Molecular Bases of Disease
Temperature instability of a mutation at a multidomain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome
ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited.Lack of ApoE inhibits ADan amyloidosis in a mouse model of familial Danish dementia
The Apolipoprotein E-ε4 allele (APOE-ε4) is the strongest genetic risk factor for late onset Alzheimer disease (AD). ApoE plays a critical role in amyloid-β (Aβ) accumulation in AD, and genetic deletion of the murine ApoE gene in mouse models results in a decrease or inhibition of Aβ deposition. The association between the presence of ApoE and amyloid in amyloidoses suggests a more general role for ApoE in the fibrillogenesis process. However, whether decreasing levels of ApoE would attenuate amyloid pathology in different amyloidoses has not been directly addressed.Distinct neurotoxic TDP-43 fibril polymorphs are generated by heterotypic interactions with α-Synuclein
Amyloid aggregates of specific proteins constitute important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show comorbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by colocalizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids.Casein kinase 1δ/ε phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration
Aberrant cytoplasmic accumulation of an RNA-binding protein, fused in sarcoma (FUS), characterizes the neuropathology of subtypes of ALS and frontotemporal lobar degeneration, although the effects of post-translational modifications of FUS, especially phosphorylation, on its neurotoxicity have not been fully characterized. Here, we show that casein kinase 1δ (CK1δ) phosphorylates FUS at 10 serine/threonine residues in vitro using mass spectrometric analyses. We also show that phosphorylation by CK1δ or CK1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells.The molecular basis of spinocerebellar ataxia type 48 caused by a de novo mutation in the ubiquitin ligase CHIP
The spinocerebellar ataxias (SCAs) are a class of incurable diseases characterized by degeneration of the cerebellum that results in movement disorder. Recently, a new heritable form of SCA, spinocerebellar ataxia type 48 (SCA48), was attributed to dominant mutations in STIP1 homology and U box-containing 1 (STUB1); however, little is known about how these mutations cause SCA48. STUB1 encodes for the protein C terminus of Hsc70 interacting protein (CHIP), an E3 ubiquitin ligase. CHIP is known to regulate proteostasis by recruiting chaperones via a N-terminal tetratricopeptide repeat domain and recruiting E2 ubiquitin-conjugating enzymes via a C-terminal U-box domain.A familial Danish dementia rat shows impaired presynaptic and postsynaptic glutamatergic transmission
Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-β precursor protein (APP), whose mutations cause familial forms of Alzheimer's disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2bD rats).The aminoglycoside G418 hinders de novo prion infection in cultured cells
The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic.Neurological impairment caused by Schistosoma mansoni systemic infection exhibits early features of idiopathic neurodegenerative disease
Schistosomiasis, a neglected tropical disease caused by trematodes of the Schistosoma genus, affects over 250 million people around the world. This disease has been associated with learning and memory deficits in children, whereas reduced attention levels, impaired work capacity, and cognitive deficits have been observed in adults. Strongly correlated with poverty and lack of basic sanitary conditions, this chronic endemic infection is common in Africa, South America, and parts of Asia and contributes to inhibition of social development and low quality of life in affected areas.FUS contributes to mTOR-dependent inhibition of translation
The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1.MEK1/2 activity modulates TREM2 cell surface recruitment
Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer’s disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface.Microtubule affinity–regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration
Accumulation of the microtubule-associated protein tau is associated with Alzheimer's disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration.Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction
HIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes.Quantitative propagation of assembled human Tau from Alzheimer's disease brain in microfluidic neuronal cultures
Tau aggregation and hyperphosphorylation is a key neuropathological hallmark of Alzheimer's disease (AD), and the temporospatial spread of Tau observed during clinical manifestation suggests that Tau pathology may spread along the axonal network and propagate between synaptically connected neurons. Here, we have developed a cellular model that allows the study of human AD-derived Tau propagation from neuron to neuron using microfluidic devices. We show by using high-content imaging techniques and an in-house developed interactive computer program that human AD-derived Tau seeds rodent Tau that propagates trans-neuronally in a quantifiable manner in a microfluidic culture model.An astrocyte cell line that differentially propagates murine prions
Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc. Elucidating the molecular and cellular mechanisms underlying prion propagation may help to develop disease interventions. Cell culture systems for prion propagation have greatly advanced molecular insights into prion biology, but translation of in vitro to in vivo findings is often disappointing. A wider range of cell culture systems might help overcome these shortcomings.Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease
Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils.Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies
Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding.Incomplete glycosylation during prion infection unmasks a prion protein epitope that facilitates prion detection and strain discrimination
The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation.The emerging role of α-synuclein truncation in aggregation and disease
α-Synuclein (αsyn) is an abundant brain neuronal protein that can misfold and polymerize to form toxic fibrils coalescing into pathologic inclusions in neurodegenerative diseases, including Parkinson's disease, Lewy body dementia, and multiple system atrophy. These fibrils may induce further αsyn misfolding and propagation of pathologic fibrils in a prion-like process. It is unclear why αsyn initially misfolds, but a growing body of literature suggests a critical role of partial proteolytic processing resulting in various truncations of the highly charged and flexible carboxyl-terminal region.FACS-array–based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model
Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this “FACS-array” with those obtained with homogenized striatal samples (STR-array).Amyloidogenic processing of amyloid β protein precursor (APP) is enhanced in the brains of alcadein α–deficient mice
Alzheimer’s disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic β-amyloid (Aβ) peptide generated from proteolytic cleavage of β-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin (PSEN) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2).Copper stabilizes antiparallel β-sheet fibrils of the amyloid β40 (Aβ40)-Iowa variant
Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue–long β-amyloid peptide (Aβ40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aβ42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD.Chronic treatment with the complex I inhibitor MPP+ depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)
Mitochondrial dysfunction is implicated in sporadic and familial Parkinson's disease (PD). However, the mechanisms that impair homeostatic responses to mitochondrial dysfunction remain unclear. Previously, we found that chronic, low-dose administration of the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) dysregulates mitochondrial fission–fusion, mitophagy, and mitochondrial biogenesis. Given that PTEN-induced kinase 1 (PINK1) regulates mitochondrial function, dynamics, and turnover, we hypothesized that alterations in endogenous PINK1 levels contribute to depletion of mitochondria during chronic complex I injury.The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein
α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized.Metal ion coordination delays amyloid-β peptide self-assembly by forming an aggregation–inert complex
A detailed understanding of the molecular pathways for amyloid-β (Aβ) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer's disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aβ peptide and efficiently modulates Aβ fibrillization.α-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species
Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments.
