Reduction of RAD23A extends lifespan and mitigates pathology in a mouse model of TDP-43 proteinopathy

Domain: biology
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
Protein misfolding and aggregation are cardinal features of neurodegenerative disease (NDD) and they contribute to pathophysiology by both loss-of-function (LOF) and gain-of-function (GOF) mechanisms. This is well exemplified by TDP-43 which aggregates and mislocalizes in several NDDs. The depletion of nuclear TDP-43 leads to reduction in its normal function in RNA metabolism and the cytoplasmic accumulation of TDP-43 leads to aberrant protein homeostasis. A modifier screen found that loss of rad23 suppressed TDP-43 pathology in invertebrate and tissue culture models. Here we show in the TAR4 mouse model of TDP-43 pathology that genetic or antisense oligonucleotide (ASO)-mediated reduction of rad23a confers benefits on survival and behavior, histological hallmarks of disease and reduction of mislocalized and aggregated TDP-43. This results in improved function of the ubiquitin-proteasome system (UPS) and correction of transcriptomic alterations evoked by pathologic TDP-43. RAD23A-dependent remodeling of the insoluble proteome appears to be a key event driving pathology in this model. As TDP-43 pathology is prevalent in both familial and sporadic NDD, targeting RAD23A may have therapeutic potential.

Introduction
Cytoplasmic, nuclear and neuritic ubiquitinated inclusions (UBI) are a pathological hallmark of amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTD) and subtypes of other NDD1. TAR-DNA-binding protein-43 (TDP-43) was originally identified as a disease-specific core protein of a subpopulation of UBIs2. We now know that TDP-43 is a critical disease driver due to both LOF and GOF mechanisms that are linked to its mislocalization. Strategies to ameliorate pathophysiological events evoked by TDP-43 are likely to have a major therapeutic impact on ALS/FTD.

Unbiased screens for phenotypic suppressors evoked by overexpression of wild type (WT) or mutated versions of TDP-43 have shown that many biological processes are impacted, including components of the proteostasis network3–10. It has been estimated that 80% of cellular proteins are degraded by UPS11. Ubiquitinated clients can bind to the proteasome (i.e., Rpn1/PSMD2, Rpn10/PSMD4 and Rpn13/ADRM1)12 to initiate proteolysis by the 20S proteasome. An additional route of those clients to the proteasome employs “shuttle factors” such as yeast proteins RAD23 (RAD23A and RAD23B in vertebrates), Dsk2 (ubiquilins, UBQLNs 1–4 invertebrates) and Ddi1 (Ddi1 and Ddi2 in vertebrates)13. While the canonical view of RAD23 function as a shuttle factor that promotes client degradation is clearly correct for many substrates14–18, it is not the complete story. Loss of rad23 can also promote the degradation of targets such as XPC19, p5320, Sic116, polyQ-expanded ataxin 321, mutant SOD and mutant TDP-4322. This may be linked to features of: (1) the substrate, as well-folded clients brought to the proteasome by RAD23 resist degradation23 or (2) ubiquitin status, as RAD23 negatively regulates multi-ubiquitin chain assembly24,25.

In this work, we expanded on the observation that ablation of RAD23 in a Caenorhabditis elegans model of TDP-43 proteinopathy conferred benefits on locomotor and biochemical phenotypes and as does knockdown (KD) of the two vertebrate isoforms of RAD23 (RAD23A or RAD23B) in a rat neuron tissue culture model22. We reasoned that mechanistic insights into RAD23A/B actions using a TDP-43-based mouse model of ALS/FTD would further empower therapeutic opportunities.

Results
Reducing RAD23A abundance benefits a model of TDP-43 proteinopathy
To test the hypothesis that a reduction of RAD23A or RAD23B can mitigate the neurodegenerative phenotypes caused by TDP-43 accumulation in vivo, we first employed an ASO targeting approach. Potent and specific ASOs were designed targeting the mouse RAD23A or RAD23B mRNA. Using the approach established by Becker et al.26. We assessed the effects of intracerebroventricular (ICV) injection of 60μg ASO in a total volume of 3μL (targeting RAD23A or RAD23B or a scrambled sequence, “SCR”, control) at postnatal day zero (P0) on RAD23A and RAD23B mRNA levels. In the cerebral cortex, RAD23A mRNA level was reduced ~75% (ASO: RAD23A versus SCR, p<0.0001) and in the spinal cord reduced ~80% (RAD23A versus SCR, p<0.0001) (Fig. 1A). The ASO targeting RAD23A had no effect on the RAD23B mRNA level in the cerebral cortex or spinal cord (RAD23A versus SCR, p=0.71; RAD23A versus SCR, p=0.11, respectively Fig. 1B). In comparison with no ASO animals, the SCR ASO led to an ~50% increase in RAD23B mRNA (p=0.01). The ASO targeting RAD23A mRNA led to an ~80% KD of brain RAD23A protein (RAD23A versus SCR, p<0.0001, Fig. 1C, D) and no effect on RAD23B protein level (RAD23A versus SCR, p=0.68) (Fig. 1C, D). We next turned to the TAR4/4 mouse that expresses WT human TDP-43 (hTDP-43) under the control of the Thy-1 promoter27. These animals express hTDP-43 ~2 time greater than the endogenous mouse TDP-43 (mTDP-43) and develop many histological and biochemical features seen at autopsy of individuals with ALS or FTD. Hereafter, we refer to TAR4/4 mice with KD of RAD23A owing to ASOs as TAR4/4;RAD23A KD and control mice receiving the scrambled sequence ASO as TAR4/4;RAD23A SCR. In comparison with the TAR4/4;RAD23A SCR mice, the TAR4/4;RAD23A KD males and females had a ~9 day extension in lifespan (~50% increase in lifespan, hazard ratio (HR) 10.15, p<0.0001) (Fig. 1E). The effects in males were a ~7 day increase in lifespan (HR=7.12, p<0.0001) and females a ~10 day increase in lifespan (HR=29.12, p<0.0001) (Supplementary Fig. 1A, B). To validate these findings, we ran a second independent cohort of TAR4/4 mice and found an identical pro-survival effect of knockdown of RAD23A in both female and male mice (Supplementary Fig. 1C–E). The TAR4/4 animals develop several abnormal motor phenotypes that worsen over time, and KD of RAD23A slowed the development of the gait impairment, tremor and kyphosis phenotypes and hindlimb clasping (Fig. 1F–I and Supplementary Fig. 1F).

We wondered if the complete elimination of RAD23A would have an even more robust pro-survival effect on the TAR4/4 mouse. We generated mice with a RAD23A null allele using CRISPR/Cas9; RAD23A-/- have no detectable RAD23A protein while RAD23A+/- animals have an ~40% reduction in RAD23A protein (WT versus RAD23A+/-, p<0.0001) (Fig. 1J, K). In comparison with WT animals, there was a ~10% and ~20% increase in RAD23B protein in the RAD23A+/- and RAD23A-/- mice, respectively (RAD23a+/- versus RAD23a+/+, p=0.06 and RAD23a-/- versus RAD23a+/+, p=0.017 Fig. 1K). TAR4/4;RAD23A+/- display an ~5 days increase in lifespan (compared with TAR4/4;RAD23A+/+ HR=9.49, p<0.0001) (Fig. 1L). To our surprise, there was no lifespan extension in the TAR4/4;RAD23A-/- mice (p=0.26). This observation may indicate that either: (1) germline loss of RAD23A evokes compensatory changes in the proteostasis network that mitigate the effects of postnatal reduction of RAD23A, or (2) only a low level of RAD23A is needed to suppress TAR4/4 phenotypes. Future study of a conditional KO allele of RAD23A could resolve this issue.

We wondered if reducing RAD23B would have similar survival benefits to RAD23A knockdown. We developed an ASO targeting RAD23B and administration of 60ug ICV, led to a ~85% reduction in the rad23b mRNA (in comparison with untreated or SCR ASO treated animals, p<0.0001) and no significant changes in the abundance of the rad23a mRNA (p=0.10) (Supplemental Fig. 2A). This dosing regimen reduced RAD23B protein abundance ~80% (SCR versus RAD23B, p=0.0001) and had no effect on RAD23A protein (Supplemental Fig. 2C, D). In comparison with the SCR ASO delivered ICV at identical concentration, KD of RAD23B led to an ~12 day shortening of lifespan (HR=13.55, p<0.0001) (Supplemental Fig. 2E). We entertained the possibility that excessive reduction of RAD23B might be adverse, and thus we studied a lower dosage of ASO (15ug). Administration of the lower ASO dose targeting RAD23B led to a ~65% reduction in rad23b mRNA (in comparison with untreated or SCR ASO-treated animals, p<0.0001) and no significant changes in the rad23a mRNA (p=0.40) (Supplemental Fig. 2B). This dosing regimen reduced RAD23B protein abundance ~65% (SCR versus RAD23B, p=0.0001) and had no effect on RAD23A protein (SCR versus RAD23A, p=0.30) (Supplemental Fig. 2C, D). In comparison with the SCR ASO delivered ICV at an identical concentration, less KD of RAD23B led to an ~5 days shortening of lifespan (HR=13.55, p<0.0001) (Supplemental Fig. 2E). These results suggest TAR4/4 phenotypes are exacerbated by reduction RAD23B protein abundance in a dose-dependent manner.

Since reducing RAD23A extends the lifespan of TAR4/4 mice, we wondered whether this also mitigated immunohistological (IHC) pathologies displayed by these mice. Our histopathological studies were quantitatively aligned with prior work27 (Fig. 2A). We saw a ~30% reduction in NeuN (+) neurons in the motor cortex of TAR4/4;RAD23A SCR in comparison with TAR4/4;RAD23A KD mice (p=0.0035). We saw a ~275% increase in GFAP (+) cells in motor cortex of TAR4/4;RAD23A SCR in comparison with TAR4/4;RAD23A KD mice (p=0.0008). Finally we saw a ~40% increase in Iba1 (+) cells in motor cortex of TAR4/4;RAD23A SCR in comparison with TAR4/4;RAD23A KD mice (p=0.0001). KD of rad23a had no effect on NeuN, GFAP or Iba1 cells in the WT animals. (Fig. 2A). These histological effects were associated with an increase in the GFAP transcript in the TAR4/4;RAD23A SCR mice in comparison with WT animals that is reduced in the TAR4/4;RAD23A KD mice (Fig. 2B). Microglial activation by human TPD-43 was seen by increased expression of the microglial markers IBA1 and CD68 (Fig. 2C, D) although rad23a KD did not have a statistically significant impact. Furthermore, we found cleaved caspase 3 in the cerebral cortex of TAR4/4;RAD23A SCR mice, but not in WT mice, and this was reduced by ~50% with RAD23A KD (p=0.0007) (Fig. 2E–G). Together, these observations reveal neuron death along with astro- and microgliosis in the cerebral cortex of the TAR4/4 mice and these pathologies are blunted by KD of RAD23A.

Transcriptomic effects of human TDP-43 are RAD23A-dependent
Normally, TDP-43 is observed almost exclusively in the nucleus, and pathologic depletion of nuclear TDP-43 causes RNA processing LOF phenotypes. Since mislocalization of TDP-43 has been reported in the TAR4/4 mice27 we wondered if this affected RNA biology and a potential relationship to rad23a. To this end we undertook a transcriptomic interrogation of four experimental groups of animals: (1) Group I - TAR4/4;RAD23A SCR versus WT;RAD23A SCR revealed 5979 differentially expressed genes (DEGs; 3770 up, 2209 down) representing the “disease genes” (Supplementary Data 1), and (2) Group II - TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR revealed 2177 DEGs (531 up, 1646 down) representing “treatment genes” (Supplemental Data 2 and Fig. 3A, B). Rad23a displayed the lowest adjusted P-value (3.99E-48) and lowest log2 fold-change −1.6 in the Group 2 DEGs comparison, confirming its successful knockdown (Fig. 3B). The abundance of mouse tardbp transcript was reduced ~15% in the TAR4/4 compared with WT mice, a predictable consequence of TDP-43 autoregulation in keeping with an observed increase in Tardbp intron-7 exclusion, but there was no independent effect of RAD23A KD on Tardbp expression (Supplemental Fig. 3).

