The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Mitochondria are associated with various cellular activities critical to homeostasis, particularly in the nervous system. The plastic architecture of the mitochondrial network and its dynamic structure play crucial roles in ensuring that varying energetic demands are rapidly met to maintain neuronal and axonal energy homeostasis. Recent evidence associates aging and neurodegeneration with anomalous neuronal metabolism as age‐dependent alterations of neuronal metabolism are now believed to occur prior to neurodegeneration. The brain has a high energy demand, which makes it particularly sensitive to mitochondrial dysfunction. Distinct cellular events causing oxidative stress or disruption of metabolism and mitochondrial homeostasis can trigger a neuropathology. This review explores the bioenergetic hypothesis for the neurodegenerative pathomechanisms, discussing factors leading to age‐related brain hypometabolism and its contribution to cognitive decline. Recent research on the mitochondrial network in healthy nervous system cells, its response to stress, and how it is affected by pathology, as well as current contributions to novel therapeutic approaches will be highlighted.

Background: Alzheimer’s disease (AD) is the most common neurodegenerative disorder, neuropathologically characterized by aggregates of β-amyloid peptides, which deposit as senile plaques, and of TAU protein, which forms neurofibrillary tangles. It is now widely accepted that neuroinflammation is implicated in AD pathogenesis. Method: Indeed, inflammatory mediators, such as cytokines and chemokines (chemotactic cytokines) can impact on the Alzheimer´s amyloid precursor protein by affecting its expression levels and amyloidogenic processing and/or β -amyloid aggregation. Additionally, cytokines and chemokines can influence kinases’ activities, leading to abnormal TAU phosphorylation. To date there is no cure for AD, but several therapeutic strategies have been directed to prevent neuroinflammation. Anti-inflammatory, but also anti-amyloidogenic compounds, such as flavonoids were shown to favourably modulate some pathological events associated with neurodegeneration. Conclusion: This review focuses on the role of cytokines and chemokines in AD-associated pathologies, and summarizes the potential anti-inflammatory therapeutic approaches aimed at preventing or slowing down disease progression.

Background: Retrograde transport of several transmembrane proteins from endosomes to the trans-Golgi network (TGN) occurs via Rab 5-containing endosomes, mediated by clathrin and the recently characterized retromer complex. This complex and one of its putative sorting receptor components, SorLA, were reported to be associated to late onset Alzheimer's disease (AD). The pathogenesis of this neurodegenerative disorder is still elusive, although accumulation of amyloidogenic Abeta is a hallmark. This peptide is generated from the sucessive β- and γ- secretase proteolysis of the Alzheimer's amyloid precursor protein (APP), events which are associated with endocytic pathway compartments. Therefore, APP targeting and time of residence in endosomes would be predicted to modulate Abeta levels. However, the formation of an APP- and retromer-containing protein complex with potential functions in retrieval of APP from the endosome to the TGN had, to date, not been demonstrated directly. Further, the motif(s) in APP that regulate its sorting to the TGN have not been characterized. Results: Through the use of APP-GFP constructs, we show that APP containing endocytic vesicles targeted for the TGN, are also immunoreactive for clathrin-, Rab 5- and VPS35. Further, they frequently generate protruding tubules near the TGN, supporting an association with a retromer-mediated pathway. Importantly, we show for the first time, that mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. The phosphomimetic APP S655E displays decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life. Conclusions: We reported for the first time the importance of APP phosphorylation on S655 in regulating its retromer-mediated sorting to the TGN or lysosomes. Significantly, the data are consistent with known interactions involving the retromer, SorLA and APP. Further, these findings add to our understanding of APP targeting and potentially contribute to our knowledge of sporadic AD pathogenesis representing putative new targets for AD therapeutic strategies.