Chemistry and Function of Glycosaminoglycans in the Nervous System.

Proteoglycans, and especially their GAG components, participate in numerous biologically significant interactions with growth factors, chemokines, morphogens, guidance molecules, survival factors, and other extracellular and cell-surface components. These interactions are often critical to the basic developmental processes of cellular proliferation and differentiation, as well as to both the onset of disease sequelae and prevention of disease progression. In many tissues, proteoglycans and especially their glycosaminoglycan (GAG) components are mediators of these processes. The GAG family is characterized by covalently linked repeating disaccharides forming long unbranched polysaccharide chains. Thus far in higher eukaryotes, the family consists of chondroitin sulfate (CS), heparin/heparan sulfate (HS), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronan (HA). All GAG chains (except HA) are characteristically modified by varying amounts of esterified sulfate. One or more GAG chains are usually found in nature bound to polypeptide backbones in the form of proteoglycans; HA is the exception. In the nervous system, GAG/proteoglycan-mediated interactions participate in proliferation and synaptogenesis, neural plasticity, and regeneration. This review focuses on the structure, chemistry and function of GAGs in nervous system development, disease, function and injury response.

Surveying the experience of postdocs in the United States before and during the COVID-19 pandemic.

In the interest of advocating for the postdoctoral community in the United States (US), we compared the results of surveys of postdocs carried out in 2019 and in late 2020. We found that respondents' mental health and wellness were significantly impacted by the pandemic irrespective of their gender, race, citizenship, or other identities. Career trajectories and progression were also affected, as respondents reported being less confident about achieving career goals, and having more negative perceptions of the job market compared to before the pandemic. Postdocs working in the US on temporary visas reported experiencing increased stress levels due to changes in immigration policy. Access to institutional Postdoctoral Offices or Associations positively impacted well-being and helped mitigate some of the personal and professional stresses caused by the pandemic.

Roles of Chondroitin Sulfate Proteoglycans as Regulators of Skeletal Development.

The extracellular matrix (ECM) is critically important for most cellular processes including differentiation, morphogenesis, growth, survival and regeneration. The interplay between cells and the ECM often involves bidirectional signaling between ECM components and small molecules, i.e., growth factors, morphogens, hormones, etc., that regulate critical life processes. The ECM provides biochemical and contextual information by binding, storing, and releasing the bioactive signaling molecules, and/or mechanical information that signals from the cell membrane integrins through the cytoskeleton to the nucleus, thereby influencing cell phenotypes. Using these dynamic, reciprocal processes, cells can also remodel and reshape the ECM by degrading and re-assembling it, thereby sculpting their environments. In this review, we summarize the role of chondroitin sulfate proteoglycans as regulators of cell and tissue development using the skeletal growth plate model, with an emphasis on use of naturally occurring, or created mutants to decipher the role of proteoglycan components in signaling paradigms.

Mapping Proteoglycan Function Using Novel Genetic Strategies.

Multiple intrinsic and extrinsic factors contribute to stem and neuronal precursor cell maintenance and/or differentiation. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. The complexity and diversity of the glycan structure of proteoglycans make their functional characterization a challenging task. In order to test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation, we have taken a loss-of-function approach by developing a library of both biosynthetic and degradative enzymes to specifically remodel the ECM.

Brain transcriptome analysis of a CLN2 mouse model as a function of disease progression.

BACKGROUND: Neuronal ceroid lipofuscinoses, (NCLs or Batten disease) are a group of inherited, early onset, fatal neurodegenerative diseases associated with mutations in 13 genes. All forms of the disease are characterized by lysosomal accumulation of fluorescent storage material, as well as profound neurodegeneration, but the relationship of the various genes' function to a single biological process is not obvious. In this study, we used a well-characterized mouse model of classical late infantile NCL (cLINCL) in which the tripeptidyl peptidase 1 (Tpp1) gene is disrupted by gene targeting, resulting in loss of detectable TPP1 activity and leading to progressive neurological phenotypes including ataxia, increased motor deficiency, and early death. METHODS: In order to identify genes and pathways that may contribute to progression of the neurodegenerative process, we analyzed forebrain/midbrain and cerebellar transcriptional differences at 1, 2, 3 and 4 months of age in control and TPP1-deficient mice by global RNA-sequencing. RESULTS: Progressive neurodegenerative inflammatory responses involving microglia, astrocytes and endothelial cells were observed, accompanied by activation of leukocyte extravasation signals and upregulation of nitric oxide production and reactive oxygen species. Several astrocytic (i.e., Gfap, C4b, Osmr, Serpina3n) and microglial (i.e., Ctss, Itgb2, Itgax, Lyz2) genes were identified as strong markers for assessing disease progression as they showed increased levels of expression in vivo over time. Furthermore, transient increased expression of choroid plexus genes was observed at 2 months in the lateral and fourth ventricle, highlighting an early role for the choroid plexus and cerebrospinal fluid in the disease pathology. Based on these gene expression changes, we concluded that neuroinflammation starts, for the most part, after 2 months in the Tpp1-/- brain and that activation of microglia and astrocytes occur more rapidly in cerebellum than in the rest of the brain; confirming increased severity of inflammation in this region. CONCLUSIONS: These findings have led to a better understanding of cLINCL pathological onset and progression, which may aid in development of future therapeutic treatments for this disease.

