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The protein FAM134B is an endoplasmic reticulum (ER)-resident receptor that facilitates ER autophagy, and downregulation of this protein (mutations of which are also known to cause sensory neuropathy in humans) results in expanded ER structures and degeneration of mouse sensory neurons. ER-phagy receptors mapped The endoplasmic reticulum (ER) is a complex network of membranes involved in protein and lipid synthesis, ion homeostasis, protein quality control and organelle communication. It is also a source of membrane-bounded vesicles called autophagosomes, the vehicles for the self-digesting cellular process of autophagy. Two papers published online this week [in this issue of Nature] show how the ER itself is targeted for degradation by autophagy — a process that could ensure constant ER turnover in response to cellular requirements. Ivan Dikic and coworkers find the protein FAM134B is an ER-resident receptor that facilitates 'ER-phagy'. Downregulation of this protein — mutations of which can cause sensory neuropathy in humans — resulted in expanded ER structures and degeneration of mouse sensory neurons. Hitoshi Nakatogawa and colleagues show that the same phenomenon is conserved in yeast, where Atg40 is enriched in the cortical and cytoplasmic ER, loading these ER subdomains into autophagosomes. A further ER-phagy receptor, Atg39, localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of a part of the nucleus, thus ensuring cell survival under nitrogen-deprived conditions. The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication 1 . Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process 2 , 3 , 4 , 5 , 6 , 7 , 8 . However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy (‘ER-phagy’). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans 9 are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB₁ cannabinoid receptors (CB₁Rs) are enriched in the axonal growth cones of γ-aminobutyric acid-containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB₁R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB₁Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

Bronchial asthma is a chronic inflammatory disease with rising prevalence worldwide. Apart from the immunological role of the tricyclic antidepressant amitriptyline in bronchial asthma, there is emerging evidence that inhaled amitriptyline directly reduces acute bronchoconstriction. However, the mechanism by which amitriptyline influences bronchial tone remains poorly understood. To influence bronchoconstriction, rat precision-cut lung slices treated with varying concentrations of amitriptyline (0–5 µM) and incubated with inhibitors targeting different signaling pathways. Amitriptyline reduces acetylcholine- and serotonin-induced bronchoconstriction. Neither the muscarinic antagonist ipratropium nor the phospholipase C inhibitor U73122, nor the protein kinase C inhibitor chelerythrine diminished the effect of amitriptyline. Inhibition of calcium sensitizing and induction failed to alter amitriptyline’s effect on bronchoconstriction. Caveolae—as part of the plasma membrane—display a microenvironment, where regulation of signal transduction takes place. Similar to methyl ß cyclodextrin (MBCD), a common substance to destroy caveolae, amitriptyline dramatically reduced the number of caveolae in lung tissue. However, unlike MBCD, this effect could not be explained by cholesterol depletion alone, as cholesterol repletion did not reverse amitriptyline’s effect. Furthermore, neither simvastatin (a lipid lowering agent) nor cytochalasin D (an inhibitor of actin polymerization), influenced the inhibitory effect of amitriptyline on bronchoconstriction. In conclusion, amitriptyline inhibits bronchoconstriction independently of direct receptor binding or interaction. It also reduces the total number of caveolae without effects on cholesterol lowering pathways or actin depolymerization. A more general mechanism seems likely, as inhibition of single signal transduction pathways failed. Further studies are required to elucidate the underlying mechanisms.

Calcium-binding proteins (CaBPs) are known to modulate neuronal excitability and calcium signaling, and they may play a role in the imbalances of excitation and inhibition of temporal lobe epilepsy (TLE). While parvalbumin and calretinin are well-characterized CaBPs, N-Terminal EF-Hand Calcium-Binding Protein 1 (NECAB1) remains understudied in epilepsy, despite its association with neurodegenerative conditions. In this study, we used fluorescent immunolabeling to determine the distribution of NECAB1, as well as its co-expression with parvalbumin and calretinin, in brain regions associated with the epileptic circuitry using a kainic acid-induced TLE model. Additionally, we examined the impact of levetiracetam and brivaracetam on NECAB1 expression. In our study, NECAB1-positive cells were prominently localized to the paraventricular nucleus of the thalamus (PVT), endopiriform nucleus (EPN), and amygdala in healthy brain regions involved in epileptic circuitry. A NECAB1–calretinin co-expressing subpopulation was detected in the amygdala, PVT, and hippocampus but was nearly absent in the EPN. In chronic epilepsy, NECAB1 expression was significantly upregulated in the PVT and bilaterally in the amygdala. These findings suggest that NECAB1 upregulation may compensate for epileptic hyperexcitability, potentially contributing to circuit remodeling via thalamocortical regulation and interneuron diversity. Levetiracetam and brivaracetam treatments partially reduced the NECAB1 density increase in TLE, indicating a modulatory effect on NECAB1 expression.

