γ-Secretase: a horseshoe structure brings good luck.

The intramembrane protease γ-secretase is a key player in signaling and Alzheimer's disease, but its structural features have remained obscure. A structure reported recently reveals a horseshoe-shaped arrangement of 19 transmembrane helices and an extracellular domain positioned for substrate recognition. This advance bodes well for a finer resolution before long.

Dynamic reversibility of α-synuclein serine-129 phosphorylation is impaired in synucleinopathy models.

α-Synuclein phosphorylation at serine-129 (pS129) is a widely used surrogate marker of pathology in Parkinson's disease and other synucleinopathies. However, we recently demonstrated that phosphorylation of S129 is also a physiological activator of synaptic transmission. In a feed-forward fashion, neuronal activity triggers reversible pS129. Here, we show that Parkinson's disease-linked missense mutations in SNCA impact activity-dependent pS129. Under basal conditions, cytosol-enriched A30P, H50Q, and G51D mutant forms of α-synuclein exhibit reduced pS129 levels in rat primary cortical neurons. A53T pS129 levels are similar to wild-type, and E46K pS129 levels are higher. A30P and E46K mutants show impaired reversibility of pS129 after stimulation. For the engineered profoundly membrane-associated α-synuclein mutant "3K" (E35K + E46K + E61K), de-phosphorylation was virtually absent after blocking stimulation, implying that reversible pS129 is severely compromised. Importantly, pS129 excess resulting from proteasome inhibition is also associated with reduced reversibility by neuronal inhibition, kinase inhibition, or phosphatase activation. Our findings suggest that perturbed pS129 dynamics are probably a shared characteristic of pathology-associated α-synuclein, with possible implications for synucleinopathy treatment and diagnosis.

Giving Alzheimer's the old one-two.

A dual goal for treating Alzheimer's disease (AD) is to decrease deposition of neurotoxic amyloid beta-peptide in the brain and to boost repair of damaged neurons. Donmez et al. (2010) now show that SIRT1 may mediate both processes by deacetylating the transcription factor retinoic acid receptor beta, a potential new therapeutic target for AD.

The Parkinson-Associated Toxin Paraquat Shifts Physiological α-Synuclein Tetramers toward Monomers That Can Be Calpain-Truncated and Form Oligomers.

Abnormal aggregation of α-synuclein (αS) is thought to initiate neuronal dysfunction and death in Parkinson disease (PD). In addition to higher-molecular-weight, oligomeric, and polymeric forms of αS associated with neurotoxicity and disease, recent findings indicate the occurrence of physiological tetrameric assemblies in healthy neurons in culture and in brain. Herein, the PD-associated neurotoxin paraquat reduced physiological tetramers and led to calpain-truncated monomers and an approximately 70-kDa apparent oligomer different in size from physiological αS multimers. These truncated and oligomeric forms could also be generated by calpain cleavage of pure, recombinant human αS in vitro. Moreover, they were detected in the brains of tetramer-abrogating, E46K-amplified (3K) mice that model PD. These results indicate that paraquat triggers membrane damage and aberrant calpain activity that can induce a pathologic shift of tetramers toward an excess of full-length and truncated monomers, the accumulation of αS oligomers, and insoluble cytoplasmic αS puncta. The findings suggest that an environmental precipitant of PD can alter αS tetramer/monomer equilibrium, as already shown for several genetically caused forms of PD.

Activation of ß2-adrenergic receptors prevents AD-type synaptotoxicity via epigenetic mechanisms.

We previously reported that prolonged exposure to an enriched environment (EE) enhances hippocampal synaptic plasticity, with one of the significant mechanistic pathways being activation of ß2-adrenergic receptor (ß2-AR) signaling, thereby mitigating the synaptotoxic effects of soluble oligomers of amyloid ß-protein (oAß). However, the detailed mechanism remained elusive. In this work, we recorded field excitatory postsynaptic potentials (fEPSP) in the CA1 region of mouse hippocampal slices treated with or without toxic Aß-species. We found that pharmacological activation of ß2-AR, but not ß1-AR, selectively mimicked the effects of EE in enhancing LTP and preventing oAß-induced synaptic dysfunction. Mechanistic analyses showed that certain histone deacetylase (HDAC) inhibitors mimicked the benefits of EE, but this was not seen in ß2-AR knockout mice, suggesting that activating ß2-AR prevents oAß-mediated synaptic dysfunction via changes in histone acetylation. EE or activation of ß-ARs each decreased HDAC2, whereas Aß oligomers increased HDAC2 levels in the hippocampus. Further, oAß-induced inflammatory effects and neurite degeneration were prevented by either ß2-AR agonists or certain specific HDAC inhibitors. These preclinical results suggest that activation of ß2-AR is a novel potential therapeutic strategy to mitigate oAß-mediated features of AD.

Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Uncovering elevated tau TPP motif phosphorylation in the brain of Alzheimer's disease patients.

INTRODUCTION: A wide array of post-translational modifications of the tau protein occurs in Alzheimer's disease (AD) and they are critical to pathogenesis and biomarker development. Several promising tau markers, pT181, pT217, and pT231, rely on increased phosphorylation within a common molecular motif threonine-proline-proline (TPP). METHODS: We validated new and existing antibodies against pT217, pT231, pT175, and pT181, then combined immunohistochemistry (IHC) and immunoassays (ELISA) to broadly examine the phosphorylation of the tau TPP motif in AD brains. RESULTS: The tau burden, as examined by IHC and ELISA, correlates to Braak stages across all TPP sites. Moreover, we observed regional variability across four TPP motif phosphorylation sites in multiple brains of sporadic AD patients. DISCUSSION: We conclude that there is an elevation of TPP tau phosphorylation in AD brains as disease advances. The regional variability of pTPP tau suggests that examining different phosphorylation sites is essential for a comprehensive assessment of tau pathology.

A calcium-sensitive antibody isolates soluble amyloid-ß aggregates and fibrils from Alzheimer's disease brain.

Aqueously soluble oligomers of amyloid-ß peptide may be the principal neurotoxic forms of amyloid-ß in Alzheimer's disease, initiating downstream events that include tau hyperphosphorylation, neuritic/synaptic injury, microgliosis and neuron loss. Synthetic oligomeric amyloid-ß has been studied extensively, but little is known about the biochemistry of natural oligomeric amyloid-ß in human brain, even though it is more potent than simple synthetic peptides and comprises truncated and modified amyloid-ß monomers. We hypothesized that monoclonal antibodies specific to neurotoxic oligomeric amyloid-ß could be used to isolate it for further study. Here we report a unique human monoclonal antibody (B24) raised against synthetic oligomeric amyloid-ß that potently prevents Alzheimer's disease brain oligomeric amyloid-ß-induced impairment of hippocampal long-term potentiation. B24 binds natural and synthetic oligomeric amyloid-ß and a subset of amyloid plaques, but only in the presence of Ca2+. The amyloid-ß N terminus is required for B24 binding. Hydroxyapatite chromatography revealed that natural oligomeric amyloid-ß is highly avid for Ca2+. We took advantage of the reversible Ca2+-dependence of B24 binding to perform non-denaturing immunoaffinity isolation of oligomeric amyloid-ß from Alzheimer's disease brain-soluble extracts. Unexpectedly, the immunopurified material contained amyloid fibrils visualized by electron microscopy and amenable to further structural characterization. B24-purified human oligomeric amyloid-ß inhibited mouse hippocampal long-term potentiation. These findings identify a calcium-dependent method for purifying bioactive brain oligomeric amyloid-ß, at least some of which appears fibrillar.

Identification of the Aß37/42 peptide ratio in CSF as an improved Aß biomarker for Alzheimer's disease.

INTRODUCTION: Identifying CSF-based biomarkers for the ß-amyloidosis that initiates Alzheimer's disease (AD) could provide inexpensive and dynamic tests to distinguish AD from normal aging and predict future cognitive decline. METHODS: We developed immunoassays specifically detecting all C-terminal variants of secreted amyloid ß-protein and identified a novel biomarker, the Aß 37/42 ratio, that outperforms the canonical Aß42/40 ratio as a means to evaluate the γ-secretase activity and brain Aß accumulation. RESULTS: We show that Aß 37/42 can distinguish physiological and pathological status in (1) presenilin-1 mutant vs wild-type cultured cells, (2) AD vs control brain tissue, and (3) AD versus cognitively normal (CN) subjects in CSF, where 37/42 (AUC 0.9622) outperformed 42/40 (AUC 0.8651) in distinguishing CN from AD. DISCUSSION: We conclude that the Aß 37/42 ratio sensitively detects presenilin/γ-secretase dysfunction and better distinguishes CN from AD than Aß42/40 in CSF. Measuring this novel ratio alongside promising phospho-tau analytes may provide highly discriminatory fluid biomarkers for AD.

Plasma NT1-tau and Aß42 correlate with age and cognitive function in two large Down syndrome cohorts.

