TTR (transthyretin) leads the autophagy disaster relief team against TARDBP/TDP-43 proteinopathy.

TTR (transthyretin) strikes a neuroprotective function in the prevention of amyloid-ß (Aß) deposition in Alzheimer disease (AD). Perturbation of the stringently controlled TARDBP/TDP-43 (TAR DNA binding protein) expression gives rise to cytoplasmic aggregation, characterized by TARDBP proteinopathy affiliated with several neurological disorders, including frontotemporal lobar degeneration with TARDBP pathology (FTLD-TDP) and amyotrophic lateral sclerosis/ALS. Proposedly, TTR can maintain cellular proteostasis susceptible to TARDBP aggregates and initiate its removal. Herein, we disclose that TTR upregulated in response to excessive TARDBP causes TARDBP aggregation in FTLD-TDP and co-accumulates with it. Moreover, TTR expression increases with age in FTLD-TDP but shows a downward decline in the elderly. TTR promotes macroautophagy/autophagy activity and facilitates aggregated TARDBP degradation via autophagy. Compellingly, TTR binds to ATF4 and boosts its nuclear import for autophagy upregulation. Therefore, TTR directs autophagy teamwork in bi-directional regulation through enhancing autophagy activity via ATF4 and chaperoning aggregated TARDBP to phagophores for degradation.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ER: endoplasmic reticulum; FTLD-TDP: frontotemporal lobar degeneration with TARDBP pathology; TARDBP/TDP-43: TAR DNA binding protein; TTR: transthyretin; UPR: unfolded protein response.

Transthyretin attenuates TDP-43 proteinopathy by autophagy activation via ATF4 in FTLD-TDP.

TAR DNA-binding protein-43 (TDP-43) proteinopathies are accompanied by the pathological hallmark of cytoplasmic inclusions in the neurodegenerative diseases, including frontal temporal lobar degeneration-TDP and amyotrophic lateral sclerosis. We found that transthyretin accumulates with TDP-43 cytoplasmic inclusions in frontal temporal lobar degeneration-TDP human patients and transgenic mice, in which transthyretin exhibits dramatic expression decline in elderly mice. The upregulation of transthyretin expression was demonstrated to facilitate the clearance of cytoplasmic TDP-43 inclusions through autophagy, in which transthyretin induces autophagy upregulation via ATF4. Of interest, transthyretin upregulated ATF4 expression and promoted ATF4 nuclear import, presenting physical interaction. Neuronal expression of transthyretin in frontal temporal lobar degeneration-TDP mice restored autophagy function and facilitated early soluble TDP-43 aggregates for autophagosome targeting, ameliorating neuropathology and behavioural deficits. Thus, transthyretin conducted two-way regulations by either inducing autophagy activation or escorting TDP-43 aggregates targeted autophagosomes, suggesting that transthyretin is a potential modulator therapy for neurological disorders caused by TDP-43 proteinopathy.

Structure-Based Analysis of Protein Cluster Size for Super-Resolution Microscopy in the Nervous System.

To overcome the diffraction limit and resolve target structures in greater detail, far-field super-resolution techniques such as stochastic optical reconstruction microscopy (STORM) have been developed, and different STORM algorithms have been developed to deal with the various problems that arise. In particular, the effect of the local structure is an important issue. For objects with closely correlated distributions, simple Gaussian-based localization algorithms often used in STORM imaging misinterpret overlapping point spread functions (PSFs) as one, which limits the ability of super-resolution imaging to resolve nanoscale local structures and leads to inaccurate length measurements. The STORM super-resolution images of biological specimens from the cluster-forming proteins in the nervous system were reconstructed for localization-based analysis. Generally, the localization of each fluorophore was determined by two-dimensional Gaussian function fitting. Further, the physical shape of the cluster structure information was incorporated into the size parameter of the localization structure analysis in order to generate structure-based fitting algorithms. In the present study, we proposed a novel, structure-based, super-resolution image analysis method: structure-based analysis (SBA), which combines a structural function and a super-resolution localization algorithm. Using SBA, we estimated the size of fluorescent beads, inclusion proteins, and subtle synaptic structures in both wide-field and STORM images. The results show that SBA has a comparable and often superior performance to the commonly used full width at half maximum (FWHM) parameter. We demonstrated that SBA is able to estimate molecular cluster sizes in far-field super-resolution STORM images, and that SBA was comparable and often superior to FWHM. We also certified that SBA provides size estimations that corroborate previously published electron microscopy data.

