Global and local mechanisms sustain axonal proteostasis of transmembrane proteins.

The control of neuronal protein homeostasis or proteostasis is tightly regulated both spatially and temporally, assuring accurate and integrated responses to external or intrinsic stimuli. Local or autonomous responses in dendritic and axonal compartments are crucial to sustain function during development, physiology and in response to damage or disease. Axons are responsible for generating and propagating electrical impulses in neurons, and the establishment and maintenance of their molecular composition are subject to extreme constraints exerted by length and size. Proteins that require the secretory pathway, such as receptors, transporters, ion channels or cell adhesion molecules, are fundamental for axonal function, but whether axons regulate their abundance autonomously and how they achieve this is not clear. Evidence supports the role of three complementary mechanisms to maintain proteostasis of these axonal proteins, namely vesicular transport, local translation and trafficking and transfer from supporting cells. Here, we review these mechanisms, their molecular machineries and contribution to neuronal function. We also examine the signaling pathways involved in local translation and their role during development and nerve injury. We discuss the relative contributions of a transport-controlled proteome directed by the soma (global regulation) versus a local-controlled proteome based on local translation or cell transfer (local regulation).

Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.

Brain regions affected by Alzheimer disease (AD) display well-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction. Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment ß (C99), generated by cleavage of APP by ß-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement in AD and the earliest initiator of synaptic plasticity and long-term memory impairment. The aim of the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosome formation or block the fusion of autophagosomes to endolysosomal compartments caused an increase in C99 levels. We also found that inhibition of autophagosome formation by depletion of Atg5 led to higher levels of C99 and to its massive accumulation in the lumen of enlarged perinuclear, lysosomal-associated membrane protein 1 (LAMP1)-positive organelles. In contrast, activation of autophagosome formation, either by starvation or by inhibition of the mammalian target of rapamycin, enhanced lysosomal clearance of C99. Altogether, our results indicate that autophagosomes are key organelles to help avoid C99 accumulation preventing its deleterious effects.-González, A. E., Muñoz, V. C., Cavieres, V. A., Bustamante, H. A., Cornejo, V.-H., Januário, Y. C., González, I., Hetz, C., daSilva, L. L., Rojas-Fernández, A., Hay, R. T., Mardones, G. A., Burgos, P. V. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.

IRE1 signaling exacerbates Alzheimer's disease pathogenesis.

Altered proteostasis is a salient feature of Alzheimer's disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress and abnormal protein aggregation. ER stress triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis or eliminate terminally damaged cells. IRE1 is an ER-located kinase and endoribonuclease that operates as a major stress transducer, mediating both adaptive and proapoptotic programs under ER stress. IRE1 signaling controls the expression of the transcription factor XBP1, in addition to degrade several RNAs. Importantly, a polymorphism in the XBP1 promoter was suggested as a risk factor to develop AD. Here, we demonstrate a positive correlation between the progression of AD histopathology and the activation of IRE1 in human brain tissue. To define the significance of the UPR to AD, we targeted IRE1 expression in a transgenic mouse model of AD. Despite initial expectations that IRE1 signaling may protect against AD, genetic ablation of the RNase domain of IRE1 in the nervous system significantly reduced amyloid deposition, the content of amyloid ß oligomers, and astrocyte activation. IRE1 deficiency fully restored the learning and memory capacity of AD mice, associated with improved synaptic function and improved long-term potentiation (LTP). At the molecular level, IRE1 deletion reduced the expression of amyloid precursor protein (APP) in cortical and hippocampal areas of AD mice. In vitro experiments demonstrated that inhibition of IRE1 downstream signaling reduces APP steady-state levels, associated with its retention at the ER followed by proteasome-mediated degradation. Our findings uncovered an unanticipated role of IRE1 in the pathogenesis of AD, offering a novel target for disease intervention.

The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein.

Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease.

Spike transmission failures in axons from cortical neurons in vivo.