Grant application outcomes for biomedical researchers who participated in the National Research Mentoring Network's Grant Writing Coaching Programs.

BACKGROUND: A diverse research workforce is essential for catalyzing biomedical advancements, but this workforce goal is hindered by persistent sex and racial/ethnic disparities among investigators receiving research grants from the National Institutes of Health (NIH). In response, the NIH-funded National Research Mentoring Network implemented a Grant Writing Coaching Program (GCP) to provide diverse cohorts of early-career investigators across the United States with intensive coaching throughout the proposal development process. We evaluated the GCP's national reach and short-term impact on participants' proposal submissions and funding outcomes. METHODS: The GCP was delivered as six similar but distinct models. All models began with an in-person group session, followed by a series of coaching sessions over 4 to 12 months. Participants were surveyed at 6-, 12- and 18-months after program completion to assess proposal outcomes (submissions, awards). Self-reported data were verified and supplemented by searches of public repositories of awarded grants when available. Submission and award rates were derived from counts of participants who submitted or were awarded at least one grant proposal in a category (NIH, other federal, non-federal). RESULTS: From June 2015 through March 2019, 545 investigators (67% female, 61% under-represented racial/ethnic minority, URM) from 187 different institutions participated in the GCP. Among them, 324 (59% of participants) submitted at least one grant application and 134 (41% of submitters) received funding. A total of 164 grants were awarded, the majority being from the NIH (93, 56%). Of the 74 R01 (or similar) NIH research proposals submitted by GCP participants, 16 have been funded thus far (56% to URM, 75% to women). This 22% award rate exceeded the 2016-2018 NIH success rates for new R01s. CONCLUSION: Inter- and intra-institutional grant writing coaching groups are a feasible and effective approach to supporting the grant acquisition efforts of early-career biomedical investigators, including women and those from URM groups.

Leveraging a collaborative consortium model of mentee/mentor training to foster career progression of underrepresented postdoctoral researchers and promote institutional diversity and inclusion.

Changing institutional culture to be more diverse and inclusive within the biomedical academic community is difficult for many reasons. Herein we present evidence that a collaborative model involving multiple institutions of higher education can initiate and execute individual institutional change directed at enhancing diversity and inclusion at the postdoctoral researcher (postdoc) and junior faculty level by implementing evidence-based mentoring practices. A higher education consortium, the Big Ten Academic Alliance, invited individual member institutions to send participants to one of two types of annual mentor training: 1) "Mentoring-Up" training for postdocs, a majority of whom were from underrepresented groups; 2) Mentor Facilitator training-a train-the-trainer model-for faculty and senior leadership. From 2016 to 2019, 102 postdocs and 160 senior faculty and administrative leaders participated. Postdocs reported improvements in their mentoring proficiency (87%) and improved relationships with their PIs (71%). 29% of postdoc respondents transitioned to faculty positions, and 85% of these were underrepresented and 75% were female. 59 out of the 120 faculty and administrators (49%) trained in the first three years provided mentor training on their campuses to over 3000 undergraduate and graduate students, postdocs and faculty within the project period. We conclude that early stage biomedical professionals as well as individual institutions of higher education benefited significantly from this collaborative mentee/mentor training model.

Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth.

The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.

The Role of Dot1l in Prenatal and Postnatal Murine Chondrocytes and Trabecular Bone.

