SNCA/PARK1 encodes α-synuclein, which is associated with familial Parkinson\'s disease. Despite its abundance in presynaptic terminals, the aggregation mechanism of α-synuclein and its relationship with Parkinson\'s disease have not yet been elucidated. Moreover, the ultrastructures of α-synuclein localization sites in neuronal presynaptic terminals remain unclear. Therefore, we herein generated transgenic mice expressing human α-synuclein tagged with mKate2 (hSNCA-mKate2 mice). These mice exhibited normal growth and fertility and had no motor dysfunction relative to their wild-type littermates, even at one year old. α-Synuclein-mKate2 accumulated in presynaptic terminals, particularly between Purkinje cells in the cerebellum and neurons in cerebellar nuclei. α-Synuclein-mKate2 was associated with the presynaptic marker, synaptophysin. In-resin CLEM and immunoelectron or electron microscopy revealed that α-synuclein-mKate2 localized on the surface of synaptic vesicles that were tightly arranged and assembled to form large synaptic pools in the cerebellum with negligible effects on the active zone. These results suggest that α-synuclein-associated ultrastructures in the presynaptic terminals of hSNCA-mKate2 mice reflect the structures of α-synuclein-assembled synaptic vesicle pools, and the size of vesicle pools increased. This transgenic mouse model will be a valuable tool for studying α-synuclein-associated synaptic vesicle pools.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motoneuron degeneration, and effective clinical treatments are lacking. In this study, we evaluated whether intranasal delivery of mesenchymal stem cell-derived small extracellular vesicles (sEVs) is a strategy for ALS therapy using SOD1G93A mice. In vivo tracing showed that intranasally-delivered sEVs entered the central nervous system and were extensively taken up by spinal neurons and some microglia. SOD1G93A mice that intranasally received sEV administration showed significant improvements in motor performances and survival time. After sEV administration, pathological changes, including spinal motoneuron death and synaptic denervation, axon demyelination, neuromuscular junction degeneration and electrophysiological defects, and mitochondrial vacuolization were remarkably alleviated. sEV administration attenuated the elevation of proinflammatory cytokines and glial responses. Proteomics and transcriptomics analysis revealed upregulation of the complement and coagulation cascade and NF-ĸB signaling pathway in SOD1G93A mouse spinal cords, which was significantly inhibited by sEV administration. The changes were further confirmed by detecting C1q and NF-ĸB expression using Western blots. In conclusion, intranasal administration of sEVs effectively delays the progression of ALS by inhibiting neuroinflammation and overactivation of the complement and coagulation cascades and NF-ĸB signaling pathway and is a potential option for ALS therapy.

TAVO101 is a humanized anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody under clinical development for the treatment of atopic dermatitis (AD) and other allergic inflammatory conditions. The crystallizable fragment region of the antibody was engineered for half-life extension and attenuated effector functions. This Phase 1, double-blinded, randomized, placebo-controlled study assessed the safety, tolerability, pharmacokinetics, and immunogenicity of TAVO101 in healthy adult subjects in seven ascending dose cohorts. Subjects received a single intravenous administration of TAVO101 or placebo with a 195-day follow-up. TAVO101 was safe and well tolerated. The incidences and severities of treatment-emergent adverse events were mostly mild and comparable between the active and placebo groups, with no trends of dose relationship. There were no severe adverse events, deaths, or treatment-related withdrawals. TAVO101 exhibited a linear pharmacokinetic profile, low clearance, and a median elimination half-life of 67 days in healthy subjects. All TAVO101-treated subjects tested negative for anti-drug antibodies. To support development in AD, TAVO101 was studied in an oxazolone-induced AD model in hTSLP transgenic mice and demonstrated efficacy. This long-acting anti-TSLP antibody has the potential for stronger and sustained allergic inflammatory disease control. The results from this study warranted further clinical development of TAVO101 in patients.

The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRβ-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.

Lhx3 is a LIM-homeodomain transcription factor that affects body size in mammals by regulating the secretion of pituitary hormones. Akita, Shiba Inu, and Mame Shiba Inu dogs are Japanese native dog breeds that have different body sizes. To determine whether Lhx3 plays a role in the differing body sizes of these three dog breeds, we sequenced the Lhx3 gene in the three breeds, which led to the identification of an SNP in codon 280 (S280N) associated with body size. The allele frequency at this SNP differed significantly between the large Akita and the two kinds of smaller Shiba dogs. To validate the function of this SNP on body size, we introduced this change into the Lhx3 gene of mice. Homozygous mutant mice (S279N+/+) were found to have significantly increased body lengths and weights compared to heterozygous mutant (S279N+/-) and wild-type (S279N-/-) mice several weeks after weaning. These results demonstrate that a nonsynonymous substitution in Lhx3 plays an important role in regulating body size in mammals.