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons (MNs) and their target muscles. Misfolded proteins which often form intracellular aggregates are a pathological hallmark of ALS. Disruption of the functional interplay between protein degradation (ubiquitin proteasome system and autophagy) and RNA-binding protein homeostasis has recently been suggested as an integrated model that merges several ALS-associated proteins into a common pathophysiological pathway. The E102Q mutation in one such candidate gene, the endoplasmic reticulum (ER) chaperone Sigma receptor-1 (SigR1), has been reported to cause juvenile ALS. Although loss of SigR1 protein contributes to neurodegeneration in several ways, the molecular mechanisms underlying E102Q-SigR1-mediated neurodegeneration are still unclear. In the present study, we showed that the E102Q-SigR1 protein rapidly aggregates and accumulates in the ER and associated compartments in transfected cells, leading to structural alterations of the ER, nuclear envelope and mitochondria and to subsequent defects in proteasomal degradation and calcium homeostasis. ER defects and proteotoxic stress generated by E102Q-SigR1 aggregates further induce autophagy impairment, accumulation of stress granules and cytoplasmic aggregation of the ALS-linked RNA-binding proteins (RBPs) matrin-3, FUS, and TDP-43. Similar ultrastructural abnormalities as well as altered protein degradation and misregulated RBP homeostasis were observed in primary lymphoblastoid cells (PLCs) derived from E102Q-SigR1 fALS patients. Consistent with these findings, lumbar α -MNs of both sALS as well as fALS patients showed cytoplasmic matrin-3 aggregates which were not co-localized with pTDP-43 aggregates. Taken together, our results support the notion that E102Q-SigR1-mediated ALS pathogenesis comprises a synergistic mechanism of both toxic gain and loss of function involving a vicious circle of altered ER function, impaired protein homeostasis and defective RBPs.

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Fragile X syndrome, the most commonly known genetic cause of autism, is due to loss of the fragile X mental retardation protein, which regulates signal transduction at metabotropic glutamate receptor-5 in the brain. Fragile X mental retardation protein deletion in mice enhances metabotropic glutamate receptor-5-dependent long-term depression in the hippocampus and cerebellum. Here we show that a distinct type of metabotropic glutamate receptor-5-dependent long-term depression at excitatory synapses of the ventral striatum and prefrontal cortex, which is mediated by the endocannabinoid 2-arachidonoyl- sn -glycerol, is absent in fragile X mental retardation protein-null mice. In these mutants, the macromolecular complex that links metabotropic glutamate receptor-5 to the 2-arachidonoyl- sn -glycerol-producing enzyme, diacylglycerol lipase-α (endocannabinoid signalosome), is disrupted and metabotropic glutamate receptor-5-dependent 2-arachidonoyl- sn -glycerol formation is compromised. These changes are accompanied by impaired endocannabinoid-dependent long-term depression. Pharmacological enhancement of 2-arachidonoyl- sn -glycerol signalling normalizes this synaptic defect and corrects behavioural abnormalities in fragile X mental retardation protein-deficient mice. The results identify the endocannabinoid signalosome as a molecular substrate for fragile X syndrome, which might be targeted by therapy. Fragile X syndrome is a major genetic cause of autism and is caused by loss of the fragile X mental retardation protein. In a mouse model of fragile X syndrome, Jung et al . show that an absence of neuronal endocannabinoid signalling is responsible for the neurophysiological and behavioural defects.

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.

Gain-of-function mutations in the human SCN11A -encoded voltage-gated Na + channel Na V 1.9 cause severe pain disorders ranging from neuropathic pain to congenital pain insensitivity. However, the entire spectrum of the Na V 1.9 diseases has yet to be defined. Applying whole-exome sequencing we here identify a missense change (p.V1184A) in Na V 1.9, which leads to cold-aggravated peripheral pain in humans. Electrophysiological analysis reveals that p.V1184A shifts the voltage dependence of channel opening to hyperpolarized potentials thereby conferring gain-of-function characteristics to Na V 1.9. Mutated channels diminish the resting membrane potential of mouse primary sensory neurons and cause cold-resistant hyperexcitability of nociceptors, suggesting a mechanistic basis for the temperature dependence of the pain phenotype. On the basis of direct comparison of the mutations linked to either cold-aggravated pain or pain insensitivity, we propose a model in which the physiological consequence of a mutation, that is, augmented versus absent pain, is critically dependent on the type of Na V 1.9 hyperactivity. A mutation in the sodium channel Nav1.9 has been identified in a family and shown to associate with cold-aggravated pain. Here, the authors characterize the electrophysiological consequences of this mutation and propose a mechanism for the pain that the individuals experience.

A growing number of hereditary neuropathies have been assigned to causative gene defects in recent years. The study of human nerve biopsy samples has contributed substantially to the discovery of many of these neuropathy genes. Genotype–phenotype correlations based on peripheral nerve pathology have provided a comprehensive picture of the consequences of these mutations. Intriguingly, several gene defects lead to distinguishable lesion patterns that can be studied in nerve biopsies. These characteristic features include the loss of certain nerve fiber populations and a large spectrum of distinct structural changes of axons, Schwann cells and other components of peripheral nerves. In several instances the lesion patterns are directly or indirectly linked to the known functions of the mutated gene. The present review is designed to provide an overview on these characteristic patterns. It also considers other aspects important for the manifestation and pathology of hereditary neuropathies including the role of inflammation, effects of chemotherapeutic agents and alterations detectable in skin biopsies.