Intriguingly, 1468 DEGs showed reversible expression patterns: genes upregulated in TAR4/4;RAD23A SCR were downregulated in TAR4/4;RAD23A KD, and genes that were down-regulated in TAR4/4;RAD23A SCR were upregulated in TAR4/4;RAD23A KD (Supplemental Data 3). This provides evidence that RAD23A KD significantly blunts the toxic effects of hTDP43 on the transcriptome. This is well illustrated by the principal component analysis and unsupervised hierarchical clustering heatmap. These analysis reveal that experimental groups are distinct both in terms of up and down-regulated transcripts (Fig. 3C, D, Supplemental Figure 4) and reversable in a RAD23A-dependent manner. We assessed changes in the abundance of 15 transcripts by qPCR and validated the RNAseq results through this independent method (Supplemental Data 4). These observations indicate that: (1) the ASO targeting RAD23A was potent, (2) expression of human TDP-43 in the TAR4/4 mouse led to autoregulation of the mouse tardbp transcript independent of RAD23A KD, and (3) expression of human TDP-43 in mice has profound transcriptional effects in a RAD23A-dependent manner.

We next conducted gene ontology analysis and identified 25 GO terms exhibiting reversed enrichment patterns in that they were downregulated in TAR4/4;RAD23A SCR in comparison to WT;RAD23A SCR, and upon Rad23A KD, they were upregulated (Fig. 3E) (Supplementary Data 5). These GO terms were associated with neural function, neural development, mitochondrial function, and mitochondria biogenesis. Aggrephagy (but not sumoylation) was also downregulated in TAR4/4;RAD23a SCR (in comparison with WT;RAD23A SCR) and upregulated in TAR4/4;RAD23a KD mice. This reaction might be predicted to promote favorable conditions for the clearance of protein aggregates.

Insolubility and aggregation of TDP-43 is RAD23A-dependent
GOF phenotypes evoked by TDP-43 are dose-dependent27, and so we wondered whether the beneficial effects of RAD23A KD influenced TDP-43 abundance. There was ~45% reduction of hTDP-43 expression in the TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR brain (p=0.0014) as well an ~35% reduction of total (mouse plus human TDP-43, m+hTDP-43) expression in the brain (p=0.0026) (Fig. 4A–C). We did not detect a difference in the abundance of the hTDP-43 transcript in TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR mice (cortex or spinal cord, p=0.14 and 0.69, respectively) (Fig. 4D). To examine the possibility that alterations in TDP-43 abundance are compartment-specific, we generated biochemical cytoplasmic and nuclear (C/N) fractions from individual animals, immunoblotted for hTDP-43 (and compartment markers) and determined the ratio of C/N TDP43 on a per animal basis. In comparison with SCR ASO treatment, knockdown of rad23a led to a ~50% decrease in the C/N ratio of hTDP-43 (p=0.02) (Fig. 4E). The observed RAD23A KD-dependent decrease in C/N TDP-43 might be part of the mechanism for remediation of the transcriptomic derangement observed in the TAR4/4 brain (Fig. 3).

In the brains of patients with ALS/FTD, a minor fraction of TDP-43 misfolds, oligomerizes and aggregates into cytotoxic species that are enriched in a sarkosyl detergent-insoluble fraction28. We found hTDP-43 in both the sarkosyl-soluble and -insoluble fractions of the TAR4/4 mouse brain. KD of RAD23A led to an ~50% reduction of sarkosyl-soluble and an ~65% reduction of sarkosyl-insoluble hTDP-43 (p<0.0001 and p<0.0001, respectively, Fig. 4F, H, H i ii). Total TDP-43 (m+h) shows similar effects; KD of RAD23A led to an ~55% reduction of sarkosyl-soluble and an ~60% reduction of sarkosyl-insoluble m+hTDP-43 (SCR versus RAD23A ASO p<0.0001 and p<0.0001, respectively) (Fig. 4F, I, I i ii). The C-terminal 25 kDa fragment of TDP-43 is a toxic species in humans and is found in the TAR4/4 mice. KD of RAD23A reduced the abundance of the full length TDP-43 by ~40% (TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR, p<0.0001) and the 25 kDa fragment by ~60% (TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR, p=0.0006) (Fig. 4G, J, J i ii). These results suggest that the benefits of RAD23A KD in the TAR4/4 mice are associated with reduced accumulation of toxic forms of hTDP-43.

In a complementary approach for assessing TDP-43 pathology2,28–31, we performed IHC for TDP-43 and phosphoTDP-43 on WT and TAR4/4 brains with and without RAD23A KD. For these studies we used a mouse monoclonal antibody that exclusively recognizes human TDP-43 and a rabbit monoclonal antibody that recognizes phosphorylated serine 409 and 410 of human TDP-4331- this allowed for simultaneous visualization of TDP-43 and phosphoTDP-43 (Supplemental Fig. 5A). In TAR4/4 brains, TDP-43 inhomogeneously filled the nucleus and also condensed into discrete puncta; the nuclear TDP-43 (+) puncta (1–5 puncta/nucleus) were seen in ~50% of cells. In addition, cytoplasmic TDP-43(+) puncta were detectable. TDP-43 IHC signal was neither seen in the brains of WT animals using this anti-TDP-43 antibody nor in no-primary-antibody control staining of TAR4/4 or WT tissue. Cytoplasmic punctate phosphoTDP-43 staining that co-localized with TDP-43 puncta was present in the TAR4/4 (but not WT or no primary antibody controls) brain. Inhomogeneous nuclear phosphoTDP-43 staining was seen and considered non-specific. In comparison with SCR ASO-treated TAR4/4 mice, the brains of mice treated with the ASO targeting rad23a demonstrated an ~75% reduction of number of cells with TDP-43(+) puncta (4.3±0.74 v. 0.95±0.38, p<0.0001) without a change in the number of puncta/nucleus (2.0±0.15 v 2.3±0.32, p=0.36). In addition, we saw a ~50% reduction in the number of cytoplasmic TDP-43(+):phosphoTDP-43(+) puncta in the TAR4/4 brains when RAD23A was KD (28.8±1.8 v. 14.0±1.4, p<0.0001, (Supplemental Fig. 5B–D). Both the biochemical (Fig. 4E) and IHC observations (Supplemental Fig. 5) support the view that RAD23A KD reduces non-nuclear TDP-43 in the TAR4/4 mouse brain. Since these different approaches report technique-specific outputs, a direct and quantitative comparison should not be drawn.

TDP-43 corruption of proteostasis is RAD23A-dependent
RAD23A has two ubiquitin-associated (UBA) domains and a ubiquitin-like (UBL) domain. The UBA domains act as ubiquitin receptors, binding ubiquitinated clients and shuttling them to the proteasome for degradation through the association of the UBL domain with the proteasome. Since the characteristic signature of pathological TDP-43 includes ubiquitinated aggregates it is possible the presence or absence of RAD23A protein influences the ubiquitome and general proteasomal function. We found a more prominent IHC signal for ubiquitin in the cerebral cortex of TAR4/4 in comparison to WT animals, in both the nucleus and proximal dendrites (Supplemental Fig. 6). By immunoblot, total ubiquitin levels was ~8 times higher in the TAR4/4;RAD23A SCR versus WT;RAD23A SCR brain lysates in both the soluble as well as insoluble fractions (p<0.0001 and p=0.0012, respectively). KD of RAD23A reduced total ubiquitin levels ~60% and ~45% in the insoluble and soluble fractions (RAD23A targeting ASO versus SCR, p =0.0012 and p<0.0001, respectively) (Fig. 5A–C). Elevated levels of total ubiquitin in the TAR4/4 mice might indicate that proteasomal degradation of ubiquitinated clients was impaired in a RAD23A-dependent manner.

We developed a reductionist assay to investigate the effects of aggregates on proteasome function. We monitored degradation kinetics of the fluorescent LLVY-AMC proteasome substrate by affinity-purified proteasomes in the presence of soluble and insoluble fractions of cell lysates expressing hTDP-43 (Fig. 5D, E). We used cell lysates from HEK293 cells engineered to inducibly-express hemagglutanin (HA)-tagged mutant TDP-43 (Q331K) as these cells develop sarkosyl-insoluble TDP-43. After induction of HA-mutTDP-43, sarkosyl-soluble and -insoluble fractions were generated, normalized to TDP-43 levels and added to proteasomes along with LLVY-AMC. MG-132-sensitive proteasomal activity was seen in this assay and addition of the sarkosyl-soluble fraction of mutTDP-43 expressing cells led to a modest (not statistically significant) decrease in proteasome activity. In contrast, the sarkosyl-insoluble fraction of mutTDP-43 expressing cells inhibited proteasomal protease activity at all time points and was ~60% lower at the one hour time point (p=0.0008, Fig. 5D, E, statistical analysis in Supplemental Data 6). Immunoblots of purified proteasomes reveal a robust signal for proteosome components PSMD4, PSMA4, PSMB6, and, interestingly, RAD23A (Fig. 5F). Next, we assayed proteosomal protease activity of TAR4/4 brain lysates using three different fluorescent substrates; LLVY-AMC and two longer peptides (e.g., RPPGFSAFK and KKVAPYPME) that are cleaved only by 26S proteasomes and report “trypsin-like” and “caspase-like” activities, respectively32. TAR4/4 brain lysate degradation of LLVY-AMC was inhibited by MG132 and KKVAPYPME by increasing salt concentrations (which disrupt the interaction between the 20S catalytic particle from the 19S regulatory particle), thus ensuring that these substrates report ubiquitin-dependent proteasomal activity (Supplemental Figure 7). TAR4/4 lysates displayed reduced levels of proteolysis of all three substrates (TAR4/4;RAD23A SCR versus WT;RAD23A SCR ~30%, ~15%, 25%, p<0.0001, Figure 5G i–iii). These observations support the view that hTDP-43 evoked a sarkosyl-insoluble cellular fraction that inhibits the 26S proteasome (Fig. 5A–C).

TDP-43 inhibition of the proteasome is RAD23A-dependent
Previously, it had been shown that neurodegenerative disease-causing proteins can lead to inhibition of proteasome function33–39. Such proteins include tau40, PrPSc34 and the glycine-alanine dipeptide repeat protein39. It had been proposed that a physical interaction (either direct or indirect) of disease proteins with the proteasome was the basis of activity inhibition. Since RAD23A binds both ubiquitinated substrates and the proteasome directly, we wondered whether RAD23A tethers ubiquitinated aggregates to the proteasome in a manner that inhibits proteasome function. This model predicts that the proteasome would be found in the insoluble fraction of TAR4/4 brain lysates in a RAD23A-dependent manner. When we probed lysates from the TAR4/4 mouse (± RAD23A KD) for components of the proteasome, we detected PSMA3 (20S subunit alpha 3), PSMC2 (19S subunit, ATPase 3) and PSMC5 (19S subunit, ATPase 5) that were more abundant in the sarkosyl-insoluble, but not soluble, fraction (Fig. 5H–K); ~270%, 170% and 190% respectively in the TAR4/4;RAD23A SCR versus WT;RAD23A SCR animals (p=0.0003, p<0.0001 and p<0.0001, respectively). Nuclear factor erythroid-derived 2-related factor 1 (Nrf1/NFE2L1) is the master regulator of proteasome subunit expression upon proteasome impairment41 and its abundance was ~10x higher in the brain TAR4/4;RAD23A SCR versus WT;RAD23A SCR animals (p<0.001). Knockdown of rad23a reduced the partitioning of the three proteasome proteins into the insoluble fraction by 40%, 20% and 20% respectively (p=0.0067, p=0.0082 and p=0.0044, respectively) (Fig. 5I–K) and reduced Nrf1 levels ~50% (p<0.0001, Supplementary Fig. 8). Thus, our results indicate that proteasome subunits incorporate into sarkosyl-insoluble aggregates evoked by hTDP-43 in a RAD23A-dependent manner.

The reduced sequestration of proteasome components in the insoluble fraction in RAD23A KD brains support the notion that this treatment caused the production of more functional proteasomes in the soluble fraction of cell lysates. We assayed proteasomal proteolytic activity using fluorescent reporters and determined that the TAR4/4 lysates displayed reduced levels of proteolysis of the three reporters in comparison with WT brain lysates. KD of RAD23A restored proteolytic activity ~20%, ~10% and ~30% (TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR, p=0.0007, p=0.0035 and p<0.0001, respectively) (Fig. 5G i–iii). These observations suggest that the burden of non-degraded ubiquitinated proteins is high in the TAR4/4 brain for two, potentially distinct, reasons: (1) the sarkosyl-insoluble fraction of TDP-43 overexpressing cells directly inhibits the proteolytic activity of the proteasome, and (2) RAD23A promotes the partitioning of proteasomes into insoluble aggregates where they display impaired proteolytic function.

