Concomitant Neuronal Tau Deposition and FKBP52 Decrease Is an Early Feature of Different Human and Experimental Tauopathies.

BACKGROUND: Pathological tau proteins constitute neurofibrillary tangles that accumulate in tauopathies including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and familial frontotemporal lobar degeneration (FTLD-Tau). We previously showed that the FKBP52 immunophilin interacts functionally with tau and strongly decreases in AD brain neurons in correlation with tau deposition. We also reported that FKBP52 co-localizes with autophagy-lysosomal markers and an early pathological tau isoform in AD neurons, suggesting its involvement in autophagic tau clearance. OBJECTIVE: Our objective was to evaluate if differences in neuronal FKBP52 expression levels and subcellular localization might be detected in AD, PSP, familial FTLD-Tau, and in the hTau-P301 S mouse model compared to controls. METHODS: Cell by cell immunohistofluorescence analyses and quantification of FKBP52 were performed on postmortem brain samples of some human tauopathies and on hTau-P301 S mice spinal cords. RESULTS: We describe a similar FKBP52 decrease and its localization with early pathological tau forms in the neuronal autophagy-lysosomal pathway in various tauopathies and hTau-P301 S mice. We find that FKBP52 decreases early during the pathologic process as it occurs in rare neurons with tau deposits in the marginally affected frontal cortex region of AD Braak IV brains and in the spinal cord of symptomless 1-month-old hTau-P301 S mice. CONCLUSION: As FKBP52 plays a significant role in cellular signaling and conceivably in tau clearance, our data support the idea that the prevention of FKBP52 decrease or the restoration of its normal expression at early pathologic stages might represent a new potential therapeutic approach in tauopathies including AD, familial FTLD-Tau, and PSP.

FKBP52 in Neuronal Signaling and Neurodegenerative Diseases: A Microtubule Story.

The FK506-binding protein 52 (FKBP52) belongs to a large family of ubiquitously expressed and highly conserved proteins (FKBPs) that share an FKBP domain and possess Peptidyl-Prolyl Isomerase (PPIase) activity. PPIase activity catalyzes the isomerization of Peptidyl-Prolyl bonds and therefore influences target protein folding and function. FKBP52 is particularly abundant in the nervous system and is partially associated with the microtubule network in different cell types suggesting its implication in microtubule function. Various studies have focused on FKBP52, highlighting its importance in several neuronal microtubule-dependent signaling pathways and its possible implication in neurodegenerative diseases such as tauopathies (i.e., Alzheimer disease) and alpha-synucleinopathies (i.e., Parkinson disease). This review summarizes our current understanding of FKBP52 actions in the microtubule environment, its implication in neuronal signaling and function, its interactions with other members of the FKBPs family and its involvement in neurodegenerative disease.

Decrease of neuronal FKBP4/FKBP52 modulates perinuclear lysosomal positioning and MAPT/Tau behavior during MAPT/Tau-induced proteotoxic stress.

Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis.Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/ßIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system.

Oral nomegestrol acetate and transdermal 17-beta-estradiol for preventing post-partum relapses in multiple sclerosis: The POPARTMUS study.

BACKGROUND: Sex steroids could explain the course of multiple sclerosis (MS) in pregnancy. OBJECTIVE: To compare the annualized relapse rate (ARR) 12 weeks post-partum in women treated with nomegestrol acetate (NOMAc) and 17-beta-estradiol (E2) versus placebo. METHODS: POPARTMUS is a randomized, proof-of-concept trial in women with MS, receiving oral NOMAc 10 mg/day and transdermal estradiol 75 µg/week, or placebo. RESULTS: Recruitment was stopped prematurely due to slow inclusions (n = 202). No treatment effect was observed on ARR after 12 weeks (sex steroids = 0.90 (0.58-1.39), placebo = 0.97 (0.63-1.50) (p = 0.79)). CONCLUSION: POPARTMUS failed showing efficacy of a NOMAc-E2 combination in preventing post-partum relapses.

Bioinspired Hybrid Fluorescent Ligands for the FK1 Domain of FKBP52.

