Two key pathological hallmarks of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are the accumulation of misfolded protein aggregates and the chronic progressive neuroinflammation that they trigger. Numerous original research and reviews have provided a comprehensive understanding of how aggregated proteins (amyloid β, pathological tau, and α-synuclein) contribute to the disease, including driving sterile inflammation, in part, through the aggregation of multi-protein inflammasome complexes and the ASC speck [composed of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), Apoptosis-associated speck-like protein containing a C-terminal caspase activation and recruitment domain (ASC), and inflammatory caspase-1] involved in innate immunity. Here, we provide a unique perspective on the crosstalk between the aggregation-prone proteins involved in AD/PD and the multi-protein inflammasome complex/ASC speck that fuels feed-forward exacerbation of each other, driving neurodegeneration. Failed turnover of protein aggregates (both AD/PD related aggregates and the ASC speck) by protein degradation pathways, prionoid propagation of inflammation by the ASC speck, cross-seeding of protein aggregation by the ASC speck, and pro-aggregatory cleavage of proteins by caspase-1 are some of the mechanisms that exacerbate disease progression. We also review studies that provide this causal framework and highlight how the ASC speck serves as a platform for the propagation and spreading of inflammation and protein aggregation that drives AD and PD.

The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer’s disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.

BackgroundHyperphosphorylation and aggregation of tau is a pathological hallmark of Alzheimer’s disease and related tauopathies. Pathologically modified tau (pTau) displays varying degree of gain‐of‐toxic functions, in addition to loss of its normal homeostatic function in neurons. Several prior studies have suggested occurrence of innate immune activation in brain regions that spatio‐temporally overlap with pTau burden in brains of various tauopathies. Recent studies have suggested that once tau is hyperphosphorylated and misfolded, the neurons tend to expel pTau to the extracellular space, which is taken up by other neurons and results in trans‐neuronal propagation of pTau. We made a compelling discovery that en route to other neurons, pTau can also interact with and serve as an initial trigger to lead to microglial activation and neuroinflammation (Cell Reports 2021). However, it is not unclear if pTau driven innate immune activation involve canonical NF‐κB pathway.MethodsFirst, we determined the levels of NF‐κB activation in autopsy brains of different tauopathies via Western blot analyses for markers of NF‐κB. Second, we performed bulk‐RNA sequence analyses of human primary microglia treated with paired‐helical filaments (PHFs) purified from human tauopathy brains. Third, we crossed PS19 mouse model of tauopathy to NF‐κB‐GFP‐Luciferase reporter mice (Luc) to generate PS19/Luc+ mice and performed IVIS® live image analyses at different time points. Finally, we treated Luc expressing BV2 cells, primary microglia, and bone marrow derived macrophages (BMDMs) with pTau and validated nuclear localization of NF‐κB via Cellomics® high‐content microscopy.ResultsPhosphorylated NF‐κB p65 (Ser536) levels were significantly high in the autopsy brains of Corticobasal degeneration (CBD), Progressive supranuclear Palsy (PSP) and Pick’s disease (PiD) compared to healthy controls. RNA‐seq and bioluminescence analyses suggested human brain‐derived pTau induced NF‐κB activation in microglial cells and BMDMs. PS19/Luc+ mice displayed increased NF‐κB activation with age.ConclusionsTogether, these results suggest NF‐κB activation in the brains of human tauopathy and that pTau can directly activate NF‐κB in various model systems. Neutralization of pTau via immunotherapy and determining if it has any effect on NF‐κB activation is in progress.

Split fluorescent proteins have been engineered for various purposes, in each case signaling their spontaneous reconstitution by fluorescence. By combining split protein reconstitution and computational protein design, we have constructed a circularly permuted and truncated variant of green fluorescent protein (GFP) in which the seventh beta strand has been left out and the sites around it computationally designed to accommodate a peptide from influenza hemagglutinin. We call this a ''leave-one-out'' GFP biosensor (LOO-GFP). A LOO-GFP was designed using DEEdesign, selected by plate screening a bacterial library in the presence of the influenza peptide target, and was found to have seven point mutations. But binding was weak (9mM) and was at the expense of stability. The weakened, partially folded protein aggregated in the absence of its target. Furthermore, the aggregated biosensor fluoresced more than its monomeric peptide-bound form. In this work, the LOO-GFP was rationally redesigned to fold more robustly and bind the target tighter. Modeling of the GFP folding pathway suggested that one of the seven mutations, F83W, interfered with the closing of the beta barrel. Mutating this residue back to a F indeed, along with several other rationally justified changes followed by re-screening, produced several biosensor sequences with slower unfolding rates, a positive binding signal, and higher chromophore maturation efficiency. We also observed a blue-shift in the excitation spectrum, and lower background fluorescence in the unbound state. Kd was unchanged. In parallel experiments, LOO-GFP biosensors were genetically fused to fibers formed by the Drosophila protein ultrabithorax (Ubx), and were found to be absent any background fluorescence in the unbound state, but recovered fluorescence when exposed to the target peptide. Implications for the design of biosensing materials are discussed. Glucokinase (GCK) is the rate-limiting enzyme for glucose metabolism in glucose-sensing tissues, including hypothalamic nuclei. Post-translational regulation of GCK by receptor-mediated signaling pathways has been identified in the liver and the pancreas, and is critical for the maintenance of glucose homeostasis; yet, it is unclear if a similar regulation of GCK occurs in hypothalamic neurons. Using the hypothalamically-derived, glucosensing GT1-7 neuronal cell line, the existence of a receptor-mediated signaling cascade culminating in the S-nitrosylation and activation of GCK is demonstrated. GCK activity is assessed by either measuring NAD(P)H autofluorescence while raising extracellular glucose, or through expression of a FRET GCK biosensor. Treatment of GT1-7 cells with isoproterenol, a G s GPCR agonist, augments a glucose-dependent rise in NAD(P)H autofluorescence, and activates the GCK biosensor. Applying the nitric oxide synthase inhibitor L-NAME impedes the metabolic effect of isoproterenol. Additionally, incorporation of an S-nitrosylation-blocking V367M mutation into the biosensor prevents GCK activation by isoproterenol....