Since hTDP-43 has a predilection to misfold and aggregate, it might initiate a cascade of protein aggregation → proteasome sequestration → reduced proteasome function → more protein aggregation. The role of RAD23A in this vicious cycle might be to link TDP-43 with proteasomes. If this was true, a physical association of TDP-43 and RAD23A should be demonstrable, and we studied this in several ways. First, immunocytochemical staining of in vitro neurons overexpressing LacZ or mutant TDP43 was performed. In LacZ-expressing neurons, endogenous mouse TDP-43 and RAD23A were homogenously distributed in the nucleus without apparent co-localization. In mutant A315T TDP43-expressing neurons, heterologous TDP-43 localized into nuclear puncta while endogenous RAD23A re-distributes into cytoplasmic puncta. No co-localization was apparent (Supplementary Fig. 9A). Second, in a split luciferase complementation assay (NanoLuc Binary Technology (NanoBiT) protein-protein interaction test) we found evidence for TDP-43 homodimer but no evidence for TDP-43/RAD23A heterodimer formation (Supplementary Fig. 9B–D). Third, we heterologously expressed epitope-tagged proteins (e.g., HA-Ubiquitin, FLAG-TDP-43 and V5-RAD23A), treated with proteasome inhibitor MG132 or vehicle and immunoprecipitated (IPed) with anti-epitope tag or irrelevant IgG beads (Fig. 5L). IP of TDP-43 pulled down TDP-43 and a high molecular weight smear of ubiquitin only after MG132 (+) but not RAD23A. In the reciprocal experiment, IP of RAD23A pulled down RAD23A, and in long exposure imaging, there is a faint band for TDP-43 in the MG132 (+) lysates. Finally, IP of ubiquitin pulled down a smear of high molecular weight ubiquitinated proteins as well as probable monoubiquitinated TDP-43, but only in the MG132 (+) lysates. Probing for RAD23A reveals a faint band in the MG132 lysates consistent with RAD23A binding some ubiquitinated proteins (as expected) or ubiquitinated RAD23A itself. These biochemical studies indicated that a minor fraction of overexpressed TDP-43 physically associates with RAD23A upon small molecule inhibition of the proteasome. In sum, the data argue against a physical link of RAD23A to ubiquitinated TDP-43.

RAD23A sculpts the TDP-43-evoked insoluble proteome
We posit that KD of RAD23A leads to a qualitative alteration in the composition of the sarkosyl-insoluble fraction of cell lysates evoked by TDP-43. This would be consistent with prior mass spectrometry interrogation of the insoluble proteome in NDD which have shown enrichment of protein homeostasis machinery (i.e., proteasome subunits37,42–44, components of aggresomes37,42 and autophagy37,45) and nuclear complexes/splicing37,42,43,45. To test this idea in our system, we used stable heavy isotope (15N)-based quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics to compare the insoluble proteome of TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR brain lysates (Fig. 6A). We quantified 6,343 total proteins and the majority were not elevated in one experimental group. 304 proteins were differentially modulated; 146 in the SCR-treated brains and 158 in the RAD23A KD brains (at ≥1.5 fold change and p-value <0.05). Representative MS1 spectra are shown in Fig. 6B. A t-distributed stochastic neighbor embedding (tSNE) plot showed that the two experimental groups were distinct, indicating that differentially enriched proteins were consistent among experimental groups (Fig. 6C). A volcano plot illustrates differentially insoluble proteins as a function of abundance and statistical significance (Fig. 6D, E, Supplementary Data 7). Interestingly, ASO-RAD23A treatment led several RAD23A binding proteins, such as Basp1 and Hspa8 to accumulate in the insoluble fraction. Consistent with our immunoblot data, TDP-43 was more enriched in the SCR ASO-treated versus RAD23A ASO-treated samples. Similarly, multiple examples of proteasome components and interacting proteins (i.e., PSMD2, HSPA2, SNAP25, DNAJ, ENO1, etc.)46,47 were elevated in the SCR ASO-treated versus RAD23A-treated samples, consistent with RAD23A-dependent sequestration of proteasomes as described above (Fig. 5G–I). The composition of the insoluble proteome was notably distinct from the differentially expressed transcripts (Supplementary Fig. 10), indicating that partitioning of proteins into aggregates did not lead to alterations in cognate transcript abundance. More broadly, these results are evidence that RAD23A governs the molecular composition of the insoluble proteome evoked by expression of TDP-43.

A GSEA of the proteins with significantly different levels in the insoluble proteome ± RAD23A was performed (Fig. 6F, Supplemental Data 7). A prominent set of proteins that were higher in TAR4/4;RAD23A SCR versus TAR4/4;RAD23A KD were cytoskeletal and motor proteins (including septins 4, 5, 7, 8, actin, cortactin, dynamin 1, 2, dynactin 4 and Kinesin 2a). These proteins are part of functional protein association networks as revealed in GeneMania protein-protein-interaction (PPI) network analysis (Fig. 6G). This observation was consistent with the known cytoskeletal abnormalities as cardinal features of NDDs and likely of pathophysiological significance. The prominent set of differential proteins in the TAR4/4;RAD23A KD versus TAR4/4;RAD23A SCR were also ribosomal (including Rpl 1, 3-8, 10-15), and the PPI network analysis revealed dense physical interactions (Fig. 6H). If this enrichment coincided with a general reduction in protein synthesis, this might be beneficial by better aligning nascent protein synthesis with protein folding capacity. RAD23A influenced the composition of the insoluble proteome of evoked by hTDP-43, and this likely contributes to the beneficial actions of reducing RAD23A levels.

Methods
Mouse crosses
All procedures related to the animal study performed here were approved by the Northwestern University Feinberg School of Medicine Institutional Animal Use and Care Committee. All experiments were conducted in accordance with the USDA Animal Welfare Act and PHS Policy on Humane Care and Use of Laboratory Animals (IACUC protocol IS00017615). Up to 5 animals per cage were housed together under a 12 hr light/dark cycle. TAR4 mice (TDP-43 Tg/+) were purchased from Jackson Laboratory (Stock #012836, B6;SJL-Tg(Thy1-TARDBP)4Singh/J). They were maintained on a B5/SJL hybrid background breeding with B6/SJLF1/J(JAX Stock #100012). Genotyping was performed as published27 and as described in the JAX protocol. RAD23A knockout mice were generated in the R.G.K. lab, described in detail below. They were back crossed in the C57Bl6 background > 8 generations. The line was maintained by mating RAD23A+/- with non-littermate C57Bl6 mice. Experimental animals for the ASO studies were generated by intercrossing non-sibling TDP-43 Tg/+ with genotype establish post hoc. For the studies of genetic manipulation of RAD23A levels, the Rad23+/- mice were bred to TDP-43 Tg/+ to generate mice with genotypes RAD23A+/+; TDP-43 Tg/Tg, RAD23A+/-;TDP-43 Tg/Tg and RAD23A-/-;TDP-43 Tg/Tg. We predicted an effect size, if it existed, would mimic that seen by Becker et al.26 and therefore, instead of a power analysis, with simply estimated that ~20 animals per genotype would be needed in the lifespan, gait interrogations and the analysis of other phenotypes. The ASO survival study was performed on two independently generated cohorts of animals created more than one year apart.

Generation of the RAD23A knockout allele
RAD23A KO mice (genotype as RAD23A-/-) were generated using CRISPR/CAS9 targeting nucleotides in NC_000074.7, exon 7 to create a stop codon. The nucleotides flanking the edit were “cagcaggggagaaccccctg(GA/C)gttcctgcgggaccagcctc” with GA edited to C. Genotyping was performed with primers 5’-CTGAACATGGTTCTGTCCAG-3’ and 5’-CCCCTAAGGAACTGGGAAG-3’ to amplify a 339 bp PCR product, which was then digested with BstN1 to release a 268bp fragment specific for RAD23A KO. A Western blot was also performed to confirm the loss of RAD23A at the protein level in transgenic mice.

ASOs validation and administration
ASOs that selectively target either mouse Rad23a or Rad23b were designed, synthesized and screened by Ionis Pharmaceuticals. Synthesis and purification of all chemically modified oligonucleotides were performed as previously described57. The ASOs are 20 nucleotides in length, in which the central gap segment has ten 2′-deoxyribonucleotides that are flanked on either side by five 2’ MOE modified nucleotides. Internucleotide linkages are phosphorothioate interspersed with phosphodiester, and all cytosine residues are 5′-methylcytosines. The following ASOs with high efficiency and well tolerance were selected for this study—RAD23A, CCTTGCTTATTATACCAAAC; and RAD23B, ACATATTATTTCAGACGCAC. One ASO with a scramble sequence (SCR), CCTATAGGACTATCCAGGAA, was included as a negative control.

Postnatal day zero (P0) neonates were used for ICV injection with 15-60μg ASOs in 3μl sterile phosphate-buffered saline PBS as described. The most effective and well-tolerated dose of each ASO was determined empirically. In brief, pups were transferred to an ice-cold metal plate to induce hypothermia anesthesia and the head was cleaned with a 70% ethanol cotton swab prior to injection. The injection site was identified as 2/5 of the distance from the lambda suture to each eye. A 32 Gauge Hamilton syringe was held perpendicular to the surface of the skull, and the needle was penetrated the skin and skull to a depth of ~3 mm. ASOs were slowly delivered into the lateral ventricle by depressing the plunger. The injection was conducted on both sides with 1.5μl administrated into each hemisphere. Before returning to the nest, the pup was transferred onto a warming pad until body temperature and color normalized, and the animal was moving freely. Half pups of the litter were randomly chosen for RAD23A or RAD23B ASO injection, while the other half received the scramble sequence ASO.

To obviate bias, ICV injections were performed with the operator blind to genotype and whether the ASO was SCR sequence or designed to target RAD23A or RAD23B. Three microliters of ASO were loaded in a coded manner into 8-strip PCR tubes by experimentalist 1. Experimentalist 2 then took the 8-strip PCR tube and loaded the syringe for injection of newborn mice. Each mouse received a tattoo to allow for linking of the animal to ASO injection and subsequent genotyping (Aramis Laboratory animal micro tattoo system, Ketchum #623). These animals were then used for behavioral phenotypes and lifespan studies. Upon euthanasia, a tail snip was obtained and PCR genotyping performed. The coordination of genotype with ASO treatment was not revealed until the data was analyzed.

The tissue samples for RNAseq analysis were obtained by experimentalist 1 and analyzed by a second experimentalist, blind to genotype and ASO. Once the RNAseq data was acquired and analyzed, the animal groups were revealed to experimentalist 2.

Care of the TAR4/4 (TDP-43 Tg/Tg) mice
Breeding pairs were feed on high-fat breeder chow (11.4% fat; Inotiv #7904). If TAR4/4 pups reached the humane endpoint before P21, they were removed euthanasia. If not, pups were weaned separately into new cages at P21 to avoid injury of the TAR4/4 mice by their littermates or parents. To ensure adequate nutrition to support the survival of compromised TAR4/4, wet food Diet Gel® 76A (w/ Wheat Protein) from ClearH2O was provided on the cage floor in addition to the moisturized chow and bottled water. Food was changed every day to keep fresh. This ensured that all mice were able of healthful survival before reaching the humane endpoint.

Phenotype scoring and humane euthanasia endpoint determination
Behavior and lifespan study were performed as described26. Behavioral features we studied include hindlimb clasping, gait and kyphosis. The assessment of animal behavior was performed blind to the animal genotype or treatment (ASO SCR versus targeting RAD23A or RAD23B). Behavioral assessments were made on animals placed on a warm, textured surface not in the cage setting. The gait impairment scoring system measures motor dysfunction, and as demonstrated by Becker et al. suitable for the study of young mice. Animals that walk normally without visible impairment were assigned a score of 0; animals walking with a slight tremor or limp were assigned a score of 1; animals walking with severe tremor, severe limp, lowered pelvis, or feet splayed away from the midline (“duck feet”) were assigned a score of 2; animals displaying troubled forward movement, minimal joint movement, and poor utilization to generate forward movement and stayed upright with difficulty (or dragged their abdomen on the examination surface) were assigned a score of 3; animals that were unable to maintain an upright posture and unable to right within 30sec on 3 trials was assigned a score of 4. Behavioral assessments were typically made every other day from 14 of life. Once a score of three was reached, the animals were assessed daily. Animals that reached the score of 4 had reached the humane pre-specified endpoint for the lifespan study and were euthanized.

