Proteomic Identification of ALDOA as a Pathogenic TDP-43 Interaction Partner in ALS

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis (ALS), while wild-type TDP-43 is a pathological hallmark of patients with sporadic ALS and frontotemporal lobar degeneration (FTLD). Various in vitro and in vivo studies have also demonstrated toxicity of both mutant and wild-type TDP-43 to neuronal cells. To study the potential additional toxicity incurred by mutant TDP-43 in vivo, we generated mutant human TDP-43 (p.M337V) transgenic mouse lines driven by the Thy-1.2 promoter (Mt-TAR) and compared them in the same experimental setting to the disease phenotype observed in wild-type TDP-43 transgenic lines (Wt-TAR) expressing comparable TDP-43 levels. Overexpression of mutant TDP-43 leads to a worsened dose-dependent disease phenotype in terms of motor dysfunction, neurodegeneration, gliosis, and development of ubiquitin and phosphorylated TDP-43 pathology. Furthermore, we show that cellular aggregate formation or accumulation of TDP-43 C-terminal fragments (CTFs) are not primarily responsible for development of the observed disease phenotype in both mutant and wild-type TDP-43 mice.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-013-8427-5) contains supplementary material, which is available to authorized users.

Curcumin abolishes mutant TDP-43 induced excitability in a motoneuron-like cellular model of ALS

Mutant TAR DNA-binding protein-43 induces oxidative injury in motor neuron-like cell

Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43) is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation) induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, which ultimately leads to paralysis and death. Mutation of TAR DNA binding protein 43 (TDP-43) has been linked to the development of an inherited form of ALS. Existing TDP-43 transgenic animals develop a limited loss of motor neurons and therefore do not faithfully reproduce the core phenotype of ALS. Here, we report the creation of multiple lines of transgenic rats in which expression of ALS-associated mutant human TDP-43 is restricted to either motor neurons or other types of neurons and skeletal muscle and can be switched on and off. All of these rats developed progressive paralysis reminiscent of ALS when the transgene was switched on. Rats expressing mutant TDP-43 in motor neurons alone lost more spinal motor neurons than rats expressing the disease gene in varying neurons and muscle cells, although these rats all developed remarkable denervation atrophy of skeletal muscles. Intriguingly, progression of the disease was halted after transgene expression was switched off; in rats with limited loss of motor neurons, we observed a dramatic recovery of motor function, but in rats with profound loss of motor neurons, we only observed a moderate recovery of motor function. Our finding suggests that mutant TDP-43 in motor neurons is sufficient to promote the onset and progression of ALS and that motor neuron degeneration is partially reversible, at least in mutant TDP-43 transgenic rats.

Characterization of β-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy

Bioenergetic abnormalities and metabolic dysfunction occur in amyotrophic lateral sclerosis (ALS) patients and genetic mouse models. However, whether metabolic dysfunction occurs early in ALS pathophysiology linked to different ALS genes remains unclear. Here, we investigated AMP-activated protein kinase (AMPK) activation, which is a key enzyme induced by energy depletion and metabolic stress, in neuronal cells and mouse models expressing mutant superoxide dismutase 1 (SOD1) or TAR DNA binding protein 43 (TDP-43) linked to ALS. AMPK phosphorylation was sharply increased in spinal cords of transgenic SOD1G93A mice at disease onset and accumulated in cytoplasmic granules in motor neurons, but not in pre-symptomatic mice. AMPK phosphorylation also occurred in peripheral tissues, liver and kidney, in SOD1G93A mice at disease onset, demonstrating that AMPK activation occurs late and is not restricted to motor neurons. Conversely, AMPK activity was drastically diminished in spinal cords and brains of presymptomatic and symptomatic transgenic TDP-43A315T mice and motor neuronal cells expressing different TDP-43 mutants. We show that mutant TDP-43 induction of the AMPK phosphatase, protein phosphatase 2A (PP2A), is associated with AMPK inactivation in these ALS models. Furthermore, PP2A inhibition by okadaic acid reversed AMPK inactivation by mutant TDP-43 in neuronal cells. Our results suggest that mutant SOD1 and TDP-43 exert contrasting effects on AMPK activation which may reflect key differences in energy metabolism and neurodegeneration in spinal cords of SOD1G93A and TDP-43A315T mice. While AMPK activation in motor neurons correlates with progression in mutant SOD1-mediated disease, AMPK inactivation mediated by PP2A is associated with mutant TDP-43-linked ALS.

