Molecular and Cellular Neuroscience最新文献

Microglia are immune cells that play important roles in the formation of the innate immune response within the central nervous system (CNS). The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a multiple protein complex that is crucial for innate immunity, and excessive activation of the inflammasome for various reasons contributes to the pathogenesis of neurodegenerative diseases (NDs). β₂-adrenoceptor agonists have become the focus of attention in studies on NDs due to the high synthesis of β₂-adrenoceptors in the central nervous system (CNS). Promising results have been obtained from these studies targeting anti-inflammatory and neuroprotective effects. Formoterol is an effective, safe for long-term use, and FDA-approved β₂-adrenoceptor agonist with demonstrated anti-inflammatory features in the CNS. In this study, we researched the effects of formoterol on LPS/ATP-stimulated NLRP3 inflammasome activation, pyroptosis, NF-κB, autophagy, and ESCRT-III-mediated plasma membrane repair pathways in the N9 microglia cells. The results showed that formoterol, through the IκBα/NF-κB axis, significantly inhibited NLRP3 inflammasome activation, reduced the level of active caspase-1, secretion of IL-1β and IL-18 proinflammatory cytokine levels, and the levels of pyroptosis. Additionally, we showed that formoterol activates autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair, which are significant pathways in the inhibition of NLRP3 inflammasome activation and pyroptosis. Our study suggests that formoterol efficaciously prevents the NLRP3 inflammasome activation and pyroptosis in microglial cells regulation through IκBα/NF-κB, autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair.

Background

Tau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies.

Methods

To understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons.

Results

FTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases.

Conclusions

Our results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.

Hypertension-induced brain renin-angiotensin system (RAS) activation and neuroinflammation are hallmark neuropathological features of neurodegenerative diseases. Previous studies from our lab have shown that inhibition of ACE/Ang II/AT1R axis (by AT1R blockers or ACE inhibitors) reduced neuroinflammation and accompanied neurodegeneration via up-regulating adult hippocampal neurogenesis. Apart from this conventional axis, another axis of RAS also exists i.e., ACE2/Ang (1–7)/MasR axis, reported as an anti-hypertensive and anti-inflammatory. However, the role of this axis has not been explored in hypertension-induced glial activation and hippocampal neurogenesis in rat models of hypertension. Hence, in the present study, we examined the effect of ACE2 activator, Diminazene aceturate (DIZE) at 2 different doses of 10 mg/kg (non-antihypertensive) and 15 mg/kg (antihypertensive dose) in renovascular hypertensive rats to explore whether their effect on glial activation, neuroinflammation, and neurogenesis is either influenced by blood-pressure. The results of our study revealed that hypertension induced significant glial activation (astrocyte and microglial), neuroinflammation, and impaired hippocampal neurogenesis. However, ACE2 activation by DIZE, even at the low dose prevented these hypertension-induced changes in the brain. Mechanistically, ACE2 activation inhibited Ang II levels, TRAF6-NFκB mediated inflammatory signaling, NOX4-mediated ROS generation, and mitochondrial dysfunction by upregulating ACE2/Ang (1–7)/MasR signaling. Moreover, DIZE-induced activation of the ACE2/Ang (1–7)/MasR axis upregulated Wnt/β-catenin signaling, promoting hippocampal neurogenesis during the hypertensive state. Therefore, our study demonstrates that ACE2 activation can effectively prevent glial activation and enhance hippocampal neurogenesis in hypertensive conditions, regardless of its blood pressure-lowering effects.

Alzheimer's disease (AD) is the most common form of dementia and characterized by extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tau tangles and neurodegeneration. Over 80 % of AD patients also exhibit cerebral amyloid angiopathy (CAA). CAA is a cerebrovascular disease caused by deposition of Aβ in the walls of cerebral blood vessels leading to vessel damage and impairment of normal blood flow. To date, different studies suggest that platelet function, including activation, adhesion and aggregation, is altered in AD due to vascular Aβ deposition. For example, the transgenic AD model mice APP23 mice that exhibit CAA and parenchymal Aβ plaques, show pre-activated platelets in the blood circulation and increased platelet integrin activation leading to a pro-thrombotic phenotype in these mice late stages of AD. However, it is still an open question whether or not platelets exhibit changes in their activation profile before they are exposed to vascular Aβ deposits. Therefore, the present study examined platelets from middle-aged transgenic APP23 mice at the age of 8–10 months. At this age, APP23 mice show amyloid plaques in the brain parenchyma but not in the vasculature. Our analyses show that these APP23 mice have unaltered platelet numbers and size, and unaltered surface expression of glycoproteins. However, the number of dense granules in transgenic platelets was increased while the release was unaltered. Male, but not female APP23 mice, exhibited reduced platelet activation after stimulation of the thrombin receptor PAR4 and decreased thrombus stability on collagen under flow conditions ex vivo compared to control mice. In an arterial thrombosis model in vivo, male APP23 mice showed attenuated occlusion of the injured artery compared to controls. These findings provide clear evidence for early changes in platelet activation and thrombus formation in male mice before development of overt CAA. Furthermore, reduced platelet activation and thrombus formation suggest sex-specific differences in platelet physiology in AD that has to be considered in future studies of platelets and their role in AD.

