Total and Phosphorylated Cerebrospinal Fluid Tau in the Differential Diagnosis of Sporadic Creutzfeldt-Jakob Disease and Rapidly Progressive Alzheimer’s Disease

Background: CSF total-tau (t-tau) became a standard cerebrospinal fluid biomarker in Alzheimer’s disease (AD). In parallel, extremely elevated levels were observed in Creutzfeldt-Jakob disease (CJD). Therefore, tau is also considered as an alternative CJD biomarker, potentially complicating the interpretation of results. We investigated CSF t-tau and the t-tau/phosphorylated tau181 ratio in the differential diagnosis of sCJD and rapidly-progressive AD (rpAD). In addition, high t-tau concentrations and associated tau-ratios were explored in an unselected laboratory cohort. Methods: Retrospective analyses included n = 310 patients with CJD (n = 205), non-rpAD (n = 65), and rpAD (n = 40). The diagnostic accuracies of biomarkers were calculated and compared. Differential diagnoses were evaluated in patients from a neurochemistry laboratory with CSF t-tau >1250 pg/mL (n = 199 out of 7036). Results: CSF t-tau showed an AUC of 0.942 in the discrimination of sCJD from AD and 0.918 in the discrimination from rpAD. The tau ratio showed significantly higher AUCs (p < 0.001) of 0.992 versus non-rpAD and 0.990 versus rpAD. In the neurochemistry cohort, prion diseases accounted for only 25% of very high CSF t-tau values. High tau-ratios were observed in CJD, but also in non-neurodegenerative diseases. Conclusions: CSF t-tau is a reliable biomarker for sCJD, but false positive results may occur, especially in rpAD and acute encephalopathies. The t-tau/p-tau ratio may improve the diagnostic accuracy in centers where specific biomarkers are not available.     

Mendelian randomization identifies blood metabolites previously linked to midlife cognition as causal candidates in Alzheimer’s disease

There are currently no disease-modifying treatments for Alzheimer’s disease (AD), and an understanding of preclinical causal biomarkers to help target disease pathogenesis in the earliest phases remains elusive. Here, we investigated whether 19 metabolites previously associated with midlife cognition—a preclinical predictor of AD—translate to later clinical risk, using Mendelian randomization (MR) to tease out AD-specific causal relationships. Summary statistics from the largest genome-wide association studies (GWASs) for AD and metabolites were used to perform bidirectional univariable MR. Bayesian model averaging (BMA) was additionally performed to address high correlation between metabolites and identify metabolite combinations that may be on the AD causal pathway. Univariable MR indicated four extra-large high-density lipoproteins (XL.HDL) on the causal pathway to AD: free cholesterol (XL.HDL.FC: 95% CI = 0.78 to 0.94), total lipids (XL.HDL.L: 95% CI = 0.80 to 0.97), phospholipids (XL.HDL.PL: 95% CI = 0.81 to 0.97), and concentration of XL.HDL particles (95% CI = 0.79 to 0.96), significant at an adjusted P < 0.009. MR–BMA corroborated XL.HDL.FC to be among the top three causal metabolites, in addition to total cholesterol in XL.HDL (XL.HDL.C) and glycoprotein acetyls (GP). Both XL.HDL.C and GP demonstrated suggestive univariable evidence of causality (P < 0.05), and GP successfully replicated within an independent dataset. This study offers insight into the causal relationship between metabolites demonstrating association with midlife cognition and AD. It highlights GP in addition to several XL.HDLs—particularly XL.HDL.FC—as causal candidates warranting further investigation. As AD pathology is thought to develop decades prior to symptom onset, expanding on these findings could inform risk reduction strategies.