INTRODUCTION: People with Down syndrome (DS) often develop Alzheimer's disease (AD). Here, we asked whether ultrasensitive plasma immunoassays for a tau N-terminal fragment (NT1-tau) and Aß isoforms predict cognitive impairment. METHODS: Plasma NT1-tau, Aß37 , Aß40 , and Aß42 levels were measured in a longitudinal discovery cohort (N = 85 participants, 220 samples) and a cross-sectional validation cohort (N = 239). We developed linear models and predicted values in the validation cohort. RESULTS: Discovery cohort linear mixed models for NT1-tau, Aß42 , and Aß37:42 were significant for age; there was no main effect of time. In cross-sectional models, NT1-tau increased and Aß42 decreased with age. NT1-tau predicted cognitive and functional scores. The discovery cohort linear model for NT1-tau predicted levels in the validation cohort. DISCUSSION: NT1-tau correlates with age and worse cognition in DS. Further validation of NT1-tau and other plasma biomarkers of AD neuropathology in DS cohorts is important for clinical utility.

Hydrophilic loop 1 of Presenilin-1 and the APP GxxxG transmembrane motif regulate γ-secretase function in generating Alzheimer-causing Aß peptides.

γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aß) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aß products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aß peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aß peptides.

Aß oligomers from human brain impair mossy fiber LTP in CA3 of hippocampus, but activating cAMP-PKA and cGMP-PKG prevents this.

Early cognitive impairment in Alzheimer's disease may result in part from synaptic dysfunction caused by the accumulation oligomeric assemblies of amyloid ß-protein (Aß). Changes in hippocampal function seem critical for cognitive impairment in early Alzheimer's disease (AD). Diffusible oligomers of Aß (oAß) have been shown to block canonical long-term potentiation (LTP) in the CA1 area of hippocampus, but whether there is also a direct effect of oAß on synaptic transmission and plasticity at synapses between mossy fibers (axons) from the dentate gyrus granule cells and CA3 pyramidal neurons (mf-CA3 synapses) is unknown. Studies in APP transgenic mice have suggested an age-dependent impairment of mossy fiber LTP. Here we report that although endogenous AD brain-derived soluble oAß had no effect on mossy-fiber basal transmission, it strongly impaired paired-pulse facilitation in the mossy fiber pathway and presynaptic mossy fiber LTP (mf-LTP). Selective activation of both ß1 and ß2 adrenergic receptors and their downstream cAMP/PKA signaling pathway prevented oAß-mediated inhibition of mf-LTP. Unexpectedly, activation of the cGMP/PKG signaling pathway also prevented oAß-impaired mf-LTP. Our results reveal certain specific pharmacological targets to ameliorate human oAß-mediated impairment at the mf-CA3 synapse.

Generation and application of semi-synthetic p-Tau181 calibrator for immunoassay calibration.

Evidence suggests that plasma levels of tau protein phosphorylated at specific residues such as p-T181, p-T217, and p-T231 can be used as biomarkers for Alzheimer's disease (AD) diagnosis and prognosis. Accurate tools to calibrate immunoassays (calibrators) to precisely detect phosphorylated residues on tau protein will provide important gains in reliability and specificity. This study sought to establish a method to generate those accurate calibrators. We generated a semi-synthetic (chimeric) p-Tau181 calibrator by coupling a recombinant tau fragment (residues 1-174) with a synthetic peptide containing a single phosphorylated residue (p-T181) via thioester bond formation. The generation of a semi-synthetic protein containing both the N-terminal region of tau and the pT181 epitope was demonstrated by mobility shift assays using CBB staining and immunoblotting with N-terminal and pT181-specific antibodies. p-Tau 181 assays performed with the novel calibrator on multiple platforms revealed LLoQs as low as 0.14 pg/ml. Our facile and inexpensive method generates a semi-synthetic tau pT181 calibrator suitable for different immunoassay platforms. The same method can easily be adapted to other AD-relevant phospho-epitopes such as pT217 and pT231.

A Stearoyl-Coenzyme A Desaturase Inhibitor Prevents Multiple Parkinson Disease Phenotypes in α-Synuclein Mice.

OBJECTIVE: Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease-modifying treatments are approved. A key therapeutic target in PD is α-synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl-coenzyme A desaturase (SCD) inhibitor ("5b") that prevented αS-positive vesicular inclusions and cytotoxicity in cultured human neurons. METHODS: Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild-type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD-like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD-related lipid changes were quantified in 5b- versus placebo-treated mice. Outcomes were compared to effects of crossing 3K to SCD1-/- mice. RESULTS: 5b treatment reduced αS hyperphosphorylation in E46K-expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD-like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K-resistant lipid-rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation. INTERPRETATION: Prolonged reduction of brain SCD activity prevented PD-like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α-synucleinopathies. ANN NEUROL 2021;89:74-90.

An ultra-sensitive immunoassay detects and quantifies soluble Aß oligomers in human plasma.

