Ubiquitin-positive Inclusions Research Articles

Progressive neuronal inclusion formation and axonal degeneration in CHMP2B mutant transgenic mice

Mutations in the charged multivesicular body protein 2B (CHMP2B) gene cause frontotemporal lobar degeneration. The mutations lead to C-terminal truncation of the CHMP2B protein. We generated Chmp2b knockout mice and transgenic mice expressing either wild-type or C-terminally truncated mutant CHMP2B. The transgenic CHMP2B mutant mice have decreased survival and show progressive neurodegenerative changes including gliosis and increasing accumulation of p62- and ubiquitin-positive inclusions. The inclusions are negative for the TAR DNA binding protein 43 and fused in sarcoma proteins, mimicking the inclusions observed in patients with CHMP2B mutation. Mice transgenic for mutant CHMP2B also develop an early and progressive axonopathy characterized by numerous amyloid precursor protein-positive axonal swellings, implicating altered axonal function in disease pathogenesis. These findings were not observed in Chmp2b knockout mice or in transgenic mice expressing wild-type CHMP2B, indicating that CHMP2B mutations induce degenerative changes through a gain of function mechanism. These data describe the first mouse model of dementia caused by CHMP2B mutation and provide new insights into the mechanisms of CHMP2B-induced neurodegeneration.

Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72

Numerous kindreds with familial frontotemporal dementia and/or amyotrophic lateral sclerosis have been linked to chromosome 9, and an expansion of the GGGGCC hexanucleotide repeat in the non-coding region of chromosome 9 open reading frame 72 has recently been identified as the pathogenic mechanism. We describe the key characteristics in the probands and their affected relatives who have been evaluated at Mayo Clinic Rochester or Mayo Clinic Florida in whom the hexanucleotide repeat expansion were found. Forty-three probands and 10 of their affected relatives with DNA available (total 53 subjects) were shown to carry the hexanucleotide repeat expansion. Thirty-six (84%) of the 43 probands had a familial disorder, whereas seven (16%) appeared to be sporadic. Among examined subjects from the 43 families (n = 63), the age of onset ranged from 33 to 72 years (median 52 years) and survival ranged from 1 to 17 years, with the age of onset <40 years in six (10%) and >60 in 19 (30%). Clinical diagnoses among examined subjects included behavioural variant frontotemporal dementia with or without parkinsonism (n = 30), amyotrophic lateral sclerosis (n = 18), frontotemporal dementia/amyotrophic lateral sclerosis with or without parkinsonism (n = 12), and other various syndromes (n = 3). Parkinsonism was present in 35% of examined subjects, all of whom had behavioural variant frontotemporal dementia or frontotemporal dementia/amyotrophic lateral sclerosis as the dominant clinical phenotype. No subject with a diagnosis of primary progressive aphasia was identified with this mutation. Incomplete penetrance was suggested in two kindreds, and the youngest generation had significantly earlier age of onset (>10 years) compared with the next oldest generation in 11 kindreds. Neuropsychological testing showed a profile of slowed processing speed, complex attention/executive dysfunction, and impairment in rapid word retrieval. Neuroimaging studies showed bilateral frontal abnormalities most consistently, with more variable degrees of parietal with or without temporal changes; no case had strikingly focal or asymmetric findings. Neuropathological examination of 14 patients revealed a range of transactive response DNA binding protein molecular weight 43 pathology (10 type A and four type B), as well as ubiquitin-positive cerebellar granular neuron inclusions in all but one case. Motor neuron degeneration was detected in nine patients, including five patients without ante-mortem signs of motor neuron disease. While variability exists, most cases with this mutation have a characteristic spectrum of demographic, clinical, neuropsychological, neuroimaging and especially neuropathological findings.