More than 50 million people worldwide currently live with dementia, and with an aging world population, this figure is expected to increase to more than 152 million by 2050 (World Alzheimer Report 2018). The most common dementia type is Alzheimer’s disease (AD), characterized by impaired everyday function, severe cognitive decline—particularly working, episodic, and declarative memory (1)—and a range of neuropsychiatric symptoms (2). It represents a major source of global morbidity and mortality and poses significant human and economic costs (3).Disappointingly, AD drug development has proven difficult, with a 99.6% failure rate in the decade of 2002 to 2012, and this rate continues at the same low level today (4). Numerous reasons have been proposed as to why such clinical trials have failed, including incomplete understanding of true causal mechanisms and a failure to intervene early enough in the pathological cascade. It is therefore necessary to discover biomarkers that can identify individuals at high risk of developing AD and at the earliest possible stages of pathology onset. Moreover, it is important for these to be potentially modifiable so as to offer targets for preventative or therapeutic strategies.Metabolomics represents one avenue that may give a deeper insight into AD etiology. Metabolites are small molecules (<1,500 atomic mass units) with a role in metabolism (5). As the products of many biological processes, they sit at the end of the systems biology pathway and therefore represent effective intermediate phenotypes to a given disease because of their proximity to the clinical endpoint (6, 7). Due to 1) their noninvasive nature of measurement, 2) the fact that they are potentially modifiable through diet and lifestyle, and 3) the ability of many to cross the blood brain barrier, blood metabolites are both practical and valuable markers of biological processes and disease states in dementia (8).Markers of lipid metabolism have received particular attention in this context, as the impairment of lipid metabolism has been associated with AD (5, 8–11) and beta-amyloid (Aβ) burden (12, 13). Relevant to early intervention, they have also been associated with cognitive performance and brain function during normal aging (14, 15). Recently, using a large British population-based birth cohort, we investigated associations between 233 blood metabolites and both memory and processing speed at 60 to 64 y of age as well as changes in these cognitive domains from 60 to 64 to 69 y old. Associations with several metabolite classes were observed, including fatty acids (FAs), various compositions of high-density lipoproteins (HDLs), and glycoprotein acetyls (GP) (16).However, it is not yet established whether these metabolites are causally associated with dementia and AD. Using knowledge from these preclinical associations to investigate translatability to later AD risk could hold special utility in informing early treatment intervention, particularly if a causal relationship can be shown. This study therefore aims to expand our observational findings and assess whether 19 blood metabolites previously associated with late midlife cognition causally associate with later clinical AD status. Both univariable and Bayesian multivariable Mendelian randomization (MR) approaches are harnessed to interrogate independent as well as group associations, and a range of sensitivity analyses are performed to further scrutinize results. Identifying candidate blood metabolites, which are detectable preclinically and on the causal pathway to later AD diagnosis, will aid in facilitating further research into early intervention strategies and more targeted therapeutics.     

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation

In neurodegenerative diseases including Alzheimer’s and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here, we present the 3.4-Å resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryomicroscopy (cryo-EM). The structure reveals the familiar in-register cross-β amyloid scaffold but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNase is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients’ brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature.

The two pathological hallmarks of Alzheimer’s disease (AD) are extracellular Aβ plaques and intracellular tau neurofibrillary tangles that accompany neuron loss in the brain (1). The deposition of tau aggregates in the brain is also the pathological hallmark of dozens of dementias and movement disorders known as tauopathies, including progressive supranuclear palsy (PSP), Pick’s disease (PiD), chronic traumatic encephalopathy (CTE), and corticobasal degeneration (CBD) (2). In each of these conditions, monomeric tau proteins stack to form amyloid fibrils, but the trigger for fibrillization is poorly understood (3). Identical copies of tau stack into long β-sheets, which in turn mate together tightly to form steric zippers; these fibrils are a common feature of all amyloid diseases (4).Cofactors are necessary to form and stabilize tau fibrils in vitro and in vivo. In vitro, fibrillization of recombinant tau requires cofactors such as heparin, RNA, or arachidonic acid (5–7). Recent electron cryomicroscopy (cryo-EM) structures of heparin-induced tau filaments show they are heterogeneous and different from those of AD or PiD, which have larger fibril cores with different structures (8–10). In vivo, many cofactors are known to associate with neurofibrillary tangles in AD. These cofactors have been shown to stimulate AD-like phosphorylation of recombinant tau (5, 11, 12), and some of these cofactors are important for seeding activity in vitro (3, 5, 13). Cryo-EM structures of tau filaments extracted from the brains of patients with CTE, CBD, and PSP reveal residual density attributed to unknown cofactors: hydrophobic in CTE and anionic in CBD (14–16). These cofactors may stabilize particular tau fibril polymorphs (17) and, together with posttranslational modifications, govern which fibril polymorph dominates in the human brain (3, 18, 19).RNA is implicated as a cofactor in the formation of fibrils of RNA binding proteins (RBPs) associated with amyotrophic lateral sclerosis, such as FUS, TDP-43, and hnRNPA1. These RBPs play important roles in gene expression by forming ribonucleoprotein complexes and participating in RNA processing steps including alternative splicing, stress granule formation, and RNA degradation (20, 21). RBPs such as FUS and hnRNPA1 condense to form functional granules by liquid–liquid phase separation. Further aggregation, sometimes facilitated by RBP mutations, leads to pathological amyloid formation and accelerates development of neurodegenerative disease (2, 22–24). Notably, these proteins contain low-complexity domains (LCDs), which further contribute to phase separation (22, 23, 25, 26) by a mechanism that is not yet clear.RNA also induces tau to condense into a separate phase similar to RBPs (27) despite the fact that tau is not a bona fide RBP, nor does it contain an LCD. RNA forms a metastable complex with tau (18). RNA’s negatively charged phosphate backbone is thought to interact with the positive charge of the tau molecule to promote its aggregation (28, 29). These aggregates have pathological consequences. For example, RNA-containing tau aggregates in cell culture and mouse brains have been shown to alter pre-mRNA splicing (30). Moreover, crowding of tau molecules in the condensed phase facilitates tau stacking in a cross-β fashion to produce amyloid fibrils (6, 31). Similarly, RNA accelerates prion propagation in vitro and in vivo (32).To learn how RNA interacts with tau at the atomic level and triggers fibril formation, we determined the cryo-EM structure of the fibril of recombinant full-length tau induced by RNA. With this structure and associated biochemical experiments we addressed several questions: 1) Which tau residues compose the RNA binding site? 2) Why is the C-terminal region of tau poorly ordered in tau fibrils extracted from autopsied brains of patients with tauopathies? 3) How does RNA facilitate reversibility in fibril assembly and modulate fibril stability? 4) What role does RNA play in nucleating or deterring (33) formation of pathological tau fibril polymorphs?     

