Apolipoprotein E imbalance in the cerebrospinal fluid of Alzheimer's disease patients.

OBJECTIVE: The purpose of this study was to examine the levels of cerebrospinal fluid (CSF) apolipoprotein E (apoE) species in Alzheimer's disease (AD) patients. METHODS: We analyzed two CSF cohorts of AD and control individuals expressing different APOE genotypes. Moreover, CSF samples from the TgF344-AD rat model were included. Samples were run in native- and SDS-PAGE under reducing or non-reducing conditions (with or without ß-mercaptoethanol). Immunoprecipitation combined with mass spectrometry or western blotting analyses served to assess the identity of apoE complexes. RESULTS: In TgF344-AD rats expressing a unique apoE variant resembling human apoE4, a ~35-kDa apoE monomer was identified, increasing at 16.5 months compared with wild-types. In humans, apoE isoforms form disulfide-linked dimers in CSF, except apoE4, which lacks a cysteine residue. Thus, controls showed a decrease in the apoE dimer/monomer quotient in the APOE ε3/ε4 group compared with ε3/ε3 by native electrophoresis. A major contribution of dimers was found in APOE ε3/ε4 AD cases, and, unexpectedly, dimers were also found in ε4/ε4 AD cases. Under reducing conditions, two apoE monomeric glycoforms at 36 kDa and at 34 kDa were found in all human samples. In AD patients, the amount of the 34-kDa species increased, while the 36-kDa/34-kDa quotient was lower compared with controls. Interestingly, under reducing conditions, a ~100-kDa apoE complex, the identity of which was confirmed by mass spectrometry, also appeared in human AD individuals across all APOE genotypes, suggesting the occurrence of aberrantly resistant apoE aggregates. A second independent cohort of CSF samples validated these results. CONCLUSION: These results indicate that despite the increase in total apoE content the apoE protein is altered in AD CSF, suggesting that function may be compromised.

Proceedings of workshop: "Neuroglycoproteins in health and disease", INNOGLY cost action.

The Cost Action "Innovation with glycans: new frontiers from synthesis to new biological targets" (INNOGLY) hosted the Workshop "Neuroglycoproteins in health and disease", in Alicante, Spain, on March 2022. This event brought together an european group of scientists that presented novel insights into changes in glycosylation in diseases of the central nervous system and cancer, as well as new techniques to study protein glycosylation. Herein we provide the abstracts of all the presentations.

Increased P2×2 receptors induced by amyloid-ß peptide participates in the neurotoxicity in alzheimer's disease.

Amyloid beta peptide (Aß) is tightly associated with the physiopathology of Alzheimer's Disease (AD) as one of the most important factors in the evolution of the pathology. In this context, we previously reported that Aß increases the expression of ionotropic purinergic receptor 2 (P2×2R). However, its role on the cellular and molecular Aß toxicity is unknown, especially in human brain of AD patients. Using cellular and molecular approaches in hippocampal neurons, PC12 cells, and human brain samples of patients with AD, we evaluated the participation of P2×2R in the physiopathology of AD. Here, we reported that Aß oligomers (Aßo) increased P2×2 levels in mice hippocampal neurons, and that this receptor increases at late Braak stages of AD patients. Aßo also increases the colocalization of APP with Rab5, an early endosomes marker, and decreased the nuclear/cytoplasmic ratio of Fe65 and PGC-1α immunoreactivity. The overexpression in PC12 cells of P2×2a, but not P2×2b, replicated these changes in Fe65 and PGC-1α; however, both overexpressed isoforms increased levels of Aß. Taken together, these data suggest that P2×2 is upregulated in AD and it could be a key potentiator of the physiopathology of Aß. Our results point to a possible participation in a toxic cycle that increases Aß production, Ca2+ overload, and a decrease of PGC-1α. These novel findings put the P2×2R as a key novel pharmacological target to develop new therapeutic strategies to treat Alzheimer's Disease.

Decreased generation of C-terminal fragments of ApoER2 and increased reelin expression in Alzheimer's disease.

Increasing evidence indicates that altered reelin signaling could contribute to synaptic dysfunction in Alzheimer's disease (AD). We found that reelin protein and mRNA levels were increased in the AD brain (particularly at advanced Braak stages in apolipoprotein E4 noncarriers), compared with that of control subjects. The ß-amyloid (Aß) protein impairs reelin activity and increases reelin expression through a mechanism that is not yet understood. To explore that mechanism, we examined the effect of Aß aa 1-42 (Aß42) on DNA methylation of the RELN promoter and the processing of reelin receptor apolipoprotein E receptor 2 (ApoER2) in differentiated SH-SY5Y cells because ApoER2 C-terminal fragments (CTFs), generated after reelin binding, regulate reelin expression. We found that Aß42 decreased nuclear levels of DNA-methyltransferase 1. However, RELN promoter methylation did not change in Aß42-treated cells or in AD brain extracts. Instead, the levels of ApoER2-CTF appeared significantly lower in Aß42-treated cells and in AD extracts from advanced Braak stages of apolipoprotein E4 noncarriers. Our data show that ApoER2-CTF levels are decreased, whereas reelin expression is increased in AD brain at advanced Braak stages and after Aß treatment, supporting the view that ApoER2-CTF exerts a modulatory role on reelin expression.-Mata-Balaguer, T., Cuchillo-Ibañez, I., Calero, M., Ferrer, I., Sáez-Valero, J. Decreased generation of C-terminal fragments of ApoER2 and increased reelin expression in Alzheimer's disease.

HNK-1 Carrier Glycoproteins Are Decreased in the Alzheimer's Disease Brain.

