Comparative Hippocampal Proteome and Phosphoproteome in a Niemann-Pick, Type C1 Mouse Model Reveal Insights into Disease Mechanisms.

Niemann-Pick disease, type C (NPC) is a neurodegenerative, lysosomal storage disorder in individuals carrying two mutated copies of either the NPC1 or NPC2 gene. Consequently, impaired cholesterol recycling and an array of downstream events occur. Interestingly, in NPC, the hippocampus displays lysosomal lipid storage but does not succumb to progressive neurodegeneration as significantly as other brain regions. Since defining the neurodegeneration mechanisms in this disease is still an active area of research, we use mass spectrometry to analyze the overall proteome and phosphorylation pattern changes in the hippocampal region of a murine model of NPC. Using 3 week old mice representing an early disease time point, we observed changes in the expression of 47 proteins, many of which are consistent with the previous literature. New to this study, changes in members of the SNARE complex, including STX7, VTI1B, and VAMP7, were identified. Furthermore, we identified that phosphorylation of T286 on CaMKIIα and S1303 on NR2B increased in mutant animals, even at the late stage of the disease. These phosphosites are crucial to learning and memory and can trigger neuronal death by altering protein-protein interactions.

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

KEY POINTS: This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. ABSTRACT: Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Modeling rare diseases with induced pluripotent stem cell technology.

Rare diseases, in totality, affect a significant proportion of the population and represent an unmet medical need facing the scientific community. However, the treatment of individuals affected by rare diseases is hampered by poorly understood mechanisms preventing the development of viable therapeutics. The discovery and application of cellular reprogramming to create novel induced pluripotent stem cell models of rare diseases has revolutionized the rare disease community. Through developmental and functional analysis of differentiated cell types, these stem cell models carrying patient-specific mutations have become an invaluable tool for rare disease research. In this review article, we discuss the reprogramming of samples from individuals affected with rare diseases to induced pluripotent stem cells, current and future applications for this technology, and how integration of genome editing to rare disease research will help to improve our understanding of disease pathogenesis and lead to patient therapies.

SRC tyrosine kinases regulate neuronal differentiation of mouse embryonic stem cells via modulation of voltage-gated sodium channel activity.

Voltage-gated Na(+) channel activity is vital for the proper function of excitable cells and has been indicated in nervous system development. Meanwhile, the Src family of non-receptor tyrosine kinases (SFKs) has been implicated in the regulation of Na(+) channel activity. The present investigation tests the hypothesis that Src family kinases influence neuronal differentiation via a chronic regulation of Na(+) channel functionality. In cultured mouse embryonic stem (ES) cells undergoing neural induction and terminal neuronal differentiation, SFKs showed distinct stage-specific expression patterns during the differentiation process. ES cell-derived neuronal cells expressed multiple voltage-gated Na(+) channel proteins (Nav) and underwent a gradual increase in Na(+) channel activity. While acute inhibition of SFKs using the Src family inhibitor PP2 suppressed the Na(+) current, chronic inhibition of SFKs during early neuronal differentiation of ES cells did not change Nav expression. However, a long-lasting block of SFK significantly altered electrophysiological properties of the Na(+) channels, shown as a right shift of the current-voltage relationship of the Na(+) channels, and reduced the amplitude of Na(+) currents recorded in drug-free solutions. Immunocytochemical staining of differentiated cells subjected to the chronic exposure of a SFK inhibitor, or the Na(+) channel blocker tetrodotoxin, showed no changes in the number of NeuN-positive cells; however, both treatments significantly hindered neurite outgrowth. These findings suggest that SFKs not only modulate the Na(+) channel activation acutely, but the tonic activity of SFKs is also critical for normal development of functional Na(+) channels and neuronal differentiation or maturation of ES cells.

Dishevelled localization and function are differentially regulated by structurally distinct sterols.

The Dishevelled (DVL) family of proteins form supramolecular protein and lipid complexes at the cytoplasmic interface of the plasma membrane to regulate tissue patterning, proliferation, cell polarity, and oncogenic processes through DVL-dependent signaling, such as Wnt/ß-catenin. While DVL binding to cholesterol is required for its membrane association, the specific structural requirements and cellular impacts of DVL-sterol association are unclear. We report that intracellular sterols which accumulate within normal and pathological conditions cause aberrant DVL activity. In silico and molecular analyses suggested orientation of the ß- and α-sterol face within the DVL-PDZ domain regulates DVL-sterol binding. Intracellular accumulation of naturally occurring sterols impaired DVL2 plasma membrane association, inducing DVL2 nuclear localization via Foxk2. Changes to intracellular sterols also selectively impaired DVL2 protein-protein interactions This work identifies sterol specificity as a regulator of DVL signaling, suggests intracellular sterols cause distinct impacts on DVL activity, and supports a role for intracellular sterol homeostasis in cell signaling.