We used the kyphosis and tremor scoring system developed by Becker et al.26. For kyphosis, animal without visible dorsal curvature of the spine and who are able to easily straighten its spine while walking were assigned a score of 0; Score 1 means a animals with mild kyphosis but still able to straighten its spine while walking were assigned score of 1; animals with a persistent but mild kyphosis and unable to completely straighten its spine most of the time were assigned a score of 2; animals with pronounced kyphosis at all times were assigned a score of 3. For tremor, animals with no detectable shaking were assigned a score of 0; Score 1, animals with very mild shaking or occasional shaking were assigned a score of 1; animals with severe but not continuous shaking were assigned a score of 2; shaking severely but not continuously; animals with continuous severe shaking were assigned a score of 3. Hindlimb clasping was assessed by the gently grasping the mouse near the tail base and lifted to observe the hindlimb position for 10 sec. Score 0 means both hindlimbs are consistently splayed out; Score 1 means one hindlimb is retracted toward the abdomen for half time of suspension or more while another hindlimb still splaying outward all the time; Score 2 means both hindlimbs are loosely retracted for half the time of suspension or more; Score 3 means both hindlimbs are closely or tightly retracted for half the time of suspension or more.

Mouse tissue collection
Deeply anesthetized mice were exsanguinated by cardiac laceration, and the central nervous system (brain and spinal cord) were rapidly dissected out of the corpse. For biochemical studies, the brain was regionally subdivided (e.g., cortex, hippocampus and remaining regions), frozen in dry ice and stored wrapped in tin foil with a coded designation. For transcriptomic and proteomic studies, one half of the brain was subjected western blot to confirm the efficacy of ASO treatment. For immunohistological studies, the brain was fixed by immersion in freshly prepared 4% paraformaldehyde in 0.1 M pH=7.4 phosphate buffer overnight and then washed extensively in phosphate-buffered saline (0.1M phosphate pH=7.4 buffer+0.9% sodium chloride) prior to further processing.

Total RNA extraction
The motor cortices of WT, TAR4/4 and Rad23 ASOs-treated mice were dissected from the left hemispheres of mice. The RNASeq library preparation and sequencing were carried out by the Genewiz facility (https://www.genewiz.com/en-GB/). Total RNA was extracted (Qiagen cat. No. 1023539), quantified (Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA) and RNA integrity was checked with TapeStation 4200. RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (NEB, Ipswich, MA, USA). Briefly, mRNA was enriched with Oligo(dT) beads and fragmented according to the manufacturer’s instructions before first-strand and second-strand cDNA synthesis. cDNA fragments were end-repaired, adenylated at 3’ ends, ligated with universal adapters, followed by indexing and enrichment by limited-cycle PCR. Libraries were validated using the NGS Kit on an Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), quantified (Qubit 4.0 Fluorometer), multiplexed and loaded on flow cells for sequencing (Illumina NovaSeq 6000) using a 2×150 paired-end configuration v1.5. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7. Raw sequence data (.bcl files) was converted into fastq files and demultiplexed (Illumina bcl2fastq program version 2.20). One mismatch was allowed for index sequence identification.

RNA-Seq data analysis
Low-quality reads (Phred<20) and adapters were removed using Trim-galore (V0.6.7) with its standard parameters (https://github.com/FelixKrueger/TrimGalore.com/fenderglass/Flye). Afterwards, reads were aligned to the GRCm38/mm10 build using RNA STAR Gapped-read mapper (V-2.7.11a+galaxy1 or V-2.7.8a+galaxy1)58. The alignment was carried out using standard parameters without transcript guidance (GTF) using the Galaxy platform59. To quantify expressed genes, we counted mapped reads on exons (GRCm38.99.gtf) using featureCounts (V 2.0.1+galaxy2)60. The differentially expressed genes between the two conditions were measured using DSeq261. Lowly expressed genes (reads <10) were filtered, and samples were normalized using the median of ratio method. A parametric fit was used to reduce the influence of false-positive outliers on fold change and p-value. All genes with an adjusted p-value (FDR) of less than 0.05 were considered to be differentially expressed. For the downstream analysis, read counts of all the samples was normalized by DESeq2 using the same median ratio method as discussed earlier. The rlog of normalized read counts was used to perform principal component analysis (PCA), gene set enrichment analysis (GSEA) and hierarchical clustering. GSEA was performed using GSEA_4.3.2, M2.all.v2023.2mm curated genes and normalized read counts62. All genes with normalized read counts >1 were included in the analysis. PCA, Volcano plot and heatmaps were generated using R-language (https://www.r-project.org/help.html.

The mouse and human Tardbp genes (exons -2, -3, and the coding region of exon 6) share over 90% identity. This means that RNASeq reads from human Tardbp also align to the mouse genome (data not shown). To prevent misestimation of mouse Tardbp expression from human TARDBP reads, we calculated mouse tardbp expression using reads aligned to the 3’ UTR of the mouse Tardbp. We first normalized the absolute read counts to counts per million and then estimated the relative tardbp expression. The exclusion of intron-7 (exitron) results in an unstable tardbp transcript that degrades rapidly. Thus, intron-7 exclusion serves as a readout for tardbp autoregulation.

TritonX-solubility
Protein was extracted from frozen tissues as indicated in the main text. Tissues were snap-frozen and were weighted, which was recorded as the initial weight. All subsequent volumes were based on this initial weight. For general western blotting, tissues were homogenized by 50 strokes in a Kontes with 10 Vol RIPA buffer supplemented with 1X protease inhibitor (Sigma) and 1X phosphatase inhibitor (Phospho-Stop, Roche). The homogenates were incubated on ice for 10 min and then centrifugated at 18,000×g for 20 min at 4°C. The supernatant was saved as TritonX-soluble fraction, and the pellet was washed with the extraction buffer and centrifugated again as above. The second round pellet was dissolved in 2 Vol urea buffer (30 mM Tris, pH 8.5, 4% CHAPS, 7m urea, 2m thiourea) as TritonX-insoluble fraction.

Immunoblotting
Approximately 30μg protein was heated at 85oC for 5 min with 1X SDS loading buffer. The samples were loaded onto 10% or 12.5% SDS-PAGE gel, depending on the size of the target proteins. After it, the protein was transferred overnight onto a nitrocellulose membrane. The membranes were then blocked, probed overnight at 4oC with the primary antibodies, washed 3 times in TBST, and incubated with appropriate secondary antibodies conjugated with Alex Fluor-680 or -800. All the antibodies used in this study was listed in Supplemental data 8. Imaging was realized with the Li-Cor Odyssey scanner, and protein quantification was performed in Image Studio Lite Version 5.2.

Cytoplasmic and nuclear fractionation
Nucleocytoplasmic fractionation was conducted as described26,27,63,64. Briefly, tissue was homogenized with Kontes in 10 vol/wt hypotonic buffer (10 mM Hepes, 10mm NaCl, 1mm KH PO , 5mm NaHCO , 5mm EDTA, 1m CaCl , 0.5mm MgCl with 1X protease inhibitor and 1 X phosphatase inhibitor). After homogenization, 0.5 vol/wt 2.5m sucrose was added into the lysate before a second round homogenization. The homogenate was centrifuged at 6300×g for 10 min. The supernatant was saved as the cytoplasmic fraction. The pellet was washed three times in pre-cold TSE buffer (10 mM Tris, pH 7.5, 300mm sucrose, 1mm EDTA, 0.1% NP-40) by resuspending and then centifuging at 4000×g for 5 min. The pellet was dissolved in 2% SDS buffer and was saved as the nuclear fraction.

HA-mutTDP43Q331K Flp-In T-Rex cell line
HA tagged human Q331K mutant TDP-43 was cloned in the pcDNA5/FRT/TO plasmid (Invitrogen™ V652020) and sequence verified. HEK293 Flp-In T-REx cells (ThermoFisher R78007) were cultured in DMEM (Gibco™ 11965092), 10% Tetracycline free FBS (Gibco™ A4736201), 1x penicillin-streptomycin (Gibco™ 15140122), 1X GlutaMAX™ (Gibco™ 35050061), 15μg/mL Blasticidin S (Gibco™ A1113903) and 100μg/mL zeocin (Gibco™ R25001). To create 65,66 a stable inducible cell line, 3×105 cells were plated per well of a 6-well plate and transfected when 70% confluent 48h later with 125 ng of pcDNA5/FRT/TO–HA-Q331K TDP-43, 1.125μg pOG44 (Invitrogen™ V600520), and 6.25μL Lipofectamine 2000 (Invitrogen™ 11668500) per well. After 48h, cells were re-plated in media without zeocin and with 150μg/mL hygromycin B (Gibco™ 10687010) for selection. Cells were selected for 3 weeks before re-plating for expansion and screening for successful integration via immunoblotting.

SarkoSpin
SarkoSpin was performed as described before56,67. Tissues were homogenized in homogenization solubilization (HS) buffer (10mm Tris, pH 7.5, 150mm NaCl, 0.1mM EDTA, 1mm DTT, 1X protease inhibitor (Roche) and Phosphatase inhibitor (Roche)). Samples were then diluted in 1 Vol of HS buffer with 4% Sarkosyl containing 2μ/μl benzonase and 4mm MgCl2. After incubating at 37oC for 45min under constant shaking at 600 rpm, brain homogenates were further diluted with 0.5 Vol HS buffer containing 0.5% Sarkosyl. Samples were then centrifugated at 21,000×g for 30min to separate Sarkosyl-soluble and -insoluble fractions. Supernatants were collected in a new tube and saved as the Sarkosyl-soluble fraction. Pellets were washed by resuspeneding in HS buffer with 2% Sarkosyl and spinning down again at 21,000×g for 30min to obtain the Sarkosyl-insoluble fraction, which were dissolved in 2% SDS for western blot analysis.

Immunohistochemistry
Immunohistochemistry was performed according to standard protocol68. Paraffin-embedded 6 µm-thick sections were deparaffinized in xylene and rehydrated in graded alcohol concentrations. Antigen retrieval was performed in 1% citrate buffer heat retrieval solution, pH 6.0 (Antigen Decloaker, Biocare Medical) using an electric pressure cooker at 121°C for 20min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20min. To reduce nonspecific signals, sections were blocked with 1% Bovine Serum Albumin (BSA) in 1x Phosphate-buffered saline (PBS). When staining for NeuN, GFAP, Iba1 or ubiquitin, sections were incubated with primary antibodies diluted in blocking buffer overnight at 4°C in a humidified chamber. Sections were incubated with peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) diluted in a blocking buffer for 2h at room temperature. 3,3′-Diaminobenzidine (DAB) solution was prepared by dissolving a DAB tablet (Sigma-Aldrich D4293) in 5 ml of deionized water containing 0.01% hydrogen peroxide. Sections were finally treated simultaneously with the DAB solution for 2-3min at room temperature. The following antibodies were used: 1:500 rabbit NeuN (Abcam, ab104225), 1:200 mouse Ubiquitin (Enzo, BML-PW0930), 1:200 rabbit Glial Fibrillary Acidic Protein (Dako, Z0334), 1:200 rabbit Iba1 (Wako, 019-19741). We used fluorescently tagged secondary antibodies for imaging TDP-43 and phospho-TDP43. The following antibodies were used: Mouse anti-TDP-43 (Abnova, 1:500), Rabbit anti-phospho-TDP-43 Ser 409/410 (DSHB, 1:500), anti-Rabbit Alexa Fluor 555, anti-mouse Alexa Fluor 647. Hoechst 33342 diluted 1:1000 from stock was included during secondary antibody incubation and coversliped with FluoroGel (Electron Microscopy Supplies).

To quantify brightfield immunohistologically labeled cells in tissues, a reticle was placed into one eyepiece (Klarmann Rulings, KR-427) and oriented such that a 3×15mm rectangle was perpendicular to the lamina of the motor cortex. Cell number in two non-contiguous rectangles was determined, and tissues were derived from 4+ independently treated animals. Masks were placed on the slides so that the cell counter was blind to genotype and ASO treatment.

To quantify fluorescently labeled immunohistology-labeled cells all images were obtained with the identical photomultiplier and laser intensity settings, and the data acquirer was blind to genotype and experimental manipulation. Tissue sections were imaged on a Leica DMI 4000B confocal microscope with a 63x lens. For Supplemental Fig. 4B, C, a standard rectangle was positioned over layers IV/V of the motor cortex such that ~10 Hoechst label nuclei were enclosed. Nuclei were scored for the presence or absence of punctata, and if found, the number/nucleus was noted. At least two non-contiguous rectangular areas were scored per tissue section, and at least two sections per experimental animal were interrogated. For Supplemental Fig. 4D, the number of TDP-43(+):phosphoTDP-43(+) double-positive puncta was determined per high-power field (hpf). At least two non-contiguous hpfs were scored per tissue section, and at least two sections per experimental animal were interrogated. Four to five animals were experimental group were studied.

