Microglia rescue neurons from aggregate-induced neuronal dysfunction and death through tunneling nanotubes.

Microglia are crucial for maintaining brain health and neuron function. Here, we report that microglia establish connections with neurons using tunneling nanotubes (TNTs) in both physiological and pathological conditions. These TNTs facilitate the rapid exchange of organelles, vesicles, and proteins. In neurodegenerative diseases like Parkinson's and Alzheimer's disease, toxic aggregates of alpha-synuclein (α-syn) and tau accumulate within neurons. Our research demonstrates that microglia use TNTs to extract neurons from these aggregates, restoring neuronal health. Additionally, microglia share their healthy mitochondria with burdened neurons, reducing oxidative stress and normalizing gene expression. Disrupting mitochondrial function with antimycin A before TNT formation eliminates this neuroprotection. Moreover, co-culturing neurons with microglia and promoting TNT formation rescues suppressed neuronal activity caused by α-syn or tau aggregates. Notably, TNT-mediated aggregate transfer is compromised in microglia carrying Lrrk22(Gly2019Ser) or Trem2(T66M) and (R47H) mutations, suggesting a role in the pathology of these gene variants in neurodegenerative diseases.

Characterizing microglial senescence: Tau as a key player.

The highest risk factor for the development of neurodegenerative diseases like tauopathies is aging. Many physiological decrements underlying aging are linked to cellular senescence. Senescent cells are characterized by an irreversible growth arrest and formation of a senescence-associated secretory phenotype (SASP), a proinflammatory secretome that modifies the cellular microenvironment and contributes to tissue deterioration. Microglia, the innate immune cells in the brain, can enter a senescent state during aging. In addition, senescent microglia have been identified in the brains of tau-transgenic mice and patients suffering from tauopathies. While the contribution of senescent microglia to the development of tauopathies and other neurodegenerative diseases is a growing area of research, the effect of tau on microglial senescence remains elusive. Here, we exposed primary microglia to 5 and 15 nanomolar (nM) of monomeric tau for 18 h, followed by a recovery period of 48 h. Using multiple senescence markers, we found that exposure to 15 nM, but not 5 nM of tau increased levels of cell cycle arrest and a DNA damage marker, induced loss of the nuclear envelope protein lamin B1 and the histone marker H3K9me3, impaired tau clearance and migration, altered the cell morphology and resulted in formation of a SASP. Taken together, we show that exposure to tau can lead to microglial senescence. As senescent cells were shown to negatively impact tau pathologies, this suggests the presence of a vicious circle, which should be further investigated in the future.

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.

Microglial NLRP3 Inflammasome Activation upon TLR2 and TLR5 Ligation by Distinct α-Synuclein Assemblies.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder and is characterized by the formation of cellular inclusions inside neurons that are rich in an abnormal form of the protein α-synuclein (α-syn). Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of signaling transduction pathways. Here, we studied activation of primary microglia isolated from wild-type mouse by distinct α-syn forms and their clearance. Internalization of α-syn monomers and oligomers efficiently activated the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome via TLR2 and TLR5 ligation, thereby acting on different signaling checkpoints. We found that primary microglia effectively engulf α-syn but hesitate in its degradation. NLRP3 inhibition by the selective inhibitor CRID3 sodium salt and NLRP3 deficiency improved the overall clearance of α-syn oligomers. Together, these data show that distinct α-syn forms exert different microglial NLRP3 inflammasome activation properties, thereby compromising its degradation, which can be prevented by NLRP3 inhibition.

Differential interaction with TREM2 modulates microglial uptake of modified Aß species.

Rare coding variants of the microglial triggering receptor expressed on myeloid cells 2 (TREM2) confer an increased risk for Alzheimer's disease (AD) characterized by the progressive accumulation of aggregated forms of amyloid ß peptides (Aß). Aß peptides are generated by proteolytic processing of the amyloid precursor protein (APP). Heterogeneity in proteolytic cleavages and additional post-translational modifications result in the production of several distinct Aß variants that could differ in their aggregation behavior and toxic properties. Here, we sought to assess whether post-translational modifications of Aß affect the interaction with TREM2. Biophysical and biochemical methods revealed that TREM2 preferentially interacts with oligomeric Aß, and that phosphorylation of Aß increases this interaction. Phosphorylation of Aß also affected the TREM2 dependent interaction and phagocytosis by primary microglia and in APP transgenic mouse models. Thus, TREM2 function is important for sensing phosphorylated Aß variants in distinct aggregation states and reduces the accumulation and deposition of these toxic Aß species in preclinical models of Alzheimer's disease.

Nitrergic modulation of neuronal excitability in the mouse hippocampus is mediated via regulation of Kv2 and voltage-gated sodium channels.

