Auditory Motion Aftereffect 1 Perception and Psychophysics. Vol. 62(5), pp. 1099–1111. Suggested running head: AUDITORY MOTION AFTEREFFECT The Auditory Motion Aftereffect: its Tuning and Specificity in the Spatial and Frequency Domains

In this paper, the auditory motion aftereffect (aMAE) was studied by using real moving sound as both the adapting and test stimulus. The real moving sound was generated by a loudspeaker mounted on a robot arm which was able to move quietly in three dimensional space. Seven subjects with normal hearing were tested. Results from Experiment 1 showed a robust and reliable negative aMAE in all the subjects involved. After listening to a sound source moving repeatedly to the right, a stationary sound source was perceived to be moving to the left. The magnitude of the aMAE tended to increase up to the highest velocity tested (<30°/sec). The tuning and specificity of this aftereffect was further studied in the spatial and frequency domains. The strength of the aftereffect depended on matching both the spatial location and the frequency content of the adapting and test stimuli. Offsetting the locations of adapting and test stimuli by 20° reduced the size of the effect by about 50%. A similar decline occurred when the frequency of the adapting and test stimuli differed by one octave

TDP-43 Inhibits NF-κB Activity by Blocking p65 Nuclear Translocation

<div><p>TDP-43 (TAR DNA binding protein 43) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that has been found to play an important role in neurodegenerative diseases. TDP-43’s involvement in nuclear factor-kappaB pathways has been reported in both neurons and microglial cells. The NF-κB pathway targets hundreds of genes, many of which are involved in inflammation, immunity and cancer. p50/p65 (p50/RelA) heterodimers, as the major Rel complex in the NF-κB family, are induced by diverse external physiological stimuli and modulate transcriptional activity in almost all cell types. Both p65 and TDP-43 translocation occur through the classic nuclear transportation system. In this study, we report that TDP-43 overexpression prevents TNF-α induced p65 nuclear translocation in a dose dependent manner, and that this further inhibits p65 transactivation activity. The inhibition by TDP-43 does not occur through preventing IκB degradation but probably by competing for the nuclear transporter-importin α3 (KPNA4). This competition is dependent on the presence of the nuclear localization signal (NLS) in TDP-43. Silencing TDP-43 using a specific siRNA also increased p65 nuclear localization upon TNF-α stimulation, suggesting that endogenous TDP-43 may be a default suppressor of the NF-κB pathway. Our results indicate that TDP-43 may play an important role in regulating the levels of NF-κB activity by controlling the nuclear translocation of p65.</p></div

Overexpression of TDP-43 on p65 activation.

<p><b>A</b>. p65 (Green) is translocated into the cells’ nuclei (Blue) after 30 minutes of 10ng/ml TNF-α treatment (n = 3 repeats). <b>B</b>. MCF-7 cells were transfected with various doses (indicated on the right) of wild type TDP-43 tagged with mCherry expressing plasmids (middle panel, red) and were treated with 10ng/ml TNF-α for 30 minutes. In the top right corner, insets of higher magnification show the localization of p65 and nuclear TDP-43 staining (n≥3 independent experiments). <b>C</b>. The quantification of B. <b>D.</b> MCF-7 Cells were transfected with 4μg plasmid encoding TDP-43 tagged with mCherry or mCherry empty plasmid (control) and treated with 10ng/ml TNF-α for 30 minutes. Nuclear proteins were isolated and detected by western immunoblotting. Histone H3 was used as a nuclear marker. The relative density was the average of 3 individual experiments. <b>E</b>. MCF-7, Neuro 2a and BV2 cells were transfected with Wild type TDP-43 tagged with mCherry expressing plasmids (middle panel, red). MCF-7 and Neuro 2a cells were treated with 10ng/ml TNF-α for 30 minutes; BV2 cells were treated with 10μg/ml LPS for 30 minutes. Arrows indicate the blocked p65 nuclear translocation by Wt TDP-43 and stars indicate the normal p65 nuclear translocation with Wt TDP-43 overexpression. n≥3 independent experiments. <b>F</b>. The plasmid encoding TDP-43 tagged with mCherry or mCherry empty plasmid (control) was cotransfected along with NF-κB-luc (containing the wild type NF-κB-binding site). Cells were treated with 10ng/ml TNF-α for 30 minutes. Luciferase activity was measured after 24 hours. The plotted error bars represent mean±SEM from 3 independent experiments; *p < 0.05 compared to control, one-way ANOVA. ** p < 0.05 compared to the TNF-α treated control samples, Student's t test.</p

The blockade of NF-κB nuclear translocation by TDP-43 can be prevented by overexpression of p65.

