Identification of a Core Amino Acid Motif within the α Subunit of GABAARs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), β(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments

The green tea polyphenol (-)-epigallocatechin gallate prevents the aggregation of tau protein into toxic oligomers at substoichiometric ratios

The accumulation of amyloid-beta (Abeta) and tau aggregates is a pathological hallmark of Alzheimer's disease. Both polypeptides form fibrillar deposits, but several lines of evidence indicate that Abeta and tau form toxic oligomeric aggregation intermediates. Depleting such structures could thus be a powerful therapeutic strategy. We generated a fragment of tau (His-K18DeltaK280) that forms stable, toxic, oligomeric tau aggregates in vitro. We show that (-)-epigallocatechin gallate (EGCG), a green tea polyphenol that was previously found to reduce Abeta aggregation, inhibits the aggregation of tau K18DeltaK280 into toxic oligomers at ten- to hundred-fold substoichiometric concentrations, thereby rescuing toxicity in neuronal model cells

Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the A90V sequence variant

The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product.CC BY 4.0</p

Wild-Type, but Not Mutant N296H, Human Tau Restores Aβ-Mediated Inhibition of LTP in Tau−/− mice

Microtubule associated protein tau (MAPT) is involved in the pathogenesis of Alzheimer's disease and many forms of frontotemporal dementia (FTD). We recently reported that Aβ-mediated inhibition of hippocampal long-term potentiation (LTP) in mice requires tau. Here, we asked whether expression of human MAPT can restore Aβ-mediated inhibition on a mouse Tau−/− background and whether human tau with an FTD-causing mutation (N296H) can interfere with Aβ-mediated inhibition of LTP. We used transgenic mouse lines each expressing the full human MAPT locus using bacterial artificial chromosome technology. These lines expressed all six human tau protein isoforms on a Tau−/− background. We found that the human wild-type MAPT H1 locus was able to restore Aβ42-mediated impairment of LTP. In contrast, Aβ42 did not reduce LTP in slices in two independently generated transgenic lines expressing tau protein with the mutation N296H associated with frontotemporal dementia (FTD). Basal phosphorylation of tau measured as the ratio of AT8/Tau5 immunoreactivity was significantly reduced in N296H mutant hippocampal slices. Our data show that human MAPT is able to restore Aβ42-mediated inhibition of LTP in Tau−/− mice. These results provide further evidence that tau protein is central to Aβ-induced LTP impairment and provide a valuable tool for further analysis of the links between Aβ, human tau and impairment of synaptic function

D-sorbitol induces the formation of a 35 kDa cleavage product of TDP-43 that is localized in the cytosol.

<p>(A) Western blot of TDP-43 protein (C-terminal antibody) after treatment of HeLa cells with 0.4 M D-sorbitol or 1 mM hydrogen peroxide for one hour. (B), (C) Densitometry analysis of (B) full-length TDP-43, (C) 35 kDa fragment of TDP-43 (CTF35). Densitometry results represent mean signal ± SEM (N = 4). (D) Detection of CTF35 in cytoplasmic (Cyto) and nuclear (Nuc) fractions of HeLa cells after treatment with D-sorbitol or hydrogen peroxide. (E) Immunofluorescence detection of cytoplasmic TDP-43 after D-sorbitol treatment. Scale bar = 15 μm. (F) Quantification of cytoplasmic TDP-43 immunofluorescence signal. Results represent mean TDP-43 immunofluorescence signal in the cytoplasm as a percentage of total TDP-43 signal in the cell ± SEM (N = 4). ** p < 0.01, *** p < 0.001.</p

Sorbitol-dependent TDP-43 relocalization is reversible and not dependent on Nup153 cleavage.

<p>(A) Western blot of nuclear pore complex protein Nup153, nuclear lamina component lamin B1, procaspases -3 and -9 and their activated forms caspase-3 and caspase-9 in HeLa cells treated with sorbitol after pre-treatment with DMSO control or caspase inhibitor Z-VAD-FMK. α-tubulin was used as a loading control. (B) Immunofluorescence staining of nuclear-cytoplasmic shuttling proteins TDP-43 (green) and HuR (red) in sorbitol-stressed HeLa cells after 0 min, 15 min or 60 min rescue in normal medium. Scale bar = 30 μm. (C) Quantification of cytoplasmic TDP-43 and HuR protein in sorbitol-treated HeLa cells after 0 min, 15 min or 60 min rescue (N = 3). Results represent mean percentage of cytoplasmic signal ± SEM;** p < 0.01, *** p < 0.001, **** p < 0.0001.</p

TDP-43 domain structure with caspase and calpain cleavage sites.

<p>(A) Schematic of the full-length, 414 amino acid long TDP-43 protein. NLS = nuclear localization signal, RRM1/RRM2 = RNA recognition motifs, NES = nuclear export signal. The A90V variant and the M337V mutation are indicated in red. CS = (putative) caspase cleavage site, CL = (putative) calpain cleavage site. (B) Table of TDP-43 caspase and calpain cleavage sites described in the literature.</p

Inhibition of caspases prevents the formation of CTF35 of TDP-43, but does not prevent relocalization of the full-length protein to the cytosol.

<p>(A) TDP-43 Western blot of samples treated with or without D-sorbitol after pre-treatment with caspase inhibitor Z-VAD-FMK or DMSO control. α-tubulin was used as a loading control. FL = full-length TDP-43, CTF35 = 35 kDa C-terminal fragment of TDP-43. (B) Densitometry quantification of the 35 kDa fragment of TDP-43 after sorbitol stress in the absence or presence of caspase inhibitor (N = 5). (C) Immunofluorescence staining of nuclear and cytoplasmic TDP-43 using a C-terminal TDP-43 antibody. Cells were treated with or without D-sorbitol after pre-treatment with caspase inhibitor. Scale bar = 15 μm. (D) Quantification of nuclear and cytoplasmic TDP-43 fluorescence signals (N = 3). (E) Western blot of nuclear and cytoplasmic fractions of stressed HeLa cells. GAPDH was used as a control for the cytoplasmic fractions, histone H3 was chosen to confirm enrichment of the nuclear fraction. All results represent mean ± SEM; ** p < 0.01, *** p < 0.001.</p

The osmotic and oxidative stressor D-sorbitol triggers cleavage of endogenous TDP-43 in HeLa cells.

<p>Untransfected HeLa cells expressing endogenous TDP-43 were treated with 10 μM MG132 for 4 h, 1 mM hydrogen peroxide (H₂O₂) for 1 h, 0.5 mM sodium arsenite (SA) for 30 min, 1 μM thapsigargin (TH) for 1 h, 0.4 M D-sorbitol (Sorb) for 1 h or with respective H₂O and DMSO solvent controls. Western blot detection using a polyclonal TDP-43 antibody revealed a truncated product of TDP-43 with an approximate molecular weight of 35 kDa (CTF35, indicated by arrowhead) after treatment with D-sorbitol, but not with the other stressors.</p