FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

Quantitative comparison of presenilin protein expression reveals greater activity of PS2-γ-secretase.

γ-secretase processing of amyloid precursor protein (APP) has long been of interest in the pathological progression of Alzheimer's disease (AD) due to its role in the generation of amyloid-ß. The catalytic component of the enzyme is the presenilins of which there are two homologues, Presenilin-1 (PS1) and Presenilin-2 (PS2). The field has focussed on the PS1 form of this enzyme, as it is typically considered the more active at APP processing. However, much of this work has been completed without appropriate consideration of the specific levels of protein expression of PS1 and PS2. We propose that expression is an important factor in PS1- and PS2-γ-secretase activity, and that when this is considered, PS1 does not have greater activity than PS2. We developed and validated tools for quantitative assessment of PS1 and PS2 protein expression levels to enable the direct comparison of PS in exogenous and endogenous expression systems, in HEK-293 PS1 and/or PS2 knockout cells. We show that exogenous expression of Myc-PS1-NTF is 5.5-times higher than Myc-PS2-NTF. Quantitating endogenous PS protein levels, using a novel PS1/2 fusion standard we developed, showed similar results. When the marked difference in PS1 and PS2 protein levels is considered, we show that compared to PS1-γ-secretase, PS2-γ-secretase has equal or more activity on APP and Notch1. This study has implications for understanding the PS1- and PS2-specific contributions to substrate processing, and their potential influence in AD pathogenesis.

Aging and brain free cholesterol concentration on amyloid-ß peptide accumulation in guinea pigs.

Alzheimer's disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid-ß (Aß40 and Aß42) peptides, Mdr1 and APP (amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9-40 weeks old guinea pigs. Protein expression levels of P-gp (Abcb1) and Cyp46a1 (24(S)-hydroxylase) for Aß40, and Aß42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low-density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aß efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aß peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aß peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)-hydroxycholesterol metabolite revealed a decreasing trend from 20 weeks old toward 40 weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aß accumulation. The decreases in P-gp and Lrp1 protein levels should further exacerbate the accumulation of Aß peptides in guinea pig brain.

SARS-CoV-2 Nucleocapsid Protein Induces Tau Pathological Changes That Can Be Counteracted by SUMO2.

Neurologic manifestations are an immediate consequence of SARS-CoV-2 infection, the etiologic agent of COVID-19, which, however, may also trigger long-term neurological effects. Notably, COVID-19 patients with neurological symptoms show elevated levels of biomarkers associated with brain injury, including Tau proteins linked to Alzheimer's pathology. Studies in brain organoids revealed that SARS-CoV-2 alters the phosphorylation and distribution of Tau in infected neurons, but the mechanisms are currently unknown. We hypothesize that these pathological changes are due to the recruitment of Tau into stress granules (SGs) operated by the nucleocapsid protein (NCAP) of SARS-CoV-2. To test this hypothesis, we investigated whether NCAP interacts with Tau and localizes to SGs in hippocampal neurons in vitro and in vivo. Mechanistically, we tested whether SUMOylation, a posttranslational modification of NCAP and Tau, modulates their distribution in SGs and their pathological interaction. We found that NCAP and Tau colocalize and physically interact. We also found that NCAP induces hyperphosphorylation of Tau and causes cognitive impairment in mice infected with NCAP in their hippocampus. Finally, we found that SUMOylation modulates NCAP SG formation in vitro and cognitive performance in infected mice. Our data demonstrate that NCAP induces Tau pathological changes both in vitro and in vivo. Moreover, we demonstrate that SUMO2 ameliorates NCAP-induced Tau pathology, highlighting the importance of the SUMOylation pathway as a target of intervention against neurotoxic insults, such as Tau oligomers and viral infection.

Alzheimer's disease brain-derived extracellular vesicles reveal altered synapse-related proteome and induce cognitive impairment in mice.