Regulation of neuronal activity is a necessity for communication and information transmission. Many regulatory processes which have been studied provide a complex picture of how neurons can respond to permanently changing functional requirements. One such activity-dependent mechanism involves signaling mediated by nitric oxide (NO). Within the brain, NO is generated in response to neuronal NO synthase (nNOS) activation but NO-dependent pathways regulating neuronal excitability in the hippocampus remain to be fully elucidated. This study was set out to systematically assess the effects of NO on ion channel activities and intrinsic excitabilities of pyramidal neurons within the CA1 region of the mouse hippocampus. We characterized whole-cell potassium and sodium currents, both involved in action potential (AP) shaping and propagation and determined NO-mediated changes in excitabilities and AP waveforms. Our data describe a novel signaling by which NO, in a cGMP-independent manner, suppresses voltage-gated Kv2 potassium and voltage-gated sodium channel activities, thereby widening AP waveforms and reducing depolarization-induced AP firing rates. Our data show that glutathione, which possesses denitrosylating activity, is sufficient to prevent the observed nitrergic effects on potassium and sodium channels, whereas inhibition of cGMP signaling is also sufficient to abolish NO modulation of sodium currents. We propose that NO suppresses both ion channel activities via redox signaling and that an additional cGMP-mediated component is required to exert effects on sodium currents. Both mechanisms result in a dampened excitability and firing ability providing new data on nitrergic activities in the context of activity-dependent regulation of neuronal function following nNOS activation.

Inhibition of neuroinflammatory nitric oxide signaling suppresses glycation and prevents neuronal dysfunction in mouse prion disease.

Several neurodegenerative diseases associated with protein misfolding (Alzheimer's and Parkinson's disease) exhibit oxidative and nitrergic stress following initiation of neuroinflammatory pathways. Associated nitric oxide (NO)-mediated posttranslational modifications impact upon protein functions that can exacerbate pathology. Nonenzymatic and irreversible glycation signaling has been implicated as an underlying pathway that promotes protein misfolding, but the direct interactions between both pathways are poorly understood. Here we investigated the therapeutic potential of pharmacologically suppressing neuroinflammatory NO signaling during early disease progression of prion-infected mice. Mice were injected daily with an NO synthase (NOS) inhibitor at early disease stages, hippocampal gene and protein expression levels of oxidative and nitrergic stress markers were analyzed, and electrophysiological characterization of pyramidal CA1 neurons was performed. Increased neuroinflammatory signaling was observed in mice between 6 and 10 wk postinoculation (w.p.i.) with scrapie prion protein. Their hippocampi were characterized by enhanced nitrergic stress associated with a decline in neuronal function by 9 w.p.i. Daily in vivo administration of the NOS inhibitor L-NAME between 6 and 9 w.p.i. at 20 mg/kg prevented the functional degeneration of hippocampal neurons in prion-diseased mice. We further found that this intervention in diseased mice reduced 3-nitrotyrosination of triose-phosphate isomerase, an enzyme involved in the formation of disease-associated glycation. Furthermore, L-NAME application led to a reduced expression of the receptor for advanced glycation end-products and the diminished accumulation of hippocampal prion misfolding. Our data suggest that suppressing neuroinflammatory NO signaling slows functional neurodegeneration and reduces nitrergic and glycation-associated cellular stress.

ß-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia.

Alzheimer's disease is the world's most common neurodegenerative disorder. It is associated with neuroinflammation involving activation of microglia by ß-amyloid (Aß) deposits. Based on previous studies showing apoptosis-associated speck-like protein containing a CARD (ASC) binding and cross-seeding extracellular Aß, we investigate the propagation of ASC between primary microglia and the effects of ASC-Aß composites on microglial inflammasomes and function. Indeed, ASC released by a pyroptotic cell can be functionally built into the neighboring microglia NOD-like receptor protein (NLRP3) inflammasome. Compared with protein-only application, exposure to ASC-Aß composites amplifies the proinflammatory response, resulting in pyroptotic cell death, setting free functional ASC and inducing a feedforward stimulating vicious cycle. Clustering around ASC fibrils also compromises clearance of Aß by microglia. Together, these data enable a closer look at the turning point from acute to chronic Aß-related neuroinflammation through formation of ASC-Aß composites.

Neuroimmune Connections in Aging and Neurodegenerative Diseases.

In recent years, the inter-relationship between the innate immune system and the central nervous system (CNS) has moved to the forefront of biomedical research, with the discovery that these two physiological systems modulate each other by a steady mutual interaction. During normal brain aging, but also under certain pathological conditions, this crosstalk can go beyond physiological control, resulting in an unresolved inflammatory response of the CNS-resident immune cells that might initiate and propagate the progression of severe tissue damage and neurodegeneration. In this review, we focus on the impact of CNS-resident cells of the innate immune system for the development of neurodegenerative diseases, review immune pathway genes that have been identified, and discuss the vicious cycle between inflammation and neurodegeneration.

NLRP3 inflammasome activation drives tau pathology.

Alzheimer's disease is characterized by the accumulation of amyloid-beta in plaques, aggregation of hyperphosphorylated tau in neurofibrillary tangles and neuroinflammation, together resulting in neurodegeneration and cognitive decline1. The NLRP3 inflammasome assembles inside of microglia on activation, leading to increased cleavage and activity of caspase-1 and downstream interleukin-1ß release2. Although the NLRP3 inflammasome has been shown to be essential for the development and progression of amyloid-beta pathology in mice3, the precise effect on tau pathology remains unknown. Here we show that loss of NLRP3 inflammasome function reduced tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. Tau activated the NLRP3 inflammasome and intracerebral injection of fibrillar amyloid-beta-containing brain homogenates induced tau pathology in an NLRP3-dependent manner. These data identify an important role of microglia and NLRP3 inflammasome activation in the pathogenesis of tauopathies and support the amyloid-cascade hypothesis in Alzheimer's disease, demonstrating that neurofibrillary tangles develop downstream of amyloid-beta-induced microglial activation.