Systemic and network functions of the microtubule-associated protein tau: Implications for tau-based therapies.

Tau is a microtubule-associated neuronal protein, whose primary role was long thought to regulate axonal microtubule assembly. Tau is subject to many posttranslational modifications and can aggregate into neurofibrillary tangles, which are considered to be a hallmark of several neurodegenerative diseases collectively called "tauopathies". The most common tauopathy is Alzheimer's disease, where tau pathology correlates with sites of neurodegeneration. Tau belongs to the class of intrinsically disordered proteins, which are known to interact with many partners and are considered to be involved in various signaling, regulation and recognition processes. Thus more recent evidence indicates that tau functionally interacts with many proteins and different cellular structures, which may have an important physiological role and may be involved in neurodegenerative processes. Furthermore, tau can be released from neurons and exert functional effects on other cells. This review article weighs the evidence that tau has subtle but important systemic effects on neuronal network function by maintaining physiological neuronal transmission and synaptic plasticity, which are possibly independent from tau's microtubule modulating activities. Implications for tau-based therapeutic approaches are discussed.

Sema-3A indirectly disrupts the regeneration process of goldfish optic nerve after controlled injury.

BACKGROUND: Neurons of adult mammalian CNS are prevented from regenerating injured axons due to formation of a non-permissive environment. The retinal ganglion cells (RGC), which are part of the CNS, share this characteristic. In sharp contrast, the RGC of lower vertebrates, such as fish, are capable of re-growing injured optic nerve axons, and achieve, through a complex multi-factorial process, functional vision after injury. Semaphorin-3A (sema-3A), a member of the class 3 semaphorins known for its repellent and apoptotic activities, has previously been shown to play a key role in the formation of a non-permissive environment after CNS injury in mammalians. METHODS: The expression of sema-3A and its effect on regenerative processes in injured gold fish retina and optic nerve were investigated in this study. Unilateral optic nerve axotomy or crush was induced in goldfish. 2 microl sema-3A was injected intraviterally 48 hours post injury. Neuronal viability was measured using the lipophilic neurotracer dye 4-Di-10-Asp. Axonal regeneration was initiated using the anterograde dye dextran. Retinas and optic nerves were collected at intervals of 2, 3, 7, 14 and 28 days after the procedure. Using Western blot and immunohistochemical analysis, the expression levels of semaphorin-3A, axonal regeneration, the removal of myelin debris and macrophage invasion were studied. RESULTS: We found a decrease in sema-3A levels in the retina at an early stage after optic nerve injury, but no change in sema-3A levels in the injured optic nerve. Intravitreal injection of sema-3A to goldfish eye, shortly after optic nerve injury, led to destructive effects on several pathways of the regenerative processes, including the survival of retinal ganglion cells, axonal growth, and clearance of myelin debris from the lesion site by macrophages. CONCLUSIONS: Exogenous administration of sema-3A in fish indirectly interferes with the regeneration process of the optic nerve. The findings corroborate our previous findings in mammals, and further validate sema-3A as a key factor in the generation of a non-permissive environment after transection of the optic nerve.

Negative impact of statins on oligodendrocytes and myelin formation in vitro and in vivo.

Statins are widely prescribed drugs in cardiovascular diseases. Recent studies also demonstrated anti-inflammatory and immunomodulatory properties of statins by modulating the activity of small GTPases. Statins are thus considered as potential therapeutic drug for the inflammatory demyelinating disease multiple sclerosis (MS). However, little is known about the effects of statins on myelin-forming oligodendrocytes. Here, we show that statins hamper process and myelin formation in vitro by interfering with Ras and Rho signaling in mature oligodendrocytes and provide evidence that statins impair ongoing remyelination in vivo. Our findings may have significant implications for the application of statins in MS patients and in other demyelinating diseases of the CNS.

Features and functions of oligodendrocytes and myelin proteins of lower vertebrate species.