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer’s disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.

Apolipoprotein E (ApoE) is a 299-amino acid protein involved in lipid transport and cholesterol homeostasis (1, 2) that plays a key role in Alzheimer’s disease (AD). The polymorphic nature of human APOE allows for encoding three variants (ApoE2, ApoE3, and ApoE4) (3) that have dramatic functional differences, even though it is only a single amino acid change that differentiates ApoE3 from ApoE2 (R158C) and ApoE4 (C112R) (4). The most striking example is ApoE4, which is recognized as the major genetic risk factor for AD (5–9), with individuals who are homozygous for the ε4 allele having up to 15-fold higher probability of developing late-onset AD (10, 11). In contrast, ApoE3 appears to have no impact on the progression of AD, while ApoE2 has been proposed to be protective toward the disease (12). A current hypothesis is that these functional differences stem from structural changes imposed upon ApoE by this single residue substitution and thus having a potential impact on its interaction with AD factors, such as amyloid-beta plaques and neurofibrillary tangles (13, 14). In both the cardiovascular and the central nervous systems, ApoE is prevalently associated noncovalently with lipids as part of lipoproteins, and the single residue substitutions are known to alter its interaction with specific lipoprotein populations (15). From a biochemical point of view, previous work from Garai et al. suggests that only the monomeric form—not the oligomers—is competent for high-affinity lipid binding (16). Therefore, understanding the monomeric structure of ApoE is key to unmasking the mechanisms controlling its interaction with lipids. In addition, recent experiments have found that ApoE expressed by microglia and astrocytes can also occur in poorly lipidated and nonlipidated forms (17). However, a structural characterization of monomeric ApoE in its lipid-free states remains elusive. One major obstacle is posed by the high propensity of ApoE to form oligomers (18), which hampers the investigation of the monomeric form (SI Appendix, Fig. S1). A second challenge is the disordered nature of numerous short segments of the protein, which have been proposed to be flexible and confer structural heterogeneity (19) rendering these regions invisible to conventional structural biology methods.ApoE comprises four different regions: the N-terminal tail (residues 1 to 23), the four-helix bundle (24 to 167) (20–22), the hinge region (168 to 205), and the C-terminal domain (206 to 299) (Fig. 1). Current conformational models (19, 23) of the monomeric lipid-free ApoE agree on the structure of the four-helix bundle (20–22), but they disagree on the configurations of the hinge and C-terminal regions and their orientation with respect to the four-helix bundle. Ensemble Förster resonance energy transfer (FRET) and Electron Paramagnetic Resonance (EPR) studies (24) suggest that ApoE4 forms a close contact between the four-helix bundle and the C-terminal domain, whereas ApoE3 explores more open conformations. This is at odds with the compact set of structures determined by Nuclear Magnetic Resonance (NMR) on a monomeric ApoE3-like variant (22). Recent Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) experiments identified isoform-dependent differences in solvent accessibility of the four-helix bundle, hinting that single amino acid substitutions affect the ability of the C-terminal domain to shield specific regions of the four-helix bundle (19). However, the interpretation of ensemble FRET, EPR (24), and HDX-MS experiments (19) is complicated by the fact that measurements were performed under conditions in which the protein is a stable tetramer (16, 19) and, therefore, are not representative of the conformations of the protein in its monomeric form. The same limitation applies to previous investigations of the folding stability of the protein domains (16, 25–27) and its interaction with lipids (24–29), where ApoE was studied at concentrations that favor either dimer or tetramer conformations (16, 24, 28, 29).Open in a separate windowFig. 1.Protein structural regions and single-molecule constructs of full-length ApoE4. (A) Schematic representation of the secondary structure content in ApoE4 based on the NMR structure (Protein Data Bank (PDB): 2L7B) of the ApoE3-like variant with corresponding designations and identification of the major protein domains: N-terminal tail (gray), four-helix bundle (teal), hinge region (green), and C-terminal domain (light purple). Helical notations are reported for each helix. Labeling positions are identified on the linear sequence by green and red dots (the color scheme is only indicative of FRET labels and not of residue labeling for a specific fluorophore). Yellow dots identify the mutations associated with ApoE3 and ApoE2 variants. Position A86C is located in the random coil between helices H2 and H3 as previously defined (20, 30) and serves as a common reference point to investigate the folded N-terminal domain from two different perspectives. When paired with position A5C (ApoE4_5,86), which is situated upstream of the start of the H1 helix, A86C monitors the conformational properties and folding stability of the N-terminal tail. When paired with position G165C (ApoE4_86,165), which is located at the end of the H4 helix, A86C provides a readout for the folding of the four-helix bundle (22, 30). Positions G182C and A241C (ApoE4_182,241) allow monitoring the behavior of the hinge domain with respect to the C terminus, while positions S223C and A291C (ApoE4_223,291) provide information on the structural properties of the C-terminal domain. Finally, probe positions located at A86C and A241C (ApoE4_86,241) allow us to monitor long-range interactions between the N- and C-terminal domains. (B) One hundred and eighty-degree rotated views of the monomeric ApoE3-like variant NMR structure (PDB: 2L7B) highlighting labeling positions shown in orange. Structure color differentiates the major protein domains described in A.Here, we circumvent these experimental difficulties by harnessing single-molecule fluorescence spectroscopy, an approach that enables working at sufficiently low protein concentrations to avoid oligomerization and directly access the protein in its monomeric form (SI Appendix, Fig. S1). Single-molecule FRET provides a direct readout on the conformations and stability of specific domains within full-length ApoE4 in both the lipid-free and lipid-bound states. We further complement single-molecule observations with molecular dynamics (MD) simulations to obtain an atomically detailed representation of protein conformations that is consistent with our experimental data.     