Osteoarthritis and osteoporosis are widely prevalent and have far-reaching public health implications. There is increasing evidence that epigenetics, in particular, histone 3 lysine 79 methyltransferase DOT1L, plays an important role in the cartilage and bone biology. In this study, we evaluated the role of Dot1l in the articular cartilage, growth plate, and trabecular bone utilizing conditional KO mouse models. We generated chondrocyte-specific constitutive and inducible conditional Dot1l KO mouse lines using Col2a1-Cre and Acan-CreER systems. Prenatal deletion of Dot1l in mouse chondrocytes led to perinatal mortality, accelerated ossification, and dysregulation of Col10a1 expression. Postnatal deletion of Dot1l in mouse chondrocytes resulted in trabecular bone loss decreased extracellular matrix production, and disruption of the growth plate. In addition, pharmacological inhibition of DOT1L in a progeria mouse model partially rescued the abnormal osseous phenotype. In conclusion, Dot1l is important in maintaining the growth plate, extracellular matrix production, and trabecular bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Comparisons and Approaches of PREP Programs at Different Stages of Maturity: Challenges, Best Practices and Benefits.

The Postbaccalaureate Research Education Programs (PREP) are designed to provide research training and educational opportunities for recent baccalaureate graduates from targeted groups defined by NIH who would benefit by academic enhancements between the completion of undergraduate studies and admission to a PhD program. These programs offer exposure to the biomedical science community in a way that helps post-undergraduate individuals visualize future careers as well-trained, enthusiastic leaders in biomedical research who represent and will promote diversity in science. Specifically, PREPs provide the preparation and skills required for entrance into, and successful completion of, a PhD program via in-depth exposure to a research setting, which helps to refine the post-undergraduate's research interests, assists in providing a realistic understanding of the end results one can expect from research, and offers a forum for discussion with lab peers and mentors about possible career paths. Beyond the lab, PREPs offer programmatic activities to develop analytical, writing, and oral presentation skills necessary for a competitive graduate school application and success in graduate school thereafter. Individual mentoring increases the post-undergraduate's confidence and familiarity with members of the research community, so that pursuit of a PhD becomes a realistic and less-intimidating path. Interventions and developmental activities are matched to the background preparation, research experience, and learning style of each post-undergraduate. As with all training programs, there is no perfect model and each program must fit in and adapt to their respective institutional environments and cultures. Thus, in this article, we provide perspectives and approaches developed by a long-standing program in existence almost since the beginning of the PREP program along with one PREP at an early stage of maturity, having just been through one renewal.

Global Brain Transcriptome Analysis of a Tpp1 Neuronal Ceroid Lipofuscinoses Mouse Model.

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.

United States National Postdoc Survey results and the interaction of gender, career choice and mentor impact.

The postdoctoral community is an essential component of the academic and scientific workforce, but a lack of data about this community has made it difficult to develop policies to address concerns about salaries, working conditions, diversity and career development, and to evaluate the impact of existing policies. Here we present comprehensive survey results from 7,603 postdocs based at 351 US academic and non-academic (e.g. hospital, industry and government lab) institutions in 2016. In addition to demographic and salary information, we present multivariate analyses on factors influencing postdoc career plans and satisfaction with mentorship. We further analyze gender dynamics and expose wage disparities. Academic research positions remain the predominant career choice, although women and US citizens are less likely than their male and non-US citizen counterparts to choose academic research positions. Receiving mentorship training has a significant positive effect on postdoc satisfaction with mentorship. Quality of and satisfaction with postdoc mentorship also appear to heavily influence career choice.

Proteoglycans in brain development and pathogenesis.

Proteoglycans are diverse, complex extracellular/cell surface macromolecules composed of a central core protein with covalently linked glycosaminoglycan (GAG) chains; both of these components contribute to the growing list of important bio-active functions attributed to proteoglycans. Increasingly, attention has been paid to the roles of proteoglycans in nervous tissue development due to their highly regulated spatio/temporal expression patterns, whereby they promote/inhibit neurite outgrowth, participate in specification and maturation of various precursor cell types, and regulate cell behaviors like migration, axonal pathfinding, synaptogenesis and plasticity. These functions emanate from both the environments proteoglycans create around cells by retaining ions and water or serving as scaffolds for cell shaping or motility, and from dynamic interactions that modulate signaling fields for cytokines, growth factors and morphogens, which may bind to either the protein or GAG portions. Also, genetic abnormalities impacting proteoglycan synthesis during critical steps of brain development and response to environmental insults and injuries, as well as changes in microenvironment interactions leading to tumors in the central nervous system, all suggest roles for proteoglycans in behavioral and intellectual disorders and malignancies.