Somatosensory Cortical Electrical Stimulation After Reperfusion Attenuates Ischemia/Reperfusion Injury of Rat Brain.

Objective: Ischemic stroke is an important cause of death and disability worldwide. Early reperfusion by thrombolysis or thrombectomy has improved the outcome of acute ischemic stroke. However, the therapeutic window for reperfusion therapy is narrow, and adjuvant therapy for neuroprotection is demanded. Electrical stimulation (ES) has been reported to be neuroprotective in many neurological diseases. In this study, the neuroprotective effect of early somatosensory cortical ES in the acute stage of ischemia/reperfusion injury was evaluated. Methods: In this study, the rat model of transient middle cerebral artery occlusion was used to explore the neuroprotective effect and underlying mechanisms of direct primary somatosensory (S1) cortex ES with an electric current of 20 Hz, 2 ms biphasic pulse, 100 µA for 30 min, starting at 30 min after reperfusion. Results: These results showed that S1 cortical ES after reperfusion decreased infarction volume and improved functional outcome. The number of activated microglia, astrocytes, and cleaved caspase-3 positive neurons after ischemia/reperfusion injury were reduced, demonstrating that S1 cortical ES alleviates inflammation and apoptosis. Brain-derived neurotrophic factor (BDNF) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were upregulated in the penumbra area, suggesting that BDNF/TrkB signals and their downstream PI3K/Akt signaling pathway play roles in ES-related neuroprotection. Conclusion: This study demonstrates that somatosensory cortical ES soon after reperfusion can attenuate ischemia/reperfusion injury and is a promising adjuvant therapy for thrombolytic treatment after acute ischemic stroke. Advanced techniques and devices for high-definition transcranial direct current stimulation still deserve further development in this regard.

TDP-43 proteinopathy impairs mRNP granule mediated postsynaptic translation and mRNA metabolism.

Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules. Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy. Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

Solution structure and backbone dynamics of the DNA-binding domain of FOXP1: insight into its domain swapping and DNA binding.

FOXP1 belongs to the P-subfamily of forkhead transcription factors and contains a conserved forkhead DNA-binding domain. According to size exclusion chromatography analysis, the forkhead domain of FOXP1 existed as a mixture of monomer and dimer. The dissociation constants of the forkhead domain of wild-type, C61S, and C61Y mutants of FOXP1 were 27.3, 28.8, and 332.0 µM, respectively. In contrast, FOXP1 A39P mutant formed only a monomer. NMR analysis also showed that FOXP1 C61S and C61Y mutants existed as a mixture. The solution structure of FOXP1 A39P/C61Y mutant was similar to the X-ray structure of the FOXP2 monomer. Comparison of backbone dynamics of FOXP1 A39P/C61Y and C61Y mutants showed that the residues preceding helix 3, the hinge region, exhibited the largest conformational exchange in FOXP1 monomer. The A39 residue of FOXP1 dimer has a lower order parameter with internal motion on the ps-ns timescale, suggesting that the dynamics of the hinge region of FOXP1 are important in the formation of the swapped dimer. The analysis also showed that the residues exhibiting the motions on the ps-ns and µs-ms timescales were located at the DNA-binding surface of FOXP1, suggesting the interactions between FOXP1 and DNA may be highly dynamic.

Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3.

Acid sensing ion channels (ASICs) are family of proteins predominantly present in the central and peripheral nervous system. They are known to play important roles in the pathophysiology of pain and ischemic stroke. APETx2 is a potent and selective inhibitor of ASIC3-containing channels and was isolated from sea anemone Anthopleura elegantissima. To facilitate the study on the molecular determinants of ASIC3-ligand interactions, we expressed recombinant APETx2 in the Pichia pastoris (P. pastoris) expression system and purified it to homogeneity. Recombinant APETx2 produced in P. pastoris inhibited the acid-evoked ASIC3 current with the IC(50) value of 37.3 nM. The potency of recombinant toxin is similar to that of native APETx2. The sequential assignment and structure analysis of APETx2 were obtained by 2D and 3D (15)N-edited NMR spectra. Our NMR data suggests that APETx2 produced in P. pastoris retained its native fold. The results presented here provide the first direct evidence that highly disulfide bonded peptide inhibitor of ASIC3, APETx2, can be expressed in P. pastoris with correct fold and high yield. We also showed that the R17A mutant exhibited a decrease in activity, suggesting the feasibility of the use of this expression system to study the interactions between APETx2 and ASIC3. These evidences may serve as the basis for understanding the selectivity and activity of APETx2.

