Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription.

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aß deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aß pathology, rendering it a potential contributor to AD risk and pathogenesis.

Alzheimer's-Causing Mutations Shift Aß Length by Destabilizing γ-Secretase-Aßn Interactions.

Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aß peptides. The shift in Aß length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aß peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aß. In contrast, E-Aßn stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aß production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.

Concerted type I interferon signaling in microglia and neural cells promotes memory impairment associated with amyloid ß plaques.

The principal signals that drive memory and cognitive impairment in Alzheimer's disease (AD) remain elusive. Here, we revealed brain-wide cellular reactions to type I interferon (IFN-I), an innate immune cytokine aberrantly elicited by amyloid ß plaques, and examined their role in cognition and neuropathology relevant to AD in a murine amyloidosis model. Using a fate-mapping reporter system to track cellular responses to IFN-I, we detected robust, Aß-pathology-dependent IFN-I activation in microglia and other cell types. Long-term blockade of IFN-I receptor (IFNAR) rescued both memory and synaptic deficits and resulted in reduced microgliosis, inflammation, and neuritic pathology. Microglia-specific Ifnar1 deletion attenuated the loss of post-synaptic terminals by selective engulfment, whereas neural Ifnar1 deletion restored pre-synaptic terminals and decreased plaque accumulation. Overall, IFN-I signaling represents a critical module within the neuroinflammatory network of AD and prompts concerted cellular states that are detrimental to memory and cognition.

Medin co-aggregates with vascular amyloid-ß in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.

APP substrate ectodomain defines amyloid-ß peptide length by restraining γ-secretase processivity and facilitating product release.

Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases generates amyloid-ß (Aß) peptides and defines the proportion of short-to-long Aß peptides, which is tightly connected to Alzheimer's disease (AD) pathogenesis. Here, we study the mechanism that controls substrate processing by γ-secretases and Aß peptide length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of γ-secretases processive cleavage by destabilizing enzyme-substrate interactions. We show that increasing hydrophobicity, via mutation or ligand binding, at APPC99 -ECD attenuates substrate-driven product release and rescues the effects of Alzheimer's disease-associated pathogenic γ-secretase and APP variants on Aß length. In addition, our study reveals that APPC99 -ECD facilitates the paradoxical production of longer Aßs caused by some γ-secretase inhibitors, which act as high-affinity competitors of the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by γ-secretases and suggest it as a sweet spot for the potential design of APP-targeting compounds selectively promoting its processing by these enzymes.

The amyloid state of proteins in human diseases.

Amyloid fibers and oligomers are associated with a great variety of human diseases including Alzheimer's disease and the prion conditions. Here we attempt to connect recent discoveries on the molecular properties of proteins in the amyloid state with observations about pathological tissues and disease states. We summarize studies of structure and nucleation of amyloid and relate these to observations on amyloid polymorphism, prion strains, coaggregation of pathogenic proteins in tissues, and mechanisms of toxicity and transmissibility. Molecular studies have also led to numerous strategies for biological and chemical interventions against amyloid diseases.

Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model.

Alzheimer's disease (AD) results in cognitive decline and altered network activity, but the mechanisms are unknown. We studied human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Electroencephalographic recordings in hAPP mice revealed spontaneous epileptiform discharges, indicating network hypersynchrony, primarily during reduced gamma oscillatory activity. Because this oscillatory rhythm is generated by inhibitory parvalbumin (PV) cells, network dysfunction in hAPP mice might arise from impaired PV cells. Supporting this hypothesis, hAPP mice and AD patients had decreased levels of the interneuron-specific and PV cell-predominant voltage-gated sodium channel subunit Nav1.1. Restoring Nav1.1 levels in hAPP mice by Nav1.1-BAC expression increased inhibitory synaptic activity and gamma oscillations and reduced hypersynchrony, memory deficits, and premature mortality. We conclude that reduced Nav1.1 levels and PV cell dysfunction critically contribute to abnormalities in oscillatory rhythms, network synchrony, and memory in hAPP mice and possibly in AD.

Alzheimer mechanisms and therapeutic strategies.

There are still no effective treatments to prevent, halt, or reverse Alzheimer's disease, but research advances over the past three decades could change this gloomy picture. Genetic studies demonstrate that the disease has multiple causes. Interdisciplinary approaches combining biochemistry, molecular and cell biology, and transgenic modeling have revealed some of its molecular mechanisms. Progress in chemistry, radiology, and systems biology is beginning to provide useful biomarkers, and the emergence of personalized medicine is poised to transform pharmaceutical development and clinical trials. However, investigative and drug development efforts should be diversified to fully address the multifactoriality of the disease.

Capturing alterations of intracellular-extracellular lactate distribution in the brain using diffusion-weighted MR spectroscopy in vivo.