Glial cell responses in a murine multifactorial perinatal brain injury model.

The impact of traumatic brain injury during the perinatal period, which coincides with glial cell (astrocyte and oligodendrocyte) maturation was assessed to determine whether a second insult, e.g., increased inflammation due to remote bacterial exposure, exacerbates the initial injury's effects, possibly eliciting longer-term brain damage. Thus, a murine multifactorial injury model incorporating both mechanisms consisting of perinatal penetrating traumatic brain injury, with or without intraperitoneal injection of lipopolysaccharide (LPS), an analog of remote pathogen exposure has been developed. Four days after injury, gene expression changes for different cell markers were assessed using mRNA in situ hybridization (ISH) and qPCR. Astrocytic marker mRNA levels increased in the stab-alone and stab-plus-LPS treated animals indicating reactive gliosis. Activated microglial/macrophage marker levels, increased in the ipsilateral sides of stab and stab-plus LPS animals by P10, but the differences resolved by P15. Ectopic expression of glial precursor and neural stem cell markers within the cortical injury site was observed by ISH, suggesting that existing precursors and neural stem cells migrate into the injured areas to replace the cells lost in the injury process. Furthermore, single exposure to LPS concomitant with acute stab injury affected the oligodendrocyte population in both the injured and contralateral uninjured side, indicating that after compromise of the blood-brain barrier integrity, oligodendrocytes become even more susceptible to inflammatory injury. This multifactorial approach should lead to a better understanding of the pathogenic sequelae observed as a consequence of perinatal brain insult/injury, caused by combinations of trauma, intrauterine infection, hypoxia and/or ischemia in humans.

Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain.

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-ß-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

Mapping proteoglycan functions with glycosidases.

The intrinsic and extrinsic factors that contribute to stem and neuronal precursor cell maintenance and/or differentiation remain poorly understood. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. We have taken a loss-of-function approach, by developing a library of bacterial lyases and sulfatases to specifically remodel the ECM and test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation.

Aggrecan is required for growth plate cytoarchitecture and differentiation.

The proteoglycan aggrecan is a prominent component of the extracellular matrix in growth plate cartilage. A naturally occurring, recessive, perinatally lethal mutation in the aggrecan core protein gene, cmd(bc) (Acan(cmd-Bc)), that deletes the entire protein-coding sequence provided a model in which to characterize the phenotypic and morphologic effects of aggrecan deletion on skeletal development. We also generated a novel transgenic mouse, Tg(COL2A1-ACAN), that has the chick ACAN coding sequence driven by the mouse COL2A1 promoter to enable the production of cmd(bc)/cmd(bc); Tg(COL2A1-ACAN) rescue embryos. These were used to assess the impact of aggrecan on growth plate organization, chondrocyte survival and proliferation, and the expression of mRNAs encoding chondrocyte differentiation markers and growth factors. Homozygous mutant (cmd(bc)/cmd(bc)) embryos exhibited severe defects in all skeletal elements with deformed and shortened (50%) limb elements. Expression of aggrecan in rescue embryos reversed the skeletal defects to varying degrees with a 20% increase in limb element length and near-full reversal (80%) of size and diameter of the ribcage and vertebrae. Aggrecan-null growth plates were devoid of matrix and lacked chondrocyte organization and differentiation, while those of the rescue embryos exhibited matrix production concomitant with partial zonation of chondrocytes having proliferative and hypertrophic morphologies. Deformation of the trachea, likely the cause of the mutation's lethality, was reduced in the rescue embryos. Aggrecan-null embryos also had abnormal patterns of COL10A1, SOX9, IHH, PTCH1, and FGFR3 mRNA expression in the growth plate. Expression of chick aggrecan in the rescue embryos notably increased COLX expression, accompanied by the reappearance of a hypertrophic zone and IHH expression. Significantly, in transgenic rescue embryos, the cell death and decreased proliferation phenotypes exhibited by the mutants were reversed; both were restored to wild-type levels. These findings suggest that aggrecan has a major role in regulating the expression of key growth factors and signaling molecules during development of cartilaginous tissue and is essential for proper chondrocyte organization, morphology, and survival during embryonic limb development.