The E3 ubiquitin ligase TRIM11 mediates the degradation of congenital central hypoventilation syndrome-associated polyalanine-expanded PHOX2B

Expansions of a polyalanine (polyA) stretch in the coding region of the PHOX2B gene cause congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by the absence of adequate control of autonomic breathing. Expansion of polyA in PHOX2B leads to protein misfolding and accumulation into inclusions. The mechanisms that regulate mutant protein degradation and turnover have been poorly elucidated. Here, we investigate the regulation of degradation of wild-type and polyA-expanded PHOX2B. We show that expanded PHOX2B is targeted for degradation through the ubiquitin-proteasome system, resulting in lowered levels of the mutant protein relative to its wild-type counterpart. Moreover, we show that mutant PHOX2B forms ubiquitin-positive inclusions, which sequester wild-type PHOX2B. This sequestration correlates with reduced transcriptional activity of endogenous wild-type protein in neuroblastoma cells. Finally, we show that the E3 ubiquitin ligase TRIM11 plays a critical role in the clearance of mutant PHOX2B through the proteasome. Importantly, clearance of mutant PHOX2B by TRIM11 correlates with a rescue of PHOX2B transcriptional activity. We propose that CCHS is partially caused by a dominant-negative effect of expanded PHOX2B due to the retention of the wild-type protein in pathogenic aggregates. Our results demonstrate that TRIM11 is a novel modifier of mutant PHOX2B toxicity and represents a potential therapeutic target for CCHS.

Different 8-Hydroxyquinolines Protect Models of TDP-43 Protein, α-Synuclein, and Polyglutamine Proteotoxicity through Distinct Mechanisms

No current therapies target the underlying cellular pathologies of age-related neurodegenerative diseases. Model organisms provide a platform for discovering compounds that protect against the toxic, misfolded proteins that initiate these diseases. One such protein, TDP-43, is implicated in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In yeast, TDP-43 expression is toxic, and genetic modifiers first discovered in yeast have proven to modulate TDP-43 toxicity in both neurons and humans. Here, we describe a phenotypic screen for small molecules that reverse TDP-43 toxicity in yeast. One group of hit compounds was 8-hydroxyquinolines (8-OHQ), a class of clinically relevant bioactive metal chelators related to clioquinol. Surprisingly, in otherwise wild-type yeast cells, different 8-OHQs had selectivity for rescuing the distinct toxicities caused by the expression of TDP-43, α-synuclein, or polyglutamine proteins. In fact, each 8-OHQ synergized with the other, clearly establishing that they function in different ways. Comparative growth and molecular analyses also revealed that 8-OHQs have distinct metal chelation and ionophore activities. The diverse bioactivity of 8-OHQs indicates that altering different aspects of metal homeostasis and/or metalloprotein activity elicits distinct protective mechanisms against several neurotoxic proteins. Indeed, phase II clinical trials of an 8-OHQ has produced encouraging results in modifying Alzheimer disease. Our unbiased identification of 8-OHQs in a yeast TDP-43 toxicity model suggests that tailoring 8-OHQ activity to a particular neurodegenerative disease may be a viable therapeutic strategy.

Comparison of phosphorylated TDP-43-positive inclusions in oculomotor neurons in patients with non-ALS and ALS disorders

TDP-43 has been identified as a major component of the pathological inclusions in most forms of frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). In the present study, paraffin sections of the midbrain in 112 patients with various non-ALS disorders and 27 patients with sporadic ALS were immunostained with antibody against phosphorylated TDP-43 (pTDP-43). pTDP-43-positive inclusions in oculomotor neurons were detected in 18 of 112 patients with non-ALS disorders (16.1%). The appearance of the inclusions showed fine filamentous structures rather than the skein-like inclusions seen in the anterior horn cells of ALS spinal cords. The incidence was increased in the age range of 80–89years old (10/37 cases; 27.0%), in which 6 of 10 cases demonstrated AD pathology in the temporal lobes. Twenty-seven ALS patients were examined and the findings were compared with those of non-ALS patients. There were 13 cases demonstrating pTDP-43-positive inclusions (48.1%) which showed stronger immunoreactivities in ALS cases. This is the first report demonstrating fine filamentous pTDP-43-positive inclusions in oculomotor neurons in non-ALS disorders. Although the mechanisms underlying pTDP-43 in oculomotor neurons are currently unknown, its detection is of interest, and the expression may occur not only in ALS but also during the aging process.

