Dysregulation of Translation in TDP-43 Proteinopathies: Deficits in the RNA Supply Chain and Local Protein Production.

Local control of gene expression provides critical mechanisms for regulating development, maintenance and plasticity in the nervous system. Among the strategies known to govern gene expression locally, mRNA transport and translation have emerged as essential for a neuron's ability to navigate developmental cues, and to establish, strengthen and remove synaptic connections throughout lifespan. Substantiating the role of RNA processing in the nervous system, several RNA binding proteins have been implicated in both developmental and age dependent neurodegenerative disorders. Of these, TDP-43 is an RNA binding protein that has emerged as a common denominator in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and related disorders due to the identification of causative mutations altering its function and its accumulation in cytoplasmic aggregates observed in a significant fraction of ALS/FTD cases, regardless of etiology. TDP-43 is involved in multiple aspects of RNA processing including splicing, transport and translation. Given that one of the early events in disease pathogenesis is mislocalization from the nucleus to the cytoplasm, several studies have focused on elucidating the pathogenic role of TDP-43 in cytoplasmic translation. Here we review recent findings describing TDP-43 translational targets and potential mechanisms of translation dysregulation in TDP-43 proteinopathies across multiple experimental models including cultured cells, flies, mice and patient derived neurons.

TDP-43 Proteinopathy Causes Broad Metabolic Alterations including TCA Cycle Intermediates and Dopamine Levels in Drosophila Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. Here, we used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, we performed global metabolomic profiling in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. Here, we report alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. We also show that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43G298S, and we find that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700µÎ´6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700µÎ´6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700µÎ´6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6.

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.

To Be or Not To Be Toxic-Is RNA Association With TDP-43 Complexes Deleterious or Protective in Neurodegeneration?

TAR DNA binding protein (TDP-43) is a nucleic acid binding protein associated with insoluble cytoplasmic aggregates in several neurodegenerative disorders, including 97% of the ALS cases. In healthy individuals, TDP-43 is primarily localized to the nucleus; it can shuttle between the nucleus and the cytoplasm, and is involved in several aspects of RNA processing including transcription, splicing, RNA stability, transport, localization, stress granule (SG) formation, and translation. Upon stress, TDP-43 aggregates in the cytoplasm and associates with several types of RNA and protein assemblies, resulting in nuclear depletion of TDP-43. Under conditions of prolonged stress, cytoplasmic TDP-43 undergoes liquid-liquid phase separation (LLPS) and becomes less mobile. Evidence exists to support a scenario in which insoluble TDP-43 complexes sequester RNA and/or proteins causing disturbances in both ribostasis and proteostasis, which in turn contribute to neurodegeneration. However, the relationship between RNA binding and TDP-43 toxicity remains unclear. Recent studies provide conflicting views on the role of RNA in TDP-43 toxicity, with some finding RNA as a toxic factor whereby RNA binding contributes to TDP-43 toxicity, while others find RNA to be a protective factor that inhibits TDP-43 aggregation. Here we review and discuss these recent reports, which ultimately highlight the importance of understanding the heterogeneity of TDP-43 assemblies and collectively point to solubilizing TDP-43 as a potential therapeutic strategy.