ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD+ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.

A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells.

Impaired redox metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies that can perturb redox metabolism in various cellular or organismal backgrounds. Most studies involving impaired redox metabolism have focused on oxidative stress; consequently, less is known about the settings where there is an overabundance of NADH reducing equivalents, termed reductive stress. Here we introduce a soluble transhydrogenase from Escherichia coli (EcSTH) as a novel genetically encoded tool to promote reductive stress in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially targeted version (mitoEcSTH), robustly elevated the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determined that metabolic and transcriptomic signatures of the NADH reductive stress are cellular background specific. Collectively, our novel genetically encoded tool represents an orthogonal strategy to promote reductive stress.

WDR47 protects neuronal microtubule minus ends from katanin-mediated severing.

Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization.

Neuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1-4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

Feedback-Driven Assembly of the Axon Initial Segment.

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood. Here, we show that Ankyrin-G acts as a scaffold interacting with End-Binding (EB) proteins and membrane proteins such as Neurofascin-186 to recruit TRIM46-positive microtubules to the plasma membrane. Using in vitro reconstitution and cellular assays, we demonstrate that TRIM46 forms parallel microtubule bundles and stabilizes them by acting as a rescue factor. TRIM46-labeled microtubules drive retrograde transport of Neurofascin-186 to the proximal axon, where Ankyrin-G prevents its endocytosis, resulting in stable accumulation of Neurofascin-186 at the AIS. Neurofascin-186 enrichment in turn reinforces membrane anchoring of Ankyrin-G and subsequent recruitment of TRIM46-decorated microtubules. Our study reveals feedback-based mechanisms driving AIS assembly.

TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment.

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.

MAP7D2 Localizes to the Proximal Axon and Locally Promotes Kinesin-1-Mediated Cargo Transport into the Axon.

The motor protein kinesin-1 plays an important role in polarized sorting of transport vesicles to the axon. However, the mechanism by which the axonal entry of kinesin-1-dependent cargo transport is regulated remains unclear. Microtubule-associated protein MAP7 (ensconsin in Drosophila) is an essential kinesin-1 cofactor and promotes kinesin-1 recruitment to microtubules. Here, we found that MAP7 family member MAP7D2 concentrates at the proximal axon, where it overlaps with the axon initial segment and interacts with kinesin-1. Depletion of MAP7D2 results in reduced axonal cargo entry and defects in axon development and neuronal migration. We propose a model in which MAP7D2 in the proximal axon locally promotes kinesin-1-mediated cargo entry into the axon.

Feedback-Driven Mechanisms between Microtubules and the Endoplasmic Reticulum Instruct Neuronal Polarity.

Establishment of neuronal polarity depends on local microtubule (MT) reorganization. The endoplasmic reticulum (ER) consists of cisternae and tubules and, like MTs, forms an extensive network throughout the entire cell. How the two networks interact and control neuronal development is an outstanding question. Here we show that the interplay between MTs and the ER is essential for neuronal polarity. ER tubules localize within the axon, whereas ER cisternae are retained in the somatodendritic domain. MTs are essential for axonal ER tubule stabilization, and, reciprocally, the ER is required for stabilizing and organizing axonal MTs. Recruitment of ER tubules into one minor neurite initiates axon formation, whereas ER retention in the perinuclear area or disruption of ER tubules prevent neuronal polarization. The ER-shaping protein P180, present in axonal ER tubules, controls axon specification by regulating local MT remodeling. We propose a model in which feedback-driven regulation between the ER and MTs instructs neuronal polarity.

The HAUS Complex Is a Key Regulator of Non-centrosomal Microtubule Organization during Neuronal Development.

Neuron morphology and function are highly dependent on proper organization of the cytoskeleton. In neurons, the centrosome is inactivated early in development, and acentrosomal microtubules are generated by mechanisms that are poorly understood. Here, we show that neuronal migration, development, and polarization depend on the multi-subunit protein HAUS/augmin complex, previously described to be required for mitotic spindle assembly in dividing cells. The HAUS complex is essential for neuronal microtubule organization by ensuring uniform microtubule polarity in axons and regulation of microtubule density in dendrites. Using live-cell imaging and high-resolution microscopy, we found that distinct HAUS clusters are distributed throughout neurons and colocalize with γ-TuRC, suggesting local microtubule nucleation events. We propose that the HAUS complex locally regulates microtubule nucleation events to control proper neuronal development.

Developmental regulation of hippocampal long-term depression by cofilin-mediated actin reorganization.

Long lasting synaptic plasticity involves both functional and morphological changes, but how these processes are molecularly linked to achieve coordinated plasticity remains poorly understood. Cofilin is a common target of multiple signaling pathways at the synapse and is required for both functional and spine plasticity, but how it is regulated is unclear. In this study, we investigate whether the involvement of cofilin in plasticity is developmentally regulated by examining the role of cofilin in hippocampal long-term depression (LTD) in both young (2 weeks) and mature (2 months) mice. We show that both total protein level of cofilin and its activation undergo significant changes as the brain matures, so that although the amount of cofilin decreases significantly in mature mice, its regulation by protein phosphorylation becomes increasingly important. Consistent with these biochemical data, we show that cofilin-mediated actin reorganization is essential for LTD in mature, but not in young mice. In contrast to cofilin, the GluA2 interactions with NSF and PICK1 appear to be required in both young and mature mice, indicating that AMPAR internalization is a common key mechanism for LTD expression regardless of the developmental stages. These results establish the temporal specificity of cofilin in LTD regulation and suggest that cofilin-mediated actin reorganization may serve as a key mechanism underlying developmental regulation of synaptic and spine plasticity. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

p21-activated kinase 1 restricts tonic endocannabinoid signaling in the hippocampus.

PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not 2-arachidonoylglycerol (2-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-2 (COX-2), and COX-2 inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-2, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies.

PAK1 regulates cortical development via promoting neuronal migration and progenitor cell proliferation.

BACKGROUND: p21-activated kinase 1 (PAK1) is a serine/threonine kinase known to be activated by the Rho family small GTPases and to play a key role in cytoskeletal reorganization, spine morphology and synaptic plasticity. PAK1 is also implicated in a number of neurodevelopmental and neurodegenerative diseases, including autism, intellectual disability and Alzheimer's disease. However, the role of PAK1 in early brain development remains unknown. RESULTS: In this study, we employed genetic manipulations to investigate the role of PAK1 in the cerebral cortical development in mice. We showed that compared to the wild type littermates, PAK1 knockout mice have a reduction in the number of pyramidal neurons in several layers of the cerebral cortex, which is associated with a smaller pool of neural progenitor cells and impaired neuronal migration. CONCLUSION: These results suggest that PAK1 regulates cortical development by promoting the proliferation of neural progenitor cells and facilitating the migration of these neurons to specific regions of the cortex.