Juvenile Neuronal Ceroid Lipofuscinosis Research Articles

Genetic and Cellular Basis of Impaired Phagocytosis and Photoreceptor Degeneration in CLN3 Disease.

CLN3 Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease induced pluripotent stem cell-RPE cells show defective phagocytosis of photoreceptor outer segment (POS). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3Δ7-8/Δ7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3Δ7-8/Δ7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3Δ7-8/Δ7-8 and wild-type miniswine eyes were carried out at 6, 36, or 48 months of age. CLN3Δ7-8/Δ7-8 RPE (CLN3 RPE) displayed decreased POS binding and consequently decreased uptake of POS compared with isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3Δ7-8/Δ7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36months of age and was followed by almost complete loss of photoreceptors at 48months of age. CLN3Δ7-8/Δ7-8 mutation (which affects ≤85% of patients) affects both RPE and POS and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.

Increased SNAI2 expression and defective collagen adhesion in cells with pediatric dementia, juvenile ceroid lipofuscinosis

Dementia-related neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), are known to be caused by accumulation of toxic proteins. However, the molecular mechanisms that cause neurodegeneration and its biophysical effects on cells remain unclear. In this study, we used juvenile neuronal ceroid lipofuscinosis (JNCL), a pediatric dementia with a clear etiology of mutations in ceroid lipofuscinosis neuronal 3 (CLN3), to explore the changes in cell adhesion, a biophysical process that regulates neuronal development and survival. We used JNCL cerebral organoid gene expression datasets to identify the biological pathways that affect neural development, and found enriched gene expression in the epithelial-mesenchymal transition (EMT) pathway and increased expression of its inducer snail family transcriptional repressor 2 (SNAI2). A cell adhesion assay using lymphoblasts from patients with JNCL revealed defective adhesion to cell culture plates, glass surfaces, collagen type I, and neuroblast-like cells. To determine whether inhibition of EMT could improve the cell adhesion of JNCL lymphoblasts, we used all-trans retinoic acid, a well-known EMT inhibitor and inducer of neural differentiation. In JNCL lymphoblasts, ATRA treatment enhanced adhesion to collagen type I and these effects were abolished by Ca2+ chelator. These results provide new insights into the role of CLN3 and cell adhesion in the pathogenesis of NDD.

TRPML1 activation ameliorates lysosomal phenotypes in CLN3 deficient retinal pigment epithelial cells

Mutations in the lysosomal membrane protein CLN3 cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL). Activation of the lysosomal ion channel TRPML1 has previously been shown to be beneficial in several neurodegenerative disease models. Here, we tested whether TRPML1 activation rescues disease-associated phenotypes in CLN3-deficient retinal pigment epithelial (ARPE-19 CLN3-KO) cells. ARPE-19 CLN3-KO cells accumulate LAMP1 positive organelles and show lysosomal storage of mitochondrial ATPase subunit C (SubC), globotriaosylceramide (Gb3), and glycerophosphodiesters (GPDs), whereas lysosomal bis(monoacylglycero)phosphate (BMP/LBPA) lipid levels were significantly decreased. Activation of TRPML1 reduced lysosomal storage of Gb3 and SubC but failed to restore BMP levels in CLN3-KO cells. TRPML1-mediated decrease of storage was TFEB-independent, and we identified TRPML1-mediated enhanced lysosomal exocytosis as a likely mechanism for clearing storage including GPDs. Therefore, ARPE-19 CLN3-KO cells represent a human cell model for CLN3 disease showing many of the described core lysosomal deficits, some of which can be improved using TRPML1 agonists.

Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease.

CLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3 Δ 7-8/ Δ 7-8 ( CLN3 ) Yucatan miniswine was also used to study the impact of CLN3 Δ 7-8/ Δ 7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3 Δ 7/8 and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age. CLN3 Δ 7-8/ Δ 7-8 RPE ( CLN3 RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3 Δ 7-8/ Δ 7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age. CLN3 Δ 7-8/ Δ 7-8 mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.

