Cyclic Nucleotide-Specific Optogenetics Highlights Compartmentalization of the Sperm Flagellum into cAMP Microdomains.

Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations.

The nonlysosomal glucosylceramidase ß2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren-like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein-protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.

Identification of a feedback loop involving ß-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease.

The lysosomal acid ß-glucosidase GBA1 and the non-lysosomal ß-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of ß-glucosidase activity in general.

Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.

The non-lysosomal ß-glucosidase GBA2 is a non-integral membrane-associated protein at the endoplasmic reticulum (ER) and Golgi.

GBA1 and GBA2 are both ß-glucosidases, which cleave glucosylceramide (GlcCer) to glucose and ceramide. GlcCer is a main precursor for higher order glycosphingolipids but might also serve as intracellular messenger. Mutations in the lysosomal GBA1 underlie Gaucher disease, the most common lysosomal storage disease in humans. Knocking out the non-lysosomal GBA2 in mice results in accumulation of GlcCer outside the lysosomes in various tissues (e.g. testis and liver) and impairs sperm development and liver regeneration. However, the underlying mechanisms are not well understood. To reveal the physiological function of GBA2 and, thereby, of the non-lysosomal GlcCer pool, it is important to characterize the localization of GBA2 and its activity in different tissues. Thus, we generated GBA2-specific antibodies and developed an assay that discriminates between GBA1 and GBA2 without the use of detergent. We show that GBA2 is not, as previously thought, an integral membrane protein but rather a cytosolic protein that tightly associates with cellular membranes. The interaction with the membrane, in particular with phospholipids, is important for its activity. GBA2 is localized at the ER and Golgi, which puts GBA2 in a key position for a lysosome-independent route of GlcCer-dependent signaling. Furthermore, our results suggest that GBA2 might affect the phenotype of Gaucher disease, because GBA2 activity is reduced in Gba1 knock-out fibroblasts and fibroblasts from a Gaucher patient. Our results provide the basis to understand the mechanism for GBA2 function in vivo and might help to unravel the role of GBA2 during pathogenesis of Gaucher disease.