Chromosomal localization of human β globin gene on human chromosome 11 in somatic cell hybrids

We have successfully used a DNA.cDNA molecular hybridization assay to directly determine the presence or absence of human beta globin gene sequences in 20 human-mouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human beta globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human beta globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human beta globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT-4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT-4. 157-BNPT-4 contained the human beta globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human gamma globin cDNA; two lines positive for human beta globin gene sequences also contained human gamma globin gene sequences while one line was negative for both beta and gamma gene sequences. On the basis of these results, the human beta and gamma globin genes have been assigned to human chromosome 11.     

Integrin αvβ1 promotes infection by human metapneumovirus

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for αvβ1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with αv or β1 cDNAs confers hMPV infectivity, whereas reduction of αv and β1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that αvβ1 integrin is a functional receptor for hMPV.     

Crosstalk between Gαi- and Gαq-coupled receptors is mediated by Gβγ exchange

Activation of Galpha(i)-coupled receptors often causes enhancement of the inositol phosphate signal triggered by Galpha(q)-coupled receptors. To investigate the mechanism of this synergistic receptor crosstalk, we studied the Galpha(i)-coupled adenosine A(1) and alpha(2C) adrenergic receptors and the Galpha(q)-coupled bradykinin B(2) and a UTP-preferring P2Y receptor. Stimulation of either Galpha(i)-coupled receptor expressed in COS cells increased the potency and the efficacy of inositol phosphate production by bradykinin or UTP. Likewise, overexpression of Gbeta(1)gamma(2) resulted in a similar increase in potency and efficacy of bradykinin or UTP. In contrast, these stimuli did not affect the potency of direct activators of Galpha(q); a truncated Gbeta(3) mutant had no effect on the receptor-generated signals whereas signals generated at the G-protein level were still enhanced. This suggests that the Gbetagamma-mediated signal enhancement occurs at the receptor level. Almost all possible combinations of Gbeta(1-3) with Ggamma(2-7) were equally effective in enhancing the signals of the B(2) and a UTP-preferring P2Y receptor, indicating a very broad specificity of this synergism. The enhancement of the bradykinin signal by (i) Galpha(i)-activating receptor ligands or (ii) cotransfection of Gbetagamma was suppressed when the B(2) receptor was replaced by a B(2)Gbeta(2) fusion protein. Gbetagamma enhanced the B(2) receptor-stimulated activation of G-proteins as determined by GTPgammaS-induced decrease in high affinity agonist binding and by B(2) receptor-enhanced [(35)S]GTPgammaS binding. These findings support the concept that Gbetagamma exchange between Galpha(i)- and Galpha(q)-coupled receptors mediates this type of receptor crosstalk.     

Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice

Transgenic overexpression of Galphaq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed beta-adrenergic receptor (betaAR) function. The role of betaAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the betaAR kinase (betaARK), which acts to inhibit the kinase, or concomitant overexpression of the beta2AR at low (approximately 30-fold, Galphaq/beta2ARL), moderate (approximately 140-fold, Galphaq/beta2ARM), and high (approximately 1,000-fold, Galphaq/beta2ARH) levels above background betaAR density. Expression of the betaARK inhibitor had no effect on the phenotype, consistent with the lack of increased betaARK levels in Galphaq mice. In marked contrast, Galphaq/beta2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and alpha-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized beta2AR/AC signal cannot be excluded. Higher expression of receptors in Galphaq/beta2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With approximately 1,000-fold overexpression, the majority of Galphaq/beta2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of betaAR signaling is deleterious in heart failure, selective enhancement by overexpressing the beta2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.     

Combined transgenic expression of α-galactosidase and α1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galα(1,3)Gal

Hyperacute rejection of pig organs by humans involves the interaction of Galα(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galα(1,3)Gal were investigated by overexpression of human lysosomal α-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human α-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galα(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human α-galactosidase showed only a 15–25% reduction in binding to natural human anti-Galα(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galα(1,3)Gal and degalactosylation results in the exposure of N-acetyllactosamine residues and potential new xenoepitopes, using α-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human α1,2-fucosyltransferase as a transgene had ≈90% reduced Galα(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing α-galactosidase and α1,2-fucosyltransferase on Galα(1,3)Gal levels. Galα(1,3)Gal-positive COS cells expressing α1,3-galactosyltransferase, α1,2-fucosyltransferase, and α-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, α1,2-fucosyltransferase and α-galactosidase effectively reduced the expression of Galα(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galα(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.     