The axons containing arginine vasopressin (AVP) from the hypothalamus innervate a variety of structures including the cerebral cortex, thalamus, hippocampus and amygdala. A plethora amount of evidence indicates that activation of the V_1a subtype of the vasopressin receptors facilitates anxiety-like and fear responses. As an essential structure involved in fear and anxiety responses, the amygdala, especially the lateral nucleus of amygdala (LA), receives glutamatergic innervations from the auditory cortex and auditory thalamus where high density of V_1a receptors have been detected. However, the roles and mechanisms of AVP in these two important areas have not been determined, which prevents the understanding of the mechanisms whereby V_1a activation augments anxiety and fear responses. Here, we used coronal brain slices and studied the effects of AVP on neuronal activities of the auditory cortical and thalamic neurons. Our results indicate that activation of V_1a receptors excited both auditory cortical and thalamic neurons. In the auditory cortical neurons, AVP increased neuronal excitability by depressing multiple subtypes of inwardly rectifying K⁺ (Kir) channels including the Kir2 subfamily, the ATP-sensitive K⁺ channels and the G protein-gated inwardly rectifying K⁺ (GIRK) channels, whereas activation of V_1a receptors excited the auditory thalamic neurons by depressing the Kir2 subfamily of the Kir channels as well as activating the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and a persistent Na⁺ channel. Our results may help explain the roles of V_1a receptors in facilitating fear and anxiety responses.

Categories: Cell Physiology.

Two sphingosine kinase isoforms, sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2), synthesize the lipid sphingosine-1-phosphate (S1P) by phosphorylating sphingosine. SPHK1 is a cytoplasmic kinase, and SPHK2 is localized to the nucleus and other organelles. In the cytoplasm, the SPHK1/S1P pathway modulates autophagy and protein ubiquitination, among other processes. In the nucleus, the SPHK2/S1P pathway regulates transcription. Here, we hypothesized that the SPHK2/S1P pathway governs protein ubiquitination in neurons. We found that ectopic expression of SPHK2 increases ubiquitinated substrate levels in cultured neurons and pharmacologically inhibiting SPHK2 decreases protein ubiquitination. With mass spectrometry, we discovered that inhibiting SPHK2 affects lipid and synaptic protein networks as well as a ubiquitin-dependent protein network. Several ubiquitin-conjugating and hydrolyzing proteins, such as the E3 ubiquitin-protein ligases HUWE1 and TRIP12, the E2 ubiquitin-conjugating enzyme UBE2Z, and the ubiquitin-specific proteases USP15 and USP30, were downregulated by SPHK2 inhibition. Using RNA sequencing, we found that inhibiting SPHK2 altered lipid and neuron-specific gene networks, among others. Genes that encode the corresponding proteins from the ubiquitin-dependent protein network that we discovered with mass spectrometry were not affected by inhibiting SPHK2, indicating that the SPHK2/S1P pathway regulates ubiquitination at the protein level. We also show that both SPHK2 and HUWE1 were upregulated in the striatum of a mouse model of Huntington's disease, the BACHD mice, indicating that our findings are relevant to neurodegenerative diseases. Our results identify SPHK2/S1P as a novel regulator of protein ubiquitination networks in neurons and provide a new target for developing therapies for neurodegenerative diseases.

