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Mammalian central nervous system (CNS) neurons do not regenerate after injury due to the inhibitory environment formed by the glial scar, largely constituted by myelin debris. The use of biomaterials to bridge the lesion area and the creation of an environment favoring axonal regeneration is an appealing approach, currently under investigation. This work aimed at assessing the suitability of three candidate polymers - poly(ε-caprolactone), poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) (11∶89 mol%) and poly(trimethylene carbonate) - with the final goal of using these materials in the development of conduits to promote spinal cord regeneration. Poly(L-lysine) (PLL) coated polymeric films were tested for neuronal cell adhesion and neurite outgrowth. At similar PLL film area coverage conditions, neuronal polarization and axonal elongation was significantly higher on P(TMC-CL) films. Furthermore, cortical neurons cultured on P(TMC-CL) were able to extend neurites even when seeded onto myelin. This effect was found to be mediated by the glycogen synthase kinase 3β (GSK3β) signaling pathway with impact on the collapsin response mediator protein 4 (CRMP4), suggesting that besides surface topography, nanomechanical properties were implicated in this process. The obtained results indicate P(TMC-CL) as a promising material for CNS regenerative applications as it promotes axonal growth, overcoming myelin inhibition.

In Alzheimer’s disease (AD), β-amyloid (Aβ) plaques are tightly enveloped by microglia processes, but the significance of this phenomenon is unknown. Here we show that microglia constitute a barrier with profound impact on plaque composition and toxicity. Using high-resolution confocal and in vivo two-photon imaging in AD mouse models, we demonstrate that this barrier prevents outward plaque expansion and leads to compact plaque microregions with low Aβ42 affinity. Areas uncovered by microglia are less compact but have high Aβ42 affinity, leading to the formation of protofibrillar Aβ42 hotspots that are associated with more severe axonal dystrophy. In ageing, microglia coverage is reduced leading to enlarged protofibrillar Aβ42 hotspots and more severe neuritic dystrophy. CX3CR1 gene deletion or anti-Aβ immunotherapy causes expansion of microglia coverage and reduced neuritic dystrophy. Failure of the microglia barrier and the accumulation of neurotoxic protofibrillar Aβ hotspots may constitute novel therapeutic and clinical imaging targets for AD. In Alzheimer’s disease (AD), β-amyloid plaques are tightly enveloped by microglia but the significance of this phenomenon is unknown. Here the authors used confocal and in vivo two-photon imaging in AD mouse models and revealed that microglia constitute a barrier that seems to prevent the formation of neurotoxic hotspots of protofibrillar β-amyloid.

Extracellular deposits of amyloid-β (Aβ) in the form of plaques are one of the main pathological hallmarks of Alzheimer’s disease (AD). Over the years, many different Aβ plaque morphologies such as neuritic plaques, dense cored plaques, cotton wool plaques, coarse-grain plaques, and diffuse plaques have been described in AD postmortem brain tissues, but correlation of a given plaque type with AD progression or AD symptoms is not clear. Furthermore, the exact trigger causing the development of one Aβ plaque morphological subtype over the other is still unknown. Here, we review the current knowledge about neuritic plaques, a subset of Aβ plaques surrounded by swollen or dystrophic neurites, which represent the most detrimental and consequential Aβ plaque morphology. Neuritic plaques have been associated with local immune activation, neuronal network dysfunction, and cognitive decline. Given that neuritic plaques are at the interface of Aβ deposition, tau aggregation, and local immune activation, we argue that understanding the exact mechanism of neuritic plaque formation is crucial to develop targeted therapies for AD.

The main organelles of the secretory and endocytic pathways—the endoplasmic reticulum (ER) and endosomes, respectively—are connected through contact sites whose numbers increase as endosomes mature 1 , 2 , 3 . One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase 4 , whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein 5 or mediate endosome fission 6 . Cholesterol transfer and Ca 2+ exchange have been proposed as additional functions of such sites 2 , 3 . However, the compositions, activities and regulations of ER–endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth 7 , forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE–ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER–LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth. Repeated contacts between the endoplasmic reticulum (ER) and a subset of endosomes called late endosomes (LEs) is shown to promote microtubule-dependent translocation of LEs to the cell periphery and their subsequent fusion with the plasma membrane to induce outgrowth of neuronal protrusions. Role of endosomes in neuronal protrusion outgrowth The endoplasmic reticulum (ER) makes contact with various other cellular organelles including endosomes. Although the functional significance of ER–endosome contact sites is known, the composition, activity and regulation of these sites is poorly explored. Harald Stenmark and co-workers provide a glimpse into these enigmatic aspects. They show that the ER protein protrudin makes contact with the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P) on a subset of endosomes called late endosomes (LEs). This allows transfer of the microtubule motor protein kinesin-1 from protrudin to the motor adaptor FYCO1 on LEs. Thus repeated ER–LE contacts promote microtubule-dependent translocation of LEs to the cell periphery and their subsequent fusion with the plasma membrane to induce neurite outgrowth of neuronal protrusions.

In Alzheimer’s disease, accumulation of amyloid-β (Aβ) peptide is thought to cause formation of neurofibrillary tangles composed of hyperphosphorylated tau protein, which correlates with neuronal loss and cognitive impairment, but the mechanism linking Aβ and tau pathologies is unknown. Dystrophic neurites, which surround Aβ plaques and accumulate phosphorylated tau and other proteins, may play a role in seeding and spreading of pathologic tau. Here, we investigate the novel hypothesis that improved membrane repair capacity decreases dystrophic neurite formation by protecting axons from Aβ-induced membrane damage. Using a ratiometric calcium sensor and a FRET-based calpain cleavage sensor, we demonstrate that dystrophic neurites in 5XFAD mice have elevated resting calcium levels and calpain activity because of putative membrane damage. Annexin A6, a plasma membrane repair in muscle and neurons, is present at plasma membrane of neurons and dystrophic neurites in murine and human brains. Overexpression of annexin A6 in brains of 5XFAD mice decreased size and quantity of dystrophic neurites and accumulation of phospho-tau181, an early biomarker of amyloid pathology. Phospho-tau231, another early amyloid biomarker, and phosphorylated of tau kinases, c-jun N-terminal kinase (JNK) and Calmodulin Kinase II (CaMKII) accumulate in dystrophic neurites in the brains of amyloid pathology mice and humans with AD, suggesting that dystrophic neurites are sites of active tau phosphorylation. Overexpression of dominant-negative annexin A6 in 5XFAD mice increased dystrophic neurites and phospho-tau181. Intracerebral injection of recombinant annexin A6 in 5XFAD and APP-NLGF knock-in mice resulted in localization of recombinant A6 to membranes of dystrophic neurites, suggesting therapeutic potential of recombinant annexin A6 for AD. In conclusion, dystrophic neurites have Aβ-induced membrane damage characterized by calcium elevation, calpain activation, and accumulation of tau kinases and phosphorylated tau. Overexpression of annexin A6 reduces dystrophic neurites and phospho-tau accumulation, suggesting that annexin A6-mediated membrane repair may represent a novel therapeutic approach for AD.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems 1 – 5 . Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)—one such nociceptor-produced neuropeptide—directly increases the exhaustion of cytotoxic CD8 + T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8 + T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8 + T cells, Ramp1 −/ − CD8 + T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1 -deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1 -expressing CD8 + T cells were more exhausted than their RAMP1 -negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8 + T cells. Melanoma cells interact with pain-mediating sensory neurons by increasing their release of the neuropeptide CGRP, which increases the exhaustion of CD8 + T cells and thus promotes the survival of cancer cells.

A new pathway for the processing of β-amyloid precursor protein (APP) is described in which η-secretase activity, in part mediated by the MT5-MMP metalloproteinase, cleaves APP, and further processing by ADAM10 and BACE1 generates proteolytic fragments capable of inhibiting long-term potentiation in the hippocampus. Neuronal inhibition by APP by-products Michael Willem et al . describe a previously unknown pathway for the processing of β-amyloid precursor protein (APP) in which η-secretase cleaves APP to yield a soluble C-terminal fragment termed CTF-η. The soluble fragment, sAPP-η can be further processed by ADAM10 and BACE1 to generate the peptides Aη-α and Aη-β respectively, which are capable of inhibiting long-term potentiation in the hippocampus. The relevant η-secretase activity is largely due to the membrane-bound matrix metalloproteinase, MT5-MMP, whose activity is enriched in dystrophic neurites in a mouse model of Alzheimer's disease and in the brains of Alzheimer's patients. Genetic or pharmacological inhibition of BACE1 results in increased accumulation of both CTF-η and Aη-α. This work suggests that BACE 1-based therapies may result in the generation of another potentially toxic substance (Aη-α) and that therapeutic inhibition of BACE1 activity requires careful titration. Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-β peptide 1 . Two principal physiological pathways either prevent or promote amyloid-β generation from its precursor, β-amyloid precursor protein (APP), in a competitive manner 1 . Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo 2 . Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-β fragments generated by the α- and β-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (β-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504–505 of APP 695 , releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-β). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo , long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

