Neuronal cell cycle reentry events in the aging brain are more prevalent in neurodegeneration and lead to cellular senescence.

Increasing evidence indicates that terminally differentiated neurons in the brain may recommit to a cell cycle-like process during neuronal aging and under disease conditions. Because of the rare existence and random localization of these cells in the brain, their molecular profiles and disease-specific heterogeneities remain unclear. Through a bioinformatics approach that allows integrated analyses of multiple single-nucleus transcriptome datasets from human brain samples, these rare cell populations were identified and selected for further characterization. Our analyses indicated that these cell cycle-related events occur predominantly in excitatory neurons and that cellular senescence is likely their immediate terminal fate. Quantitatively, the number of cell cycle re-engaging and senescent neurons decreased during the normal brain aging process, but in the context of late-onset Alzheimer's disease (AD), these cells accumulate instead. Transcriptomic profiling of these cells suggested that disease-specific differences were predominantly tied to the early stage of the senescence process, revealing that these cells presented more proinflammatory, metabolically deregulated, and pathology-associated signatures in disease-affected brains. Similarly, these general features of cell cycle re-engaging neurons were also observed in a subpopulation of dopaminergic neurons identified in the Parkinson's disease (PD)-Lewy body dementia (LBD) model. An extended analysis conducted in a mouse model of brain aging further validated the ability of this bioinformatics approach to determine the robust relationship between the cell cycle and senescence processes in neurons in this cross-species setting.

Clinical characteristics of unvaccinated or incompletely vaccinated children with neurological manifestations due to SARS-CoV-2 Omicron infection.

Omicron generally causes milder disease than previous strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially in fully vaccinated individuals. However, incompletely vaccinated children may develop Omicron-related complications such as those affecting the central nervous system. To characterize the spectrum of clinical manifestations of neuro-COVID and to identify potential biomarkers associated with clinical outcomes, we recruited 15 children hospitalized for Omicron-related neurological manifestations in three hospitals in Hong Kong (9 boys and 6 girls aged 1-13 years). All were unvaccinated or incompletely vaccinated. Fourteen (93.3%) were admitted for convulsion, including benign febrile seizure (n = 7), complex febrile seizure (n = 2), seizure with fever (n = 3), and recurrent breakthrough seizure (n = 2), and the remaining nonconvulsive patient developed encephalopathic state with impaired consciousness. None of the seven children with benign febrile seizure and six of eight children with other neurological manifestations had residual deficits at 9-month follow-up. SARS-CoV-2 RNA was undetectable in the cerebrospinal fluid (CSF) specimens of seven patients who underwent lumbar puncture. Spike-and-wave/sharp waves affecting the frontal lobes were detected in four of seven (57.1%) patients who underwent electroencephalogram. Children with Omicron-related neurological manifestations had significantly higher blood levels of IL-6 (p < 0.001) and CHI3L1 (p = 0.022) than healthy controls, and higher CSF levels of IL-6 (p = 0.002) than children with non-COVID-19-related febrile illnesses. Higher CSF-to-blood ratios of IL-8 and CHI3L1 were associated with longer length of stay, whereas higher ratios of IL-6 and IL-8 were associated with higher blood tau level. The role of CSF:blood ratio of IL-6, IL-8, and CHI3L1 as prognostic markers for neuro-COVID should be further evaluated.

Cross-talk between DNA damage response and the central carbon metabolic network underlies selective vulnerability of Purkinje neurons in ataxia-telangiectasia.

Cerebellar ataxia is often the first and irreversible outcome in the disease of ataxia-telangiectasia (A-T), as a consequence of selective cerebellar Purkinje neuronal degeneration. A-T is an autosomal recessive disorder resulting from the loss-of-function mutations of the ataxia-telangiectasia-mutated ATM gene. Over years of research, it now becomes clear that functional ATM-a serine/threonine kinase protein product of the ATM gene-plays critical roles in regulating both cellular DNA damage response and central carbon metabolic network in multiple subcellular locations. The key question arises is how cerebellar Purkinje neurons become selectively vulnerable when all other cell types in the brain are suffering from the very same defects in ATM function. This review intended to comprehensively elaborate the unexpected linkages between these two seemingly independent cellular functions and the regulatory roles of ATM involved, their integrated impacts on both physical and functional properties, hence the introduction of selective vulnerability to Purkinje neurons in the disease will be addressed.