The myelin-forming cells in the central nervous system (CNS) of lower vertebrate species, in particular those of fish, profoundly differ from their mammalian counterparts in their biochemical phenotype in that they express Po-like glycoproteins as major myelin protein constituents instead of proteolipid protein, while in their overall cellular structure and their cell lineage relationships, they closely resemble mammalian oligodendrocytes. While molecular biology in the past has allowed to appropriately classify the major myelin proteins synthesized by fish oligodendrocytes, heterologous expression studies are expected to give a deeper insight into the particular features and the conserved functions of these proteins required for myelin formation and maintenance in fish. It is hoped that this approach will also help to improve our understanding of the molecular processes underlying the unique capacity of fish oligodendrocytes for remyelination after injury in the CNS. This survey may stimulate neuroscientists to engage into this exciting field.

Molecular cloning and partial functional characterization of Tsha3--a novel modulatory potassium channel alpha-subunit of trout CNS.

A novel Shaker-related potassium channel subunit termed Tsha3 that is widely expressed in the CNS of trout was PCR-cloned and sequenced: its deduced amino acid sequence showed an extended N-terminal domain with a high proportion of negatively charged residues and possessed highest similarity with KCNA10, a human epithelial potassium channel. Upon heterologous expression in Sf21 cells, homomeric Tsha3 did not yield voltage-activated potassium channels but produced only ohmic currents that reversed at -15 mV. After co-expression with Tsha1, a novel outward rectifier current was generated that differed from homomeric Tsha1 by its slower kinetics of activation, its partial current inactivation, and its partial blockade by 5 mM TEA as well as 1 microM DTX. Co-immunoprecipitation studies using anti-Tsha3 antibodies confirmed that Tsha3 tightly bound with Tsha1 in co-infected Sf21 cells. As revealed from GFP- and DsRed-labeling studies, the pattern of distribution of Tsha1 was profoundly altered after co-infection with Tsha3 subunits.

The 36K protein of zebrafish CNS myelin is a short-chain dehydrogenase.

Previous studies identified homologues to mammalian myelin genes expressed in the teleost central nervous system (CNS), including myelin basic protein (MBP), protein zero (P0), and a member of the proteolipid protein family, DM20. In addition, an uncharacterized 36-kDa (36K) protein is a major component of teleost myelin, but is not a major component of myelin in other species. In the present study, we sought to better understand myelin proteins and myelination in one teleost, zebrafish, by molecular characterization of the zebrafish 36K protein. Purified zebrafish CNS myelin was isolated and the amino acid sequences of peptides present in the 36-kDa band were determined by mass spectrometry. These sequences matched a previously uncharacterized EST in The Institute for Genome Research (TIGR) zebrafish database that is related to the short-chain dehydrogenase/reductase (SDR) protein family. In vitro expression of the zebrafish 36K cDNA in Neuro 2a cells resulted in a protein product that was recognized by a 36K polyclonal antibody. The zebrafish 36K mRNA and protein expression patterns were determined and correlated to other known myelin gene expression profiles. In addition, we determined by in situ hybridization that a human 36K homologue (FLJ13639) is expressed in oligodendrocytes and neurons in the adult human cortex. This study identified a major myelin protein in zebrafish, 36K, as a member of the SDR superfamily; an expression pattern similar to other myelin genes was demonstrated.

Maturation of spiking activity in trout retinal ganglion cells coincides with upregulation of Kv3.1- and BK-related potassium channels.