INTRODUCTION: Extracellular vesicles (EVs) have been implicated in the spread of neuropathology in Alzheimer's disease (AD), but their involvement in behavioral outcomes linked to AD remains to be determined. METHODS: EVs isolated from post mortem brain tissue from control, AD, or frontotemporal dementia (FTD) donors, as well as from APP/PS1 mice, were injected into the hippocampi of wild-type (WT) or a humanized Tau mouse model (hTau/mTauKO). Memory tests were carried out. Differentially expressed proteins in EVs were assessed by proteomics. RESULTS: Both AD-EVs and APP/PS1-EVs trigger memory impairment in WT mice. We further demonstrate that AD-EVs and FTD-EVs carry Tau protein, present altered protein composition associated with synapse regulation and transmission, and trigger memory impairment in hTau/mTauKO mice. DISCUSSION: Results demonstrate that AD-EVs and FTD-EVs have negative impacts on memory in mice and suggest that, in addition to spreading pathology, EVs may contribute to memory impairment in AD and FTD. HIGHLIGHTS: Aß was detected in EVs from post mortem AD brain tissue and APP/PS1 mice. Tau was enriched in EVs from post mortem AD, PSP and FTD brain tissue. AD-derived EVs and APP/PS1-EVs induce cognitive impairment in wild-type (WT) mice. AD- and FTD-derived EVs induce cognitive impairment in humanized Tau mice. Proteomics findings associate EVs with synapse dysregulation in tauopathies.

Wild-type sTREM2 blocks Aß aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aß aggregation.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

Combination of human tau and islet amyloid polypeptide exacerbates metabolic dysfunction in transgenic mice.

Amyloid plaques and neurofibrillary tangles composed of hyperphosphorylated tau are important contributors to Alzheimer's disease (AD). Tau also impacts pancreatic beta cell function and glucose homeostasis. Amyloid deposits composed of islet amyloid polypeptide (IAPP) are a pathological feature of type 2 diabetes (T2D). The current study investigates the role of human tau (hTau) in combination with human IAPP (hIAPP) as a potential mechanism connecting AD and T2D. Transgenic mice expressing hTau and hIAPP in the absence of murine tau were generated to determine the impact of these pathological factors on glucose metabolism. Co-expression of hIAPP and hTau resulted in mice with increased hyperglycaemia, insulin resistance, and glucose intolerance. The hTau-hIAPP mice also exhibited reduced beta cell area, increased amyloid deposition, impaired insulin processing, and reduced insulin content in islets. Tau phosphorylation also increased after stimulation with high glucose. In addition, brain insulin content and signalling were reduced, and tau phosphorylation was increased in these animals. These data support a link between tau and IAPP amyloid, which seems to act co-ordinately to impair beta cell function and glucose homeostasis, and suggest that the combined pathological actions of these proteins may be a potential mechanism connecting AD and T2D. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Amyloid-ß toxicity modulates tau phosphorylation through the PAX6 signalling pathway.

The molecular link between amyloid-ß plaques and neurofibrillary tangles, the two pathological hallmarks of Alzheimer's disease, is still unclear. Increasing evidence suggests that amyloid-ß peptide activates multiple regulators of cell cycle pathways, including transcription factors CDKs and E2F1, leading to hyperphosphorylation of tau protein. However, the exact pathways downstream of amyloid-ß-induced cell cycle imbalance are unknown. Here, we show that PAX6, a transcription factor essential for eye and brain development which is quiescent in adults, is increased in the brains of patients with Alzheimer's disease and in APP transgenic mice, and plays a key role between amyloid-ß and tau hyperphosphorylation. Downregulation of PAX6 protects against amyloid-ß peptide-induced neuronal death, suggesting that PAX6 is a key executor of the amyloid-ß toxicity pathway. Mechanistically, amyloid-ß upregulates E2F1, followed by the induction of PAX6 and c-Myb, while Pax6 is a direct target for both E2F1 and its downstream target c-Myb. Furthermore, PAX6 directly regulates transcription of GSK-3ß, a kinase involved in tau hyperphosphorylation and neurofibrillary tangles formation, and its phosphorylation of tau at Ser356, Ser396 and Ser404. In conclusion, we show that signalling pathways that include CDK/pRB/E2F1 modulate neuronal death signals by activating downstream transcription factors c-Myb and PAX6, leading to GSK-3ß activation and tau pathology, providing novel potential targets for pharmaceutical intervention.

