HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

Background: STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Results: Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). Conclusions: HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

Neuronal paxillin and drebrin mediate BDNF-induced force transduction and growth cone turning in a soft-tissue-like environment.

Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms underlying chemotropism-directed axonal growth cone movement in extremely soft environments remain unclear. Here, we show that drebrin, a growth cone T-zone protein, modulates growth cone turning in response to brain-derived neurotrophic factor (BDNF) coated on a soft substrate. Structurally, axonal growth cones of rodent hippocampal neurons grown on 0.1 kPa hydrogels possess an expanded T zone in which drebrin is highly integrated with both F-actin and microtubules. Biochemically, we identify paxillin as interacting with drebrin in cells grown on 0.1 kPa hydrogels but not on glass coverslips. When grown on 0.1 kPa substrates, growth cones asymmetrically exposed to BDNF-bound stripes exhibit enhanced paxillin-drebrin interaction on the side facing the stripes, an activity that is PKA and AAK1 dependent but independent of Src kinase. Functionally, we show that BDNF-induced growth cone turning and force generation on soft substrates require drebrin phosphorylation and paxillin-drebrin association.

Dysosmia Is a Predictor of Motor Function and Quality of Life in Patients with Parkinson's Disease.

(1) Background: The correlation between dysosmia with quality of life (QoL) in patients with PD was rarely reported. The study aimed to examine the effect of dysosmia on motor function and QoL in PD. (2) Methods: This cross-sectional study, performed between October 2016 and February 2021, recorded the traditional Chinese version of the University of Pennsylvania Smell Identification Test (UPSIT), the Montreal Cognitive Assessment (MoCA), the Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS UPDRS), and the 39-item Parkinson's Disease Questionnaire (PDQ-39) in patients with PD. UPSIT = 19 was applied to separate the total anosmia and non-anosmia groups. (3) Results: 243 patients with PD were recruited. The total anosmia group had higher MDS UPDRS total, part II, and part III scores than the non-anosmia group. They also had worse scores on the dimensions of activities of daily living (ADL) and cognition of the PDQ-39 than the non-anosmia group. The UPSIT score correlated MDS UPDRS part III score (p < 0.0001), PDQ-39 ADL quartile (p = 0.0202), and Dopamine transporter scan (p = 0.0082) in the linear regression. (4) Conclusions: Dysosmia in PD predicted a phenotype with defective motor function, ADL, and cognition QoL. The findings supported the olfactory transmission of α-synuclein to the cortices, substantia nigra.

Age-Stratified Risk of Dementia in Parkinson's Disease: A Nationwide, Population-Based, Retrospective Cohort Study in Taiwan.

Introduction: Parkinson's disease (PD) manifests with dominant motor symptoms and a wide range of non-motor symptoms (NMS). Dementia is one of the most disabling and exhausting NMS throughout the clinical course. We conducted a population-based, age-stratified, retrospective cohort study to investigate the incidence rate and risk of dementia of patients with newly diagnosed PD, and linked to the clinicopathological PD subtypes. Methods: Patients with newly diagnosed PD (PD group, n = 760) and control subjects (non-PD group, n = 3,034) were selected from the Taiwan's National Health Insurance Research Database from January 2001 to December 2005. The dementia incidence rate and dementia-free survival rate were calculated. Results: The overall dementia incidence rate was 17.5 and 5.7 per 1,000 person-years in PD and non-PD groups, respectively. The PD group had a significantly higher overall risk of dementia than controls (p < 0.001). The younger PD patients had a lower dementia incidence rate than the older PD patients, but a higher dementia risk compared to the same age of controls (<60 years, adjusted HR 6.55, 95% CI 1.56-27.48, p = 0.010). The dementia-free survival rate was significantly lower in the PD group compared to the non-PD group during follow-up (p < 0.001). Conclusion: In our study, the older age of onset in PD patients resulted in a higher incidence rate of dementia. In the young age of PD patients, the incidence rate of dementia was lower than the older PD patients, but the dementia risk was higher than controls of the same age. These findings possibly implied that there were different pathogenesis and pathologies causing dementia in younger and older PD patients.

Is there a close association of depression with either constipation or dysosmia in Parkinson's disease?

