Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons.

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide repeat expansion disorders.

The response of nutrient cycle, microbial community abundance and metabolic function to nitrogen fertilizer in rhizosphere soil of Phellodendron chinense Schneid seedlings.

Nitrogen (N) as an essential macronutrient affects the soil nutrient cycle, microbial community abundance, and metabolic function. However, the specific responses of microorganisms and metabolic functions in rhizosphere soil of Phellodendron chinense Schneid seedlings to N addition remain unclear. In this study, four treatments (CK, N5, N10 and N15) were conducted, and the soil physicochemical properties, enzyme activities, microbial community abundances and diversities, metabolism, and gene expressions were investigated in rhizosphere soil of P. chinense Schneid. The results showed that N addition significantly decreased rhizosphere soil pH, among which the effect of N10 treatment was better. N10 treatment significantly increased the contents of available phosphorus (AP), available potassium (AK), ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3--N) and sucrase (SU) activity, as well as fungal diversity and the relative expression abundances of amoA and phoD genes in rhizosphere soil, but observably decreased the total phosphorus (TP) content, urease (UR) activity and bacterial diversity, among which the pH, soil organic matter (SOM), AP, NH4+-N and NO3--N were the main environmental factors for affecting rhizosphere soil microbial community structure based on RDA and correlation analyses. Meanwhile, N10 treatment notably enhanced the absolute abundances of the uracil, guanine, indole, prostaglandin F2α and γ-glutamylalanine, while reduced the contents of D-phenylalanine and phenylacetylglycine in rhizosphere soil of P. chinense Schneid seedlings. Furthermore, the soil available nutrients represented a significant correlation with soil metabolites and dominant microorganisms, suggesting that N10 addition effectively regulated microbial community abundance and metabolic functions by enhancing nutrient cycle in the rhizosphere soil of P. chinense Schneid seedlings.

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation.

A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.

Antibody Therapy Targeting RAN Proteins Rescues C9 ALS/FTD Phenotypes in C9orf72 Mouse Model.

The intronic C9orf72 G4C2 expansion, the most common genetic cause of ALS and FTD, produces sense- and antisense-expansion RNAs and six dipeptide repeat-associated, non-ATG (RAN) proteins, but their roles in disease are unclear. We generated high-affinity human antibodies targeting GA or GP RAN proteins. These antibodies cross the blood-brain barrier and co-localize with intracellular RAN aggregates in C9-ALS/FTD BAC mice. In cells, α-GA1 interacts with TRIM21, and α-GA1 treatment reduced GA levels, increased GA turnover, and decreased RAN toxicity and co-aggregation of proteasome and autophagy proteins to GA aggregates. In C9-BAC mice, α-GA1 reduced GA as well as GP and GR proteins, improved behavioral deficits, decreased neuroinflammation and neurodegeneration, and increased survival. Glycosylation of the Fc region of α-GA1 is important for cell entry and efficacy. These data demonstrate that RAN proteins drive C9-ALS/FTD in C9-BAC transgenic mice and establish a novel therapeutic approach for C9orf72 ALS/FTD and other RAN-protein diseases.

Balanced Activity between Kv3 and Nav Channels Determines Fast-Spiking in Mammalian Central Neurons.

Fast-spiking (FS) neurons can fire action potentials (APs) up to 1,000 Hz and play key roles in vital functions such as sound location, motor coordination, and cognition. Here we report that the concerted actions of Kv3 voltage-gated K+ (Kv) and Na+ (Nav) channels are sufficient and necessary for inducing and maintaining FS. Voltage-clamp analysis revealed a robust correlation between the Kv3/Nav current ratio and FS. Expressing Kv3 channels alone could convert ∼30%-60% slow-spiking (SS) neurons to FS in culture. In contrast, co-expression of either Nav1.2 or Nav1.6 together with Kv3.1 or Kv3.3, but not alone or with Kv1.2, converted SS to FS with 100% efficiency. Furthermore, RNA-sequencing-based genome-wide analysis revealed that the Kv3/Nav ratio and Kv3 expression levels strongly correlated with the maximal AP frequencies. Therefore, FS is established by the properly balanced activities of Kv3 and Nav channels and could be further fine-tuned by channel biophysical features and localization patterns.

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons.

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

Suppression of Inflammatory Demyelinaton and Axon Degeneration through Inhibiting Kv3 Channels.