Differential interaction with TREM2 modulates microglial uptake of modified Aß species.

Rare coding variants of the microglial triggering receptor expressed on myeloid cells 2 (TREM2) confer an increased risk for Alzheimer's disease (AD) characterized by the progressive accumulation of aggregated forms of amyloid ß peptides (Aß). Aß peptides are generated by proteolytic processing of the amyloid precursor protein (APP). Heterogeneity in proteolytic cleavages and additional post-translational modifications result in the production of several distinct Aß variants that could differ in their aggregation behavior and toxic properties. Here, we sought to assess whether post-translational modifications of Aß affect the interaction with TREM2. Biophysical and biochemical methods revealed that TREM2 preferentially interacts with oligomeric Aß, and that phosphorylation of Aß increases this interaction. Phosphorylation of Aß also affected the TREM2 dependent interaction and phagocytosis by primary microglia and in APP transgenic mouse models. Thus, TREM2 function is important for sensing phosphorylated Aß variants in distinct aggregation states and reduces the accumulation and deposition of these toxic Aß species in preclinical models of Alzheimer's disease.

Tau ablation in mice leads to pancreatic ß cell dysfunction and glucose intolerance.

The microtubule-associated protein tau is highly expressed in pancreatic islets. Abnormally phosphorylated tau aggregates assemble into neurofibrillary tangles linked to Alzheimer's disease pathology and has also been found in islets of patients with type 2 diabetes. However, the significance of tau in islet function remains relatively unexplored. Therefore, we investigated the role of tau on ß cell function and glucose homeostasis using tau knockout (tauKO) mice. TauKO mice were hyperglycemic and glucose intolerant at an early age. Islet insulin content was reduced and proinsulin levels were significantly elevated in tauKO mice, resulting in impaired glucose-stimulated insulin secretion. Loss of tau also resulted in increased epididymal fat mass and leptin levels, reduced glucose production, and insulin resistance at later ages, leading to complete onset of diabetes. Transgenic expression of human tau in islets was unable to rescue those defects in glucose regulation, indicating structural and/or functional differences between mouse and human tau. Cumulatively, these results suggest an important role for tau in the proper maintenance of pancreatic ß cell function and glucose homeostasis.-Wijesekara, N., Gonçalves, R. A., Ahrens, R., De Felice, F. G., Fraser, P. E. Tau ablation in mice leads to pancreatic ß cell dysfunction and glucose intolerance.

SUMO1 impact on Alzheimer disease pathology in an amyloid-depositing mouse model.

Small ubiquitin-related modifiers (SUMOs) conjugated or bound to target proteins can affect protein trafficking, processing and solubility. SUMOylation has been suggested to play a role in the amyloid plaque and neurofibrillary tangle pathology of Alzheimer disease (AD) and related neurodegenerative diseases. The current study examines the impact of SUMO1 on processing of the amyloid precursor protein (APP) leading to the production and deposition of the amyloid-ß (Aß) peptide. An in vivo model of these pathways was developed by the generation of double transgenic mice over-expressing human SUMO1 and a mutant APP. The SUMO1-APP transgenics displayed normal APP processing but, at later ages, exhibited increased insoluble Aß and plaque density accompanied by increased dendritic spine loss, more pronounced synaptic and cognitive deficits. These findings suggest a potential impairment in Aß clearance as opposed to increased amyloid production. Examination of microglia indicated a reduction in the SUMO1-APP transgenics which is a possible mechanism for the SUMO1-mediated increase in amyloid load. These findings suggest an indirect activity of SUMO1 possibly in the removal of Aß plaques rather than a direct impact on amyloid generation.

Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes ß-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

Parkinsonism due to A53E α-synuclein gene mutation: Clinical, genetic, epigenetic, and biochemical features.