Developmental changes in membrane excitability and the potassium channel profile were monitored in acutely isolated trout retinal ganglion cells by patch-clamp recording in combination with single-cell RT-PCR. During embryonic development in the egg, a sustained above-threshold stimulation of ganglion cells elicited in most cases only a single spike response. After hatching, the proportion of multiply spiking cells increased strongly and the ability of spike frequency coding was acquired. This was accompanied by the occurrence of a highly tetraethylammonium (TEA)- and quinine-sensitive delayed rectifier current, which gradually masked a rapidly inactivating A-type potassium current that was predominant at earlier stages. Pharmacology of the delayed rectifier current closely matched those of recombinant Traw1, a Kv3.1-related potassium channel in trout. The appearance of this current correlated closely with initial expression of Traw1 and Traw2 channel transcripts, as revealed by multiplex single-cell RT-PCR, whereas mRNA, encoding Shaker-related channel genes in trout (termed Tsha1-Tsha4), were already detectable at early embryonic stages. Iberiotoxin-sensitive, calcium-activated potassium currents (BK) were extremely low before hatching, but increased significantly thereafter. These developmental changes in potassium channel expression occurred after the arrival of retinal fibers in the optic tectum and the initiation of synapse formation in the visual center. It is suggested that early expressed Shaker-related potassium channels could act to influence neuronal differentiation, whereas proper neuronal signaling requires expression of Kv3.1- and BK-related potassium channels.

Molecular cloning, tissue expression, and partial characterization of the major fish CNS myelin protein 36k.

A full-length cDNA clone encoding the major structural protein of trout CNS myelin 36K was isolated and sequenced. The deduced amino acid sequence did not reveal a putative transmembrane domain and exhibited no structural homology with any of the known myelin proteins. 36K instead shared characteristic structural elements with enzymes of the short-chain dehydrogenase family. The highest similarity in the database (60%), however, was obtained with a human protein of unknown function. By Northern blotting, a single mRNA species of about 2 kb was identified, which was expressed in brain tissue but not in liver. By in situ hybridization, a selective labeling of myelinating glial cells in the trout CNS but not in the PNS was revealed. The developmental appearance of the 36K transcript closely coincided with a period of active myelin deposition in most regions of the trout brain. As a first step in elucidating the structural and biochemical role of 36K for myelin formation and maintenance, we have overexpressed it in Escherichia coli as a soluble His-tag fusion protein and purified it in high yield by Ni+-chelated affinity chromatography. By SDS-PAGE, a single band of the expected molecular size was revealed, which heavily cross-reacted with polyclonal antibodies generated against the native protein. The results of circular dichroism spectroscopy are compatible with a betaalphabeta-barrel structure (Rossman fold), confirming the results of computer-assisted secondary structure predictions.

A novel conus peptide ligand for K+ channels.

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.

Pattern of structural differentiation in the optic nerve of trout (Salmo gairdneri).

The development of the trout optic nerve is quantitatively described from early ontogenesis into adulthood. The nerve is oval in cross section until stage 34, thereafter the formation of vertically aligned parallel folds can be observed and thus the unique shape of a folded ribbon is gradually attained. Quantitative measurements revealed a linear increase in cross sectional area, caused in part by the formation of new folds and in part by an increase in size of the preexisting ones. We attribute the continuous expansion of individual folds to an increase in fiber size subsequent to myelination rather than to the addition of new fibers. The total number of glial cells increased concomitantly per fold.Myelinogenesis starst at stage 33 with the ensheathement of axons beginning at the dorsal edge of the primary fold and follows a highly ordered pattern throughout development, strictly succeeding neural outgrowth. The functional significance of this pattern is discussed.

Quantitative morphogenetic investigations on fine structural changes in the optic tectum of the rainbow trout (Salmo gairdneri) during ontogenesis.

The morphogenetic differentiation of synapses of the optic tectum of the rainbow trout was investigated at different stages of development (from hatching to adult) and compared with the improvement in visual discrimination (minimum separable). (1) The main phase of synaptogenesis (increase in number of synapses, length of contact zone and number of vesicles) begins about one week after hatching and continues up to the age of one month, when the larvae start swimming freely. (2) Myelination begins 26 days after hatching and induces the end of the synaptogenesis period. (3) The visual discrimination (minimum separable) of trout larvae improves from 30 degrees of arc on the 10th day after hatching to 1 degree on day 30, then to about 14 to 18 min of arc in the adult. The results are discussed with special reference to previous biochemical investigations on changes in the ganglioside composition of the trout brain during comparable periods of development.