Neuronal double-stranded DNA accumulation induced by DNase II deficiency drives tau phosphorylation and neurodegeneration.

BACKGROUND: Deoxyribonuclease 2 (DNase II) plays a key role in clearing cytoplasmic double-stranded DNA (dsDNA). Deficiency of DNase II leads to DNA accumulation in the cytoplasm. Persistent dsDNA in neurons is an early pathological hallmark of senescence and neurodegenerative diseases including Alzheimer's disease (AD). However, it is not clear how DNase II and neuronal cytoplasmic dsDNA influence neuropathogenesis. Tau hyperphosphorylation is a key factor for the pathogenesis of AD. The effect of DNase II and neuronal cytoplasmic dsDNA on neuronal tau hyperphosphorylation remains unclarified. METHODS: The levels of neuronal DNase II and dsDNA in WT and Tau-P301S mice of different ages were measured by immunohistochemistry and immunolabeling, and the levels of DNase II in the plasma of AD patients were measured by ELISA. To investigate the impact of DNase II on tauopathy, the levels of phosphorylated tau, phosphokinase, phosphatase, synaptic proteins, gliosis and proinflammatory cytokines in the brains of neuronal DNase II-deficient WT mice, neuronal DNase II-deficient Tau-P301S mice and neuronal DNase II-overexpressing Tau-P301S mice were evaluated by immunolabeling, immunoblotting or ELISA. Cognitive performance was determined using the Morris water maze test, Y-maze test, novel object recognition test and open field test. RESULTS: The levels of DNase II were significantly decreased in the brains and the plasma of AD patients. DNase II also decreased age-dependently in the neurons of WT and Tau-P301S mice, along with increased dsDNA accumulation in the cytoplasm. The DNA accumulation induced by neuronal DNase II deficiency drove tau phosphorylation by upregulating cyclin-dependent-like kinase-5 (CDK5) and calcium/calmodulin activated protein kinase II (CaMKII) and downregulating phosphatase protein phosphatase 2A (PP2A). Moreover, DNase II knockdown induced and significantly exacerbated neuron loss, neuroinflammation and cognitive deficits in WT and Tau-P301S mice, respectively, while overexpression of neuronal DNase II exhibited therapeutic benefits. CONCLUSIONS: DNase II deficiency and cytoplasmic dsDNA accumulation can initiate tau phosphorylation, suggesting DNase II as a potential therapeutic target for tau-associated disorders.

Thrombomodulin reduces α-synuclein generation and ameliorates neuropathology in a mouse model of Parkinson's disease.

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD. Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1ß, which is subsequently secreted into the extracellular space. This secreted IL-1ß then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.

ArhGAP11A mediates amyloid-ß generation and neuropathology in an Alzheimer's disease-like mouse model.

Amyloid-ß (Aß) plays an important role in the neuropathology of Alzheimer's disease (AD), but some factors promoting Aß generation and Aß oligomer (Aßo) neurotoxicity remain unclear. We here find that the levels of ArhGAP11A, a Ras homology GTPase-activating protein, significantly increase in patients with AD and amyloid precursor protein (APP)/presenilin-1 (PS1) mice. Reducing the ArhGAP11A level in neurons not only inhibits Aß generation by decreasing the expression of APP, PS1, and ß-secretase (BACE1) through the RhoA/ROCK/Erk signaling pathway but also reduces Aßo neurotoxicity by decreasing the expressions of apoptosis-related p53 target genes. In APP/PS1 mice, specific reduction of the ArhGAP11A level in neurons significantly reduces Aß production and plaque deposition and ameliorates neuronal damage, neuroinflammation, and cognitive deficits. Moreover, Aßos enhance ArhGAP11A expression in neurons by activating E2F1, which thus forms a deleterious cycle. Our results demonstrate that ArhGAP11A may be involved in AD pathogenesis and that decreasing ArhGAP11A expression may be a promising therapeutic strategy for AD treatment.

The immunodominant and neutralization linear epitopes for SARS-CoV-2.

Although vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulate the 3D structures and predict the B cell epitopes on the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches and validate epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induce antibody production, six of these are immunodominant epitopes in individuals, and 23 are conserved within SARS-CoV-2, SARS-CoV, and bat coronavirus RaTG13. We find that the immunodominant epitopes of individuals with domestic (China) SARS-CoV-2 are different from those of individuals with imported (Europe) SARS-CoV-2, which may be caused by mutations on the S (G614D) and N proteins. Importantly, we find several epitopes on the S protein that elicit neutralizing antibodies against D614 and G614 SARS-CoV-2, which can contribute to vaccine design against coronaviruses.

