BACE2 beyond ß-processing of APP, its neuroprotective role in cerebrovascular endothelium.

Several proteases are involved in the proteolytic processing of the amyloid precursor protein (APP) generating the amyloidogenic Aß peptide, which can act as the triggering pathological effector of Alzheimer's disease (AD). Among these proteases, the ß-site amyloid precursor protein cleaving enzyme 2 (BACE2) is of particular interest because it was first proposed as an alternative ß-secretase to its homolog BACE1; however, accumulating evidence suggests that BACE2 acts as a non-amyloidogenic α-secretase and exerts neuroprotective effects. In this issue of J Neurochem, Katusic et al. present an interesting article reporting that BACE2 plays a role in preservation of cerebral vascular endothelial nitric oxide synthase (eNOS) function, thus exerting protective functions. Their data support that the process is mediated by the large soluble non-amyloidogenic APP fragment sAPPα through the γ-aminobutyric acid type B receptor 1, which enhances the expression of a major transcription factor for eNOS gene expression in endothelial cells, the Krüppel-like factor 2. These protective functions of BACE2 contrast with the pathogenic role of BACE1 as a key player in the AD amyloidogenic pathway. Indeed, many efforts have been invested in BACE1 inhibitors as potential disease modifiers for AD. Unfortunately, the results in clinical trials have been disappointing. In this scenario, a better understanding of the functions of BACE2, as well as the selectivity of BACE1 inhibitors with respect to other ß-secretases (mainly BACE2), is crucial for the development of new therapeutic agents. Furthermore, specific cellular targeting should also be considered to improve such therapies due to the diverse balance of secretases targeting APP and the complex cross-talk between them and the generated APP fragments.

No evidence for cognitive decline or neurodegeneration in strain-matched Grin3a knockout mice.

N-methyl-D-aspartate (NMDA) receptor (NMDAR) dysregulation is thought to contribute to impaired cognition and neurodegeneration in a variety of brain disorders. In a recent article, Zhong et al. proposed that deficiency of the NMDAR subunit GluN3A may be a primary pathogenic factor in sporadic Alzheimer´s disease (AD) based on evidence for degenerative excitotoxicity and cognitive impairment in aging mice lacking GluN3A. Because the result appeared to be at odds with earlier work where genetic GluN3A deletion enhanced learning in younger mice, we have now compared wild-type and GluN3A knockout mice at later life stages using a congenic mouse strain. Rather than age-dependent cognitive decline or neurodegeneration, we find that the enhanced performance of young adult GluN3A knockouts in memory tasks persists during aging. In sum, our analysis does not support the hypothesis that GluN3A loss underlies cognitive impairment in AD..

Validity of CSF alpha-synuclein to predict psychosis in prodromal Alzheimer's disease.

Background: Alzheimer's disease (AD) accompanied by psychotic symptoms (PS) has a poor prognosis and may be associated with imbalances in key neural proteins such as alpha-synuclein (AS). Aim: The aim of the study was to evaluate the diagnostic validity of AS levels in the cerebrospinal fluid (CSF) as a predictor of the emergence of PS in patients with prodromal AD. Materials and methods: Patients with mild cognitive impairment were recruited between 2010 and 2018. Core AD biomarkers and AS levels were measured in CSF obtained during the prodromal phase of the illness. All patients who met the NIA-AA 2018 criteria for AD biomarkers received treatment with anticholinesterasic drugs. Follow-up evaluations were conducted to assess patients for the presence of psychosis using current criteria; the use of neuroleptic drugs was required for inclusion in the psychosis group. Several comparisons were made, taking into account the timing of the emergence of PS. Results: A total of 130 patients with prodromal AD were included in this study. Of these, 50 (38.4%) met the criteria for PS within an 8-year follow-up period. AS was found to be a valuable CSF biomarker to differentiate between the psychotic and non-psychotic groups in every comparison made, depending on the onset of PS. Using an AS level of 1,257 pg/mL as the cutoff, this predictor achieved at least 80% sensitivity. Conclusion: To our knowledge, this study represents the first time that a CSF biomarker has shown diagnostic validity for prediction of the emergence of PS in patients with prodromal AD.

ADAM10 Gene Variants in AD Patients and Their Relationship to CSF Protein Levels.

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.

Transient Changes in the Plasma of Astrocytic and Neuronal Injury Biomarkers in COVID-19 Patients without Neurological Syndromes.

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.

Presenilin 1 Modulates Acetylcholinesterase Trafficking and Maturation.

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.

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.

Serum angiotensin-converting enzyme 2 as a potential biomarker for SARS-CoV-2 infection and vaccine efficacy.

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.

Altered Balance of Reelin Proteolytic Fragments in the Cerebrospinal Fluid of Alzheimer's Disease Patients.

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 Aß42-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.

The apolipoprotein receptor LRP3 compromises APP levels.

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 ß-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 Aß42 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 (sAPPß) pathways, as well as Aß 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.

Relation between Alpha-Synuclein and Core CSF Biomarkers of Alzheimer's Disease.

Background: Alzheimer's disease (AD) is characterized by the presence of ß-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 < 0.05) and p-tau181p and α-syn (ρ = 0.571; p value < 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.

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.

Plasma ACE2 species are differentially altered in COVID-19 patients.

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.

Tau phosphorylation by glycogen synthase kinase 3ß modulates enzyme acetylcholinesterase expression.

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-3ß (GSK3ß) 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 GSK3ß 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 GSK3ß 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.

α-Secretase nonsense mutation (ADAM10 Tyr167*) in familial Alzheimer's disease.

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 sAPPß 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 Aß42, 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 sAPPß levels remained unchanged. Interestingly, sporadic AD cases had a similar decrease in CSF ADAM10 levels to that of mutants, though their sAPPα and sAPPß levels resembled those of controls. Therefore, a decreased sAPPα/sAPPß 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.

Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer's disease.

BACKGROUND: The amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes alternative proteolytic processing. Its processing through the amyloidogenic pathway originates a large sAPPß ectodomain fragment and the ß-amyloid peptide, while non-amyloidogenic processing generates sAPPα and shorter non-fibrillar fragments. Hence, measuring sAPPα and sAPPß 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 sAPPß fragments and their membrane-tethered C-terminal fragments CTFα and CTFß. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether Aß42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPPα and sAPPß 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 sAPPß protein levels remained unchanged, as did those of CTFα and CTFß. We studied the glycosylation of the brain sAPPα and sAPPß 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 sAPPß 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 sAPPß 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 sAPPß from CHO-PS70 cells were also altered in cells treated with the Aß peptide. CONCLUSION: Our analysis of the lectin binding to sAPPα and sAPPß 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.

Elevated Plasma Reelin Levels in Children With Autism.

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.

Characterization of Cerebrospinal Fluid BACE1 Species.

The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the main brain ß-secretase responsible for the amyloidogenic processing of the amyloid precursor protein (APP). Previous studies have suggested that cerebrospinal fluid (CSF) ß-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 Aß42, total tau, and P-tau CSF biomarker levels. We found that ß-secretase activity was higher in the CSF of AD subjects than in that of the controls. We found that the majority of the ß-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.

Pre-analytical stability of novel cerebrospinal fluid biomarkers.

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.

Measurement of CSF α-synuclein improves early differential diagnosis of mild cognitive impairment due to Alzheimer's disease.

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. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.