Hybrid Membrane-Coated Nanoparticles for Precise Targeting and Synergistic Therapy in Alzheimer's Disease.

The blood brain barrier (BBB) limits the application of most therapeutic drugs for neurological diseases (NDs). Hybrid cell membrane-coated nanoparticles derived from different cell types can mimic the surface properties and functionalities of the source cells, further enhancing their targeting precision and therapeutic efficacy. Neuroinflammation has been increasingly recognized as a critical factor in the pathogenesis of various NDs, especially Alzheimer's disease (AD). In this study, a novel cell membrane coating is designed by hybridizing the membrane from platelets and chemokine (C-C motif) receptor 2 (CCR2) cells are overexpressed to cross the BBB and target neuroinflammatory lesions. Past unsuccessful endeavors in AD drug development underscore the challenge of achieving favorable outcomes when utilizing single-mechanism drugs.Two drugs with different mechanisms of actions into liposomes are successfully loaded to realize multitargeting treatment. In a transgenic mouse model for familial AD (5xFAD), the administration of these drug-loaded hybrid cell membrane liposomes results in a significant reduction in amyloid plaque deposition, neuroinflammation, and cognitive impairments. Collectively, the hybrid cell membrane-coated nanomaterials offer new opportunities for precise drug delivery and disease-specific targeting, which represent a versatile platform for targeted therapy in AD.

Both gain- and loss-of-function variants of KCNA1 are associated with paroxysmal kinesigenic dyskinesia.

KCNA1 is the coding gene for Kv1.1 voltage-gated potassium-channel α subunit. Three variants of KCNA1 have been reported to manifest as paroxysmal kinesigenic dyskinesia (PKD), but the correlation between them remains unclear due to the phenotypic complexity of KCNA1 variants as well as the rarity of PKD cases. Using the whole exome sequencing followed by Sanger sequencing, we screen for potential pathogenic KCNA1 variants in patients clinically diagnosed with paroxysmal movement disorders and identify three previously unreported missense variants of KCNA1 in three unrelated Chinese families. The proband of one family (c.496G>A, p.A166T) manifests as episodic ataxia type 1, and the other two (c.877G>A, p.V293I and c.1112C>A, p.T371A) manifest as PKD. The pathogenicity of these variants is confirmed by functional studies, suggesting that p.A166T and p.T371A cause a loss-of-function of the channel, while p.V293I leads to a gain-of-function with the property of voltage-dependent gating and activation kinetic affected. By reviewing the locations of PKD-manifested KCNA1 variants in Kv1.1 protein, we find that these variants tend to cluster around the pore domain, which is similar to epilepsy. Thus, our study strengthens the correlation between KCNA1 variants and PKD and provides more information on genotype-phenotype correlations of KCNA1 channelopathy.

Identification and functional characterization of novel variants of MAPT and GRN in Chinese patients with frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common cause of dementia after Alzheimer's disease, characterized by distinct changes in behavior, personality, and language. Our study performed whole exome sequencing and repeat-primed PCR analysis in 29 unrelated FTD patients. Consequently, 2 known pathogenic variants (MAPT: p.P301L; TBK1: p.I450Kfs), and 4 novel variants (MAPT: p.R406Q, p.D430H, p.A330D; GRN: c.350-2A>G) were identified. The functional analysis results showed that phosphorylated tau levels were higher in cells expressing p.R406Q and p.D430H tau than those expressing wild-type tau, especially at the Thr205, Thr231, and Ser396 phosphorylation epitopes. Besides, the p.R406Q and p.D430H variants of MAPT impaired the ability of tau to bind to the microtubules and increased tau self-aggregation. Furthermore, we found that the c.350-2A>G variant caused exon 5 skipping. Our results showed that p.R406Q, p.D430H, and c.350-2A>G variants were classified as pathogenic. Finally, we summarized the clinical characterization of patients carrying pathogenic variants of MAPT in the East Asia populations. Our results broaden the genetic spectrum of FTD with MAPT and GRN variants.

