PMC3655113_01
PMC3655113_02
Structural MRI comparison. MR images of Patient 1 (A, B), an apoE epsilon4 homozygote, taken 1 year after initial presentation, showed gray matter atrophy in FTLD-vulnerable regions, including the lateral and medial prefrontal cortex, anterior cingulate cortex, and frontoinsulae (A, arrow), as well as thinning of the anterior corpus callosum (B, arrowhead) and ex vacuo ventricular enlargement. MR images of Patient 2 (C, D), an apoE epsilon3 homozygote, taken 2 years after initial presentation, showed less significant atrophy in these regions. R = right, L = left.
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F1
Patient 2 (Figure 1). Patient 2 had focal precentral gyrus atrophy, consistent with her predominantly motor presentation.
Histological and biochemical analysis. Immunohistochemistry with an antibody to human transactive response DNA-binding protein 43 (TDP-43) (polyclonal rabbit, Proteintech Group, #10782-2-AP, 1:2000) revealed neuronal cytoplasmic inclusions with absence of nuclear staining (A, arrowhead; arrow indicates adjacent neuron with normal nuclear TDP-43 staining) in the anterior cingulate cortex (ACC) (A, Patient 1), hippocampal dentate gyrus (DG) granule cells (B, Patient 1, C, Patient 2) and in bulbar motor nuclei, including the nucleus ambiguous (D, Patient 1) and spinal accessory nucleus (E, Patient 2). Western blotting of detergent-solubilized pellet fractions of the ACC (10 mug of protein per lane) with anti-human apoE (polyclonal goat, Calbiochem, 1:8000) revealed similar amounts of full-length apoE in both patients; however, Patient 1's ACC contained more apoE fragments and high-molecular-weight complexes (F, left panel). Membranes were stripped and re-probed with anti-human TDP-43 (1:600). Several high-molecular-weight complexes positive for apoE also contained TDP-43; these apoE-TDP-43 complexes were more abundant in Patient 1's ACC (F, right panel). Interactions between apoE and TDP-43 were confirmed by co-immunoprecipitation (G). Protein lysates (50 mug/reaction) from high detergent buffer samples (4% SDS) were incubated with anti-human TDP-43 antibody (2 mul/reaction) and magnetic beads pre-coated with Protein A (Pierce #21348, 50 mul/reaction) at 4 C for 12 hours. The precipitated proteins were analyzed by western blotting with anti-human apoE (1:4000) to demonstrate interactions between the two proteins and with anti-human TDP-43 antibody (1:600) to demonstrate pull-down of TDP-43. SDS-stable apoE-TDP-43 complexes were found at ~54 kDa.
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F2
Histological examination revealed neuronal cytoplasmic inclusions containing TDP-43 in the anterior cingulate cortex (ACC), hippocampal dentate gyrus, substantia nigra, and bulbar motor nuclei, confirming the diagnoses of FTLD-TDP and MND in both patients (Figure 2A-E). However, apoE fragments and larger complexes in ACC were more abundant in Patient 1 (Figure 2F). TDP-43. The biochemical interaction between these proteins was confirmed by co-immunoprecipitation (Figure 2G).