Interaction between magnesium and methylglyoxal in diabetic polyneuropathy and neuronal models.

OBJECTIVE: The lack of effective treatments against diabetic sensorimotor polyneuropathy demands the search for new strategies to combat or prevent the condition. Because reduced magnesium and increased methylglyoxal levels have been implicated in the development of both type 2 diabetes and neuropathic pain, we aimed to assess the putative interplay of both molecules with diabetic sensorimotor polyneuropathy. METHODS: In a cross-sectional study, serum magnesium and plasma methylglyoxal levels were measured in recently diagnosed type 2 diabetes patients with (n = 51) and without (n = 184) diabetic sensorimotor polyneuropathy from the German Diabetes Study baseline cohort. Peripheral nerve function was assessed using nerve conduction velocity and quantitative sensory testing. Human neuroblastoma cells (SH-SY5Y) and mouse dorsal root ganglia cells were used to characterize the neurotoxic effect of methylglyoxal and/or neuroprotective effect of magnesium. RESULTS: Here, we demonstrate that serum magnesium concentration was reduced in recently diagnosed type 2 diabetes patients with diabetic sensorimotor polyneuropathy and inversely associated with plasma methylglyoxal concentration. Magnesium, methylglyoxal, and, importantly, their interaction were strongly interrelated with methylglyoxal-dependent nerve dysfunction and were predictive of changes in nerve function. Magnesium supplementation prevented methylglyoxal neurotoxicity in differentiated SH-SY5Y neuron-like cells due to reduction of intracellular methylglyoxal formation, while supplementation with the divalent cations zinc and manganese had no effect on methylglyoxal neurotoxicity. Furthermore, the downregulation of mitochondrial activity in mouse dorsal root ganglia cells and consequently the enrichment of triosephosphates, the primary source of methylglyoxal, resulted in neurite degeneration, which was completely prevented through magnesium supplementation. CONCLUSIONS: These multifaceted findings reveal a novel putative pathophysiological pathway of hypomagnesemia-induced carbonyl stress leading to neuronal damage and merit further investigations not only for diabetic sensorimotor polyneuropathy but also other neurodegenerative diseases associated with magnesium deficiency and impaired energy metabolism.

Hepatic encephalopathy is linked to alterations of autophagic flux in astrocytes.

BACKGROUND: Hepatic encephalopathy (HE) is a severe neuropsychiatric syndrome caused by various types of liver failure resulting in hyperammonemia-induced dysfunction of astrocytes. It is unclear whether autophagy, an important pro-survival pathway, is altered in the brains of ammonia-intoxicated animals as well as in HE patients. METHODS: Using primary rat astrocytes, a co-culture model of primary mouse astrocytes and neurons, an in vivo rat HE model, and post mortem brain samples of liver cirrhosis patients with HE we analyzed whether and how hyperammonemia modulates autophagy. FINDINGS: We show that autophagic flux is efficiently inhibited after administration of ammonia in astrocytes. This occurs in a fast, reversible, time-, dose-, and ROS-dependent manner and is mediated by ammonia-induced changes in intralysosomal pH. Autophagic flux is also strongly inhibited in the cerebral cortex of rats after acute ammonium intoxication corroborating our results using an in vivo rat HE model. Transglutaminase 2 (TGM2), a factor promoting autophagy, is upregulated in astrocytes of in vitro- and in vivo-HE models as well as in post mortem brain samples of liver cirrhosis patients with HE, but not in patients without HE. LC3, a commonly used autophagy marker, is significantly increased in the brain of HE patients. Ammonia also modulated autophagy moderately in neuronal cells. We show that taurine, known to ameliorate several parameters caused by hyperammonemia in patients suffering from liver failure, is highly potent in reducing ammonia-induced impairment of autophagic flux. This protective effect of taurine is apparently not linked to inhibition of mTOR signaling but rather to reducing ammonia-induced ROS formation. INTERPRETATION: Our data support a model in which autophagy aims to counteract ammonia-induced toxicity, yet, as acidification of lysosomes is impaired, possible protective effects thereof, are hampered. We propose that modulating autophagy in astrocytes and/or neurons, e.g. by taurine, represents a novel strategy to treat liver diseases associated with HE. FUNDING: Supported by the DFG, CRC974 "Communication and Systems Relevance in Liver Injury and Regeneration", Düsseldorf (Project number 190586431) Projects A05 (DH), B04 (BG), B05 (NK), and B09 (ASR).

Regulated Dynamic Trafficking of Neurexins Inside and Outside of Synaptic Terminals.

Synapses depend on trafficking of key membrane proteins by lateral diffusion from surface populations and by exocytosis from intracellular pools. The cell adhesion molecule neurexin (Nrxn) plays essential roles in synapses, but the dynamics and regulation of its trafficking are unknown. Here, we performed single-particle tracking and live imaging of transfected, epitope-tagged Nrxn variants in cultured rat and mouse wild-type or knock-out neurons. We observed that structurally larger αNrxn molecules are more mobile in the plasma membrane than smaller ßNrxns because αNrxns displayed higher diffusion coefficients in extrasynaptic regions and excitatory or inhibitory terminals. We found that well characterized interactions with extracellular binding partners regulate the surface mobility of Nrxns. Binding to neurexophilin-1 (Nxph1) reduced the surface diffusion of αNrxns when both molecules were coexpressed. Conversely, impeding other interactions by insertion of splice sequence #4 or removal of extracellular Ca(2+) augmented the mobility of αNrxns and ßNrxns. We also determined that fast axonal transport delivers Nrxns to the neuronal surface because Nrxns comigrate as cargo on synaptic vesicle protein transport vesicles (STVs). Unlike surface mobility, intracellular transport of ßNrxn(+) STVs was faster than that of αNrxns, but both depended on the microtubule motor protein KIF1A and neuronal activity regulated the velocity. Large spontaneous fusion of Nrxn(+) STVs occurred simultaneously with synaptophysin on axonal membranes mostly outside of active presynaptic terminals. Surface Nrxns enriched at synaptic terminals where αNrxns and Nxph1/αNrxns recruited GABAAR subunits. Therefore, our results identify regulated dynamic trafficking as an important property of Nrxns that corroborates their function at synapses. SIGNIFICANCE STATEMENT: Synapses mediate most functions in our brains and depend on the precise and timely delivery of key molecules throughout life. Neurexins (Nrxns) are essential synaptic cell adhesion molecules that are involved in synaptic transmission and differentiation of synaptic contacts. In addition, Nrxns have been linked to neuropsychiatric diseases such as autism. Because little is known about the dynamic aspects of trafficking of neurexins to synapses, we investigated this important question using single-molecule tracking and time-lapse imaging. We identify distinct differences between major Nrxn variants both in surface mobility and during intracellular transport. Because their dynamic behavior is highly regulated, for example, by different binding activities, these processes have immediate consequences for the function of Nrxns at synapses.

Fast Three-Dimensional Single-Particle Tracking in Natural Brain Tissue.

Observation of molecular dynamics is often biased by the optical very heterogeneous environment of cells and complex tissue. Here, we have designed an algorithm that facilitates molecular dynamic analyses within brain slices. We adjust fast astigmatism-based three-dimensional single-particle tracking techniques to depth-dependent optical aberrations induced by the refractive index mismatch so that they are applicable to complex samples. In contrast to existing techniques, our online calibration method determines the aberration directly from the acquired two-dimensional image stream by exploiting the inherent particle movement and the redundancy introduced by the astigmatism. The method improves the positioning by reducing the systematic errors introduced by the aberrations, and allows correct derivation of the cellular morphology and molecular diffusion parameters in three dimensions independently of the imaging depth. No additional experimental effort for the user is required. Our method will be useful for many imaging configurations, which allow imaging in deep cellular structures.

The Sushi domains of GABAB receptors function as axonal targeting signals.

GABA(B) receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. Two receptor subtypes, GABA(B(1a,2)) and GABA(B(1b,2)), are formed by the assembly of GABA(B1a) and GABA(B1b) subunits with GABA(B2) subunits. The GABA(B1b) subunit is a shorter isoform of the GABA(B1a) subunit lacking two N-terminal protein interaction motifs, the sushi domains. Selectively GABA(B1a) protein traffics into the axons of glutamatergic neurons, whereas both the GABA(B1a) and GABA(B1b) proteins traffic into the dendrites. The mechanism(s) and targeting signal(s) responsible for the selective trafficking of GABA(B1a) protein into axons are unknown. Here, we provide evidence that the sushi domains are axonal targeting signals that redirect GABA(B1a) protein from its default dendritic localization to axons. Specifically, we show that mutations in the sushi domains preventing protein interactions preclude axonal localization of GABA(B1a). When fused to CD8alpha, the sushi domains polarize this uniformly distributed protein to axons. Likewise, when fused to mGluR1a the sushi domains redirect this somatodendritic protein to axons, showing that the sushi domains can override dendritic targeting information in a heterologous protein. Cell surface expression of the sushi domains is not required for axonal localization of GABA(B1a). Altogether, our findings are consistent with the sushi domains functioning as axonal targeting signals by interacting with axonally bound proteins along intracellular sorting pathways. Our data provide a mechanistic explanation for the selective trafficking of GABA(B(1a,2)) receptors into axons while at the same time identifying a well defined axonal delivery module that can be used as an experimental tool.

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function.

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.

Differential compartmentalization and distinct functions of GABAB receptor variants.

GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.

The RXR-type endoplasmic reticulum-retention/retrieval signal of GABAB1 requires distant spacing from the membrane to function.

Functional gamma-aminobutyric acid type B (GABA(B)) receptors are normally only observed upon coexpression of GABA(B1) with GABA(B2) subunits. A C-terminal arginine-based endoplasmic reticulum (ER) retention/retrieval signal, RSRR, prevents escape of unassembled GABA(B1) subunits from the ER and restricts surface expression to correctly assembled heteromeric receptors. The RSRR signal in GABA(B1) is proposed to be shielded by C-terminal coiled-coil interaction of the GABA(B1) with the GABA(B2) subunit. Here, we investigated whether the RSRR motif in GABA(B1) remains functional when grafted to ectopic sites. We found that the RSRR signal in GABA(B1) is inactive in any of the three intracellular loops but remains functional when moved within the distal zone of the C-terminal tail. C-terminal deletions that position the RSRR signal closer to the plasma membrane drastically reduce its effectiveness, supporting that proximity to the membrane restricts access to the RSRR motif. Functional ectopic RSRR signals in GABA(B1) are efficiently inactivated by the GABA(B2) subunit in the absence of coiled-coil dimerization, supporting that coiled-coil interaction is not critical for release of the receptor complex from the ER. The data are consistent with a model in which removal of RSRR from its active zone rather than its direct shielding by coiled-coil dimerization triggers forward trafficking. Because arginine-based intracellular retention signals of the type RXR, where X represents any amino acid, are used to regulate assembly and surface transport of several multimeric complexes, such a mechanism may apply to other proteins as well.