The Role of α-Synuclein Oligomers in Parkinson's Disease.

α-synuclein (α-syn) is a protein associated with the pathogenesis of Parkinson's disease (PD), the second most common neurodegeneration disease with no effective treatment. However, how α-syn drives the pathology of PD remains elusive. Recent studies suggest that α-syn oligomers are the primary cause of neurotoxicity and play a critical role in PD. In this review, we discuss the process of α-syn oligomers formation and the current understanding of the structures of oligomers. We also describe seed and propagation effects of oligomeric forms of α-syn. Then, we summarize the mechanism by which α-syn oligomers exert neurotoxicity and promote neurodegeneration, including mitochondrial dysfunction, endoplasmic reticulum stress, proteostasis dysregulation, synaptic impairment, cell apoptosis and neuroinflammation. Finally, we investigate treatment regimens targeting α-syn oligomers at present. Further research is needed to understand the structure and toxicity mechanism of different types of oligomers, so as to provide theoretical basis for the treatment of PD.

A novel rapid modularized hepatitis B core virus-like particle-based platform for personalized cancer vaccine preparation via fixed-point coupling.

Personalized cancer vaccine which targets neoepitopes shows great promise for cancer treatment. However, rapid preparation is a critical challenge for clinical application of personalized cancer vaccine. Genetic recombination and chemical modification are a time-consuming "trial and error" pattern for making vaccines. Here we first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of hepatitis B core protein (HBc) (1-183). The resulted recombinant protein HBc(1-183)-SpyCatcher (HBc(1-183)-S) assembled to virus-like particles (VLPs) and readily bound to SpyTag conjugated with OVA epitope peptides by just mixing, forming HBc(1-183)-S-OVA. HBc(1-183)-S-OVA VLPs effectively induced dendritic cell maturation. Our further results indicated that HBc(1-183)-S-OVA VLPs vaccination inhibited tumor growth in both prophylactic and treatment ways in E.G7-OVA tumor bearing mice by generating significant OVA-specific cytotoxic T lymphocyte responses. Our study provides a simple, rapid, efficient and universal HBc-based platform for the preparation of personalized cancer vaccine.

Naturally occurring autoantibodies against α-synuclein rescues memory and motor deficits and attenuates α-synuclein pathology in mouse model of Parkinson's disease.

It has been suggested that aggregation of α-synuclein (α-syn) into oligomers leads to neurodegeneration in Parkinson's disease (PD), but intravenous immunoglobulin (IVIG) which contains antibodies against α-syn monomers and oligomers fails to treat PD mouse model. The reason may be because IVIG contains much low level of antibodies against α-syn, and of which only a small part can penetrate the blood-brain barrier, resulting in an extremely low level of effective antibodies in the brain, and limiting the beneficial effect of IVIG on PD mice. Here, we first isolated naturally occurring autoantibodies against α-syn (NAbs-α-syn) from IVIG. Our further investigation results showed that NAbs-α-syn inhibited α-syn aggregation and attenuated α-syn-induced cytotoxicity in vitro. Compared with vehicles, NAbs-α-syn significantly attenuated the memory and motor deficits by reducing the levels of soluble α-syn, total human α-syn and α-syn oligomers, decreasing the intracellular p-α-synser129 deposits and axonal pathology, inhibiting the microgliosis and astrogliosis, as well as the production of proinflammatory cytokines, increasing the levels of PSD95, synaptophysin and TH in the brain of A53T transgenic mice. These findings suggest that NAbs-α-syn overcomes the deficiency of IVIG and exhibits a promising therapeutic potential for the treatment of PD.

Hepatitis B core VLP-based mis-disordered tau vaccine elicits strong immune response and alleviates cognitive deficits and neuropathology progression in Tau.P301S mouse model of Alzheimer's disease and frontotemporal dementia.

