RNA-binding deficient TDP-43 drives cognitive decline in a mouse model of TDP-43 proteinopathy

Reviewer #1 (Public Review):

With this work, the authors address a central question regarding the potential consequences of post-translational modifications for the pathogenesis of neurodegenerative diseases. Phosphorylation and mislocalization of the RNA binding protein TDP43 are characteristic of ~50% of frontotemporal lobar degeneration (FTLD), as well as >95% of amyotrophic lateral sclerosis (ALS). To determine if acetylation is a primary, disease-driving event, they generated a TDP-43 mutant harboring an acetylation-mimicking mutation (K145Q). Animals carrying the acetylation-mimic mutation (K145Q) displayed key pathological features of disease, including more cytoplasmic TDP43 and impaired TDP43 splicing activity, together with behavioral phenotypes reminiscent of FTLD.

This is a well-written and well-illustrated manuscript, with clear and convincing findings. The observations are significant and emphasize the importance of post-translational modifications to TDP-43 function and disease phenotypes. In addition, the TDP43(K145Q) mice may prove to be a valuable model for studying TDP-43-related mechanisms of neurodegeneration and therapeutic strategies.

However, as it stands it is challenging to determine if any or all of the phenotypes are a direct consequence of interrupted RNA binding by TDP-43, rather than acetylation per se. Furthermore, all the results are obtained using an acetylation-mimic mutation that may simply be disrupting a key residue involved in RNA binding by TDP43, instead of mirroring acetylation itself, which in theory is a reversible modification. Lastly, it remains unknown why TDP43(K145Q) mice developed features of FTLD, but not ALS, despite the fact that TDP-43 acetylation was found in ALS tissue and not FTLD.

Reviewer #2 (Public Review):

This paper extends prior work demonstrating the importance of K145 acetylation of TDP-43 as a post-translational modification that impacts its RNA-binding capacity and may contribute to pathology in FTLD-ALS. The main strengths of this paper are the generation of a novel mouse model, using CRISPR gene editing, in which an acetylation-mimetic mutation (K to Q) is introduced at position 145. Behavioral, biochemical, and genetic analyses indicate that these mice display phenotypes relevant to TDP-43-associated disease and will be a valuable contribution to the field. While most of the data are rigorous and clearly presented, several weaknesses should be addressed to strengthen the manuscript and further characterize the phenotype of mutant mice.

Reviewer #3 (Public Review):

Numerous experimental models are phenotyped in this manuscript including mouse neurons, iPSC-derived human neurons, knock-in mice, and knock-in iPSCs. Expression of acetylation-mimic or acetylation-null TDP-43 protein is achieved either with overexpression or CRISPR-Cas9-based knock-in. A complex phenotype is observed including loss of TDP-43 function (reduced autoregulation, increased cryptic splicing) and a gain of TDP-43 (increased insoluble TDP-43 protein). These correlate with downstream neurobehavioral changes which are most consistent with a cortical/hippocampal phenotype without a motor phenotype. Post-translational modifications of disease-associated proteins are thought to contribute to neurodegenerative disease pathogenesis, and this study succeeds in demonstrating that TDP-43 acetylation results in downstream molecular and behavioral phenotypes.

There are numerous additional strengths. TDP-43 acetylation is a post-translational modification that is known to be associated with TDP-43 inclusions that are characteristic of human diseases. An important strength is the rigorous use of multiple different experimental models (rodent cells, iPSC-derived neurons, mice, overexpression, knock-in) with overall consistent results. Moreover, multiple orthogonal endpoints are presented including histology/cytology/immunostaining, biochemistry, molecular biology, and neurobehavioral assays. As TDP-43 acetylation is known to block RNA binding, these novel cellular and mouse models represent interesting albeit complex tools to study the functional consequences of a partial loss of function. As TDP-43 regulates its own expression (i.e. autoregulation), the complexity lies at least in part due to the loss of RNA binding leading to a functional loss of TDP-43 function which includes the increased expression of the TARDBP transcript and TDP-43 protein.

Conceptually, there is a disconnect in that the mouse model exhibits primarily a cortical/hippocampal phenotype more akin to frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), while TDP-43 acetylation is only seen in ALS tissues and not in FTLD-TDP tissues because most of the pathologic protein in the latter is N-terminally truncated (i.e. the acetylation site is not present). That being said, there is no mouse model which completely and faithfully recapitulates the human disease, and this mouse model avoids overt overexpression (increased TDP-43 protein expression stemming from altered autoregulation) and avoids the use of synthetic/artificial mutation (such as mutation of the TDP-43 nuclear localization signal).

In terms of the CNS phenotype, it is difficult to interpret the reduced density of NeuN-positive neurons in the mouse model as a neurodegenerative phenotype. The reduction in NeuN density but not overall cellular density is only suggestive of neurodegeneration (as opposed to, for example, a developmental phenotype) without more rigorous stereological approaches that take into account potential volumetric changes. Indeed, the absence of astrocytosis and microgliosis argues against neurodegeneration.

Some of the loss of function measures (CFTR construct splicing, shift in SORT1 protein) are subtle, although RNA sequencing clearly shows many splicing aberrations including cryptic splicing events which overall supports that a loss of TDP-43 function is observed.

Finally, there are multiple instances where multiple measurements are made on a few biological replicates. ANOVA or t-tests are not appropriate in these instances (lack of independence).