A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.     

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261–414 aa) with K_D values of 6.2 nM and 8.7 nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170–414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.     

Evolutionarily Conserved Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A/B Proteins Functionally Interact with Human and Drosophila TAR DNA-binding Protein 43 (TDP-43)

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.     

Identification of Genetic Modifiers of TDP-43 Neurotoxicity in Drosophila

Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the TARDBP gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included SpindleB and the Notch target Hey, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin Nup50 increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.     

Sustained Expression of TDP-43 and FUS in Motor Neurons in Rodent's Lifetime

TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) are two highly conserved ribonucleoproteins. Pathogenic mutations of the TDP-43 or the FUS gene are all linked to amyotrophic lateral sclerosis (ALS) that is characterized by progressive degeneration of motor neurons. To better understand the correlation of ALS disease genes with the selectivity of chronic motor neuron degeneration, we examined the longitudinal expression of the TDP-43 and the FUS genes in C57BL6 mice and in Sprague-Dawley rats. TDP-43 and FUS were robustly and ubiquitously expressed in the postnatal mice and rats, but were markedly decreased in the adult rodents. In adulthood, TDP-43 and FUS proteins were even undetectable in peripheral organs including skeletal muscles, liver, and kidney, but were constantly expressed at substantial levels in the central nervous system. Motor neurons expressed the TDP-43 and the FUS genes at robust levels throughout rodent''s lifetime. Moreover, TDP-43 and FUS were accumulated in the cytoplasm of motor neurons in aged animals. Our findings suggest that TDP-43 and FUS play an important role in development and that constant and robust expression of the genes in motor neurons may render the neurons vulnerable to pathogenic mutation of the TDP-43 or the FUS gene. To faithfully model the pathology of TDP-43- or FUS gene mutations in rodents, we must replicate the expression patterns of the TDP-43 and the FUS gene in animals.     

TDP-1/TDP-43 potentiates human α-Synuclein (HASN) neurodegeneration in Caenorhabditis elegans

TAR DNA binding protein (TDP-43) is a DNA/RNA binding protein whose pathological role in amyotrophic lateral sclerosis (ALS) and frontal temporal lobe dementia (FTLD) via formation of protein aggregates is well established. In contrast, knowledge concerning its interactions with other neuropathological aggregating proteins is poorly understood. Human α-synuclein (HASN) elicits dopaminergic neuron degeneration via protein aggregation in Parkinson's disease. HASN protein aggregates are also found in TDP-43 lesions and colocalize in Lewy Body Dementia (LBD). To better understand the interactions of TDP-43 and HASN, we investigated the effects of genetic deletion of tdp-1, the Caenorhabditis elegans ortholog of human TDP-43, as well as overexpression of TDP-43, in transgenic models overexpressing HASN^WT and HASN^A53T. Tdp-1 deletion improved the posture, movement, and developmental delay observed in transgenic animals pan-neuronally overexpressing HASN^A53T, and attenuated the loss and impairment of dopaminergic neurons caused by HASN^A53T or HASN^WT overexpression. Tdp-1 deletion also led to a decrease in protein level, mRNA level and aggregate formation of HASN in living animals. RNA-seq studies suggested that tdp-1 supports expression of lysosomal genes and decreases expression of genes involved in heat shock. RNAi demonstrated that heat shock proteins can mediate HASN neuropathology. Co-overexpression of both human TDP-43 and HASN^WT resulted in locomotion deficits, shorter lifespan, and more severe dopaminergic neuron impairments compared to single transgenes. Our results suggest TDP-1/TDP-43 potentiates HASN mediated neurodegeneration in C. elegans. This study indicates a multifunctional role for TDP-1/TDP-43 in neurodegeneration involving HASN.     

Exploring the aggregation-prone regions from structural domains of human TDP-43

TDP-43 (transactive- response DNA binding protein) amazes structural biologist as its aberrant ubiquitinated cytosolic inclusions is largely involved in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). An important question in TDP-43 research is to identify the structural region mediating the formation of cytoplasmic pathological aggregates. In this study, we attempted to delineate the aggregation-prone sequences of the structural domain of TDP-43. Here, we investigated the self-assembly of peptides of TDP-43 using aggregation prediction algorithms, Zipper DB and AMYLPRED2. The three aggregation-prone peptides identified were from N-terminal domain (²⁴GTVLLSTV³¹), and RNA recognition motifs, RRM1 (¹²⁸GEVLMVQV¹³⁵) and RRM2 (²⁴⁷DLIIKGIS²⁵⁴). Furthermore, the amyloid fibril forming propensities of these peptides were analyzed through different biophysical techniques and molecular dynamics simulation. Our study shows the different aggregation ability of conserved stretches in structural domain of TDP-43 that will possibly induce full-length aggregation of TDP-43 in vivo. The peptide form RRM2 demonstrates the higher intrinsic amyloid forming propensity and suggests that RRM2 might form the structural core of TDP-43 aggregation seen in vivo. The results of this study would help in designing peptide based inhibitors of TDP-43 aggregation.     

CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1

The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy–endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2B^Intron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43–mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2B^Intron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.     

Inflammation Induces TDP-43 Mislocalization and Aggregation

TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43^A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.     

