Vipers of the Middle East: A Rich Source of Bioactive Molecules.

Snake venom serves as a tool of defense against threat and helps in prey digestion. It consists of a mixture of enzymes, such as phospholipase A2, metalloproteases, and l-amino acid oxidase, and toxins, including neurotoxins and cytotoxins. Beside their toxicity, venom components possess many pharmacological effects and have been used to design drugs and as biomarkers of diseases. Viperidae is one family of venomous snakes that is found nearly worldwide. However, three main vipers exist in the Middle Eastern region: Montivipera bornmuelleri, Macrovipera lebetina, and Vipera (Daboia) palaestinae. The venoms of these vipers have been the subject of many studies and are considered as a promising source of bioactive molecules. In this review, we present an overview of these three vipers, with a special focus on their venom composition as well as their biological activities, and we discuss further frameworks for the exploration of each venom.

HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tau pathology.

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.

ZEB2 zinc-finger missense mutations lead to hypomorphic alleles and a mild Mowat-Wilson syndrome.

Mowat-Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive facial gestalt-multiple congenital anomaly syndrome, commonly associating microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects, urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to haplo-insufficiency through premature stop codons or large gene deletions. Only three missense mutations have been reported so far; none of which resides in a known functional domain of ZEB2. In this study, we report and analyze the functional consequences of three novel missense mutations, p.Tyr1055Cys, p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger (C-ZF) domain of ZEB2. Patients' phenotype included the facial gestalt of MWS and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies showed that all the three mutations prevented binding and repression of the E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the zebrafish morphant technology, we performed rescue experiments using wild-type (WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue, correlating with the severity of patients' phenotype, was observed. Our data provide evidence that these missense mutations cause a partial loss of function of ZEB2, suggesting that its role is not restricted to repression of E-cadherin. Functional domains other than C-ZF may play a role in early embryonic development. Finally, these findings broaden the clinical spectrum of ZEB2 mutations, indicating that MWS ought to be considered in patients with lesser degrees of ID and a suggestive facial gestalt, even in the absence of congenital malformation.

Spatacsin and spastizin act in the same pathway required for proper spinal motor neuron axon outgrowth in zebrafish.

Hereditary spastic paraplegias (HSPs) are rare neurological conditions caused by degeneration of the long axons of the cerebrospinal tracts, leading to locomotor impairment and additional neurological symptoms. There are more than 40 different causative genes, 24 of which have been identified, including SPG11 and SPG15 mutated in complex clinical forms. Since the vast majority of the causative mutations lead to loss of function of the corresponding proteins, we made use of morpholino-oligonucleotide (MO)-mediated gene knock-down to generate zebrafish models of both SPG11 and SPG15 and determine how invalidation of the causative genes (zspg11 and zspg15) during development might contribute to the disease. Micro-injection of MOs targeting each gene caused locomotor impairment and abnormal branching of spinal cord motor neurons at the neuromuscular junction. More severe phenotypes with abnormal tail developments were also seen. Moreover, partial depletion of both proteins at sub-phenotypic levels resulted in the same phenotypes, suggesting for the first time, in vivo, a genetic interaction between these genes. In conclusion, the zebrafish orthologues of the SPG11 and SPG15 genes are important for proper development of the axons of spinal motor neurons and likely act in a common pathway to promote their proper path finding towards the neuromuscular junction.

Heparan Sulfate as a Therapeutic Target in Tauopathies: Insights From Zebrafish.

Microtubule-associated protein tau (MAPT) hyperphosphorylation and aggregation, are two hallmarks of a family of neurodegenerative disorders collectively referred to as tauopathies. In many tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and Pick's disease, tau aggregates are found associated with highly sulfated polysaccharides known as heparan sulfates (HSs). In AD, amyloid beta (Aß) peptide aggregates associated with HS are also characteristic of disease. Heparin, an HS analog, promotes misfolding, hyperphosphorylation and aggregation of tau protein in vitro. HS also provides cell surface receptors for attachment and uptake of tau seeds, enabling their propagation. These findings point to HS-tau interactions as potential therapeutic targets in tauopathies. The zebrafish genome contains genes paralogous to MAPT, genes orthologous to HS biosynthetic and chain modifier enzymes, and other genes implicated in AD. The nervous system in the zebrafish bears anatomical and chemical similarities to that in humans. These homologies, together with numerous technical advantages, make zebrafish a valuable model for investigating basic mechanisms in tauopathies and identifying therapeutic targets. Here, we comprehensively review current knowledge on the role of HSs in tau pathology and HS-targeting therapeutic approaches. We also discuss novel insights from zebrafish suggesting a role for HS 3-O-sulfated motifs in tau pathology and establishing HS antagonists as potential preventive agents or therapies for tauopathies.

Surfen and oxalyl surfen decrease tau hyperphosphorylation and mitigate neuron deficits in vivo in a zebrafish model of tauopathy.

