Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice.

A reduction in proteasome activity, loss of synapses and increased neuroinflammation in the brain are hallmarks of aging and many neurodegenerative disorders, including Alzheimer's disease (AD); however, whether proteasome dysfunction is causative to neuroinflammation remains less understood. In this study, we investigated the impact of 26S proteasome deficiency on neuroinflammation in the Psmc1 knockout (KO) mice deficient in a 19S proteasome subunit limited to the forebrain region. Our results revealed that impaired 26S proteasome led to reduced learning and memory capability and overt neuroinflammation in the synapses of the Psmc1 KO brain at eight weeks of age. Moreover, pronounced neuroinflammation was also found in the whole brain cortex, which was confirmed by increased levels of several key immune response-related proteins, including Stat1, Trem2 and NF-κB, and by activation of astrocytes and microglia in the KO brain. To validate NF-κB mediating neuroinflammation, we administered a selective NF-κB inhibitor to the KO animals at 5 weeks of age for three weeks, and then, animal behaviors and neuroinflammation were assessed when they reached eight weeks of age. Following the treatment, the KO mice exhibited improved behaviors and reduced neuroinflammation compared to the control animals. These data indicate that impaired 26S proteasome causes AD-like cognitive deficiency and induces neuroinflammation mediated largely by NF-κB. These results may aid development of effective therapeutics and better understanding of the pathogenesis of AD and many other neurodegenerative disorders where impaired proteasome is consistently coupled with neuroinflammation.

Pathogenic and therapeutic role of exosomes in neurodegenerative disorders.

Neurodegenerative disorders affect millions of people worldwide, and the prevalence of these disorders is only projected to rise as the number of people over 65 will drastically increase in the coming years. While therapies exist to aid in symptomatic relief, effective treatments that can stop or reverse the progress of each neurodegenerative disease are lacking. Recently, research on the role of extracellular vesicles as disease markers and therapeutics has been intensively studied. Exosomes, 30-150 nm in diameter, are one type of extracellular vesicles facilitating cell-to-cell communication. Exosomes are thought to play a role in disease propagation in a variety of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Accordingly, the exosomes derived from the patients are an invaluable source of disease biomarkers. On the other hand, exosomes, especially those derived from stem cells, could serve as a therapeutic for these disorders, as seen by a rapid increase in clinical trials investigating the therapeutic efficacy of exosomes in different neurological diseases. This review summarizes the pathological burden and therapeutic approach of exosomes in neurodegenerative disorders. We also highlight how heat shock increases the yield of exosomes while still maintaining their therapeutic efficacy. Finally, this review concludes with outstanding questions that remain to be addressed in exosomal research.

Mesenchymal and Neural Stem Cell-Derived Exosomes in Treating Alzheimer's Disease.

As the most common form of dementia and a progressive neurodegenerative disorder, Alzheimer's disease (AD) affects over 10% world population with age 65 and older. The disease is neuropathologically associated with progressive loss of neurons and synapses in specific brain regions, deposition of amyloid plaques and neurofibrillary tangles, neuroinflammation, blood-brain barrier (BBB) breakdown, mitochondrial dysfunction, and oxidative stress. Despite the intensive effort, there is still no cure for the disorder. Stem cell-derived exosomes hold great promise in treating various diseases, including AD, as they contain a variety of anti-apoptotic, anti-inflammatory, and antioxidant components. Moreover, stem cell-derived exosomes also promote neurogenesis and angiogenesis and can repair damaged BBB. In this review, we will first outline the major neuropathological features associated with AD; subsequently, a discussion of stem cells, stem cell-secreted exosomes, and the major exosome isolation methods will follow. We will then summarize the recent data involving the use of mesenchymal stem cell- or neural stem cell-derived exosomes in treating AD. Finally, we will briefly discuss the challenges, perspectives, and clinical trials using stem cell-derived exosomes for AD therapy.

Heat Shock-Induced Extracellular Vesicles Derived from Neural Stem Cells Confer Marked Neuroprotection Against Oxidative Stress and Amyloid-ß-Caused Neurotoxicity.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and a leading cause of dementia. Although the amyloid-ß (Aß) peptide is deemed a crucial driver of AD, there are no effective therapeutics available to treat Aß-caused neurotoxicity. Extracellular vesicles (EVs) are membrane-bound small particles mediating intercellular traffic of nucleic acids, lipids, proteins, and metabolites. Exosomes are a subtype of EVs with a size range of 30-150 nm in diameter. Stem cell-derived EVs are a potential therapeutic for AD, while EVs isolated from normal stem cell cultures generally have a low yield. Here, we studied the EVs secreted by the rat neural stem cells in the presence of heat shock (HS) stimulus. Nanoparticle tracking analysis confirmed that HS-derived EVs exhibit significantly higher concentration and larger diameter in comparison to the non-heat shock (NHS)-derived EVs. Mass spectrometric studies of EV proteins revealed that HS-derived EVs contained fewer diverse proteins than NHS-derived exosomes. GO enrichment analysis of the proteins suggested that the top two biological functions of the proteins in HS-derived EVs are involved in the negative regulation of apoptotic process and positive modulation of DNA repair. Importantly, the therapeutic efficacy of the NHS- and HS-derived EVs were tested in a cell culture model of AD: HS-derived EVs exhibited greater neuroprotection against not only oxidative stress but also amyloid-ß (Aß) induced neurotoxicity compared to NHS-derived EVs. Moreover, HS-derived EVs were also able to dramatically attenuate Aß-induced apoptosis and oxidative stress. These data indicate that in response to HS, neural stem cells increase EV production and alter EV morphology and cargo to confer better neuroprotection against oxidative stress and Aß-caused neurotoxicity, suggesting that HS-induced EVs from neural stem cells can be a therapeutic agent for AD and possibly other neurological disorders.

