Nadia Soussi-Yanicostas

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 Alzheimers 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.

A novel role for anosmin-1 in the adhesion and migration of oligodendrocyte precursors

At embryonic stages of development, oligodendrocyte precursors (OPCs) generated in the preoptic area colonize the entire optic nerve (ON). Different factors controlling migration of ON OPCs have been identified, including secreted growth factors, morphogens and guidance cues, as well as cell adhesion molecules. We have shown previously that the soluble form of the extracellular matrix (ECM) protein anosmin‐1, impairs OPC migration induced by FGF‐2. In the present work, we show that anosmin‐1 is expressed by both migrating OPCs and axons of the retinal ganglion cells in the embryonic ON. In vitro, we observe that OPC migration is strongly impaired by contact with anosmin‐1 when used as a substrate and, in contrast to previous results, this effect is independent of FGF‐2/FGFR1 signaling. We also show that OPCs preferentially adhere to anosmin‐1 when compared with other ECM molecules used as substrates, and that when the endogenous anosmin‐1 expressed by OPCs is blocked, OPC adhesion to all the different substrates (including anosmin‐1), is significantly reduced. This novel effect of anosmin‐1 on cell adhesion is also independent of FGF‐2/FGFR1. We finally demonstrate that the blockade of the endogenous anosmin‐1 expressed by OPCs impairs their migration. Our data suggest that the endogenous anosmin‐1 expressed by OPCs is necessary for the correct adhesion of these cells to the different components of the ECM (including anosmin‐1 itself), contributing to the migration of these cells.

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.

Prokineticin 2 Expression Is Associated with Neural Repair of Injured Adult Zebrafish Telencephalon

Prokineticin 2 (PROK2) is a secreted protein that regulates diverse biological processes including olfactory bulb neurogenesis in adult mammals. However, its precise role in this process is as yet not fully understood. Because it is well known that adult teleost fish, including zebrafish, display an intense proliferative activity in several brain regions, we took advantage of this feature to analyze the distribution of PROK2 transcripts in the adult zebrafish brain and during injury-induced telencephalon (TC) regeneration. First, we characterized the zebrafish PROK2 gene and showed that its transcription takes place in almost all proliferating areas previously identified in adult zebrafish brain. Moreover, in TC, PROK2 transcription was mainly restricted to neurons. Next, using a novel model of TC injury in adult zebrafish, we observed that TC lesion induced a dramatic increase in cell proliferation within the injured hemisphere in regions located both adjacent and distal to injury sites. Moreover, our data strongly suggest that cell proliferation was followed by the migration of newly generated neurons toward injury sites. In addition, we observed a transient over-expression of PROK2 transcripts, which was detected in cells surrounding the lesion during the very first days post injury, and, a few days later, in broad cell rows extending from cortical regions of the TC toward injury sites. PROK2 over-expression was no longer detected when the regeneration process was close to completion, showing that ectopic PROK2 transcription paralleled neuronal regeneration. Taken together, our results suggest that in adult zebrafish brain, PROK2 may play a role in both constitutive and injury-induced neurogenesis.

Dynamic roles of FGF-2 and Anosmin-1 in the migration of neuronal precursors from the subventricular zone during pre- and postnatal development

FGF-2 and Anosmin-1 are diffusible proteins which act in cell proliferation and/or migration during CNS development. We describe their developmental expression patterns in the subventricular zone (SVZ) of the forebrain and the neuronal precursors (NPs) that migrate from this neurogenic site towards the olfactory bulb, forming the rostral migratory stream (RMS). The analysis is carried out before (E14), during (E17, P5) and after (P15) the peaks of migration along the RMS and before this acquires its mature conformation. At all these stages, FGF-2 exerts a FGFR1-mediated motogenic effect on NPs and induces the proliferation of SVZ astrocytes (putatively type B cells from triads), and Anosmin-1 works as a typical chemotropic agent for the NPs (mediated by FGFR1 at P5-P15). Altogether, our results are consistent with the notion that FGF-2 increases cell proliferation in the SVZ and would be the motogenic cue which feeds the migration of the newly produced NPs once generated, from early development (E14) and at least until P15, while Anosmin-1 cooperates in this migration attracting the NPs. In this sense, both cues should be considered as two of the first to be chronologically identified as actors in the formation of the RMS.