<p>MCF-7 cells were transfected with wild type TDP-43 expressing plasmids (top row) alone or cotransfected with TDP-43 and p65 expressing plasmids (bottom row). Then MCF-7 cells were treated with TNF-α for 30 minutes. The p65 was found co-localized with TDP-43 in the nuclei (bottom row). n = 3 independent experiments.</p

The NLS mutated TDP-43 does not affect p65 nuclear translocation.

<p>MCF-7 cells were transfected with TDP-43 ΔNLS expressing plasmids (middle panel, Red). After 30 minutes of TNF-α treatment, labeled p65 (Left panel, Green) was found in the nuclei. n = 3 independent experiments.</p

The competition of importin α3 (KPNA4) occurs in both the cytoplasm and nucleus.

<p>The nuclear and cytoplasmic proteins of MCF-7 cells were separated after overexpression of wild type TDP-43 and TNF-α stimulation. The top panel shows the protein levels of total TDP-43 in each fraction. N: nucleus. C: cytoplasm. The bottom shows that TDP-43 interacted with importin α3 (KPNA4). p < 0.05, Student's t test.</p

Overexpressing TDP-43 inhibits NF-κB activity by competing for the nuclear transporter.

<p><b>A</b>. Cells were transfected with wild type TDP-43 expressing plasmids (tagged with mCherry) or mCherry empty plasmid (control) and were then treated with 10ng/ml TNF-α for 30 minutes. Cell lysates were subjected to coimmunoprecipitation and western blot for the presence of p65 and TDP-43. 10% cell lysate were reserved as input. p65, TDP-43 and importin α3 (KPNA4) were used to detect input. n = 3 repeats. <b>B</b>. The relative density of p65 as shown in A (IP), *p<0.05. <b>C</b>. The relative density of wild type TDP-43 as shown in A (IP), *p<0.05.</p

Overexpression of TDP-43 accelerates IκB degradation.

<p>Cells were transfected with wild type TDP-43 expressing plasmids (tagged with mCherry) or mCherry empty plasmid (control) and were treated with 10ng/ml TNF-α for the indicated periods. Total proteins were extracted for Western blot analysis using antibodies as indicated. n = 3 independent cultures; <b>A</b>. 30 min after TNF-α treatment, the degradation of IκB occurred in both control group and TDP-43 overexpression group. <b>B</b>. Overexpression of TDP-43 facilitates the degradation of IκB within 5 minutes after TNF-α treatment.</p

Schematic hypothetical modes for TDP-43 and p65 competition.

<p>The presence of TDP-43 in the cytosol may compete with p65 for importin α3 (cytoplasmic mode) and therefore to inhibit the nuclear localization of p65. An alternative mode (nuclear mode) shows that nuclear TDP-43 may occupy importin α3 to prevent its exit and therefore to reduce the availability of free importin α3 in the cytosol for p65 nuclear translocation.</p

Reducing TDP-43 aggregation does not prevent its cytotoxicity

Background: TAR DNA-binding protein 43 (TDP-43) is a protein that is involved in the pathology of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). In patients with these neurodegenerative diseases, TDP-43 does not remain in its normal nuclear location, but instead forms insoluble aggregates in both the nucleus and cytoplasm of affected neurons. Results We used high density peptide array analysis to identify regions in TDP-43 that are bound by TDP-43 itself and designed candidate peptides that might be able to reduce TDP-43 aggregation. We found that two of the synthetic peptides identified with this approach could effectively inhibit the formation of TDP-43 protein aggregates in a concentration-dependent manner in HeLa cells in which a mutated human TDP-43 gene was overexpressed. However, despite reducing aggregation, these peptides did not reduce or prevent cell death. Similar results were observed in HeLa cells treated with arsenite. Again we found reduced aggregation, in this case of wild type TDP-43, but no difference in cell death. Conclusions Our results suggest that TDP-43 aggregation is associated with the cell death process rather than being a direct cause.Non UBCMedicine, Faculty ofReviewedFacult