Amyloid precursor protein (APP) and amyloid ß (Aß) interact with cell adhesion molecules: Implications in Alzheimer's disease and normal physiology.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder in which dysfunction and loss of synapses and neurons lead to cognitive impairment and death. Accumulation and aggregation of neurotoxic amyloid-ß (Aß) peptides generated via amyloidogenic processing of amyloid precursor protein (APP) is considered to play a central role in the disease etiology. APP interacts with cell adhesion molecules, which influence the normal physiological functions of APP, its amyloidogenic and non-amyloidogenic processing, and formation of Aß aggregates. These cell surface glycoproteins also mediate attachment of Aß to the neuronal cell surface and induce intracellular signaling contributing to Aß toxicity. In this review, we discuss the current knowledge surrounding the interactions of cell adhesion molecules with APP and Aß and analyze the evidence of the critical role these proteins play in regulating the processing and physiological function of APP as well as Aß toxicity. This is a necessary piece of the complex AD puzzle, which we should understand in order to develop safe and effective therapeutic interventions for AD.

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi.

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.

Intersectin associates with synapsin and regulates its nanoscale localization and function.

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Age-dependent loss of parvalbumin-expressing hippocampal interneurons in mice deficient in CHL1, a mental retardation and schizophrenia susceptibility gene.

In humans, deletions/mutations in the CHL1/CALL gene are associated with mental retardation and schizophrenia. Juvenile CHL1-deficient (CHL1(-/-) ) mice have been shown to display abnormally high numbers of parvalbumin-expressing (PV(+) ) hippocampal interneurons and, as adults, display behavioral traits observed in neuropsychiatric disorders. Here, we addressed the question whether inhibitory interneurons and synaptic plasticity in the CHL1(-/-) mouse are affected during brain maturation and in adulthood. We found that hippocampal, but not neocortical, PV(+) interneurons were reduced with age in CHL1(-/-) mice, from a surplus of +27% at 1 month to a deficit of -20% in adulthood compared with wild-type littermates. This loss occurred during brain maturation, correlating with microgliosis and enhanced interleukin-6 expression. In parallel with the loss of PV(+) interneurons, the inhibitory input to adult CA1 pyramidal cells was reduced and a deficit in short- and long-term potentiation developed at CA3-CA1 excitatory synapses between 2 and 9 months of age in CHL1(-/-) mice. This deficit could be abrogated by a GABAA receptor agonist. We propose that region-specific aberrant GABAergic synaptic connectivity resulting from the mutation and a subsequently enhanced synaptic elimination during brain maturation lead to microgliosis, increase in pro-inflammatory cytokine levels, loss of interneurons, and impaired synaptic plasticity. Close homolog of L1-deficient (CHL1(-/-) ) mice have abnormally high numbers of parvalbumin (PV)-expressing hippocampal interneurons in juvenile animals, but in adult animals a loss of these cells is observed. This loss correlates with an increased density of microglia (M), enhanced interleukin-6 (IL6) production and a deficit in short- and long-term potentiation at CA3-CA1 excitatory synapses. Furthermore, adult CHL1(-/-) mice display behavioral traits similar to those observed in neuropsychiatric disorders of humans.

Simple computational technique to quantify nuclear shape asymmetry.

The nucleus of an eukaryotic cell is a membrane-bound organelle containing a major part of the cellular genome. Nuclear shape is controlled by forces generated in the cytoskeleton, nuclear envelope and matrix of the nucleus and may change when the balance of these forces is disturbed. In certain cases, such changes may be indicative of cell pathology. Nuclear shape feature is being commonly addressed in both experimental research and diagnostics; nevertheless its symmetry-related aspects receive little attention. This article introduces a technique allowing to estimate nuclear shape asymmetry in digital images captured from cyto- or histological preparations. Implemented in a software package, this technique quantifies the asymmetry using two scenarios. The first one presumes the identification of nuclear pixels laying outside the largest inscribed circle. According to the second scenario, the algorithm searches for nuclear pixels lacking pixel-partners symmetric with respect to the nuclear area's centroid. In both cases, the proportion of "asymmetric" pixels is used to estimate the feature of interest. The technique was validated on images of cell nuclei having distinctive shape phenotypes. A conclusion was made that shape asymmetry feature may be useful accessory to the toolbox of nuclear morphometry.

Nitric oxide mediates local activity-dependent excitatory synapse development.

