Caveolin-1 Phosphorylation Is Essential for Axonal Growth of Human Neurons Derived From iPSCs.

Proper axonal growth and guidance is essential for neuron differentiation and development. Abnormal neuronal development due to genetic or epigenetic influences can contribute to neurological and mental disorders such as Down syndrome, Rett syndrome, and autism. Identification of the molecular targets that promote proper neuronal growth and differentiation may restore structural and functional neuroplasticity, thus improving functional performance in neurodevelopmental disorders. Using differentiated human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs), the present study demonstrates that during early stage differentiation of human NPCs, neuron-targeted overexpression constitutively active Rac1 (Rac1CA) and constitutively active Cdc42 (Cdc42CA) enhance expression of P-Cav-1, T-Cav-1, and P-cofilin and increases axonal growth. Similarly, neuron-targeted over-expression of Cav-1 (termed SynCav1) increases axonal development by increasing both axon length and volume. Moreover, inhibition of Cav-1(Y14A) phosphorylation blunts Rac1/Cdc42-mediated both axonal growth and differentiation of human NPCs and SynCav1(Y14A)-treated NPCs exhibited blunted axonal growth. These results suggest that: (1) SynCav1-mediated dendritic and axonal growth in human NPCs is dependent upon P-Cav-1, (2) P-Cav-1 is necessary for proper axonal growth during early stages of neuronal differentiation, and (3) Rac1/Cdc42CA-mediated neuronal growth is in part dependent upon P-Cav-1. In conclusion, Cav-1 phosphorylation is essential for human neuronal axonal growth during early stages of neuronal differentiation

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis

Array atomic force microscopy for real-time multiparametric analysis.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale biological and physical systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. Conventional AFMs only permit sequential single-point analysis; widespread adoption of array AFMs for simultaneous multipoint study is challenging owing to the intrinsic limitations of existing technological approaches. Here, we describe a prototype dispersive optics-based array AFM capable of simultaneously monitoring multiple probe-sample interactions. A single supercontinuum laser beam is utilized to spatially and spectrally map multiple cantilevers, to isolate and record beam deflection from individual cantilevers using distinct wavelength selection. This design provides a remarkably simplified yet effective solution to overcome the optical cross-talk while maintaining subnanometer sensitivity and compatibility with probe-based sensors. We demonstrate the versatility and robustness of our system on parallel multiparametric imaging at multiscale levels ranging from surface morphology to hydrophobicity and electric potential mapping in both air and liquid, mechanical wave propagation in polymeric films, and the dynamics of living cells. This multiparametric, multiscale approach provides opportunities for studying the emergent properties of atomic-scale mechanical and physicochemical interactions in a wide range of physical and biological networks

Age-Dependent Behavioral and Metabolic Assessment of AppNL−G−F/NL−G−F Knock-in (KI) Mice

Mitochondria play a crucial role in Alzheimer's disease (AD) onset and progression. Traditional transgenic AD mouse models which were widely used in the past decades share a common limitation: The overexpression of APP and overproduction of amyloid-beta (Aβ) are accompanied by other APP peptide fragments, which could introduce artificial and non-clinically relevant phenotypes. Here, we performed an in-depth and time-resolved behavioral and metabolic characterization of a clinically relevant AD mouse model engineered to express normal physiological levels of APP harboring humanized Swedish (K670N/M671L), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations (AppNL−G−F/NL−G−F), termed APP knock-in (APPKI) mice. Our result showed that APPKI mice exhibited fear learning deficits at 6-m age and contextual memory deficit at 12-m age. Histopathological analysis revealed mild amyloidosis (6E10) accompanied by microgliosis (Iba1) as early as 3 months, which progressed significantly together with significant astrocytosis at 6 and 12 m. We further analyzed hippocampal mitochondrial dysfunction by multiple assays, while 3-m APPKI mice brain mitochondrial function remains a similar level as WT mice. Significant mitochondrial dysfunction characterized by decreased ATP production and higher membrane potential with subsequent overproduction of reactive oxygen species (ROS) was observed in mitochondria isolated from 7-m APPKI mice hippocampal tissue. Morphologically, these mitochondria were larger in volume with a decreased level of mitochondrial fusion protein mitofusin-2 (MFN2). At 12 months, APPKI mice exhibit a significantly decreased total mitochondrial oxygen consumption rate (OCR) in isolated hippocampal mitochondria detected by high-resolution respirometry. These data indicate early mitochondrial dysfunction in the brain at pre-symptomatic age in the AppNL−G−F/NL−G−mice, which may play a key role in the progression of the disease. Moreover, the identified behavioral and bioenergetic alterations in this clinically relevant AD mouse model provide a valuable tool to optimize the temporal component for therapeutic interventions to treat AD

Genes Influencing Circadian Differences in Blood Pressure in Hypertensive Mice

Essential hypertension is a common multifactorial heritable condition in which increased sympathetic outflow from the central nervous system is involved in the elevation in blood pressure (BP), as well as the exaggerated morning surge in BP that is a risk factor for myocardial infarction and stroke in hypertensive patients. The Schlager BPH/2J mouse is a genetic model of hypertension in which increased sympathetic outflow from the hypothalamus has an important etiological role in the elevation of BP. Schlager hypertensive mice exhibit a large variation in BP between the active and inactive periods of the day, and also show a morning surge in BP. To investigate the genes responsible for the circadian variation in BP in hypertension, hypothalamic tissue was collected from BPH/2J and normotensive BPN/3J mice at the ‘peak’ (n = 12) and ‘trough’ (n = 6) of diurnal BP. Using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays, validation by quantitative real-time PCR and a statistical method that adjusted for clock genes, we identified 212 hypothalamic genes whose expression differed between ‘peak’ and ‘trough’ BP in the hypertensive strain. These included genes with known roles in BP regulation, such as vasopressin, oxytocin and thyrotropin releasing hormone, as well as genes not recognized previously as regulators of BP, including chemokine (C-C motif) ligand 19, hypocretin and zinc finger and BTB domain containing 16. Gene ontology analysis showed an enrichment of terms for inflammatory response, mitochondrial proton-transporting ATP synthase complex, structural constituent of ribosome, amongst others. In conclusion, we have identified genes whose expression differs between the peak and trough of 24-hour circadian BP in BPH/2J mice, pointing to mechanisms responsible for diurnal variation in BP. The findings may assist in the elucidation of the mechanism for the morning surge in BP in essential hypertension