Paroxysmal Kinesigenic Dyskinesia Caused by 16p11.2 Microdeletion and Related Clinical Features.

BACKGROUND AND OBJECTIVES: Isolated paroxysmal kinesigenic dyskinesia (PKD) is mainly caused by PRRT2 variants and TMEM151A variants. Patients with proximal 16p11.2 microdeletion (16p11.2MD) (including PRRT2) often have neurodevelopmental phenotypes, whereas a few patients have PKD. Here, we aimed to identify 16p11.2MD in patients with PKD and describe the related phenotypes. METHODS: Whole-exome sequencing and bioinformatics analysis of copy number variant (CNV) were performed in patients with PKD carrying neither PRRT2 nor TMEM151A variant. Quantitative PCR and low-coverage whole-genome sequencing verified the CNV. RESULTS: We identified 9 sporadic patients with PKD and 16p11.2MD (∼535 kb), accounting for 9.6% (9/94) of our patients. Together with 9 previously reported patients with PKD and 16p11.2MD, we found that 16p11.2MD was de novo in 11 of 12 tested patients and inherited from a parent in the other patient. And 80% (12/15) of these patients had a mild language delay, 64.3% (9/14) had compromised learning ability, 42.9% (6/14) had a mild motor delay, and 50% (6/12) had abnormal neuroimaging findings. No severe autism disorders were observed. DISCUSSION: Mild developmental problems may be overlooked. A detailed inquiry of developmental history and CNV testing are necessary to distinguish patients with 16p11.2MD from isolated PKD.

Features Differ Between Paroxysmal Kinesigenic Dyskinesia Patients with PRRT2 and TMEM151A Variants.

BACKGROUND: Mutations in proline-rich transmembrane protein 2 (PRRT2) are the major cause of paroxysmal kinesigenic dyskinesia (PKD). We recently reported transmembrane protein 151A (TMEM151A) mutations caused PKD. Herein, we aimed to conduct phenotypic comparisons of patients with PKD carrying PRRT2 variants, carrying TMEM151A variants, and carrying neither the PRRT2 nor TMEM151A variant. METHODS: Sanger sequencing of PRRT2 and TMEM151A was performed, and phenotypic characteristics were analyzed. RESULTS: In a cohort of 131 PKD probands (108 without PRRT2 variants and 23 newly recruited), five novel TMEM151A variants were identified and one (c.647C > A) occurred de novo. Together with our previous studies, PRRT2 and TMEM151A variants accounted for 34.7% (85/245) and 6.9% (17/245) of PKD probands, respectively. Compared with patients carrying PRRT2 variants, those with TMEM151A variants tended to exbibit dystonia with shorter durations, have no history of benign infantile epilepsy, and have residual attacks/aura when treated with carbamazepine/oxcarbazepine. CONCLUSIONS: Patients with TMEM151A variants have different features from patients with PRRT2 variants. © 2022 International Parkinson and Movement Disorder Society.

A novel frameshift ACTN2 variant causes a rare adult-onset distal myopathy with multi-minicores.

INTRODUCTION: Distal myopathies are a group of rare muscle disorders characterized by selective or predominant weakness in the feet and/or hands. In 2019, ACTN2 gene was firstly identified to be a cause of a new adult-onset distal muscular dystrophy calling actininopathy and another distinctly different myopathy, named multiple structured core disease (MsCD). Thus, the various phenotypes and limited mutations in ACTN2-related myopathy make the genotype-phenotype correlation hard to understand. AIMS: To investigate the clinical features and histological findings in a Chinese family with distal myopathy. Whole exome sequencing and several functional studies were performed to explore the pathogenesis of the disease. RESULTS: We firstly identified a novel frameshift variant (c.2504delT, p.Phe835Serfs*66) within ACTN2 in a family including three patients. The patients exhibited adult-onset distal myopathy with multi-minicores, which, interestingly, was more like a combination of MsCD and actininopathy. Moreover, functional analysis using muscle samples revealed that the variant significantly increased the expression level of α-actinin-2 and resulted in abnormal Z-line organization of muscle fiber. Vitro studies suggested aggregate formations might be involved in the pathogenesis of the disease. CONCLUSION: Our results expanded the phenotypes of ACTN2-related myopathy and provided helpful information to clarify the molecular mechanisms.