A recessive CLN3 variant is responsible for delayed-onset retinal degeneration in Hereford cattle.

Thirteen American Hereford cattle were reported blind with presumed onset when ~12-mo-old. All blind cattle shared a common ancestor through both the maternal and paternal pedigrees, suggesting a recessive genetic origin. Given the pedigree relationships and novel phenotype, we characterized the ophthalmo-pathologic changes associated with blindness and identified the responsible gene variant. Ophthalmologic examinations of 5 blind cattle revealed retinal degeneration. Histologically, 2 blind cattle had loss of the retinal photoreceptor layer. Whole-genome sequencing (WGS) of 7 blind cattle and 9 unaffected relatives revealed a 1-bp frameshift deletion in ceroid lipofuscinosis neuronal 3 (CLN3; chr25 g.26043843del) for which the blind cattle were homozygous and their parents heterozygous. The identified variant in exon 16 of 17 is predicted to truncate the encoded protein (p. Pro369Argfs*8) battenin, which is involved in lysosomal function necessary for photoreceptor layer maintenance. Of 462 cattle genotyped, only blind cattle were homozygous for the deletion. A query of WGS data of > 5,800 animals further revealed that the variant was only observed in related Hereford cattle. Mutations in CLN3 are associated with human juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which results in early-onset retinal degeneration and lesions similar to those observed in our cases. Our data support the frameshift variant of CLN3 as causative of blindness in these Hereford cattle, and provide additional evidence of the role of this gene in retinal lesions, possibly as a model for human non-syndromic JNCL.

CLN3 deficiency leads to neurological and metabolic perturbations during early development.

Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

Assessing the integrity of auditory sensory memory processing in CLN3 disease (Juvenile Neuronal Ceroid Lipofuscinosis (Batten disease)): an auditory evoked potential study of the duration-evoked mismatch negativity (MMN)

BackgroundWe interrogated auditory sensory memory capabilities in individuals with CLN3 disease (juvenile neuronal ceroid lipofuscinosis), specifically for the feature of “duration” processing. Given decrements in auditory processing abilities associated with later-stage CLN3 disease, we hypothesized that the duration-evoked mismatch negativity (MMN) of the event related potential (ERP) would be a marker of progressively atypical cortical processing in this population, with potential applicability as a brain-based biomarker in clinical trials.MethodsWe employed three stimulation rates (fast: 450 ms, medium: 900 ms, slow: 1800 ms), allowing for assessment of the sustainability of the auditory sensory memory trace. The robustness of MMN directly relates to the rate at which the regularly occurring stimulus stream is presented. As presentation rate slows, robustness of the sensory memory trace diminishes. By manipulating presentation rate, the strength of the sensory memory trace is parametrically varied, providing greater sensitivity to detect auditory cortical dysfunction. A secondary hypothesis was that duration-evoked MMN abnormalities in CLN3 disease would be more severe at slower presentation rates, resulting from greater demand on the sensory memory system.ResultsData from individuals with CLN3 disease (N = 21; range 6–28 years of age) showed robust MMN responses (i.e., intact auditory sensory memory processes) at the medium stimulation rate. However, at the fastest rate, MMN was significantly reduced, and at the slowest rate, MMN was not detectable in CLN3 disease relative to neurotypical controls (N = 41; ages 6–26 years).ConclusionsResults reveal emerging insufficiencies in this critical auditory perceptual system in individuals with CLN3 disease.

Treatment of non-epileptic episodes of anxious, fearful behavior in adolescent juvenile neuronal ceroid lipofuscinosis (CLN3 disease).

