hiPSC-derived GRN-deficient astrocytes delay spiking activity of developing neurons.

Frontotemporal dementia (FTD) refers to a group of neurodegenerative disorders that are characterized by pathology predominantly localized to the frontal and temporal lobes. Approximately 40% of FTD cases are familial, and up to 20% of these are caused by heterozygous loss of function mutations in the gene encoding for progranulin (PGRN), GRN. The mechanisms by which loss of PGRN leads to FTD remain incompletely understood. While astrocytes and microglia have long been linked to the neuropathology of FTD due to mutations in GRN (FTD-GRN), a primary mechanistic role of these supporting cells have not been thoroughly addressed. In contrast, mutations in MAPT, another leading cause of familial FTD, greatly alters astrocyte gene expression leading to subsequent non-cell autonomous effects on neurons, suggesting similar mechanisms may be present in FTD-GRN. Here, we utilized human induced pluripotent stem cell (hiPSC)-derived neural tissue carrying a homozygous GRN R493X-/- knock-in mutation to investigate in vitro whether GRN mutant astrocytes have a non-cell autonomous effect on neurons. Using microelectrode array (MEA) analysis, we demonstrate that the development of spiking activity of neurons cultured with GRN R493X-/- astrocytes was significantly delayed compared to cultures with WT astrocytes. Histological analysis of synaptic markers in these cultures showed an increase in GABAergic synaptic markers and a decrease in glutamatergic synaptic markers during this period when activity was delayed. We also demonstrate that this effect may be due in-part to soluble factors. Overall, this work represents one of the first studies investigating astrocyte-induced neuronal pathology in GRN mutant hiPSCs, and supports the hypothesis of astrocyte involvement in the early pathophysiology of FTD.

Simultaneous imaging of redox states in dystrophic neurites and microglia at Aß plaques indicate lysosome accumulation not microglia correlate with increased oxidative stress.

The inter-relationship between microglia dynamics and oxidative stress (Ox-stress) in dystrophic neurites (DNs) at Alzheimer's Disease (AD) plaques may contribute to the pathological changes in neurons. We developed new in vivo imaging strategies to combine EGFP expression in microglia with neuronal expression of genetically encoded ratiometric redox sensors (rogRFP2 or roGFP1), and immunohistochemistry to investigate how microglia influence Ox-stress at amyloid plaques in 5xFAD AD mice. By simultaneously imaging microglia morphology and neuronal Ox-stress over time in vivo and in fixed brains we found that microglia preferentially enwrapped DNs exhibiting the greatest degree of Ox-stress. After microglia were partially depleted with the CSF1 receptor antagonist PLX3397, Ox-stress in DNs increased in a manner that was inversely correlated to the extent of coverage of the adjacent Aß plaques by the remaining microglia. These data suggest that microglia do not create Ox-stress at Aß plaques but instead create protective barriers around Aß plaques possibly reducing the spread of Aß. Intracranial injection of Aß was sufficient to induce neuronal Ox-stress suggesting it to be the initial trigger of Ox-stress generation. Although Ox-stress is increased in DNs, neuronal survival is enhanced following microglia depletion indicating complex and multifactorial roles of microglia with both neurotoxic and neuroprotective components. Increased Ox-stress of DNs was correlated with higher LAMP1 and ubiquitin immunoreactivity supporting proposed mechanistic links between lysosomal accumulation in DNs and their intrinsic generation of Ox-stress. Our results suggest protective as well as neurotoxic roles for microglia at plaques and that the generation of Ox-stress of DNs could intrinsically be generated via lysosomal disruption rather than by microglia. In Brief: Simultaneous imaging of microglia and neuronal Ox-stress revealed a double-edged role for microglia in 5xFAD mice. Plaque associated microglia were attracted to and enwrapped Aß plaques as well as the most highly oxidized DNs. After partial depletion of microglia, DNs were larger with greater levels of Ox-stress. Despite increased Ox-stress after microglia removal neuronal survival improved. Greater Ox-stress was correlated with increased levels of LAMP1 and ubiquitin thereby linking lysosome accumulation and Ox-stress in DNs.

A novel bio-inspired strategy to prevent amyloidogenesis and synaptic damage in Alzheimer's disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects millions of people worldwide. AD pathogenesis is intricate. It primarily involves two main molecular players-amyloid-ß (Aß) and tau-which actually have an intrinsic trend to generate molecular assemblies that are toxic to neurons. Incomplete knowledge of the molecular mechanisms inducing the onset and sustaining the progression of the disease, as well as the lack of valid models to fully recapitulate the pathogenesis of human disease, have until now hampered the development of a successful therapy for AD. The overall experience with clinical trials with a number of potential drugs-including the recent outcomes of studies with monoclonal antibodies against Aß-seems to indicate that Aß-targeting is not effective if it is not accompanied by an efficient challenge of Aß neurotoxic properties. We took advantage from the discovery of a naturally-occurring variant of Aß (AßA2V) that has anti-amyloidogenic properties, and designed a novel bio-inspired strategy for AD based on the intranasal delivery of a six-mer peptide (Aß1-6A2V) retaining the anti-amyloidogenic abilities of the full-length AßA2V variant. This approach turned out to be effective in preventing the aggregation of wild type Aß and averting the synaptic damage associated with amyloidogenesis in a mouse model of AD. The results of our preclinical studies inspired by a protective model already existing in nature, that is the human heterozygous AßA2V carriers which seem to be protected from AD, open the way to an unprecedented and promising approach for the prevention of the disease in humans.

Parallels between the Developing Vascular and Neural Systems: Signaling Pathways and Future Perspectives for Regenerative Medicine.