The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies

The degraded, misfolded C terminus of TAR DNA-binding protein-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. However, the precise mechanism of pathological cleavage of the TAR DNA-binding protein-43 remains unknown. Here we show that the TAR DNA-binding protein-43 C-terminal protein physically interacts with itself or with the cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of the TAR DNA-binding protein-43, including subcellular localisation and exon skipping of the cystic fibrosis transmembrane conductance regulator. Significantly, mutants with a failure to engage in prion-like interactions are processed into an ~24-kDa C-terminal fragment of the TAR DNA-binding protein-43. The estimated cleavage site of degraded TAR DNA-binding protein-43 fragments corresponds to the pathological cleavage site identified in patients with the TAR DNA-binding protein-43 proteinopathies. Consistently, epigallocatechin gallate constrains prion-like interactions, attenuating pathological-like degradation. Thus, the native folding of TAR DNA-binding protein-43 C terminus acts as a guardian of pathogenesis, which is directly associated with loss-of-function.

Mechanisms and models of TDP-43 proteinopathies

Background Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Results To explore the pathogenic properties of mutant forms of TDP-43, we generated and characterized two mouse lines expressing human TDP-43 carrying the M337V mutation (hTDP-43M337V). We found hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, intranuclear and cytoplasmic phosphorylated TDP43 aggregates were frequently detected, and the levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased. Overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice. Conclusion While overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity.

Functional Annotation of Small Noncoding RNAs Target Genes Provides Evidence for a Deregulated Ubiquitin-Proteasome Pathway in Spinocerebellar Ataxia Type 1

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by the expansion of CAG repeats in the ataxin 1 (ATXN1) gene. In affected cerebellar neurons of patients, mutant ATXN1 accumulates in ubiquitin-positive nuclear inclusions, indicating that protein misfolding is involved in SCA1 pathogenesis. In this study, we functionally annotated the target genes of the small noncoding RNAs (ncRNAs) that were selectively activated in the affected brain compartments. The primary targets of these RNAs, which exhibited a significant enrichment in the cerebellum and cortex of SCA1 patients, were members of the ubiquitin-proteasome system. Thus, we identified and functionally annotated a plausible regulatory pathway that may serve as a potential target to modulate the outcome of neurodegenerative diseases.

Animal Models of Parkinson's Disease 2012

Parkinson's disease (PD) is a prevalent movement disorder, which is characterized by age-dependent degeneration of dopaminergic neurons in the midbrain. Recent advances in PD study have suggested that multiple nervous systems are affected as well as the nigrostriatal system, involving decline of cognitive function, sleep disturbance, mood change, and dysfunction of the autonomic nervous system. Multifactorial causes of PD are hypothesized. Neurotoxins including artificial compounds, pesticides, heavy metals as well as dopamine itself have been proposed to be environmental risk factors of PD. Recent genome-wide genetic and mutational studies have revealed various genetic risk factors and SNPs while microglial activation in the affected regions have emerged to be involved in the disease development as a local microenvironmental factor. One of the pathological hallmarks of PD is the appearance of ubiquitin-positive cytoplasmic inclusions called Lewy bodies (LBs) in the affected regions. A presynaptic protein α-synuclein, which is the first identified monogenic PD gene product, is a major component of LBs. Understanding of the neuropathological mechanism underlying the formation of LBs is a key aspect of PD study. Newly identified monogenic PD gene products, leucine-rich repeat kinase 2 (LRRK2), ATPase type 13A2, and Vps35 are implicated in vesicle transport, endosomal—autophagic and lysosomal pathways. Dysregulation of these gene products might be involved in abnormal protein turnover associated with the LB formation.

Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking

TDP-43 is the major disease protein in ubiquitin-positive inclusions of amyotrophic lateral sclerosis and frontotemporal lobar degeneration (FTLD) characterized by TDP-43 pathology (FTLD-TDP). Accumulation of insoluble TDP-43 aggregates could impair normal TDP-43 functions and initiate disease progression. Thus, it is critical to define the signalling mechanisms regulating TDP-43 since this could open up new avenues for therapeutic interventions. Here, we have identified a redox-mediated signalling mechanism directly regulating TDP-43. Using in vitro and cell-based studies, we demonstrate that oxidative stress promotes TDP-43 cross-linking via cysteine oxidation and disulphide bond formation leading to decreased TDP-43 solubility. Biochemical analysis identified several cysteine residues located within and adjacent to the second RNA-recognition motif that contribute to both intra- and inter-molecular interactions, supporting TDP-43 as a target of redox signalling. Moreover, increased levels of cross-linked TDP-43 species are found in FTLD-TDP brains, indicating that aberrant TDP-43 cross-linking is a prominent pathological feature of this disease. Thus, TDP-43 is dynamically regulated by a redox regulatory switch that links oxidative stress to the modulation of TDP-43 and its downstream targets.