Progressive hypertrophy and heart failure in β1-adrenergic receptor transgenic mice

Stimulation of cardiac beta1-adrenergic receptors is the main mechanism that increases heart rate and contractility. Consequently, several pharmacological and gene transfer strategies for the prevention of heart failure aim at improving the function of the cardiac beta-adrenergic receptor system, whereas current clinical treatment favors a reduction of cardiac stimulation. To address this controversy, we have generated mice with heart-specific overexpression of beta1-adrenergic receptors. Their cardiac function was investigated in organ bath experiments as well as in vivo by cardiac catheterization and by time-resolved NMR imaging. The transgenic mice had increased cardiac contractility at a young age but also developed marked myocyte hypertrophy (3.5-fold increase in myocyte area). This increase was followed by progressive heart failure with functional and histological deficits typical for humans with heart failure. Contractility was reduced by approximately 50% in 35-week-old mice, and ejection fraction was reduced down to a minimum of approximately 20%. We conclude that overexpression of beta1-adrenergic receptors in the heart may lead to a short-lived improvement of cardiac function, but that increased beta1-adrenergic receptor signalling is ultimately detrimental.     

Rescue of cardiac α-actin-deficient mice by enteric smooth muscle γ-actin

The muscle actins in higher vertebrates display highly conserved amino acid sequences, yet they show distinct expression patterns. Thus, cardiac α-actin, skeletal α-actin, vascular smooth muscle α-actin, and enteric smooth muscle γ-actin comprise the major actins in their respective tissues. To assess the functional and developmental significance of cardiac α-actin, the murine (129/SvJ) cardiac α-actin gene was disrupted by homologous recombination. The majority (≈56%) of the mice lacking cardiac α-actin do not survive to term, and the remainder generally die within 2 weeks of birth. Increased expression of vascular smooth muscle and skeletal α-actins is observed in the hearts of newborn homozygous mutants and also heterozygotes but apparently is insufficient to maintain myofibrillar integrity in the homozygous mutants. Mice lacking cardiac α-actin can be rescued to adulthood by the ectopic expression of enteric smooth muscle γ-actin using the cardiac α-myosin heavy chain promoter. However, the hearts of such rescued cardiac α-actin-deficient mice are extremely hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenically expressed enteric smooth muscle γ-actin reduces cardiac contractility in wild-type and heterozygous mice. These results demonstrate that alterations in actin composition in the fetal and adult heart are associated with severe structural and functional perturbations.     

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (E_δ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by E_δ and the TCR α enhancer, and that it prevents E_δ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.     

Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF–RARα and NPM–RARα

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARalpha and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF-RARalpha and NPM-RARalpha. PLZF-RARalpha transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM-RARalpha transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF-RARalpha transgenic mice, those from NPM-RARalpha transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 microM, 0.1 microM, and 1.0 microM for RARalpha-RXRalpha, NPM-RARalpha, and PML-RARalpha, respectively, but not observed for PLZF-RARalpha even in the presence of 10 microM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF-RARalpha and NPM-RARalpha and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Visualization of alpha9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome

The alpha9 acetylcholine receptor (alpha9 AChR) is specifically expressed in hair cells of the inner ear and is believed to be involved in synaptic transmission between efferent nerves and hair cells. Using a recently developed method, we modified a bacterial artificial chromosome containing the mouse alpha9 AChR gene with a reporter gene encoding green fluorescent protein (GFP) to generate transgenic mice. GFP expression in transgenic mice recapitulated the known temporal and spatial expression of alpha9 AChR. However, we observed previously unidentified dynamic changes in alpha9 AChR expression in cochlear and vestibular sensory epithelia during neonatal development. In the cochlea, inner hair cells persistently expressed high levels of alpha9 AChR in both the apical and middle turns, whereas both outer and inner hair cells displayed dynamic changes of alpha9 AChR expression in the basal turn. In the utricle, we observed high levels of alpha9 AChR expression in the striolar region during early neonatal development and high levels of alpha9 AChR in the extrastriolar region in adult mice. Further, simultaneous visualization of efferent innervation and alpha9 AChR expression showed that dynamic expression of alpha9 AChR in developing hair cells was independent of efferent contacts. We propose that alpha9 AChR expression in developing auditory and vestibular sensory epithelia correlates with maturation of hair cells and is hair-cell autonomous.     

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1α (SDF-1α) and SDF-1β are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1α (SDF-1α) and 1β (SDF-1β), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34⁺ progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1α confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1α and SDF-1β likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4⁺ cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1α and SDF-1β are involved.     

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo

GBA1 mutations that encode lysosomal β-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson’s disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Gaucher disease (GD) is a lysosomal storage disorder caused by loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase). GCase degrades glycosphingolipids (GSLs), including glucosylceramides (GluCers), into glucose and ceramide, and GCase mutations result in the accumulation of GluCer in lysosomes of various tissues. Heterozygote carriers of the same loss-of-function GCase mutations are estimated to be at 5- to 10-fold higher risk for developing Parkinson’s disease (PD) or Lewy body dementia (1). In GD, significant variability exists in the clinical and pathological presentation, resulting in three main GD subtypes (2). Type 1 GD is characterized by visceral abnormalities, including enlarged liver and spleen and bone marrow dysfunction, leading to thrombocytopenia but without neurodegeneration and α-synuclein (α-syn) pathology (3). Types 2 and 3 demonstrate similar visceral symptoms but with additional extensive neuronal loss, α-syn pathology in the form of classical Lewy bodies, and neurological dysfunction (3, 4). As life expectancy of type 1 GD has increased because of enzyme replacement therapy, a higher percentage of patients develop PD symptoms with age (5), suggesting that aging could contribute to the penetrance of GBA1 mutations. The dramatic phenotypic heterogeneity suggests that GD is not a simple, monogenic disease but a complex disorder that is influenced by both genetic and nongenetic modifiers. Although the factors that contribute to clinical and pathological variability in GD are not known, genetic modifiers have been identified that associate with GD severity, including CLN8 and SCARB2 (6, 7). Within PD patients that harbor GBA1 mutations (GBA-PD), the search for genetic modifiers has shown that synergism may exist with the SNCA gene that encodes α-syn and CTSB that encodes lysosomal cathepsin B (8). Variants in lysosomal cathepsins could influence the severity of α-syn accumulation, since, under physiological or pathological conditions, α-syn can be degraded by the lysosome (9–11) and is a direct substrate of cathepsin B and L (12).An additional factor that may contribute to phenotypic variability in GD is the accumulation of specific GluCer subtypes with particular acyl chain lengths. GluCer and other GSLs exist as a family of lipid isoforms differentiated by the length of the N-acyl fatty acid moiety linked to the sphingoid base. GluCer chains range from C14 to C26 in the brain; however, C18 and C24:1 are the predominant species (13). Studies of neuronopathic GD (nGD) brain or mouse models showed intraneuronal accumulation of multiple GluCer species that correlated with neuroinflammation (14–19), and some cases demonstrate selective accumulation of long-chain GluCers in nGD (20). Our recent work in PD patient midbrain neurons showed that inhibition of wild-type (wt) GCase, caused by α-syn, resulted in the selective accumulation of long-chain-length GluCers, including C22 and C24:1, while C14, 16, and C18 were unchanged (21). Together, these data indicate that GluCer accumulation plays an important role in neurodegeneration induced by GBA1 mutations; however, the specific contributions of distinct GluCer species have not been examined.Here, we extend our studies on the role of GSLs in α-syn aggregation to further define conditions that are required to induce pathology and neurological dysfunction. We previously showed that α-syn exists as monomers and high–molecular weight (HMW) oligomers under physiological conditions in human midbrain cultures (22). In vitro, we found that GluCer mildly induced aggregation of α-syn monomers but primarily acted on physiological oligomers to convert them into toxic oligomers and fibrillar inclusions (22). α-syn accumulation can be prevented or reversed by reducing GSLs with GluCer synthase inhibitors (GCSi) in both GD and PD patient cultures, as well as in mouse models (22–24). While this work suggests a close relationship between GCase function and α-syn pathology, additional factors must exist that create a permissive environment for α-syn accumulation. Indeed, studies that used newborn mice or embryonic primary neuron cultures treated with the GCase inhibitor, conduritol beta epoxide (CBE), have shown no changes in α-syn despite reduced GCase activity (25–27). However, other studies that use matured neuron cultures, neuronal cell lines, or adult mice have shown that CBE dramatically induces α-syn aggregates (22, 28–31). We used an in vivo GD model and induced pluripotent stem cell (iPSC)–derived patient midbrain cultures to identify specific conditions that are required to induce α-syn pathology, providing possible explanations for the variable neurological penetrance in patients that harbor GBA1 mutations.