Clinical and genetic characteristics of Chinese patients with reducing body myopathy.

Reducing body myopathy (RBM) is a rare myopathy characterized by reducing bodies (RBs) in morphological presentation. The clinical manifestations of RBM present a wide clinical spectrum, varying from infantile lethal form through childhood and adult benign forms. FHL1 gene is the causative gene of RBM. To date, only 6 Chinese RBM patients have been reported. Here, we reported the clinical presentations and genetic findings of 3 Chinese RBM patients from two families. Two novel pathogenic variants, c.395G>A and c.401_402insGAC, were identified by whole exome sequencing. Furthermore, by reviewing previous studies, we revealed that most RBM patients manifested with an early onset, symmetric, progressive limb-girdle and axial muscle weakness with joint contractures, rigid spine or scoliosis except familial female patients who exhibited asymmetric benign muscle involvements. Our results provide insightful information to help better diagnose and understand the disease.

Bi-allelic loss of function variants in COX20 gene cause autosomal recessive sensory neuronopathy.

Sensory neuronopathies are a rare and distinct subgroup of peripheral neuropathies, characterized by degeneration of the dorsal root ganglia neurons. About 50% of sensory neuronopathies are idiopathic and genetic causes remain to be clarified. Through a combination of homozygosity mapping and whole exome sequencing, we linked an autosomal recessive sensory neuronopathy to pathogenic variants in the COX20 gene. We identified eight unrelated families from the eastern Chinese population carrying a founder variant c.41A>G (p.Lys14Arg) within COX20 in either a homozygous or compound heterozygous state. All patients displayed sensory ataxia with a decrease in non-length-dependent sensory potentials. COX20 encodes a key transmembrane protein implicated in the assembly of mitochondrial complex IV. We showed that COX20 variants lead to reduction of COX20 protein in patient's fibroblasts and transfected cell lines, consistent with a loss-of-function mechanism. Knockdown of COX20 expression in ND7/23 sensory neuron cells resulted in complex IV deficiency and perturbed assembly of complex IV, which subsequently compromised cell spare respiratory capacity and reduced cell proliferation under metabolic stress. Consistent with mitochondrial dysfunction in knockdown cells, reduced complex IV assembly, enzyme activity and oxygen consumption rate were also found in patients' fibroblasts. We speculated that the mechanism of COX20 was similar to other causative genes (e.g. SURF1, COX6A1, COA3 and SCO2) for peripheral neuropathies, all of which are functionally important in the structure and assembly of complex IV. Our study identifies a novel causative gene for the autosomal recessive sensory neuronopathy, whose vital function in complex IV and high expression in the proprioceptive sensory neuron further underlines loss of COX20 contributing to mitochondrial bioenergetic dysfunction as a mechanism in peripheral sensory neuron disease.

TGM6 might not be a specific causative gene for spinocerebellar ataxia resulting from genetic analysis and functional study.

OBJECTIVE: To investigate whether TGM6 is a specific causative gene for spinocerebellar ataxia type 35 (SCA35). MATERIALS AND METHODS: The next-generation sequencing (NGS) data consisted of 47 SCA, 762 non-SCA patients and 2827 normal controls were analyzed. The allele frequencies of low frequent and deleterious TGM6 variants were compared. Functional studies were performed in five widely distributed variants (V314M, R342Q, P347L, V391M, L517W). RESULTS: Two TGM6 detrimental variants were identified in one SCA patient, 14 in non-SCA patients and 43 in normal controls, the allele frequencies of TGM6 variants did not differ among the SCA and other controls. Seven reported pathogenic variants (c.7 + 1G > T, c.331C > T, c.1171G > A, c.1478C > T, c.1528G > C, c.1550 T > G and c.1722_1724delAGA) were identified in patients with various neurologic diseases or normal controls. All the 5 widely distributed variants led to destabilization and significantly reduction of enzymatic activity of TG6 as the reported pathogenic mutations. CONCLUSIONS: TGM6 might not be a specific causative gene for SCA35, the relevant clinical consult or diagnostic should be pay more attention.

