TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein which forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, altered neuronal dendritic morphology is a common theme among several neurological disorders and is thought to precede neurodegeneration. We previously found that both TDP-43 overexpression (OE) and knockdown (KD) result in reduced dendritic branching of cortical neurons. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as an approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 RNA targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescues defects in dendritic branching. Finally, we demonstrate, using RNA sequencing, that TDP-43 OE and KD cause similar changes in the abundance of specific messenger RNAs, consistent with their ability to produce similar morphological defects. Our data therefore provide a mechanism by which TDP-43 dysfunction interferes with dendritic branching, and may define pathways for therapeutic intervention in neurodegenerative diseases.

The Metastasis Suppressor Protein Nme1 Is a Concentration-Dependent Modulator of Ca2+/Calmodulin-Dependent Protein Kinase II.

Nucleoside diphosphate kinases (Nmes or NDPKs) have been implicated in a multitude of cellular processes, including an important role in metastasis suppression, and several enzymatic activities have been assigned to the Nme family. Nevertheless, for many of these processes, it has not been possible to establish a strong connection between Nme enzymatic activity and the relevant biological function. We hypothesized that, in addition to its known enzymatic functions, members of the Nme family might also regulate signaling cascades by acting on key signal transducers. Accordingly, here we show that Nme1 directly interacts with the calcium/calmodulin-dependent kinase II (CaMKII). Using purified proteins, we monitored the phosphorylation of a number of CaMKII substrates and determined that at nanomolar levels Nme1 enhances the phosphorylation of T-type substrates; this modulation shifts to inhibition at low micromolar concentrations. Specifically, the autophosphorylation of CaMKII at Thr286 is completely inhibited by 2 µM Nme1, a feature that distinguishes Nme1 from other known endogenous CaMKII inhibitors. Importantly, CaMKII inhibition does not require phosphotransfer activity by Nme1 because the kinase-dead Nme1 H118F mutant is as effective as the wild-type form of the enzyme. Our results provide a novel molecular mechanism whereby Nme1 could modulate diverse cellular processes in a manner that is independent of its known enzymatic activities.

The Ras-like GTPase Rem2 is a potent inhibitor of calcium/calmodulin-dependent kinase II activity.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue-specific manner. For example, in heart muscle, CaMKII regulates Ca2+ homeostasis, whereas in neurons, CaMKII regulates activity-dependent dendritic remodeling and long-term potentiation (LTP), a neurobiological correlate of learning and memory. Previously, we identified the GTPase Rem2 as a critical regulator of dendrite branching and homeostatic plasticity in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously unknown Rem2 function. Our results suggest that Rem2 inhibition involves interaction with both the CaMKII hub domain and substrate recognition domain. Moreover, we found that Rem2-mediated inhibition of CaMKII regulates dendritic branching in cultured hippocampal neurons. Lastly, we report that substitution of two key amino acid residues in the Rem2 N terminus (Arg-79 and Arg-80) completely abolishes its ability to inhibit CaMKII. We propose that our biochemical findings will enable further studies unraveling the functional significance of Rem2 inhibition of CaMKII in cells.

TDP-43 misexpression causes defects in dendritic growth.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) share overlapping genetic causes and disease symptoms, and are linked neuropathologically by the RNA binding protein TDP-43 (TAR DNA binding protein-43 kDa). TDP-43 regulates RNA metabolism, trafficking, and localization of thousands of target genes. However, the cellular and molecular mechanisms by which dysfunction of TDP-43 contributes to disease pathogenesis and progression remain unclear. Severe changes in the structure of neuronal dendritic arbors disrupt proper circuit connectivity, which in turn could contribute to neurodegenerative disease. Although aberrant dendritic morphology has been reported in non-TDP-43 mouse models of ALS and in human ALS patients, this phenotype is largely unexplored with regards to TDP-43. Here we have employed a primary rodent neuronal culture model to study the cellular effects of TDP-43 dysfunction in hippocampal and cortical neurons. We show that manipulation of TDP-43 expression levels causes significant defects in dendritic branching and outgrowth, without an immediate effect on cell viability. The effect on dendritic morphology is dependent on the RNA-binding ability of TDP-43. Thus, this model system will be useful in identifying pathways downstream of TDP-43 that mediate dendritic arborization, which may provide potential new avenues for therapeutic intervention in ALS/FTD.