Increased Active OMI/HTRA2 Serine Protease Displays a Positive Correlation with Cholinergic Alterations in the Alzheimer's Disease Brain.

OMI/HTRA2 (high-temperature requirement serine protease A2) is a mitochondrial serine protease involved in several cellular processes, including autophagy, chaperone activity, and apoptosis. Few studies on the role of OMI/HTRA2 in Alzheimer's disease (AD) are available, but none on its relationship with the cholinergic system and neurotrophic factors as well as other AD-related proteins. In this study, immunohistochemical analyses revealed that AD patients had a higher cytosolic distribution of OMI/HTRA2 protein compared to controls. Quantitative analyses on brain extracts indicated a significant increase in the active form of OMI/HTRA2 in the AD brain. Activated OMI/HTRA2 protein positively correlated with stress-associated read-through acetylcholinesterase activity. In addition, α7 nicotinic acetylcholine receptor gene expression, a receptor also known to be localized on the outer membrane of mitochondria, showed a strong correlation with OMI/HTRA2 gene expression in three different brain regions. Interestingly, the activated OMI/HTRA2 levels also correlated with the activity of the acetylcholine-biosynthesizing enzyme, choline acetyltransferase (ChAT); with levels of the neurotrophic factors, NGF and BDNF; with levels of the soluble fragments of amyloid precursor protein (APP); and with gene expression of the microtubule-associated protein tau in the examined brain regions. Overall, the results demonstrate increased levels of the mitochondrial serine protease OMI/HTRA2, and a coherent pattern of association between the activated form of OMI/HTRA2 and several key proteins involved in AD pathology. In this paper, we propose a new hypothetical model to highlight the importance and needs of further investigation on the role of OMI/HTRA2 in the mitochondrial function and AD.

Soluble Aß42 Acts as Allosteric Activator of the Core Cholinergic Enzyme Choline Acetyltransferase.

Two major questions in the field of Alzheimer-type dementia remain elusive. One is the native function of amyloid-ß (Aß) peptides and the other is an early deficit in the central cholinergic network. Nevertheless, recent evidence suggests that Aß peptides are involved in the regulation of acetylcholine (ACh) homeostasis either by allosteric activation of ACh-degrading cholinesterases or by inhibiting the high-affinity choline uptake transporter. In the current study, we report that Aß peptides, in particular Aß42, allosterically enhances the catalytic rate of the core-cholinergic enzyme choline acetyltransferase (ChAT), responsible for biosynthesis of ACh. Detailed in vitro enzyme kinetic analysis indicated that both soluble Aß40 and Aß42 enhanced the catalytic efficiency of ChAT by ∼21% and 26% at physiological concentration ranges found in human cerebrospinal fluid (CSF). Further analyses indicated that activation of ChAT by Aß was highly specific. Intriguingly, Aß42 exhibited an EC50 of activation potency at 10-fold lower concentrations compared to Aß40. The activation was persistent even in the presence of a physiological Aß 40/42 mixture ratio, expected in human CSF. In conclusion, we report for the first time that Aß42 peptide acts as allosteric enhancers of ACh-biosynthesizing enzyme ChAT. Together with two previous observations, this points to a complex molecular cross-talk between Aß and the enzymatic machinery involved in maintaining cellular, synaptic and extra-synaptic ACh homeostasis, warranting further investigation.

Perforin Promotes Amyloid Beta Internalisation in Neurons.

Studies on the mechanisms of neuronal amyloid-ß (Aß) internalisation are crucial for understanding the neuropathological progression of Alzheimer's disease (AD). We here investigated how extracellular Aß peptides are internalised and focused on three different pathways: (i) via endocytic mechanisms, (ii) via the receptor for advanced glycation end products (RAGE) and (iii) via the pore-forming protein perforin. Both Aß40 and Aß42 were internalised in retinoic acid differentiated neuroblastoma (RA-SH-SY5Y) cells. A higher concentration was required for Aß40 (250 nM) compared with Aß42 (100 nM). The internalised Aß40 showed a dot-like pattern of distribution whereas Aß42 accumulated in larger and distinct formations. By confocal microscopy, we showed that Aß40 and Aß42 co-localised with mitochondria, endoplasmic reticulum (ER) and lysosomes. Aß treatment of human primary cortical neurons (hPCN) confirmed our findings in RA-SH-SY5Y cells, but hPCN were less sensitive to Aß; therefore, a 20 (Aß40) and 50 (Aß42) times higher concentration was needed for inducing uptake. The blocking of endocytosis completely inhibited the internalisation of Aß peptides in RA-SH-SY5Y cells and hPCN, indicating that this is a major pathway by which Aß enters the cells. In addition, the internalisation of Aß42, but not Aß40, was reduced by 55 % by blocking RAGE. Finally, for the first time we showed that pore formation in cell membranes by perforin led to Aß internalisation in hPCN. Understanding how Aß is internalised sheds light on the pathological role of Aß and provides further ideas of inhibitory strategies for preventing Aß internalisation and the spreading of neurodegeneration in AD.

