Amyloid-beta induced CA1 pyramidal cell loss in young adult rats is alleviated by systemic treatment with FGL, a neural cell adhesion molecule-derived mimeticPeptide

Increased levels of neurotoxic amyloid-beta in the brain are a prominent feature of Alzheimer’s disease. FG-Loop (FGL), a neural cell adhesion molecule-derived peptide that corresponds to its second fibronectin type III module, has been shown to provide neuroprotection against a range of cellular insults. In the present study impairments in social recognition memory were seen 24 days after a 5 mg/15 µl amyloid-beta(25–35) injection into the right lateral ventricle of the young adult rat brain. This impairment was prevented if the animal was given a systemic treatment of FGL. Unbiased stereology was used to investigate the ability of FGL to alleviate the deleterious effects on CA1 pyramidal cells of the amyloid-beta(25–35) injection. NeuN, a neuronal marker (for nuclear staining) was used to identify pyramidal cells, and immunocytochemistry was also used to identify inactive glycogen synthase kinase 3beta (GSK3β) and to determine the effects of amyloid-beta(25–35) and FGL on the activation state of GSK3β, since active GSK3β has been shown to cause a range of AD pathologies. The cognitive deficits were not due to hippocampal atrophy as volume estimations of the entire hippocampus and its regions showed no significant loss, but amyloid-beta caused a 40% loss of pyramidal cells in the dorsal CA1 which was alleviated partially by FGL. However, FGL treatment without amyloid-beta was also found to cause a 40% decrease in CA1 pyramidal cells. The action of FGL may be due to inactivation of GSK3β, as an increased proportion of CA1 pyramidal neurons contained inactive GSK3β after FGL treatment. These data suggest that FGL, although potentially disruptive in nonpathological conditions, can be neuroprotective in disease-like conditions

N-methyl-d-aspartate receptor independent changes in expression of polysialic acid-neural cell adhesion molecule despite blockade of homosynaptic long-term potentiation and heterosynaptic long-term depression in the awake freely behaving rat dentate gyrus

Investigations examining the role of polysialic acid (PSA) on the neural cell adhesion molecule (NCAM) in synaptic plasticity have yielded inconsistent data. Here, we addressed this issue by determining whether homosynaptic long-term potentiation (LTP) and heterosynaptic long-term depression (LTD) induce changes in the distribution of PSA-NCAM in the dentate gyrus (DG) of rats in vivo. In addition, we also examined whether the observed modifications were initiated via the activation of N-methyl-d-aspartate (NMDA) receptors. Immunocytochemical analysis showed an increase in PSA-NCAM positive cells both at 2 and 24 h following high-frequency stimulation of either medial or lateral perforant paths, leading to homosynaptic LTP and heterosynaptic LTD, respectively, in the medial molecular layer of the DG. Analysis of sub-cellular distribution of PSA-NCAM by electron microscopy showed decreased PSA dendritic labelling in LTD rats and a sub-cellular relocation towards the spines in LTP rats. Importantly, these modifications were found to be independent of the activation of NMDA receptors. Our findings suggest that strong activation of the granule cells up-regulates PSA-NCAM synthesis which then incorporates into activated synapses, representing NMDA-independent plastic processes that act synergistically on LTP/LTD mechanisms without participating in their expression

Expression of chemokines and their receptors by human brain endothelium: Implications for multiple sclerosis

Leukocyte migration into the CNS is mediated by chemokines, expressed on the surface of brain endothelium. This study investigated the production of chemokines and expression of chemokine receptors by human brain endothelial cells (HBEC), in vitro and in situ in multiple sclerosis tissue. Four chemokines (CCL2, CCL5, CXCL8 and CXCL10), were demonstrated in endothelial cells in situ, which was reflected in the chemokine production by primary HBEC and a brain endothelial cell line, hCEMC/D3. CXCL8 and CCL2 were constitutively released and increased in response to TNF and/or IFN . CXCL10 and CCL5 were undetectable in resting cells but were secreted in response to these cytokines. TNF strongly increased the production of CCL2, CCL5 and CXCL8, while IFN up-regulated CXCL10 exclusively. CCL3 was not secreted by HBECs and appeared to be confined to astrocytes in situ. The chemokine receptors CXCR1 and CXCR3 were expressed by HBEC both in vitro and in situ, and CXCR3 was up-regulated in response to cytokine stimulation in vitro. By contrast, CXCR3 expression was reduced in silent MS lesions. Brain endothelium expresses particularly high levels of CXCL10 and CXCL8, which may account for the predominant TH1-type inflammatory reaction seen in chronic conditions such as multiple sclerosis

Average social recognition ratio on day 24.