The protein FKBP52 is a steroid hormone receptor coactivator likely involved in neurodegenerative disease. A series of small, water-soluble, bioinspired, pseudopeptidic fluorescent ligands for the FK1 domain of this protein are described. The design is such that engulfing of the ligand in the pocket of this domain is accompanied by hydrogen-bonding of the dansyl chromophore which functions as both an integral part of the ligand and a fluorescent reporter. Binding is concomitant with a significant wavelength shift and an enhancement of the ligand fluorescence signal. Excitation of FK1 domain native tryptophan residues in the presence of bound ligand results in Förster resonance energy transfer. Variation of key ligand residues within the short sequence was undertaken, and the interaction of the resulting library with the protein was measured by techniques including isothermal calorimetry analysis, fluorescence, and FRET quenching, and a range of Kd values were determined. Cocrystallization of a protein ligand complex at 2.30 Å resolution provided detailed information at the atomic scale, while also providing insight into native substrate binding.

Steroids and Brain, a Rising Bio-Medical Domain: a Perspective.

Some newly described steroid-related compounds, also found in the rest of the body, are formed and active in the central nervous system, particularly in the brain. Some are of pharmacological and physiopathological interest. We specifically report on two compounds, "MAP4343," a new neurosteroid very efficient antidepressant, and "FKBP52," a protein component of hetero-oligomeric steroid receptors that we found involved in cerebral function, including in Alzheimer's disease.

Neurosteroids: non-genomic pathways in neuroplasticity and involvement in neurological diseases.

Neurosteroids are neuroactive brain-born steroids. They can act through non-genomic and/or through genomic pathways. Genomic pathways are largely described for steroid hormones: the binding to nuclear receptors leads to transcription regulation. Pregnenolone, Dehydroepiandrosterone, their respective sulfate esters and Allopregnanolone have no corresponding nuclear receptor identified so far whereas some of their non-genomic targets have been identified. Neuroplasticity is the capacity that neuronal networks have to change their structure and function in response to biological and/or environmental signals; it is regulated by several mechanisms, including those that involve neurosteroids. In this review, after a description of their biosynthesis, the effects of Pregnenolone, Dehydroepiandrosterone, their respective sulfate esters and Allopregnanolone on their targets will be exposed. We then shall highlight that neurosteroids, by acting on these targets, can regulate neurogenesis, structural and functional plasticity. Finally, we will discuss the therapeutic potential of neurosteroids in the pathophysiology of neurological diseases in which alterations of neuroplasticity are associated with changes in neurosteroid levels.

Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibromatosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its endogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and trimethoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr-/- mice during myelin development. AHR ablation leads to locomotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr-/- mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased ß-catenin levels and stimulated the binding of ß-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myelination and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors.

Identification of the Tau phosphorylation pattern that drives its aggregation.

Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.

The stress response factor daf-16/FOXO is required for multiple compound families to prolong the function of neurons with Huntington's disease.

Helping neurons to compensate for proteotoxic stress and maintain function over time (neuronal compensation) has therapeutic potential in aging and neurodegenerative disease. The stress response factor FOXO3 is neuroprotective in models of Huntington's disease (HD), Parkinson's disease and motor-neuron diseases. Neuroprotective compounds acting in a FOXO-dependent manner could thus constitute bona fide drugs for promoting neuronal compensation. However, whether FOXO-dependent neuroprotection is a common feature of several compound families remains unknown. Using drug screening in C. elegans nematodes with neuronal expression of human exon-1 huntingtin (128Q), we found that 3ß-Methoxy-Pregnenolone (MAP4343), 17ß-oestradiol (17ßE2) and 12 flavonoids including isoquercitrin promote neuronal function in 128Q nematodes. MAP4343, 17ßE2 and isoquercitrin also promote stress resistance in mutant Htt striatal cells derived from knock-in HD mice. Interestingly, daf-16/FOXO is required for MAP4343, 17ßE2 and isoquercitrin to sustain neuronal function in 128Q nematodes. This similarly applies to the GSK3 inhibitor lithium chloride (LiCl) and, as previously described, to resveratrol and the AMPK activator metformin. Daf-16/FOXO and the targets engaged by these compounds define a sub-network enriched for stress-response and neuronally-active pathways. Collectively, these data highlights the dependence on a daf-16/FOXO-interaction network as a common feature of several compound families for prolonging neuronal function in HD.