Patterns of Cerebrospinal Fluid Alzheimer’s Dementia Biomarkers in People Living with HIV: Cross-Sectional Study on Associated Factors According to Viral Control,Neurological Confounders and Neurocognition

People living with HIV (PLWH) age with an excess burden of comorbidities that may increase the incidence of age-related complications. There is controversy surrounding the hypothesis that HIV can accelerate neurodegeneration and Alzheimer’s dementia (AD). We performed a retrospective study to analyze the distribution of cerebrospinal fluid (CSF) AD biomarkers (beta amyloid 1–42 fragment, tau, and phosphorylated tau) in adult PLWH (on cART with undetectable viremia, n = 136, with detectable viremia, n = 121, and with central nervous system CNS disorders regardless of viremia, n = 72) who underwent a lumbar puncture between 2008 to 2018; HIV-negative controls with AD were included (n = 84). Five subjects (1.5%) presented CSF biomarkers that were compatible with AD: one was diagnosed with AD, whereas the others showed HIV encephalitis, multiple sclerosis, cryptococcal meningitis, and neurotoxoplasmosis. Regardless of confounders, 79.6% of study participants presented normal CSF AD biomarkers. Isolated abnormalities in CSF beta amyloid 1–42 (7.9%) and tau (10.9%) were associated with age, biomarkers of intrathecal injury, and inflammation, although no HIV-specific feature was associated with abnormal CSF patterns. CSF levels of AD biomarkers very poorly overlapped between HIV-positive clinical categories and AD controls. Despite the correlations with neurocognitive performance, the inter-relationship between amyloid and tau proteins in PLWH seem to differ from that observed in AD subjects; the main driver of the isolated increase in tau seems represented by non-specific CNS inflammation, whereas the mechanisms underlying isolated amyloid consumption remain unclear.     

Alzheimer’s-related endosome dysfunction in Down syndrome is Aβ-independent but requires APP and is reversed by BACE-1 inhibition

An additional copy of the β-amyloid precursor protein (APP) gene causes early-onset Alzheimer’s disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or β-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (βCTF), all of which elevate the levels of βCTFs. Expression of a mutant form of APP that cannot undergo β-cleavage had no effect on endosomes. Pharmacological inhibition of APP γ-secretase, which markedly reduced Aβ production but raised βCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the βCTF of APP, and exclude Aβ and the αCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.     

Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Parkinson’s disease (PD) is characterized by accumulation of α-synuclein (αSyn; encoded by the SNCA gene) (1). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal cell death in part via microglial activation (2, 3). Moreover, misfolded proteins in general are thought to interact with brain microglia, triggering microglial activation that contributes to neurodegenerative disorders, although microglial phagocytosis may also initially clear aberrant proteins to afford some degree of protection (2, 4). Additionally, in Alzheimer’s disease (AD), amyloid-β peptide (Aβ) is thought to trigger similar processes in microglia (5–7); however, the mechanism for this trigger is still poorly understood.Microglial cells contribute to neuroinflammation, specifically that mediated by the inflammasome. In particular, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with several neurodegenerative disorders, although other types of inflammation may also be important in this regard (8). The NLRP3 inflammasome is a multiprotein complex that responds to cell stress and pathogenic stimuli to promote activation of caspase-1, which in turn mediates maturation and release of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18 (9–11). NLRP3 inflammasome activation is a two-step process, involving an initial priming step and a secondary trigger. Priming involves a proinflammatory stimulus, such as endotoxin, a ligand for Toll-like receptor 4 (TLR4), that increases the abundance of NLRP3 and promotes de novo synthesis of pro–IL-1β via nuclear factor κB (11). The secondary trigger promotes inflammasome complex assembly and caspase-1 activation that in turn mediates the cleavage of pro–IL-1β and subsequent release of mature IL-1β. There are various secondary triggers, including adenosine triphosphate (ATP), microparticles, and bacterial toxins, all of which somehow lead to mitochondrial damage and release of oxidized mitochondrial DNA (11). Neuroinflammation has been reported in both human PD and AD brains (12–15), and NLRP3 inflammasome activation in particular has been observed in mouse models of PD and AD (7, 16). Importantly, in these PD models, dopaminergic (DA) neurons in the substantia nigra are resistant to damage in NLRP3-deficient mice compared with wild-type (WT) mice (16). Interestingly, a recent report identified an NLRP3 polymorphism that confers decreased risk in PD (17). Several groups have reported that fibrillar αSyn can activate the NLRP3 inflammasome in mice and in human monocytes (18–22), but it remains unknown if human brain microglia can be activated in this manner. Critically, antibodies targeting misfolded proteins are being tested in human clinical trials for several neurodegenerative diseases, including AD and PD; however, it is still unclear how antibodies to αSyn might affect this inflammatory response. In this study, we characterized the response of human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) to oligomeric/fibrillar αSyn in vitro and in vivo, using engraftment of hiMG in humanized mice. We used these immunocompromised mice because they prevent human cell rejection and express three human genes that support human cell engraftment (23). We show that αSyn and, even more so, αSyn–antibody complexes activate the NLRP3 inflammasome. Moreover, this process is further sensitized by the presence of Aβ and its cognate antibodies. These observations are of heightened interest because recent studies have shown that both misfolded Aβ and αSyn are present in several neurodegenerative disorders such as AD and Lewy body dementia (LBD), a form of dementia that can occur in the setting of PD (24–26).     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

Alzheimer’s disease related single nucleotide polymorphisms and correlation with intracerebral hemorrhage incidence

Apolipoprotein E alleles have been associated with both Alzheimer’s disease (AD) and intracerebral hemorrhage (ICH). In addition, ICH is associated with a markedly high risk of subsequent dementia compared to other subtypes of stroke. We sought to evaluate if other genetic markers for AD were also associated with ICH. We examined whether published AD risk single nucleotide polymorphisms (SNPs) and haplotypes were associated with ICH utilizing genome-wide association study data from 2 independent studies (genetic and environmental risk factors for hemorrhagic stroke [GERFHS] study and genetics of cerebral hemorrhage with anticoagulation [GOCHA]). Analyses included evaluation by location of ICH. GERFHS and GOCHA cohorts contained 745 ICH cases and 536 controls for analysis. The strongest association was on 1q32 near Complement receptor type 1 (CR1), where rs6701713 was associated with all ICH (P = .0074, odds ratio [OR] = 2.07) and lobar ICH (P = .0073, OR = 2.80). The 51 most significant 2-SNP haplotypes associated with lobar ICH were identified within the Clusterin (CLU) gene. We identified that variation within CR1 and CLU, previously identified risk factors for AD, and are associated with an increased risk for ICH driven primarily by lobar ICH. Previous work implicated CR1 and CLU in cerebral amyloid clearance, the innate immune system, and cellular stress response.     