Impact of Cardiovascular Diseases on Ischemic Stroke Outcomes.

Stroke induces complex pathological cascades in the affected brain area, leading to brain injury and functional disability. To fight against cerebral ischemia/reperfusion-induced neuronal death, numerous neuroprotective strategies and reagents have been studied. However, translation of these neuroprotective drugs to clinical trials has been unsuccessful. To date, the tissue plasminogen activator is still the only FDA-approved drug for treating ischemic stroke. Thus, it is obligatory to identify and validate additional therapeutic strategies for stroke. A stroke rarely occurs without any other pathophysiological condition; but instead, it often has multi-morbidity conditions, one of which is cardiac disease. Indeed, up to half of the stroke cases are associated with cardiac and large artery diseases. As an adequate blood supply is essential for the brain to maintain its normal function, any pathophysiological alterations in the heart are frequently implicated in stroke outcomes. In this review, we summarize some of the cardiovascular factors that influence stroke outcomes and propose that considering these factors in designing stroke therapies should enhance success in clinical trials. We also highlight the recent advances regarding the potential effect of protein aggregates in a peripheral organ, such as in the heart, on ischemic stroke-caused brain injury and functional recovery. Including these and other comorbidity factors in the future therapeutic strategy designs should facilitate translational success toward developing effective combinational therapies for the disorder.

Peripherally misfolded proteins exacerbate ischemic stroke-induced neuroinflammation and brain injury.

BACKGROUND: Protein aggregates can be found in peripheral organs, such as the heart, kidney, and pancreas, but little is known about the impact of peripherally misfolded proteins on neuroinflammation and brain functional recovery following ischemic stroke. METHODS: Here, we studied the ischemia/reperfusion (I/R) induced brain injury in mice with cardiomyocyte-restricted overexpression of a missense (R120G) mutant small heat shock protein, αB-crystallin (CryABR120G), by examining neuroinflammation and brain functional recovery following I/R in comparison to their non-transgenic (Ntg) littermates. To understand how peripherally misfolded proteins influence brain functionality, exosomes were isolated from CryABR120G and Ntg mouse blood and were used to treat wild-type (WT) mice and primary cortical neuron-glia mix cultures. Additionally, isolated protein aggregates from the brain following I/R were isolated and subjected to mass-spectrometric analysis to assess whether the aggregates contained the mutant protein, CryABR120G. To determine whether the CryABR120G misfolding can self-propagate, a misfolded protein seeding assay was performed in cell cultures. RESULTS: Our results showed that CryABR120G mice exhibited dramatically increased infarct volume, delayed brain functional recovery, and enhanced neuroinflammation and protein aggregation in the brain following I/R when compared to the Ntg mice. Intriguingly, mass-spectrometric analysis of the protein aggregates isolated from CryABR120G mouse brains confirmed presence of the mutant CryABR120G protein in the brain. Importantly, intravenous administration of WT mice with the exosomes isolated from CryABR120G mouse blood exacerbated I/R-induced cerebral injury in WT mice. Moreover, incubation of the CryABR120G mouse exosomes with primary neuronal cultures induced pronounced protein aggregation. Transduction of CryABR120G aggregate seeds into cell cultures caused normal CryAB proteins to undergo dramatic aggregation and form large aggregates, suggesting self-propagation of CryABR120G misfolding in cells. CONCLUSIONS: These results suggest that peripherally misfolded proteins in the heart remotely enhance neuroinflammation and exacerbate brain injury following I/R likely through exosomes, which may represent an underappreciated mechanism underlying heart-brain crosstalk.

Disrupted blood-brain barrier in 5×FAD mouse model of Alzheimer's disease can be mimicked and repaired in vitro with neural stem cell-derived exosomes.

Alzheimer's disease (AD) is a devastating neurodegenerative disease and is associated with blood-brain barrier (BBB) disruption. AD mice and cell culture models play an essential role in understanding AD pathogenesis and validation of therapeutic reagents. One of the commonly used AD mice is the 5 × FAD mouse and previous studies have shown that BBB leakage occurs at 9 months of age in the mice. However, it remains unknown whether disrupted BBB also occurs in young animals and whether AD-caused BBB impairment can be replicated and further corrected in a cell culture model. Here, we examine BBB breakdown in the 5 × FAD mouse model at different ages including both pre-symptomatic and post-symptomatic ages and test an in vitro BBB model established with the 5 × FAD primary cerebral endothelial cells. Moreover, with the BBB in vitro model, we also examined the therapeutic effect of human neural stem cells (NSCs)-derived exosomes on AD-caused BBB leakage. Our result indicated that BBB breakdown in the 5 × FAD mice occurred at 4 months of age, which could be mimicked with an in vitro BBB model. Importantly, we further demonstrated that treatment of the in vitro BBB model with NSCs-derived exosomes reversed AD-caused BBB deficiency. The information should be useful for researchers to determine which ages of the AD mice should be employed in specific in vivo and in vitro studies and the data also suggest that AD-caused BBB disruption can be corrected at least by NSC-derived exosomes.