The propagation of action potentials along axons is traditionally considered reliable due to the high safety factor for axonal spike transmission. However, numerical simulations suggest that high-frequency spikes could fail to invade distal axonal branches. To explore this experimentally in vivo, we used an axonal-targeted calcium indicator to image action potentials at axonal terminal branches in the superficial layers of mouse somatosensory cortical neurons. We activated axons with an extracellular electrode, varying stimulation frequencies, and analyzed the images to computationally extract axonal morphologies and associated calcium responses. We found that axonal boutons have higher calcium accumulations than their axonal shafts, as was reported in vitro. However, contrary to previous in vitro results, our data reveal spike failures at high spike frequencies in a significant subset of branches as a function of branching geometry. These findings suggest that axonal morphologies could contribute to signal processing in the cortex.

Spike transmission failures in axons from mouse cortical pyramidal neurons in vivo.

The propagation of action potentials along axons is traditionally considered to be reliable, as a consequence of the high safety factor of action potential propagation. However, numerical simulations have suggested that, at high frequencies, spikes could fail to invade distal axonal branches. Given the complex morphologies of axonal trees, with extensive branching and long-distance projections, spike propagation failures could be functionally important. To explore this experimentally in vivo, we used an axonal-targeted calcium indicator to image action potentials at axonal terminal branches in superficial layers from mouse somatosensory cortical pyramidal neurons. We activated axons with an extracellular electrode, varying stimulation frequencies, and computationally extracted axonal morphologies and associated calcium responses. We find that axonal boutons have higher calcium accumulations than their parent axons, as was reported in vitro. But, contrary to previous in vitro results, our data reveal spike failures in a significant subset of branches, as a function of branching geometry and spike frequency. The filtering is correlated with the geometric ratio of the branch diameters, as expected by cable theory. These findings suggest that axonal morphologies contribute to signal processing in the cortex.

Role of the unfolded protein response in organ physiology: lessons from mouse models.

The endoplasmic reticulum (ER) is a key subcellular compartment involved in the folding and maturation of around one-third of the total proteome. Accumulation of misfolded proteins in the ER lumen engages a signal transduction pathway known as unfolded protein response (UPR) that feedback to recover ER homeostasis or to trigger apoptosis of irreversible damaged cells. The UPR is initiated by three main stress sensors including protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring protein 1α (IRE1α), which reprogram the genome through the control of downstream transcription factors. In this article, the authors have reviewed most relevant studies uncovering the physiological function of the UPR in different organs and tissues based on the phenotypes observed after genetic manipulation of the pathway in vivo. Biomedical applications of targeting the UPR on a disease context are also discussed.

Transport and localization on dendrite-inspired flat band linear photonic lattices.

The capacity of a physical system to transport and localize energy or information is usually linked to its spatial configuration. This is relevant for integration and transmission of signals as performed, for example, by the dendrites of neuronal cells. Inspired by recent works on the organization of spines on the surface of dendrites and how they promote localization or propagation of electrical impulses in neurons, here we propose a linear photonic lattice configuration to study how the geometric features of a dendrite-inspired lattice allows for the localization or propagation of light on a completely linear structure. We show that by increasing the compression of the photonic analogue of spines and thus, by increasing the coupling strength of the spines with the main chain (the "photonic dendrite"), flat band modes become prevalent in the system, allowing spatial localization in the linear - low energy - regime. Furthermore, we study the inclusion of disorder in the distribution of spines and show that the main features of ordered systems persist due to the robustness of the flat band states. Finally, we discuss if the photonic analog, having evanescent interactions, may provide insight into linear morphological mechanisms at work occurring in some biological systems, where interactions are of electric and biochemical origin.

Voltage compartmentalization in dendritic spines in vivo.

Dendritic spines mediate most excitatory neurotransmission in the nervous system, so their function must be critical for the brain. Spines are biochemical compartments but might also electrically modify synaptic potentials. Using two-photon microscopy and a genetically encoded voltage indicator, we measured membrane potentials in spines and dendrites from pyramidal neurons in the somatosensory cortex of mice during spontaneous activity and sensory stimulation. Spines and dendrites were depolarized together during action potentials, but, during subthreshold and resting potentials, spines often experienced different voltages than parent dendrites, even activating independently. Spine voltages remained compartmentalized after two-photon optogenetic activation of individual spine heads. We conclude that spines are elementary voltage compartments. The regulation of voltage compartmentalization could be important for synaptic function and plasticity, dendritic integration, and disease states.