The discriminative capacity of CSF ß-amyloid 42 and Tau in neurodegenerative diseases in the Chinese population.

INTRODUCTION: In the past few years, the ß-amyloid 42 peptide and tau protein in cerebrospinal fluid (CSF) have become primary diagnostic biomarkers in differentiating Alzheimer's disease (AD) and cognitive normal controls. As we know, several neurodegenerative diseases have been reported to overlap with AD in neuropathology and clinical symptoms. To examine the discriminative utility of these biomarkers in AD and other neurodegenerative diseases, we measured them in a cohort of Chinese population. METHODS: We measured CSF Aß42, t-tau and p-tau181 by ELISA tests and calculated the ratios of t-tau/Aß42 and p-tau181/Aß42 in 240 Chinese Han patients with AD (n = 82), frontotemporal dementia (FTD, n = 20), Huntington's disease (HD, n = 27), multiple system atrophy (MSA, n = 24), spinocerebellar ataxia type-3 (SCA3, n = 27), amyotrophic lateral sclerosis (ALS, n = 36) and controls (n = 24). RESULTS: As expected, all biomarkers showed high discriminative capacity between AD and non-AD groups (p < .05) except for the elevated CSF t-tau in FTD (p > .05). Comparing with the controls, tau related biomarkers significantly elevated in the FTD (p < .001) and MSA (p < .05) groups. Surprisingly, comparing with controls, we found that CSF Aß42 increased remarkably in the SCA3 (p < .05), HD and ALS groups (p < .001), achieving a high specificity, respectively. CONCLUSION: To our best knowledge, this is the first comprehensive study in the Han Chinese population that confirmed the discriminative utility of CSF Aß42 and tau biomarkers between AD and other neurodegenerative diseases.

Functional study and pathogenicity classification of PRRT2 missense variants in PRRT2-related disorders.

AIMS: PRRT2 variants are associated with various paroxysmal disorders. To date, more than 90 PRRT2 variants have been reported in PRRT2-related disorders. Lack of functional study in majority of missense variants makes their pathogenicity uncertain. We aim to evaluate the clinical significance of PRRT2 missense variants by performing in vitro experiments. METHODS: We systematically reviewed PRRT2-related disorders and summarized reported PRRT2 missense variants. Protein expression and subcellular localization of mutant PRRT2 were investigated in mammal cells. American College of Medical Genetics and Genomics (ACMG) guidelines were used to analyze the pathogenicity of PRRT2 missense variants. RESULTS: A total of 29 PRRT2 missense variants were identified in PRRT2-related disorders. Ten variants were observed to affect both subcellular localization and protein level, three variants only affect membrane localization, and two variants only affect protein level. According to ACMG guidelines, 15 variants were finally classified as "likely pathogenic", three as "benign", three as "likely benign", and eight as "uncertain significance" variants. The likely pathogenic variants were concentrated in the C-terminal of PRRT2. CONCLUSIONS: The pathogenicity of eight uncertain significance variants needs further investigation. C-terminal of PRRT2 is crucial for its physiological function.

Mutation screening in Chinese patients with familial Alzheimer's disease by whole-exome sequencing.

