Neuronal sensitivity to TDP-43 overexpression is dependent on timing of induction

Ubiquitin-immunoreactive neuronal inclusions composed of TAR DNA binding protein of 43 kDa (TDP-43) are a major pathological feature of frontotemporal lobar degeneration (FTLD-TDP). In vivo studies with TDP-43 knockout mice have suggested that TDP-43 plays a critical, although undefined role in development. In the current report, we generated transgenic mice that conditionally express wild-type human TDP-43 (hTDP-43) in the forebrain and established a paradigm to examine the sensitivity of neurons to TDP-43 overexpression at different developmental stages. Continuous TDP-43 expression during early neuronal development produced a complex phenotype, including aggregation of phospho-TDP-43, increased ubiquitin immunoreactivity, mitochondrial abnormalities, neurodegeneration and early lethality. In contrast, later induction of hTDP-43 in the forebrain of weaned mice prevented early death and mitochondrial abnormalities while yielding salient features of FTLD-TDP, including progressive neurodegeneration and ubiquitinated, phospho-TDP-43 neuronal cytoplasmic inclusions. These results suggest that neurons in the developing forebrain are extremely sensitive to TDP-43 overexpression and that timing of TDP-43 overexpression in transgenic mice must be considered when distinguishing normal roles of TDP-43, particularly as they relate to development, from its pathogenic role in FTLD-TDP and other TDP-43 proteinopathies. Finally, our adult induction of hTDP-43 strategy provides a mouse model that develops critical pathological features that are directly relevant for human TDP-43 proteinopathies

Global Transcriptional Response of <i>Methylorubrum extorquens</i> to Formaldehyde Stress Expands the Role of EfgA and Is Distinct from Antibiotic Translational Inhibition

The potency and indiscriminate nature of formaldehyde reactivity upon biological molecules make it a universal stressor. However, some organisms such as Methylorubrum extorquens possess means to rapidly and effectively mitigate formaldehyde-induced damage. EfgA is a recently identified formaldehyde sensor predicted to halt translation in response to elevated formaldehyde as a means to protect cells. Herein, we investigate growth and changes in gene expression to understand how M. extorquens responds to formaldehyde with and without the EfgA-formaldehyde-mediated translational response, and how this mechanism compares to antibiotic-mediated translation inhibition. These distinct mechanisms of translation inhibition have notable differences: they each involve different specific players and in addition, formaldehyde also acts as a general, multi-target stressor and a potential carbon source. We present findings demonstrating that in addition to its characterized impact on translation, functional EfgA allows for a rapid and robust transcriptional response to formaldehyde and that removal of EfgA leads to heightened proteotoxic and genotoxic stress in the presence of increased formaldehyde levels. We also found that many downstream consequences of translation inhibition were shared by EfgA-formaldehyde- and kanamycin-mediated translation inhibition. Our work uncovered additional layers of regulatory control enacted by functional EfgA upon experiencing formaldehyde stress, and further demonstrated the importance this protein plays at both transcriptional and translational levels in this model methylotroph

Neuropathology of 25 month old iTDP-43^8A mice.

<p>Immunohistochemical detection of ubiquitin revealed rare cells bearing increased ubiquitin staining in the cortex of iTDP-43^8A mice (arrows, A) that was absent in NT animals (B, scale bar = 200 µm). Staining was detected in both nucleus and cytoplasm of affected cells (inset in A, scale bar = 10 µm). (C) In iTDP-43^8A animals hTDP-43 was predominantly nuclear, some cells displaying cytoplasmic localization without aggregation. Cytoplasmic localization was observed in NT and iTDP-43^8A mice using antibodies to total TDP-43 (tTDP-43 Ab1) and phosphorylated forms of TDP-43 (p403/404, p409/410).</p

Biochemistry of iTDP-43^14A brain lysates at P5.

<p>(A) Western blotting using two antibodies to total TDP-43 (tTDP-43 Ab1 and tTDP-43 Ab2) demonstrated increased levels of low molecular weight species at 35 kDa (arrow) and 25 kDa (arrowhead) in iTDP-43^14A mice relative to NT mice. These species were not observed using antibodies to the C-terminus (405–414) or N-terminus (3–12) of TDP-43. (B) Western blot analysis of high salt (HS), myelin floatation buffer (MFB), sarkosyl (SARK) and urea fractions using antibody to human TDP-43. Note that human TDP-35 (arrow) is present in the urea fraction but is absent from MFB and SARK fractions, N = non-transgenic, T = iTDP-43^14A. (C) Antibody to murine Tdp-43 demonstrated reduction of mTdp-43 in brain compared to NT mice. (D) Quantification of blot in (C), **<i>p</i><0.01, unpaired two tailed <i>t-</i>tes<i>t</i>.</p

Early degenerative phenotype in iTDP-43^14A mice at P5 in the absence of FTLD-like TDP-43 aggregation.

<p>(A) Monoclonal antibody to human TDP-43 showed expression at P5 remained restricted to previously characterized regions of hippocampus, cortex and striatum. (B) Western blotting of brain lysate of P5 non-transgenic (NT) and iTDP-43^14A demonstrated increased levels of activated caspase 3 in iTDP-43^14A mice. (C) Abundant caspase 3 immunoreactivity in the cortex of iTDP-43^14A mice that was virtually absent in NT mice, suggestive of elevated cell death in iTDP43^14A compared to NT mice. iTDP-43^14A mice were also characterized by increased ubiquitin staining in the upper layers of the cortex compared to NT mice, which upon higher magnification appeared to be completely diffuse and cytoplasmic. (D) Immunohistochemistry for hTDP-43 and p403/404 and immunofluorescence using antibodies to total TDP-43 and p409/410 TDP-43. Significant amounts of cytoplasmic hTDP-43 were observed in iTDP-43 mice (arrowheads). Note that this cytoplasmic staining was also observed in NT mice (arrowheads) with antibodies to total TDP-43 (tTDP-43 Ab1) and TDP-43 phosphorylated at 409/410 (p409/410). Scale bars in D = 50 µm.</p

Expression of human TDP-43 in iTDP-43^14A and iTDP-43^8A mice in the postnatal period.

<p>Immunohistochemical detection of hTDP-43 expression in cortex (CTX), hippocampus (HIP) and striatum (STR) in iTDP-43^14A (A) and iTDP-43^8A (B). Western analysis of organs demonstrated specificity of hTDP-43 expression to the brain in both iTDP-43^14A (C) and iTDP-43^8A (D) (SC = spinal cord, He = heart, Lu = lung, Li = liver, Ki = kidney, St = stomach, SM = skeletal muscle, Sp = spleen, Br = brain). (E) Brain weight measurement of non-transgenic (NT) and iTDP-43^14A mice at postnatal stages until 2 months of age (P60) (*<i>p</i><0.05, **<i>p</i><0.01, *** p<0.001, unpaired two tailed <i>T-test</i>). (F) Expression of hTDP-43 at indicated postnatal time points for iTDP-43^14A. (G) Expression of hTDP-43 at indicated postnatal time points for iTDP-43^14A (14) compared to iTDP-43^8A (8).</p