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The pathogenic aggregation of misfolded prion protein (PrP) in axons underlies prion disease pathologies. The molecular mechanisms driving axonal misfolded PrP aggregate formation leading to neurotoxicity are unknown. We found that the small endolysosomal guanosine triphosphatase (GTPase) Arl8b recruits kinesin-1 and Vps41 (HOPS) onto endosomes carrying misfolded mutant PrP to promote their axonal entry and homotypic fusion toward aggregation inside enlarged endomembranes that we call endoggresomes. This axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) mechanism forms pathologic PrP endoggresomes that impair calcium dynamics and reduce neuronal viability. Inhibiting ARESTA diminishes endoggresome formation, rescues calcium influx, and prevents neuronal death. Our results identify ARESTA as a key pathway for the regulation of endoggresome formation and a new actionable antiaggregation target to ameliorate neuronal dysfunction in the prionopathies.

Simple Summary: Poultry are unable to synthesize arginine. Thus, it is essential to include enough arginine in poultry feed. One can save some arginine by means of providing the molecules in which arginine is needed as a building block. However, the question is how much arginine can these kinds of molecules replace in poultry feed. Herein, these questions are addressed in two trials comparing guanidinoacetic acid and arginine. Moreover, the energy-saving potential of guanidinoacetic acid is evaluated. Optimal performance in broiler chickens was achieved when birds were provided with higher dietary arginine content compared to the available recommendation tables. Supplementation of either arginine or guanidinoacetic acid with a low-arginine feed alleviated the lower performance. However, more guanidinoacetic acid was needed to provide the same level of growth attained with arginine (1 to 0.57 ratio). Reduction of dietary energy by 50 and 100 kcal/kg did not significantly influence the performance of birds at all stages of growth. Moreover, guanidinoacetic acid had no energy-sparing effect. This study helps the industry to accurately meet the arginine and energy requirements of broiler chickens. Two 35-day trials were conducted to determine the arginine (Arg) requirement of broiler chickens and the Arg and energy-sparing effects of guanidinoacetic acid (GAA). In experiment 1, a low-Arg diet (basal diet) was supplemented with increasing levels (0.06–0.61%) of L-Arg or GAA. In experiment 2, a diet meeting the energy and amino acid requirements of broiler chickens served as the positive control (PC). Two negative control (NC) groups were assigned by reducing either 50 (NC1) or 100 (NC2) kcal nitrogen-corrected metabolizable energy. Test groups were supplemented with 0.06% GAA for NC1 and 0.12% GAA for NC2 to compensate for the lower energy in the feed. The low dietary Arg concentration (starter: 1.02%, grower: 0.88%, finisher: 0.75%) significantly reduced overall performance (p < 0.05). Supplementation of either L-Arg or GAA with a low-Arg diet both alleviated the lower performance (p < 0.05). However, more GAA was needed to provide the same level of growth attained with L-Arg at an equivalency rate of 1 GAA to 0.57 Arg. Reduction of dietary energy by 50 and 100 kcal did not significantly influence the performance of birds at all stages of growth. Moreover, no effect of GAA supplementation at 0.06% or 0.12% was observed. [ABSTRACT FROM AUTHOR]

Purpose: Gelsolin amyloidosis (AGel), also known as familial amyloidosis, Finnish type (FAF), is an autosomal, dominant, incurable disease caused by a point mutation (G654A/T) in the gelsolin (GSN) gene. The mutation results in loss of a Ca2+-binding site in the second gelsolin domain. Subsequent incorrect folding exposes a cryptic furin cleavage site, leading to the formation of a 68-kDa C-terminal cleavage product (C68) in the trans-Golgi network. This C68 fragment is cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) during secretion into the extracellular environment, releasing 8- and 5-kDa amyloidogenic peptides. These peptides aggregate and cause disease-associated symptoms. We set out to investigate whether AGel-specific nanobodies could be used to monitor amyloidogenic gelsolin buildup.Procedures: Three nanobodies (FAF Nb1-3) raised against the 8-kDa fragment were screened as AGel amyloid imaging agents in WT and AGel mice using 99mTc-based single-photon emission computed tomography (SPECT)/X-ray tomography (CT), biodistribution analysis, and immunofluorescence (IF). The quantitative characteristics were analyzed in a follow-up study with a Nb11-expressing mouse model.Results: All three nanobodies possess the characteristics desired for a 99mTc-based SPECT/CT imaging agent, high specificity and a low background signal. FAF Nb1 was identified as the most potent, based on its superior signal-to-noise ratio and signal specificity. As a proof of concept, we implemented 99mTc-FAF Nb1 in a follow-up study of the Nb11-expressing AGel mouse model. Using biodistribution analysis and immunofluorescence, we demonstrated the validity of the data acquired via 99mTc-FAF Nb1 SPECT/CT.Conclusion: These findings demonstrate the potential of this nanobody as a non-invasive tool to image amyloidogenic gelsolin deposition and assess the therapeutic capacity of AGel therapeutics currently under development. We propose that this approach can be extended to other amyloid diseases, thereby contributing to the development of specific therapies. [ABSTRACT FROM AUTHOR]

Dystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.