Correction: Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source.

[This corrects the article DOI: 10.1371/journal.pone.0065818.].

Non-conventional Axonal Organelles Control TRPM8 Ion Channel Trafficking and Peripheral Cold Sensing.

TRPM8 is the main ion channel responsible for cold transduction in the somatosensory system. Nerve terminal availability of TRPM8 determines cold sensitivity, but how axonal secretory organelles control channel delivery remains poorly understood. Here we examine the distribution of TRPM8 and trafficking organelles in cold-sensitive peripheral axons and disrupt trafficking by targeting the ARF-GEF GBF1 pharmacologically or the small GTPase RAB6 by optogenetics. In axons of the sciatic nerve, inhibition of GBF1 interrupts TRPM8 trafficking and increases association with the trans-Golgi network, LAMP1, and Golgi satellites, which distribute profusely along the axonal shaft. Accordingly, both TRPM8-dependent ongoing activity and cold-evoked responses reversibly decline upon GBF1 inhibition in nerve endings of corneal cold thermoreceptors. Inhibition of RAB6, which also associates to Golgi satellites, decreases cold-induced responses in vivo. Our results support a non-conventional axonal trafficking mechanism controlling the availability of TRPM8 in axons and cold sensitivity in the peripheral nervous system.

Golgi bypass for local delivery of axonal proteins, fact or fiction?

Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus.

The axonal endoplasmic reticulum: One organelle-many functions in development, maintenance, and plasticity.

The endoplasmic reticulum (ER) is highly conserved in eukaryotes and neurons. Indeed, the localization of the organelle in axons has been known for nearly half a century. However, the relevance of the axonal ER is only beginning to emerge. In this review, we discuss the structure of the ER in axons, examining the role of ER-shaping proteins and highlighting reticulons. We analyze the multiple functions of the ER and their potential contribution to axonal physiology. First, we examine the emerging roles of the axonal ER in lipid synthesis, protein translation, processing, quality control, and secretory trafficking of transmembrane proteins. We also review the impact of the ER on calcium dynamics, focusing on intracellular mechanisms and functions. We describe the interactions between the ER and endosomes, mitochondria, and synaptic vesicles. Finally, we analyze available proteomic data of axonal preparations to reveal the dynamic functionality of the ER in axons during development. We suggest that the dynamic proteome and a validated axonal interactome, together with state-of-the-art methodologies, may provide interesting research avenues in axon physiology that may extend to pathology and regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 181-208, 2018.

ER stress signaling and neurodegeneration: At the intersection between Alzheimer's disease and Prion-related disorders.

Alzheimer's and Prion diseases are two neurodegenerative conditions sharing different pathophysiological characteristics. Disease symptoms are associated with the abnormal accumulation of protein aggregates, which are generated by the misfolding and oligomerization of specific proteins. Recent functional studies uncovered a key role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in the occurrence of synaptic dysfunction and neurodegeneration in Prion-related disorders and Alzheimer's disease. Here we review common pathological features of both diseases, emphasizing the link between amyloid formation, its pathogenesis and alterations in ER proteostasis. The potential benefits of targeting the UPR as a therapeutic strategy is also discussed.

The unfolded protein response in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by synaptic dysfunction and accumulation of amyloid-beta (Aß) peptide, which are responsible for the progressive loss of memory. The mechanisms involved in neuron dysfunction in AD remain poorly understood. Recent evidence implicates the participation of adaptive responses to stress within the endoplasmic reticulum (ER) in the disease process, via a pathway known as the unfolded protein response (UPR). Here, we review the findings suggesting a functional role of ER stress in the etiology of AD. Possible therapeutic strategies to mitigate ER stress in the context of AD are discussed.

Proliferation of murine midbrain neural stem cells depends upon an endogenous sonic hedgehog (Shh) source.

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.