The structural events associated with the binding of divalent cations to beta-bungarotoxin.

In order to address the mechanism why the Ca2+ was crucial for the manifestation of the phospholipase A2 (PLA2) activity of beta-bungarotoxin (beta-BuTx), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of beta-BuTx. Substitution Mg2+ or Sr2+ for Ca2+ in the substrate solution was found to cause a decrease in the PLA2 activity to approximately 15 or 6% of that in the presence of Ca2+. However, only marginally detectable PLA2 activity was observed with the addition of Ba2+. The nonpolarity of 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of beta-BuTx markedly increased with the binding of cations to beta-BuTx. The negative ellipticity noted with the CD spectra of beta-BuTx increased upon the binding of cations too. With the exception of Ba2+, the order of the ability of cations to enhance the intensity of ANS fluorescence or increase the increment of negative ellipticity was Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. However, the energy transfer from Trp fluorescence emission to ANS was most effective upon the addition of Ca2+. Moreover, the extent of glutaraldehyde crosslinking between A chain and B chain decreased in the presence of cations. Nevertheless, the binding affinities of beta-BuTx for the four cations were similar. These results, together with the findings that the ANS molecule binds at the active site of the A chain in beta-BuTx, suggest that the binding of Ca2+ to beta-BuTx induces subtly conformational changes occurred at the active site for exerting the activity of beta-BuTx. Moreover, the change in the gross conformation induced by the binding of Ca2+ may affect the interaction between A chain and B chain, and consequently the activity of beta-BuTx as well.

Lys-64 of the A chain is involved in the enzymatic activity and neurotoxic effect of beta-bungarotoxin.

Two beta-bungarotoxin isotoxins BM12 and BM13 were isolated from Bungarus multicinctus (Taiwan banded krait) venom by sequential chromatography on ion-exchange and reverse phase columns. The two toxins have the same A chain, but different B chains. Different phospholipase A2 activity and different potencies in inhibiting the spontaneous enhancement of spontaneous synaptic current frequency and muscle contraction were observed for BM12 and BM13. Nevertheless, modification of Lys-64 in the A chain of BM12 and BM13 similarly reduced in their phospholipase A2 activity and toxicity. The modified derivatives retained their affinity with Ca2+ and their conformation as deduced by CD. These results suggest that Lys-64 of the A chain is involved in the phospholipase A2 activity and in the neurotoxic effect of beta-bungarotoxin.

Both A chain and B chain of beta-bungarotoxin are functionally involved in the facilitation of spontaneous transmitter release in Xenopus nerve-muscle cultures.

In the present study, Xenopus nerve-muscle cultures were used to explore the functional roles of A chain (a phospholipase A(2) subunit) and B chain (a non-phospholipase A(2) subunit) of Bungarus multicinctus beta-bungarotoxin. It was found that beta-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve-muscle cultures. Modification of beta-bungarotoxin with pyridoxal-5'-phosphate or substitution of Ca(2+) with Ba(2+) in buffer abolished the phospholipase A(2) activity of beta-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A(2) activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of beta-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve-muscle cultures.

The organization of the genes encoding the A chains of beta-bungarotoxins: evidence for the skipping of exon.

Bungarus multicinctus (Taiwan banded krait) beta-bungarotoxins consist of two dissimilar polypeptide chains, A and B. The A chain is structurally homologous to phospholipase A(2) (PLA(2)) enzymes. The structural organization of the genes encoding A1, A2 and A8 chains are reported in this study. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with their cDNAs revealed that A1 chain gene organized with four exons and three introns, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponding to the first exon of A1 chain gene is skipped instead of the inclusion of intronic sequence adjacent to the second exon. The resulting A2 chain mRNA encoded a 25 residue signal peptide, which is different from A1 chain mRNA with a 27 residue signal peptide. Nevertheless, expression of the A chain genes was partly regulated by a common mechanism as evidenced by sequence conservation of their promoter region and consensus transcriptional factor binding-sites inside this region. 5'-RACE analyses revealed that A chain mRNAs with 27 residue signal peptide represented the predominant species in the preparation of B. multicinctus venom gland mRNAs. Comparative analyses on PLA(2) genes and cDNAs suggest that this is the first report on the skipping of exon which changes the signal peptide sequence of snake venom proteins.