Pregnancy outcome for fetuses with increased nuchal translucency but normal karyotype.

OBJECTIVE: To investigate pregnancy outcome for fetuses with nuchal translucency (NT) ≥3.5 mm but normal karyotype in the Stockholm (Sweden) area. METHODS: A retrospective population-based cohort study. From 2006 to 2012, fetal NT was measured in 55123 singleton pregnancies. There were 341 pregnancies with NT thickness ≥3.5 mm; 139 had a normal karyotype, 164 had an abnormal karyotype and 38 were removed from the study. Pregnancy outcome was defined as adverse (termination of pregnancy [TOP], miscarriage [MC], intrauterine fetal death [IUFD], or delivery of a child with structural defects or genetic disorders), or favourable (delivery of a child without any structural defects or genetic disorders diagnosed before discharge). RESULTS: Of the 139 high NT pregnancies with normal karyotype, 110 (79.2%) resulted in live births, one (0.7%) IUFD, 23 (16.5%) TOP and five (3.6%) MC. The risk of an adverse pregnancy outcome increased with increasing NT. Structural fetal defects were found in 28 (19.5%) of pregnancies undergoing second trimester ultrasound screening, of which seven resulted in live births and 21 were terminated. The most common structural defect was cardiac defects. CONCLUSIONS: Adverse pregnancy outcome increased with increasing NT, even with normal karyotype, however, the prognosis is good if the second trimester ultrasound screening is normal.

High apolipoprotein E in cerebrospinal fluid of patients with Lewy body disorders is associated with dementia.

Apolipoprotein E ε4 allele (APOE ε4) increases the apolipoprotein E (apoE) protein levels in Alzheimer's disease (AD) cerebrospinal fluid (CSF). Thus, we hypothesized that apoE levels were also associated with the APOE genotype, and amyloid-ß (Aß)-associated clinical, functional, and imaging parameters in patients with Lewy body-associated disorders (LBD). Indeed, similar to AD, patients with LBD displayed high CSF apoE levels (greatest in patients with dementia with LBD), and this was linked to APOE ε4. High CSF apoE protein correlated positively with CSF soluble amyloid precursor protein, total tau, and cortical and striatal Pittsburgh compound B retention; and correlated negatively with CSF Aß42, cognitive tests scores, and glucose uptake ratio in the temporal and parietal cortices. APOE ε4-triggered accumulation of apoE in CSF is related to Aß-associated clinical and functional imaging parameters in LBD. Accordingly, therapeutic strategies aimed at reducing apoE levels in the brain should be explored not only in AD but also in LBD, particularly when accompanied with dementia.

Disruption of neocortical histone H3 homeostasis by soluble Aß: implications for Alzheimer's disease.

Amyloid-ß peptide (Aß) fragment misfolding may play a crucial role in the progression of Alzheimer's disease (AD) pathophysiology as well as epigenetic mechanisms at the DNA and histone level. We hypothesized that histone H3 homeostasis is disrupted in association with the appearance of soluble Aß at an early stage in AD progression. We identified, localized, and compared histone H3 modifications in multiple model systems (neural-like SH-SY5Y, primary neurons, Tg2576 mice, and AD neocortex), and narrowed our focus to investigate 3 key motifs associated with regulating transcriptional activation and inhibition: acetylated lysine 14, phosphorylated serine 10 and dimethylated lysine 9. Our results in vitro and in vivo indicate that multimeric soluble Aß may be a potent signaling molecule indirectly modulating the transcriptional activity of DNA by modulating histone H3 homeostasis. These findings reveal potential loci of transcriptional disruption relevant to AD. Identifying genes that undergo significant epigenetic alterations in response to Aß could aid in the understanding of the pathogenesis of AD, as well as suggesting possible new treatment strategies.

Transgenic mice as a model for Alzheimer's disease.

During the last few decades, numerous stable transgenic mouse strains have been developed in order to mimic a range of Alzheimer's disease (AD)-related pathologies. Although none of the models fully replicates the human disease, the models have been a key feature in translational research, providing significant insights into the pathophysiology of AD. They have also been widely used in the preclinical testing of potential therapies. The choice of transgenic mouse model, as well as the stage of Aß pathology, significantly contributes to the outcome of the studies. Therefore, it is important to combine studies in different transgenic mouse models and detailed in vitro experiments to obtain a complete understanding of the origin of the disease, the actual sequences of early pathological events as well as being able to evaluate the effects of new drugs in the treatment of AD.

Characterization of the brain ß-amyloid isoform pattern at different ages of Tg2576 mice.

BACKGROUND: Although genetic and biochemical studies have suggested a cardinal role for ß-amyloid (Aß) in Alzheimer's disease, the underlying mechanism(s) of how Aß induces neurodegeneration is still unclear. Our objective was to investigate the consequences of Aß, especially on tau phosphorylation at specific epitopes important for Alzheimer's disease. METHODS: We used cortices from Tg2576 mice at 7 days to 15 months of age. RESULTS: MALDI-TOF MS revealed an age-dependent shift in the Aß isoform pattern. Young animals displayed high cortical levels of the shorter Aß isoforms (Aß1-16 and Aß1-17) compared to 15-month-old Tg2576 mice which mainly expressed Aß1-40 and Aß1-42. The Aß1-42 showed an age-dependent increase, whereas total Aß1-40 levels remained constant. The highest levels of TBS-soluble Aß oligomers were found at 90 days of age. Brain Aß build-up did not affect the phosphorylation of tau at the epitopes investigated. CONCLUSIONS: This study provides new information about age-dependent Aß isoforms and oligomers as well as their effect on site-specific tau phosphorylation in this transgenic mouse model. Our observations suggest that the different human Aß isoforms do not directly cause increased tau phosphorylation and that the cognitive deficits seen in this mouse model are only related to the Aß overexpression.

Effect of huprine X on ß-amyloid, synaptophysin and α7 neuronal nicotinic acetylcholine receptors in the brain of 3xTg-AD and APPswe transgenic mice.

BACKGROUND: Several studies implicate acetylcholinesterase (AChE) in the pathogenesis of Alzheimer's disease (AD), raising the question of whether inhibitors of AChE also might act in a disease-modifying manner. Huprine X (HX), a reversible AChE inhibitor hybrid of tacrine and huperzine A, has shown to affect the amyloidogenic process in vitro. In this study, the aim was to investigate whether HX could affect the AD-related neuropathology in vivo in two mouse models. METHODS: Tg2576 (K670M/N671L) (APPswe) and 3xTg-AD (K670M/N671L, PS1M146V, tauP301L) mice were treated with HX (0.12 µmol/kg, i.p., 21 days) or saline at 6-7 months. Human ß-amyloid (Aß) was measured by ELISA, synaptophysin by Western blot and α7 neuronal nicotinic acetylcholine receptors (nAChRs) were analyzed by [(125)I]α-bungarotoxin autoradiography. RESULTS: Treatment with HX reduced insoluble Aß1-40 (about 40%) in the hippocampus of 3xTg-AD mice, while showing no effect in APPswe mice. Additionally, HX markedly increased cortical synaptophysin levels (about 140%) and decreased (about 30%) the levels of α7 nAChRs in the caudate nucleus of 3xTg-AD mice, while increasing (about 10%) hippocampal α7 nAChRs in APPswe mice. CONCLUSION: The two mouse models react differently to HX treatment, possibly due to their differences in brain neuropathology. The modulation of Aß and synaptophysin by HX in 3xTg-AD mice might be due to its suggested interaction with the peripheral anionic site on AChE, and/or via cholinergic mechanisms involving activation of cholinergic receptors. Our results provide further evidence that drugs targeting AChE affect some of the fundamental processes that contribute to neurodegeneration, but whether HX might act in a disease-modifying manner in AD patients remains to be proven.