A possible association between depression and either the severity of constipation or dysosmia in Parkinson's disease (PD) patients was investigated in this cross-sectional study. One-hundred six patients who had the history of PD for less than 5 years were recruited. Depression was measured using the Beck Depression Inventory-II (BDI-II), and our patients were divided into depressive and non-depressive groups (DP: BDI-II ≥ 14; n = 22 and NDP: BDI-II < 14; n = 84). Olfactory dysfunction was assessed by the University of Pennsylvania Smell Identification Test (UPSIT). Constipation severity was defined by stool softener dosage and amount. Statistical analyses with one-tailed T- or chi-squared test, odds ratios (OR), and beta-coefficient were used to determine significant differences. Total scores based on the Unified Parkinson's Disease Rating Scale (UPDRS) were significantly higher in the DP group. A significant relationship was observed between PD patients with depression and severe constipation; PD patients with depression were more likely to present with severe constipation (OR 5.81; 95% CI 1.24-27.29, p = 0.026, adjusted for age and gender); but the significance became marginal after adjusted for age, gender and UPDRS part 3 (OR 4.46, 95% CI 0.93-21.33; p = 0.061). However, no association between olfactory dysfunction and depression was detected. There were significant positive correlations between BDI-II scores and severe constipation (ß ± SE 7.65 ± 2.02; p = < 0.001, adjusted for age and gender; ß ± SE 7.06 ± 2.04; p = 0.001, adjusted for age, gender, and UPDRS-3). Besides, we detected a marginally significant correlation that PD patients with higher BDI-II scores tended to present more severe motor symptoms. Olfactory dysfunction seemed to be less relevant to BDI-II scores. Based on our findings, we speculate that depression may be more closely related to brainstem nuclei than to the limbic pathway.

Proteomics analysis of altered proteins in kidney of mice with aristolochic acid nephropathy using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method.

Aristolochic acid (AA) causes interstitial renal fibrosis, called aristolochic acid nephropathy (AAN). There is no specific indicator for diagnosing AAN, so this study aimed to investigate the biomarkers for AAN using a proteomics method. The C3H/He female mice were given ad libitum AA-distilled water (0.5 mg/kg/day) and distilled water for 56 days in the AA and normal groups, respectively. The AA-induced proteins in the kidney were investigated using a proteomics study, including fluorogenic derivatization with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, followed by high-performance liquid chromatography analysis and liquid chromatography tandem mass spectrometry with a MASCOT database searching system. There were two altered proteins, thrombospondin type 1 (TSP1) and G protein-coupled receptor 87 (GPR87), in the kidney of AA-group mice on day 56. GPR87, a tumorigenesis-related protein, is reported for the first time in the current study. The renal interstitial fibrosis was certainly induced in the AA-group mice under histological examination. Based on the results of histological examination and the proteomics study, this model might be applied to AAN studies in the future. TSP1 might be a novel biomarker for AAN, and the further role of GPR87 leading to AA-induced tumorigenesis should be researched in future studies.

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties.

Stage-Dependent Axon Transport of Proteasomes Contributes to Axon Development.

Axon extension at the growing tip requires elevated local protein supply, with a capability sustainable over long axons in varying environments. The exact mechanisms, however, remain elusive. Here we report that axon-promoting factors elicited a retrograde transport-dependent removal of proteasomes from nascent axon terminals, thereby increasing protein stability at axon tips. Such an effect occurred through phosphorylation of a dynein-interacting proteasome adaptor protein Ecm29. During the transition from immature neurites to nascent axons in cultured hippocampal neurons, live-cell imaging revealed a significant increase of the retrograde axonal transport of fluorescently labeled 20S proteasomes. This retrograde proteasome transport depended on neuron stage and increased with axon lengths. Blockade of retrograde transport caused accumulation of proteasomes, reduction of axon growth, and attenuation of growth-associated Par6 at the axon tip of newly polarized neurons. Our results delineate a regulatory mechanism that controls proteasome abundance via preferential transport required for axon development in newborn neurons.

Relay of cyclin-dependent kinases in the regulation of axonal growth.

One of the most perplexing problems in neuronal morphogenesis is how local polarity signals echo genetic instructions to establish structural and functional asymmetry of neuronal compartments, i.e., axons, dendrites, and synapses. However studying these phenomena is complicated because both genes and the local environment influence the phenotype of developing neurons. Cell cycle-associated nuclear transcription regulators involved in axon extension, for example Cdk12 and Cdk13, thus provide ideal models for connecting spatially separated events at specific developmental time points.