Immunocytochemistry
Mixed spinal cord neuron cultures growing on an astrocyte monolayer were prepared as previously described69–72. At fourteen days in vitro, cultures were infected with recombinant herpes simplex viruses (HSVs) engineered to express beta-galactosidase or A315T mutant human TDP-43 (fabrication of viruses – NeuroTools, BRAIN Initiative Viral Vector Core, University of North Carolina), and two days later, the cultures were fixed with freshly prepared 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Neuron/astrocyte coverslips were blocked in phosphate buffered saline (PBS) containing 2% bovine serum albumin then incubated with primary antibodies (Altas Antibodies, rabbit polyclonal anti-human RAD23A, HPA026418, 1:250 and Abnova, mouse monoclonal anti-TDP-43, H00023435-M01, 1:400) overnight, washed with PBS, incubated with species-specific Alexa fluor conjugated secondary antibodies for 4h, washed again and mounted with Fluoro-Gel mounting media (Electron Microscopy Supplies). Coverslips were imaged on a Leica DMI 4000B confocal microscope with a 63x lens. Once the laser and photomultiplier settings were established for suitable imaging, they were applied without modification to all coverslips.

To create figures, the above images were moved in Photoshop 2025. Once images were assembled into a multi-panel presentation, sometimes minor changes in brightness or contrast adjustments were made for readability. When the images from two experimental conditions were presented, any adjustment in brightness or contrast was yoked. For example, if the green channel was adjusted, a single box was placed over the images from condition one and condition two; any adjustment made to brightness or contrast was thus made to two both images simultaneously. Images from experimental condition one were never adjusted without the co-adjustment of the images from condition two.

NanoLuc Binary Technology (NanoBiT) protein-protein interaction analysis
For NanoBiT analysis, human TDP-43 and human RAD23A were subcloned into pBiT2.1-TK-Small BiT (SmBiT) or pBiT1.1-TK-Large BiT (LgBiT) vector (Promega), with SmBiT/LgBiT tags positioned at the N- or C-termini. HEK293T cells were transfected with 4μg of total plasmid DNA (2μg each of SmBiT and LgBiT DNAs) in a 6-well culture plate using Lipofectamine 3000 (Thermo Fisher Scientific). At 24h post-transfection, the cells were transferred to 96-well, clear-bottom plates at a density of 1×105 cells per well and maintained for an additional 16h. The culture medium was then removed and replaced with fresh Opti-MEM, followed by the addition of 25μL of 1x Nano-Glo Live Cell Luciferase Reagent (Promega) diluted in LCS buffer (Promega) to each well. Luminescence measurements were performed using a GloMax Discover Microplate Reader (Promega) with an integration time of 1000ms.

RNA extraction and RT-qPCR
Motor cortex tissues were dissected in RNase-free conditions from freshly culled mice and flash frozen for gene transcription analysis73 here from different-treated mice. RT-qPCR was conducted as reported before74. Briefly, total RNA was extracted with the RNeasy® Plus Mini Kit (Qiagen #74134) following the manufacturer’s instructions. RNA integrity and quantity was determined by NanoDrop. Only those with an A260/A280 ratio above 1.95 were used for the following reverse transcription. The RNA was converted to cDNA using qScript cDNA SuperMix (Quantbio #95048-100). Real-time qPCR was performed on StepOnePlus Real-Time PCR System (Applied Biosystems™) with Power SYBR™ Green PCR Master Mix (Thermofisher #4368706) and primers as listed in Supplemental data 8. The amplification conditions comprised 50oC for 2m, 95oC for 10min, followed by 40 cycles of “95oC for 10s, 60oC for 1min”. Each reaction was performed in triplicate. GAPDH and β-actin were included as housekeeping genes, and their average Ct values were used for normalization. The relative transcript levels were calculated by the method of ΔΔCt.

Proteasome activity assay
For measuring proteasome activity32, brain cortex tissues were homogenized in proteasome lysis buffer (40 mM Tris, pH 7.5, 50 mM NaCl, 2mM β-mercaptoethanol, 2mM ATP, 5mM MgCl , 10% glycerol) to whole cell extracts. The lysates were briefly sonicated and then centrifugated at 21,000×g for 20min at 4oC. Protein concentration of the supernatants containing cellular proteasomes were measured with the BCA assay and was adjusted to 5mg/ml for hydrolysis assay as follows. Whole proteasome activity was assessed with various fluorogenic substrates (all from Enzo Life Sciences), in reaction buffer (50μM Tris-HCl, pH 7.5, 1mg/ml BSA, 1mM ATP and 1mM DTT) at 37oC for 30min, including 50μM Suc-LLVY-Amc 20μM Mca-RPPGFSAFK(Dnp) and 20μM Mca-KKVAPYPME-Dap(Dnp)–NH2. Fluorescence was measured using a BioTek Synergy II plate reader with the excitation and emission (Ex/Em) filters at 360/460nm for Amc and 320/405nm for Mca.

To evaluate the effect of insoluble TDP-43 on proteasome activity, a suc-LLVY-AMC hydrolysis assay was conducted for the indicated time periods using 10nM affinity-purified proteasomes75 with sarkosyl-soluble or -insoluble fractions from doxycycline (Dox)-inducible HEK293 cells. Cells were pre-treated with 2.5μg/mL Dox for 24h to induce TDP43-Q331K overexpression, and a modified SarkoSpin method was used to separate soluble and insoluble fractions of whole-cell lysates. The volumes of these fractions were adjusted so that the band intensities were equal in immunoblotting. Protein concentrations were determined using the Bradford method for soluble samples and gel-based TDP43 immunoblotting for insoluble samples. Similar experiments were performed using the inducible HEK293 cells after RAD23A was knocked down by siRNA.

MS sample preparation
We performed stable isotope labeling in mammals (SILAM) labeling as previously described76,77. Briefly, C57/BL6 mice were fed with a 15N-rich Spirulina-based food (MF-Spirulina N-IR; Cambridge Isotopes) for 12 weeks after weaning. The 15N protein enrichment was calculated based on the shape of the peptide isotope envelope78. In the “heavy” brains, at least 97% of proteins were highly enriched with 15N. The anterior half of the cerebrum that contains the motor cortex was homogenized in 500μL of fresh, ice cold 0.32M sucrose, 10mM HEPES+protease inhibitors using Precellys beads (4000rpmx15s, one cycle). BCA protein determination was performed and 200ug of homogenate was combined 1:1 with 200μg of 15N brain homogenate that was processed in parallel79 and the total volume was brought up to 400μL with homogenization solution. Forty μL of 10% SDS was added to each sample and equilibrated on a rotator at 4oC. for 30min prior to centrifugation at 100,000g for 60min. The supernatant was discarded and the protein pellets were resuspended in 8M urea (Thermo Fisher Scientific, Cat# 29700) prepared in 100mM ammonium bicarbonate solution (Fluka, Cat# 09830) and processed with ProteaseMAX (Promega, Cat# V2072) according to the manufacturer’s protocol. The samples were reduced with 5mM Tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich, Cat# C4706; vortexed for 1h at RT), alkylated in the dark with 10mM iodoacetamide (IAA, Sigma-Aldrich, Cat# I1149; 20min at RT), diluted with 100mM ABC, and quenched with 25mM TCEP. Samples were diluted with 100mM ammonium bicarbonate solution, and digested with Trypsin (1:50, Promega, Cat# V5280) for overnight incubation at 37°C with intensive agitation. The next day, the reaction was quenched by adding 1% trifluoroacetic acid (TFA, Fisher Scientific, O4902-100). The samples were desalted using Peptide Desalting Spin Columns (Thermo Fisher Scientific, Cat# 89882). All samples were vacuum centrifuged to dry.

Tandem Mass spectrometry
Three micrograms of each fraction or sample were auto-sampler loaded with an UltiMate 3000 HPLC pump onto a vented Acclaim Pepmap 100, 75μm x 2cm, nanoViper trap column coupled to a nanoViper analytical column (Thermo Fisher Scientific, Cat#: 164570, 3µm, 100Å, C18, 0.075mm, 500mm) with stainless steel emitter tip assembled on the Nanospray Flex Ion Source with a spray voltage of 2000 V. An Orbitrap Fusion (Thermo Fisher Scientific) was used to acquire all the MS spectral data. Buffer A contained 94.785% H O with 5% ACN and 0.125% FA, and buffer B contained 99.875% ACN with 0.125% FA. The chromatographic run was for 2h in total with the following profile: 2–8% for 6, 8–24% for 64, 24–36% for 20, 36–55% for 10, 55–95% for 10, 95% for 10 mins.

We used the CID-MS2 method for this experiment. Briefly, ion transfer tube temp=300°C, Easy-IC internal mass calibration, default charge state=2 and cycle time=3s. Detector type set to Orbitrap, with 60K resolution, with wide quad isolation, mass range=normal, scan range=300–1500 m/z, max injection time=50ms, AGC target=200,000, microscans=1, S-lens RF level=60, without source fragmentation, and datatype=positive and centroid. MIPS was set as on, included charge states=2–6 (reject unassigned). Dynamic exclusion enabled with n=1 for 30s and 45s exclusion duration at 10 ppm for high and low. Precursor selection decision=most intense, top 20, isolation window=1.6, scan range=auto normal, first mass=110, collision energy 30%, CID, Detector type=ion trap, OT resolution=30K, IT scan rate=rapid, max injection time=75ms, AGC target=10,000, Q=0.25, injections for all available parallelizable time.

MS data analysis and quantification
Protein identification/quantification and analysis were performed with Integrated Proteomics Pipeline-IP2 (Bruker, Madison, WI. http://www.integratedproteomics.com/) using ProLuCID80, DTASelect281, Census and Quantitative Analysis. Spectrum raw files were extracted into MS1, MS2 files using RawConverter (http://fields.scripps.edu/downloads.php). The tandem mass spectra (raw files from the same sample were searched together) were searched against UniProt mouse (downloaded on 07-29-2023) protein databases82 and matched to sequences using the ProLuCID/SEQUEST algorithm (ProLuCID version 3.1) with 50 ppm peptide mass tolerance for precursor ions and 600 ppm for fragment ions. The search space included all fully and half-tryptic peptide candidates within the mass tolerance window with no-miscleavage constraint, assembled, and filtered with DTASelect2 through IP2. To estimate protein probabilities and false-discovery rates (FDR) accurately, we used a target/decoy database containing the reversed sequences of all the proteins appended to the target database82. Each protein identified was required to have a minimum of one peptide of a minimal length of six amino acid residues. After the peptide/spectrum matches were filtered, we estimated that the peptide FDRs were ≤1% for each sample analysis. Resulting protein lists include subset proteins to allow for consideration of all possible protein isoforms implicated by at least three given peptides identified from the complex protein mixtures. Static modification: 57.02146 C for carbamidomethylation. Differential modification: 42.0106 for acetylation at N-terminal.

We quantified the SILAM data as described previously83. For MS1-based quantification, Census first calculates the elemental compositions and corresponding isotopic distributions for both the unlabeled and labeled peptides. All isotopes with greater than 5% of the calculated isotope cluster base peak abundance were used. MS1 files were used to generate chromatograms from the m/z range surrounding both the unlabeled and labeled precursor peptides. The core of Census is a linear least-squares correlation that is used to calculate the ratio (i.e., slope of the line) and closeness of fit (i.e., correlation coefficient [r]) between the data points of the unlabeled and labeled ion chromatograms. In this study, only peptide ratios with the coefficient correlation values (r2) greater than 0.5 were used for further analysis. The Grubbs test (p-value <0.01) was used to remove outlier peptides. Final protein ratios were calculated according to the peptide levels and generated with QuantCompare, which uses Log2-fold change on the biological replicates. ANOVA was performed on peptide levels to assess statistical significance.

Software
Spyder (MIT, Python 3.7) was used for MS data analyses. RStudio (version 1.2.1335) was used for MS data visualization. The Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) was utilized for protein functional annotation analysis. GeneMania (https://genemania.org/) was employed for generating protein-protein interaction networks. Cytoscape (version 3.9.1) was used for constructing and visualizing the networks.

