transcription factors in the human fetal brain from the early embryonic period (5 gestational weeks, gw) until midgestation (24 gw). Our in vitro evidence indicated that a subpopulation of human oligodendrocytes may have dorsal origin, from cortical radial glia cells, in addition to their ventral telencephalic origin. Furthermore, we demonstrated that the regulation of myelination in the human fetal brain includes positive and negative regulators. Chemokines, such as CXCL1, abundant in proliferative zones during brain development and in regions of remyelination in adult, are discussed in the view of their potential roles in stimulating oligodendrocyte development. Other signals are inhibitory and may include, but are not limited to, polysialic acid modification of the neural cell adhesion molecule on axons. Overall, important differences in temporal and spatial distribution and regulatory signals for oligodendrocyte differentiation exist in the human brain. Those differences may underlie the unique susceptibility of humans to demyelinating diseases, such as MS.

Understanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an in vitro injury model, the mixed culture of cortical astrocytes and microglia, and observed that without TnC microglial cells tend to populate gap area in greater numbers and proliferate more, whereas astrocytes build up in the border region to promote faster gap closure. Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-α and IL-1β upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of β1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area.

Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC’s different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain.