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ADAM10 is the main α-secretase acting in the non-amyloidogenic processing of APP. We hypothesized that certain rare ADAM10 variants could increase the risk for AD by conferring the age-related downregulation of α-secretase. The ADAM10 gene was sequenced in 103 AD cases (82% familial) and 96 cognitively preserved nonagenarians. We examined rare variants (MAF < 0.01) and determined their potential association in the AD group with lower CSF protein levels, as analyzed by means of ELISA, and Western blot (species of 50 kDa, 55 kDa, and 80 kDa). Rare variants were found in 15.5% of AD cases (23% early-onset, 8% late-onset) and in 12.5% of nonagenarians, and some were group-specific. All were intronic variants except Q170H, found in three AD cases and one nonagenarian. The 3′UTR rs74016945 (MAF = 0.01) was found in 6% of the nonagenarians (OR 0.146, p = 0.057). Altogether, ADAM10 total levels or specific species were not significantly different when comparing AD with controls or carriers of rare variants versus non-carriers (except a Q170H carrier exhibiting low levels of all species), and did not differ according to the age at onset or APOE genotype. We conclude that ADAM10 exonic variants are uncommon in AD cases, and the presence of rare intronic variants (more frequent in early-onset cases) is not associated with decreased protein levels in CSF.

This article belongs to the Special Issue Molecular Advances in Alzheimer's Disease., In Alzheimer’s disease (AD), the reduction in acetylcholinesterase (AChE) enzymatic activity is not paralleled with changes in its protein levels, suggesting the presence of a considerable enzymatically inactive pool in the brain. In the present study, we validated previous findings, and, since inactive forms could result from post-translational modifications, we analyzed the glycosylation of AChE by lectin binding in brain samples from sporadic and familial AD (sAD and fAD). Most of the enzymatically active AChE was bound to lectins Canavalia ensiformis (Con A) and Lens culinaris agglutinin (LCA) that recognize terminal mannoses, whereas Western blot assays showed a very low percentage of AChE protein being recognized by the lectin. This indicates that active and inactive forms of AChE vary in their glycosylation pattern, particularly in the presence of terminal mannoses in active ones. Moreover, sAD subjects showed reduced binding to terminal mannoses compared to non-demented controls, while, for fAD patients that carry mutations in the PSEN1 gene, the binding was higher. The role of presenilin-1 (PS1) in modulating AChE glycosylation was then studied in a cellular model that overexpresses PS1 (CHO-PS1). In CHO-PS1 cells, binding to LCA indicates that AChE displays more terminal mannoses in oligosaccharides with a fucosylated core. Immunocytochemical assays also demonstrated increased presence of AChE in the trans-Golgi. Moreover, AChE enzymatic activity was higher in plasmatic membrane of CHO-PS1 cells. Thus, our results indicate that PS1 modulates trafficking and maturation of AChE in Golgi regions favoring the presence of active forms in the membrane., This study was funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants PI17/00261 to M.S.G.-A.), co-financed by Fondo Europeo de Desarrollo Regional and through CIBERNED, ISCIII, Spain. M.-Á.C.-G. was supported by a GVA-Predoctoral fellowship (grant ACIF2019/242) from Generalitat Valenciana, Spain.

This article belongs to the Special Issue Latest Advances in Neuroscience., The levels of several glial and neuronal plasma biomarkers have been found to increase during the acute phase in COVID-19 patients with neurological symptoms. However, replications in patients with minor or non-neurological symptoms are needed to understand their potential as indicators of CNS injury or vulnerability. Plasma levels of glial fibrillary acidic protein (GFAP), neurofilament light chain protein (NfL), and total Tau (T-tau) were determined by Single molecule array (Simoa) immunoassays in 45 samples from COVID-19 patients in the acute phase of infection [moderate (n = 35), or severe (n = 10)] with minor or non-neurological symptoms; in 26 samples from fully recovered patients after ~2 months of clinical follow-up [moderate (n = 23), or severe (n = 3)]; and in 14 non-infected controls. Plasma levels of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), were also determined by Western blot. Patients with COVID-19 without substantial neurological symptoms had significantly higher plasma concentrations of GFAP, a marker of astrocytic activation/injury, and of NfL and T-tau, markers of axonal damage and neuronal degeneration, compared with controls. All these biomarkers were correlated in COVID-19 patients at the acute phase. Plasma GFAP, NfL and T-tau levels were all normalized after recovery. Recovery was also observed in the return to normal values of the quotient between the ACE2 fragment and circulating full-length species, following the change noticed in the acute phase of infection. None of these biomarkers displayed differences in plasma samples at the acute phase or recovery when the COVID-19 subjects were sub-grouped according to occurrence of minor symptoms at re-evaluation 3 months after the acute episode (so called post-COVID or “long COVID”), such as asthenia, myalgia/arthralgia, anosmia/ageusia, vision impairment, headache or memory loss. Our study demonstrated altered plasma GFAP, NfL and T-tau levels in COVID-19 patients without substantial neurological manifestation at the acute phase of the disease, providing a suitable indication of CNS vulnerability; but these biomarkers fail to predict the occurrence of delayed minor neurological symptoms., This work was supported by grants from the Fondo de Investigaciones Sanitarias (PI19-01359, co-funded by the Fondo Europeo de Desarrollo Regional, FEDER “Investing in your future”), CIBERNED (Instituto de Salud Carlos III, Spain), by the Instituto de Investigación Sanitaria y Biomédica de Alicante (ISABIAL; grant 2020-0308) and from the Direcció General de Ciència I Investigació, Generalitat Valenciana (AICO/2021/308). MPL is supported by a BEFPI fellowship from the Generalitat Valenciana. HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532), the European Union’s Horizon Europe research and innovation programme under grant agreement No 101053962, Swedish State Support for Clinical Research (#ALFGBG-71320), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862), the AD Strategic Fund and the Alzheimer’s Association (#ADSF-21-831376-C, #ADSF-21-831381-C, and #ADSF-21-831377-C), the Bluefield Project, the Olav Thon Foundation, the Erling-Persson Family Foundation, Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden (#FO2022-0270), the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 860197 (MIRIADE), the European Union Joint Programme–Neurodegenerative Disease Research (JPND2021-00694), and the UK Dementia Research Institute at UCL (UKDRI-1003).

[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 β-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., This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI15/00665 and PI19-01359, co-funded by the Fondo Europeo de Desarrollo Regional, FEDER “Investing in your future”), from the CIBERNED (Instituto de Salud Carlos III, Spain) and from the Direcció General de Ciència I Investigació, Generalitat Valenciana (AICO/2021/308). We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723). MPL is supported by a BEFPI fellowship from the Generalitat Valenciana. This study was also supported by the FIS, Contratos para la intensificación de la actividad investigadora en el SNS from Instituto de Salud Carlos III (PI14/01126, PI17/01019, PI20/01473, and INT16/00171 to JF; PI18/00435 to DA; PI14/1561, PI17/01896, and PI20/1330 to AL). This work was also supported by the CIBERNED program (Program 1, Alzheimer Disease to AL and SIGNAL study, www.signalstudy.es), the National Institutes of Health (NIA grants 1R01AG056850-01A1, R21AG056974, and R01AG061566 to JF), and Departament de Salut de la Generalitat de Catalunya, Pla Estratègic de Recerca i Innovació en Salut (SLT002/16/00408 to AL). This work was also supported by Generalitat de Catalunya (SLT006/17/00119 to JF and SLT006/17/00125 to DA). This study was also supported by the Spanish Ministry of Science, Innovation and Universities MICINN/FEDER (PID2019-107738RB-I00, to FA, a fellowship to ISD and the “Maria Maeztu Unit of Excellence Program”) and Instituto de Salud Carlos III (PI18/00893 to GS, co-funded by ERDF, “A way to make Europe”). HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532); the European Research Council (#681712); the Swedish State Support for Clinical Research (#ALFGBG-720931); the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862); the AD Strategic Fund and the Alzheimer’s Association (#ADSF-21-831376-C, #ADSF-21-831381-C, and #ADSF-21-831377-C); the Olav Thon Foundation; the Erling-Persson Family Foundation; Stiftelsen för Gamla Tjänarinnor; Hjärnfonden, Sweden (#FO2019-0228); the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 860197 (MIRIADE); the European Union Joint Programme – Neurodegenerative Disease Research (JPND2021-00694); and the UK Dementia Research Institute at UCL (UKDRI-1003). KB is supported by the Swedish Research Council (#2017-00915); the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615); the Swedish Alzheimer Foundation (#AF-742881); Hjärnfonden, Sweden (#FO2017-0243); the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement (#ALFGBG-715986); the European Union Joint Program for Neurodegenerative Disorders (JPND2019-466-236); the National Institute of Health (NIH), USA (grant #1R01AG068398-01); and the Alzheimer’s Association 2021 Zenith Award (ZEN-21-848495).

Various species of the SARS-CoV-2 host cell receptor, the angiotensin-converting enzyme 2 (ACE2), are present in serum, which may result from virus entry and subsequent proteolytic processing of the membrane receptor. We have recently demonstrated changes of particular ACE2 species in virus infected humans, either cleaved fragments or circulating full-length species. Here, we further explore the potential of serum ACE2 as a biomarker to test SARS-CoV-2 infection and vaccine efficacy in virus susceptible transgenic K18-hACE2 mice expressing human ACE2. First, in serum samples derived from K18-hACE2 mice challenged with a lethal dose of SARS-CoV-2, we observed an increase in the levels of cleaved ACE2 fragment at day 2 post-challenge, which may represent the subsequent proteolytic processing through virus entry. These elevated levels were maintained until the death of the animals at day 6 post-challenge. The circulating full-length ACE2 form displayed a sizable peak at day 4, which declined at day 6 post-challenge. Noticeably, immunization with two doses of the MVA-CoV2-S vaccine candidate prevented ACE2 cleaved changes in serum of animals challenged with a lethal dose of SARS-CoV-2. The efficacy of the MVA-CoV2-S was extended to vaccinated mice after virus re-challenge. These findings highlight that ACE2 could be a potential serum biomarker for disease progression and vaccination against SARS-CoV-2., This study was funded in part by the Instituto de Investigación Sanitaria y Biomédica de Alicante (ISABIAL; grant 2020-0308), the Direcció General de Ciència I Investigació, Generalitat Valenciana (AICO/2021/308), and by the Instituto de Salud Carlos III (ISCIII, grants PI19-01359), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER, “Investing in your future”) and through CIBERNED, ISCIII. We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723). This research was also supported by Fondo COVID-19 grant COV20/00151 (Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII)), Fondo Supera COVID-19 grant (Crue Universidades-Banco Santander), and Spanish Research Council (CSIC) grant 202120E079 (to JG-A); CSIC grant 2020E84, Ferrovial, and MAPFRE donations (to ME); and Spanish Ministry of Science and Innovation (MCIN)/Spanish Research Agency (AEI)/10.13039/501100011033 grant (PID2020-114481RB-I00; to JG-A and ME). This research work was also funded by the European Commission- NextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) (to JG-A and ME). MPL is supported by a BEFPI scholarship from the Generalitat Valenciana. JGA and ME acknowledges financial support from the Spanish State Research Agency, AEI/10.13039/501100011033, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2013-0347, SEV-2017-0712).

Early View: Online Version of Record before inclusion in an issue., In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co‐localizes with hyperphosphorylated tau (P‐tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg‐VLW, here we examined whether modulating phosphorylated tau levels by over‐expressing wild‐type human tau and glycogen synthase kinase‐3β (GSK3β) influences AChE expression. In SH‐SY5Y neuroblastoma cells expressing higher levels of P‐tau, AChE activity and protein increased by (20% ± 2%) and (440% ± 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergic ACHE‐T variant, for which the protein and transcript levels increased ~60% and ~23%, respectively. Moreover, in SH‐SY5Y cells differentiated into neurons by exposure to retinoic acid (10 µM), over‐expression of GSK3β and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 ± 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3β inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 ± 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib‐treated patients. Moreover, CSF levels of P‐tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P‐tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD., MACG is supported by a GVA-Predoctoral fellowship from Generalitat Valenciana, Spain. This study was funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants CP11/00067 and PI17/00261 to MSGA); co-financed by Fondo Europeo de Desarrollo Regional and through CIBERNED, ISCIII, Spain.

[Background]: Members of the low-density lipoprotein (LDL) receptor family are involved in endocytosis and in transducing signals, but also in amyloid precursor protein (APP) processing and β-amyloid secretion. ApoER2/LRP8 is a member of this family with key roles in synaptic plasticity in the adult brain. ApoER2 is cleaved after the binding of its ligand, the reelin protein, generating an intracellular domain (ApoER2-ICD) that modulates reelin gene transcription itself. We have analyzed whether ApoER2-ICD is able to regulate the expression of other LDL receptors, and we focused on LRP3, the most unknown member of this family. We analyzed LRP3 expression in middle-aged individuals (MA) and in cases with Alzheimer’s disease (AD)-related pathology, and the relation of LRP3 with APP., [Methods]: The effects of full-length ApoER2 and ApoER2-ICD overexpression on protein levels, in the presence of recombinant reelin or Aβ42 peptide, were evaluated by microarray, qRT-PCRs, and western blots in SH-SY5Y cells. LRP3 expression was analyzed in human frontal cortex extracts from MA subjects (mean age 51.8±4.8 years) and AD-related pathology subjects [Braak neurofibrillary tangle stages I–II, 68.4±8.8 years; III–IV, 80.4 ± 8.8 years; V–VI, 76.5±9.7 years] by qRT-PCRs and western blot; LRP3 interaction with other proteins was assessed by immunoprecipitation. In CHO cells overexpressing LRP3, protein levels of full-length APP and fragments were evaluated by western blots. Chloroquine was employed to block the lysosomal/autophagy function., [Results]: We have identified that ApoER2 overexpression increases LRP3 expression, also after reelin stimulation of ApoER2 signaling. The same occurred following ApoER2-ICD overexpression. In extracts from subjects with AD-related pathology, the levels of LRP3 mRNA and protein were lower than those in MA subjects. Interestingly, LRP3 transfection in CHO-PS70 cells induced a decrease of full-length APP levels and APP-CTF, particularly in the membrane fraction. In cell supernatants, levels of APP fragments from the amyloidogenic (sAPPα) or non-amyloidogenic (sAPPβ) pathways, as well as Aβ peptides, were drastically reduced with respect to mock-transfected cells. The inhibitor of lysosomal/autophagy function, chloroquine, significantly increased full-length APP, APP-CTF, and sAPPα levels., [Conclusions]: ApoER2/reelin signaling regulates LRP3 expression, whose levels are affected in AD; LRP3 is involved in the regulation of APP levels., This work was supported by grants from the Fondo de Investigaciones Sanitarias (PI15/00665 and PI19-01359, co-funded by the Fondo Europeo de Desarrollo Regional, FEDER “Investing in your future”),CIBERNED (Instituto de Salud Carlos III, Spain) and from the Direcció General de Ciència i Investigació, Generalitat Valenciana (AICO/2021/308). We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723).

Background: Alzheimer’s disease (AD) is characterized by the presence of β-amyloid plaques and neurofibrillary tangles, while Lewy body dementia (LBD) is characterized by α-synuclein (α-syn) inclusions. Some authors examine α-syn protein in the neurodegeneration process of AD and propose to consider cerebrospinal fluid (CSF) α-syn as a possible additional biomarker to the so-called “core” of AD. Objective: To determine whether there is a correlation between α-syn levels and “core” AD biomarkers in patients with mild cognitive impairment (MCI). Materials and methods: In total, 81 patients in the early stages of MCI were selected from the outpatient dementia consultation in Alicante General Hospital. Using a cross-sectional case–control design, patients were analyzed in four groups: stable MCI (MCIs, n = 25), MCI due to AD (MCI-AD, n = 32), MCI due to LBD (MCI-LBD, n = 24) and a control group of patients with acute or chronic headache (Ctrl, n = 18). Correlation between CSF protein levels in the different groups was assessed by the Rho Spearman test. Results: We found positive correlations between T-tau protein and α-syn (ρ = 0.418, p value &lt, 0.05) and p-tau181p and α-syn (ρ = 0.571, 0.05) exclusively in the MCI-AD group. Conclusion: The correlation found between α-syn and tau proteins in the first stages of AD support the involvement of α-syn in the pathogenesis of AD. This result may have clinical and diagnostic implications, as well as help to apply the new concept of “precision medicine” in patients with MCI.

This article belongs to the Special Issue Molecular Advances in Alzheimer's Disease., Reelin binds to the apolipoprotein E receptor apoER2 to activate an intracellular signaling cascade. The proteolytic cleavage of reelin follows receptor binding but can also occur independently of its binding to receptors. This study assesses whether reelin proteolytic fragments are differentially affected in the cerebrospinal fluid (CSF) of Alzheimer’s disease (AD) subjects. CSF reelin species were analyzed by Western blotting, employing antibodies against the N- and C-terminal domains. In AD patients, we found a decrease in the 420 kDa full-length reelin compared with controls. In these patients, we also found an increase in the N-terminal 310 kDa fragment resulting from the cleavage at the so-called C-t site, whereas the 180 kDa fragment originated from the N-t site remained unchanged. Regarding the C-terminal proteolytic fragments, the 100 kDa fragment resulting from the cleavage at the C-t site also displayed increased levels, whilst the one resulting from the N-t site, the 250 kDa fragment, decreased. We also detected the presence of an aberrant reelin species with a molecular mass of around 500 kDa present in AD samples (34 of 43 cases), while it was absent in the 14 control cases analyzed. These 500 kDa species were only immunoreactive to N-terminal antibodies. We validated the occurrence of these aberrant reelin species in an Aβ42-treated reelin-overexpressing cell model. When we compared the AD samples from APOE genotype subgroups, we only found minor differences in the levels of reelin fragments associated to the APOE genotype, but interestingly, the levels of fragments of apoER2 were lower in APOE ε4 carriers with regards to APOE ε3/ε3. The altered proportion of reelin/apoER2 fragments and the occurrence of reelin aberrant species suggest a complex regulation of the reelin signaling pathway, which results impaired in AD subjects., This work was supported by grants from the Fondo de Investigaciones Sanitarias (PI15/00665 and PI19-01359, co-funded by the Fondo Europeo de Desarrollo Regional, FEDER “Investing in your future”), CIBERNED (Instituto de Salud Carlos III, Spain), and the Direcció General de Ciència I Investigació, Generalitat Valenciana (AICO/2021/308). We also acknowledge the financial support from the Spanish Ministerio de Economía y Competitividad through the “Severo Ochoa” Program for Centers of Excellence in R&D (SEV-2017-0723). M.P.L. was supported by a BEFPI fellowship from the Generalitat Valenciana. H.Z. is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532); the European Research Council (#681712); Swedish State Support for Clinical Research (#ALFGBG-720931); the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862); the AD Strategic Fund and the Alzheimer’s Association (#ADSF-21-831376-C, #ADSF-21-831381-C, and #ADSF-21-831377-C); the Olav Thon Foundation; the Erling-Persson Family Foundation; Stiftelsen för Gamla Tjänarinnor; Hjärnfonden, Sweden (#FO2019-0228); the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie (grant agreement no. 860197; MIRIADE); and the UK Dementia Research Institute at UCL. K.B. is supported by the Swedish Research Council (#2017-00915); the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615); the Swedish Alzheimer Foundation (#AF-742881); Hjärnfonden, Sweden (#FO2017-0243); the Swedish state under the agreement between the Swedish government and the County Councils; the ALF-agreement (#ALFGBG-715986); the European Union Joint Program for Neurodegenerative Disorders (JPND2019-466-236); the National Institute of Health (NIH), USA, (grant #1R01AG068398-01); and the Alzheimer’s Association 2021 Zenith Award (ZEN-21-848495).

Amyloid beta peptide (Aβ) 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 Aβ increases the expression of ionotropic purinergic receptor 2 (P2×2R). However, its role on the cellular and molecular Aβ 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 Aβ oligomers (Aβo) increased P2×2 levels in mice hippocampal neurons, and that this receptor increases at late Braak stages of AD patients. Aβo 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 Aβ. Taken together, these data suggest that P2×2 is upregulated in AD and it could be a key potentiator of the physiopathology of Aβ. Our results point to a possible participation in a toxic cycle that increases Aβ 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., This work was supported by the FONDECYT [grant numbers 1161078, 1200908 (JF)], and by the Instituto de Salud Carlos III [grant numbers PI15/00665, PI19-01359] (co-financed by the Fondo Europeo de Desarrollo Regional, “Investing in your future”) (JSV). PAG is a PhD student of CONICYT [grant number 21160392].

Background. Members of the low-density lipoprotein (LDL) receptor family are involved in endocytosis and in transducing signals, but also in APP (amyloid precursor protein) processing and β-amyloid secretion. ApoER2/LRP8 is a member of this family with key roles in synaptic plasticity in the adult brain. ApoER2 is cleaved after the binding of its ligand, the reelin protein, generating an intracellular domain (ApoER2-ICD) that modulates reelin gene transcription itself. In this work, we have analysed whether ApoER2-ICD is able to regulate the expression of other members of the LDL receptor family. We focused on LRP3, the most unknown member of the LDL receptor family, whose precise physiological role and potential participation in pathological processes such as Alzheimer’s disease (AD) are still unknown.Methods. The effects of full-length ApoER2 and ApoER2-ICD overexpression on protein levels, in presence of recombinant reelin or Ab42 peptide, were evaluated by a microarray, qRT-PCRs and western blots. The expression of LRP3 was analysed in human frontal cortex extracts from AD and non-demented subjects by qRT-PCRs and western blot; and LRP3 interaction with other proteins was assessed by immunoprecipitation. In CHO cells overexpressing LRP3, protein levels of full-length APP and fragments were evaluated by western blots. Results. We have identified that ApoER2 overexpression increases LRP3 expression. Stimulation of ApoER2 signaling by reelin increased LRP3 levels, and the same occurred following ApoER2-ICD overexpression. In human frontal cortex extracts we demonstrate that LRP3 interacts with apolipoprotein E and APP. In extracts from AD subjects, the levels of LRP3 mRNA and protein were lower than those in control subjects. Interestingly, LRP3 transfection in CHO-PS70 cells induced a decrease of full-length APP levels and APP-CTF, and in the supernatant, levels of soluble APP fragments from the amyloidogenic (sAPPa) or non-amyloidogenic (sAPPβ) pathway, as well as Aβ peptides, were drastically reduced respect to mock-transfected cells.Limitations. There is a scarce knowledge of LRP3 physiological function as a neuronal receptor.Conclusion. We describe that LRP3 expression is regulated via ApoER2/reelin signaling, and its levels are affected in AD; similarly to other LDL receptors, LRP3 is involved in APP expression.

Studies are needed to identify useful biomarkers to assess the severity and prognosis of COVID-19 disease, caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) virus. Here, we examine the levels of various plasma species of the SARS-CoV-2 host receptor, the angiotensin-converting enzyme 2 (ACE2), in patients at different phases of the infection. Human plasma ACE2 species were characterized by immunoprecipitation and western blotting employing antibodies against the ectodomain and the C-terminal domain, using a recombinant human ACE2 protein as control. In addition, changes in the cleaved and full-length ACE2 species were also examined in serum samples derived from humanized K18-hACE2 mice challenged with a lethal dose of SARS-CoV-2. ACE2 immunoreactivity was present in human plasma as several molecular mass species that probably comprise truncated (70 and 75 kDa) and full-length forms (95, 100, 130, and 170 kDa). COVID-19 patients in the acute phase of infection (n = 46) had significantly decreased levels of ACE2 full-length species, while a truncated 70-kDa form was marginally higher compared with non-disease controls (n = 26). Levels of ACE2 full-length species were in the normal range in patients after a recovery period with an interval of 58-70 days (n = 29), while the 70-kDa species decreased. Levels of the truncated ACE2 species served to discriminate between individuals infected by SARS-CoV-2 and those infected with influenza A virus (n = 17). In conclusion, specific plasma ACE2 species are altered in patients with COVID-19 and these changes normalize during the recovery phase. Alterations in ACE2 species following SARS-CoV-2 infection warrant further investigation regarding their potential usefulness as biomarkers for the disease process and to asses efficacy during vaccination., This study was funded in part by the Instituto de Investigación Sanitaria y Biomédica de Alicante (ISABIAL; grants 190258 and 2020-0308) and by the Instituto de Salud Carlos III (ISCIII, grants PI19-01359), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER, “Investing in your future”) and through CIBERNED, ISCIII. We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723). Work at CNB and CISA is funded by the Spanish Health Ministry, ISCIII, Fondo COVID-19 grant COV20/00151, and Fondo Supera COVID-19 (Crue Universidades-Banco Santander) (to JGA). MACG is supported by BEFPI fellowship from the Generalitat Valenciana. HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532), the European Research Council (#681712), and Swedish State Support for Clinical Research (#ALFGBG-720931).

[Background]: The disintegrin metalloproteinase 10 (ADAM10) is the main α-secretase acting in the non-amyloidogenic processing of APP. Some ADAM10 gene variants have been associated with higher susceptibility to develop late-onset AD, though clear clinical-genetic correlates remain elusive., [Methods]: Clinical-genetic and biomarker study of a first family with early- and late-onset AD associated with a nonsense ADAM10 mutation (p.Tyr167*). CSF analysis included AD core biomarkers, as well as Western blot of ADAM10 species and sAPPα and sAPPβ peptides. We evaluate variant’s pathogenicity, pattern of segregation, and further screened for the p.Tyr167* mutation in 197 familial AD cases from the same cohort, 200 controls from the same background, and 274 AD cases from an independent Spanish cohort., [Results]: The mutation was absent from public databases and segregated with the disease. CSF Aβ42, total tau, and phosphorylated tau of affected siblings were consistent with AD. The predicted haploinsufficiency effect of the nonsense mutation was supported by (a) ADAM10 isoforms in CSF decreased around 50% and (b) 70% reduction of CSF sAPPα peptide, both compared to controls, while sAPPβ levels remained unchanged. Interestingly, sporadic AD cases had a similar decrease in CSF ADAM10 levels to that of mutants, though their sAPPα and sAPPβ levels resembled those of controls. Therefore, a decreased sAPPα/sAPPβ ratio was an exclusive feature of mutant ADAM10 siblings. The p.Tyr167* mutation was not found in any of the other AD cases or controls screened., [Conclusions]: This family illustrates the role of ADAM10 in the amyloidogenic process and the clinical development of the disease. Similarities between clinical and biomarker findings suggest that this family could represent a genetic model for sporadic late-onset AD due to age-related downregulation of α-secretase. This report encourages future research on ADAM10 enhancers., This work was supported by grants from the Ministry of Sciences and Technology, (SAF2010-18277), Instituto de Salud Carlos III (FIS14/00099), Direcció General d’Universitat, Investigació i Ciència, GVA (AICO/2018/090), and FEDER funds, Spain. MSGA is supported by a Miguel Servet II Grant from Instituto de Salud Carlos III (CPII16/00011).

[Background]: The amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes alternative proteolytic processing. Its processing through the amyloidogenic pathway originates a large sAPPβ ectodomain fragment and the β-amyloid peptide, while non-amyloidogenic processing generates sAPPα and shorter non-fibrillar fragments. Hence, measuring sAPPα and sAPPβ has been proposed as a means to identify imbalances between the amyloidogenic/non-amyloidogenic pathways in the brain of Alzheimer’s disease (AD) patients. However, to date, no consistent changes in these proteolytic fragments have been identified in either the brain or cerebrospinal fluid of AD individuals., [Methods]: In frontal cortex homogenates from AD patients (n = 7) and non-demented controls (NDC; n = 7), the expression of total APP mRNA and that of the APP isoforms generated by alternative splicing, APP695 and APP containing the Kunitz protease inhibitor (KPI), was analyzed by qRT-PCR using TaqMan and SYBR Green probes. The balance between the amyloidogenic/non-amyloidogenic pathways was examined in western blots estimating the sAPPα and sAPPβ fragments and their membrane-tethered C-terminal fragments CTFα and CTFβ. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether Aβ42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPPα and sAPPβ in brain extracts and CHO-PS70 culture media by lectin-binding assays., [Results]: In the cortex of AD patients, we detected an increase in total APP mRNA relative to the controls, due to an increase in both the APP695 and APP-KPI variants. However, the sAPPα or sAPPβ protein levels remained unchanged, as did those of CTFα and CTFβ. We studied the glycosylation of the brain sAPPα and sAPPβ using lectins and pan-specific antibodies to discriminate between the fragments originated from neuronal APP695 and glial/KPI variants. Lectin binding identified differences in the glycosylation of sAPPβ species derived from the APP695 and APP-KPI variants, probably reflecting their distinct cellular origin. Moreover, the lectin-binding pattern differed in the sAPPα and sAPPβ originated from all the variants. Finally, when the lectin-binding pattern was compared between AD and NDC groups, significant differences were evident in sAPPα glycosylation. Lectin binding of the soluble sAPPα and sAPPβ from CHO-PS70 cells were also altered in cells treated with the Aβ peptide., [Conclusion]: Our analysis of the lectin binding to sAPPα and sAPPβ suggests that glycosylation dictates the proteolytic pathway for APP processing. Differences between the demented and controls indicate that changes in glycosylation may influence the generation of the different APP fragments and, consequently, the pathological progression of AD., This study was funded in part by the Direcció General d’Universitat, Investigació i Ciència, GVA (AICO/2018/090), Agència Valenciana de la Innovació-Generalitat Valenciana, within the framework of the collaboration agreement (INNCON/2020/8-UCIE Instituto de Neurociencias), and by the Instituto de Salud Carlos III (ISCIII, grants PI15/00665 and PI19-01359), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER, “Investing in your future”), and through CIBERNED, ISCIII. We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723).

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders involving age-dependent gene dysregulation. Reelin is a glycoprotein that varies its expression throughout lifetime and controls cortical patterning and synaptogenesis. Brain and plasma reelin levels have been reported to be low in adults with autism; as well as in children with autism, but only when compared to control adults. Therefore, reelin expression levels in children with autism are unclear. For this reason, we compared plasma reelin levels in children with autism and children without autism (non-ASD) of similar ages to evaluate reelin expression in ASD during childhood. Plasma samples from 19 non-ASD (8.9 ± 0.8 years) and 40 children with autism (7.5 ± 0.5 years) were analyzed. We found that 50% of the children with autism displayed similar plasma reelin levels to the non-ASD group. However, the remaining 50% expressed more than 30 times more reelin compared to non-ASD levels. We also show that male children with autism displayed significantly higher reelin levels than females. The clinical presentation of this subgroup could not be distinguished from that of children with autism. Epilepsy or attention-deficit/hyperactivity disorder (ADHD) was not associated to reelin levels. We conclude that the high levels of plasma reelin might be an important hallmark in a subset of children with autism, previously unnoticed. As we could not find any correlation between reelin levels and ASD clinical presentations, our results may indicate transient reelin increases in the plasma or the characterization of a group of ASD individuals with a different pathophysiology., This work was supported by grants from Fondo de Investigaciones Sanitarias (PI15/00665 and PI19-01359, co-funded by the Fondo Europeo de Desarrollo Regional, FEDER "Investing in your future"), by the Direcció General d’Universitat, Investigació i Ciència, GVA (AICO/2018/090) and through CIBERNED (Instituto de Salud Carlos III, Spain). We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723).

We recently demonstrated that soluble forms of the amyloid-β protein precursor (sAβPP) assemble into multimeric complexes in cerebrospinal fluid (CSF), which contributes to the underestimation of specific sAβPP species when assessed by ELISA. To circumvent this issue, we analyzed by SDS-PAGE large fragments of sAβPP and their variants in the CSF from Alzheimer's disease (AD; n = 20) and control (n = 20) subjects, probing with specific antibodies against particular domains. Similar levels of sAβPPα and sAβPPβ protein were found in CSF samples from AD and controls, yet there appeared to be a shift in the balance of the soluble full-length AβPP (sAβPPf) species in AD samples, with a decrease in the proportion of the lower (∼100 kDa) band relative to the upper (∼120 kDa) band. Similar differences were observed in the contribution of the major KPI-immunoreactive AβPP species. CSF samples also displayed differences in the correlations of AβPP species with classical AD biomarkers, particularly with respect to the Aβ42 peptide. The differences reveal alterations that probably reflect pathophysiological changes in the brain., This study was funded in part by the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project, by the Instituto de Salud Carlos III (ISCIII grants PI11/03026 to JSV, PI11/02425 and PI14/01126 to JF, PI11/03035 and PI14/1561 to AL, PI08/0036 and PI12/00013 to RSV, and PI11/03023 to JLM), co-financed by the Fondo Europeo de Desarrollo Regional, under the aegis of JPND, and through CIBERNED, ISCIII. This work was also supported by the Fundació Catalana de Síndrome de Down and by a “Marató TV3” grant (20141210 to JF) and a grant from the Griffols Foundation, and the Torsten Söderberg Foundation, Sweden (to KB).

Stability of the cerebrospinal fluid (CSF) composition under different pre-analytical conditions is relevant for the diagnostic potential of biomarkers. Our aim was to examine the pre-analytical stability of promising CSF biomarkers that are currently evaluated for their discriminative use in various neurological diseases. Pooled CSF was aliquoted and experimentally exposed to delayed storage: 0, 1, 2, 4, 24, 72, or 168 h at 4 °C or room temperature (RT), or 1–4 months at −20 °C; or up to 7 freeze/thaw (f/t) cycles, before final storage at −80 °C. Eleven CSF biomarkers were screened using immunoassays, liquid chromatography, or enzymatic methods. Levels of neurogranin (truncP75), chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1) and homovanillic acid (HVA) levels were not affected by the applied storage conditions. 3-Methoxy-4-hydroxyphenylglycol (MHPG) levels linearly and strongly decreased after 4 h at RT (−10%) or 24 h at 4 °C (−27%), and with 6% after every f/t cycle. 5-Methyltetrahydrofolate (5-MTHF) (−29% after 1 week at RT) and 5-hydroxyindoleacetic acid levels (5-HIAA) (−16% after 1 week at RT) were reduced and 3,4-dihydroxyphenylacetic acid (DOPAC) levels (+22% after 1 week at RT) increased, but only after >24 h at RT. Ten out of eleven potential CSF novel biomarkers showed very limited change under common storage and f/t conditions, suggesting that these CSF biomarkers can be trustfully tested under the pre-analytical conditions present across different cohorts., This research was financially supported by Biobanking and BioMolecular resources Research Infrastructure the Netherlands (BBMRI-NL), a research infrastructure financed by the Dutch Government (NWO 184.021.007) under project CP2013-68. This study commenced within the BIOMARKAPD program of the EU Joint Programme – Neurodegenerative Disease Research (JPND). Project supported by the European Regional Development Fund (ERDF) through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., under the projects POCI-01-0145-FEDER-007440, POCI-01-0145-FEDER-016428, and POCI-01-0145-FEDER-016795, and by The National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125.

Reelin is a glycoprotein associated with synaptic plasticity and neurotransmission. The malfunctioning of reelin signaling in the brain is likely to contribute to the pathogenesis of Alzheimer's disease (AD). Reelin binding to Apolipoprotein E receptor 2 (ApoER2) activates downstream signaling and induces the proteolytic cleavage of ApoER2, resulting in the generation of soluble fragments. To evaluate the efficiency of reelin signaling in AD, we have quantified the levels of reelin and soluble ectodomain fragments of ApoER2 (ectoApoER2) in the cerebrospinal fluid (CSF). CSF from sporadic AD patients (sAD; n = 14, age 54–83 years) had lower levels of ecto-ApoER2 (~31% reduction; p = .005) compared to those in the age-matched controls (n = 10, age 61–80), and a higher reelin/ecto-ApoER2 ratio. In contrast, autosomal dominant AD patients, carriers of PSEN1 mutations (ADAD; n = 7, age 31–49 years) had higher ecto-ApoER2 levels (~109% increment; p = .001) and a lower reelin/ecto-ApoER2 ratio than the non-mutation carriers from the same families (n = 7, age 25–47 years). Our data suggest that the levels of ecto-ApoER2 in CSF could be a suitable read-out of an impaired reelin signaling in AD, but also indicate differences between sAD and ADAD., This study was funded in part by the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project, by the Instituto de Salud Carlos III (ISCIII grants PI11/03026 and PI15/00665 to JSV, PI08/0036 and PI12/00013 to RSV, and PI11/03023 to JLM), by the Direcció General d'Universitat, Investigació i Ciència, GVA (AICO/2018/090 to JSV), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER, “Investing in your future”), and through CIBERNED, ISCIII.

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the main brain β-secretase responsible for the amyloidogenic processing of the amyloid precursor protein (APP). Previous studies have suggested that cerebrospinal fluid (CSF) β-secretase activity may be a candidate diagnostic biomarker for Alzheimer’s disease (AD), but biochemical characterization of BACE1 protein in CSF is needed. CSF samples from 19 AD patients and 19 age-matched non-AD controls (n = 19) were classified according to their Aβ42, total tau, and P-tau CSF biomarker levels. We found that β-secretase activity was higher in the CSF of AD subjects than in that of the controls. We found that the majority of the β-secretase activity in the CSF, measured using a peptide substrate homologous to the BACE1 cleavage site, was not inhibited by specific BACE1 inhibitors. We defined enzymatic activity attributable specifically to BACE1 as the activity that was blocked by the specific inhibitors, which is still higher in AD subjects. BACE1 protein levels were characterized by lectin binding, immunoprecipitation, blue native-PAGE, and western blotting using antibodies against specific protein domains. BACE1 was found to be present in human CSF as a mature form of ~ 70 kDa that probably comprised truncated and full-length species, and also as an immature form of ~ 50 kDa that retains the prodomain. CSF-BACE1 was found to assemble into hetero-complexes containing distinct species. Immunoblotting with an antibody against the C-terminus of BACE1 revealed significantly higher levels of the 70-kDa full-length BACE1, while the 50 kDa immature form remained unaltered. When the 70-kDa species was probed with an antibody against the N-terminus of BACE1 (which does not discriminate between truncated and full-length forms), no increase in immunoreactivity was observed, suggesting that truncated forms of BACE1 do not increase in AD. In conclusion, the complexity of BACE1 species in CSF has to be taken into consideration when determining BACE1 activity and protein levels in CSF as biomarkers of AD., This study was funded in part by the Direcció General d’Universitat, Investigació i Ciència, GVA (AICO/2018/090), by the EU BIOMARKAPD-Joint Programme on Neurodegenerative Diseases (JPND) and the Instituto de Salud Carlos III (ISCIII grant PI11/03026), by the ISCIII (PI15/00665), co-financed by the Fondo Europeo de Desarrollo Regional under the aegis of JPND), and through CIBERNED, ISCIII (FEDER, “Investing in your future”). HZ is a Wallenberg Academy Fellow supported by grants from the European Research Council, the Swedish Research Council and the UK Dementia Research Institute at UCL. KB holds the Torsten Söderberg Professorship in Medicine at the Royal Swedish Academy of Sciences, and is supported by the Swedish Research Council (#2017-00915), the Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden (#FO2017-0243), and a grant (#ALFGBG-715986) from the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement.

Previous studies have indicated the potential of cerebrospinal fluid (CSF) α‐synuclein (α‐syn) to be an additional biomarker for improving differential diagnosis of Alzheimer’s disease (AD). We evaluated α‐syn diagnostic performance across a well‐characterized patient cohort with long‐term follow‐up. For this purpose, CSF α‐syn levels were determined in 25 subjects diagnosed with stable mild cognitive impairment (stable MCI; n = 25), 27 MCI cases due to AD (MCI‐AD; n = 32), 24 MCI cases due to Lewy body disease (MCI‐LBD; n = 24) and control subjects (Ctrl; n = 18). CSF α‐syn levels discriminate between the four groups. There were higher α‐syn levels in MCI‐AD patients and lower levels in MCI‐LBD patients. The combination of α‐syn and P‐tau resulted in a specificity of 99% and a sensitivity of 97% for MCI‐AD. MCI‐AD patients with early psychotic symptoms (n = 9) displayed a trend towards a decrease in P‐tau and α‐syn compared to the MCI‐AD patients without psychotic symptoms (n = 23). We conclude that adding CSF α‐syn to central core AD biomarkers improves an early differential diagnosis of MCI‐AD from other forms of MCI., This study was funded in part by the Direcció General d’Universitat, Investigació i Ciència, GVA (AICO/2018/090 to JSV), co‐financed by the Fondo Europeo de Desarrollo Regional, and through CIBERNED, ISCIII (FEDER, ‘Investing in your future’). MSGA is supported by a Miguel Servet II Grant from Instituto de Salud Carlos III (CPII1600011).

ErbB4 is a transmembrane receptor tyrosine kinase that binds to neuregulins to activate signaling. Proteolytic cleavage of ErbB4 results in release of soluble fragments of ErbB4 into the interstitial fluid. Disruption of the neuregulin-ErbB4 pathway has been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). This study assesses whether soluble proteolytic fragments of the ErbB4 ectodomain (ecto-ErbB4) can be detected in cerebrospinal fluid (CSF) and plasma, and if the levels are altered in ALS. Immunoprecipitation combined with mass spectrometry or western blotting analyses confirmed the presence of ecto-ErbB4 in human CSF. Several anti-ErbB4-reactive bands, including a 55 kDa fragment, were detected in CSF. The bands were generated in the presence of neuregulin-1 (Nrg1) and were absent in plasma from ErbB4 knockout mice. Ecto-ErbB4 levels were decreased in CSF from ALS patients (n = 20) and ALS with concomitant frontotemporal dementia patients (n = 10), compared to age-matched controls (n = 13). A similar decrease was found for the short ecto-ErbB4 fragments in plasma of the same subjects. Likewise, the 55-kDa ecto-ErbB4 fragments were decreased in the plasma of the two transgenic mouse models of ALS (SOD1G93A and TDP-43A315T). Intracellular ErbB4 fragments were decreased in the frontal cortex from SOD1G93A mice, indicating a reduction in Nrg-dependent induction of ErbB4 proteolytic processing, and suggesting impaired signaling. Accordingly, overexpression of Nrg1 induced by an adeno-associated viral vector increased the levels of the ecto-ErbB4 fragment in the SOD1G93A mice. We conclude that the determination of circulating ecto-ErbB4 fragments could be a tool to evaluate the impairment of the ErbB4 pathway and may be a useful biomarker in ALS., This work was supported by grants from the Fondo de Investigaciones Sanitarias (PI15/00665 to JSV and PI15/01618 to RRG), cooperative project 2015-01 from CIBERNED (Instituto de Salud Carlos III, Spain) to XN and JSV, all co-funded by the Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, “Una manera de hacer Europa”; grants TV3201428-10 to XN and 201437 to RRG of Fundació La Marato-TV3; and grant #20289 of AFM-Telethon to XN. HZ is a Wallenberg Academy Fellow and is supported by grants from the Swedish and European Research Councils and the UK Dementia Research Institute at UCL. KB holds the Torsten Söderberg Professorship in Medicine at the Royal Swedish Academy of Sciences. We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723).

