TBN improves motor function and prolongs survival in a TDP-43M337V mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are serious neurodegenerative diseases. Although their pathogenesis is unclear, the abnormal accumulation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological feature that exists in almost all patients. Thus far, there is no drug that can cure ALS/FTLD. Tetramethylpyrazine nitrone (TBN) is a derivative of tetramethylapyrazine, derived from the traditional Chinese medicine Ligusticum chuanxiong, which has been widely proven to have therapeutic effects on models of various neurodegenerative diseases. TBN is currently under clinical investigation for several indications including a Phase II trial of ALS. Here, we explored the therapeutic effect of TBN in an ALS/FTLD mouse model. We injected the TDP-43 M337V virus into the striatum of mice unilaterally and bilaterally, and then administered 30 mg/kg TBN intragastrically to observe changes in behavior and survival rate of mice. The results showed that in mice with unilateral injection of TDP-43M337V into the striatum, TBN improved motor deficits and cognitive impairment in the early stages of disease progression. In mice with bilateral injection of TDP-43M337V into the striatum, TBN not only improved motor function but also prolonged survival rate. Moreover, we show that its therapeutic effect may be through activation of the Akt/mTOR/GSK-3ß and AMPK/PGC-1α/Nrf2 signaling pathways. In summary, TBN is a promising agent for the treatment of ALS/FTLD.

Frontal lobe 1H MR spectroscopy in asymptomatic and symptomatic MAPT mutation carriers.

OBJECTIVE: To determine the frontal lobe proton magnetic resonance spectroscopy (1H MRS) abnormalities in asymptomatic and symptomatic carriers of microtubule-associated protein tau (MAPT) mutations. METHODS: We recruited patients with MAPT mutations from 5 individual families, who underwent single voxel 1H MRS from the medial frontal lobe at 3T (n = 19) from the Longitudinal Evaluation of Familial Frontotemporal Dementia Subjects (LEFFTDS) Study at the Mayo Clinic site. Asymptomatic MAPT mutation carriers (n = 9) had Frontotemporal Lobar Degeneration Clinical Dementia Rating Sum of Boxes (FTLD-CDR SOB) score of zero, and symptomatic MAPT mutation carriers (n = 10) had a median FTLD-CDR SOB score of 5. Noncarriers from healthy first-degree relatives of the patients were recruited as controls (n = 25). The demographic aspects and 1H MRS metabolite ratios were compared by use of the Fisher exact test for sex and linear mixed models to account for within-family correlations. We used Tukey contrasts for pair-wise comparisons. RESULTS: Asymptomatic MAPT mutation carriers had lower neuronal marker N-acetylaspartate (NAA)/creatine (Cr) (p = 0.001) and lower NAA/myo-inositol (mI) (p = 0.026) than noncarriers after adjustment for age. Symptomatic MAPT mutation carriers had lower NAA/Cr (p = 0.01) and NAA/mI (p = 0.01) and higher mI/Cr (p = 0.02) compared to noncarriers after adjustment for age. Furthermore, NAA/Cr (p = 0.006) and NAA/mI (p < 0.001) ratios decreased, accompanied by an increase in mI/Cr ratio (p = 0.001), as the ages of carriers approached and passed the age at symptom onset. CONCLUSION: Frontal lobe neurochemical alterations measured with 1H MRS precede the symptom onset in MAPT mutation carriers. Frontal lobe 1H MRS is a potential biomarker for early neurodegenerative processes in MAPT mutation carriers.

Combined Transcriptomics and Proteomics in Frontal Cortex Area 8 in Frontotemporal Lobar Degeneration Linked to C9ORF72 Expansion.