Neurovascular disorders, which involve the vascular and nervous systems, are common. Research on such disorders usually focuses on either vascular or nervous components, without looking at how they interact. Adopting a neurovascular perspective is essential to improve current treatments. Therefore, comparing molecular processes known to be involved in both systems separately can provide insight into promising areas of future research. Since development and regeneration share many mechanisms, comparing signaling molecules involved in both the developing vascular and nervous systems and shedding light to those that they have in common can reveal processes, which have not yet been studied from a regenerative perspective, yet hold great potential. Hence, this review discusses and compares processes involved in the development of the vascular and nervous systems, in order to provide an overview of the molecular mechanisms, which are most promising with regards to treatment for neurovascular disorders. Vascular endothelial growth factor, semaphorins, and ephrins are found to hold the most potential, while fibroblast growth factor, bone morphogenic protein, slits, and sonic hedgehog are shown to participate in both the developing vascular and nervous systems, yet have not been studied at the neurovascular level, therefore being of special interest for future research.

Microglial Heterogeneity and Its Potential Role in Driving Phenotypic Diversity of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly recognized as a highly heterogeneous disorder occurring under distinct clinical and neuropathological phenotypes. Despite the molecular determinants of such variability not being well defined yet, microglial cells may play a key role in this process by releasing distinct pro- and/or anti-inflammatory cytokines, potentially affecting the expression of the disease. We carried out a neuropathological and biochemical analysis on a series of AD brain samples, gathering evidence about the heterogeneous involvement of microglia in AD. The neuropathological studies showed differences concerning morphology, density and distribution of microglial cells among AD brains. Biochemical investigations showed increased brain levels of IL-4, IL-6, IL-13, CCL17, MMP-7 and CXCL13 in AD in comparison with control subjects. The molecular profiling achieved by measuring the brain levels of 25 inflammatory factors known to be involved in neuroinflammation allowed a stratification of the AD patients in three distinct "neuroinflammatory clusters". These findings strengthen the relevance of neuroinflammation in AD pathogenesis suggesting, in particular, that the differential involvement of neuroinflammatory molecules released by microglial cells during the development of the disease may contribute to modulate the characteristics and the severity of the neuropathological changes, driving-at least in part-the AD phenotypic diversity.

An SPRi-based biosensor pilot study: Analysis of multiple circulating extracellular vesicles and hippocampal volume in Alzheimer's disease.

One of the main hurdles in the study of Alzheimer's Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscale particles released by body cells, studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), ß-Amyloid and ganglioside M1. As results, significant variations in the relative amount and cargoes of specific brain-derived populations of EVs were observed comparing EVs coming from AD patients and healthy subjects, finding the main differences in the activation phenotype of microglia EVs, in the lipid moieties on generic EVs and in the ß-Amyloid expression on surfaces of neuronal EVs. Besides, the demonstrated correlation of SPRi data with Magnetic Resonance Imaging analysis, provided support for using the SPRi-based biosensor for the evaluation of neurodegeneration detecting and characterizing circulating EVs as peripheral biomarkers for the diagnosis and monitoring of progression and rehabilitation treatments in AD patients.

Curcumin and Novel Synthetic Analogs in Cell-Based Studies of Alzheimer's Disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is associated with the most common type of dementia and is characterized by the presence of deposits of the protein fragment amyloid beta (Aß) in the brain. The natural product mixture of curcuminoids that improves certain defects in innate immune cells of AD patients may selectively enhance Aß phagocytosis by alteration of gene transcription. In this work, we evaluated the protective effects of curcuminoids in cells from AD patients by investigating the effect on NF-κB and BACE1 signaling pathways. These results were compared to the gene expression profile of the clearance of Aß. The minor curcumin constituent, bisdemethoxycurcumin (BDC) showed the most potent protective action to decrease levels of NF-κB and BACE1, decrease the inflammatory cascade and diminish Aß aggregates in cells from AD patients. Moreover, mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (MGAT3) and vitamin D receptor (VDR) gene mRNAs were up-regulated in peripheral blood mononuclear cells from AD patients treated with BDC. BDC treatment impacts both gene expression including Mannosyl (Beta-1,4-)-Glycoprotein Beta-1,4-N-Acetylglucosaminyltransferase, Vitamin D and Toll like receptor mRNA and Aß phagocytosis. The observation of down-regulation of BACE1 and NF-κB following administration of BDC to cells from AD patients as a model system may have utility in the treatment of asymptomatic AD patients.

Molecular subtypes of Alzheimer's disease.

Protein misfolding and aggregation is a central feature of several neurodegenerative disorders including Alzheimer's disease (AD), in which assemblies of amyloid ß (Aß) peptides accumulate in the brain in the form of parenchymal and/or vascular amyloid. A widely accepted concept is that AD is characterized by distinct clinical and neuropathological phenotypes. Recent studies revealed that Aß assemblies might have structural differences among AD brains and that such pleomorphic assemblies can correlate with distinct disease phenotypes. We found that in both sporadic and inherited forms of AD, amyloid aggregates differ in the biochemical composition of Aß species. These differences affect the physicochemical properties of Aß assemblies including aggregation kinetics, resistance to degradation by proteases and seeding ability. Aß-amyloidosis can be induced and propagated in animal models by inoculation of brain extracts containing aggregated Aß. We found that brain homogenates from AD patients with different molecular profiles of Aß are able to induce distinct patterns of Aß-amyloidosis when injected into mice. Overall these data suggest that the assembly of mixtures of Aß peptides into different Aß seeds leads to the formation of distinct subtypes of amyloid having distinctive physicochemical and biological properties which result in the generation of distinct AD molecular subgroups.