Oxidative stress induced by glutathione depletion reproduces pathological modifications of TDP-43 linked to TDP-43 proteinopathies

TAR DNA-binding protein 43 (TDP-43) is a major component of ubiquitin-positive inclusion of TDP-43 proteinopathies including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions, which is now referred to as FTLD-TDP. TDP-43 in the aberrant inclusion is known to be hyperphosphorylated at C-terminal sites, to be truncated at the N-terminal region, and to re-distribute from nucleus to cytoplasm or neurite. The pathogenic role of these modifications, however, has not been clarified. Furthermore, there is no evidence about the initial cause of these modifications. Herein we show that ethacrynic acid (EA), which is able to increase cellular oxidative stress through glutathione depletion, induces TDP-43 C-terminal phosphorylation at serine 403/404 and 409/410, insolubilization, C-terminal fragmentation, and cytoplasmic distribution in NSC34 cells and primary cortical neurons. In the investigation using a nonphosphorylable mutant of TDP-43, there was no evidence that C-terminal phosphorylation of TDP-43 contributes to its solubility or distribution under EA induction. Our findings suggest that oxidative stress induced by glutathione depletion is associated with the process of the pathological TDP-43 modifications and provide new insight for TDP-43 proteinopathies.

Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila

Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, we have generated new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein. We found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, we showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, our data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies.

TDP‐43 toxicity is mediated by the unfolded protein response‐unrelated induction of C/EBP homologous protein expression

Transactive response DNA-binding protein-43 (TDP-43) neuronal toxicity plays an essential role in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. In our previous study, we showed that low-grade overexpression of TDP-43, which is thought to mimic the gain-of-function of TDP-43, caused neuronal death, mediated by the upregulation of Bim and the downregulation of Bcl-xL in vitro. In this study, we show that TDP-43 overexpression caused the upregulation of C/EBP-homologous protein (CHOP) and that disruption of the CHOP gene markedly attenuated TDP-43-induced cell death. These results indicate that increases in CHOP expression contribute to TDP-43-induced cell death. We also show that the TDP-43-induced upregulation of CHOP expression is mediated by both the upregulation of the mRNA level of CHOP and the attenuation of thedegradation of CHOP, which is independent on the PERK/eIF2α/ATF4 or other pathway related to the unfolded protein response (UPR) to endoplasmic reticulum stress. This study provides the first example of the CHOP-mediated cell death that is independent of the UPR. © 2011 Wiley Periodicals, Inc.

Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice

BackgroundAbnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Researchers have identified 44 mutations in the TARDBP gene that encode TDP-43 as causative for cases of sporadic and familial ALS http://www.molgen.ua.ac.be/FTDMutations/. Certain mutant forms of TDP-43, such as M337V, are associated with increased low molecular weight (LMW) fragments compared to wild-type (WT) TDP-43 and cause neuronal apoptosis and developmental delay in chick embryos. Such findings support a direct link between altered TDP-43 function and neurodegeneration.ResultsTo explore the pathogenic properties of the M337V mutation, we generated and characterized two mouse lines expressing human TDP-43 (hTDP-43M337V) carrying this mutation. hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, and intranuclear and cytoplasmic phosphorylated TDP-43 aggregates were frequently detected. The levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased in the transgenic mice. Moreover, overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP-43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice.ConclusionOur novel TDP-43M337V mouse model indicates that overexpression of hTDP-43M337V alone is toxic in vivo. Because overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, the mechanisms causing pathogenesis in the mutant model remain unknown. However, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity. The mutant model will serve as a valuable tool in the development of future studies designed to uncover pathways associated with TDP-43 neurotoxicity and the precise roles TDP-43 RNA targets play in neurodegeneration.