INTRODUCTION: Evidence strongly suggests that soluble oligomers of amyloid beta protein (oAß) help initiate the pathogenic cascade of Alzheimer's disease (AD). To date, there have been no validated assays specific for detecting and quantifying oAß in human blood. METHODS: We developed an ultrasensitive oAß immunoassay using a novel capture antibody (71A1) with N-terminal antibody 3D6 for detection that specifically quantifies soluble oAß in the human brain, cerebrospinal fluid (CSF), and plasma. RESULTS: Two new antibodies (71A1; 1G5) are oAß-selective, label Aß plaques in non-fixed AD brain sections, and potently neutralize the synaptotoxicity of AD brain-derived oAß. The 71A1/3D6 assay showed excellent dilution linearity in CSF and plasma without matrix effects, good spike recovery, and specific immunodepletion. DISCUSSION: We have created a sensitive, high throughput, and inexpensive method to quantify synaptotoxic oAß in human plasma for analyzing large cohorts of aged and AD subjects to assess the dynamics of this key pathogenic species and response to therapy.

A critical appraisal of the pathogenic protein spread hypothesis of neurodegeneration.

There has been an explosion in the number of papers discussing the hypothesis of 'pathogenic spread' in neurodegenerative disease - the idea that abnormal forms of disease-associated proteins, such as tau or α-synuclein, physically move from neuron to neuron to induce disease progression. However, whether inter-neuronal spread of protein aggregates actually occurs in humans and, if so, whether it causes symptom onset remain uncertain. Even if pathogenic spread is proven in humans, it is unclear how much this would alter the specific therapeutic approaches that are in development. A critical appraisal of this increasingly popular hypothesis thus seems both important and timely.

Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity.

BACKGROUND: Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation. OBJECTIVES: We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism. METHODS: Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes. RESULTS: APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients. CONCLUSIONS: Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.

A mechanistic hypothesis for the impairment of synaptic plasticity by soluble Aß oligomers from Alzheimer's brain.

It is increasingly accepted that early cognitive impairment in Alzheimer's disease results in considerable part from synaptic dysfunction caused by the accumulation of a range of oligomeric assemblies of amyloid ß-protein (Aß). Most studies have used synthetic Aß peptides to explore the mechanisms of memory deficits in rodent models, but recent work suggests that Aß assemblies isolated from human (AD) brain tissue are far more potent and disease-relevant. Although reductionist experiments show Aß oligomers to impair synaptic plasticity and neuronal viability, the responsible mechanisms are only partly understood. Glutamatergic receptors, GABAergic receptors, nicotinic receptors, insulin receptors, the cellular prion protein, inflammatory mediators, and diverse signaling pathways have all been suggested. Studies using AD brain-derived soluble Aß oligomers suggest that only certain bioactive forms (principally small, diffusible oligomers) can disrupt synaptic plasticity, including by binding to plasma membranes and changing excitatory-inhibitory balance, perturbing mGluR, PrP, and other neuronal surface proteins, down-regulating glutamate transporters, causing glutamate spillover, and activating extrasynaptic GluN2B-containing NMDA receptors. We synthesize these emerging data into a mechanistic hypothesis for synaptic failure in Alzheimer's disease that can be modified as new knowledge is added and specific therapeutics are developed.

Environmental enrichment prevents Aß oligomer-induced synaptic dysfunction through mirna-132 and hdac3 signaling pathways.

As the most common cause of progressive cognitive decline in humans, Alzheimer's disease (AD) has been intensively studied, but the mechanisms underlying its profound synaptic dysfunction remain unclear. Here we confirm that exposing wild-type mice to an enriched environment (EE) facilitates signaling in the hippocampus that promotes long-term potentiation (LTP). Exposing the hippocampus of mice kept in standard housing to soluble Aß oligomers impairs LTP, but EE can fully prevent this. Mechanistically, the key molecular features of the EE benefit are an upregulation of miRNA-132 and an inhibition of histone deacetylase (HDAC) signaling. Specifically, soluble Aß oligomers decreased miR-132 expression and increased HDAC3 levels in cultured primary neurons. Further, we provide evidence that HDAC3 is a direct target of miR-132. Overexpressing miR-132 or injecting an HDAC3 inhibitor into mice in standard housing mimics the benefits of EE in enhancing hippocampal LTP and preventing hippocampal impairment by Aß oligomers in vivo. We conclude that EE enhances hippocampal synaptic plasticity by upregulating miRNA-132 and reducing HDAC3 signaling in a way that counteracts the synaptotoxicity of human Aß oligomers. Our findings provide a rationale for prolonged exposure to cognitive novelty and/or epigenetic modulation to lessen the progressive effects of Aß accumulation during human brain aging.