Analysis of ER stress and unfolded protein response markers in <i>nclf</i> mice.

<p>Cerebellar extracts from wild-type and <i>nclf</i> mice of 4, 12, 21, and 43 weeks of age were analyzed for eIF2alpha phosphorylation and for Hsp70 and BiP expression by western blotting. No major differences were detectable between the genotypes. Tubulin was used as loading control. Representative blots of three mice of different litters are shown.</p

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

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

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