Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration

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