BACKGROUND: Frontotemporal lobar degeneration with TDP-43 immunoreactive inclusions (FTLD-TDP) may appear as sporadic (sFTLD-TDP) or linked to mutations in various genes including expansions of the non-coding region of C9ORF72 (c9FTLD). OBJECTIVE: Analysis of differential mRNA and protein expression in the frontal cortex in c9FLTD and evaluation with previous observations in frontal cortex in sFTLD-TDP and amyotrophic lateral sclerosis with TDP-43 inclusions. METHODS: Microarray hybridization and mass spectrometry-based quantitative proteomics followed by RT-qPCR, gel electrophoresis, and western blotting in frontal cortex area 8 in 19 c9FTLD cases and 14 age- and gender-matched controls. RESULTS: Microarray hybridization distinguish altered gene transcription related to DNA recombination, RNA splicing regulation, RNA polymerase transcription, myelin synthesis, calcium regulation, and ubiquitin-proteasome system in c9FTLD; proteomics performed in the same tissue samples pinpoints abnormal protein expression involving apoptosis, inflammation, metabolism of amino acids, metabolism of carbohydrates, metabolism of membrane lipid derivatives, microtubule dynamics, morphology of mitochondria, neuritogenesis, neurotransmission, phagocytosis, receptor-mediated endocytosis, synthesis of reactive oxygen species, and calcium signaling in c9FTLD. CONCLUSION: Transcriptomics and proteomics, as well as bioinformatics processing of derived data, reveal similarly altered pathways in the frontal cortex in c9FTLD, but different RNAs and proteins are identified by these methods. Combined non-targeted '-omics' is a valuable approach to deciphering altered molecular pathways in FTLD provided that observations are approached with caution when assessing human postmortem brain samples.

FDG-PET underscores the key role of the thalamus in frontotemporal lobar degeneration caused by C9ORF72 mutations.

C9ORF72 mutations are the most common cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). MRI studies have investigated structural changes in C9ORF72-associated FTLD (C9FTLD) and provided first insights about a prominent involvement of the thalamus and the cerebellum. Our multicenter, 18F-fluorodeoxyglucose positron-emission tomography study of 22 mutation carriers with FTLD, 22 matched non-carriers with FTLD, and 23 cognitively healthy controls provided valuable insights into functional changes in C9FTLD: compared to non-carriers, mutation carriers showed a significant reduction of glucose metabolism in both thalami, underscoring the key role of the thalamus in C9FTLD. Thalamic metabolism did not correlate with disease severity, duration of disease, or the presence of psychotic symptoms. Against our expectations we could not demonstrate a cerebellar hypometabolism in carriers or non-carriers. Future imaging and neuropathological studies in large patient cohorts are required to further elucidate the central role of the thalamus in C9FTLD.

mTh1 driven expression of hTDP-43 results in typical ALS/FTLD neuropathological symptoms.

Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models.

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation.

Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-ß2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration.

Withania somnifera Reverses Transactive Response DNA Binding Protein 43 Proteinopathy in a Mouse Model of Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration.

Abnormal cytoplasmic mislocalization of transactive response DNA binding protein 43 (TARDBP or TDP-43) in degenerating neurons is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Our previous work suggested that nuclear factor kappa B (NF-κB) may constitute a therapeutic target for TDP-43-mediated disease. Here, we investigated the effects of root extract of Withania somnifera (Ashwagandha), an herbal medicine with anti-inflammatory properties, in transgenic mice expressing a genomic fragment encoding human TDP-43A315T mutant. Ashwagandha extract was administered orally to hTDP-43A315T mice for a period of 8 weeks starting at 64 and 48 weeks of age for males and females, respectively. The treatment of hTDP-43A315T mice ameliorated their motor performance on rotarod test and cognitive function assessed by the passive avoidance test. Microscopy examination of tissue samples revealed that Ashwagandha treatment of hTDP-43A315T mice improved innervation at neuromuscular junctions, attenuated neuroinflammation, and reduced NF-κB activation. Remarkably, Ashwagandha treatment reversed the cytoplasmic mislocalization of hTDP-43 in spinal motor neurons and in brain cortical neurons of hTDP-43A315T mice and it reduced hTDP-43 aggregation. In vitro evidence is presented that the neuronal rescue of TDP-43 mislocalization may be due to the indirect effect of factors released from microglial cells exposed to Ashwagandha. These results suggest that Ashwagandha and its constituents might represent promising therapeutics for TDP-43 proteinopathies.

The truncated C-terminal RNA recognition motif of TDP-43 protein plays a key role in forming proteinaceous aggregates.