Economic considerations in the management of Alzheimer’s disease

Alzheimer’s disease is a devastating chronic disease that significantly increases healthcare costs and affects the quality of life (QoL) of the afflicted patients and their caregivers. Population aging and other demographic changes may further increase the already staggering costs of this devastating disease. While few pharmacoeconomic studies have used a prospective health economics design to assess resource utilization, most studies showed beneficial treatment effects and suggested potential savings in healthcare costs and reductions in caregiver burden. Various degrees of cost savings have been reported depending on the type of economic model, treatment evaluated, and region used in the studies. Direct comparisons of the results are difficult because different methods have been used in these evaluations. The preference of patients and families for home care for as long as possible suggests that promoting noninstitutional care for these patients should become a priority. Continued home care for patients under pharmacological treatment may reduce caregiver burden, healthcare costs, and ultimately improve patients’ and caregivers’ QoL.     

The effect of socioeconomic status on mortality among Alzheimer’s disease patients: A nationwide population-based cohort study in Korea

To investigate the effects of household income level on all-cause mortality in patients with Alzheimer’s disease (AD). Data was obtained from 7,937 participants in the Korean National Health Insurance cohort who was newly diagnosed with Alzheimer’s disease using the anti-dementia medication between 2003 and 2013. All individuals were followed-up until December 2013 or death, whichever came first. Individual income was estimated from the national health insurance premium. Information on mortality was obtained from the Korean National Statistical Office. Cox proportional hazard models were used to compare mortality rates between different income groups after adjusting for possible confounding risk factors. Of 7937 participants, 2292 AD patients (28.9%) died. Those with low, middle-low, middle income level were likely to have more increasing risk of mortality (HR 1.142 [1.022–1.276], HR 1.211 [1.045–1.402], and HR 1.158 [1.009–1.328], respectively), compared to those with high income level. The findings of this study indicate that AD patients with low income level have higher risk for mortality. Promotion of targeted policies and priority support for these groups may help reduce the mortality rate in this vulnerable group.     

Homocysteine fibrillar assemblies display cross-talk with Alzheimer’s disease β-amyloid polypeptide