Conjoint pathologic cascades mediated by ALS/FTLD-U linked RNA-binding proteins TDP-43 and FUS

The RNA-binding proteins TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) play central roles in neurodegeneration associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Normally localized in the nucleus, in sites affected by ALS and FTLD-U they are mislocalized to the cytoplasm and form cytoplasmic inclusions. TDP-43 and FUS are transported to the nucleus in a Ran-GTPase-dependent manner via nuclear import receptors, but they also contribute to the formation of stress granules (SGs), which are intracytoplasmic structures incorporating RNA. C-terminal truncations of TDP-43 eliminate the nuclear transport signal and cause mislocalization of the protein to the cytoplasm, where it accumulates and forms SGs. ALS-associated FUS mutations impair nuclear transport and cause mislocalization of FUS to the cytoplasm, where it also contributes to assembly of SGs. Furthermore, the ALS susceptibility factor ataxin-2, recently identified as a potent modifier of TDP-43 toxicity, is also a predicted cytoplasmic RNA-binding protein and a constituent protein of SGs, suggesting that it is a part of the common pathologic cascade formed by TDP-43 and FUS. Thus, we propose that excessive mislocalization of the RNA-binding proteins TDP-43, FUS, and ataxin-2 into the cytoplasm leads to impairment of the RNA quality control system, forming the core of the ALS/FTLD-U degenerative cascade. In this review, we discuss the molecular basis of the novel disease spectrum of ALS/FTLD-U, including the neurodegenerative mechanism of the cytoplasmic RNA-binding proteins TDP-43 and FUS and the possibility of a novel therapeutic strategy.

Frontotemporal Dementia: From Mendelian Genetics Towards Genome Wide Association Studies

Frontotemporal lobar degeneration is the most common cause of dementia of non-Alzheimer's type worldwide. It manifests, clinically, with behavioural changes and language impairment and is pathologically associated with tau- or ubiquitin-positive inclusions detected in neurons and glial cells of the frontal and temporal lobes in the brain. Genetic variations in the microtubule-associated protein tau and progranulin genes explain almost 50% of familial cases, whilst variations in TAR DNA-binding protein, charged multivescicular body protein 2B, valosin-containing protein and fused in sarcoma genes contribute to <5% of cases. The rapidly developing investigative techniques available to geneticists such as genome-wide association studies, whole-exome sequencing and, soon, whole-genome sequencing promise to contribute to the unravelling of the genetic architecture of this complex disease and, in the future, to the development of more sensitive, accurate and effective diagnostic and treatment measures.

Crystal Structure of the Ubiquitin-associated (UBA) Domain of p62 and Its Interaction with Ubiquitin

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.

Rodent Models of TDP-43 Proteinopathy: Investigating the Mechanisms of TDP-43-Mediated Neurodegeneration

Since the identification of phosphorylated and truncated transactive response DNA-binding protein 43 (TDP-43) as a primary component of ubiquitinated inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions, much effort has been directed towards ascertaining how TDP-43 contributes to the pathogenesis of disease. As with other protein misfolding disorders, TDP-43-mediated neuronal death is likely caused by both a toxic gain and loss of TDP-43 function. Indeed, the presence of cytoplasmic TDP-43 inclusions is associated with loss of nuclear TDP-43. Moreover, post-translational modifications of TDP-43, including phosphorylation, ubiquitination, and cleavage into C-terminal fragments, may bestow toxic properties upon TDP-43 and cause TDP-43 dysfunction. However, the exact neurotoxic TDP-43 species remain unclear, as do the mechanism(s) by which they cause neurotoxicity. Additionally, given our incomplete understanding of the roles of TDP-43, both in the nucleus and the cytoplasm, it is difficult to truly appreciate the detrimental consequences of aberrant TDP-43 function. The development of TDP-43 transgenic animal models is expected to narrow these gaps in our knowledge. The aim of this review is to highlight the key findings emerging from TDP-43 transgenic animal models and the insight they provide into the mechanisms driving TDP-43-mediated neurodegeneration.