TDP-43 is the major pathological protein identified in the cellular inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenic forms of TDP-43 are processed C-terminal fragments containing a truncated RNA-recognition motif (RRM2) and a glycine-rich region. Although extensive studies have focused on this protein, it remains unclear how the dimeric full-length TDP-43 is folded and assembled and how the processed C-terminal fragments are misfolded and aggregated. Here, using size-exclusion chromatography, pulldown assays, and small angle x-ray scattering, we show that the C-terminal-deleted TDP-43 without the glycine-rich tail is sufficient to form a head-to-head homodimer primarily via its N-terminal domain. The truncated RRM2, as well as two ß-strands within the RRM2, form fibrils in vitro with a similar amyloid-negative staining property to those of TDP-43 pathogenic fibrils in diseases. In addition to the glycine-rich region, the truncated RRM2, but not the intact RRM2, plays a key role in forming cytoplasmic inclusions in neuronal cells. Our data thus suggest that the process that disrupts the dimeric structure, such as the proteolytic cleavage of TDP-43 within the RRM2 that removes the N-terminal dimerization domain, may produce unassembled truncated RRM2 fragments with abnormally exposed ß-strands, which can oligomerize into high-order inclusions.

TDP-43 subtypes are associated with distinct atrophy patterns in frontotemporal dementia.

BACKGROUND: We sought to describe the antemortem clinical and neuroimaging features among patients with frontotemporal lobar degeneration with TDP-43 immunoreactive inclusions (FTLD-TDP). METHODS: Subjects were recruited from a consecutive series of patients with a primary neuropathologic diagnosis of FTLD-TDP and antemortem MRI. Twenty-eight patients met entry criteria: 9 with type 1, 5 with type 2, and 10 with type 3 FTLD-TDP. Four patients had too sparse FTLD-TDP pathology to be subtyped. Clinical, neuropsychological, and neuroimaging features of these cases were reviewed. Voxel-based morphometry was used to assess regional gray matter atrophy in relation to a group of 50 cognitively normal control subjects. RESULTS: Clinical diagnosis varied between the groups: semantic dementia was only associated with type 1 pathology, whereas progressive nonfluent aphasia and corticobasal syndrome were only associated with type 3. Behavioral variant frontotemporal dementia and frontotemporal dementia with motor neuron disease were seen in type 2 or type 3 pathology. The neuroimaging analysis revealed distinct patterns of atrophy between the pathologic subtypes: type 1 was associated with asymmetric anterior temporal lobe atrophy (either left- or right-predominant) with involvement also of the orbitofrontal lobes and insulae; type 2 with relatively symmetric atrophy of the medial temporal, medial prefrontal, and orbitofrontal-insular cortices; and type 3 with asymmetric atrophy (either left- or right-predominant) involving more dorsal areas including frontal, temporal, and inferior parietal cortices as well as striatum and thalamus. No significant atrophy was seen among patients with too sparse pathology to be subtyped. CONCLUSIONS: FTLD-TDP subtypes have distinct clinical and neuroimaging features, highlighting the relevance of FTLD-TDP subtyping to clinicopathologic correlation.

Quantitative proton magnetic resonance spectroscopy detects abnormalities in dorsolateral prefrontal cortex and motor cortex of patients with frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disease of the frontal and temporal neocortex. The single most common pathology underlying FTLD is neuronal degeneration with ubiquitin-positive but tau-negative inclusions consisting of Tar DNA binding proteins (TDP-43). Inclusions containing TDP-43 in neurons are also the most common pathology underlying motor neuron disease (MND). The present study tested the hypothesis that abnormal metabolite patterns within the dorsolateral prefrontal cortex (DLPFC) as well as the motor cortex (MC) may be observed in FTLD patients without motor disorders, using proton magnetic resonance spectroscopy ((1)H MRS). Twenty-six FTLD patients with cognitive damage and ten controls underwent multivoxel (1)H MRS. Absolute concentrations of N-acetyl aspartate (NAA), creatine (Cr), choline (Cho) and myo-inositol (mI) were measured from the DLPFC, the MC and the parietal cortex (PC, an internal control). Statistical analyses were performed for group differences between FTLD patients and controls. Comparisons were also made across brain regions (PC and DLPFC; PC and MC) within FTLD patients. Significant reductions in NAA and Cr along with increased Cho and mI were observed in the DLPFC of FTLD patients compared to controls. Significantly lower NAA and higher Cho were also observed in the MCs of patients as compared to controls. Within the FTLD patients, both the MC and the DLPFC exhibited significantly decreased NAA and elevated Cho compared to the PC. However, only the DLPFC had significantly lower Cr and higher mI. Abnormal metabolite pattern from the MC supports the hypothesis that FTLD and MND may be closely linked.