Molecular Mechanisms of Synaptotoxicity and Neuroinflammation in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, which is clinically associated with a global cognitive decline and progressive loss of memory and reasoning. According to the prevailing amyloid cascade hypothesis of AD, increased soluble amyloid-β (Aβ) oligomer levels impair the synaptic functions and augment calcium dyshomeostasis, neuroinflammation, oxidative stress as well as the formation of neurofibrillary tangles at specific brain regions. Emerging new findings related to synaptic dysfunction and initial steps of neuroinflammation in AD have been able to delineate the underlying molecular mechanisms, thus reinforcing the development of new treatment strategies and biomarkers for AD beyond the conventional Aβ- and tau-targeted approaches. Particularly, the identification and further characterization of disease-associated microglia and their RNA signatures, AD-associated novel risk genes, neurotoxic astrocytes, and in the involvement of complement-dependent pathway in synaptic pruning and loss in AD have set the outstanding basis for further preclinical and clinical studies. Here, we discuss the recent development and the key findings related to the novel molecular mechanisms and targets underlying the synaptotoxicity and neuroinflammation in AD

Molecular mechanisms of synaptotoxicity and neuroinflammation in Alzheimer’s disease

Abstract Alzheimer’s disease (AD) is the most common neurodegenerative disorder, which is clinically associated with a global cognitive decline and progressive loss of memory and reasoning. According to the prevailing amyloid cascade hypothesis of AD, increased soluble amyloid-β (Aβ) oligomer levels impair the synaptic functions and augment calcium dyshomeostasis, neuroinflammation, oxidative stress as well as the formation of neurofibrillary tangles at specific brain regions. Emerging new findings related to synaptic dysfunction and initial steps of neuroinflammation in AD have been able to delineate the underlying molecular mechanisms, thus reinforcing the development of new treatment strategies and biomarkers for AD beyond the conventional Aβ- and tau-targeted approaches. Particularly, the identification and further characterization of disease-associated microglia and their RNA signatures, AD-associated novel risk genes, neurotoxic astrocytes, and in the involvement of complement-dependent pathway in synaptic pruning and loss in AD have set the outstanding basis for further preclinical and clinical studies. Here, we discuss the recent development and the key findings related to the novel molecular mechanisms and targets underlying the synaptotoxicity and neuroinflammation in AD

The Alzheimer's disease-associated protective Plc gamma 2-P522R variant promotes immune functions

Background: Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer's disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in thePLCG2gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. Methods To assess the role of the protective variant in the context of immune cell functions, we generated a Plc gamma 2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. Results Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plc gamma 2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLC gamma 2-P522R, but not in PLC gamma 2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plc gamma 2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plc gamma 2-P522R KI mice. Conclusion The AD-associated protective Plc gamma 2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLC gamma 2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD

FTLD patient–derived fibroblasts show defective mitochondrial function and accumulation of p62

Abstract Frontotemporal lobar degeneration (FTLD) is a clinically, genetically, and neuropathologically heterogeneous group of neurodegenerative syndromes, leading to progressive cognitive dysfunction and frontal and temporal atrophy. C9orf72 hexanucleotide repeat expansion (C9-HRE) is the most common genetic cause of FTLD, but pathogenic mechanisms underlying FTLD are not fully understood. Here, we compared cellular features and functional properties, especially related to protein degradation pathways and mitochondrial function, of FTLD patient–derived skin fibroblasts from C9-HRE carriers and non-carriers and healthy donors. Fibroblasts from C9-HRE carriers were found to produce RNA foci, but no dipeptide repeat proteins, and they showed unchanged levels of C9orf72 mRNA transcripts. The main protein degradation pathways, the ubiquitin–proteasome system and autophagy, did not show alterations between the fibroblasts from C9-HRE-carrying and non-carrying FTLD patients and compared to healthy controls. An increase in the number and size of p62-positive puncta was evident in fibroblasts from both C9-HRE carriers and non-carriers. In addition, several parameters of mitochondrial function, namely, basal and maximal respiration and respiration linked to ATP production, were significantly reduced in the FTLD patient–derived fibroblasts from both C9-HRE carriers and non-carriers. Our findings suggest that FTLD patient–derived fibroblasts, regardless of whether they carry the C9-HRE expansion, show unchanged proteasomal and autophagic function, but significantly impaired mitochondrial function and increased accumulation of p62 when compared to control fibroblasts. These findings suggest the possibility of utilizing FTLD patient–derived fibroblasts as a platform for biomarker discovery and testing of drugs targeted to specific cellular functions, such as mitochondrial respiration.

BV-2 microglial cells overexpressing C9orf72 hexanucleotide repeat expansion produce DPR proteins and show normal functionality but no RNA foci

Abstract Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia

Protective Alzheimer's disease-associated APP A673T variant predominantly decreases sAPPβ levels in cerebrospinal fluid and 2D/3D cell culture models

The rare A673T variant was the first variant found within the amyloid precursor protein (APP) gene conferring protection against Alzheimer's disease (AD). Thereafter, different studies have discovered that the carriers of the APP A673T variant show reduced levels of amyloid beta (Aβ) in the plasma and better cognitive performance at high age. Here, we analyzed cerebrospinal fluid (CSF) and plasma of APP A673T carriers and control individuals using a mass spectrometry-based proteomics approach to identify differentially regulated targets in an unbiased manner. Furthermore, the APP A673T variant was introduced into 2D and 3D neuronal cell culture models together with the pathogenic APP Swedish and London mutations. Consequently, we now report for the first time the protective effects of the APP A673T variant against AD-related alterations in the CSF, plasma, and brain biopsy samples from the frontal cortex. The CSF levels of soluble APPβ (sAPPβ) and Aβ42 were significantly decreased on average 9–26% among three APP A673T carriers as compared to three well-matched controls not carrying the protective variant. Consistent with these CSF findings, immunohistochemical assessment of cortical biopsy samples from the same APP A673T carriers did not reveal Aβ, phospho-tau, or p62 pathologies. We identified differentially regulated targets involved in protein phosphorylation, inflammation, and mitochondrial function in the CSF and plasma samples of APP A673T carriers. Some of the identified targets showed inverse levels in AD brain tissue with respect to increased AD-associated neurofibrillary pathology. In 2D and 3D neuronal cell culture models expressing APP with the Swedish and London mutations, the introduction of the APP A673T variant resulted in lower sAPPβ levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFβ and Aβ42 were detected in some of these models. Our findings emphasize the important role of APP-derived peptides in the pathogenesis of AD and demonstrate the effectiveness of the protective APP A673T variant to shift APP processing towards the non-amyloidogenic pathway in vitro even in the presence of two pathogenic mutations