Recent advances in immunotherapeutic approaches to the treatment of Alzheimer's disease (AD) have increased the importance of understanding the exact binding preference of each amyloid-beta (Aβ) antibody employed, since this determines both efficacy and risk for potentially serious adverse events known as amyloid-related imaging abnormalities. Lecanemab is a humanized IgG1 antibody that was developed to target the soluble Aβ protofibril conformation. The present study prepared extracts of post mortem brain samples from AD patients and non-demented elderly controls, characterized the forms of Aβ present, and investigated their interactions with lecanemab. Brain tissue samples were homogenized and extracted using tris-buffered saline. Aβ levels and aggregation states in soluble and insoluble extracts, and in fractions prepared using size-exclusion chromatography or density gradient ultracentrifugation, were analyzed using combinations of immunoassay, immunoprecipitation (IP), and mass spectrometry. Lecanemab immunohistochemistry was also conducted in temporal cortex. The majority of temporal cortex Aβ (98 %) was in the insoluble extract. Aβ42 was the most abundant form present, particularly in AD subjects, and most soluble Aβ42 was in soluble aggregated protofibrillar structures. Aβ protofibril levels were much higher in AD subjects than in controls. Protofibrils captured by lecanemab-IP contained high levels of Aβ42 and lecanemab bound to large, medium, and small Aβ42 protofibrils in a concentration-dependent manner. Competitive IP showed that neither Aβ40 monomers nor Aβ40-enriched fibrils isolated from cerebral amyloid angiopathy reduced lecanemab's binding to Aβ42 protofibrils. Immunohistochemistry showed that lecanemab bound readily to Aβ plaques (diffuse and compact) and to intraneuronal Aβ in AD temporal cortex. Taken together, these findings indicate that while lecanemab binds to Aβ plaques, it preferentially targets soluble aggregated Aβ protofibrils. These are largely composed of Aβ42, and lecanemab binds less readily to the Aβ40-enriched fibrils found in the cerebral vasculature. This is a promising binding profile because Aβ42 protofibrils represent a key therapeutic target in AD, while a lack of binding to monomeric Aβ and cerebral amyloid deposits should reduce peripheral antibody sequestration and minimize risk for adverse events.

Amyloid beta (Aβ) peptides, which aggregate to form neocortical plaques in Alzheimer's disease, exist in states that range from soluble monomers and oligomers/protofibrils to insoluble fibrillar amyloid. The present study evaluated the effects of mAb158, a mouse monoclonal antibody version of lecanemab that preferentially binds to soluble Aβ protofibrils, in aged transgenic mice (Tg2576) with Aβ pathology. Female Tg2576 mice (12 months old) received weekly intraperitoneal mAb158 (35 mg/kg) or vehicle for 4 weeks or for 18 weeks, with or without a subsequent 12-week off-treatment period. Aβ protofibril levels were significantly lower in mAb158-treated animals at both 4 and 18 weeks, while longer treatment duration (18 weeks) was required to observe significantly lower Aβ42 levels in insoluble brain fractions and lower Aβ plaque load. Following the off-treatment period, comparison of the vehicle- and mAb158-treated mice demonstrated that the Aβ protofibril levels, insoluble Aβ42 levels and Aβ plaque load remained significantly lower in mAb158-treated animals, as compared with age-matched controls. However, there was a significant increase of brain accumulation of both the Aβ protofibril levels, insoluble Aβ42 levels and Aβ plaque load after treatment cessation. Thus, repeated mAb158 treatment of aged Tg2576 mice first reduced Aβ protofibril levels within 4 weeks of treatment, which then was followed by a reduction of amyloid plaque pathology within 18 weeks of treatment. These effects were maintained 12 weeks after the final dose, indicating that mAb158 had a disease-modifying effect on the Aβ pathology in this mouse model. In addition, brain accumulation of both Aβ protofibril levels and amyloid pathology progressed after discontinuation of the treatment which supports the importance of continued treatment with mAb158 to maintain the effects on Aβ pathology.

As the main players in the central nervous system (CNS), neurons dominate most life activities. However, after accidental trauma or neurodegenerative diseases, neurons are unable to regenerate themselves. The loss of this important role can seriously affect the quality of life of patients, ranging from movement disorders to disability and even death. There is no suitable treatment to prevent or reverse this process. Therefore, the regeneration of neurons after loss has been a major clinical problem and the key to treatment. Replacing the lost neurons by transdifferentiation of other cells is the only viable approach. Although much progress has been made in stem cell therapy, ethical issues, immune rejection, and limited cell sources still hinder its clinical application. In recent years, somatic cell reprogramming technology has brought a new dawn. Among them, astrocytes, as endogenously abundant cells homologous to neurons, have good potential and application value for reprogramming into neurons, having been reprogrammed into neurons in vitro and in vivo in a variety of ways.