Unexpected central role of the androgen receptor in the spontaneous regeneration of myelin.

Lost myelin can be replaced after injury or during demyelinating diseases in a regenerative process called remyelination. In the central nervous system (CNS), the myelin sheaths, which protect axons and allow the fast propagation of electrical impulses, are produced by oligodendrocytes. The abundance and widespread distribution of oligodendrocyte progenitors (OPs) within the adult CNS account for this remarkable regenerative potential. Here, we report a key role for the male gonad, testosterone, and androgen receptor (AR) in CNS remyelination. After lysolecithin-induced demyelination of the male mouse ventral spinal cord white matter, the recruitment of glial fibrillary acidic protein-expressing astrocytes was compromised in the absence of testes and testosterone signaling via AR. Concomitantly, the differentiation of OPs into oligodendrocytes forming myelin basic protein (MBP)+ and proteolipid protein-positive myelin was impaired. Instead, in the absence of astrocytes, axons were remyelinated by protein zero (P0)+ and peripheral myelin protein 22-kDa (PMP22)+ myelin, normally only produced by Schwann cells in the peripheral nervous system. Thus, testosterone favors astrocyte recruitment and spontaneous oligodendrocyte-mediated remyelination. This finding may have important implications for demyelinating diseases, psychiatric disorders, and cognitive aging. The testosterone dependency of CNS oligodendrocyte remyelination may have roots in the evolutionary history of the AR, because the receptor has evolved from an ancestral 3-ketosteroid receptor through gene duplication at the time when myelin appeared in jawed vertebrates.

A ß-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II ß-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein.

Caspase-cleaved Tau-D(421) is colocalized with the immunophilin FKBP52 in the autophagy-endolysosomal system of Alzheimer's disease neurons.

Pathologic modifications of the Tau protein leading to neurofibrillary tangle (NFT) formation are a common feature of a wide range of neurodegenerative diseases known as tauopathies, which include Alzheimer's disease (AD). We previously showed that the immunophilin FKBP52 physically and functionally interacts with Tau, and we recently reported that FKBP52 levels are abnormally low in AD patients' brains. To decipher the mechanism of FKBP52 decrease in AD brains, we performed multiple labeling immunohistofluorescence and lysosomal purification using postmortem brain samples of healthy controls (n = 8) and AD (n = 20) patients. Confocal analysis revealed that FKBP52 localizes to the endolysosomal system. We also report FKBP52 colocalization with the truncated Tau-D(421) in the autophagy-endolysosomal system in some AD neurons and that the decrease of FKBP52 correlates with NFT formation. Additional experiments of autophagy inhibition in Tau-inducible SH-SY5Y cells allowed demonstrating FKBP52 release in the extracellular milieu. Our findings point out the possibility that FKBP52 could be abnormally released from NFTs negative neurons in AD brains in correlation with the early pathologic Tau-D(421) neuronal accumulation.

Effects of the Synthetic Neurosteroid: 3ß-Methoxypregnenolone (MAP4343) on Behavioral and Physiological Alterations Provoked by Chronic Psychosocial Stress in Tree Shrews.

BACKGROUND: Most currently available active antidepressant drugs are selective serotonin/noradrenaline reuptake inhibitors. However, as their clinical efficacy is not immediate, long-term administration is often accompanied by substantial side effects, and numerous patients remain non- or partial responders. We have recently found that the synthetic neurosteroid derivative 3ß-methoxypregnenolone, which binds to the microtubule-associated protein-2, can provide a novel therapeutic approach in experimental model of depressive disorders in rats. To further validate the antidepressant-like efficacy of 3ß-methoxypregnenolone, we investigated effects of a longer treatment (4-week oral administration; 50mg/kg/d) in a nonrodent species, the tree shrew, exposed to psychosocial stress that elicits close-to-human alterations observed in patients with depressive disorders. METHODS: During the experimental period, physiological parameters were registered, including core body temperature and electroencephalogram, while animals were videotaped to analyze their avoidance behavior. Morning urine samples were collected for measurements of cortisol and noradrenaline levels. RESULTS: We found that treatment with 3ß-methoxypregnenolone abolished stress-triggered avoidance behavior and prevented hormone hypersecretion, hypothermia, and sleep disturbances, further suggesting its antidepressant-like efficacy. Comparative treatment with fluoxetine also prevented some of the physiological alterations, while the hypersecretion of cortisol and sleep disturbances were not or partially restored by fluoxetine, suggesting a better efficacy of 3ß-methoxypregnenolone. Alpha-tubulin isoforms were measured in hippocampi: we found that 3ß-methoxypregnenolone reversed the specific decrease in acetylation of α-tubulin induced by psychosocial stress, while it did not modify the psychosocial stress-elicited reduction of tyrosinated α-tubulin. CONCLUSIONS: Taken together, these data strongly suggest a potent antidepressant-like effect of 3ß-methoxypregnenolone on translational parameters.