BACKGROUND: Truncated mis-disordered tau protein plays an important role in the pathogenesis of Alzheimer's disease (AD) and frontotemporal dementia (FTD). Tau294-305, an epitope in the truncated tau, is essential for pathological tau-tau interaction and aggregation. A tau294-305-targeted approach may have beneficial effects in the treatment of AD and FTD. METHODS: In this study, we genetically fused tau294-305 epitope to the hepatitis B virus core protein (HBc) major immunodominant region (MIR) (with the resultant protein termed T294-HBc), and we subcutaneously immunized a Tau.P301S transgenic mouse model of FTD and AD with T294-HBc four times. The levels and characteristics of antibodies induced by T294-HBc were determined by enzyme-linked immunosorbent assay. The effect of T294-HBc on the cognitive deficits of Tau.P301S mice was tested using the Morris water maze test, novel object recognition, and a Y-maze test. Western blot analysis and IHC were applied to measure the effect of T294-HBc on tau pathologies and neuroinflammation in the mouse brains. RESULTS: The results showed that T294-HBc self-assembled into HBc chimeric virus-like particles (VLPs) with tau294-305 displayed on the surface and that it induced high antibody titers specifically against the mis-disordered truncated tau. Further investigation showed that these antibodies simultaneously bound to microtubule-binding regions 1-4 (MTBR1-4) [tau263-274, tau294-305, tau325-336, tau357-368 and tau294-305(P301S)]. Moreover, T294-HBc VLP vaccination significantly ameliorated memory and cognitive decline; reduced the levels of AT8-positive tau, truncated tau monomer, and oligomer; attenuated microgliosis and astrogliosis; and rescued synaptic deficits in Tau.P301S transgenic mice. CONCLUSIONS: T294-HBc VLP vaccine elicited strong immune response and alleviated cognitive deficits and neuropathology progression in Tau.P301S mice, indicating that the T294-HBc VLP vaccine has promising therapeutic potential for the treatment of AD and FTD.

Efficient co-delivery of neo-epitopes using dispersion-stable layered double hydroxide nanoparticles for enhanced melanoma immunotherapy.

Cancer immunotherapy has shown tremendous progresses in recent years for various cancers and layered double hydroxide (LDH) nanoparticles are demonstrated as effective adjuvants for protein-based vaccines. This research further shows that the colloidal stability of LDH-based vaccines significantly influences the therapeutic efficacy and LDH nanoparticles are able to adjuvant multiple tumor-associated antigen peptides to provoke strong cell-mediated immune responses for effective inhibition of cancer growth. The LDH-based multi-target therapeutic vaccines were constructed by assembling epitope peptides and CpG onto LDH nanoparticles. Using melanoma as the model cancer and Tyrosinase-related protein 2 (Trp2) peptide as the model antigen, we demonstrated that dispersion-stable LDH-based vaccine induced stronger cytotoxic T-lymphocyte (CTL) responses and significantly inhibited tumor growth in comparison with aggregated LDH-based vaccine. We further constructed multi-target dispersion-stable LDH-based vaccine by co-loading Trp2, two mutated epitopes (M27 and M30) and CpG, which showed remarkable inhibition of melanoma growth. These results suggest that dispersion-stable LDH nanoparticles are an ideal platform to load multi-antigens and immune stimulants as effective personalized therapeutic cancer vaccines.

A vaccine with Aß oligomer-specific mimotope attenuates cognitive deficits and brain pathologies in transgenic mice with Alzheimer's disease.

BACKGROUND: ß-Amyloid peptide (Aß) oligomers are initial factors used to induce Alzheimer's disease (AD) development, and Aß monomers have normal physiological function. The antibodies or vaccines against Aß monomers have serious problems, such as side effects and low curative effects. Therefore, it is essential to specifically target Aß oligomers rather than monomers for the treatment of AD. METHODS: The mimotopes of Aß oligomers were obtained by panning the phage-displayed random peptide libraries using oligomer-specific antibodies as targets and expressed on the surface of EBY100 Saccharomyces cerevisiae to generate yeast cell base vaccines. One vaccine (AOE1) induced antibodies specifically against Aß oligomers and was selected for further study. The APP/PS1 mice were subcutaneously immunized with AOE1 eight times. The levels and characteristics of antibodies induced by AOE1 were determined by enzyme-linked immunosorbent assay. The effect of AOE1 on the cognitive deficits of AD mice was tested by novel object recognition (NOR) and Y-maze. Dot blot analysis, Western blot analysis, and immunohistochemistry were applied to measure the effects of AOE1 on Aß pathologies, neuroinflammation, and microhemorrhages in the brains of AD mice. RESULTS: Eight mimotope candidates of Aß oligomers were selected and expressed on EBY100 S. cerevisiae. Only AOE1 vaccine containing mimotope L2 induced antibodies that specifically recognized Aß42 oligomers rather than monomers. AOE1 immunization significantly increased the AD mice's exploration times for the novel object in the NOR test and the choices for new arms in the Y-maze test, and it reduced levels of Aß oligomers and glial activation in the AD mouse brains. No activation of Aß-specific T cells and microhemorrhages was observed in their brains following AOE1 vaccination. CONCLUSIONS: AOE1 is the first vaccine applying the oligomer-specific mimotope as an immunogen, which could induce antibodies with high specificity to Aß oligomers. AOE1 immunization attenuated Aß pathologies and cognitive deficits in AD mice, decreased the overactivation of glial cells, and did not induce microhemorrhage in the brains of AD mice. These findings suggest that AOE1 may be a safer and more effective vaccine for AD treatment.

