TDP-43 causes neurotoxicity and cytoskeletal dysfunction in primary cortical neurons

TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24–72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy

The zinc finger/RING domain protein Unkempt regulates cognitive flexibility

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory

Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo

Intracellular inclusions of the TAR-DNA binding protein 43 (TDP-43) have been reported in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD-TDP). Rare mutations in TARDBP have been linked to both ALS and FTD-TDP suggesting that TDP-43 dysfunction is mechanistic in causing disease. TDP-43 is a predominantly nuclear protein with roles in regulating RNA transcription, splicing, stability and transport. In ALS, TDP-43 aberrantly accumulates in the cytoplasm of motor neurons where it forms aggregates. However it has until recently been unclear whether the toxic effects of TDP-43 involve recruitment to motor axons, and what effects this might have on axonal growth and integrity. Here we use chick embryonic motor neurons, in vivo and in vitro, to model the acute effects of TDP-43. We show that wild-type and two TDP-43 mutant proteins cause toxicity in chick embryonic motor neurons in vivo. Moreover, TDP-43 is increasingly mislocalised to axons over time in vivo, axon growth to peripheral targets is truncated, and expression of neurofilament-associated antigen is reduced relative to control motor neurons. In primary spinal motor neurons in vitro, a progressive translocation of TDP-43 to the cytoplasm occurs over time, similar to that observed in vivo. This coincides with the appearance of cytoplasmic aggregates, a reduction in the axonal length, and cellular toxicity, which was most striking for neurons expressing TDP-43 mutant forms. These observations suggest that the capacity of spinal motor neurons to produce and maintain an axon is compromised by dysregulation of TDP-43 and that the disruption of cytoskeletal integrity may play a role in the pathogenesis of ALS and FTD-TDP

C9orf72 poly GA RAN-translated protein plays a key role in amyotrophic lateral sclerosis via aggregation and toxicity

An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD

TDP-43 affects anterograde transport in cortical neurons.

<p>(A) Example of a cortical neuron co-transfected with EB3-RFP, live imaged to show EB3 ‘comets’ (arrows) (A’) Higher power view. EB3 moves in an anterograde manner towards the growing ends of axon. (B)–(I) Example kymographs of neurons expressing GFP, or co-transfected with GFP and TDP isoforms, at 48 and 72H, as labelled. Distance along the X-axis and time along the Y-axis. Scale bar = 10 µm, except in A’ (J) Quantification of the velocity of EB3-RFP comets in the axons of transfected neurons. Graphs represent means and standard error of (n = 3). No significant differences were observed.</p

TDP-43 is mislocalised in cortical neurons.

<p>Rat cortical neurons were transfected with GFP control (A—D), and GFP-tagged TDP-43 wild-type and mutant isoforms (E—P) and analysed at 24–144 hours (H). Immunostained with anti-GFP antibodies. Scale bar = 10 µm. Insets (E’–P’)—higher power views of the corresponding panels above, converted to black and white pixels. Scale bar = 10 µm.</p

Cytoskeletal integrity is compromised over time in cortical neurons.

<p>Rat cortical neurons were transfected with GFP control, or GFP-tagged TDP-43 wild-type and mutant isoforms as labelled and analysed at 24, 72 H and 144 H. Immunostaining with anti-GFP (green) and anti-tyrosinated tubulin (red) antibodies. Cellular fragmentation was observed at 144 H (bottom right panel). Scale bar = 10 µm.</p

TDP-43 mislocalisation visualised with anti-TDP-43 antibodies.

<p>Rat cortical neurons were transfected with GFP control, and GFP-tagged TDP-43 wild-type and mutant isoforms as labelled and analysed at 72 H and 144 H. Immunostaining with anti-GFP (green) and anti-TDP-43 antibodies (red). Scale bar– 10 µm. Insets—higher power views of corresponding panels to the left, stained with anti-TDP-43 antibodies. Scale bar = 10 µm.</p

TDP-43 affects axonal growth <i>in vitro</i>.

<p>(A) Quantitation of axon lengths of transfected neurons at time-points as labelled (n = 3 experiments; 100 neurons per experiment). Data is presented as means and standard errors. Significance were compared by ANOVA between constructs at same time points (no significance—p > 0.05; * P < 0.01; ** P < 0.001; *** P < 0.0001). (B)–(M) typical cortical neuron growth cones stained with anti-tyrosinated tubulin antibodies at timepoints and with constructs as labelled. Scale bar = 10 µm. (N) Quantitation of mean growth cone area at 3 different time points as labelled (70 neurons per construct per experiment, 3 experiments). Data is presented as mean and standard error. Significance denoted as for (A).</p