Learning related paradigms play an important role in shaping the development and specificity of synaptic networks, notably by regulating mechanisms of spine growth and pruning. The molecular events underlying these synaptic rearrangements remain poorly understood. Here we identify NO signaling as a key mediator of activity-dependent excitatory synapse development. We find that chronic blockade of NO production in vitro and in vivo interferes with the development of hippocampal and cortical excitatory spine synapses. The effect results from a selective loss of activity-mediated spine growth mechanisms and is associated with morphological and functional alterations of remaining synapses. These effects of NO are mediated by a cGMP cascade and can be reproduced or prevented by postsynaptic expression of vasodilator-stimulated phosphoprotein phospho-mimetic or phospho-resistant mutants. In vivo analyses show that absence of NO prevents the increase in excitatory synapse density induced by environmental enrichment and interferes with the formation of local clusters of excitatory synapses. We conclude that NO plays an important role in regulating the development of excitatory synapses by promoting local activity-dependent spine-growth mechanisms.

Heterozygosity for the mutated X-chromosome-linked L1 cell adhesion molecule gene leads to increased numbers of neurons and enhanced metabolism in the forebrain of female carrier mice.

Mutations in the X-chromosomal L1CAM gene lead to severe neurological deficits. In this study, we analyzed brains of female mice heterozygous for L1 (L1+/-) to gain insights into the brain structure of human females carrying one mutated L1 allele. From postnatal day 7 onward into adulthood, L1+/- female mice show an increased density of neurons in the neocortex and basal ganglia in comparison to wild-type (L1+/+) mice, correlating with enhanced metabolic parameters as measured in vivo. The densities of astrocytes and parvalbumin immunoreactive interneurons were not altered. No significant differences between L1+/- and L1+/+ mice were seen for cell proliferation in the cortex during embryonic days 11.5-15.5. Neuronal differentiation as estimated by analysis of doublecortin-immunoreactive cortical cells of embryonic brains was similar in L1+/- and L1+/+ mice. Interestingly, at postnatal days 3 and 5, apoptosis was reduced in L1+/- compared to L1+/+ mice. We suggest that reduced apoptosis leads to increased neuronal density in adult L1+/- mice. In conclusion, L1+/- mice display an unexpected phenotype that is not an intermediate between L1+/+ mice and mice deficient in L1 (L1-/y), but a novel phenotype which is challenging to understand regarding its underlying molecular and cellular mechanisms.

Patterns of papillary thyroid carcinoma cells analyzed in fine-needle aspiration smears may reveal changes in tumor cell behavior.

Fine-needle aspiration (FNA) cytology is widely used to examine thyroid lesions. However, its diagnostic accuracy is limited by the narrow choice of cytopathologic markers indicative of invasive/metastatic powers of a tumor. The aim of this study was to identify features that may serve as such indicators. We have examined FNA smears of 50 histologically proven papillary thyroid carcinoma (PTC) cases applying computer-assisted morphometry to assess patterns formed by PTC cells. Cytokeratine (CK) 8 immunocytochemistry was used to verify the epithelial origin of cells under study. All analyzed smears contained blood, histiocyte-like cells and CK8-positive follicular cells occurring both as single cells and in monolayer cell sheets. In 60% of cases we revealed cell sheets displaying two distinct cell patterns. The first one (pattern R) consisted of moderately pleomorphic, rather regularly arranged cells having an amphophilic cytoplasm. The second one (pattern I) was formed by highly pleomorphic cells with a basophilic cytoplasm. Patterns R and I were clearly different in cell size and shape as well as in nuclear size and shape. These patterns were never observed within the same cell sheet indicating that they may be formed by different subclones of tumor cells. Thus, it can be concluded that PTC frequently displays two definitely different cell patterns. We think that these patterns have a potential to serve as indicators for early events of an invasive/metastatic process. It remains to be seen whether the simultaneous occurrence of these patterns is a PTC-specific feature.

NCAM/spectrin complex disassembly results in PSD perforation and postsynaptic endocytic zone formation.

