Jane Cooper

Short Fibrils Constitute the Major Species of Seed-Competent Tau in the Brains of Mice Transgenic for Human P301S Tau

The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. SIGNIFICANCE STATEMENT The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimers disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein conformers from TgP301S tau mice and show that seed-competent tau species comprise small fibrils capable of seeding tau pathology in cell and animal models. Characterization of seed-competent tau gives insight into disease mechanisms and therapeutic interventions.

LY393558, a 5-hydroxytryptamine reuptake inhibitor and 5-HT1B/1D receptor antagonist: effects on extracellular levels of 5-hydroxytryptamine in the guinea pig and rat

The stimulation of terminal 5-HT(1B/1D) autoreceptors limits the effects of selective serotonin reuptake inhibitors on extracellular levels of 5-hydroxytryptamine (5-HT, serotonin) in vivo. Microdialysis studies show that acute oral administration of LY393558-a 5-HT reuptake inhibitor and antagonist at both the human 5-HT(1B) and 5-HT(1D) receptor-in the dose range 1-20 mg/kg, increases extracellular levels of 5-HT in both the guinea pig hypothalamus and rat frontal cortex. In both species, the levels of 5-HT that were attained were higher than following an acute, maximally effective dose of fluoxetine (20 mg/kg orally), reaching approximately 1500% in the guinea pig hypothalamus and 700% in the rat frontal cortex. In both species, the response to LY393558 (10 mg/kg p.o.) was impulse dependent, being absent in the presence of tetrodotoxin delivered at 1 microM via the microdialysis probe. The sensitivity to tetrodotoxin contrasted with the effects seen with DL-fenfluramine. Studies in rats showed that the microdialysate 5-HT concentration achieved in the frontal cortex after an acute challenge with LY393558 (5 mg/kg p.o.) was significantly greater than following a chronic regime of fluoxetine treatment (10 mg/kg/day orally for 21 days). Moreover, in rats chronically treated with LY393558 (5 mg/kg/day orally for 21 days), the mean basal concentration, 24 h after the final pretreatment dose, was of the same magnitude as that following chronic fluoxetine. However, in contrast to the response seen in fluoxetine-pretreated animals, a challenge dose of LY393558 still elicited a further increase in extracellular 5-HT in LY393558-pretreated animals. LY393558 is a potent 5-HT reuptake inhibitor and 5-HT(1B/1D) receptor antagonist. Microdialysis studies show that acute oral administration increases extracellular levels of 5-HT, by an impulse-dependent mechanism, above those produced by a maximally effective dose of fluoxetine, and in rats to levels only achieved following chronic fluoxetine treatment. Its neurochemical profile in vivo suggests that it may be a more effective antidepressant with the potential for producing an earlier onset of clinical activity than selective serotonin reuptake inhibitors.

Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF.

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinsons Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the worlds major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.

Ventral subiculum administration of the somatostatin receptor agonist MK-678 increases dopamine levels in the nucleus accumbens.

Somatostatin (or somatotropin-release inhibitory factor, SRIF) binding and in situ hybridisation studies have indicated a high expression of receptor subtypes throughout the rat brain and, in particular, in subregions of the hippocampus and subiculum. In vitro, somatostatin and related peptides, including seglitide (MK-678), hyperpolarize subicular neurones of the burst firing type-a response, which may have functional consequences for their output. One major projection from the subiculum is to the nucleus accumbens. The functional consequence of somatostatin receptor stimulation in the ventral subiculum has been assessed by measuring extracellular levels of dopamine in the ipsilateral nucleus accumbens. In anaesthetised rats, administration of seglitide (MK-678), a somatostatin analogue with selectivity for the SRIF-1 receptor (comprising somatostatin sst2, sst3 and sst5 subtypes) significantly increased extracellular levels of dopamine in the ipsilateral nucleus accumbens shell. The result suggests that hyperpolarization of subicular neurones by MK-678 may lead to activation of the subiculo-accumbens projection system, and an associated increase in dopaminergic function.

TRANSECTION OF TARGETED AXONAL PATHWAYS INHIBITS NETWORK SPREAD OF TAU PATHOLOGY IN A P301S MODEL OF TAU PROPAGATION

classified older individuals into disease stages using deciles of whole-brain PIB distribution volume ratio (DVR), assuming that those with lower whole-brain DVRs are at earlier stages. We identified epicenters by finding ROIs that met the following criteria: (1) the PIB DVR in the ROI is greater than the average of its neighbors on the MST, (2) in the successive stage average PIB DVR in the neighbors increases, and (3) the ROI is not near another epicenter on the MST. Results:At all stages of PIB progression we found a strong significant positive correlation of regional PIB DVR with the average PIB DVR in neighboring regions at the successive stage. Moreover, with each 5 or 10 percentile increase in whole brain DVR we found a progressively stronger positive correlation of regional PIB DVRwith increased PIB DVR in neighboring regions relative to the initial stage. Finally, we identified distinct epicenters of PIB accumulation in the left posterior cingulate, left rostral anterior cingulate, left putamen, right pars opercularis, right suparmarginal, right lateral orbitofrontal, and right pericalcarine cortex which this approach identified as arising independently from one another. Conclusions:PIB appears to arise from multiple epicenters based on patterns of normal brain connectivity.

CHARACTERISATION OF TAU SPECIES INVOLVED IN TAU SEEDING AND SPREAD IN CELLULAR AND ANIMAL MODELS

Background:The neurofibrillary tangle is a pathological hallmark of Alzheimer’s disease (AD) and primarily consists of hyperphosphorylated tau protein (pTau). pTau first appears in the entorhinal cortex in the presymptomatic stage, then gradually disseminates to the hippocampal region around the onset of clinical symptoms of AD. Halting this tau spread in the asympomatic stage is a promising therapeutic approach for AD. The exosome is a small vesicle of 50-100 nm in diameter, enriched in ceramide, and is suggested to contain neuropathogenic proteins, such as prion, a-synuclein, and recently tau proteins. A growing body of evidence suggests that microglia contribute to tauopathy-related pathogenesis in both human and animal models. We hypothesize that microglia transduce tau aggregates into nearby neuronal cells via exosomal secretion, and that inhibition of the exosome synthesis or secretory pathway reduces tau dissemination. Methods: Adeno-associated virus serotype 6 expressing FTDP-17-linked mutation of tau protein was stereotaxically injected into the entorhinal cortical region of C57BL/6 mice, and the animals were sacrificed at 7 and 28 days post injection. The brain specimens were examined for tau accumulation in the hippocampal region. The animals were also systemically treated with specific inhibitors of neutral sphingomyelinase-2 to block exosome synthesis, or colony stimulating factor 1 receptor (CSF1R) to deplete microglia. Results:We found that human tau propagate from entorhinal cortical neurons to dentate granular cells after AAV injection, and that this propagation is sensitive to the inhibition of exosome synthesis or microglial depletion. We also found that tau-containing exosomes isolated from microglia efficiently transduce tau protein to neurons in vitro and in vivo. Finally, these results were reproduced in P301S tau mice (PS19) treated with these compounds. Conclusions: These results demonstrate that exosome secretion from microglia play a significant role in propagation of tau protein from entorhinal cortex to hippocampal neurons. Our findings could lead to an entirely novel paradigm for delaying the progression of disease in AD and other tauopathies such as frontotemporal dementia and chronic traumatic encephalopathy.