The development of neuroprotective and repair strategies for treating progressive multiple sclerosis (MS) requires new insights into axonal injury. 4-aminopyridine (4-AP), a blocker of voltage-gated K+ (Kv) channels, is used in symptomatic treatment of progressive MS, but the underlying mechanism remains unclear. Here we report that deleting Kv3.1-the channel with the highest 4-AP sensitivity-reduces clinical signs in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. In Kv3.1 knockout (KO) mice, EAE lesions in sensory and motor tracts of spinal cord were markedly reduced, and radial astroglia were activated with increased expression of brain derived neurotrophic factor (BDNF). Kv3.3/Kv3.1 and activated BDNF receptors were upregulated in demyelinating axons in EAE and MS lesions. In spinal cord myelin coculture, BDNF treatment promoted myelination, and neuronal firing via altering channel expression. Therefore, suppressing Kv3.1 alters neural circuit activity, which may enhance BNDF signaling and hence protect axons from inflammatory insults.

Polarity of varicosity initiation in central neuron mechanosensation.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca2+/Na+-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca2+ increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.

Physiological and pathological functions of mechanosensitive ion channels.

Rapid sensation of mechanical stimuli is often mediated by mechanosensitve ion channels. Their opening results from conformational changes induced by mechanical forces. It leads to membrane permeation of selected ions and thereby to electrical signaling. Newly identified mechanosensitive ion channels are emerging at an astonishing rate, including some that are traditionally assigned for completely different functions. In this review, we first provide a brief overview of ion channels that are known to play a role in mechanosensation. Next, we focus on three representative ones, including the transient receptor potential channel V4 (TRPV4), Kv1.1 voltage-gated potassium (Kv) channel, and Piezo channels. Their structures, biophysical properties, expression and targeting patterns, and physiological functions are highlighted. The potential role of their mechanosensation in related diseases is further discussed. In sum, mechanosensation appears to be achieved in a variety of ways by different proteins and plays a fundamental role in the function of various organs under normal and abnormal conditions.

Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons.

Action potentials (APs) propagating along axons require the activation of voltage-gated Na(+) (Nav) channels. How Nav channels are transported into axons is unknown. We show that KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with small interfering RNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing AP firing. Live-cell imaging showed that fluorescently tagged AnkG or Nav1.2 cotransported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav, but not Kv1.2, channels in mouse cerebellum. These results indicate that AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport before anchoring them to the AIS and nodes of Ranvier.

Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels.

The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency.

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.

Clustering and activity tuning of Kv1 channels in myelinated hippocampal axons.

Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr458) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr458 mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.

Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding.

Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here, we report that KIF5/kinesin I transports Kv3.1 voltage-gated K(+) (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull-down assays with exogenously expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Overexpression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by RNA interference significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn(2+)-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, coexpression of the YFP (yellow fluorescent protein)-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels colocalize and comove with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons.

Polarized targeting of L1-CAM regulates axonal and dendritic bundling in vitro.

Proper axonal and dendritic bundling is essential for the establishment of neuronal connections and the synchronization of synaptic inputs, respectively. Cell adhesion molecules of the L1-CAM (L1-cell adhesion molecule) family regulate axon guidance and fasciculation, neuron migration, dendrite morphology, and synaptic plasticity. It remains unclear how these molecules play so many different roles. Here we show that polarized axon-dendrite targeting of an avian L1-CAM protein, NgCAM (neuron-glia cell adhesion molecule), can regulate the switch of bundling of the two major compartments of rat hippocampal neurons. Using a new in-vitro model for studying neurite-neurite interactions, we found that expressed axonal NgCAM induced robust axonal bundling via the trans-homophilic interaction of immunoglobulin domains. Interestingly, dendritic bundling was induced by the dendritic targeting of NgCAM, caused by either deleting its fibronectin repeats or blocking activities of protein kinases. Consistent with the NgCAM results, expression of mouse L1-CAM also induced axonal bundling and blocking kinase activities disrupted its axonal targeting. Furthermore, the trans-homophilic interaction stabilized the bundle formation, probably through recruiting NgCAM proteins to contact sites and promoting guided axon outgrowth. Taken together, our results suggest that precise localization of L1-CAM is important for establishing proper cell-cell contacts in neural circuits.

Dynamics of Kv1 channel transport in axons.

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K(+) channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvbeta2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvbeta2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvbeta2 mutants with disrupted either Kv1.2-Kvbeta2 binding or Kvbeta2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvbeta2. Furthermore, fluorescently tagged Kv1.2 and Kvbeta2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvbeta2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons.