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The microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-020-02254-3.

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response., The role of Endonuclease G in autophagy remains unclear. Here the authors report that ENDOG is released from mitochondria during starvation and promotes autophagy by suppressing mTOR signaling and activating DNA damage response.

The protein tyrosine kinase Pyk2 is encoded by PTK2B, a novel Alzheimer's disease (AD) susceptibility variant, with the PTK2B risk allele being associated with increased mRNA levels, suggestive of increased Pyk2 levels. However, the role of Pyk2, a member of the focal adhesion kinase (FAK) family, in AD pathology and its regulation are largely unknown. To address this, we generated mice with neuronal expression of human Pyk2. Because we had previously reported an association of Pyk2 and hyperphosphorylated tau (a hallmark feature of AD) in human tau transgenic pR5 mice, we also generated Pyk2/tau double-transgenic mice, which exhibit increased tyrosine phosphorylation and accumulation of tau. We further demonstrated that Pyk2 colocalizes, interacts with, and phosphorylates tau in vivo and in vitro. Importantly, although Pyk2 interacts with the established tyrosine-directed tau kinase Fyn, we identified an increased Pyk2 activity in mice which constitutively overexpress Fyn (FynCA), and a decreased activity in mice lacking Fyn (FynKO). Together, our study reveals a novel role for Pyk2 as a direct tyrosine kinase of tau that is active downstream of Fyn. Our analysis may enhance the understanding of how the PTK2B risk allele contributes to tauopathy.

The cause of protein accumulation in neurodegenerative disease is incompletely understood. In Alzheimer's disease (AD), the axonally enriched protein Tau forms hyperphosphorylated aggregates in the somatodendritic domain. Consequently, a process of subcellular relocalization driven by Tau phosphorylation and detachment from microtubules has been proposed. Here, we reveal an alternative mechanism of de novo protein synthesis of Tau and its hyperphosphorylation in the somatodendritic domain, induced by oligomeric amyloid‐β (Aβ) and mediated by the kinase Fyn that activates the ERK/S6 signaling pathway. Activation of this pathway is demonstrated in a range of cellular systems, and in vivo in brains from Aβ‐depositing, Aβ‐injected, and Fyn‐overexpressing mice with Tau accumulation. Both pharmacological inhibition and genetic deletion of Fyn abolish the Aβ‐induced Tau overexpression via ERK/S6 suppression. Together, these findings present a more cogent mechanism of Tau aggregation in disease. They identify a prominent role for neuronal Fyn in integrating signal transduction pathways that lead to the somatodendritic accumulation of Tau in AD.

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes. Electronic supplementary material The online version of this article (10.1186/s40478-018-0514-4) contains supplementary material, which is available to authorized users.

Of 10 variation sites between sequences of amyloid-resistant porcine islet amyloid polypeptide (pIAPP) and amyloid-prone human IAPP (hIAPP), seven locate within residues 17–29, the most amyloidogenic fragment within hIAPP. To investigate how these variations affect amyloidogenicity, 26 IAPP(17–29) or IAPP(20–29) variants were synthesized and their secondary structures, amyloidogenicity, oligomerization and cytotoxicity were studied. Our results indicated that pIAPP fragments are refractory to amyloid formation and significantly less cytotoxic compared with hIAPP fragments. A novel stable dimer was observed in pIAPP(20–29) solution, whereas hIAPP(20–29) exists mostly as monomers and trimers. Among all human to porcine substitutions, S20R caused the most prolonged lag time and significantly attenuated cytotoxicity. The different oligomerization and amyloidogenic properties of hIAPP and pIAPP fragments are discussed.Structured summarypIAPP and pIAPP bind: shown by molecular sieving (view interactions 1, 2)hIAPP and hIAPP bind: shown by molecular sieving (view interactions 1, 2)

Alzheimer's disease is characterized by the accumulation of amyloid-β (Aβ) and Tau in the brain. In mature neurons, Tau is concentrated in the axon and found at lower levels in the dendrite where it is required for targeting Fyn to the spines. Here Fyn mediates Aβ toxicity, which is vastly abrogated when Tau is either deleted or a truncated form of Tau (Tau1-255) is co-expressed. Interestingly, MAP2, a microtubule-binding protein with mainly dendritic localization that shares Fyn-binding motifs with Tau, does not mediate Aβ's synaptic toxicity in the absence of Tau. Here we show in hippocampal neurons that endogenous Tau enters the entire spine, albeit at low levels, whereas MAP2 only enters its neck or is restricted to the dendritic shaft. Based on an extensive mutagenesis study, we also reveal that the spine localization of Tau is facilitated by deletion of the microtubule-binding repeat domain. When distinct phosphorylation sites (AT180–T231/S235, 12E8–S262/S356, PHF1–S396/S404) were pseudophosphorylated (with glutamic acid, using alanine replacements as controls), Tau targeting to spines was markedly increased, whereas the pseudophosphorylation of the late phospho-epitope S422 had no effect. In determining the role physiological Fyn has in the spine localization of Tau, we found that neither were endogenous Tau levels reduced in Fyn knockout compared with wild-type synaptosomal brain fractions nor was the spine localization of over-expressed pseudophosphorylated or P301L Tau. This demonstrates that although Fyn targeting to the spine is Tau dependent, elevated levels of phosphorylated Tau or P301L Tau can enter the spine in a Fyn-independent manner.

Poly(ADP-ribose) polymerase (PARP) inhibitors have neuroprotective effects after retinal ischemia and reperfusion (I/R) injury, but mechanisms of this action are not clear. A second generation PARP inhibitor, GPI 15427, was administrated to mice to investigate the possible mechanisms underlying its neuroprotective effects after retinal I/R injury. Ischemia was induced by increasing intraocular pressure to 80–90 mm Hg for 60 min followed by reperfusion, and mice were treated with GPI 15427 (40 mg/kg−1 day−1, orally) 2 days before or 1 day after injury. Histopathology caused by the retinal I/R injury was estimated by TUNEL assay and histological analyses. Relative gene expressions were evaluated by RT-PCR, Western blotting and immunohistological studies. GPI 15427 inhibited the retinal I/R-induced PARP activation and glial cell activation. GPI 15427 also significantly inhibited the I/R-induced neurodegeneration, as well as increase in TUNEL-positive cells. I/R-induced PERK-eIF2α-CHOP activation and Bip over-expression were inhibited by GPI 15427, while it did not suppress I/R-induced CHOP over-expression and degeneration of retinal capillaries. Our results suggest that GPI 15427 inhibited retinal I/R-induced neurodegeneration and glial cell activation, and this was associated with an effect of the drug to suppress PERK-eIF2α-CHOP activation and Bip over-expression. These results provide evidence that GPI 15427 inhibits retinal I/R injury at least in part via inhibition of endoplasmic reticulum stress.