Traumatic brain injury enhances neuroinflammation and lesion volume in caveolin deficient mice

BACKGROUND: Traumatic brain injury (TBI) enhances pro-inflammatory responses, neuronal loss and long-term behavioral deficits. Caveolins (Cavs) are regulators of neuronal and glial survival signaling. Previously we showed that astrocyte and microglial activation is increased in Cav-1 knock-out (KO) mice and that Cav-1 and Cav-3 modulate microglial morphology. We hypothesized that Cavs may regulate cytokine production after TBI. METHODS: Controlled cortical impact (CCI) model of TBI (3 m/second; 1.0 mm depth; parietal cortex) was performed on wild-type (WT; C57Bl/6), Cav-1 KO, and Cav-3 KO mice. Histology and immunofluorescence microscopy (lesion volume, glia activation), behavioral tests (open field, balance beam, wire grip, T-maze), electrophysiology, electron paramagnetic resonance, membrane fractionation, and multiplex assays were performed. Data were analyzed by unpaired t tests or analysis of variance (ANOVA) with post-hoc Bonferroni’s multiple comparison. RESULTS: CCI increased cortical and hippocampal injury and decreased expression of MLR-localized synaptic proteins (24 hours), enhanced NADPH oxidase (Nox) activity (24 hours and 1 week), enhanced polysynaptic responses (1 week), and caused hippocampal-dependent learning deficits (3 months). CCI increased brain lesion volume in both Cav-3 and Cav-1 KO mice after 24 hours (P < 0.0001, n = 4; one-way ANOVA). Multiplex array revealed a significant increase in expression of IL-1β, IL-9, IL-10, KC (keratinocyte chemoattractant), and monocyte chemoattractant protein 1 (MCP-1) in ipsilateral hemisphere and IL-9, IL-10, IL-17, and macrophage inflammatory protein 1 alpha (MIP-1α) in contralateral hemisphere of WT mice after 4 hours. CCI increased IL-2, IL-6, KC and MCP-1 in ipsilateral and IL-6, IL-9, IL-17 and KC in contralateral hemispheres in Cav-1 KO and increased all 10 cytokines/chemokines in both hemispheres except for IL-17 (ipsilateral) and MIP-1α (contralateral) in Cav-3 KO (versus WT CCI). Cav-3 KO CCI showed increased IL-1β, IL-9, KC, MCP-1, MIP-1α, and granulocyte-macrophage colony-stimulating factor in ipsilateral and IL-1β, IL-2, IL-9, IL-10, and IL-17 in contralateral hemispheres (P = 0.0005, n = 6; two-way ANOVA) compared to Cav-1 KO CCI. CONCLUSION: CCI caused astrocyte and microglial activation and hippocampal neuronal injury. Cav-1 and Cav-3 KO exhibited enhanced lesion volume and cytokine/chemokine production after CCI. These findings suggest that Cav isoforms may regulate neuroinflammatory responses and neuroprotection following TBI

Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease

An Aged Canid with Behavioral Deficits Exhibits Blood and Cerebrospinal Fluid Amyloid Beta Oligomers

Many of the molecular and pathological features associated with human Alzheimer disease (AD) are mirrored in the naturally occurring age-associated neuropathology in the canine species. In aged dogs with declining learned behaviour and memory the severity of cognitive dysfunction parallels the progressive build up and location of Aβ in the brain. The main aim of this work was to study the biological behaviour of soluble oligomers isolated from an aged dog with cognitive dysfunction through investigating their interaction with a human cell line and synthetic Aβ peptides. We report that soluble oligomers were specifically detected in the dog’s blood and cerebrospinal fluid via anti-oligomer- and anti-Aβ specific binders. Importantly, our results reveal the potent neurotoxic effects of the dog’s cerebrospinal fluid on cell viability and the seeding efficiency of the cerebrospinal fluid-borne soluble oligomers on the thermodynamic activity and the aggregation kinetics of synthetic human Aβ. The value of further characterising the naturally occurring Alzheimer-like neuropathology in dogs using genetic and molecular tools is discussed

Opportunities, barriers, and recommendations in down syndrome research

Recent advances in medical care have increased life expectancy and improved the quality of life for people with Down syndrome (DS). These advances are the result of both pre-clinical and clinical research but much about DS is still poorly understood. In 2020, the NIH announced their plan to update their DS research plan and requested input from the scientific and advocacy community. The National Down Syndrome Society (NDSS) and the LuMind IDSC Foundation worked together with scientific and medical experts to develop recommendations for the NIH research plan. NDSS and LuMind IDSC assembled over 50 experts across multiple disciplines and organized them in eleven working groups focused on specific issues for people with DS. This review article summarizes the research gaps and recommendations that have the potential to improve the health and quality of life for people with DS within the next decade. This review highlights many of the scientific gaps that exist in DS research. Based on these gaps, a multidisciplinary group of DS experts has made recommendations to advance DS research. This paper may also aid policymakers and the DS community to build a comprehensive national DS research strategy