Activation of ER Stress and Autophagy Induced by TDP-43 A315T as Pathogenic Mechanism and the Corresponding Histological Changes in Skin as Potential Biomarker for ALS with the Mutation

TAR DNA binding protein 43 (TDP-43) A315T mutation (TDP-43^A315T) has been found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) as a disease causing mutation with enhanced protein aggregation, formation of protease-resistant fragments, and neurotoxicity. However, the molecular mechanisms for its pathogenic effects are largely unknown. In this study, we demonstrate that TDP-43^A315T enhanced neuronal toxicity via activating endoplasmic reticulum (ER) stress-mediated apoptosis in SH-SY5Y cells. Moreover, autophagy was activated by overexpression of TDP-43^A315T in a self-defensive manner to decrease neuronal toxicity. Inhibition of autophagy attenuates TDP-43^A315T induced neuronal cell death. Furthermore, the expression levels of TDP-43, ER chaperone 78 kDa glucose-regulated protein (GRP-78), and autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) in the skin tissues from ALS patients with TDP-43^A315T mutation were markedly higher than those from the healthy control. Thus, our findings provide new molecular evidence for TDP-43^A315T neuropathology. In addition, the pathological change in the skin tissues of the patients with TDP-43^A315T mutation can be used as a quick diagnostic biomarker.     

Cytoplasmic mislocalization and mitochondrial colocalization of TDP-43 are common features between normal aged and young mice

Transactive response DNA binding protein 43 (TDP-43) pathologies have been well recognized in various neurodegenerative disorders including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). However, there have been limited studies on whether there are any TDP-43 alterations in normal aging. We investigated TDP-43 distribution in different brain regions in normal aged (n = 3 for 26- or 36-month-old) compared to young (n = 3 for 6- or 12-month-old) mice. In both normal aged and young mice, TDP-43 and phosphorylated TDP-43 (pTDP-43) demonstrated a unique pattern of distribution in neurons in some specific brain regions including the pontine nuclei, thalamus, CA3 region of the hippocampus, and orbital cortex. This pattern was demonstrated on higher magnification of high-resolution double fluorescence images and confocal microscopy as mislocalization of TDP-43 and pTDP-43, characterized by neuronal nuclear depletion and cytoplasmic accumulation in these brain regions, as well as colocalization between TDP-43 or pTDP-43 and mitochondria, similar to what has been described previously in neurodegenerative disorders. All these findings were identical in both normal aged and young mice. In summary, TDP-43 and pTDP-43 mislocalization from nucleus to cytoplasm and their colocalization with mitochondria in the specific brain regions are present not only in aging, but also in young healthy states. Our findings provide a new insight for the role of TDP-43 proteinopathy in health and diseases, and that aging may not be a critical factor for the development of TDP-43 proteinopathy in subpopulations of neurons.Impact statementDespite increasing evidence implicating the important role of TDP-43 in the pathogenesis of a wide range of age-related neurodegenerative diseases, there is limited study of TDP-43 proteinopathy and its association with mitochondria during normal aging. Our findings of cytoplasmic accumulation of TDP-43 that is highly colocalized with mitochondria in neurons in selective brain regions in young animals in the absence of neuronal loss provide a novel insight into the development of TDP-43 proteinopathy and its contribution to neuronal loss.     

Transgenic Rat Model of Neurodegeneration Caused by Mutation in the TDP Gene

TDP-43 proteinopathies have been observed in a wide range of neurodegenerative diseases. Mutations in the gene encoding TDP-43 (i.e., TDP) have been identified in amyotrophic lateral sclerosis (ALS) and in frontotemporal lobe degeneration associated with motor neuron disease. To study the consequences of TDP mutation in an intact system, we created transgenic rats expressing normal human TDP or a mutant form of human TDP with a M337V substitution. Overexpression of mutant, but not normal, TDP caused widespread neurodegeneration that predominantly affected the motor system. TDP mutation reproduced ALS phenotypes in transgenic rats, as seen by progressive degeneration of motor neurons and denervation atrophy of skeletal muscles. This robust rat model also recapitulated features of TDP-43 proteinopathies including the formation of TDP-43 inclusions, cytoplasmic localization of phosphorylated TDP-43, and fragmentation of TDP-43 protein. TDP transgenic rats will be useful for deciphering the mechanisms underlying TDP-43–related neurodegenerative diseases.     

New insights and therapeutic opportunities for progranulin-deficient frontotemporal dementia

Frontotemporal dementia (FTD) is the second most common form of dementia. It affects the frontal and temporal lobes of the brain and has a highly heterogeneous clinical representation with patients presenting with a wide range of behavioral, language, and executive dysfunctions. Etiology of FTD is complex and consists of both familial and sporadic cases. Heterozygous mutations in the GRN gene, resulting in GRN haploinsufficiency, cause progranulin (PGRN)-deficient FTD characterized with cytoplasmic mislocalization of TAR DNA-binding protein 43 kDa (TDP-43) aggregates. GRN codes for PGRN, a secreted protein that is also localized in the endolysosomes and plays a critical role in regulating lysosomal homeostasis. How PGRN deficiency modulates immunity and causes TDP-43 pathology and FTD-related neurodegeneration remains an active area of intense investigation. In the current review, we discuss some of the significant progress made in the past two years that links PGRN deficiency with microglial-associated neuroinflammation, TDP-43 pathology, and lysosomal dysfunction. We also review the opportunities and challenges toward developing therapies and biomarkers to treat PGRN-deficient FTD.     

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.     

Mitochondrial Dysfunction and Decrease in Body Weight of a Transgenic Knock-in Mouse Model for TDP-43

The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43^A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43^A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.