Background: Tauopathies comprise a family of neurodegenerative disorders including Alzheimer's disease for which there is an urgent and unmet need for disease-modifying treatments. Tauopathies are characterized by pathological tau hyperphosphorylation, which has been shown to correlate tightly with disease progression and memory loss in patients suffering from Alzheimer's disease. We recently demonstrated an essential requirement for 3-O-sulfated heparan sulfate in pathological tau hyperphosphorylation in zebrafish, a prominent model organism for human drug discovery. Here, we investigated whether in vivo treatment with surfen or its derivatives oxalyl surfen and hemisurfen, small molecules with heparan sulfate antagonist properties, could mitigate tau hyperphosphorylation and neuronal deficits in a zebrafish model of tauopathies. Results: In vivo treatment of Tg[HuC::hTauP301L; DsRed] embryos for 2 days with surfen or oxalyl surfen significantly reduced the accumulation of the pThr181 tau phospho-epitope measured by ELISA by 30% and 51%, respectively. Western blot analysis also showed a significant decrease of pThr181 and pSer396/pSer404 in embryos treated with surfen or oxalyl surfen. Immunohistochemical analysis further confirmed that treatment with surfen or oxalyl surfen significantly decreased the AT8 tau epitope in spinal motoneurons. In addition, in vivo treatment of Tg[HuC::hTauP301L; DsRed] embryos with surfen or oxalyl surfen significantly rescued spinal motoneuron axon-branching defects and, as a likely consequence, the impaired stereotypical touch-evoked escape response. Importantly, treatment with hemisurfen, a surfen derivative devoid of heparan sulfate antagonist activity, does not affect tau hyperphosphorylation, nor neuronal or behavioural deficits in Tg[HuC::hTauP301L; DsRed] embryos. Conclusion: Our findings demonstrate for the first time that surfen, a well-tolerated molecule in clinical settings, and its derivative, oxalyl surfen, could mitigate or delay neuronal defects in tauopathies, including Alzheimer's disease.

What is the evidence that tau pathology spreads through prion-like propagation?

Emerging experimental evidence suggests that the spread of tau pathology in the brain in Tauopathies reflects the propagation of abnormal tau species along neuroanatomically connected brain areas. This propagation could occur through a "prion-like" mechanism involving transfer of abnormal tau seeds from a "donor cell" to a "recipient cell" and recruitment of normal tau in the latter to generate new tau seeds. This review critically appraises the evidence that the spread of tau pathology occurs via such a "prion-like" mechanism and proposes a number of recommendations for directing future research. Recommendations for definitions of frequently used terms in the tau field are presented in an attempt to clarify and standardize interpretation of research findings. Molecular and cellular factors affecting tau aggregation are briefly reviewed, as are potential contributions of physiological and pathological post-translational modifications of tau. Additionally, the experimental evidence for tau seeding and "prion-like" propagation of tau aggregation that has emerged from cellular assays and in vivo models is discussed. Propagation of tau pathology using "prion-like" mechanisms is expected to incorporate several steps including cellular uptake, templated seeding, secretion and intercellular transfer through synaptic and non-synaptic pathways. The experimental findings supporting each of these steps are reviewed. The clinical validity of these experimental findings is then debated by considering the supportive or contradictory findings from patient samples. Further, the role of physiological tau release in this scenario is examined because emerging data shows that tau is secreted but the physiological function (if any) of this secretion in the context of propagation of pathological tau seeds is unclear. Bona fide prions exhibit specific properties, including transmission from cell to cell, tissue to tissue and organism to organism. The propagation of tau pathology has so far not been shown to exhibit all of these steps and how this influences the debate of whether or not abnormal tau species can propagate in a "prion-like" manner is discussed. The exact nature of tau seeds responsible for propagation of tau pathology in human tauopathies remains controversial; it might be tightly linked to the existence of tau strains stably propagating peculiar patterns of neuropathological lesions, corresponding to the different patterns seen in human tauopathies. That this is a property shared by all seed-competent tau conformers is not yet firmly established. Further investigation is also required to clarify the relationship between propagation of tau aggregates and tau-induced toxicity. Genetic variants identified as risks factors for tauopathies might play a role in propagation of tau pathology, but many more studies are needed to document this. The contribution of selective vulnerability of neuronal populations, as an alternative to prion-like mechanisms to explain spreading of tau pathology needs to be clarified. Learning from the prion field will be helpful to enhance our understanding of propagation of tau pathology. Finally, development of better models is expected to answer some of these key questions and allow for the testing of propagation-centred therapies.

Tau Hyperphosphorylation and Oxidative Stress, a Critical Vicious Circle in Neurodegenerative Tauopathies?

Hyperphosphorylation and aggregation of the microtubule-associated protein tau in brain, are pathological hallmarks of a large family of neurodegenerative disorders, named tauopathies, which include Alzheimer's disease. It has been shown that increased phosphorylation of tau destabilizes tau-microtubule interactions, leading to microtubule instability, transport defects along microtubules, and ultimately neuronal death. However, although mutations of the MAPT gene have been detected in familial early-onset tauopathies, causative events in the more frequent sporadic late-onset forms and relationships between tau hyperphosphorylation and neurodegeneration remain largely elusive. Oxidative stress is a further pathological hallmark of tauopathies, but its precise role in the disease process is poorly understood. Another open question is the source of reactive oxygen species, which induce oxidative stress in brain neurons. Mitochondria have been classically viewed as a major source for oxidative stress, but microglial cells were recently identified as reactive oxygen species producers in tauopathies. Here we review the complex relationships between tau pathology and oxidative stress, placing emphasis on (i) tau protein function, (ii) origin and consequences of reactive oxygen species production, and (iii) links between tau phosphorylation and oxidative stress. Further, we go on to discuss the hypothesis that tau hyperphosphorylation and oxidative stress are two key components of a vicious circle, crucial in neurodegenerative tauopathies.