Statistical analysis and reproducibility
We used Microsoft Office Excel and GraphPad Prism version 9.0 or 9.3.1 to analyze data and plot graphs. Data were expressed as mean ± standard error of the mean (SEM). For comparisons among 3 groups, one-way ANOVA with Tukey’s multiple comparison test was used, and for comparisons between 2 groups, two-tailed, unpaired Student t-tests were used. P≤0.05 was considered significant. With regard to animal survival studies, a power calculation is based on the prediction of effect size of 10 days longer survival (e.g., longer in the treated v. control ALS mouse) and a standard deviation in survival of 3 days. Using a two tailed t test with an alpha error probability of 0.05, power (1–β error probability) of 0.9, and an allocation ratio of 1, a sample size of 11 per group is needed (using G*Power 3.1). Since a smaller effect size or more variance is possible, we will make our sample size 15+ animals per group.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript “Reduction of RAD23A extends lifespan and mitigates pathology in TDP-43 mice” follows up on earlier studies showing that ablation of RAD23 in a C. elegans model and RAD23A in a rat neuron tissue culture model of TDP-43 proteinopathy leads to a suppression the disease phenotype. Here the authors show that reducing RAD23 protein levels in a mouse model of TDP-43 proteinopathy (TAR4/4) via injecting ASOs or genetically via crossbreeding with a RAD23A KO mouse increases the lifespan, reduces behavioral phenotypes, accumulation of insoluble TDP-43, and rescues neuronal loss. Further experiments demonstrate improved UPS function and correction of transcriptomic alterations, perhaps by reducing TDP-43 protein levels. Proteomics comparison of the insoluble protein fraction also suggests RAD23A-dependent changes to the insoluble proteome appears as a key driver of pathology in this model.\nThis is a very-well written and very interesting manuscript that addresses an important question in the field. Experiments are extensive, including the generation of RAD23A and B KO mouse models, and are generally well performed and presented, spanning different approaches and models. One weakness is the lack of truly physiological animal models, but this is a general problem in the field. This study provides some insight into a toxic gain of function phenotype, at least in this mouse model, and how it can be partially rescued. While not providing clarity for the mechanistic role of RAD23A in regulating protein degradation/aggregation/unfolding, it nevertheless provides important clues for further studies and nominates RAD23 ablation as a promising target for therapy development. Despite its strengths, there are several issues that should be addressed, as listed below.\nMajor comments:\nA major issue to be addressed is the lack of IHC to investigate TDP-43 neuropathological hallmarks in this mouse model and their modulation by RAD23A knock-down. Rescue is shown for behavior and neuronal loss, and biochemical studies, but it lacks confirmation on a neuropathological level by staining for TDP43 and pTDP-43 in mouse tissue. The manuscript also should provide IHC staining for Iba1 and CD68, instead of just mRNA levels. Some IHC images, including P62 and ubiquitin staining, are of rather modest quality, with high background and poor tissue preservation.\nThere is a critical lack of information about experimental details of the proteomics experiments. What was the source for heavy isotope-labeled 15N brain lysate? How was it generated? How were these SILAC-like data analyzed?\nMinor comments:\nFigures often contain vastly different font sizes from hardly readable to very large. Readability could be improved by making them more similar.\nWhile mRNA of rad23b does not go down after ASO administration, the protein levels do so dramatically (Fig 1B vs 1M). The authors should comment on it.\nJudging by the nucleo-cytoplasmic fractionation, nuclear TDP-43 also goes down. Immunofluorescence would help to see if there are fewer nuclear aggregates, with the possibility of even a functional rescue.\nIn the IP experiments, bands from IgG chains if present should be indicated (bands in V5 staining?)\n“In parallel motor cortex the 15N fully-labeled mouse was homogenized and protein content determined.” -> maybe revise to e.g. “In parallel the 15N fully-labeled mouse motor cortex was homogenized and protein content determined.”", "reviewer_2": "In this work, Guo et al describe that a modifier of TDP-43 pathology, Rad23, can improve the survival, behaviour, and the histological hallmarks of disease in the very aggressive TAR4/4 mouse model. Overall, the authors have generated interesting phenotypic effects in the mice that have been treated wither with ASOs against the two Rad23 isoforms but also following ablation of just Rad23 through CRISPR/cas9. The RNA sequencing analyses of these different groups has shown that many altered pathways (some already described to play a role in the pathology) can be recovered after Rad23 KD and this correlates well with the increase in lifespan. In general, this part of the work is very interesting although very descriptive.\nOne of the most interesting observations of the manuscript refers to the levels of TDP-43 expression in the brains of the TAR4/4;RAD23AKD versus TAR4/4;RAD23ASCR mice. In this respect, it would therefore be very interesting to know what happens to the autoregulatory mechanism of TDP-43 and whether Rad23 could be directly involved in these changes (or whether the mechanism through which Rad23 primarily exerts its protective effect occurs through the change in the nuclear/cytoplasmic ratio of hTDP-43).\nAnother issue that should be clarified regards the direct interaction between TDP-43 and RAD23. The authors confirm that this interaction should not really occur using flagged proteins and in vitro data- However, do the two protein immunolocalize in IHC studies of the brain or they also fail to do this in the mouse brains?. It could be that in vivo conditions some other cellular factor may act as a bridge between these two proteins.\nFinally, it would be interesting to know how the GO terms obtained from the proteomic analysis performed in Figure 6 compares with the transcriptomic analysis in Figure 3E. Is there any correlation between some of the terms in the two types of analyses?.", "reviewer_3": "Building on previous work in C. elegans and primary neurons from the same lab (Jablonski et al, J Neuroscience 2015), Guo et al tested the efficacy of ASOs targeting RAD23A and RAD23B in a very fast-progressing TDP-43 mouse model (TAR4/4). RAD23A shuttles ubiquitinated proteins to the proteasome for degradation and is also involved in DNA repair by regulating XPC at DNA lesions. Guo et al. show that Rad23a ASO improves survival and motor deficits, whereas Rad23b ASO shortens survival. In addition, genetic knockout of RAD23A showed some benefits in heterozygotes but not in homozygotes. There is no good explanation for the discrepancy between RAD23A ASO and KO and the mechanism of action of RAD23A ASO is not understood. Rad23a knockdown appears to alter TDP-43 solubility and the overall composition of the insoluble fraction in TDP-43 mouse models, but the data appear to be preliminary.\nWhile RAD23A ASOs have potential translational value and are worth pursuing further, the novel mechanistic insights from this manuscript are limited. For example, comparing RAD23A vs. RAD23B knockdown may help to find correlates of efficacy. It is also unclear why RAD23A overexpression appears to be beneficial for some protein aggregates, e.g. in C9orf72 models (PMID: 26998601).\nKey points:\n-The results read more like a talk than a scientific paper. However, the manuscript is difficult to read because it does not follow standard scientific writing conventions (e.g. tenses, too much discussion in the results section, missing subheadings). Figures are not numbered and especially the supplementary data are difficult to sort because the file names are not meaningful.\nFig 2: Add quantification of effects on neuronal loss and gliosis using histology. Neurofilament levels should be measured as an neurodegeneration biomakers.\nFigure 3: The whole figure looks like a first draft. It needs to be made more readable and appealing. What is \"NES\" in Figure 4E? Apparently thousands of transcripts are differentially expressed, but with small differences. 14312 DEGs make up the majority of the transcriptome. Especially for the ASO comparison in Fig. 3B, most of the genes have minimal LFC. A cutoff should be applied to focus on the most affected genes. Add gene names of highly regulated genes in volcano plots.\nFig. 4E: Explain the experimental approach in the main text and/or legend. Display gels and confirm effects on TDP-43 mislocalisation by immunofluorescence. This potentially important finding should be confirmed by a second method. I would expect the wild-type TDP-43 expressed in these mice to be predominantly nuclear. What's the difference between Figure 4E and Figure S7? Figure S7A looks like the nuclear and cytoplasmic fractions were quantified from different gels. This is not good practice. Rad23a ASO seems to cause a small shift in the TDP-43 band. Does this indicate decreased phosphorylation?\nFig. 4F/G Remove or label lane with ladder.\nFigure S8: Sort panels according to text. Are p62 puncta reduced in TAR4/4 mice after Rad23a knockdown? Include quantification\nFigure 5: Adding the insoluble fractions to other cells is an unusual approach. Overexpression of TDP-43 together with ras23a/b knockdown or overexpression in neurons may be more informative.\nFig. 6D: What are the most enriched proteins? The full dataset should be included in the supplementary material. TDP-43 is clearly not the most regulated protein by RAD23A ASO and is barely significant. What about known RAD23A interactors like ATXN3 and UBE3A? This whole figure also looks like a draft. Most of the text is too small to read."}, "author_response": "Thank you for the thoughtful reviews of our manuscript entitled “Reduction of RAD23A extends lifespan and mitigates pathology in TDP-43 mice”. We have been able to address all the issues raised by the three Reviewers. Below I will enumerate each Reviewer’s “remarks to authors” and how we have addressed these issues in the revision.\nREVIEWER COMMENTS – In bold, provided in full.\nReviewer #1 (Remarks to the Author):\n[...]\nThank you for your kind remarks.\nMajor comments:\n[...]\nIn the revised manuscript we now provide IHC staining of TDP-43 and phospho-TDP-43 and the effects of RAD23A knockdown; in addition the results are quantified (new Supplemental Figure 5). We also now provide IHC for Iba1 and the effects of RAD23A knockdown are quantified (modified Figure 2). We have provided a better bright field image of Ubiquitin staining of the WT and TAR4/4 brain (new Supplemental Figure 6).\nIn the original submission there was a single paragraph on the topic of p62 and a single supplemental figure. In ongoing studies (mostly in the pilot stages) we have determined that the topic of p62 in the TAR4/4 mouse is complex. This will require a more comprehensive set of experiments to more rigorously understand what’s going on. We feel the p62 story is beyond the scope of the current ms. With this consideration, we decided the story would not be adversely affected by removing the small amount of data on p62.\nThere is a critical lack of information about experimental details of the proteomics experiments. What was the source for heavy isotope-labeled 15N brain lysate? How was it generated? How were these SILAC-like data analyzed?\nThe reviewer is correct that important information regarding the performance and analysis of the proteomics studies was lacking. The revised manuscript was emended to include complete and accurate details as requested.\nMinor comments:\nFigures often contain vastly different font sizes from hardly readable to very large. Readability could be improved by making them more similar.\nCorrected.\nWhile mRNA of rad23b does not go down after ASO administration, the protein levels do so dramatically (Fig 1B vs 1M). The authors should comment on it.\nIn Figure 1, panel B we provide qPCR data on the abundance of the RAD23B mRNA after administration of the ASO targeting RAD23A. In Figure 1, panel M we provide western blot data on the abundance of the RAD23B protein after administration of the ASO targeting RAD23B. The apparently disparate results in these two panels of Figure 1 can be explained by the application of ASOs targeting different mRNAs (e.g. RAD23A versus RAD23B).\nThat said, in the revised manuscript we provide a more indepth analysis of the effects of the ASO targeting RAD23B. New data are presented on this topic and discussed in greater detail in the revised manuscript (new Supplemental Figure 2).\nJudging by the nucleo-cytoplasmic fractionation, nuclear TDP-43 also goes down. Immunofluorescence would help to see if there are fewer nuclear aggregates, with the possibility of even a functional rescue.\nWe now present both in vitro and in vivo immunofluorescent images of TDP-43 and the effects of RAD23A reduction (new Supplemental Figure 5 and 9).\nIn the IP experiments, bands from IgG chains if present should be indicated (bands in V5 staining?)\nDone.\n“In parallel motor cortex the 15N fully-labeled mouse was homogenized and protein content determined.” -> maybe revise to e.g. “In parallel the 15N fully-labeled mouse motor cortex was homogenized and protein content determined.”\nThe description of the 15N mouse work has been expanded to provide significantly more detail on the performance of these experiments.\nReviewer #2 (Remarks to the Author):\n[...]\nThank you for your kind remarks.\nOne of the most interesting observations of the manuscript refers to the levels of TDP-43 expression in the brains of the TAR4/4;RAD23AKD versus TAR4/4;RAD23ASCR mice. In this respect, it would therefore be very interesting to know what happens to the autoregulatory mechanism of TDP-43 and whether Rad23 could be directly involved in these changes (or whether the mechanism through which Rad23 primarily exerts its protective effect occurs through the change in the nuclear/cytoplasmic ratio of hTDP-43).\nThe TAR4 transgenic mouse was created by cloning a human cDNA encoding TARDBP after the murine Thy-1 promoter and in front of an SV40 polyadenylation sequence. The TARDBP cDNA had no introns and does not include the human 3’UTR and so autoregulation of the human transcript is unlikely (1 and Samir Kumar-Singh, personal communication). So while the reviewer’s question about RAD23A mechanism of action is interesting, the TAR4/4 mice (the basis of this report) are not a suitable substrate for such studies. We do provide experimental evidence in favor of the idea that RAD23A operates to influence the nuclear/cytoplasmic ratio of TDP-43 (revised Figure 4, panel E).