Anosmin-1a is required for fasciculation and terminal targeting of olfactory sensory neuron axons in the zebrafish olfactory system

The KAL-1 gene underlies the X-linked form of Kallmann syndrome (KS), a neurological disorder that impairs the development of the olfactory and GnRH systems. KAL-1 encodes anosmin-1, a cell matrix protein that shows cell adhesion, neurite outgrowth, and axon-guidance and -branching activities. We used zebrafish embryos as model to better understand the role of this protein during olfactory system (OS) development. First, we detected the protein in olfactory sensory neurons from 22 h post-fertilization (hpf) onward, i.e. prior their pioneer axons reached presumptive olfactory bulbs (OBs). We found that anosmin-1a depletion impaired the fasciculation of olfactory axons and their terminal targeting within OBs. Last, we showed that kal1a inactivation induced a severe decrease in the number of GABAergic and dopaminergic OB neurons. Though the phenotypes induced following anosmin-1a depletion in zebrafish embryos did not match precisely the defects observed in KS patients, our results provide the first demonstration of a direct requirement for anosmin-1 in OS development in vertebrates and stress the role of OB innervation on OB neuron differentiation.

FGFR1 and anosmin-1 underlying genetically distinct forms of Kallmann syndrome are co-expressed and interact in olfactory bulbs

Kallmann syndrome is a genetically heterogeneous developmental disease characterised by a partial or complete lack of olfactory bulb development. Two genes underlying this disease have so far been identified: the KAL-1 gene, which encodes anosmin-1, an extracellular matrix protein that promotes axonal guidance and branch formation in vitro; and KAL-2, which encodes the known FGFR1. The implication of FGFR1 and anosmin-1 in the same developmental disease led us to test whether anosmin-1 and FGFR1 interact during the development of the olfactory system. In this paper, we showed that the two proteins co-localise in the olfactory bulb during development in rat. Using cross-immunoprecipitation assays of olfactory bulb extracts, we also demonstrated that anosmin-1 and FGFR1 are comprised within the same protein complex. Moreover, we show that anosmin-1 expression in CHO transfected cells increases FGFR1 accumulation, suggesting that anosmin-1 may act as a positive extracellular regulator of FGFR1 signalling. Taken together, our findings strongly suggest that anosmin-1 is an essential component of a FGFR1 pathway that plays a key role during olfactory bulb morphogenesis.

Prokineticin receptor 2 expression identifies migrating neuroblasts and their subventricular zone transient-amplifying progenitors in adult mice

The adult subventricular zone (SVZ) contains progenitors cells, which continually give rise to new neurons that migrate along the rostral migratory stream (RMS) to the olfactory bulbs (OB). Prokineticin receptor 2 (ProKR2) is a G‐protein‐coupled receptor that plays an essential role in this migration process. However, the identity of the prokr2‐expressing cells has not yet been clearly established. Here, we have characterized in detail the identity of the prokr2‐expressing cells in the SVZ/RMS/OB pathway in adult mice. In the SVZ, accumulation of prokr2 transcripts was detected in almost all migrating neuroblasts or type A cells as well as in a large population of their precursors, the rapidly dividing type C cells. Moreover, we observed that, in dissociated SVZ cells from Mash1::GFP postnatal mice, ProKR2 protein is also present in type C and type A cells. We found that, along the RMS and in the OB, prokr2 expression was restricted to migrating type A cells and was absent in astrocytes. Finally, we observed a highly marked decrease of prokr2 expression in Mash1−/− mutant mice, suggesting that this transcription factor directly or indirectly regulates prokr2 expression. Although the expression of ProKR2 in migrating type A cells is in good agreement with the essential role played by this receptor during this migration process, its expression in a large population of their progenitors suggests an additional function for ProKR2, providing novel insights into the role of ProKR2/ProK2 signalling in adult neurogenesis. J. Comp. Neurol. 512:232–242, 2009.

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 Alzheimers 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 Alzheimers disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimers 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 Alzheimers disease.

Expression pattern of Anosmin-1 during pre- and postnatal rat brain development

Anosmin‐1 participates in the development of the olfactory and GnRH systems. Defects in this protein are responsible for both the anosmia and the hypogonadotrophic hypogonadism found in Kallmanns syndrome patients. Sporadically, these patients also manifest some neurological symptoms that are not explained in terms of the developmental defects in the olfactory system. We describe the pattern of Anosmin‐1 expression in the central nervous system during rat development using a novel antibody raised against Anosmin‐1 (Anos1). The areas with Anos1‐stained neurons and glial cells were classified into three groups: (1) areas with immunoreactivity from embryonic day 16 to postnatal day (P) 15; (2) areas with Anosmin‐1 expression only at postnatal development; (3) nuclei with immunoreactivity only at P15. Our data show that Anos1 immunoreactivity is detected in projecting neurons and interneurons within areas of the brain that may be affected in patients with Kallmanns syndrome that develop both the principal as well as sporadic symptoms. Developmental Dynamics 237:2518–2528, 2008.