While the intracellular-extracellular distribution of lactate has been suggested to play a critical role in the healthy and diseased brain, tools are lacking to noninvasively probe lactate in intracellular and extracellular spaces. Here, we show that, by measuring the diffusion of lactate with diffusion-weighted magnetic resonance (MR) spectroscopy in vivo and comparing it to the diffusion of purely intracellular metabolites, noninvasive quantification of extracellular and intracellular lactate fractions becomes possible. More specifically, we detect alterations of lactate diffusion in the APP/PS1 mouse model of Alzheimer's disease. Data modeling allows quantifying decreased extracellular lactate fraction in APP/PS1 mice as compared to controls, which is quantitatively confirmed with implanted enzyme-microelectrodes. The capability of diffusion-weighted MR spectroscopy to quantify extracellular-intracellular lactate fractions opens a window into brain metabolism, including in Alzheimer's disease.

Spatial-Temporal Mapping Reveals the Golgi as the Major Processing Site for the Pathogenic Swedish APP Mutation: Familial APP Mutant Shifts the Major APP Processing Site.

Alzheimer's disease is associated with increased levels of amyloid beta (Aß) generated by sequential intracellular cleavage of amyloid precursor protein (APP) by membrane-bound secretases. However, the spatial and temporal APP cleavage events along the trafficking pathways are poorly defined. Here, we use the Retention Using Selective Hooks (RUSH) to compare in real time the anterograde trafficking and temporal cleavage events of wild-type APP (APPwt) with the pathogenic Swedish APP (APPswe) and the disease-protective Icelandic APP (APPice). The analyses revealed differences in the trafficking profiles and processing between APPwt and the APP familial mutations. While APPwt was predominantly processed by the ß-secretase, BACE1, following Golgi transport to the early endosomes, the transit of APPswe through the Golgi was prolonged and associated with enhanced amyloidogenic APP processing and Aß secretion. A 20°C block in cargo exit from the Golgi confirmed ß- and γ-secretase processing of APPswe in the Golgi. Inhibition of the ß-secretase, BACE1, restored APPswe anterograde trafficking profile to that of APPwt. APPice was transported rapidly through the Golgi to the early endosomes with low levels of Aß production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aß production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.

Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP.

Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.

The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases.

Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic ß-amyloid (Aß)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.

Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aß pathology.

Accumulation of amyloid-beta (Aß) can lead to the formation of aggregates that contribute to neurodegeneration in Alzheimer's disease (AD). Despite globally reduced neural activity during AD onset, recent studies have suggested that Aß induces hyperexcitability and seizure-like activity during the early stages of the disease that ultimately exacerbate cognitive decline. However, the underlying mechanism is unknown. Here, we reveal an Aß-induced elevation of postsynaptic density protein 95 (PSD-95) in cultured neurons in vitro and in an in vivo AD model using APP/PS1 mice at 8 weeks of age. Elevation of PSD-95 occurs as a result of reduced ubiquitination caused by Akt-dependent phosphorylation of E3 ubiquitin ligase murine-double-minute 2 (Mdm2). The elevation of PSD-95 is consistent with the facilitation of excitatory synapses and the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induced by Aß. Inhibition of PSD-95 corrects these Aß-induced synaptic defects and reduces seizure activity in APP/PS1 mice. Our results demonstrate a mechanism underlying elevated seizure activity during early-stage Aß pathology and suggest that PSD-95 could be an early biomarker and novel therapeutic target for AD.

Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons.

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aß, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

Loss of insulin signaling in astrocytes exacerbates Alzheimer-like phenotypes in a 5xFAD mouse model.

Brain insulin signaling controls peripheral energy metabolism and plays a key role in the regulation of mood and cognition. Epidemiological studies have indicated a strong connection between type 2 diabetes (T2D) and neurodegenerative disorders, especially Alzheimer's disease (AD), linked via dysregulation of insulin signaling, i.e., insulin resistance. While most studies have focused on neurons, here, we aim to understand the role of insulin signaling in astrocytes, a glial cell type highly implicated in AD pathology and AD progression. To this end, we created a mouse model by crossing 5xFAD transgenic mice, a well-recognized AD mouse model that expresses five familial AD mutations, with mice carrying a selective, inducible insulin receptor (IR) knockout in astrocytes (iGIRKO). We show that by age 6 mo, iGIRKO/5xFAD mice exhibited greater alterations in nesting, Y-maze performance, and fear response than those of mice with the 5xFAD transgenes alone. This was associated with increased Tau (T231) phosphorylation, increased Aß plaque size, and increased association of astrocytes with plaques in the cerebral cortex as assessed using tissue CLARITY of the brain in the iGIRKO/5xFAD mice. Mechanistically, in vitro knockout of IR in primary astrocytes resulted in loss of insulin signaling, reduced ATP production and glycolic capacity, and impaired Aß uptake both in the basal and insulin-stimulated states. Thus, insulin signaling in astrocytes plays an important role in the control of Aß uptake, thereby contributing to AD pathology, and highlighting the potential importance of targeting insulin signaling in astrocytes as a site for therapeutics for patients with T2D and AD.