Recurrent non-epileptic episodes of frightened facial and body expression occur in more than half of post-adolescent patients with juvenile neuronal ceroid lipofuscinosis (JNCL, CLN3 disease). Clinically, the episodes look similar to the attacks of paroxysmal sympathetic hyperactivity (PSH) commonly seen following traumatic brain injury (TBI). The episodes occur when the patients are exposed to separation, hear loud sounds or are otherwise bothered by discomfort and as in PSH following TBI, the attacks are difficult to prevent and/or treat. Based on present knowledge of triggering factors, the neural anxiety/fear circuit, its afferent and efferent pathways and documented CLN3 disease-impact on these tracks, the current study discusses a rational approach how to prevent and/or treat the attacks. Patients with JNCL have a disturbed somatosensory modulation leading to a reduced threshold of pain; a degeneration within the neural anxiety/fear circuit leading to an imbalance of central network inhibition and excitation pathways; and finally, an, with advancing age, increasing autonomic imbalance leading to a significant dominance of the sympathetic neural system. Theoretically, there are three points of attack how to prevent or treat the episodes: (1) increase in threshold of discomfort impact; (2) modulation of imbalance of central network inhibition and excitation, and (3) restoring the balance between the sympathetic and parasympathetic neural systems prompted by a parasympathetic withdrawal. As to (1) and (2), prevention should have the greatest priority. As regards (3), research of transcutaneous vagal stimulation treatment in JNCL is warranted.

A mouse mutant deficient in both neuronal ceroid lipofuscinosis-associated proteins CLN3 and TPP1.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) and juvenile neuronal ceroid lipofuscinosis (JNCL) are inherited neurodegenerative diseases caused by mutations in the genes encoding lysosomal proteins tripeptidyl peptidase 1 (TPP1) and CLN3 protein, respectively. TPP1 is well-understood and, aided by animal models that accurately recapitulate the human disease, enzyme replacement therapy has been approved and other promising therapies are emerging. In contrast, there are no effective treatments for JNCL, partly because the function of the CLN3 protein remains unknown but also because animal models have attenuated disease and lack robust survival phenotypes. Mouse models for LINCL and JNCL, with mutations in Tpp1 and Cln3, respectively, have been thoroughly characterized but the phenotype of a double Cln3/Tpp1 mutant remains unknown. We created this double mutant and find that its phenotype is essentially indistinguishable from the single Tpp1-/- mutant in terms of survival and brain pathology. Analysis of brain proteomic changes in the single Tpp1-/- and double Cln3-/- ;Tpp1-/- mutants indicates largely overlapping sets of altered proteins and reinforces earlier studies that highlight GPNMB, LYZ2, and SERPINA3 as promising biomarker candidates in LINCL while several lysosomal proteins including SMPD1 and NPC1 appear to be altered in the Cln3-/- animals. An unexpected finding was that Tpp1 heterozygosity significantly decreased lifespan of the Cln3-/- mouse. The truncated survival of this mouse model makes it potentially useful in developing therapies for JNCL using survival as an endpoint. In addition, this model may also provide insights into CLN3 protein function and its potential functional interactions with TPP1.

Etiology of anxious and fearful behavior in juvenile neuronal ceroid lipofuscinosis (CLN3 disease).