Early-onset familial Alzheimer’s disease mutations induce both amyloid-β and tau pathologies in differentiated human neural stem cells in 3D cultures. Cell-culture model for Alzheimer's disease Although it is accepted that both aggregates of amyloid-β and neurofibrillary tangles of hyper-phosphorylated tau protein contribute to Alzheimer's disease pathology, no single model has incorporated both of these pathological events using human cells. Here, Rudolph Tanzi and colleagues find that familial Alzheimer's disease mutations in the amyloid-β precursor protein ( APP ) and presenilin 1 ( PSEN1 ) genes are able to induce robust extracellular deposition of amyloid-β, including β-amyloid plaques, in a human neural stem-cell-derived three-dimensional culture system. This provides experimental validation of the amyloid hypothesis that proposes that the amyloid-β accumulation drives tauopathy in Alzheimer's disease. The cell culture system used here can be used as a platform for studying the pathogenic mechanisms of Alzheimer's disease and for drug screening. Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles 1 . The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau 2 , 3 . However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology 4 , 5 . Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles 6 , 7 , 8 , 9 , 10 , 11 . Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

Through injection of human Alzheimer's disease (AD) brain extracts containing pathological tau protein into transgenic mouse lines harboring different levels of amyloid plaque burden, the authors find that the presence of amyloid plaques modifies endogenous pools of tau protein, creating a unique environment required for the seeding and spreading of distinct tau pathologies. Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) plaques and intracellular tau inclusions. However, the exact mechanistic link between these two AD lesions remains enigmatic. Through injection of human AD-brain-derived pathological tau (AD-tau) into Aβ plaque–bearing mouse models that do not overexpress tau, we recapitulated the formation of three major types of AD-relevant tau pathologies: tau aggregates in dystrophic neurites surrounding Aβ plaques (NP tau), AD-like neurofibrillary tangles (NFTs) and neuropil threads (NTs). These distinct tau pathologies have different temporal onsets and functional consequences on neural activity and behavior. Notably, we found that Aβ plaques created a unique environment that facilitated the rapid amplification of proteopathic AD-tau seeds into large tau aggregates, initially appearing as NP tau, which was followed by the formation and spread of NFTs and NTs, likely through secondary seeding events. Our study provides insights into a new multistep mechanism underlying Aβ plaque–associated tau pathogenesis.

Alzheimer’s disease (AD) is the most common neurodegenerative disease, but it remains an intractable condition. Its pathogenesis is predominantly attributed to the aggregation and transmission of two molecules, Aβ and tau; however, other pathological mechanisms are possible. Here, we reveal that phosphorylation of MARCKS, a submembrane protein that regulates the stability of the actin network, occurs at Ser46 prior to aggregation of Aβ and is sustained throughout the course of AD in human and mouse brains. Furthermore, HMGB1 released from necrotic or hyperexcitatory neurons binds to TLR4, triggers the specific phosphorylation of MARCKS via MAP kinases and induces neurite degeneration, the classical hallmark of AD pathology. Subcutaneous injection of a newly developed monoclonal antibody against HMGB1 strongly inhibits neurite degeneration even in the presence of Aβ plaques and completely recovers cognitive impairment in a mouse model. HMGB1 and Aβ mutually affect polymerization of the other molecule and the therapeutic effects of the anti-HMGB1 monoclonal antibody are mediated by Aβ-dependent and Aβ-independent mechanisms. We propose that HMGB1 is a critical pathogenic molecule promoting AD pathology in parallel with Aβ and tau and a new key molecular target of preclinical antibody therapy to delay the onset of AD.