ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Abstract

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43 Q331K and TDP-43 M337V ), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell typeselective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43 Q331K , but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43 Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.A myotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are progressive, adult-onset neurodegenerative diseases with overlapping clinical and pathological features (1-3). ALS is characterized by the selective loss of upper and lower motor neurons, leading to progressive fatal paralysis and muscle atrophy. A large majority (∼90%) of ALS and FTLD-U cases are without a known genetic cause. Importantly, in these sporadic cases, the appearance of ubiquitinated inclusions within the affected neurons of the nervous system characterizes both ALS and FTLD-U patients, suggesting an overlapping mechanism underlying both diseases. Biochemical characterization of brains and spinal cords from ALS and FTLD-U patients identified transactivating response region (TAR) DNA binding protein (TDP-43) as the major protein component of these ubiquitinated inclusions (4, 5). The discovery of ALS-linked mutations in the glycine-rich C-terminal domain of TDP-43 (6-8) demonstrated a pathological role of TDP-43 in both diseases. The subsequent identification of mutations in a structurally and functionally related nucleic acid binding protein, FUS/ TLS (fused in sarcoma/translocated in liposarcoma) (9, 10), further implicated defects in RNA processing in ALS pathogenesis.TDP-43 is a multifunctional nucleic acid binding protein.Within the nervous system, TDP-43 binds to >6,000 pre-mRNAs and affects the levels of ∼600 mRNAs and the splicing patterns of another 950 (11). Structura...

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

“…Importantly, the motor phenotype in homozygous LCDmut mice develops without TDP‐43 nuclear depletion, TDP‐43 inclusions or features of TDP‐43 LOF, such as the inclusion of cryptic exons or downregulation of long intron genes, showing that these phenomena are not necessary for the initial stages of neurodegeneration, as also demonstrated by others (Arnold et al , 2013). …”

“…Both TDP‐43 gain and loss of function have been proposed as possible crucial mechanisms in the pathogenesis of ALS, a devastating incurable neurodegenerative disorder, and both reduced TDP‐43 expression and TDP‐43 overexpression have detrimental effects in mice (Arnold et al , 2013; Yang et al , 2014). …”

“…Similarly, in transgenic mice, even low levels of overexpression of wild‐type (WT) or mutant TDP‐43 cause multiple RNA changes, making it difficult to identify the pathogenic RNA profile that occurs physiologically in disease (Arnold et al , 2013). …”

Motor-Coordinative and Cognitive Dysfunction Caused by Mutant TDP-43 Could Be Reversed by Inhibiting Its Mitochondrial Localization

“…In addition, although premature sudden death prevented the assessment of cognitive function in previous studies using mutant TDP-43 transgenic animal models, cortical neuron degeneration has been consistently observed. 11,14,19,20,28 Therefore, our findings in hemizygous TDP-43 M337V mice do not necessarily contradict previous findings only reporting motor dysfunction in TDP-43 transgenic mice. Unlike other transgenic animal models showing increased expression of total TDP-43, the hemizygous TDP-43 M337V model does not demonstrate significantly changed expression of total TDP-43 in the brain and spinal cord, 29 indicating very low transgene expression.…”

“…[11][12][13][14][15][16][17][18][19][20] However, either premature death before the presence of full behavior impairments or extremely aggressive disease progression in many of these animal models make the interpretation of behavioral and neuropathological measurements, especially cognitive assessment, difficult. The TDP-43 M337V transgenic (Tg) mouse (i.e., Prnp-TARDBP* M337V; The Jackson Laboratory, stock no.…”