<p>An unfamiliar juvenile rat was introduced into the cage of the test rat and 30 minutes later the juvenile was placed back in the cage. Both investigation times were recorded and the social recognition ratio was calculated. A social recognition ratio of 0.5 indicates no memory of the juvenile. Using a one sample t-test, it was found that animals treated with Aβ_25–35 followed by FGL, FGL alone or vehicles (control) had significantly lower ratios than 0.5 (P<0.01), whilst animals given only Aβ_25–35 did not. A one-way ANOVA was also performed on the individual social recognition ratios and animals given Aβ_25–35 alone had significantly greater social recognition ratios than that of any other group (*<i>P<0.05</i>). The mean ratio for each group is signified by a black diamond (± SEM, n = 4), whilst the open circles indicate individual ratios in that given group.</p

Percentage of CA1 pyramidal cells in the right dorsal hippocampus containing inactive GSK3β.

<p>Double immunocytochemistry and the optical fractionator method were used to establish the pyramidal cell density in the CA1 and also the density of pyramidal cells containing inactive GSK3β. The absolute numbers of both densities were calculated and the percentage of all the CA1 pyramidal cells that contained inactive GSK3β was established. The data was analysed using a one-way ANOVA and Tukey’s post-hoc test. Aβ_25–35+FGL rats had a significantly higher percentage of pyramidal neurons in the CA1 that contained inactive GSK3β compared with Aβ_25–35 alone rats. (*<i>P</i><0.05). Mean ± SEM, n = 4.</p

The density and total number of CA1 pyramidal cells in the right dorsal hippocampus.

<p>Immunocytochemistry (NeuN antibody in conjunction with DAB) and the optical fractionator method were used to establish cell density within the CA1. Cell density (a) was multiplied by the volume of the dorsal CA1 SP to establish total number (b). The data was analysed using a one-way ANOVA and Tukey’s post-hoc test. The Aβ_25–35+FGL group had a significantly greater density (a) and total number (b) of pyramidal cells in the CA1 SP than compared with the Aβ_25–35 alone and the FGL alone groups, whilst the control group had significantly more pyramidal cells than all of the other groups, regardless of the groups cell density. (*<i>P</i><0.05, **<i>P</i><0.01). Mean ± SEM, n = 4.</p

Immunopositive staining of CA1 pyramidal cell nuclei (NeuN) and inactive GSK3β (cytoplasm).

<p>To visualize pyramidal cells for cell counting an antibody against NeuN, a nuclear marker was used along with SG, a dark grey stain. Inactive GSK3β (GSK3βps9) in the cytoplasm of pyramidal cells in the CA1 was visualised using an antibody against GSK3βps9 along with DAB, a brown stain. A double immunopositive staining was performed to count the number of CA1 pyramidal cells that contained inactive GSK3β in the cytoplasm using antibodies against NeuN and GSK3βps9 with SG and DAB respectively. Scale Bar = 40 µm.</p

'Damaged' pyramidal cells in the right CA3.

<p>a) A section of the CA3 from a rat given Aβ_25–35 alone, showing a large number of damaged pyramidal cells (arrow indicates a group of damaged pyramidal cells). b) An enlarged image of the box in a. The arrows indicate ‘damaged’ pyramidal cells with densely toluidine blue stained, concaved cell bodies. ‘n’ is an example of a ‘healthy’ neuron. Scale bar a = 50 µm and b = 20 µm.</p

Volume estimations of the right dorsal CA3.

<p>Using a one-way ANOVA and Tukey’s post-hoc test, the volume of the dorsal CA3 in the control group was significantly larger than all the other groups (**P<0.01). Mean ± SEM, n = 4.</p