Activation of microglia in <i>nclf</i> brain.

<p>Immunohistochemical stainings of sagittal mouse brain sections showed a prominent microgliosis as assessed by the microglial marker CD68 at 54 weeks of age in <i>nclf</i> and wild-type (wt) mice. Scale bars. 500 µm. In the right panel, higher magnification images of the areas marked by the black rectangles are shown.</p

Accumulation of autophagosomes in <i>nclf</i> brain and hippocampal neurons.

<p>A) Accumulation of autophagosomes was assessed by determining the levels of the autophagic marker protein microtubule-associated protein 1 light chain 3 -II (LC3-II) in wild-type or <i>nclf</i> total mouse brain extracts at 54 weeks of age by western blotting. B) Densitometric quantification of LC3-II levels normalized to tubulin as a loading control revealed enhanced autophagosome numbers with increasing age. Data are presented as mean ±SD, n = 3 per age. Wild-type values were set to 1. C) Double-membrane autophagic vacuoles (marked by arrows) were also found in hippocampal neurons from <i>nclf</i> mice cultured for 14 days. D) For quantification of autophagic vacuoles, pictures were taken from 37 randomly selected wild-type and <i>nclf</i> neurons of two different preparations. The number of autophagic vacuoles related to the cytoplasmic area was determined. Data are presented as mean ± S.E.M. Scale bars: 1 µm.</p

Mutant GFP-Cln6 is rapidly degraded by proteasomes.

<p>BHK cells overexpressing murine wild-type or mutant p.R103PfsX62 GFP-Cln6 (mut) were labelled for 24 hours with [³⁵S]-methionine (75 µCi/ml) and either harvested or chased for 3 (lanes 1–4) and 24 hours (lanes 5–8) in the absence (–) or presence (+) of the proteasomal inhibitor epoxomicin (2 µM). GFP-Cln6 fusion proteins were immunoprecipitated, separated by SDS-PAGE (10% acrylamide) and revealed by fluorography. A representative experiment out of three is shown. The [³⁵S]-labelled bands of the presented experiment were excised from the gel, solubilized and counted. The values are given above the lanes.</p

Localized astrocytosis in <i>nclf</i> mice.

<p>Immunohistochemical staining for glial fibrillary associated protein (GFAP) revealed pronounced upregulation of this marker of astrocytosis in 21 weeks old <i>nclf</i> mice compared to age-matched wild-type controls (wt). Intense localized astrocytosis was evident in the ventral posterior (VPM/VPL) and dorsal lateral geniculate (LGNd) relay nuclei of the thalamus of <i>nclf</i> mice, with more diffuse scattered GFAP positive astrocytes present predominantly in deeper (V-VI) and more superficial (I-III) laminae of the cortical mantle. Compared to wt controls, many GFAP stained astrocytes were also evident in the caudate-putamen of <i>nclf</i> mice. Laminar boundaries are indicated by roman numerals and white dashed lines indicate the boundaries of thalamic relay nuclei. Scale bar: 100 µm.</p

Aggregates of p62 and ubiquitinated proteins in brains of <i>nclf</i> mice.

<p>A) Immunohistochemical analysis of brain sections (35 µm thickness) of 54 weeks old mouse showed p62-positive aggregates in <i>nclf</i> but not wild-type brain regions. The insets show higher magnification images of the areas marked by the white rectangles. Scale bar: 20 µm. B) p62-positive accumulations showed no colocalization with microglial marker CD68, astrocytic marker GFAP or the lysosomal membrane protein Lamp-1 as determined by immunofluorescence microscopy in sections of the olfactory bulb in 54 weeks old <i>nclf</i> mice. Scale bar: 20 µm. C) Western blot analysis confirmed increased p62 levels and show furthermore accumulation of ubiquitinated proteins in Triton X-100 insoluble fractions of <i>nclf</i> brain.</p

Storage material in <i>nclf</i> mouse brain.

<p>A) Sagittal mouse brain sections were stained with Nissl staining, showing cerebellar atrophy in the <i>nclf</i> brain. B) Autofluorescent storage material was evident in cerebellum, thalamus, hippocampus and cortex of 54 weeks old <i>nclf</i> mice but not in age-matched wild-type controls. The selected areas are shown in panel A (a, b, c, d). Scale bars: 300 µm. C) Ultrastructural analysis showed storage material in cerebellum and hippocampus of the brain of 52 weeks old <i>nclf</i> mice. Scale bar: 2 µm.</p