Juvenile neuronal ceroid lipofuscinosis (JNCL, CLN3) is a childhood-onset neurodegenerative disease with prominent symptoms comprising a pediatric dementia syndrome. As in adult dementia, behavioral symptoms like mood disturbances and anxiety are common. In contrast to in adult dementia, however, the anxious behavioral symptoms increase during the terminal phase of JNCL disease. In the present study, the current understanding of the neurobiological mechanisms of anxiety and anxious behavior in general is addressed as will a discussion of the mechanism of the anxious behavior seen in young JNCL patients. Based on developmental behavioral points of view, known neurobiological mechanisms, and the clinical presentation of the anxious behavior, a theory of its etiology is described. During the terminal phase, the cognitive developmental age of JNCL patients is below 2 years. At this stage of mental development individuals act primarily from a concrete world of consciousness and do not have the cognitive ability to encounter a normal anxiety response. Instead, they experience the evolutionary basic emotion of fear, and as the episodes typically are provoked when the adolescent JNCL patient is exposed to either loud sounds, is lifted from the ground, or separated from the mother/known caregiver, the fear can best be perceived as the developmental natural fear-response that appears in children 0-2 years of age. The efferent pathways of the neural fear circuits are mediated through autonomic, neuroendocrine, and skeletal-motor responses. The autonomic activation occurs early, is mediated through the sympathetic and parasympathetic neural systems, and as JNCL patients beyond puberty have an autonomic imbalance with a significant sympathetic hyperactivity, the activation of the autonomic nervous system results in a disproportionate high sympathetic activity resulting in tachycardia, tachypnea, excessive sweating, hyperthermia, and an increased atypical muscle activity. The episodes are thus phenotypically similar to what is seen as Paroxysmal Sympathetic Hyperactivity (PSH) following an acute traumatic brain injury. As in PSH, treatment is difficult and so far, no consensus of a treatment algorithm exists. Use of sedative and analgesic medication and minimizing or avoiding provocative stimuli may partly reduce the frequency and intensity of the attacks. Transcutaneous vagal nerve stimulation might be an option worth to investigate rebalancing the sympathetic-parasympathetic disproportion.

The Batten disease protein CLN3 is important for stress granules dynamics and translational activity

The assembly of membrane-less organelles such as stress granules (SGs) is emerging as central in helping cells rapidly respond and adapt to stress. Following stress sensing, the resulting global translational shutoff leads to the condensation of stalled mRNAs and proteins into SGs. By reorganizing cytoplasmic contents, SGs can modulate RNA translation, biochemical reactions, and signaling cascades to promote survival until the stress is resolved. While mechanisms for SG disassembly are not widely understood, the resolution of SGs is important for maintaining cell viability and protein homeostasis. Mutations that lead to persistent or aberrant SGs are increasingly associated with neuropathology and a hallmark of several neurodegenerative diseases. Mutations in CLN3 are causative of juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease affecting children also known as Batten disease. CLN3 encodes a transmembrane lysosomal protein implicated in autophagy, endosomal trafficking, metabolism, and response to oxidative stress. Using a HeLa cell model lacking CLN3, we now show that CLN3KO is associated with an altered metabolic profile, reduced global translation, and altered stress signaling. Furthermore, loss of CLN3 function results in perturbations in SG dynamics, resulting in assembly and disassembly defects, and altered expression of the key SG nucleating factor G3BP1. With a growing interest in SG-modulating drugs for the treatment of neurodegenerative diseases, novel insights into the molecular basis of CLN3 Batten disease may reveal avenues for disease-modifying treatments for this debilitating childhood disease.

