Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process.

Palmitoyl protein thioesterase (PPT) localizes into synaptosomes and synaptic vesicles in neurons: implications for infantile neuronal ceroid lipofuscinosis (INCL).

A deficiency of palmitoyl protein thioesterase (PPT) leads to the neurodegenerative disease infantile neuronal ceroid lipofuscinosis (INCL), which is characterized by an almost complete loss of cortical neurons. PPT expressed in COS-1 cells is recognized by the mannose-6-phosphate receptor (M6PR) and is routed to lysosome, but a substantial fraction of PPT is secreted. We have here determined the neuronal localization of PPT by confocal microscopy, cryoimmunoelectron microscopy and cell fractionation. In mouse primary neurons and brain tissue, PPT is localized in synaptosomes and synaptic vesicles but not in lysosomes. Furthermore, in polarized epithelial Caco-2 cells, PPT is localized exclusively to the basolateral site, in contrast to the classical lysosomal enzyme, aspartylglucosaminidase (AGA), which is localized in the apical site. The current data imply that PPT has a role outside the lysosomes in the brain and may be associated with synaptic functioning. This finding opens a new route to study the neuropathological events associated with INCL.

Role of adenine nucleotide translocator 1 in mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

The neuronal ceroid lipofuscinosis CLN8 membrane protein is a resident of the endoplasmic reticulum.

Progressive epilepsy with mental retardation (EPMR) is a new member of the neuronal ceroid lipofuscinoses (NCLs). The CLN8 gene underlying EPMR was recently identified. It encodes a novel 286 amino acid transmembrane protein that contains an endoplasmic reticulum (ER)-retrieval signal (KKRP) in its C-terminus. A homozygous mutation in the orthologous mouse gene (Cln8) underlies the phenotype of a naturally occurring NCL model, the motor neuron degeneration mouse (mnd). To characterize the product of the CLN8 gene and to determine its intracellular localization, we expressed CLN8 cDNA in BHK, HeLa and CHO cell lines. In western blotting and pulse-chase analyses an approximately 33 kDa protein that does not undergo proteolytic processing steps was detected. Using CLN8 and cell organelle specific antibodies with confocal immunofluorescence microscopy the CLN8 protein was shown to localize in the ER. Partial localization to the ER-Golgi intermediate compartment (ERGIC) was also observed. The ER-ERGIC localization was not altered in the CLN8 protein representing the EPMR mutation. However, mnd mutant protein was only found in the ER. Mutations in the ER retrieval signal KKRP resulted in localization of CLN8 to the Golgi apparatus. Taken together, these data strongly suggest that CLN8 is an ER resident protein that recycles between ER and ERGIC.

Expression of palmitoyl protein thioesterase in neurons.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder in childhood that is caused by mutations in the gene encoding lysosomal palmitoyl protein thioesterase (PPT). INCL is characterized by massive and selective loss of cortical neurons. Here we have analyzed the intracellular processing and localization of adenovirus-mediated PPT in mouse primary neurons and NGF-induced PC-12 cells. The neuronal processing of PPT was found to be similar to that observed in peripheral cells, and a significant amount of the PPT enzyme was secreted in the primary neurons. Immunofluorescence analysis of the neuronal cells infected with wild-type PPT showed a granular staining pattern in the cell soma and neuronal shafts. Interestingly, PPT was also found in the synaptic ends of the neuronal cells and the staining pattern of the enzyme colocalized to a significant extent with the synaptic markers SV2 and synaptophysin. These in vitro data correspond with the distribution of endogeneous PPT in mouse brain and suggest that PPT may not solely be a lysosomal hydrolase. The specific targeting of PPT into the neuritic shafts and nerve terminals indicates that PPT may be associated with the maintenance of synaptic function. Furthermore, since a substantial amount of PPT is secreted by neurons, it is tempting to speculate that the enzyme could also have an extracellular substrate.