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.

OBJECTIVE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses.
RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials.
CONCLUSIONS: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

The small GTPase RAS acts as a plasma membrane-anchored intracellular neurotrophin counteracting neuronal degeneration in the brain, but the underlying molecular mechanisms are largely unknown. In transgenic mice expressing constitutively activated V12-Ha-RAS selectively in neurons, proteome analysis uncovered a 70% decrease in voltage-dependent anion channel-1 (VDAC-1) in the cortex and hippocampus. We observed a corresponding reduction in the levels of mRNA splicing variant coding for plasma membrane-targeted VDAC-1 (pl-VDAC-1) while mRNA levels for mitochondrial membrane VDAC-1 (mt-VDAC-1) remained constant. In primary cortical neurons derived from V12-Ha-RAS animals, a decrease in pl-VDAC-1 mRNA levels was observed, accompanied by a concomitant reduction in the ferricyanide reductase activity associated with VDAC-1 protein. Application of MEK inhibitor U0126 to transgenic cortical neurons reconstituted pl-VDAC-1 mRNA to reach wild-type levels. Excitotoxic glutamate-induced cell death was strongly attenuated in transgenic V12-Ha-RAS overexpressing cortical cultures. Consistently, a neuroprotective effect could also be achieved in wild-type cortical cultures by the extracellular application of channel-blocking antibody targeting the N-terminus of VDAC-1. These results may encourage novel therapeutic approaches toward blocking pl-VDAC-1 by monoclonal antibody targeting for complementary treatments in transplantation and neurodegenerative disease.

Epithelial sodium channel (ENaC) is responsible for regulating Na+ homeostasis. While its physiological functions have been investigated extensively in peripheral tissues, far fewer studies have explored its functions in the brain. Since our limited knowledge of ENaC\'s distribution in the brain impedes our understanding of its functions there, we decided to explore the whole-brain expression pattern of the Scnn1a gene, which encodes the core ENaC complex component ENaCα. To visualize Scnn1a expression in the brain, we crossed Scnn1a-Cre mice with Rosa26-lsl-tdTomato mice. Brain sections were subjected to immunofluorescence staining using antibodies against NeuN or Myelin Binding Protein (MBP), followed by the acquisition of confocal images. We observed robust tdTomato fluorescence not only in the soma of cortical layer 4, the thalamus, and a subset of amygdalar nuclei, but also in axonal projections in the hippocampus and striatum. We also observed expression in specific hypothalamic nuclei. Contrary to previous reports, however, we did not detect significant expression in the circumventricular organs, which are known for their role in regulating Na+ balance. Finally, we detected fluorescence in cells lining the ventricles and in the perivascular cells of the median eminence. Our comprehensive mapping of Scnn1a-expressing cells in the brain will provide a solid foundation for further investigations of the physiological roles ENaC plays within the central nervous system.

Parkinson\'s disease (PD), the most common neurodegenerative movement disorder, is characterized by dopaminergic neuron loss in the substantia nigra pars compacta (SNpc) and intraneuronal α-synuclein (α-syn) inclusions. It is highly needed to establish a rodent model that recapitulates the clinicopathological features of PD within a short period to efficiently investigate the pathological mechanisms and test disease-modifying therapies. To this end, we analyzed three mouse lines, i.e., wild-type mice, wild-type human α-syn bacterial artificial chromosome (BAC) transgenic (BAC-SNCA Tg) mice, and A53T human α-syn BAC transgenic (A53T BAC-SNCA Tg) mice, receiving dorsal striatum injections of human and mouse α-syn preformed fibrils (hPFFs and mPFFs, respectively). mPFF injections induced more severe α-syn pathology in most brain regions, including the ipsilateral SNpc, than hPFF injections in all genotypes at 1-month post-injection. Although these Tg mouse lines expressed a comparable amount of α-syn in the brains, the mPFF-injected A53T BAC-SNCA Tg mice exhibited the most severe α-syn pathology as early as 0.5-month post-injection. The mPFF-injected A53T BAC-SNCA Tg mice showed a 38% reduction in tyrosine hydroxylase (TH)-positive neurons in the ipsilateral SNpc, apomorphine-induced rotational behavior, and motor dysfunction at 2 months post-injection. These data indicate that the extent of α-syn pathology induced by α-syn PFF injection depends on the types of α-syn PFFs and exogenously expressed α-syn in Tg mice. The mPFF-injected A53T BAC-SNCA Tg mice recapitulate the key features of PD more rapidly than previously reported mouse models, suggesting their usefulness for testing disease-modifying therapies as well as analyzing the pathological mechanisms.