In the continuing search for proteins that play a role in Alzheimer's disease (AD) and that are related to the pathological hallmarks, those that influence cognitive function and that constitute potential therapeutic targets deserve special interest. Reelin is a signaling protein that is involved in a cascade of cytoplasmic events that control tau phosphorylation and that regulate synaptic neurotransmission, plasticity, and memory. Both Reelin expression and glycosylation are modulated by amyloid-β (Aβ), suggesting that the activity of Reelin could be affected in AD and hence, its possible influence on this pathology should be taken into consideration. The levels of Reelin in the brain of AD patients appear to be altered and interestingly, disrupted Reelin signaling is associated with increased tau phosphorylation as well as with amyloid-β protein precursor processing. We discuss here the somewhat contradictory data regarding Reelin levels in AD and we evaluate the processing of the Reelin receptor, ApoER2, and other downstream events, such as the phosphorylation of the intracellular adapter Dab1. Together with brain Reelin levels, these changes may represent a relevant read-out of Reelin signaling in the human brain.

[Background] The disintegrin metalloproteinase 10 (ADAM10) is the main α-secretase acting in the non-amyloidogenic processing of the amyloid precursor protein. This study assesses whether ADAM10 is present in cerebrospinal fluid (CSF), and whether it has potential as a biomarker for Alzheimer’s disease (AD)., [Methods] ADAM10 was characterized in human CSF samples by immunoprecipitation and western blotting using antibodies specific for different domains of the protein and by ultracentrifugation in sucrose density gradients. Samples from AD patients (n = 20) and age-matched non-AD controls (n = 20) were characterized for classical CSF biomarkers, Aβ42, T-tau, or P-tau by ELISA, and assayed for soluble ADAM10 levels by western blotting., [Results] We found that ADAM10 is present in human CSF as several distinct species: an immature form retaining the prodomain (proADAM10; ~ 80 kDa), a mature unprocessed full-length form (ADAM10f; ~ 55 kDa), and a truncated large soluble form released from the membrane (sADAM10; ~ 50 kDa). Fractionation by ultracentrifugation on sucrose density gradients showed that the ADAM10f and sADAM10 species form large complexes. Immunoblotting revealed a significant decrease in ADAM10f and sADAM10 in AD CSF compared to control CSF, while proADAM10 levels remained unaltered., [Conclusions] Several forms of ADAM10 are present in CSF, mainly assembled as high-molecular weight complexes. The determination of the levels of mature forms of CSF-ADAM10 may be useful as a biomarker for AD., This study was funded in part by the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project, by the Instituto de Salud Carlos III (ISCIII grants PI11/03026 and PI15/00665), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER, “Investing in your future”), under the aegis of JPND, and through CIBERNED, ISCIII. We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723). This work was also supported by a grant from Torsten Söderberg Foundation, Sweden (to KB) and the Knut and Alice Wallenberg Foundation (to HZ)., We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

γ-Secretase inhibitors (GSIs) are potential therapeutic agents for Alzheimer’s disease (AD); however, trials have proven disappointing. We addressed the possibility that γ-secretase inhibition can provoke a rebound effect, elevating the levels of the catalytic γ-secretase subunit, presenilin-1 (PS1). Acute treatment of SH-SY5Y cells with the GSI LY-374973 (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, DAPT) augments PS1, in parallel with increases in other γ-secretase subunits nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, yet with no increase in messenger RNA expression. Over-expression of the C-terminal fragment (CTF) of APP, C99, also triggered an increase in PS1. Similar increases in PS1 were evident in primary neurons treated repeatedly (4 days) with DAPT or with the GSI BMS-708163 (avagacestat). Likewise, rats examined after 21 days administered with avagacestat (40 mg/kg/day) had more brain PS1. Sustained γ-secretase inhibition did not exert a long-term effect on PS1 activity, evident through the decrease in CTFs of APP and ApoER2. Prolonged avagacestat treatment of rats produced a subtle impairment in anxiety-like behavior. The rebound increase in PS1 in response to GSIs must be taken into consideration for future drug development., This study was funded in part by the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project by the Instituto de Salud Carlos III (ISCIII grants PI11/03026 and PI15/00665 for JSV and PI14/00566 for MSGA), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER) “Investing in your future,” and through CIBERNED (ISCIII). We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2013-0317).

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 β-amyloid (Aβ) 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 Aβ aa 1–42 (Aβ42) 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 Aβ42 decreased nuclear levels of DNA-methyltransferase 1. However, RELN promoter methylation did not change in Aβ42- treated cells or in AD brain extracts. Instead, the levels of ApoER2-CTF appeared significantly lower in Aβ42-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 Aβ 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., This work was supported by grants from the Fundación Ramión Areces, Fondo de Investigaciones Sanitarias [PI15/00665, cofunded by the Fondo Europeo de Desarrollo Regional (FEDER) “Investing in your future”], and through Centro de Investigatión Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; Instituto de Salud Carlos III, Madrid, Spain). The authors also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV- 2013–0317).

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, β-1,4-galactosyltransferase (β4GalT), 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 Aβ42 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 Aβ 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 β-amyloid protein., This study was funded in part by Consejeria de Sanidad, Generalitat Valenciana (AP-091/08) and the Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants PS09/00684 and PI11/03026 for JSV and CP11/00067 and PI14/00566 to MSGA); cofinanced by Fondo Europeo de Desarrollo Regional), the Torsten Söderberg Foundation at the Royal Swedish Academy of Sciences, and under the aegis of the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project; and through CIBERNED, ISCIII, Spain.

Acetylcholinesterase (AChE) is a key enzyme in the cholinergic nervous system and is one of the most studied proteins in the field of Alzheimer's disease (AD). Moreover, alternative functions of AChE unrelated with the hydrolysis of acetylcholine are suspected. Until now, the majority of investigations on AChE in AD pathology have been focused on the determination of its enzymatic activity level, which is depleted in the AD brain. Despite this overall decrease, AChE activity increases at the vicinity of the two hallmarks of AD, the amyloid plaques and the neurofibrillary tangles (NFT). In fact, AChE may directly interact with Aβ in a manner that increases the deposition of Aβ to form plaques. In the context of protein-protein interactions, we have recently reported that AChE can interact with presenilin-1, the catalytic component of γ-secretase, influencing its expression level and also its activity. However, the alteration of AChE protein in the AD brain has not been determined. Here, we demonstrated by Western blotting and immunohistochemistry that a prominent pool of enzymatically inactive AChE protein existed in the AD brain. The potential significance of these unexpected levels of inactive AChE protein in the AD brain was discussed, especially in the context of protein-protein interactions with β-amyloid and presenilin-1. © 2013 Springer Science+Business Media., MLC is supported by a Consolider-Predoctoral fellowship from the CSIC, Spain. This work was supported by grants from Fundación CIEN-Reina Sofía, Fondo de Investigaciones Sanitarias (FIS; Grant PS09/00684) from Spain to JSV and Fondo de Investigaciones Sanitarias (FIS; Grant CP11/00312) to MSGA

Reelin is a signaling protein that plays a crucial role in synaptic function, which expression is influenced by β-amyloid (Aβ). We show that Reelin and Aβ oligomers co-immunoprecipitated in human brain extracts and were present in the same size-exclusion chromatography fractions. Aβ treatment of cells led to increase expression of Reelin, but secreted Reelin results trapped together with Aβ aggregates. In frontal cortex extracts an increase in Reelin mRNA and in soluble and insoluble (guanidine-extractable) Reelin protein, was associated with late Braak stages of Alzheimer’s disease (AD), while expression of its receptor, ApoER2, did not change. However, Reelin-dependent induction of Dab1 phosphorylation appeared reduced in AD. In cells, Aβ reduced the capacity of Reelin to induce internalization of biotinylated ApoER2 and ApoER2 processing. Soluble proteolytic fragments of ApoER2 generated after Reelin binding can be detected in cerebrospinal fluid (CSF). Quantification of these soluble fragments in CSF could be a tool to evaluate the efficiency of Reelin signaling in the brain. These CSF-ApoER2 fragments correlated with Reelin levels only in control subjects, not in AD, where these fragments diminished. We conclude that while Reelin expression is enhanced in the Alzheimer’s brain, the interaction of Reelin with Aβ hinders its biological activity.