\nIt is possible the Reviewer is wondering about what happens to the abundance of the mouse TDP-43 mRNA in the TAR4/4 mice. In other words, does expression of human TDP-43 influence the abundance of the mouse mRNA and protein and does KD of RAD23A (which reduces the abundance of human TDP-43 protein but not mRNA – Figure 4. Panels A and D) impact mouse TDP-43 mRNA? This we are equipped to answer and the manuscript now includes these data. We interrogated the RNAseq data for abundance of reads of the human v. mouse transcript. While the coding sequences are very similar, drilling down on intron7 and the 3’UTR enabled us to determine that, in fact, the abundance of the mouse tardbp transcript is modestly reduced (~15%) in the TAR4/4 mouse brain in comparison with WT mouse brains. In addition, we determine that knockdown of RAD23A in the TAR4/4 mouse brains has no effect on the abundance of the mouse transcript. These data are presented in the revised manuscript as well as a new Supplemental Figure 3.\nAnother issue that should be clarified regards the direct interaction between TDP-43 and RAD23. The authors confirm that this interaction should not really occur using flagged proteins and in vitro data- However, do the two protein immunolocalize in IHC studies of the brain or they also fail to do this in the mouse brains?. It could be that in vivo conditions some other cellular factor may act as a bridge between these two proteins.\nIn the original submission, we explored the possibility that TDP-43 and RAD23A are part of a complex using co-immunoprecipitation experiments. To investigate this issue in greater depth we now have undertaken two additional approaches to address the issue raised by the reviewer. First, we established conditions that permit us to visualize native RAD23A in neurons grown in vitro and combined this with expression of mutant TDP-43 using viral vectors. Unfortunately we were unable to find conditions to do with immunohistologically. The immunocytochemical study shows that the two proteins do not colocalize (new Supplemental Figure 9, panel A). Second we deployed a novel protein-protein interaction platform called NanoBiT. Luciferase is split into two parts (neither of which display luciferase activity) and the parts are cloned onto potential protein-protein binding partners of interest (N- or C- termini). If the two potential binding partners physically interact, it brings the two portions of split luciferase so close such that it reconstitutes luciferase activity.\nUsing this very sensitive assay, we find that the positive control provided in the kit restores luciferase activity. We find that engineering the two portions of luciferase onto TDP-43 also restores luciferase. This is consistent with the idea that TDP-43 can multimerize, which aligns with the literature. When we engineer RAD23A to contain the luciferase fragment and co-expressed with TDP-43/luciferase chimera, it does not reconstitute luciferase activity (new Supplemental Figure 9, panels B and C). Thus these orthogonal assays provide further evidence that RAD23A and TDP-43 are not part of a complex. While it is not possible to prove a negative result, the multiple approaches we have taken argue that under the conditions we have studied, little, if any, RAD23A and TDP-43 associate (directly or indirectly) in a physical complex.\nFinally, it would be interesting to know how the GO terms obtained from the proteomic analysis performed in Figure 6 compares with the transcriptomic analysis in Figure 3E. Is there any correlation between some of the terms in the two types of analyses?.\nThis is an interesting point and thus we interrogated our transcriptomic and proteomic dataset to see if such an overlap existed. We now report that there is no overlap. It is important to note that our proteomic analysis focused exclusively on the insoluble protein fraction and this may be why there is a disconnect between the mRNA and protein levels. We discuss this in the revised ms. and the analysis is now provided in the new Supplement Figure 10\nReviewer #3 (Remarks to the Author):\n[...]\nIn the revised manuscript we directly discuss the discrepancy between the RAD23A ASO and KO on TDP-43 phenotypes (as highlighted by the reviewer). In essence there are two main possibilities. First it is possible that germline loss of RAD23A evokes compensatory changes in the proteostasis network that mitigate the effects of postnatal reduction of RAD23A. Second, it is possible that a low (non-zero) level of RAD23A is needed to suppress TAR4/4 phenotypes and complete ablation is adverse. We are planning future studies to distinguish between these possibilities and thank the reviewer for emphasizing the issue.\nWith regard to the mechanism of action of RAD23A ASO, the investigators who participated in this work share the reviewers view that mechanism is an issue. We do show that proteasomes are sequestered into the insoluble fraction of lysates evoked by overexpression of TDP-43 and this occurs in a RAD23A-dependent manner. The evidence that sequestered proteasomes are functionally hypoactive comes from our demonstration that TDP-43-evoked aggregated directly inhibit proteosome function. We hypothesize that via sequestration and direct inhibition, aggregates evoked by TDP-43 disrupts proteostasis in the disease state (Figure 5 panels A – H). Ongoing work aims to identify that region of RAD23A that links proteasomes to aggregates and if that quest is successful, we would be positioned to develop peptides or small molecules that disrupt this interaction. Such an approach might bring more clarity to the mechanism of action of loss of RAD23A. We hope the reviewer will permit that such studies are beyond the scope of the present submission.\nWith regard to the Reviewer’s question of the overall composition of the insoluble fraction in TDP-43 mouse models we performed mass spectrometric interrogation of the fraction. This is presented in Figure 6 panels A – H with an accompanying analysis in Supplemental Data 6. We hope the reviewer finds these data a reasonable interrogation of the insoluble fraction of the TDP-43 mouse brain lysate.\nWhile RAD23A ASOs have potential translational value and are worth pursuing further, the novel mechanistic insights from this manuscript are limited. For example, comparing RAD23A vs. RAD23B knockdown may help to find correlates of efficacy. It is also unclear why RAD23A overexpression appears to be beneficial for some protein aggregates, e.g. in C9orf72 models (PMID: 26998601).\nWith regard to C9ORF72, the reviewer is referring to the 2016 paper from the Petrucelli lab showing that RAD23B overexpression is beneficial. Zhang et al. study mouse and tissue culture models of C9ORF72 disease in which the diamino acid peptide repeat protein, glycine-alanine is repeated 50 times (GA50). In their somatic transgenesis model (ICV injection at P0 of AAV-GA50), they find immunohistological cytoplasmic aggregates of polyGA some of which sequester RAD23A or RAD23B (which is denoted HR23A or HR23B, respectively). The same observation is made in HEK293 cells or primary neurons that overexpress GA50. The authors argue that sequestration of HR23A or HR23B results in loss of function because a known binding partner, xeroderma pigmentosum C (XPC), is reduced ~50% in abundance. Further the authors show that in primary neurons overexpressing GA50, with coincident overexpression of RAD23B, XPC levels are restored, caspase-3 activation is decreased and GA inclusions are reduced. This important work provides evidence that “… loss of HR23 protein function contributed to poly(GA) aggregation and toxicity…”. The authors did not study the effects of directly reducing HR23.\nThe differences between this work and what we report here are significant: 1) they study GA50 and we study TDP43, 2) they overexpress RAD23B and we study the effect of reduced RAD23A, 3) they monitor GA50 toxicity in vitro by western for XPC and cleaved caspase 3 and we monitor TDP-43 toxicity in vivo by studying survival, motor behavior, IHC counts of neurons, astrocytes, microglia and a plethora of biochemical assays including RNAseq and proteomics.\nIn the revised ms. we now discuss the work by Zhang et el. and how their findings are both compatible and complementary to what we report.\nKey points:\n-The results read more like a talk than a scientific paper. However, the manuscript is difficult to read because it does not follow standard scientific writing conventions (e.g. tenses, too much discussion in the results section, missing subheadings). Figures are not numbered and especially the supplementary data are difficult to sort because the file names are not meaningful.\nWe apologize for the presentation difficulties pointed out by the Reviewer and we have made pervasive changes in the document to make the work adhere to community standards.\nFig 2: Add quantification of effects on neuronal loss and gliosis using histology. Neurofilament levels should be measured as an neurodegeneration biomakers.\nWe include now a quantification of neuronal loss, gliosis and microgliosis in the revised Figure 2. Measurement of Neurofilament levels (presumably in plasma or CSF) is an interesting idea but unfortunately we do not have the capacity to undertake this determination. We will develop such an assay for future work, thank you for the suggestion.\nFigure 3: The whole figure looks like a first draft. It needs to be made more readable and appealing.\nDone.\nWhat is \"NES\" in Figure 4E? Apparently thousands of transcripts are differentially expressed, but with small differences. 14312 DEGs make up the majority of the transcriptome. Especially for the ASO comparison in Fig. 3B, most of the genes have minimal LFC. A cutoff should be applied to focus on the most affected genes. Add gene names of highly regulated genes in volcano plots.\nWe apologize for eliding the definition of NES. It “Normalized enrichment score” and this information is now included in the figure legend. We have modified the figure according to the wishes of the review (revised Figure 2, panels A and B). Additional emendations requested by the Reviewer have been made and discussed in the Results section, Methods and Figure legends.\nFig. 4E: Explain the experimental approach in the main text and/or legend. Display gels and confirm effects on TDP-43 mislocalisation by immunofluorescence. This potentially important finding should be confirmed by a second method. I would expect the wild-type TDP-43 expressed in these mice to be predominantly nuclear. What's the difference between Figure 4E and Figure S7? Figure S7A looks like the nuclear and cytoplasmic fractions were quantified from different gels. This is not good practice. Rad23a ASO seems to cause a small shift in the TDP-43 band. Does this indicate decreased phosphorylation?\nWe thank the reviewer for drawing more attention to Figure 4 panel E and the need for a second independent method for assessing nuclear TDP-43 ±RAD23A KD. To this end we performed the following new experiments and analysis:\na) We generated cytoplasmic and nuclear biochemical fractions from individual animals and ran them side-by-side for western blotting. We then analyzed the cytoplasmic-to-nuclear ratio of TDP43 on a per animal basis. We report the results from 4 animals per experimental group (revised Figure 4 panel E with quantification).\nb) We performed immunofluorescent localization of human TDP-43 in tissue sections from the TAR4/4 mouse ± RAD23A KD and quantified the TDP-43 puncta (which potentially correspond to TDP-43 found in detergent insoluble fraction of lysates). We quantified the number of neurons with nuclear aggregates and the number of aggregates per nucleus. We do see sparse cytoplasmic human TDP43 puncta in the TAR4/4 brain tissue sections and this aligns with the original description by Wils1 (they provide a single example in Figure 2 panel C without quantification). In the other major investigation that utilized the TAR4/4 mice (Becker et al.2) there is no mention of cortical cytoplasmic TDP-43. We have limited confidence that the IHC cytoplasmic TDP-43 signal is quantifiable. In our opinion the biochemical approach is a more rigorous approach to the extant issue. Images illustrating the key findings and their quantification are presented in the new Supplemental Figure 5\nc) We performed immunofluorescent localization of human TDP-43 in primary neuron cultures that express mutant A315T TDP-43. The human TDP-43 is found in nuclear, as well as sparse cytoplasmic puncta. Considering the inherent variability in the system, we have significant doubts that the cytoplasmic puncta are a scoreable phenotype.\nWe think the best independent method for assessing nuclear TDP-43 it not histological but functional – the RNAseq data. It is clear that expression of TDP-43 in the TAR4/4 brain profoundly influences the transcriptome (revised Figure 2). The fact that KD of RAD23A normalizes the expression of many genes is the best evidence that RAD23A KD restores nuclear TDP-43 function.\nFig. 4F/G Remove or label lane with ladder.\nDone.\nFigure S8: Sort panels according to text. Are p62 puncta reduced in TAR4/4 mice after Rad23a knockdown? Include quantification\nAs enumerated above, our ongoing studies suggest that the p62 story is complex and will require substantial further experimentation to obtain a comprehensive understanding. We hope the reviewer and editor will allow us to elide the question of p62 in this manuscript and remove this single supplemental figure.\nFigure 5: Adding the insoluble fractions to other cells is an unusual approach. Overexpression of TDP-43 together with ras23a/b knockdown or overexpression in neurons may be more informative.\nThe central questions addressed in Figure 5 are: why are the total quantity of ubiquitinated proteins elevated in lysates from the TAR4/4 cortex and why does RAD23A KD blunt this? We show that the total ubiquitin smear in an anti-ubiquitin blot (in both the soluble and insoluble fractions) is heavier in the TAR4/4 brain than controls and RAD23A KD blunts this. We assay the three protease activities of the proteasome with fluorogenic substrates in TAR4/4 lysates and these activities are reduced in the TAR4/4 brain. This effect is reversed when RAD23A is KD in TAR4/4 animals. Thus proteasome dysfunction explains why the total burden of ubiquitinated substrates accumulates in the TAR4/4 brain – but why? We hypothesize that proteasomes get ensnarled into aggregates and in this locale they are hypofunctional. In support of this, we show that multiple proteasome subunits are enriched in the insoluble fraction of TAR4/4 brain lysates. And this abnormal portioning is abrogated in TAR4/4 brains with RAD23A KD. To test the idea that aggregates themselves inhibit proteasome function we took a reductionist approach – purify proteasomes, add the soluble or insoluble fraction from cells expressing mutant TDP-43 and assay protease activity with the fluorogenic substrate. We report that the insoluble fraction does, in fact, inhibit protease activity.