Chemistry and function of glycosaminoglycans in the nervous system.

The glycosaminoglycan (GAG) family is characterized by covalently linked repeating disaccharides forming long unbranched polysaccharide chains. Thus far in higher eukaryotes, the family consists of chondroitin sulfate (CS), heparin/heparan sulfate (HS), dermatan sulfate (DS), and hyaluronan (HA). All GAG chains (except HA) are characteristically modified by varying amounts of esterified sulfate. One or more GAG chains are usually found in nature bound to polypeptide backbones in the form of proteoglycans; HA is the exception and is not synthesized covalently bound to a protein. Proteoglycans, and especially their GAG components, participate in numerous biologically significant interactions with growth factors, chemokines, morphogens, guidance molecules, survival factors, and other extracellular and cell-surface components. These interactions are often critical to the basic developmental processes of cellular proliferation and differentiation, as well as to both the onset of disease sequelae and the prevention of disease progression. In the nervous system, GAG/proteoglycan-mediated interactions participate in proliferation and synaptogenesis, neural plasticity, and regeneration. This review focuses on the structure, chemistry, and function of GAGs in nervous system development, disease, and injury response.

Forward genetics defines Xylt1 as a key, conserved regulator of early chondrocyte maturation and skeletal length.

The long bones of the vertebrate body are built by the initial formation of a cartilage template that is later replaced by mineralized bone. The proliferation and maturation of the skeletal precursor cells (chondrocytes) within the cartilage template and their replacement by bone is a highly coordinated process which, if misregulated, can lead to a number of defects including dwarfism and other skeletal deformities. This is exemplified by the fact that abnormal bone development is one of the most common types of human birth defects. Yet, many of the factors that initiate and regulate chondrocyte maturation are not known. We identified a recessive dwarf mouse mutant (pug) from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. pug mutant skeletal elements are patterned normally during development, but display a ~20% length reduction compared to wild-type embryos. We show that the pug mutation does not lead to changes in chondrocyte proliferation but instead promotes premature maturation and early ossification, which ultimately leads to disproportionate dwarfism. Using sequence capture and high-throughput sequencing, we identified a missense mutation in the Xylosyltransferase 1 (Xylt1) gene in pug mutants. Xylosyltransferases catalyze the initial step in glycosaminoglycan (GAG) chain addition to proteoglycan core proteins, and these modifications are essential for normal proteoglycan function. We show that the pug mutation disrupts Xylt1 activity and subcellular localization, leading to a reduction in GAG chains in pug mutants. The pug mutant serves as a novel model for mammalian dwarfism and identifies a key role for proteoglycan modification in the initiation of chondrocyte maturation.

Neutral sphingomyelinase 2 deficiency increases hyaluronan synthesis by up-regulation of Hyaluronan synthase 2 through decreased ceramide production and activation of Akt.

Fibroblasts from the fro/fro mouse, with a deletion in the Smpd3 gene coding for the active site of neutral sphingomyelinase 2 (NSMase2), secreted increased amounts of hyaluronan (HA). This was reversed by transfection with the Smpd3 gene, suggesting a connection between sphingolipid and glycosaminoglycan metabolism. The deficiency of NSMase2 resulted in storage of sphingomyelin (SM) and cholesterol with a 50% reduction in ceramides (Cer). RT-PCR and Western blot analysis showed that increased HA secretion resulted from increased hyaluronan synthase 2 (HAS2) activity localized to sphingolipid-enriched lipid rafts. Although cholesterol levels were also elevated in lipid rafts from mouse fibroblasts deficient in lysosomal acid SMase activity (deletion of the Smpd1(-/-) gene), there was no increase in HA secretion. We then showed that in fro/fro fibroblasts, the reduced ceramide was associated with decreased phosphorylation of protein phosphatase 2A (PP2A) and increased phosphorylation of its substrate Akt-p, together with PI3K, PDK1, mTOR (mammalian target of rapamycin), and p70S6K, although PTEN was unaffected. Exogenous ceramide, as well as inhibitors of Akt (Akt inhibitor VIII), PI 3-kinase (LY294002 and wortmannin), and mTOR (rapamycin) reduced secretion of HA, whereas the NSMase2 inhibitor GW4869 increased HA synthesis and secretion. We propose that NSMase2/Cer are the key mediators of the regulation of HA synthesis, via microdomains and the Akt/mTOR pathway.