CLN7 disease is an autosomal recessive, childhood-onset neurodegenerative lysosomal storage disorder caused by the defective lysosomal membrane protein CLN7. We have disrupted the Cln7/Mfsd8 gene in mice by targeted deletion of exon 2 generating a novel knockout (KO) mouse model for CLN7 disease, which recapitulates key features of human CLN7 disease pathology. Cln7 KO mice showed increased mortality and a neurological phenotype including hind limb clasping and myoclonus. Lysosomal dysfunction in the brain of mutant mice was shown by the storage of autofluorescent lipofuscin-like lipopigments, subunit c of mitochondrial ATP synthase and saposin D and increased expression of lysosomal cathepsins B, D and Z. By immunohistochemical co-stainings, increased cathepsin Z expression restricted to Cln7-deficient microglia and neurons was found. Ultrastructural analyses revealed large storage bodies in Purkinje cells of Cln7 KO mice containing inclusions composed of irregular, curvilinear and rectilinear profiles as well as fingerprint profiles. Generalized astrogliosis and microgliosis in the brain preceded neurodegeneration in the olfactory bulb, cerebral cortex and cerebellum in Cln7 KO mice. Increased levels of LC3-II and the presence of neuronal p62- and ubiquitin-positive protein aggregates suggested that impaired autophagy represents a major pathomechanism in the brain of Cln7 KO mice. The data suggest that loss of the putative lysosomal transporter Cln7 in the brain leads to lysosomal dysfunction, impaired constitutive autophagy and neurodegeneration late in disease.

No abstract

PURPOSE. Neuronal ceroid lipofuscinoses comprise a genetically heterogeneous group of mainly childhood-onset neurodegenerative lysosomal storage disorders. Progressive loss of vision is among the typical clinical symptoms of these fatal disorders. Here, we performed a detailed analysis of retinal degeneration in mice deficient in the lysosomal membrane protein CLN7, a novel animal model of CLN7 disease.METHODS. Immunohistochemical analyses of retinas at different ages were performed to qualitatively and quantitatively characterize retinal degeneration in CLN7-deficient mice. Storage material in mutant retinas was analyzed by electron microscopy, and expression levels of various lysosomal proteins were studied using immunohistochemistry, immunoblot analyses, and quantitative real-time PCR.RESULTS. We observed an early onset and rapidly progressing degeneration of photoreceptor cells in CLN7-deficient mice, resulting in the loss of more than 70% rod photoreceptors in 4-month-old animals. The number of cone photoreceptors was not detectably altered at this age. Loss of rod photoreceptors was accompanied by reactive astrogliosis and microgliosis. Immunohistochemical and immunoblot analyses revealed accumulation of subunit c of mitochondrial ATP synthase and saposin D in mutant retinas, and electron microscopic analyses demonstrated the presence of curvilinear bodies or fingerprint-like profiles in various cell types of CLN7-deficient retinas. We also found a marked dysregulation of various lysosomal proteins in mutant retinas. CONCLUSIONS.We conclude that the retina of CLN7-deficient mice represents a useful model to elucidate the pathomechanisms ultimately leading to neurodegeneration in CLN7 disease, and to evaluate the efficacy of strategies aimed at developing treatments for this fatal neurodegenerative lysosomal storage disorder.

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

Costello syndrome (CS) is caused by heterozygous germline HRAS mutations. Most patients share the HRAS mutation c.34G>A (p.Gly12Ser) associated with the typical, relatively homogeneous phenotype. Rarer mutations occurred in individuals with an attenuated phenotype. Although many disease-associated HRAS alterations trigger constitutive activation of HRAS-dependent signalling pathways, additional pathological consequences exist. An infant with failure-to-thrive and hypertrophic cardiomyopathy had a novel de novo HRAS mutation (c.179G>T; p.Gly60Val). He showed subtle dysmorphic findings consistent with attenuated CS and died from presumed cardiac cause. Functional studies revealed that amino acid change p.Gly60Val impairs HRAS binding to effectors PIK3CA, phospholipase C1, and RAL guanine nucleotide dissociation stimulator. In contrast, interaction with effector rapidly accelerated fibrosarcoma (RAF) and regulator NF1 GTPase-activating protein was enhanced. Importantly, expression of HRAS p.Gly60Val in HEK293 cells reduced growth factor sensitivity leading to damped RAF-MAPK and phosphoinositide 3-kinases-AKT signalling response. Our data support the idea that a variable range of dysregulated HRAS-dependent signalling dynamics, rather than static activation of HRAS-dependent signal flow, may underlie the phenotypic variability in CS.