From steroid hormones to depressive states and senile dementias: New mechanistic, therapeutical and predictive approaches.

The discovery of "neurosteroids" leads to new descriptions and treatments of neuro-pathological pathologies. 3-ß-methoxy-pregnenolone may be used to treat alterations of neuro-traumatisms and cerebral lesions associated with depressive states. Protein FKBP52 is involved in the dysfunction of the tau protein in Alzheimer's disease and senile dementias. In all cases, neuronal microtubules are involved in the mechanism of lesions and their repair.

Liver X receptors alpha and beta promote myelination and remyelination in the cerebellum.

The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and ß are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/ß in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

The FK506-binding protein FKBP52 in vitro induces aggregation of truncated Tau forms with prion-like behavior.

Tauopathies, including Alzheimer's disease (AD), are neurodegenerative diseases associated with the pathologic aggregation of human brain Tau protein. Neuronal Tau is involved in microtubule (MT) formation and stabilization. We showed previously that the immunophilin FK506-binding protein of MW ∼52 kDa (FKBP52) interferes with this function of full-length Tau and provokes aggregation of a disease-related mutant of Tau. To dissect the molecular interaction between recombinant human FKBP52 and Tau, here, we study the effect of FKBP52 on a functional Tau fragment (Tau-F4, Ser(208)-Ser(324)) containing part of the proline- rich region and MT-binding repeats. Therefore, we perform MT assembly and light-scattering assays, blue native PAGE analysis, electron microscopy, and Tau seeding experiments in SH-SY5Y human neuroblastoma cells. We show that FKBP52 (6 µM) prevents MT formation generated by Tau-F4 (5 µM) and induces Tau-F4 oligomerization and aggregation. Electron microscopy analyses show granular oligomers and filaments of Tau-F4 after short-time FKBP52 incubation. We demonstrate that the terminal parts of Tau interfere with the effects of FKBP52. Finally, we find that FKBP52-induced Tau-F4 oligomers cannot only generate in vitro, direct conformational changes in full-length Tau and that their uptake into neuronal cells can equally lead to aggregation of wild-type endogenous Tau. This suggests a potential prion-like property of these particular Tau-F4 aggregates. Collectively, our results strengthen the hypothesis of FKBP52 involvement in the Tau pathogenicity process.

Immunophilin FKBP52 induces Tau-P301L filamentous assembly in vitro and modulates its activity in a model of tauopathy.

The Tau protein is the major component of intracellular filaments observed in a number of neurodegenerative diseases known as tauopathies. The pathological mutant of Tau containing a proline-to-leucine mutation at position 301 (P301L) leads to severe human tauopathy. Here, we assess the impact of FK506-binding protein with a molecular mass of ∼52 kDa (FKBP52), an immunophilin protein that interacts with physiological Tau, on Tau-P301L activity. We identify a direct interaction of FKBP52 with Tau-P301L and its phosphorylated forms and demonstrate FKBP52's ability to induce the formation of Tau-P301L oligomers. EM analysis shows that Tau-P301L oligomers, induced by FKBP52, can assemble into filaments. In the transgenic zebrafish expressing the human Tau-P301L mutant, FKBP52 knockdown is sufficient to redrive defective axonal outgrowth and branching related to Tau-P301L expression in spinal primary motoneurons. This result correlates with a significant reduction of pT181 pathological phosphorylated Tau and with recovery of the stereotypic escape response behavior. Collectively, FKBP52 appears to be an endogenous candidate that directly interacts with the pathogenic Tau-P301L and modulates its function in vitro and in vivo.