Defective intracellular transport of CLN3 is the molecular basis of Batten disease (JNCL)

Batten disease [juvenile-onset neuronal ceroid lipofuscinosis (JNCL)], the most common progressive encephalopathy of childhood, is caused by mutations in a novel lysosomal membrane protein (CLN3) with unknown function. In this study, we have confirmed the lysosomal localization of the CLN3 protein by immunoelectron microscopy by co-localizing it with soluble and membrane-associated lysosomal proteins. We have analysed the intracellular processing and localization of two mutants, 461-677del, which is present in 85% of CLN3 alleles and causes the classical JNCL, and E295K [corrected], which is a rare missense mutation associated with an atypical form of JNCL. Pulse-chase labelling and immunoprecipitation of the two mutant proteins in COS-1-cells indicated that 461-677del is synthesized as an approximately 24 kDa truncated polypeptide, whereas the maturation of E295K [corrected] resembles that of the wild-type CLN3 polypeptide. Transient expression of the two mutants in BHK cells showed that 461-677del is retained in the endoplasmic reticulum, whereas E295K [corrected] was capable of reaching the lysosomal compartment. The CLN3 polypeptides were expressed further in mouse primary neurons where the wild-type CLN3 protein was localized both in the cell soma and in neuronal extensions, whereas the 461-677del mutant was arrested in the cell soma. Interestingly, co-localization of the wild-type CLN3 and E295K [corrected] proteins with a synaptic vesicle marker indicates that the CLN3 protein might participate in synaptic vesicle transport/transmission. The data presented here provide clear evidence for a cellular distinction between classical and atypical forms of Batten disease both in neural and non-neural cells.

Expression and endocytosis of lysosomal aspartylglucosaminidase in mouse primary neurons.

Aspartylglucosaminuria (AGU) is a neurodegenerative lysosomal storage disease that is caused by mutations in the gene encoding for a soluble hydrolase, aspartylglucosaminidase (AGA). In this study, we have used our recently developed mouse model for AGU and analyzed processing, intracellular localization, and endocytosis of recombinant AGA in telencephalic AGU mouse neurons in vitro. The processing steps of AGA were found to be similar to the peripheral cells, but both the accumulation of the inactive precursor molecule and delayed lysosomal processing of the enzyme were detected. AGA was distributed to the cell soma and neuronal processes but was not found in the nerve terminals. Endocytotic capability of cultured telencephalic neurons was comparable to that of fibroblasts, and endocytosis of AGA was blocked by free mannose-6-phosphate (M6P), indicating that uptake of the enzyme was mediated by M6P receptors (M6PRs). Uptake of extracellular AGA was also studied in the tumor-derived cell lines rat pheochromocytoma (PC12) and mouse neuroblastoma cells (N18), which both endocytosed AGA poorly as compared with cultured primary neurons. Expression of cation-independent M6PRs (CI-M6PRs) in different cell lines correlated well with the endocytotic capability of these cells. Although a punctate expression pattern of CI-M6PRs was found in fibroblasts and cultured primary neurons, the expression was beyond the detection limit in PC12 and N18 cells. This indicates that PC12 and N18 are not feasible cell lines to describe neuronal uptake of mannose-6-phosphate-tagged proteins. This in vitro data will form an important basis for the brain-targeted therapy of AGU.

Adenovirus-mediated gene transfer results in decreased lysosomal storage in brain and total correction in liver of aspartylglucosaminuria (AGU) mouse.

Aspartylglucosaminuria (AGU) is a lysosomal storage disease leading to mental retardation, which is caused by deficiency of aspartylglucosaminidase (AGA). AGU is strongly enriched in the Finnish population in which one major mutation called AGU(Fin) has been identified. The molecular pathogenesis of AGU as well as the biology of the AGA enzyme have been extensively studied, thus giving a profound basis for therapeutic interventions. In this study we have performed adenovirus-mediated gene transfer to the recently produced mouse model of AGU, which exhibits similar pathophysiology as that in humans. Recombinant adenovirus vectors encoding for the human AGA and AGU(Fin) polypeptides were first applied in primary neurons of AGU mouse to demonstrate wild-type and mutant AGA expression in vitro. In vivo, both of the adenovirus vectors were injected into the tail vein of AGU mice and the expression of AGA was demonstrated in the liver. The adenovirus vectors were also injected intraventricularly into the brain of AGU mice resulting in AGA expression in the ependymal cells lining the ventricles and further, diffusion of AGA into the neighbouring neurons. Also, AGA enzyme injected intraventricularly was shown to transfer across the ependymal cell layer. One month after administration of the wild-type Ad-AGA, a total correction of lysosomal storage in the liver and a partial correction in brain tissue surrounding the ventricles was observed. After administration of the Ad-AGU virus the lysosomal storage vacuoles in liver or brain remained unchanged. These data demonstrate that the lysosomal storage in AGU can be biologically corrected and furthermore, in the brain a limited number of transduced cells can distribute AGA enzyme to the surrounding areas.