Dimerization of the Alzheimer's disease pathogenic receptor SORLA regulates its association with retromer.

SORL1, the gene encoding the large multidomain SORLA protein, has emerged as only the fourth gene that when mutated can by itself cause Alzheimer's disease (AD), and as a gene reliably linked to both the early- and late-onset forms of the disease. SORLA is known to interact with the endosomal trafficking regulatory complex called retromer in regulating the recycling of endosomal cargo, including the amyloid precursor protein (APP) and the glutamate receptor GluA1. Nevertheless, SORLA's precise structural-functional relationship in endosomal recycling tubules remains unknown. Here, we address these outstanding questions by relying on crystallographic and artificial-intelligence evidence to generate a structural model for how SORLA folds and fits into retromer-positive endosomal tubules, where it is found to dimerize via both SORLA's fibronectin-type-III (3Fn)- and VPS10p-domains. Moreover, we identify a SORLA fragment comprising the 3Fn-, transmembrane, and cytoplasmic domains that has the capacity to form a dimer, and to enhance retromer-dependent recycling of APP by decreasing its amyloidogenic processing. Collectively, these observations generate a model for how SORLA dimer (and possibly polymer) formation can function in stabilizing and enhancing retromer function at endosome tubules. These findings can inform investigation of the many AD-associated SORL1 variants for evidence of pathogenicity and can guide discovery of novel drugs for the disease.

Nasal delivery of polymeric nanoDisc mobilizes a synergy of central and peripheral amyloid-ß clearance to treat Alzheimer's disease.

The disequilibrium of amyloid ß-peptide (Aß) between the central and peripheral pools has been claimed as an initiating event in Alzheimer's disease (AD). In this study, we employ discoidal high-density lipoproteins (HDL-Disc) mimicking Aß antibody for directional flux of Aß from central to peripheral catabolism, with desirable safety and translation potential. Structurally, HDL-Disc assembly (polyDisc) is prepared with aid of chitosan derivative polymerization. After intranasal administration and response to slightly acidic nasal microenvironment, polyDisc depolymerizes into carrier-free HDL-Disc with chitosan derivatives that adhere to the mucosal layer to reversibly open tight junctions, helping HDL-Disc penetrate the olfactory pathway into brain. Thereafter, HDL-Disc captures Aß into microglia for central clearance or ferries Aß out of the brain for liver-mediated compensatory catabolism. For synergy therapy, intranasal administration of polyDisc can effectively reduce intracerebral Aß burden by 97.3% and vascular Aß burden by 73.5%, ameliorate neurologic damage, and rescue memory deficits in APPswe/PS1dE9 transgenic AD mice with improved safety, especially vascular safety. Collectively, this design provides a proof of concept for developing Aß antibody mimics to mobilize a synergy of central and peripheral Aß clearance for AD treatment.

Human Presenilin-1 delivered by AAV9 rescues impaired γ-secretase activity, memory deficits, and neurodegeneration in Psen mutant mice.

Mutations in the Presenilin (PSEN1 and PSEN2) genes are the major cause of early-onset familial Alzheimer's disease (FAD). Presenilin (PS) is the catalytic subunit of the γ-secretase complex, which cleaves type I transmembrane proteins, such as Notch and the amyloid precursor protein (APP), and plays an evolutionarily conserved role in the protection of neuronal survival during aging. FAD PSEN1 mutations exhibit impaired γ-secretase activity in cell culture, in vitro, and knockin (KI) mouse brains, and the L435F mutation is the most severe in reducing γ-secretase activity and is located closest to the active site of γ-secretase. Here, we report that introduction of the codon-optimized wild-type human PSEN1 cDNA by adeno-associated virus 9 (AAV9) results in broadly distributed, sustained, low to moderate levels of human PS1 (hPS1) expression and rescues impaired γ-secretase activity in the cerebral cortex of Psen mutant mice either lacking PS or expressing the Psen1 L435F KI allele, as evaluated by endogenous γ-secretase substrates of APP and recombinant γ-secretase products of Notch intracellular domain and Aß peptides. Furthermore, introduction of hPS1 by AAV9 alleviates impairments of synaptic plasticity and learning and memory in Psen mutant mice. Importantly, AAV9 delivery of hPS1 ameliorates neurodegeneration in the cerebral cortex of aged Psen mutant mice, as shown by the reversal of age-dependent loss of cortical neurons and elevated microgliosis and astrogliosis. These results together show that moderate hPS1 expression by AAV9 is sufficient to rescue impaired γ-secretase activity, synaptic and memory deficits, and neurodegeneration caused by Psen mutations in mouse models.