Bioenergetic abnormalities and metabolic dysfunction occur in amyotrophic lateral sclerosis (ALS) patients and genetic mouse models. However, whether metabolic dysfunction occurs early in ALS pathophysiology linked to different ALS genes remains unclear. Here, we investigated AMP-activated protein kinase (AMPK) activation, which is a key enzyme induced by energy depletion and metabolic stress, in neuronal cells and mouse models expressing mutant superoxide dismutase 1 (SOD1) or TAR DNA binding protein 43 (TDP-43) linked to ALS. AMPK phosphorylation was sharply increased in spinal cords of transgenic SOD1G93A mice at disease onset and accumulated in cytoplasmic granules in motor neurons, but not in presymptomatic mice. AMPK phosphorylation also occurred in peripheral tissues, liver and kidney, in SOD1G93A mice at disease onset, demonstrating that AMPK activation occurs late and is not restricted to motor neurons. Conversely, AMPK activity was drastically diminished in spinal cords and brains of presymptomatic and symptomatic transgenic TDP-43A315T mice and motor neuronal cells expressing different TDP-43 mutants. We show that mutant TDP-43 induction of the AMPK phosphatase, protein phosphatase 2A (PP2A), is associated with AMPK inactivation in these ALS models. Furthermore, PP2A inhibition by okadaic acid reversed AMPK inactivation by mutant TDP-43 in neuronal cells. Our results suggest that mutant SOD1 and TDP-43 exert contrasting effects on AMPK activation which may reflect key differences in energy metabolism and neurodegeneration in spinal cords of SOD1G93A and TDP-43A315T mice. While AMPK activation in motor neurons correlates with progression in mutant SOD1-mediated disease, AMPK inactivation mediated by PP2A is associated with mutant TDP-43-linked ALS.

TDP-43 is a nuclear protein involved in exon skipping and alternative splicing. Recently, TDP-43 has been identified as the pathological signature protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis. In addition, TDP-43-positive inclusions are present in Parkinson disease, dementia with Lewy bodies, and 30% of Alzheimer disease cases. Pathological TDP-43 is redistributed from the nucleus to the cytoplasm, where it accumulates. An approximately 25-kDa C-terminal fragment of TDP-43 accumulates in affected brain regions, suggesting that it may be involved in the disease pathogenesis. Here, we show that overexpression of the 25-kDa C-terminal fragment is sufficient to cause the mislocalization and cytoplasmic accumulation of endogenous full-length TDP-43 in two different cell lines, thus recapitulating a key biochemical characteristic of TDP-43 proteinopathies. We also found that TDP-43 mislocalization is associated with a reduction in the low molecular mass neurofilament mRNA levels. Notably, we show that the autophagic system plays a role in TDP-43 metabolism. Specifically, we found that autophagy inhibition increases the accumulation of the C-terminal fragments of TDP-43, whereas inhibition of mTOR, a key protein kinase involved in autophagy regulation, reduces the 25-kDa C-terminal fragment accumulation and restores TDP-43 localization. Our results suggest that autophagy induction may be a valid therapeutic target for TDP-43 proteinopathies.

Tar DNA binding protein 43 (TDP-43) mislocalization and aggregation is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. Moreover, TDP-43 mRNA was found to be upregulated by ∼2.5-fold in the spinal cord of sporadic ALS subjects. Here we have examined the effects of nerve injury in new transgenic mouse models overexpressing by approximately threefold wild-type or mutant (G348C) TDP-43 species. Four weeks after axonal crush of sciatic nerve, TDP-43 transgenic mice remained paralyzed at the injured limb unlike control mice, which had regained most of their normal mobility. In contrast to normal mice, TDP-43 transgenic mice exhibited sustained elevation of TDP-43 cytoplasmic levels in motor neurons after nerve crush, and the relocalization of TDP-43 to the nucleus was delayed by several weeks. After crush, peripherin and ubiquitin levels remained also significantly elevated in TDP-43 transgenic mice compared with control mice. Analysis of the sciatic nerve at 11 d after nerve crush showed that the number of regenerating axons in the distal portion of the lesion was considerably reduced in TDP-43 transgenic mice, especially in TDP-43(G348C) mice, which exhibited a reduction of ∼40%. In addition, markers of neuroinflammation were detected at much higher levels in TDP-43 transgenic mice. These results suggest that a deregulation of TDP-43 expression in ALS is a phenomenon that can affect the regenerative responses to neuronal injury and regrowth potential of axons.

Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS.