High levels of homocysteine are reported as a risk factor for Alzheimer’s disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated β-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between β-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, currently affecting tens of millions of individuals globally and leading to immense social and financial impacts (1, 2). Despite the vast efforts to develop a pharmacological treatment for the disease, recent clinical trials had failed (3, 4). While it is very clear that amyloid formation is associated with AD (5, 6) and genetic variations in the gene coding for the β-amyloid polypeptide are associated with early onset of the disease (7), direct targeting of the aggregation of AD-associated pathological proteins and polypeptides has so far not resulted in clinical development of any disease-modifying treatment (8–10). Therefore, there is an essential need to understand the early biological changes that may induce the diverse pathologies observed in AD and other forms of dementia in order to identify new therapeutic targets (10).In the last decades, an enormous body of research had provided important information on the amyloid cascade, oxidative stress, and inflammatory response that are involved in AD (3, 11). Specifically, the notion of β-amyloid toxicity and extracellular plaque formation as the main cause of neuronal and synaptic loss has been extensively studied (12–14). Yet, the key to AD treatment and prevention remains elusive, and the initial steps leading to the formation and accumulation of these protein aggregates and the role of nonproteinaceous agents in this process are still not understood (10). Since only 1% of AD cases result from a familial mutation and the majority of patients are sporadic (2, 4), it is important to explore new directions to understand the biological mechanisms underlying these cases. Studies of metabolite profiling in brain regions and body fluids of AD and Parkinson’s disease (PD) patients show interference with specific metabolic pathways (15–19). Homocysteine (Hcy), a noncoded amino acid, was identified as a major risk factor for AD as high plasma concentrations were associated with the progression of the disease (20–22). Higher Hcy serum concentration was also correlated with behavioral and psychological symptoms of AD (23) and was associated with changes in motor function and cognitive decline in PD as well as with a more severe cognitive impairment in elderly adults (24–26). Furthermore, it was shown that significantly decreased hippocampal and cortical volume is associated with increased Hcy plasma concentration (27).Cystathionine β-synthase (CBS) deficiency, leading to excess of Hcy, results in the hyperhomocysteinemia inborn error of metabolism (IEM) disorder characterized by severe cognitive consequences (28–30). While elevated plasma Hcy is frequently reported as a strong and independent risk factor for the development of cognitive decline and dementia, the mechanism of its involvement is elusive (20, 31–34). Interestingly, the association between Hcy and AD-related pathological proteins was also demonstrated. Hcy-rich medium was shown to be cytotoxic to hippocampal and cortical neurons, resulting in increased β-amyloid–induced cell death (35–37). In addition, Hcy was found to bind β-amyloid_1-40, thereby stimulating β-sheet structure formation to facilitate its deposition. Indeed, induced Hcy accumulation in the brains of rats caused an elevation of β-amyloid deposition (38, 39). Furthermore, Hcy increased total tau and phosphorylated tau protein levels as well as the level of tau oligomers (40). Although the involvement of Hcy accumulation in AD pathology is evidential, no mechanistic insight has so far been suggested.We have previously demonstrated that small metabolites can self-assemble into amyloid-like structures with amyloidogenic characteristics (41–44). The presence of metabolite assemblies in IEM disorders (e.g., phenylalanine in Phenylketonuria) exemplifies their physiological importance in pathologically diversified diseases. Interestingly, recent studies demonstrated that metabolite assemblies could cross-seed the aggregation of proteins under physiological conditions, thereby suggesting a possible mechanism in which accumulated metabolites interfere with protein function and folding (45, 46). Assemblies of quinolinic acid, an endogenous neurometabolite that is involved in the pathology of PD, induce the aggregation of α-synuclein both in vitro and in cell culture (45). In addition, phenylalanine preformed fibrils were shown to initiate the aggregation of several proteins under physiological conditions (46). Metabolite accumulation, structure formation, and seeding of proteins may thus underlie the unknown role of metabolites in neurodegenerative pathologies (47). Specifically, seeding of amyloidogenic proteins by preformed fibrils may be part of the mechanisms underlying the stereotypical spreading of toxic aggregates in the brains of AD patients.We recently established an in vivo yeast model for IEM disorders by genetically modifying the yeast to reflect the mutations found in patients showing accumulation of the adenine nucleobase and its derivatives (48). Yeast model systems provide a powerful platform to elucidate the pathophysiology of diseases, as well as for the screening and development of disease-modifying therapeutics (49). This model was found to be a valid system, as supported by its robust sensitivity to adenine feeding and by the fact that adenine supramolecular structures could be detected. Furthermore, the addition of a generic fibrillation-modifying polyphenolic compound rescued the toxic effect without lowering the concentration of adenine, indicating the therapeutic potential of our model for the modulation of structure formation (48, 50).Here, aiming to explore the role of Hcy in AD, we demonstrate the formation of amyloid-like fibrils by Hcy in vitro and in vivo in a yeast model. Structural characterization of the Hcy fibrils and their cytotoxic effect were both studied. In addition, we revealed that polyphenolic inhibitors could rescue the toxic effect of Hcy assemblies and inhibit its structure formation. Remarkably, immunohistochemistry allowed the detection of Hcy fibrils in the brain of AD model mice as well as the apparent interplay between Hcy and β-amyloid. Finally, we demonstrated the cross-seeding of AD-related pathological protein by Hcy assemblies. Our work suggests a research direction for the association between metabolite accumulation and the initiation of neurodegenerative processes, thus offering a path for the development of therapeutic treatments that will target the key early stages of the disease.     

Atypical Presentation of Hashimoto’s Disease in an Adolescent: Thyroid-Associated Ophthalmopathy

Hashitoxicosis is generally differentiated from Graves’ hyperthyroidism by its shorter course and absence of ophthalmopathy. In this case report, we describe an adolescent girl who presented with significant clinical findings of hyperthyroidism, a diffuse goiter with homogenously increased uptake in scintigraphy, and with ocular findings of ophthalmopathy. The thyroid stimulating hormone receptor antibody test was positive, and the family history revealed thyroid-associated ophthalmopathy. Clinical findings supported the diagnosis of Hashimoto’s disease (HD) in the follow-up period. Radioactive iodine uptake investigation was found to be a reliable method for differential diagnosis. Attention was drawn to the rarity of pediatric cases of HD who present with ophthalmopathy.     

