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.

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.

Levels of ADAM10 are reduced in Alzheimer's disease CSF.

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, Aß42, 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.

COVID-19-associated encephalitis successfully treated with combination therapy.

BACKGROUND: Acute encephalitis can occur in different viral diseases due to infection of the brain or by an immune mechanism. Severe novel coronavirus disease 2019 (COVID-19) is associated with a major immune inflammatory response with cytokine upregulation including interleukin 6 (IL-6). We report a case presenting with acute encephalitis that was diagnosed as having severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection with hyperinflammatory systemic response and recovered after therapy with immunoglobulins and cytokine blockade. CASE REPORT: A 39-year-old-man was brought to the Emergency Department with drowsiness, mental disorientation, intermittent fever and headache. A brain magnetic resonance imaging showed extensive involvement of the brain including cortical and subcortical right frontal regions, right thalamus, bilateral temporal lobes and cerebral peduncles, with no leptomeningeal enhancement. Cerebrospinal fluid (CSF) showed a leukocyte count of 20/µL (90% lymphocytes), protein level of 198 mg/dL, and glucose of 48 mg/dL. SARS-CoV-2 was detected in nasopharyngeal swabs by reverse-transcriptase-PCR (RT-PCR) but it was negative in the CSF. Remarkable laboratory findings in blood tests included low lymphocyte count and elevated ferritin, IL-6 and D-dimer. He had a complicated clinical course requiring mechanical ventilation. Intravenous immunoglobulins and cytokine blockade with tocilizumab, an IL-6 receptor antagonist, were added considering acute demyelinating encephalomyelitis. The patient made a full recovery, suggesting that it could have been related to host inflammatory response. CONCLUSION: This case report indicates that COVID-19 may present as an encephalitis syndrome mimicking acute demyelinating encephalomyelitis that could be amenable to therapeutic modulation.

Oxidative stress in Alzheimer's disease and mild cognitive impairment with high sensitivity and specificity.

Some studies have determined that oxidative stress is a decisive factor in Alzheimer's disease (AD) and even suggested that it is present in the initial phase of mild cognitive impairment (MCI). The aim of our study was to investigate the process of oxidative stress by measuring the level of malondialdehyde (MDA), the specific activity of two peripheral antioxidant defenses (superoxide dismutase (SOD) and ceruloplasmin), and the level of copper in AD and MCI patients and compare those results with healthy subjects. The sample group consisted of 36 patients with AD, 18 patients with MCI, and 33 healthy aged subjects. Blood samples were obtained from each subject. A significantly higher copper level was found in patients with AD and MCI compared to the control group. The levels of MDA showed a similar trend and were higher in patients from the AD and MCI groups than in the control group. It was found that both studied parameters had positive correlation in the whole studied population (r = 0.340; p = 0.001). A stepwise logistic regression analysis was used to identify an optimal combination of these biomarkers. The optimal biomarker combinations were MDA and SOD with area under the curve of 0.803 (0.691-0.915, CI 95%, p < 0.001) for the diagnosis of AD. The optimal cutpoint yielded 88.0% Sensitivity and 70.0% Specificity. The biomarker combinations predicted AD and were markedly superior to individual biomarkers. Our findings support the hypothesis that oxidative stress might represent a sign of AD pathology and could be an early event in the progression of MCI to AD.

CSF Presenilin-1 complexes are increased in Alzheimer's disease.

BACKGROUND: Presenilin-1 (PS1) is the active component of the amyloid precursor protein cleaving γ-secretase complex. PS1 protein is a transmembrane protein containing multiple hydrophobic regions which presence in cerebrospinal fluid (CSF) has not been measured to date. This study assesses whether PS1 and other components of the γ-secretase complex are present in CSF. RESULTS: Here, we show that PS1 is present in ventricular post-mortem and lumbar ante-mortem CSF, and plasma as 100-150-kDa hetero-complexes containing both the N- and C-terminal fragments (NTF and CTF) of the protein. Immunoprecipitation and immunoblotting with different antibodies confirmed the identity of the PS1 species. The γ-secretase components, APH-1 (anterior pharynx-defective 1) and PEN-2 (presenilin enhancer 2), as well as presenilin-2 (PS2) fragments, co-exist within these CSF complexes, while nicastrin is not detected. These CSF-PS1 complexes differ from active γ-secretase membrane-complexes, and may represent nonspecific aggregation of the PS1 protein. Levels of PS1 complexes are increased in CSF samples from autopsy-confirmed Alzheimer's disease (AD) cases and were found to be more stable than complexes in CSF from control subjects. Despite similar levels of total PS1 in CSF from probable AD patients and cognitively normal subjects, an increased proportion of highly stable PS1 complexes were observed in AD CSF. CONCLUSIONS: Our data suggest that fragments of the PS1 protein present in CSF as complexes may be useful as a biomarker for AD.

Altered levels of acetylcholinesterase in Alzheimer plasma.

BACKGROUND: Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem. PRINCIPAL FINDINGS: Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were approximately 20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G(1)+G(2) forms and not G(4) tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer's disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G(1)+G(2) forms, the subset of AChE species which are increased in Alzheimer's brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer's plasma, attributed in part to AChE-T subunits common in brain and CSF. CONCLUSION: Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.

Relationship between the efficacy of rivastigmine and apolipoprotein E (epsilon4) in patients with mild to moderately severe Alzheimer disease.

Alzheimer disease is the most common form of dementia in Western countries and the leading cause of disability in the over-65 population. Apolipoprotein E (APOE) is a multifunctional protein implied in lipid metabolism and neurobiology. Polymorphisms of the APOE gene have been associated with a variety of medical disorders, from arteriosclerosis to AD. A high frequency of the APOE epsilon4 allele has been found in patients with AD and they seem to have a higher risk of developing the disease. Various authors have suggested a possible relationship between the efficacy of cholinesterase inhibitors and the presence of the APOE epsilon4 allele. The purpose of the present study was to compare prospectively the efficacy of rivastigmine in patients with mild to moderately severe AD presenting different polymorphisms of the APOE gene on chromosome 19 and to determine if there was a difference in the response to rivastigmine treatment in AD patients with the APOE epsilon4 allele (heterozygous or homozygous) versus patients who had other forms of APOE, such as epsilon2 and epsilon3. This was an open-label, nonrandomized, multicenter study in patients over 50 years of age diagnosed with mild to moderately severe AD. The results of the analysis of this study indicate that the presence of at least one APOE epsilon4 allele does not determine a difference in the response to treatment with rivastigmine. The data indicate that knowledge of the patient's genotype is not necessary for treatment with rivastigmine. It would be interesting in the future to analyze the interaction between these 2 factors using other available anticholinesterase drugs.