Sterol dysregulation in Smith-Lemli-Opitz syndrome causes astrocyte immune reactivity through microglia crosstalk.

Owing to the need for de novo cholesterol synthesis and cholesterol-enriched structures within the nervous system, cholesterol homeostasis is critical to neurodevelopment. Diseases caused by genetic disruption of cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome, which is caused by mutations in 7-dehydrocholesterol reductase (DHCR7), frequently result in broad neurological deficits. Although astrocytes regulate multiple neural processes ranging from cell migration to network-level communication, immunological activation of astrocytes is a hallmark pathology in many diseases. However, the impact of DHCR7 on astrocyte function and immune activation remains unknown. We demonstrate that astrocytes from Dhcr7 mutant mice display hallmark signs of reactivity, including increased expression of glial fibrillary acidic protein (GFAP) and cellular hypertrophy. Transcript analyses demonstrate extensive Dhcr7 astrocyte immune activation, hyper-responsiveness to glutamate stimulation and altered calcium flux. We further determine that the impacts of Dhcr7 are not astrocyte intrinsic but result from non-cell-autonomous effects of microglia. Our data suggest that astrocyte-microglia crosstalk likely contributes to the neurological phenotypes observed in disorders of cholesterol biosynthesis. Additionally, these data further elucidate a role for cholesterol metabolism within the astrocyte-microglia immune axis, with possible implications in other neurological diseases.

Nanoarchitectonics of a Microsphere-Based Scaffold for Modeling Neurodevelopment and Neurological Disease.

Three-dimensional cellular constructs derived from pluripotent stem cells allow the ex vivo study of neurodevelopment and neurological disease within a spatially organized model. However, the robustness and utility of three-dimensional models is impacted by tissue self-organization, size limitations, nutrient supply, and heterogeneity. In this work, we have utilized the principles of nanoarchitectonics to create a multifunctional polymer/bioceramic composite microsphere system for stem cell culture and differentiation in a chemically defined microenvironment. Microspheres could be customized to produce three-dimensional structures of defined size (ranging from >100 to <350 µm) with lower mechanical properties compared with a thin film. Furthermore, the microspheres softened in solution, approaching more tissue-like mechanical properties over time. With neural stem cells (NSCs) derived from human induced pluripotent stem cells, microsphere-cultured NSCs were able to utilize multiple substrates to promote cell adhesion and proliferation. Prolonged culture of NSC-bound microspheres under differentiating conditions allowed the formation of both neural and glial cell types from control and patient-derived stem cell models. Human NSCs and differentiated neurons could also be cocultured with astrocytes and human umbilical vein endothelial cells, demonstrating application for tissue-engineered modeling of development and human disease. We further demonstrated that microspheres allow the loading and sustained release of multiple recombinant proteins to support cellular maintenance and differentiation. While previous work has principally utilized self-organizing models or protein-rich hydrogels for neural culture, the three-dimensional matrix developed here through nanoarchitectonics represents a chemically defined and robust alternative for the in vitro study of neurodevelopment and nervous system disorders.

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.

iPSC modeling of rare pediatric disorders.

Discerning the underlying pathological mechanisms and the identification of therapeutic strategies to treat individuals affected with rare neurological diseases has proven challenging due to a host of factors. For instance, rare diseases affecting the nervous system are inherently lacking in appropriate patient sample availability compared to more common diseases, while animal models often do not accurately recapitulate specific disease phenotypes. These challenges impede research that may otherwise illuminate aspects of disease initiation and progression, leading to the ultimate identification of potential therapeutics. The establishment of induced pluripotent stem cells (iPSCs) as a human cellular model with defined genetics has provided the unique opportunity to study rare diseases within a controlled environment. iPSC models enable researchers to define mutational effects on specific cell types and signaling pathways within increasingly complex systems. Among rare diseases, pediatric diseases affecting neurodevelopment and neurological function highlight the critical need for iPSC-based disease modeling due to the inherent difficulty associated with collecting human neural tissue and the complexity of the mammalian nervous system. Rare neurodevelopmental disorders are therefore ideal candidates for utilization of iPSC-based in vitro studies. In this review, we address both the state of the iPSC field in the context of their utility and limitations for neurodevelopmental studies, as well as speculating about the future applications and unmet uses for iPSCs in rare diseases.