MgAl-layered double hydroxide nanoparticles co-delivering siIDO and Trp2 peptide effectively reduce IDO expression and induce cytotoxic T-lymphocyte responses against melanoma tumor in mice.

Active immunotherapy has shown promising potential for cancer treatment. However, there still remain major challenges including induction of a potent and specific T-cell response against the endogenous antigen and retention of tumor immunity. To address these problems, we used layered double hydroxide (LDH) nanoparticles (NPs) to co-deliver tyrosinase-related protein 2 (Trp2) and indoleamine 2,3-dioxygenase siRNA (siIDO) to dendritic cells (DCs). These LDH NPs were readily taken in by DCs, and escaped from endosomes into the cytoplasm. Compared with free Trp2 peptide or siIDO, the vaccination with the LDH NPs co-delivering Trp2 and siIDO significantly inhibited tumor growth in melanoma mouse models by relieving IDO-mediated immune suppression and increasing naïve and specific T cell activation process in vivo. Thus, these LDH NPs, which have a high loading capacity for peptide or siRNA effectively protect and deliver Trp2 and siIDO, overcome the immune tolerance and strengthen T cell immunity, are potential therapeutics to enhance cancer treatment.

Naturally occurring autoantibodies against Aß oligomers exhibited more beneficial effects in the treatment of mouse model of Alzheimer's disease than intravenous immunoglobulin.

Alzheimer's disease (AD) is characterized by memory loss, intracellular neurofibrillary tangles, and extracellular plaque deposits composed of ß-amyloid (Aß). Previous reports showed that naturally occurring autoantibodies, such as intravenous immunoglobulin (IVIG), benefited patients with moderate-stage AD who carried an APOE-ε4 allele. However, the mechanism underlying the role of IVIG remains unclear. In this study, we identified naturally occurring autoantibodies against Aß oligomers (NAbs-Aßo), which were purified by Aß42 oligomer or Cibacron Blue affinity chromatography from IVIG and termed as Oli-NAbs and Blue-NAbs, respectively. Oli-NAbs and Blue-NAbs recognized Aß42 oligomers or both Aß40 and 42 oligomers, differently. Both antibodies inhibited Aß42 aggregation and attenuated Aß42-induced cytotoxicity. Compared with vehicles, Oli-NAbs, Blue-NAbs and IVIG significantly improved the memory and cognition, and reduced the soluble and oligomeric Aß levels in APPswe/PS1dE9 transgenic mice. Further investigation showed that Blue-NAbs at increased doses effectively decreased plaque burden and insoluble Aß levels, whereas Oli-NAbs significantly declined the microgliosis and astrogliosis, as well as the production of proinflammatory cytokines in vivo. Therefore, high levels of these antibodies against oligomeric Aß40 or Aß42 were required, correspondingly, to achieve the optimal effect. NAbs-Aßo could be condensed to a high concentration by affinity chromatography and its isolation from IVIG may not interfere with the normal function of conventional IVIG as its concentration is very low. Thus, the isolated NAbs-Aßo as an extra product of plasma required low cost and the enriched NAbs-Aßo may be more feasible than IVIG for the treatment of AD.

Decreasing oxidative stress and neuroinflammation with a multifunctional peptide rescues memory deficits in mice with Alzheimer disease.

Alzheimer disease (AD) is characterized by extracellular senile plaques, intracellular neurofibrillary tangles, and memory loss. Aggregated amyloid-ß (Aß), oxidative stress, and inflammation have pivotal roles in the pathogenesis of AD. Therefore, the inhibition of Aß-induced neurotoxicity, oxidative stress, and inflammation is a potential therapeutic strategy for the treatment of AD. In this study, a heptapeptide, isolated from a Ph.D.-C7C library by phage display, attenuated Aß42-induced cytotoxicity in SH-SY5Y neuroblastoma cells and reduced Aß42-induced oxidative stress by decreasing the production of reactive oxygen species and glutathione disulfide. As a result, glutathione level increased and superoxide dismutase and glutathione peroxidase activities were enhanced in vitro and in vivo. This peptide also suppressed the inflammatory response by decreasing the release of proinflammatory cytokines, such as tumor necrosis factor α and interleukin 1ß, in microglia and by reducing microgliosis and astrogliosis in AD transgenic mice. This peptide was intracerebroventricularly administered to APPswe/PS1dE9 transgenic mice. We found that this peptide significantly improved spatial memory and reduced the amyloid plaque burden and soluble and insoluble Aß levels. Our findings suggest that this multifunctional peptide has therapeutic potential for an Aß-targeted treatment of AD.