\nWe believe using whole cell lysates is a better approximation of the native situation (i.e., evaluating the effect of global proteomes on proteasome activity) than using purified Rad23a or/and TDP43. This experiment directly tests the ability of protein aggregates to inhibit proteasome activity. With all due respect to the reviewer, we would characterize this experiment as novel rather than unusual. That said, the basic approach (assay the activity of purified proteasomes when aggregates or toxic proteins are doped in) has been reported3,4.\nThe Results section has been amended to highlight the novel assay deployed and why they are useful.\nFig. 6D: What are the most enriched proteins? The full dataset should be included in the supplementary material. TDP-43 is clearly not the most regulated protein by RAD23A ASO and is barely significant. What about known RAD23A interactors like ATXN3 and UBE3A? This whole figure also looks like a draft. Most of the text is too small to read.\nWe have provided a new Supplemental Table 1, which includes the full dataset for all the quantified proteins and summarizes the alterations of previously reported RAD23A binding proteins. Additionally, Figure 6 has been significantly revised to improve the presentation."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have now addressed several issues to our satisfaction.\nHowever, there are still remaining major issues that need to be addressed.\n1) A major prior concern was the lack of IHC to investigate TDP-43 neuropathological hallmarks in this mouse model by staining for TDP43 and pTDP-43 and their rescue by RAD23A knock-down. The authors now “provide IHC staining of TDP-43 and phospho-TDP-43 and the effects of RAD23A knockdown; in addition the results are quantified (new Supplemental Figure 5).”\nThe results of the new Sup. Fig. 5 “Immunohistology for TDP-43 and phospho-TDP-43 in TAR4/4 motor cortex with and without RAD23A KD” are hard to interpret. Most problematic is the lack of wild-type mouse tissue in Suppl. Figure 5. It is described in the text but missing in the figure. This negative control is essential, since the shown nuclear phospho-TDP-43 staining is unconvincing and may be unspecific. First, phospho-TDP-43 is observed in cytoplasmic inclusions. Second, there is no good co-localization between TDP-43 and pTDP-43 staining in nuclear foci, which is also very concerning.\nThe statement that “TDP-43 histopathology in the TAR4/4 mouse recapitulates findings in human autopsy material” is hard to justify, since pTDP-43 staining shown here does not match at all TDP-43 pathology that is present in FTLD-TDP/ALS human brain tissue and characterized by loss of nuclear TDP-43 and cytoplasmic aggregation. While there is nuclear pTDP-43 staining in FTLD-TDP type D (caused by VCP mutations), the appearance of these lentiform nuclear pTDP-43 inclusions is very different from what is shown here for the TAR4/4 mice. There are several nuclear phospho-TDP-43 foci shown in Suppl. Fig. 5 that do not colocalize with total TDP-43 signal, which is indicative of unspecific staining by the pTDP-43 antibody. In response to other reviewer comments, the authors point out that the TAR4/4 model does not display cytoplasmic aggregates. Therefore, perhaps it does not display phosphorylated TDP-43 aggregates at all? To answer this question, IHC images need to be replaced with -better-quality staining that at least shows co-localisation of pTDP43 with total TDP43, if present, and needs to include wild-type control tissue to confirm specificity.\nThe text states that “Similarly, phosphoTDP-43 was only detected in the TAR4/4 brains, most often as nuclear puncta and occasionally in the neuropil. Some, but not all puncta, demonstrated co-staining for TDP-43 and phosphoTDP-43.” However, this is not evident from the presented data.\n2) Supplementary Figure 9 shows strong unspecific cytoplasmic TDP-43 staining in the control section, which raises concerns about interpreting the difference in nuclear staining to the mutant A315T TDP43-expressing neurons that do not show this background.\n3) In addition, the nucleo-cytoplasmic fractionation from Figure 4E shows a strong presence of TDP43 in the cytoplasm, while in the IHC staining it remains mostly nuclear. The authors should comment on this obvious and unexplained major difference between IHC and WB findings.", "reviewer_2": "Authors have answered well all the queries of this reviewer", "reviewer_3": "The authors address all critical points. The manuscript is ready for publication in Nature Communications."}, "author_response": "I appreciate the opportunity to respond to the critiques of our revised ms. entitled \"Reduction of RAD23A extends lifespan and mitigates pathology in TDP-43 mice\" (NCOMMS-24-56672A). I will focus on the comments made by Reviewer #1, which are provided in full below.\nReviewer #1 (Remarks to the Author):\n[...]\nThe Reviewer major concern is immunohistology (IHC) and we have undertaken a thorough review of our data and how it was presented. Additionally, we have stained new tissue blocks/samples to ensure that the results presented most accurately reflect reality. I appreciate the Reviewer’s scrutiny of this important issue.\nTo begin, the Reviewer asks for IHC of wild-type mice for TDP-43 and phosphoTDP-43. To achieve double labeling of tissue we used the Abnova mouse monoclonal (H00023435-M01) to TDP-43 and the rabbit monoclonal antibody (Developmental Studies Hybridoma Bank - DHSB) to phospho S409/410 TDP-43. The antigen used to create the Abnova mouse serum was amino acids 1-260 of human TDP-43. The antigen used to create the DSHB antibody was a 12 amino acid peptide corresponding to the region flanking S409/410 that was phosphorylated in vitro by recombinant caesin kinase 1. The specificity and tissue staining with this rabbit monoclonal antibody was described in Castellanos Otero et al.1\nFor all of our IHC we used the same concentrations of primary and secondary antibodies. When images were collected by confocal microscopy, the identical settings were used for laser intensity and photomultiplier sensitivity (all channels). All images were collected with the operator blind to genotype and antibodies (for example ± primary). By employing such rigor, we believe that the results presented in new Supplemental Figure 5 accurately represent IHC for TDP-43 and phosphoTDP-43 in TAR4/4 and WT mice.\nOn the basis of our new staining and analysis, we must modify what was stated in the first revision. First, abundant extranuclear puncta that are TDP-43(+) and phosphoTDP(+) are seen in the TAR4/4 mouse brain. We counted these puncta in the sections from the TAR4/4 mice with or without rad23A antisense oligonucleotide treatment and we find a statistically significant reduction of these puncta when rad23A is knocked down (KD). We provide representative images and the quantification. We note that in addition to these puncta there is inhomogeneous nuclear TDP-43 staining. This is not unexpected since the TAR4/4 mice were engineered to express full length human TDP-43 and some portion of human TDP-43 could reasonably be expected to be nuclear – and this is consistent with our biochemical studies described in Figur3 4. However, we also note inhomogeneous nuclear phosphoTDP-43 – this staining pattern was not found in the original work characterizing this antibody using human postmortem samples or staining of the rNLS8 mouse brain1.\nAfter discussions with Drs. Yong-Jie Zhang and Leonard Petrucelli (the senior authors on the paper describing the generation of these antibodies) this staining is thought to be non-specific. While their arguments are trenchant, this inhomogeneous nuclear phosphoTDP-43 IHC signal appears greater in the TAR4/4 brains without rad23a KD in comparison with rad23a KD. In addition, we do not see this in the WT brain. Inspection of the pictures in Supplemental Figure 5 reveals this enigmatic staining. Regardless, the manuscript has been edited to bring the attention of the reader to the punctate nuclear and cytoplasmic staining as well as non-punctate nuclear staining\nSecond, our IHC investigations of the WT mouse brain for TDP-43 (and phosphoTDP-43) are negative and are now presented. While we previously thought there was nuclear TDP-43 in these animals, further tissue staining and side-by-side comparisons with no-primary controls now convinces us that these antibodies do not stain WT tissue. I apologize for the erroneous “not-shown” data noted in the first revision.\n[...]\nIn the new figure we highlight both nuclear and extranuclear in the TAR4/4 brain that are TDP-43 (+) and phosphoTDP-43 (+). Not unexpectedly there are some puncta that are TDP-43 (+) and phosphoTDP-43 (-). Finally there are some puncta that are TDP-43 (-) and phospho TDP-43 (+). Such puncta may be artifactual.\n[...]\n2) Supplementary Figure 9 shows strong unspecific cytoplasmic TDP-43 staining in the control section, which raises concerns about interpreting the difference in nuclear staining to the mutant A315T TDP43-expressing neurons that do not show this background.\nIn Supplemental figure 9 we show immunocytochemistry of primary neurons grown in vitro that are infected with a virus to express either A315T TDP-43 or LacZ and after fixation, these coverslips were stained with antibodies that recognize human TDP-43 and RAD23A. The reviewer states that there is “strong unspecific cytoplasmic TDP-43 staining in the control section” – which likely refers to anti-TDP-43 staining of LacZ expressing neurons in vitro. With all due respect, I do not think there is strong cytoplasmic TDP-43 staining in the LacZ expressing cells. It would be possible to re-image coverslips with the laser setting such that the TDP-43 staining of LacZ expressing cells is completely black and the TDP-43 staining of the A315T expressing cells is evident (but less bright than currently presented). In my opinion, this would make the primary observations (lack of co-localization of TDP-43 and RAD23A) more difficult to appreciate.\n3) In addition, the nucleo-cytoplasmic fractionation from Figure 4E shows a strong presence of TDP43 in the cytoplasm, while in the IHC staining it remains mostly nuclear. The authors should comment on this obvious and unexplained major difference between IHC and WB findings.\nThe reviewer points out that in biochemical assays, human TDP-43 is easily detectable in a cytoplasmic subcellular fraction (panel E in Figure 4) and in IHC assays, cytoplasmic human TDP-43 is clearly less abundant than nuclear TDP-43 signal. There are a number of potential explanations for this apparent disparity. First, while the nuclear IHC TDP-43 signal is greater than the cytoplasmic TDP-43 signal, the percent of total area enclosed by nuclei is but a fraction of that enclosed by the cytoplasm. So while there is more nuclear TDP-43 per unit area in comparison with cytoplasmic TDP-43 per unit area, the total cytoplasmic area is much greater than nuclear area. Thus in this case appearances are deceptive. Second, the two techniques for monitoring TDP-43 expression are distinct and although they report on a common phenomenon, the technical differences make direct comparisons difficult. Does anyone really know the relationship between a reduced and denatured protein visualized as a band on a western blot and a fluorescent image obtained from paraformaldehyde fixed tissue that has undergone antigen retrieval at high temperature in a citrate buffer? In my opinion the two techniques provide complementary information and the disparity highlighted by the review is likely due to many issues, several of which are technical. The issue is commented upon in the revised manuscript: \nSince these different approaches report technique-specific outputs, a direct and quantitative comparison should not be drawn."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have now addressed our concerns about IHC in a thoughtful and thorough manner. I believe that these changes were important and have improved a strong manuscript by correcting a major weakness of IF data and their representation.\n\"With all due respect, I do not think there is strong cytoplasmic TDP-43 staining in the LacZ expressing cells. It would be possible to re-image coverslips with the laser setting such that the TDP-43 staining of LacZ expressing cells is completely black and the TDP-43 staining of the A315T expressing cells is evident (but less bright than currently presented). In my opinion, this would make the primary observations (lack of co-localization of TDP-43 and RAD23A) more difficult to appreciate.\"\n-> Having strong un-specific background staining (not cytoplasmic as we had stated, but in neurites and perhaps the substrate around the cells) only in the control cells is not normal and potentially problematic. There is a remaining concerns about the green haze around the LacZ expressing cells but not the high-contrast green staining w/o surrounding background in A315T expressing cells. Therefore, the authors should make absolutely sure that the same acquisition and image post-processing settings (!) were used for Suppl. Fig. 9A. (Sometimes imaging software will change display settings to boost the brightness in control images, which is not appropriate for use here)."}, "author_response": "Responses to REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\n[...]\nI thank the reviewer for the trenchant comments and I thank the Journal for giving us an opportunity to directly address the comments. The work is better for them.\n[...]\nThe additional comments by the reviewer are helpful and now I understand better the nature of his or her valid concern. In the original images, there is a green haze in the LacZ expressing cells and this is less prominent in the A315T expressing cells. The origin of the haze is technical – the original images are a merged z-stack of confocal slices. Despite identical photomultiplier settings, the background staining is more prominent in the LacZ expressing cells. To more clearly illustrate the main finding, I have elected to present a single z-stack slice through the center of the nucleus, both for the LacZ and for the A315T expressing cells. This is faithful to the data and shows better the main finding – TDP-43 and RAD23A do not co-localize. This immunocytochemical data complements the biochemistry (also presented in the same figure – panels C and D). While proving a “negative” result is always problematic (potentially impossible), we feel that together, the data in Supplemental figure 9 make a compelling case."}]

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