BACKGROUND: SNCA mutations cause autosomal dominant parkinsonism and inform our understanding of the molecular underpinnings of synucleinopathies. The most recently identified mutation, p.Ala53Glu (A53E), has only been observed in Finland. The objectives of this study were to examine clinical, genetic, epigenetic, and biochemical features of the first family outside Finland with A53E. METHODS: We examined a Canadian family with parkinsonism because of A53E using haplotype and DNA methylation analyses. We assessed aggregation properties of A53E α-synuclein in vitro. RESULTS: Family members with parkinsonism shared a common haplotype distinct from Finnish patients with A53E. Increased acceleration of DNA methylation age was accompanied by earlier age at onset in the family members. We demonstrate that A53E α-synuclein has a propensity to form oligomers and phosphorylation promotes fibrillation. CONCLUSIONS: A53E as a cause of parkinsonism is not restricted to Finnish individuals. DNA methylation may contribute to disease age at onset. A53E enriches α-synuclein oligomers and fibrils dependent on the phosphorylation state. © 2018 International Parkinson and Movement Disorder Society.

Amyloid-ß and islet amyloid pathologies link Alzheimer's disease and type 2 diabetes in a transgenic model.

Alzheimer's disease (AD) and type 2 diabetes (T2D) present a significant risk to each other. AD and T2D are characterized by deposition of cerebral amyloid-ß (Aß) and pancreatic human islet amyloid polypeptide (hIAPP), respectively. We investigated the role of amyloidogenic proteins in the interplay between these diseases. A novel double transgenic mouse model combining T2D and AD was generated and characterized. AD-related amyloid transgenic mice coexpressing hIAPP displayed peripheral insulin resistance, hyperglycemia, and glucose intolerance. Aß and IAPP amyloid co-deposition increased tau phosphorylation, and a reduction in pancreatic ß-cell mass was detected in islets. Increased brain Aß deposition and tau phosphorylation and reduced insulin levels and signaling were accompanied by extensive synaptic loss and decreased neuronal counts. Aß immunization rescued the peripheral insulin resistance and hyperglycemia, suggesting a role for Aß in T2D pathogenesis for individuals predisposed to AD. These findings demonstrate that Aß and IAPP are key factors in the overlapping pathologies of AD and T2D.-Wijesekara, N., Ahrens, R., Sabale, M., Wu, L., Ha, K., Verdile, G., Fraser, P. E. Amyloid-ß and islet amyloid pathologies link Alzheimer's disease and type 2 diabetes in a transgenic model.

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization.

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.

Validation and Characterization of a Novel Peptide That Binds Monomeric and Aggregated ß-Amyloid and Inhibits the Formation of Neurotoxic Oligomers.

Although the formation of ß-amyloid (Aß) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aß toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aß oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aß and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D'Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203-214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aß42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25-30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aß42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aß42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aß42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies.

ATP-binding cassette transporter A7 (ABCA7) loss of function alters Alzheimer amyloid processing.

The ATP-binding cassette transporter A7 (ABCA7) has been identified as a susceptibility factor of late onset Alzheimer disease in genome-wide association studies. ABCA7 has been shown to mediate phagocytosis and affect membrane trafficking. The current study examined the impact of ABCA7 loss of function on amyloid precursor protein (APP) processing and generation of amyloid-ß (Aß). Suppression of endogenous ABCA7 in several different cell lines resulted in increased ß-secretase cleavage and elevated Aß. ABCA7 knock-out mice displayed an increased production of endogenous murine amyloid Aß42 species. Crossing ABCA7-deficient animals to an APP transgenic model resulted in significant increases in the soluble Aß as compared with mice expressing normal levels of ABCA7. Only modest changes in the amount of insoluble Aß and amyloid plaque densities were observed once the amyloid pathology was well developed, whereas Aß deposition was enhanced in younger animals. In vitro studies indicated a more rapid endocytosis of APP in ABCA7 knock-out cells that is mechanistically consistent with the increased Aß production. These in vitro and in vivo findings indicate a direct role of ABCA7 in amyloid processing that may be associated with its primary biological function to regulate endocytic pathways. Several potential loss-of-function ABCA7 mutations and deletions linked to Alzheimer disease that in some instances have a greater impact than apoE allelic variants have recently been identified. A reduction in ABCA7 expression or loss of function would be predicted to increase amyloid production and that may be a contributing factor in the associated Alzheimer disease susceptibility.