Generation of a CLTA reporter human induced pluripotent stem cell line, CRMi001-A-1, using the CRISPR/Cas9 system to monitor endogenous clathrin trafficking.

The most highly studied endocytic pathway, clathrin-dependent endocytosis, mediates a wide range of fundamental processes including nutrient internalization, receptor recycling, and signal transduction. In order to model tissue specific and developmental aspects of this process, CRISPR/Cas9 genomic editing was utilized to fluorescently label the C-terminus of clathrin light chain A (CLTA) within the phenotypically normal, parental CRMi001-A human induced pluripotent stem cell line. Successfully edited cells were isolated by fluorescently activated cell sorting, remained karyotypically normal, and maintained their differentiation potential. This cell line facilitates imaging of endogenous clathrin trafficking within varied cell types.

Concise Review: Fat and Furious: Harnessing the Full Potential of Adipose-Derived Stromal Vascular Fraction.

Due to their capacity to self-renew, proliferate and generate multi-lineage cells, adult-derived stem cells offer great potential for use in regenerative therapies to stop and/or reverse degenerative diseases such as diabetes, heart failure, Alzheimer's disease and others. However, these subsets of cells can be isolated from different niches, each with differing potential for therapeutic applications. The stromal vascular fraction (SVF), a stem cell enriched and adipose-derived cell population, has garnered interest as a therapeutic in regenerative medicine due to its ability to secrete paracrine factors that accelerate endogenous repair, ease of accessibility and lack of identified major adverse effects. Thus, one can easily understand the rush to employ adipose-derived SVF to treat human disease. Perhaps faster than any other cell preparation, SVF is making its way to clinics worldwide, while critical preclinical research needed to establish SVF safety, efficacy and optimal, standardized clinical procedures are underway. Here, we will provide an overview of the current knowledge driving this phenomenon, its regulatory issues and existing studies, and propose potential unmapped applications. Stem Cells Translational Medicine 2017;6:1096-1108.

Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/ß-catenin defects in neuronal cholesterol synthesis phenotypes.

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7, which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment, behavioral abnormalities and nervous system defects, though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS, we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation, not cholesterol deficiency, is critical for SLOS-associated defects. We further identified downregulation of Wnt/ß-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs, suggesting that Wnt signaling may be a promising therapeutic target for SLOS.

Progesterone reverses the spatial memory enhancements initiated by tonic and cyclic oestrogen therapy in middle-aged ovariectomized female rats.

While some research has indicated that ovarian hormone therapy (HT) benefits memory and decreases risk of Alzheimer's disease in menopausal women, several newer studies have shown null or detrimental effects. Despite the null and negative cognitive findings, the numerous studies showing positive effects beg the question of what factors determine whether HT acts as a neuroprotectant or a risk factor for brain functioning. Using middle-aged female rats, we directly compared six HTs. We evaluated the effects of ovariectomy, tonic low-dose, tonic high-dose and biweekly cyclic estradiol treatment, as well as whether progesterone altered the effectiveness of any one of these oestrogen regimens. Animals were tested on spatial and complex cued (intramaze patterns) reference memory using variants of the Morris maze. The tonic low-dose and cyclic estradiol treatments improved spatial performance, while the addition of progesterone reversed these beneficial cognitive effects of estradiol. Additionally, all groups learned to locate the platform on the cued task; however, an egocentric circling strategy was used with sham ovary-intact and hormone-replacement groups showing the most efficient search strategy. Although the question of memory retention 8 weeks after the first cognitive assessment was addressed, a large number of animals died between the first and second test, rendering the retest uninterpretable for many group comparisons. Specifically, both doses of tonic estradiol dramatically increased the number of deaths during the 17-week experiment, while the cyclic estradiol treatment did not. Progesterone decreased the number of deaths due to tonic estradiol treatment. Our findings suggest that the dose of estradiol replacement as well as the presence of progesterone influences the cognitive outcome of estradiol treatment. Further, there appears to be a dissociation between HT effects on cognition and mortality rates.