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Synucleinopathies refer to a range of neurodegenerative diseases caused by abnormal α-synuclein (α-Syn) deposition, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Their pathogenesis is strongly linked to microglial dysfunction and neuroinflammation, which involves the leucine-rich-repeat kinase 2 (LRRK2)-regulated nuclear factor of activated T-cells (NFAT). Of the NFAT family, NFATc1 has been found to be increasingly translocated into the nucleus in α-syn stimulation. However, the specific role of NFATc1-mediated intracellular signaling in PD remains elusive in regulating microglial functions. In the current study, we crossbred LRRK2 or NFATc1 conditional knockout mice with Lyz2Cre mice to generate mice with microglia-specific deletion of LRRK2 or NFATc1, and by stereotactic injection of fibrillary α-Syn, we generated PD models in these mice. We found that LRRK2 deficiency enhanced microglial phagocytosis in the mice after α-Syn exposure and that genetic inhibition of NFATc1 markedly diminished phagocytosis and α-Syn elimination. We further demonstrated that LRRK2 negatively regulated NFATc1 in α-Syn-treated microglia, in which microglial LRRK2-deficiency facilitated NFATc1 nuclear translocation, CX3CR1 upregulation, and microglia migration. Additionally, NFATc1 translocation upregulated the expression of Rab7 and promoted the formation of late lysosomes, resulting in α-Syn degradation. In contrast, the microglial NFATc1 deficiency impaired CX3CR1 upregulation and the formation of Rab7-mediated late lysosomes. These findings highlight the critical role of NFATc1 in modulating microglial migration and phagocytosis, in which the LRRK2-NFATc1 signaling pathway regulates the expression of microglial CX3CR1 and endocytic degradative Rab7 to attenuate α-synuclein immunotoxicity.

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Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

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The disruption of nucleus accumbens (NAc) function impacts mood and learning behavior in α-Synucleinopathy, in which microglial synaptic pruning plays a pivotal role in modulating the neuropathologic progression. Available literature documents that in microglia, the activation of cannabinoid receptor 2 (CB2R) decreases inflammation, but it remains obscured regarding the roles of CB2R in microglia-mediated synaptic pruning in the NAc during the neuropathological progression of α-Synucleinopathy. We adopted the fibrillar α-Synuclein (α-Syn) treatment to characterize the effect of genetic CB2R deletion on microglial function and the signaling pathway. CB2R knockout (CB2-/-) mice and wild-type (CB2+/+) mice were divided into the α-Syn or saline treatment groups. Biochemical and microscopy approaches, including immunofluorescence, real-time PCR, and western blotting, were employed to assess the changes in homeostasis of synaptic pruning in NAc under the α-Syn-induced microglia. Moreover, the underlying mechanisms of CB2R on α-Syn induced microglial activity was assessed in vitro. After the injection of α-Syn into the NAc, distinct microglial morphological changes and M1 phenotype transformation were observed between CB2-/- and CB2+/+ mice. Meanwhile, after the α-Syn treatment, CB2-/- mice showed an increased upregulation of CD68 protein and IL-1ß mRNA but decreased brain-derived neurotrophic factor (BDNF) and TGF-ß mRNA compared with CB2+/+ mice. Additionally, CB2-/- microglia after the treatment showed a highly enriched complement 3a receptor (C3aR) producing excessive pruning of cholinergic synapses but less engulfment of dopaminergic synapses. Mechanistically, the loss of CB2R function in the α-Syn stimulation triggered c-Fos activation in microglia, but not in neurons. Further inhibition of microglial CB2R functions under α-Syn stimulation activated the phosphorylated cAMP-response element-binding protein (pCREB)-c-Fos, which was closely related to the C3aR upregulation. Our results reveal a critical and mechanistic role of CB2R in altering the microglial function and its value in the homeostasis of synaptic circuits in the NAc under the α-Syn pathology.

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Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with pronounced sleep disorders or cognitive dysfunction in neurodegenerative diseases. However, the effects of LRRK2 deficiency on sleep rhythms and sleep deprivation-related cognitive changes, and the relevant underlying mechanism, remain unrevealed. In this study, Lrrk2-/- and Lrrk2+/+ mice were subjected to normal sleep (S) or sleep deprivation (SD). Sleep recording, behavioral testing, Golgi-cox staining, immunofluorescence, and real-time PCR were employed to evaluate the impacts of LRRK2 deficiency on sleep behaviors and to investigate the underlying mechanisms. The results showed that after SD, LRRK2-deficient mice displayed lengthened NREM and shortened REM, and reported decreased dendritic spines, increased microglial activation, and synaptic endocytosis in the prefrontal cortex. Meanwhile, after SD, LRRK2 deficiency aggravated cognitive impairments, especially in the recall memory cued by fear conditioning test. Our findings evidence that LRRK2 modulates REM/NREM sleep and its deficiency may exacerbate sleep deprivation-related cognitive disorders by perturbing synaptic plasticity and microglial synaptic pruning in mice.

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Pichia pastoris, an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris. First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, ß-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.

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BACKGROUND: Spinal cord injury (SCI) triggers the primary mechanical injury and secondary inflammation-mediated injury. Neuroinflammation-mediated insult causes secondary and extensive neurological damage after SCI. Microglia play a pivotal role in the initiation and progression of post-SCI neuroinflammation. METHODS: To elucidate the significance of LRCH1 to microglial functions, we applied lentivirus-induced LRCH1 knockdown in primary microglia culture and tested the role of LRCH1 in microglia-mediated inflammatory reaction both in vitro and in a rat SCI model. RESULTS: We found that LRCH1 was downregulated in microglia after traumatic SCI. LRCH1 knockdown increased the production of pro-inflammatory cytokines such as IL-1ß, TNF-α, and IL-6 after in vitro priming with lipopolysaccharide and adenosine triphosphate. Furthermore, LRCH1 knockdown promoted the priming-induced microglial polarization towards the pro-inflammatory inducible nitric oxide synthase (iNOS)-expressing microglia. LRCH1 knockdown also enhanced microglia-mediated N27 neuron death after priming. Further analysis revealed that LRCH1 knockdown increased priming-induced activation of p38 mitogen-activated protein kinase (MAPK) and Erk1/2 signaling, which are crucial to the inflammatory response of microglia. When LRCH1-knockdown microglia were adoptively injected into rat spinal cords, they enhanced post-SCI production of pro-inflammatory cytokines, increased SCI-induced recruitment of leukocytes, aggravated SCI-induced tissue damage and neuronal death, and worsened the locomotor function. CONCLUSION: Our study reveals for the first time that LRCH1 serves as a negative regulator of microglia-mediated neuroinflammation after SCI and provides clues for developing novel therapeutic approaches against SCI.

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