Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. In Alzheimer’s disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer’s disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer’s disease (n = 8; 76.8 ± 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 ± 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P301L mutation hTau^P301L, and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-κB p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau^P301L, that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer’s disease.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes biosynthesis of progesterone (P) and all precursors of glucocorticoids, mineralocorticoids, androgens, and estrogens. Despite the broad interest raised by neurosteroids, the cellular localization of 3 beta-HSD has never been investigated in the brain. We took advantage of the availability of an antiserum raised against human placental 3 beta-HSD to determine the distribution of 3 beta-HSD-immunoreactive structures in the brain of the frog Rana ridibunda by the indirect immunofluorescence technique. Three populations of 3 beta-HSD-immunoreactive cell bodies were observed in the hypothalamus, namely, in the rostral region of the preoptic nucleus, the dorsal infundibular nucleus, and the dorsal part of the ventral infundibular nucleus. A dense network of 3 beta-HSD- immunoreactive nerve fibers was visualized in the dorsal area of the diencephalon, that is, in the lateral neuropil, the corpus geniculatus lateralis, and the nucleus posterolateralis thalami. Reversed-phase HPLC analysis of frog hypothalamic extracts combined with RIA detection showed the presence of substantial amounts of immunoreactive steroids coeluting with P and 17-hydroxyprogesterone (17OH-P). The synthesis of delta 4–3-keto-steroids in the frog hypothalamus was investigated using the pulse-chase technique with 3H-pregnenolone (3H-delta 5P) as a precursor. The formation of five tritiated metabolites of 3H-delta 5P was observed, one of which coeluted with 17OH-P. Conversion of 3H-delta 5P into this radioactive metabolite was significantly reduced by trilostane, a specific inhibitor of 3 beta-HSD. Immunodetection of newly synthesized steroids in HPLC fractions of hypothalamic extracts, using 17OH-P antibodies, revealed the existence of an immunoreactive steroid that exhibited the same retention time as synthetic 17OH-P. The present study provides the first immunocytochemical mapping of 3 beta- HSD, a key enzyme of the steroid biosynthetic pathway, in the CNS of a vertebrate. The data also demonstrate for the first time biosynthesis of neurosteroids in the brain of a nonmammalian vertebrate.

The brain has high energy requirements to maintain neuronal activity. Consequently impaired mitochondrial function will lead to disease. Normal aging is associated with several alterations in neurosteroid production and secretion. Decreases in neurosteroid levels might contribute to brain aging and loss of important nervous functions, such as memory. Up to now, extensive studies only focused on estradiol as a promising neurosteroid compound that is able to ameliorate cellular bioenergetics, while the effects of other steroids on brain mitochondria are poorly understood or not investigated at all. Thus, we aimed to characterize the bioenergetic modulating profile of a panel of seven structurally diverse neurosteroids (progesterone, estradiol, estrone, testosterone, 3α-androstanediol, DHEA and allopregnanolone), known to be involved in brain function regulation. Of note, most of the steroids tested were able to improve bioenergetic activity in neuronal cells by increasing ATP levels, mitochondrial membrane potential and basal mitochondrial respiration. In parallel, they modulated redox homeostasis by increasing antioxidant activity, probably as a compensatory mechanism to a slight enhancement of ROS which might result from the rise in oxygen consumption. Thereby, neurosteroids appeared to act via their corresponding receptors and exhibited specific bioenergetic profiles. Taken together, our results indicate that the ability to boost mitochondria is not unique to estradiol, but seems to be a rather common mechanism of different steroids in the brain. Thus, neurosteroids may act upon neuronal bioenergetics in a delicate balance and an age-related steroid disturbance might be involved in mitochondrial dysfunction underlying neurodegenerative disorders.