Chronic alcohol metabolism results in DNA repair infidelity and cell cycle-induced senescence in neurons.

Chronic binge-like drinking is a risk factor for age-related dementia, however, the lasting and irreversible effect of alcohol on the brain remains elusive. Transcriptomic changes in brain cortices revealed pro-ageing hallmarks upon chronic ethanol exposure and these changes predominantly occur in neurons. The changes are attributed to a prioritized ethyl alcohol oxidation in these cells via the NADPH-dependent cytochrome pathway. This hijacks the folate metabolism of the 1-carbon network which supports the pathway choice of DNA repair via the non-cell cycle-dependent mismatch repair networks. The lost-in-function of such results in the de-inactivation of the less preferred cell cycle-dependent homologous recombination (HR) repair, forcing these post-mitotic cells to re-engage in a cell cycle-like process. However, mature neurons are post-mitotic. Therefore, instead of successfully completing a full round of cell cycle which is necessary for the completion of HR-mediated repair; these cells are arrested at checkpoints. The resulting persistence of repair intermediates induces and promotes the nuclear accumulation of p21 and cyclin B-a trigger for permanent cell cycle exits and irreversible senescence response. Supplementation of bioactive 5-methyl tetrahydrofolate simultaneously at times with ethyl alcohol exposure supports the fidelity of the 1-carbon network and hence the activity of the mismatch repair. This prevents aberrant and irreversible cell cycle re-entry and senescence events of neurons. Together, our findings offer a direct connection between binge-drinking behaviour and its irreversible impact on the brain, which makes it a potential risk factor for dementia.

Insulin stimulates atypical protein kinase C-mediated phosphorylation of the neuronal adaptor FE65 to potentiate neurite outgrowth by activating ARF6-Rac1 signaling.

Neurite outgrowth is a fundamental process in neurons that produces extensions and, consequently, neural connectivity. Neurite damage and atrophy are observed in various brain injuries and disorders. Understanding the intrinsic pathways of neurite outgrowth is essential for developing strategies to stimulate neurite regeneration. Insulin is a pivotal hormone in the regulation of glucose homeostasis. There is increasing evidence for the neurotrophic functions of insulin, including the induction of neurite outgrowth. However, the associated mechanism remains elusive. Here, we demonstrate that insulin potentiates neurite outgrowth mediated by the small GTPases ADP-ribosylation factor 6 (ARF6) and Ras-related C3 botulinum toxin substrate 1 (Rac1) through the neuronal adaptor FE65. Moreover, insulin enhances atypical protein kinase Cι/λ (PKCι/λ) activation and FE65 phosphorylation at serine 459 (S459) in neurons and mouse brains. In vitro and cellular assays show that PKCι/λ phosphorylated FE65 at S459. Consistently, insulin potentiates FE65 S459 phosphorylation only in the presence of PKCι/λ. Phosphomimetic studies show that an FE65 S459E mutant potently activates ARF6, Rac1, and neurite outgrowth. Notably, this phosphomimetic mutation enhances the FE65-ARF6 interaction, a process that promotes ARF6-Rac1-mediated neurite outgrowth. Likewise, insulin treatment and PKCι/λ overexpression potentiate the FE65-ARF6 interaction. Conversely, PKCι/λ knockdown suppresses the stimulatory effect of FE65 on ARF6-Rac1-mediated neurite outgrowth. The effect of insulin on neurite outgrowth is also markedly attenuated in PKCι/λ knockdown neurons, in the presence and absence of FE65. Our findings reveal a novel mechanism linking insulin with ARF6-Rac1-dependent neurite extension through the PKCι/λ-mediated phosphorylation of FE65.

Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of TP53 gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape. Given the high mutation frequency of TP53 in TNBC cells, restoration of p53 function could be a potential strategy to overcome their resistance to anti-programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) therapy. Herein, we have assessed the use of Pos3Aa crystal-based platform to mediate the intracellular delivery of p53 protein to restore p53 activity in p53 null tumors and consequently augment anti-PD-1 activity. METHODS: The efficiency of Pos3Aa-p53 crystals in delivering p53 protein was evaluated using confocal imaging, immunofluorescence staining, flow cytometry and RNA-seq. The ability of Pos3Aa-p53 crystals to remodel tumor microenvironment was investigated by examining the markers of immunogenic cell death (ICD) and the expression of PD-L1, indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase 2 and type I interferon (IFN). Finally, both unilateral and bilateral 4T1 tumor mouse models were utilized to assess the efficacy of Pos3Aa-p53 crystal-mediated p53 restoration in enhancing the antitumor activity of ICIs. T cells in tumor tissues and spleens were analyzed, and the in vivo biosafety of the Pos3Aa-p53 crystal/anti-PD-1 antibody combination was also evaluated. RESULTS: Delivery of p53 protein into p53-null TNBC 4T1 cells via Pos3Aa-p53 crystals restored the p53 activity, and therefore led to the induction of ICD, activation of type I IFN signaling and upregulation of PD-L1 expression. Pos3Aa-p53 crystals significantly enhanced T cell infiltration and activation in 4T1 tumors, thereby sensitizing them to anti-PD-1 therapy. The combination of Pos3Aa-p53 crystals with anti-PD-1 antibody also induced a systemic antitumor immunity resulting in the inhibition of distal tumor growth with minimal toxicity. CONCLUSION: This study validates that p53 restoration can be an effective approach to overcome ICI resistance and demonstrates that intracellular delivery of p53 protein can be an efficient, safe and potentially universal strategy to restore p53 activity in tumors carrying TP53 mutation.

A peptide inhibitor that rescues polyglutamine-induced synaptic defects and cell death through suppressing RNA and protein toxicities.

Polyglutamine (polyQ) diseases, including spinocerebellar ataxias and Huntington's disease, are progressive neurodegenerative disorders caused by CAG triplet-repeat expansion in the coding regions of disease-associated genes. In this study, we found that neurotoxic small CAG (sCAG) RNA species, microscopic Ataxin-2 CAG RNA foci, and protein aggregates exist as independent entities in cells. Synaptic defects and neurite outgrowth abnormalities were observed in mutant Ataxin-2-expressing mouse primary cortical neurons. We examined the suppression effects of the CAG RNA-binding peptide beta-structured inhibitor for neurodegenerative diseases (BIND) in mutant Ataxin-2-expressing mouse primary cortical neurons and found that both impaired synaptic phenotypes and neurite outgrowth defects were rescued. We further demonstrated that BIND rescued cell death through inhibiting sCAG RNA production, Ataxin-2 CAG RNA foci formation, and mutant Ataxin-2 protein translation. Interestingly, when the expanded CAG repeats in the mutant Ataxin-2 transcript was interrupted with the alternative glutamine codon CAA, BIND's inhibitory effect on mutant protein aggregation was lost. We previously demonstrated that BIND interacts physically and directly with expanded CAG RNA sequences. Our data provide evidence that the BIND peptide associates with transcribed mutant CAG RNA to inhibit the formation of toxic species, including sCAG RNA, RNA foci, and polyQ protein translation and aggregation.

Low-Density Lipoprotein Receptor-Related Protein 6 Cell Surface Availability Regulates Fuel Metabolism in Astrocytes.

Early changes in astrocyte energy metabolism are associated with late-onset Alzheimer's disease (LOAD), but the underlying mechanism remains elusive. A previous study suggested an association between a synonymous SNP (rs1012672, C→T) in LRP6 gene and LOAD; and that is indeed correlated with diminished LRP6 gene expression in the frontal cortex region. The authors show that LRP6 is a unique Wnt coreceptor on astrocytes, serving as a bimodal switch that modulates their metabolic landscapes. The Wnt-LRP6 mediated mTOR-AKT axis is essential for sustaining glucose metabolism. In its absence, Wnt switches to activate the LRP6-independent Ca2+ -PKC-NFAT axis, resulting in a transcription network that favors glutamine and branched chain amino acids (BCAAs) catabolism over glucose metabolism. Exhaustion of these raw materials essential for neurotransmitter biosynthesis and recycling results in compromised synaptic, cognitive, and memory functions; priming for early changes that are frequently found in LOAD. The authors also highlight that intranasal supplementation of glutamine and BCAAs is effective in preserving neuronal integrity and brain functions, proposing a nutrient-based method for delaying cognitive and memory decline when LRP6 cell surface levels and functions are suboptimal.

CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration.

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.