The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease

Numerous studies demonstrate that neuroinflammation is a key player in the progression of Alzheimer’s disease (AD). Interleukin (IL)-1β is a main inducer of inflammation and therefore a prime target for therapeutic options. The inactive IL-1β precursor requires processing by the the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome into a mature and active form. Studies have shown that IL-1β is up-regulated in brains of patients with AD, and that genetic inactivation of the NLRP3 inflammasome improves behavioral tests and synaptic plasticity phenotypes in a murine model of the disease. In the present study, we analyzed the effect of pharmacological inhibition of the NLRP3 inflammasome using dapansutrile (OLT1177), an oral NLRP3-specific inhibitor that is safe in humans. Six-month-old WT and APP/PS1 mice were fed with standard mouse chow or OLT1177-enriched chow for 3 mo. The Morris water maze test revealed an impaired learning and memory ability of 9-mo-old APP/PS1 mice (P = 0.001), which was completely rescued by OLT1177 fed to mice (P = 0.008 to untreated APP/PS1). Furthermore, our findings revealed that 3 mo of OLT1177 diet can rescue synaptic plasticity in this mouse model of AD (P = 0.007 to untreated APP/PS1). In addition, microglia were less activated (P = 0.07) and the number of plaques was reduced in the cortex (P = 0.03) following NLRP3 inhibition with OLT1177 administration. We also observed an OLT1177 dose-dependent normalization of plasma metabolic markers of AD to those of WT mice. This study suggests the therapeutic potential of treating neuroinflammation with an oral inhibitor of the NLRP3 inflammasome.

Alzheimer’s disease (AD) and other related neurodegenerative diseases leading to dementia represent an enormous burden for the society and health economies. AD patients suffer progressive cognitive and functional deficits often for many years, which result in a heavy burden to patients, families, and the public health system. In fact, in 2015 an estimated 46.8 million people worldwide were living with dementia, which could extend to 131.5 million by 2050 (1). Rising prevalence and mortality rates in combination with a lack of effective treatments lead to enormous costs to society. Research on AD in the last decades has focused on the pathological hallmarks and cellular deposits of amyloid-β (Aβ) peptides and neurofibrils (2). Recently, there has been increased evidence supporting a central role of the immune system in the progression or even the origin of the disease (3–5). In this respect, it is noteworthy that it has been known since 1989 that levels of interleukin (IL)-1β, one of the main mediators of innate immune response, are elevated in brains of patients with AD and can be associated with the progression and onset of AD (6–11). Additionally, it was shown that the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (12, 13), a multisubunit complex important for the maturation of IL-1β, is activated by Aβ peptides, leading to an overproduction of IL-1β, neuroinflammation, and cognitive impairment (14, 15). Inhibition of the NLRP3 inflammasome and the subsequent reduced IL-1β production can be linked to a change in the phenotype of microglia, the innate immune cells in the brain. Heneka et al. (16) pointed out the important role of the NLRP3 inflammasome/caspase-1 axis in AD pathogenesis by demonstrating significant improvements (e.g., in cognition) in APP/PS1 mice (a mouse model for AD) when crossed with NLRP3^−/− animals. The APP/PS1 mice express a human amyloid precursor protein (APP) and human presenilin-1 (PS1), leading to the accumulation of Aβ peptides, neuroinflammation, and cognitive impairment (17).OLT1177 (rINN: dapansutrile) is a new chemical entity small molecule that specifically targets the NLRP3 inflammasome and prevents the activation of caspase-1 and the maturation and release of IL-1β (18). OLT1177 has been shown to be well tolerated in animals and humans (18) and is currently in phase 2 clinical studies for the treatment of inflammatory conditions, such as osteoarthritis (topical gel dosage form) and inflammatory diseases, such as acute gout flare (oral capsule dosage form), among other diseases (19).In this study, we used the APP/PS1 mouse model of AD to investigate the effects of OLT1177 as an acute, oral pharmacological intervention (17). Six-month-old WT and APP/PS1ΔE9 mice consumed ad libitum OLT1177 in feed pellets (∼0, 500, or 1,000 mg/kg/d based on feed concentrations of 0, 3.75 or 7.5 g of OLT1177 per kilogram of feed; hereafter referred to as 3.75 or 7.5 g/kg OLT1177) for the treatment duration of 3 mo. APP/PS1 mice treated with OLT1177 showed rescue effects in various assessments, ranging from improved cognitive function to overall reduction in proinflammatory cytokines in the brain, suggesting the potential benefits of pharmaceutically blocking NLRP3 signaling in AD.     

Treating senile dementia with traditional Chinese medicine

Senile dementia is a syndrome in the elderly involving deficits in memory and cognition. There has been a long history of research and medical practice in dementia in China, during which the ancient Chinese people have formed a whole theory and accumulated abundant experience in the treatment of dementia. During recent decades, with new theories and technologies being digested and integrated, progress has been made in the medical and pharmacy research on senile dementia in China. In this review, we will focus on the traditional opinion, clinical practice, and recent progress in pharmacological research in China towards the treatment of dementia. We also discuss the potential trends of global convergence.