The identification of TAR DNA-binding protein 43 (TDP-43) as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions has defined a new class of neurodegenerative conditions: the TDP-43 proteinopathies. This breakthrough was quickly followed by mutation analysis of TARDBP, the gene encoding TDP-43. Herein, we provide a review of our previously published efforts that led to the identification of 3 TARDBP mutations (p.M337V, p.N345K, and p.I383V) in familial ALS patients, two of which were novel. With over 40 TARDBP mutations now discovered, there exists conclusive evidence that TDP-43 plays a direct role in neurodegeneration. The onus is now on researchers to elucidate the mechanisms by which mutant TDP-43 confers toxicity, and to exploit these findings to gain a better understanding of how TDP-43 contributes to the pathogenesis of disease. Our biochemical analysis of TDP-43 in ALS patient lymphoblastoid cell lines revealed a substantial increase in TDP-43 truncation products, including a ~25 kDa fragment, compared to control lymphoblastoid cell lines. We discuss the putative harmful consequence of abnormal TDP-43 fragmentation, as well as highlight additional mechanisms of toxicity associated with mutant TDP-43.

Pathological TAR DNA-binding protein 43 (TDP-43) dysfunction is associated with multiple neurodegenerative disorders. However, the mechanistic link between TDP-43 dysfunction and neurodegeneration is poorly understood and likely involves a combination of genetic and environmental risk factors. A major risk factor for neurodegenerative disease is exposure to traumatic brain injury (TBI). Here, we investigated the synergistic interplay between TDP-43 dysfunction and TBI in a murine model of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). A model of TDP-43 dysfunction caused by a knock-in Q331K mutation in Tardbp was combined with a mild model of TBI. Control conditions included both WT mice and mice with sham surgery. Animals were evaluated for behavioral deficits at timepoints pre- and post-surgery. Additionally, post-mortem brain tissues were examined using RNA sequencing and mass spectrometry-based quantitative proteomics together with histological and biochemical analyses. Expression of dysfunctional TDP-43 in vivo caused deficits in multiple branches of the proteostasis network, including protein folding, protein synthesis, and protein turnover. Examples include mis-expression of chaperones and genes within the ubiquitin-proteosome pathway in mutant TDP-43 versus WT mice. Further, mutant TDP-43 expression correlated with reduced thermostability of proteins associated with the ribosome and the chaperonin containing TCP-1 complex. In response to TBI, mutant TDP-43 mice exhibited significantly worse neurological outcomes relative to WT animals. Heightened neurological deficits in mutant TDP-43 mice following TBI coincided with a robust upregulation of proteostasis- and stress-related genes at the transcript level. However, this upregulation was not detected at the protein level. Our data demonstrate that expression of dysfunctional TDP-43 leads to deficits within the proteostasis network in vivo at baseline. Despite an upregulation of proteostasis-related genes at the transcript level in mutant TDP-43 mice after TBI, mutant TDP-43 mice exhibit an impaired response to, and recovery from, brain trauma relative to their WT counterparts. Restoring proteostasis is expected to protect against the detrimental effects of TDP-43 dysfunction, especially under stress conditions that promote neurodegenerative disease.

The cytoplasmic accumulation of the nuclear TAR DNA-binding protein 43 (TDP-43) is a pathologic hallmark in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and other neurological disorders. However, most transgenic TDP-43 rodent models show predominant nuclear distribution of TDP-43 in the brain. By expressing mutant TDP-43 (M337V) in the brains of rhesus monkeys and mice, we verified that mutant TDP-43 is distributed in the cytoplasm of the monkey brain and that the majority of mutant TDP-43 remains in the nuclei of the mouse brain. The primate-specific caspase-4, but not mouse homologue caspase-11, could remove the NLS-containing N-terminal domain and generate fragmented TDP-43 that accumulates in the cytoplasm. Moreover, increased expression of caspase-4 in the monkey brain promotes the cytoplasmic accumulation of endogenous TDP-43, and suppressing caspase-4 reduces the cytoplasmic distribution of endogenous TDP-43 in cultured human neural cells. Our findings suggest that primate-specific caspase-4-mediated cleavage of TDP-43 accounts for its cytoplasmic mislocalization in the primate brains and may serve as a potential therapeutic target.

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular dysfunction of astrocytes. Whether non-cell autonomous effects occur in other forms of ALS, such as TAR DNA binding protein 43 (TDP-43)-related disease, remains unclear. Here, we characterised the impact of mutant TDP-43 expression on primary astrocytes derived from transgenic TDP-43A315T mice. Mutant TDP-43 astrocytes revealed evidence for TDP-43 pathology, shown by cytoplasmic TDP-43 inclusions and accumulation in insoluble cell fractions which was exacerbated by proteasomal inhibition. L-glutamate uptake, measured using an [3H]D-aspartate assay, was impaired in mutant TDP-43 astrocytes, while ATP accumulation was abnormal, suggesting mutant TDP-43 induced astrocytic dysfunction. Astrocyte activation coupled with spinal and cortical motor neuron loss in transgenic TDP-43A315T mice could imply non-cell autonomous effects of astrocytes in vivo. These data demonstrate mutant TDP-43-mediated cell autonomous effects on astrocytes that may contribute to motor neuron pathology in ALS.