Familial Alzheimer's disease (FAD) is characterized by a positive family history of dementia and typically occurs at an early age with an autosomal dominant pattern of inheritance. Amyloid precursor protein (APP), presenilin1 (PSEN1), and presenilin2 (PSEN2) are the major causative genes of FAD. The spectrum of mutations in patients with FAD has been investigated extensively in the Caucasian population but rarely in the Chinese population. Here, we performed whole-exome sequencing in a total of 15 unrelated Chinese patients with FAD. Among them, 12 were found to carry missense variants in APP, PSEN1, and PSEN2. Two novel variants (APP: p.D244G, p.K687Q), 3 variants not previously associated with FAD (APP: p.T297M, p.D332G; PSEN1: p.R157S), and 7 previously reported pathogenic variants (APP: p.V717I; PSEN1: p.M139I, p.T147I, p.L173W, p.F177S, p.R269H; PSEN2: p.V139M) were identified. The novel variant APP p.K687Q was classified as likely pathogenic, and the other 4 variants (APP: p.D244G, p.T297M, p.D332G; PSEN1: p.R157S) were classified as uncertain significance. Therefore, APP, PSEN1, and PSEN2 mutations account for 2 (25.0%), 5 (62.5%), and 1 (12.5%) of the genotyped cases positive for mutations, respectively. Furthermore, the genotype-phenotype correlations were described. Our findings broaden the genetic spectrum of FAD with APP, PSEN1, and PSEN2 variants.

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia.

In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.

SETD4 Regulates Cell Quiescence and Catalyzes the Trimethylation of H4K20 during Diapause Formation in Artemia.

As a prominent characteristic of cell life, the regulation of cell quiescence is important for proper development, regeneration, and stress resistance and may play a role in certain degenerative diseases. However, the mechanism underlying quiescence remains largely unknown. Encysted embryos of Artemia are useful for studying the regulation of this state because they remain quiescent for prolonged periods during diapause, a state of obligate dormancy. In the present study, SET domain-containing protein 4, a histone lysine methyltransferase from Artemia, was identified, characterized, and named Ar-SETD4. We found that Ar-SETD4 was expressed abundantly in Artemia diapause embryos, in which cells were in a quiescent state. Meanwhile, trimethylated histone H4K20 (H4K20me3) was enriched in diapause embryos. The knockdown of Ar-SETD4 reduced the level of H4K20me3 significantly and prevented the formation of diapause embryos in which neither the cell cycle nor embryogenesis ceased. The catalytic activity of Ar-SETD4 on H4K20me3 was confirmed by an in vitro histone methyltransferase (HMT) assay and overexpression in cell lines. This study provides insights into the function of SETD4 and the mechanism of cell quiescence regulation.

The RNA-editing deaminase ADAR is involved in stress resistance of Artemia diapause embryos.

The most widespread type of RNA editing, conversion of adenosine to inosine (A→I), is catalyzed by two members of the adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2. These enzymes edit transcripts for neurotransmitter receptors and ion channels during adaption to changes in the physical environment. In the primitive crustacean Artemia, when maternal adults are exposed to unfavorable conditions, they release diapause embryos to withstand harsh environments. The aim of the current study was therefore to elucidate the role of ADAR of Artemia diapause embryos in resistance to stress. Here, we identified Artemia ADAR (Ar-ADAR), which harbors a putative nuclear localization sequence (NLS) and two double-stranded RNA-binding motifs (dsRBMs) in the amino-terminal region and an adenosine deaminase (AD) domain in the carboxyl-terminal region. Western blot and immunofluorescence analysis revealed that Ar-ADAR is expressed abundantly in post-diapause embryos. Artemia (n = 200, three replicates) were tested under basal and stress conditions. We found that Ar-ADAR was significantly induced in response to the stresses of salinity and heat-shock. Furthermore, in vivo knockdown of Ar-ADAR (n = 100, three replicates) by RNA interference induced formation of pseudo-diapause embryos, which lack resistance to the stresses and exhibit high levels of apoptosis. These results indicate that Ar-ADAR contributes to resistance to stress in Artemia diapause embryos.

An La-related protein controls cell cycle arrest by nuclear retrograde transport of tRNAs during diapause formation in Artemia.