Mechanisms inducing perforation of the postsynaptic density (PSD) are poorly understood. We show that neural cell adhesion molecule- deficient (NCAM-/-) hippocampal neurons have an abnormally high percentage of synapses with perforated PSDs. The percentage of synapses with perforated PSDs is also increased in wild-type (NCAM+/+) neurons after the disruption of the NCAM/spectrin complex indicating that the NCAM-assembled spectrin cytoskeleton maintains the structural integrity of PSDs. We demonstrate that PSD perforations contain endocytic zones involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization. Induction of long-term potentiation in NCAM+/+ neurons accompanied by insertion of AMPAR into the neuronal cell surface is subsequently followed by formation of perforated synapses and AMPAR endocytosis suggesting that perforation of PSDs is important for membrane homeostasis in activated synapses. In NCAM-/- or NCAM+/+ neurons with dissociated spectrin meshwork, AMPAR endocytosis is enhanced under conditions of basal activity. An abnormally high rate of postsynaptic membrane endocytosis may thus contribute to brain pathologies associated with mutations in NCAM or spectrin.

Brain plasticity after ischemic episode.

Brain plasticity describes the potential of the organ for adaptive changes involved in various phenomena in health and disease. A substantial amount of experimental evidence, received in animal and cell models, shows that a cascade of plastic changes at the molecular, cellular, and tissue levels, is initiated in different regions of the postischemic brain. Underlying mechanisms include neurochemical alterations, functional changes in excitatory and inhibitory synapses, axonal and dendritic sprouting, and reorganization of sensory and motor central maps. Multiple lines of evidence indicate numerous points in which the process of postischemic recovery may be influenced with the aim to restore the full capacity of the brain tissue injured by an ischemic episode.

Structural features of ischemic damage in the hippocampus.

Cerebral ischemic injury resulting from either focal or global circulatory arrests in the brain is one of the major causes of death and disability in the adult population. The hippocampus, playing important roles in learning and memory, is selectively vulnerable to ischemic insults. Distinct populations of hippocampal neurons are targeted by ischemia and multiple factors, including excitotoxicity, oxidative stress, and inflammation, are responsible for their damage and death. Modifications of synapses occur very early after ischemia, reflecting related changes in synaptic transmission. These modifications structurally relate to spatial patterns formed by synaptic vesicles, geometry of postsynaptic density, and so forth. Ischemia-induced changes of synaptic contacts can be implicated in the mechanisms leading to delayed neuronal death. In this review, we summarize the available data on the structural aspects of ischemic injury of the hippocampus obtained in tissue culture and animal models and discuss pathways of neurodegeneration common for cerebral ischemia and various neurodegenerative disorders.

Early reaction of astroglial cells in rat hippocampus to streptozotocin-induced diabetes.

Diabetic patients show impaired brain functions, although underlying mechanisms remain unclear. Little is known as well about early diabetes-related changes in a brain tissue. To investigate them we analyzed the reaction of astroglial cells in the hippocampus of rats rendered diabetic by a single injection of streptozotocin (STZ). Astrocyte count, size and shape as well as levels of glial fibrillary acidic protein (GFAP) and S100b protein were assessed 3, 7 and 14 days after the STZ injection using immunohistochemistry, immuno-enzyme assay and computer-assisted image analysis. The reduced GFAP-positive cell count was found on day 3 when these cells were significantly smaller and less arborized with respect to the control. This tendency reversed on day 7 when more numerous GFAP-positive cells grew in size and became more ramified. S100b-positive cell count changes followed a contrasting pattern, elevating on days 3 and 7 and dropping on day 14. The level of cytoskeletal GFAP changed in parallel with GFAP expression revealed by immunocytochemistry, while cytosolic GFAP level started to increase only 7 days after the STZ injection. At the same time S100b level experienced an elevation on day 3 reaching the peak on day 7 and decreasing afterwards. These results suggest that the reaction of astroglial cells may be the earliest response of the brain tissue to an altered glucose metabolism playing presumably the critical role in the mechanisms underlying diabetes-related impairments of brain functions.

NCAM promotes assembly and activity-dependent remodeling of the postsynaptic signaling complex.

The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. We show that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the N-methyl-d-aspartate (NMDA) receptor and Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) in a manner not firmly or directly linked to PSD95 and alpha-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM-spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM-spectrin-associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIalpha. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIalpha to synapses after NMDA receptor activation, which is a critical process in NMDA receptor-dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM.

Ischemia-induced modifications in hippocampal CA1 stratum radiatum excitatory synapses.