The human natural killer-1 (HNK-1), 3-sulfonated glucuronic acid, is a glycoepitope marker of cell adhesion that participates in cell-cell and cell-extracellular matrix interactions and in neurite growth. Very little is known about the regulation of the HNK-1 glycan in neurodegenerative disease, particularly in Alzheimer's disease (AD). In this study, we investigate changes in the levels of HNK-1 carrier glycoproteins in AD. We demonstrate an overall decrease in HNK-1 immunoreactivity in glycoproteins extracted from the frontal cortex of AD subjects, compared with levels from non-demented controls (NDC). Immunoblotting of ventricular post-mortem and lumbar ante-mortem cerebrospinal fluid with HNK-1 antibodies indicate similar levels of carrier glycoproteins in AD and NDC samples. Decrease in HNK-1 carrier glycoproteins were not paralleled by changes in messenger RNA (mRNA) levels of the enzymes involved in the synthesis of the glycoepitope, ß-1,4-galactosyltransferase (ß4GalT), glucuronyltransferases GlcAT-P and GlcAT-S, or sulfotransferase HNK-1ST. Over-expression of amyloid precursor protein in Tg2576 transgenic mice and in vitro treatment of SH-SY5Y neuroblastoma cells with the amyloidogenic Aß42 peptide resulted in a decrease in HNK-1 immunoreactivity levels in brain and cellular extracts, whereas the levels of soluble HNK-1 glycoproteins detected in culture media were not affected by Aß treatment. HNK-1 levels remain unaffected in the brain extracts of Tg-VLW mice, a model of mutant hyperphosphorylated tau, and in SH-SY5Y cells over-expressing hyperphosphorylated wild-type tau. These results provide evidence that cellular levels of HNK-1 carrier glycoforms are decreased in the brain of AD subjects, probably influenced by the ß-amyloid protein.

The Notch intracellular domain represses CRE-dependent transcription.

Members of the cyclic-AMP response-element binding protein (CREB) transcription factor family regulate the expression of genes needed for long-term memory formation. Loss of Notch impairs long-term, but not short-term, memory in flies and mammals. We investigated if the Notch-1 (N1) exerts an effect on CREB-dependent gene transcription. We observed that N1 inhibits CREB mediated activation of cyclic-AMP response element (CRE) containing promoters in a γ-secretase-dependent manner. We went on to find that the γ-cleaved N1 intracellular domain (N1ICD) sequesters nuclear CREB1α, inhibits cAMP/PKA-mediated neurite outgrowth and represses the expression of specific CREB regulated genes associated with learning and memory in primary cortical neurons. Similar transcriptional effects were observed with the N2ICD, N3ICD and N4ICDs. Together, these observations indicate that the effects of Notch on learning and memory are, at least in part, via an effect on CREB-regulated gene expression.

ApoER2 processing by presenilin-1 modulates reelin expression.

The reelin signaling protein and its downstream components have been associated with synaptic plasticity and neurotransmission. The reelin signaling pathway begins with the binding of reelin to the transmembrane lipoprotein receptor apolipoprotein E receptor 2 (ApoER2), which in turns induces the sequential cleavage of ApoER2 by the sequential action of α- and γ-secretases. Using conditional-knockout mice of the catalytic component of the γ-secretase complex, presenilin 1 (PS1), we demonstrated increased brain ApoER2 and reelin protein and transcript levels, with no changes in the number of reelin-positive cells. Using the human SH-SY5Y neuroblastoma cell line, we showed that ApoER2 processing occurs in the presence of PS1, producing an intracellular ApoER2 C-terminal fragment. In addition, the pharmacologic inhibition of γ-secretase in SH-SY5Y cells led to increased reelin levels. Overexpression of ApoER2 decreased reelin mRNA levels in these cells. A luciferase reporter gene assay and nuclear fractionation confirmed that increased amounts of intracellular fragment of ApoER2 suppressed reelin expression at a transcriptional level. Chromatin immunoprecipitation experiments corroborated that the intracellular fragment of ApoER2 bound to the RELN promoter region. Our study suggests that PS1/γ-secretase-dependent processing of the reelin receptor ApoER2 inhibits reelin expression and may regulate its signaling.

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells.

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca(2+) during action potentials. Indeed, Ca(2+)-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K(+) currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca(2+)) Ca(2+) concentrations. Despite their low open probability under these conditions (V(50) of +146.8 mV), BK channels were responsible for more than 25% of the total K(+) efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K(+) channels. The other main source of fast voltage-dependent K(+) efflux at such a low Ca(2+) was a transient K(+) (I(A)-type) current activating with V (50) = -14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca(2+). Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca(2+) concentration, and suggest that they could do so also in the presence of extracellular Ca(2+), before Ca(2+) entering the cell facilitates their activity.

Beta-amyloid controls altered Reelin expression and processing in Alzheimer's disease.

Reelin is a glycoprotein that modulates synaptic function and plasticity in the mature brain, thereby favouring memory formation. We recently reported altered cerebral Reelin expression in Alzheimer's disease (AD). Here we demonstrate pronounced Reelin changes at protein and mRNA levels in the frontal cortex in adult Down's syndrome (DS), where the extra copy of chromosome 21 leads to overexpression of beta-amyloid. In cortical extracts of fetal DS samples we detected increased levels of the full-length Reelin and the 310-kDa fragment. Overexpression of mutant human amyloid precursor protein also led to an increase in levels of Reelin fragments in Tg2576 transgenic mice for human beta-amyloid. Finally, in vitro Abeta42 treatment of SH-SY5Y neuroblastoma cells led to increased Reelin levels. An altered pattern of Reelin glycosylation was detected in extracts from the frontal cortex of AD patients and in Abeta42-treated SH-SY5Y cells, supporting the notion that beta-amyloid triggers altered Reelin processing. These results provide evidence that Reelin expression and processing is altered in several amyloid conditions.