BACKGROUND: In eukaryotes, tRNA trafficking between the nucleus and cytoplasm is a complex process connected with cell cycle regulation. Such trafficking is therefore of fundamental importance in cell biology, and disruption of this process has grave consequences for cell viability and survival. To cope with harsh habitats, Artemia has evolved a special reproductive mode to release encysted embryos in which cell division can be maintained in a dormancy state for a long period. RESULTS: Using Artemia as a peculiar model of the cell cycle, an La-related protein from Artemia, named Ar-Larp, was found to bind to tRNA and accumulate in the nucleus, leading to cell cycle arrest and controlling the onset of diapause formation in Artemia. Furthermore, exogenous gene expression of Ar-Larp could induce cell cycle arrest in cancer cells and suppress tumor growth in a xenograft mouse model, similar to the results obtained in diapause embryos of Artemia. Our study of tRNA trafficking indicated that Ar-Larp controls cell cycle arrest by binding to tRNAs and influencing their retrograde movement from the cytoplasm to the nucleus, which is connected to pathways involved in cell cycle checkpoints. CONCLUSIONS: These findings in Artemia offer new insights into the mechanism underlying cell cycle arrest regulation, as well as providing a potentially novel approach to study tRNA retrograde movement from the cytoplasm to the nucleus.

The Transcription Factor p8 Regulates Autophagy in Response to Palmitic Acid Stress via a Mammalian Target of Rapamycin (mTOR)-independent Signaling Pathway.

Autophagy is an evolutionarily conserved degradative process that allows cells to maintain homoeostasis in numerous physiological situations. This process also functions as an essential protective response to endoplasmic reticulum (ER) stress, which promotes the removal and degradation of unfolded proteins. However, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. In this study, different types of autophagy were identified in human gastric cancer MKN45 cells in response to the stress induced by nutrient starvation or lipotoxicity in which the regulation of these pathways is mammalian target of rapamycin (mTOR)-dependent or -independent, respectively. Interestingly, we found that p8, a stress-inducible transcription factor, was enhanced in MKN45 cells treated with palmitic acid to induce lipotoxicity. Furthermore, an increase in autophagy was observed in MKN45 cells stably overexpressing p8 using a lentivirus system, and autophagy induced by palmitic acid was blocked by p8 RNAi compared with the control. Western blotting analyses showed that autophagy was regulated by p8 or mTOR in response to the protein kinase-like endoplasmic reticulum kinase/activating transcription factor 6-mediated ER stress of lipotoxicity or the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that autophagy induced by palmitic acid is mTOR-independent, but this autophagy pathway was regulated by p8 via p53- and PKCα-mediated signaling in MKN45 cells. Our findings provide insights into the role of p8 in regulating autophagy induced by the lipotoxic effects of excess fat accumulation in cells.

Two p90 ribosomal S6 kinase isoforms are involved in the regulation of mitotic and meiotic arrest in Artemia.

There are multiple isoforms of p90 ribosomal S6 kinase (RSK), which regulate diverse cellular functions such as cell growth, proliferation, maturation, and motility. However, the relationship between the structures and functions of RSK isoforms remains undetermined. Artemia is a useful model in which to study cell cycle arrest because these animals undergo prolonged diapauses, a state of obligate dormancy. A novel RSK isoform was identified in Artemia, which was termed Ar-Rsk2. This isoform was compared with an RSK isoform that we previously identified in Artemia, termed Ar-Rsk1. Ar-Rsk2 has an ERK-docking motif, whereas Ar-Rsk1 does not. Western blot analysis revealed that Ar-Rsk1 was activated by phosphorylation, which blocked meiosis in oocytes. Knockdown of Ar-Rsk1 reduced the level of phosphorylated cdc2 and thereby suppressed cytostatic factor activity. This indicates that Ar-Rsk1 regulates the cytostatic factor in meiosis. Expression of Ar-Rsk2 was down-regulated in Artemia cysts in which mitosis was arrested. Knockdown of Ar-Rsk2 resulted in decreased levels of cyclin D3 and phosphorylated histone H3, and the production of pseudo-diapause cysts. This indicates that Ar-Rsk2 regulates mitotic arrest. PLK and ERK RNAi showed that Ar-Rsk2, but not Ar-Rsk1, could be activated by PLK-ERK in Artemia. This is the first study to report that RSK isoforms with and without an ERK-docking motif regulate mitosis and meiosis, respectively. This study provides insight into the relationship between the structures and functions of RSK isoforms.