Mutations in DNAJC5, encoding cysteine-string protein alpha, cause autosomal-dominant adult-onset neuronal ceroid lipofuscinosis

Abstract

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.

Figure 1

Pedigree and Neuropathology Findings in Family P1 (A) Pedigree of the Czech family. Black symbols denote affected individuals; open symbols denote unaffected individuals. Age of onset is shown above current age or age of death (indicated by †). (B) Epifluorescence. Hippocampal pyramidal neurons with prominent lysosomal storage of autofluorescent material representing the general neurolysosomal storage pattern in the brain cortex. The autofluorescence was demonstrated with the filter block with an excitation wavelength of 400–440 nm. (C) Electron micrograph. GROD-type ultrastructure of the storage lysosomes.

Figure 2

Identification of DNAJC5 Mutations (A) A whole-genome parametric linkage analysis showing candidate regions reaching the theoretical maximum LOD scores of 2.1 attainable in this family on chromosomes 1 (1: 233,697,529–249,250,621), 4 (4: 23,561,661–28,920,119), 15 (15: 39,049,915–61,382,423; 65,139,935–67,296,086; 71,515,415–78,819,152), 20 (20:53,448,624–63,025,520), and 22 (22: 1–21,982,248). All coordinates refer to hg19. (B) Gene-expression changes in leucocytes from four affected individuals compared to those of four controls. The logarithm of the probability that the gene is differentially expressed (log odds) is plotted as a function of the logarithm of the gene-expression fold change (log fold change) between the patient and control samples. Differentially expressed genes located in the candidate regions are shown as red dots, and DNAJC5 is specifically indicated. The list of differentially expressed genes located within the linked regions is, together with log fold changes and corresponding t test values, p-values and adjusted p-values, provided in Supplemental Data. (C) Chromatograms of DNAJC5 genomic DNA sequences showing identified heterozygous mutations. (Upper panel) Sequence of an unaffected individual, (middle panel) sequence showing heterozygous mutation c.346_348delCTC in the proband from family P1, and (lower panel) sequence showing heterozygous mutation c.344T>G in the proband from family N1.

Figure 3

In Silico Analysis of Properties of the Cysteine-String Domain (A) p.Leu115Arg mutation decreases the hydrophobicity of this domain, which is needed for initial binding to the ER. (B) The p.Leu116del mutation decreases the palmitoylation score, that is, the confidence that cysteine residues adjacent to Leu116 might be efficiently palmitoylated.

Figure 4

Characterization of Mutated CSPα (A) Immunofluorescence analysis of transiently expressed EGFP-CSPα proteins in CAD5 cells showing prominent membrane localization of wild-type CSPα compared to the diffuse cytoplasmic staining and marked colocalization of mutated CSPα with endoplasmic reticulum represented by PDI and Golgi apparatus represented by Golgi-SNARE of 28 kDa (GS28). (B) Immunoblot analysis of transiently expressed EGFP-CSPα proteins showing higher levels of nonpalmitoylated protein precursors for mutant proteins compared the wild-type (wt) protein. (C) Immunoblot analysis of brain homogenates showing no soluble CSPα and the marked presence of CSPα-containing beta-mercaptoethanol (β-ME)-resistant aggregate (indicated by the asterisk) released upon hydroxylamine (HA) treatment in the affected individual (NCL4) compared to the brain homogenates of the control. (D) Immunohistochemistry analysis of CSPα in gray matter of the cerebral cortex showing, at a low field, a significant decrease of CSPα in affected individuals compared to the strong CSPα staining in the age-matched control.

Figure 5

Brain Immunohistochemistry (A–C) Detail of the CSPα staining in neuropil in the cerebral cortex that is absent in the individual with mutation p.Leu115Arg (A), decreased in the individual with mutation p.Leu116del (B), and strong in the age-matched control (C). Note the prominent neuronal storage, shown by large cell bodies, in both affected individuals. (D–F) Staining pattern of the synaptic marker synaptobrevin in neuropil in the same regions in the individual with mutation p.Leu115Arg (D), in the individual with mutation p.Leu116del (E), and in the age-matched control (F). (G and H) Cerebellar cortex of the case with p.Leu115Arg mutation. Similar to that in the cerebral cortex, CSPα staining is absent (G). This contrasts with a strong signal for synaptobrevin in the corresponding area in all three cerebellar cortical layers that are preserved adjacent to areas undergoing neurodegeneration (H). (I) Strong CSPα staining in a control cerebellum. Note that the CSPα signal in the control (I) as well as the synaptobrevin signal in the individual with mutation p.Leu115Arg (H) are confined to the well defined synaptic regions (i.e., to the dendrites in the molecular layer, to the surface of the Purkinje cells, and to the synaptic glomeruli in the granular cell layer). The scale bars represent 25 μm.