Pathological hallmarks of amyotrophic lateral sclerosis/frontotemporal lobar degeneration in transgenic mice produced with TDP-43 genomic fragments

Transactive response DNA-binding protein 43 ubiquitinated inclusions are a hallmark of amyotrophic lateral sclerosis and of frontotemporal lobar degeneration with ubiquitin-positive inclusions. Yet, mutations in TARDBP, the gene encoding these inclusions are associated with only 3% of sporadic and familial amyotrophic lateral sclerosis. Recent transgenic mouse studies have revealed a high degree of toxicity due to transactive response DNA-binding protein 43 proteins when overexpressed under the control of strong neuronal gene promoters, resulting in early paralysis and death, but without the presence of amyotrophic lateral sclerosis-like ubiquitinated transactive response DNA-binding protein 43-positive inclusions. To better mimic human amyotrophic lateral sclerosis, we generated transgenic mice that exhibit moderate and ubiquitous expression of transactive response DNA-binding protein 43 species using genomic fragments that encode wild-type human transactive response DNA-binding protein 43 or familial amyotrophic lateral sclerosis-linked mutant transactive response DNA-binding protein 43 (G348C) and (A315T). These novel transgenic mice develop many age-related pathological and biochemical changes reminiscent of human amyotrophic lateral sclerosis including ubiquitinated transactive response DNA-binding protein 43-positive inclusions, transactive response DNA-binding protein 43 cleavage fragments, intermediate filament abnormalities, axonopathy and neuroinflammation. All three transgenic mouse models (wild-type, G348C and A315T) exhibited impaired learning and memory capabilities during ageing, as well as motor dysfunction. Real-time imaging with the use of biophotonic transactive response DNA-binding protein 43 transgenic mice carrying a glial fibrillary acidic protein-luciferase reporter revealed that the behavioural defects were preceded by induction of astrogliosis, a finding consistent with a role for reactive astrocytes in amyotrophic lateral sclerosis pathogenesis. These novel transactive response DNA-binding protein 43 transgenic mice mimic several characteristics of human amyotrophic lateral sclerosis-frontotemporal lobar degeneration and they should provide valuable animal models for testing therapeutic approaches.

A comparative clinical, pathological, biochemical and genetic study of fused in sarcoma proteinopathies

Neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration are rare diseases characterized by ubiquitin-positive inclusions lacking transactive response DNA-binding protein-43 and tau. Recently, mutations in the fused in sarcoma gene have been shown to cause familial amyotrophic lateral sclerosis and fused in sarcoma-positive neuronal inclusions have subsequently been demonstrated in neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration with ubiquitinated inclusions. Here we provide clinical, imaging, morphological findings, as well as genetic and biochemical data in 14 fused in sarcoma proteinopathy cases. In this cohort, the age of onset was variable but included cases of young-onset disease. Patients with atypical frontotemporal lobar degeneration with ubiquitinated inclusions all presented with behavioural variant frontotemporal dementia, while the clinical presentation in neuronal intermediate filament inclusion disease was more heterogeneous, including cases with motor neuron disease and extrapyramidal syndromes. Neuroimaging revealed atrophy of the frontal and anterior temporal lobes as well as the caudate in the cases with atypical frontotemporal lobar degeneration with ubiquitinated inclusions, but was more heterogeneous in the cases with neuronal intermediate filament inclusion disease, often being normal to visual inspection early on in the disease. The distribution and severity of fused in sarcoma-positive neuronal cytoplasmic inclusions, neuronal intranuclear inclusions and neurites were recorded and fused in sarcoma was biochemically analysed in both subgroups. Fused in sarcoma-positive neuronal cytoplasmic and intranuclear inclusions were found in the hippocampal granule cell layer in variable numbers. Cortical fused in sarcoma-positive neuronal cytoplasmic inclusions were often 'Pick body-like' in neuronal intermediate filament inclusion disease, and annular and crescent-shaped inclusions were seen in both conditions. Motor neurons contained variable numbers of compact, granular or skein-like cytoplasmic inclusions in all fused in sarcoma-positive cases in which brainstem and spinal cord motor neurons were available for study (five and four cases, respectively). No fused in sarcoma mutations were found in any cases. Biochemically, two major fused in sarcoma species were found and shown to be more insoluble in the atypical frontotemporal lobar degeneration with ubiquitinated inclusions subgroup compared with neuronal intermediate filament inclusion disease. There is considerable overlap and also significant differences in fused in sarcoma-positive pathology between the two subgroups, suggesting they may represent a spectrum of the same disease. The co-existence of fused in sarcoma-positive inclusions in both motor neurons and extramotor cerebral structures is a characteristic finding in sporadic fused in sarcoma proteinopathies, indicating a multisystem disorder.