PMCID: PMC5013723, Alzheimer's disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). AChE is expressed as multiple splice variants, which may serve both cholinergic degradative functions and non-cholinergic functions unrelated with their capacity to hydrolyze acetylcholine. We have recently demonstrated that a prominent pool of enzymatically inactive AChE protein exists in the AD brain. In this study, we analyzed protein and transcript levels of individual AChE variants in human frontal cortex from AD patients by western blot analysis using specific anti-AChE antibodies and by quantitative real-time PCR (qRT-PCR). We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchoring subunit, proline-rich membrane anchor (PRiMA-1) in frontal cortex obtained from AD patients and non-demented controls. Interestingly, we found an increase in the protein and transcript levels of the non-cholinergic "readthrough" AChE (AChE-R) variants in AD patients compared to controls. Similar increases were detected by western blot using an antibody raised against the specific N-terminal domain, exclusive of alternative N-extended variants of AChE (N-AChE). In accordance with a subset of AChE-R monomers that display amphiphilic properties that are upregulated in the AD brain, we demonstrate that the increase of N-AChE species is due, at least in part, to N-AChE-R variants. In conclusion, we demonstrate selective alterations in specific AChE variants in AD cortex, with no correlation in enzymatic activity. Therefore, differential expression of AChE variants in AD may reflect changes in the pathophysiological role of AChE, independent of cholinergic impairment or its role in degrading acetylcholine., MLC was supported by a Consolider-Predoctoral fellowship from the CSIC, Spain. This study was funded in part by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants CP11/00067 and PI14/00566 to MSGA and PS09/00684 and PI11/03026 for JSV); co-financed by Fondo Europeo de Desarrollo Regional) and Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (Fisabio) and through CIBERNED, ISCIII, Spain. Support also comes from NIH grant AG043375 (S. Ginsberg).

10 p., 5 figures and references., 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 β-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 β-amyloid. Finally, in vitro Aβ42 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 Aβ42-treated SH-SY5Y cells, supporting the notion that β-amyloid triggers altered Reelin processing. These results provide evidence that Reelin expression and processing is altered in several amyloid conditions., This work was supported by grants from Ministerio de Ciencia e Innovación (MCI, Grant BFU2008-3980), FIS (Grant 06/0181), Fundació Obra Social Caixa Catalunya, EU-037627-AnEUploidy, and CIBERNED, ISC-III from Spain.

A number of biomarkers (e.g. Aβ, tau) has been identified in Alzheimer's disease CSF. However, none fulfils the criteria of sensitivity and specificity (> 80%) needed for the development of an accurate diagnostic test. The lack of a suitable marker has prompted the search for new CSF biomarkers. In this study, the glycosylation of CSF proteins was examined using lectin blotting. Lumbar CSF was collected ante mortem from 22 non-Alzheimer's disease and 12 probable Alzheimer's disease cases and ventricular CSF collected post mortem from 7 non-Alzheimer's disease and 16 Alzheimer's disease cases confirmed by pathologic examination. When CSF glycoproteins were stained with wheat germ agglutinin (WGA), the staining intensity was found to be significantly lower in the Alzheimer's disease group. No difference in staining was found using other lectins (Canavalia ensiformis agglutinin, Ricinus communis agglutinin, Lens culinaris agglutinin). The measurement of WGA-reactive glycoproteins in CSF may be a useful biomarker for diagnosis of Alzheimer's disease.

It has been proposed that impairment of the glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in hepatic encephalopathy. The aims of this work were to assess whether the function of this pathway and of nitric oxide synthase (NOS) are altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval shunt (PCS) and whether these alterations are due to hyperammonemia. The glutamate-nitric oxide-cGMP pathway function and NOS activation by NMDA was analysed by in vivo microdialysis in cerebral cortex of PCS and control rats and in rats with hyperammonemia without liver failure. Similar studies were done in cortical slices from these rats and in cultured cortical neurons exposed to ammonia. Basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These increases seem due to increased inducible nitric oxide synthase expression. NOS activation by NMDA is impaired in cerebral cortex in both animal models and in neurons exposed to ammonia. Chronic liver failure increases basal NOS activity, nitric oxide and cGMP but reduces activation of NOS induced by NMDA receptors activation. Hyperammonemia is responsible for both effects which will lead, independently, to alterations contributing to neurological alterations in hepatic encephalopathy.

Background: Soluble fragments of the amyloid precursor protein (APP) generated by α- and β-secretases, sAPPα and sAPPβ, have been postulated as promising new cerebrospinal fluid (CSF) biomarkers for the clinical diagnosis of Alzheimer’s disease (AD). However, the capacity of these soluble proteins to assemble has not been explored and could be relevant. Our aim is to characterize possible sAPP oligomers that could contribute to the quantification of sAPPα and sAPPβ in CSF by ELISA, as well as to characterize the possible presence of soluble full-length APP (sAPPf)., Results: We employed co-immunoprecipitation, native polyacrylamide gel electrophoresis and ultracentrifugation in sucrose density gradients to characterize sAPP oligomers in CSF. We have characterized the presence of sAPPf in CSF from NDC and AD subjects and demonstrated that all forms, including sAPPα and sAPPβ, are capable of assembling into heteromers, which differ from brain APP membrane-dimers. We measured sAPPf, sAPPα and sAPPβ by ELISA in CSF samples from AD (n = 13) and non-disease subjects (NDC, n = 13) before and after immunoprecipitation with antibodies against the C-terminal APP or against sAPPβ. We demonstrated that these sAPP heteromers participate in the quantification of sAPPα and sAPPβ by ELISA. Immunoprecipitation with a C-terminal antibody to remove sAPPf reduced by ~30% the determinations of sAPPα and sAPPβ by ELISA, whereas immunoprecipitation with an APPβ antibody reduced by ~80% the determination of sAPPf and sAPPα., Conclusions: The presence of sAPPf and sAPP heteromers should be taken into consideration when exploring the levels of sAPPα and sAPPβ as potential CSF biomarkers., This study was funded in part by the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project. This project is supported through the Instituto de Salud Carlos III (ISCIII; grants PI11/03026 to JSV and PI11/03023 to JLM) under the aegis of JPND, and through CIBERNED, ISC-III. We also acknowledge the support for the publication fee to the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

© 2015 Elsevier Inc. All rights reserved., Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinson's disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program - Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings. Recommendations for further validation experiments and improvement of the interlaboratory results obtained are given., This is an EU Joint Program – Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organizations under the aegis of JPND–www.jpnd.eu: The Alzheimer Research Foundation (SAO-FRA), Belgium (grant number S#12018); Danish Council for Strategic Research, Denmark (grant number 0603-00511B); Bundesministerium für Bildung und Forschung (BMBF), Germany (grant number OIED1203C); General Secretary of Research and Technology, Greece; Italian Ministry of Health (Ricerca Corrente), Italy; The Fundação para a Ciência e a Tecnologia, Portugal (grant numbers JPND/0004/2011, IMM/BPD/51-2013, IMM/BI/7-2013, JPND/0005/2011); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (grant numbers PI11/03026, PI11/03035; cofinanced by the Fondo Europeo de Desarrollo Regional); The Ministry of Education, Research and Sport of the Slovak Republic, Slovakia; The Slovenian Research Agency, Slovenia (grant number A3-0001); The Swedish Research Council, Sweden; Ministry of Education for Netherlands: ZonMw (The Netherlands Organisation for Health Research and Development), The Netherlands (grant number 629000002); The Scientific and Technological Research Council of Turkey (grant number 112S335).

Presented in part as a poster at 2005 Meeting EASL, Paris., Studies of the pathogenesis of hepatic encephalopathy are hampered by the lack of a satisfactory animal model. We examined the neurological features of rats after bile duct ligation fed a hyperammonemic diet (BDL+HD). Six groups were studied: sham, sham pair-fed, hyperammonemic, bile duct ligation (BDL), BDL pair fed, and BDL+HD. The BDL+HD rats were made hyperammonemic via an ammonia-containing diet that began 2 weeks after operation. One week later, the animals were sacrificed. BDL+HD rats displayed an increased level of cerebral ammonia and neuroanatomical characteristics of hepatic encephalopathy (HE), including the presence of type II Alzheimer astrocytes. Both BDL and BDL+HD rats showed activation of the inflammatory system. BDL+HD rats showed an increased amount of brain glutamine, a decreased amount of brain myo-inositol, and a significant increase in the level of brain water. In coordination tests, BDL+HD rats showed severe impairment of motor activity and performance as opposed to BDL rats, whose results seemed only mildly affected. In conclusion, the BDL+HD rats displayed similar neuroanatomical and neurochemical characteristics to human HE in liver cirrhosis. Brain edema and inflammatory activation can be detected under these circumstances., Supported by grants from Instituto de Salud Carlos III, Convocatoria de RedesTematicas (G03/155 and C03/02); Conselleria de Sanitat, Escuela Valenciana para Estudios de la Salud (BM-004/2002); Organisme Public Valencia d’Investigacio, Ajudes per a Accions Especials (21-2002), Generalitat Valenciana,GV Grupos 03/053, Junta de Comunidades de Castilla-La Mancha (GC-02-022)and Instituto de Salud Carlos III (PI03/0576).