Relatively mild ischemic episode can initiate a chain of events resulting in delayed cell death and significant lesions in the affected brain regions. We studied early synaptic modifications after brief ischemia modeled in rats by transient vessels' occlusion in vivo or oxygen-glucose deprivation in vitro and resulting in delayed death of hippocampal CA1 pyramidal cells. Electron microscopic analysis of excitatory spine synapses in CA1 stratum radiatum revealed a rapid increase of the postsynaptic density (PSD) thickness and length, as well as formation of concave synapses with perforated PSD during the first 24 h after ischemic episode, followed at the long term by degeneration of 80% of synaptic contacts. In presynaptic terminals, ischemia induced a depletion of synaptic vesicles and changes in their spatial arrangement: they became more distant from active zones and had larger intervesicle spacing compared to controls. These rapid structural synaptic changes could be implicated in the mechanisms of cell death or adaptive plasticity. Comparison of the in vivo and in vitro model systems used in the study demonstrated a general similarity of these early morphological changes, confirming the validity of the in vitro model for studying synaptic structural plasticity.

The cell adhesion molecule l1 is required for chain migration of neural crest cells in the developing mouse gut.

BACKGROUND & AIMS: During development, the enteric nervous system is derived from neural crest cells that emigrate from the hindbrain, enter the foregut, and colonize the gut. Defects in neural crest migration can result in intestinal aganglionosis. Hirschsprung's disease (congenital aganglionosis) is a human condition in which enteric neurons are absent from the distal bowel. A number of clinical studies have implicated the cell adhesion molecule L1 in Hirschsprung's disease. We examined the role of L1 in the migration of neural crest cells through the developing mouse gut. METHODS: A variety of in vitro and in vivo assays were used to examine: (1) the effect of L1 blocking antibodies or exogenous soluble L1 protein known to compromise L1 function on the rate of crest cell migration, (2) the effect of blocking L1 activity on the dynamic behavior of crest cells using time-lapse microscopy, and (3) whether the colonization of the gut by crest cells in L1-deficient mice differs from control mice. RESULTS: We show that L1 is expressed by neural crest cells as they colonize the gut. Perturbation studies show that disrupting L1 activity retards neural crest migration and increases the number of solitary neural crest cells. L1-deficient mice show a small but significant reduction in neural crest cell migration at early developmental stages, but the entire gastrointestinal tract is colonized. CONCLUSIONS: L1 is important for the migration of neural crest cells through the developing gut and is likely to be involved in the etiology of Hirschsprung's disease.

Enhanced perisomatic inhibition and impaired long-term potentiation in the CA1 region of juvenile CHL1-deficient mice.

The cell adhesion molecule, CHL1, like its close homologue L1, is important for normal brain development and function. In this study, we analysed the functional role of CHL1 in synaptic transmission in the CA1 region of the hippocampus using juvenile CHL1-deficient (CHL1-/-) and wild-type (CHL1+/+) mice. Inhibitory postsynaptic currents evoked in pyramidal cells by minimal stimulation of perisomatically projecting interneurons were increased in CHL1-/- mice compared with wild-type littermates. Also, long-term potentiation (LTP) at CA3-CA1 excitatory synapses was reduced under physiological conditions in CHL1-/- mice. This abnormality was abolished by application of a GABAA receptor antagonist, suggesting that enhanced inhibition is the cause of LTP impairment. Quantitative ultrastructural and immunohistochemical analyses revealed aberrations possibly related to the abnormally high inhibition observed in CHL1-/- mice. The length and linear density of active zones in symmetric synapses on pyramidal cell bodies, as well as number of perisomatic puncta containing inhibitory axonal markers were increased. Density and total number of parvalbumin-positive interneurons was also abnormally high. These observations and the finding that CA1 interneurons express CHL1 protein indicate that CHL1 is important for regulation of inhibitory synaptic transmission and interneuron populations in the postnatal brain. The observed enhancement of inhibitory transmission in CHL1-/- mice is in contrast to the previous finding of reduced inhibition in L1 deficient mice and indicates different functions of these two closely related molecules.

Reduced GABAergic transmission and number of hippocampal perisomatic inhibitory synapses in juvenile mice deficient in the neural cell adhesion molecule L1.