Reelin is a glycoprotein that is essential for the correct cytoarchitectonic organization of the developing CNS. Its function in the adult brain is less understood, although it has been proposed that Reelin is involved in signaling pathways linked to neurodegeneration. Here we analyzed Reelin expression in brains and cerebrospinal fluid (CSF) from Alzheimer’s disease (AD) patients and nondemented controls. We found a 40% increase in the Reelin protein levels in the cortex of AD patients compared with controls. Similar increases were detected at the Reelin mRNA transcriptional level. This expression correlates with parallel increases in CSF but not in plasma samples. Next, we examined whether CSF Reelin levels were also altered in neurological diseases, including frontotemporal dementia, progressive supranuclear palsy, and Parkinson’s disease. The Reelin 180-kDa band increased in all of the neurodegenerative disorders analyzed. Moreover, the 180-kDa Reelin levels correlated positively with Tau protein in CSF. Finally, we studied the pattern of Reelin glycosylation by using several lectins and the anti-HNK-1 antibody. Glycosylation differed in plasma and CSF. Furthermore, the pattern of Reelin lectin binding differed between the CSF of controls and in AD. Our results show that Reelin is up-regulated in the brain and CSF in several neurodegenerative diseases and that CSF and plasma Reelin have distinct cellular origins, thereby supporting that Reelin is involved in the pathogenesis of a number of neurodegenerative diseases.

Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylcholinesterase (BuChE), whereas acetylcholinesterase (AChE) has not received much attention. In the current study, liver and plasma AChE levels were investigated in rats with cirrhosis induced after 3 weeks of bile duct ligation (BDL). BDL rats showed a pronounced decrease in liver AChE levels (∼50%) compared with sham-operated (non-ligated, NL) controls; whereas liver BuChE appeared unaffected. A selective loss of tetrameric (G4) AChE was detected in BDL rats, an effect also observed in rats with carbon tetrachloride-induced cirrhosis. In accordance, SDS-PAGE analysis showed that the major 55-kd immunoreactive AChE band was decreased in BDL as compared with NL. A 65-kd band, attributed in part to inactive AChE, was increased as became the most abundant AChE subunit in BDL liver. The overall decrease in AChE activity in BDL liver was not accompanied by a reduction of AChE transcripts. The loss of G4 was also reflected by changes observed in AChE glycosylation pattern attributable to different liver AChE forms being differentially glycosylated. BDL affects AChE levels in both hepatocytes and Kupffer cells; however, altered AChE expression was mainly reflected in an alteration in hepatocyte AChE pattern. Plasma from BDL rats had approximately 45% lower AChE activity than controls, displaying decreased G4 levels and altered lectin-binding patterns. In conclusion, the liver is an important source of serum AChE; altered AChE levels may be a useful biomarker for liver cirrhosis., Supported by grants from la Caixa Foundation, Instituto de Salud Carlos III (Grants 03/0038, G03/155, C03/02) and Generalitat Valenciana (CTIDIB/ 2002/42, OPVI-21-2002 & GV04B-664) from Spain.

In this study we examined changes in acetylcholinesterase (AChE) pattern in the brain of adult Reelin Orleans (RelnOrl) homozygous mutant mice. The AChE histochemistry firstly revealed an abnormal distribution of AChE-positive cells in several areas of the reeler brain, including cortices; the strongest labelling was observed in cerebellum and hippocampus when compared with controls. Biochemical determinations demonstrated an increase of 80-90% in AChE specific activity from cerebellar and hippocampal extracts. We also report that the AChE tetrameric form (G4) was selectively increased in the RelnOrl brain. The relationship between AChE and Reelin and suggested morphogenetic functions are also discussed.

Biochemical changes in rat brain acetylcholinesterase (AChE) activity and molecular isoform distribution were studied using the middle cerebral artery occlusion model. After 24 h of permanent focal ischemia, AChE specific activity from infarcted cortex was found to decrease compared with contralateral cortex. Also a decrease in the minor monomeric (G1) form of AChE is response to ischemia was observed. Ischemia-induced AChE decrease was partially diminished by pre-treatment with dizocilpine (MK-801), a neuroprotective NMDA receptor blocker., This work has been supported, in part, from grants SAF99-0060 from CICYT, BFI2001-1565 from MCyT; G03/167 from MSCO; GC-02-019 and PAI-02-031 from JCCM and from Fundación Campollano-BSCH to V.C., BFI2001-1058 from MCyT and GC02-029 JCCM to C.G.-G. J.S.-V. was supported by a grant from the MCyT of Spain (Ramón y Cajal Program).

The identification of biochemical markers of Alzheimer's disease (AD) may help in the diagnosis of the disease. Previous studies have shown that Abeta(1-42) is decreased, and tau and phospho-tau are increased in AD cerebrospinal fluid (CSF). Our own studies have identified glycosylated isoforms of acetylcholinesterase (Glyc-AChE) and butyrylcholinesterase (Glyc-BuChE) that are increased in AD CSF. Glyc-AChE is increased in APP (SW) Tg2576 transgenic mice prior to amyloid plaque deposition, which suggests that Glyc-AChE may be an early marker of AD. The aim of this study was to determine whether Glyc-AChE or Glyc-BuChE is increased in CSF at early stages of AD and to compare the levels of these markers with those of Abeta(1-42), tau and phospho-tau. Lumbar CSF was obtained ante mortem from 106 non-AD patients, including 15 patients with mild cognitive impairment (MCI), and 102 patients with probable AD. Glyc-AChE, tau and phospho-tau were significantly increased in the CSF of AD patients compared to non-neurological disease (NND) controls. Abeta(1-42) was lower in the AD patients than in NND controls. A positive correlation was found between the levels of Glyc-AChE or Glyc-BuChE and disease duration. However, there was no clear correlation between the levels of tau, phospho-tau or Abeta(1-42) and disease duration. The results suggest that Glyc-AChE and Glyc-BuChE are unlikely to be early markers of AD, although they may have value as markers of disease progression.

Reelin is an essential glycoprotein for correct cytoarchitectonic organization during CNS development. Its function in the adult brain is far less well understood, but altered brain and blood reelin levels have been reported in some psychiatric disorders, and the possibility has been considered of an involvement of the reelin signaling pathway in neurodegeneration. Here we report, for the first time, the presence of detectable levels of reelin in rat and human cerebrospinal fluid (CSF) and show evidence for the involvement of a 180-kDa reelin fragment in two neurodegenerative disorders. This fragment was analyzed by Western blotting in CSF samples from 13 healthy control individuals and 14 frontotemporal dementia (FTD) and 20 Alzheimer's disease (AD) patients. Increased CSF 180-kDa reelin was found in FTD (161.7 ± 6.7 arbitrary units; a.u.) and AD (151.4 ± 3.8 a.u.) compared with control individuals (141.4 ± 1.2 a.u., P < 0.05). Our results strongly suggest the involvement of reelin signaling in neurodegenerative pathologies. © 2003 Wiley-Liss, Inc.

Previous studies have shown that an abnormal salt-soluble form of G1 acetylcholinesterase (AChE) is increased in the Alzheimer's disease (AD) brain. The aim of the present study was to examine changes in AChE activity in an in vivo model of β-amyloid peptide (Aβ) administration. Rats received intracerebroventricular injections of Aβ25-35 (20 μg/day for seven days). Levels of AChE were measured in cerebral cortex and cerebrospinal fluid (CSF) after two months. Aβ25-35 administration did not alter total AChE activity in the cerebral cortex or CSF. However, analysis of salt-extractable AChE isoforms revealed an increase in the proportion of G1 in both cortex and CSF, similar to that previously observed in AD patients. The results support the view that changes in AChE isoform pattern in the AD brain are a direct consequence of Aβ accumulation. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer’s disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Ab deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Ab plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble humansequence Ab was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer’s disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that

control brains, JNKs levels were assessed by western blots and Ab42 was evaluated by ELISA. In 8 AD and 9 control brains JNKs immunostainings were also carried out. CSF analysis included Ab42, tau and ptau by ELISA and JNK3 by immunoblot in 30 AD patients and 26 neurological disease controls. AD patients were followed from two to three years using MMSE scores. Results: Biochemical and histological results revealed significant increased JNK3 and phosphorylated JNK levels in AD brains and JNK3 levels correlated with Ab42 levels. Confocal microscopy showed that JNK3 was associated with Ab in senile plaques. CSF JNK3 levels were significantly enhanced in AD patients and were statistically correlated with the rate of cognitive decline. A mild enhancement of CSF JNK3 level was linked to a more accentuated slope of cognitive impairment.Conclusions:All together, brain and CSF JNK3 levels are increased in AD and CSF levels could reflect the rate of cognitive in AD patients.