Cell adhesion molecules have been implicated in neural development and hippocampal synaptic plasticity. Here, we investigated the role of the neural cell adhesion molecule L1 in regulation of basal synaptic transmission and plasticity in the CA1 area of the hippocampus of juvenile mice. We show that theta-burst stimulation (TBS) and pairing of low-frequency presynaptic stimulation with depolarization of postsynaptic CA1 pyramidal cells induced similar levels of LTP in L1-deficient and wild-type mice. The basal excitatory synaptic transmission and density of asymmetric excitatory synapses in the stratum radiatum were also normal in L1-deficient mice. Since L1 is expressed not only by principal cells but also by inhibitory interneurons, we recorded inhibitory postsynaptic currents (IPSCs) evoked in CA1 pyramidal cells by minimal stimulation of perisomatic interneurons. L1-deficient mice showed a reduction in the mean amplitude of putative unitary IPSCs, higher values of the coefficient of amplitude variation, higher number of failures in transmitter release, and a reduction in frequency but not amplitude of miniature IPSCs. The use-dependent modulation of inhibitory transmission by paired-pulse or short tetanic stimulation was, however, normal in L1-deficient mice. The physiological abnormalities correlated with a strong reduction in the density of inhibitory active zones, indicating that L1 is involved in establishing inhibitory perisomatic synapses in the hippocampus.

Technique to quantify local clustering of synaptic vesicles using single section data.

Synaptic vesicles are organelles that specialize in the storage of a neurotransmitter that continuously undergo an exo-endocytotic cycle. During this cycle vesicles change their positions within a presynaptic terminal and their numbers as well as spatial arrangement can provide insight into a neurotransmitter turnover. This article introduces a technique based on the nearest-neighbor formalism to quantify the proximity of vesicles to active zones and vesicle clustering in different regions of a terminal. The technique, implemented in a software package, uses the two-dimensional coordinates of features identified in digitized electron micrographs as an input. It has been validated in the analysis of asymmetric synapses of the rat hippocampal CA1 stratum radiatum affected by transient cerebral ischemia. It was shown that a 15-minute-long ischemic episode influenced the spatial arrangement of vesicles that were more distant from active zones and had larger intervesicle spacings with respect to the control. The latter effect was apparently stronger within 200 nm distance of active zones.

Decreased anxiety, altered place learning, and increased CA1 basal excitatory synaptic transmission in mice with conditional ablation of the neural cell adhesion molecule L1.

L1, a neural cell adhesion molecule of the immunoglobulin superfamily, is involved in neuronal migration and differentiation and axon outgrowth and guidance. Mutations in the human and mouse L1 gene result in similarly severe neurological abnormalities. To dissociate the functional roles of L1 in the adult brain from developmental abnormalities, we have generated a mutant in which the L1 gene is inactivated by cre-recombinase under the control of the calcium/calmodulin-dependent kinase II promoter. This mutant (L1fy+) did not show the overt morphological and behavioral abnormalities observed previously in constitutive L1-deficient (L1-/-) mice; however, there was an increase in basal excitatory synaptic transmission that was not apparent in L1-/- mice. Similar to L1-/- mice, no defects in short- and long-term potentiation in the CA1 region of the hippocampus were observed. Interestingly, L1fy+ mice showed decreased anxiety in the open field and elevated plus-maze, contrary to L1-/- mice, and altered place learning in the water maze, similar to L1-/- mice. Thus, mice conditionally deficient in L1 expression in the adult brain share some abnormalities, but also display different ones, as compared with L1-/- mice, highlighting the role of L1 in the regulation of synaptic transmission and behavior in adulthood.

Technique to study three-dimensional spatial arrangement of synaptic vesicles using data from single sections.

Synaptic vesicles are membrane-bound organelles storing neurotransmitters in presynaptic terminals and releasing them into the synaptic cleft. Coordinated movements of synaptic vesicles relate to synaptic function and their spatial arrangement can provide useful information about the activity of a synapse. This article presents a technique to extract quantitative information about three-dimensional (3D) spatial arrangement of synaptic vesicles from measurements performed on single ultrathin random sections of a presynaptic terminal. The technique presumes quantification of a 2D density as well as 2D spatial pattern formed by vesicle profiles using a minimum spanning tree (MST) algorithm, in digitized micrographs of a presynaptic terminal. Further, original software was used to simulate a 3D spatial arrangement of synaptic vesicles and their random sectioning. A 3D density and pattern of synaptic vesicles were used as basic input parameters of the model, while a 2D density and MST quantities for vesicle profiles served as output, model-derived parameters allowing one to compare and fit simulated distributions to experimental ones. Pilot simulations performed to check the validity of the technique have shown that a 2D density and MST quantities of vesicle profiles closely relate to a 3D density and spatial pattern of vesicles. The technique was demonstrated in the analysis of spatial distribution of synaptic vesicles in axonal terminals forming asymmetric synaptic densities in the stratum radiatum of the CA1 subfield of the murine hippocampus.