Activation of PPARα Exhibits Therapeutic Efficacy in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal inherited neurodegenerative disease of children that occurs because of defective function of the lysosomal membrane glycoprotein CLN3. JNCL features glial activation and accumulation of autofluorescent storage material containing subunit c of mitochondrial ATP synthase (SCMAS), ultimately resulting into neuronal loss. Until now, no effective therapy is available for JNCL. This study underlines the possible therapeutic importance of gemfibrozil, an activator of peroxisome proliferator-activated receptor α (PPARα) and a lipid-lowering drug approved by the Food and Drug Administration in an animal model of JNCL. Oral gemfibrozil treatment reduced microglial and astroglial activation, attenuated neuroinflammation, restored the level of transcription factor EB (TFEB; the master regulator of lysosomal biogenesis), and decreased the accumulation of storage material SCMAS in somatosensory barrel field (SBF) cortex of Cln3Δex7/8 (Cln3ΔJNCL) mice of both sexes. Accordingly, gemfibrozil treatment also improved locomotor activities of Cln3ΔJNCL mice. While investigating the mechanism, we found marked loss of PPARα in the SBF cortex of Cln3ΔJNCL mice, which increased after gemfibrozil treatment. Oral gemfibrozil also stimulated the recruitment of PPARα to the Tfeb gene promoter in vivo in the SBF cortex of Cln3ΔJNCL mice, indicating increased transcription of Tfeb in the CNS by gemfibrozil treatment via PPARα. Moreover, disease pathologies aggravated in Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCLΔPPARα) and gemfibrozil remained unable to decrease SCMAS accumulation, reduce glial activation, and improve locomotor performance of Cln3ΔJNCLΔPPARα mice. These results suggest that activation of PPARα may be beneficial for JNCL and that gemfibrozil may be repurposed for the treatment of this incurable disease.SIGNIFICANCE STATEMENT Despite intense investigations, no effective therapy is available for JNCL, an incurable inherited lysosomal storage disorder. Here, we delineate that oral administration of gemfibrozil, a lipid-lowering drug, decreases glial inflammation, normalizes and/or upregulates TFEB, and reduces accumulation of autofluorescent storage material in SBF cortex to improve locomotor activities in Cln3Δex7/8 (Cln3ΔJNCL) mice. Aggravation of disease pathology in Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCLΔPPARα) and inability of gemfibrozil to decrease SCMAS accumulation, reduce glial activation, and improve locomotor performance of Cln3ΔJNCLΔPPARα mice delineates an important role of PPARα in this process. These studies highlight a new property of gemfibrozil and indicate its possible therapeutic use in JNCL patients.

Alterations of dopaminergic signalling and metabolism in experimental CLN3 ex7/8 model of juvenile neuronal ceroid lipofuscinosis

Alterations of dopaminergic signalling and metabolism in experimental CLN3 ex7/8 model of juvenile neuronal ceroid lipofuscinosis

Top-down and bottom-up propagation of disease in the neuronal ceroid lipofuscinoses.

The Neuronal Ceroid Lipofuscinoses (NCLs) may be considered distinct neurodegenerative disorders with separate underlying molecular causes resulting from monogenetic mutations. An alternative hypothesis is to consider the NCLs as related diseases that share lipofuscin pathobiology as the common core feature, but otherwise distinguished by different a) initial anatomic location, and b) disease propagation. We have tested this hypothesis by comparing known differences in symptomatology and pathology of the CLN1 phenotype caused by complete loss of PPT1 function (i.e., the classical infantile form) and of the classical juvenile CLN3 phenotype. These two forms of NCL represent early onset and rapidly progressing vs. late onset and slowly progressing disease modalities respectively. Despite displaying similar pathological endpoints, the clinical phenotypes and the evidence of imaging and postmortem studies reveal strikingly different time courses and distributions of disease propagation. Data from CLN1 disease are indicative of disease propagation from the body, with early effects within the spinal cord and subsequently within the brainstem, the cerebral hemispheres, cerebellum and retina. In contrast, the retina appears to be the most vulnerable organ in CLN3, and the site where pathology is first present. Pathology subsequently is present in the occipital connectome of the CLN3 brain, followed by a top-down propagation in which cerebral and cerebellar atrophy in early adolescence is followed by involvement of the peripheral nerves in later adolescence/early twenties, with the extrapyramidal system also affected during this time course. The propagation of disease in these two NCLs therefore has much in common with the "Brain-first" vs. "Body-first" models of alpha-synuclein propagation in Parkinson's disease. CLN1 disease represents a "Body-first" or bottom-up disease propagation and CLN3 disease having a "Brain-first" and top-down propagation. It is noteworthy that the varied phenotypes of CLN1 disease, whether it starts in infancy (infantile form) or later in childhood (juvenile form), still fit with our proposed hypothesis of a bottom-up disease propagation in CLN1. Likewise, in protracted CLN3 disease, where both cognitive and motor declines are delayed, the initial manifestations of disease are also seen in the outer retinal layers, i.e., identical to classical Juvenile NCL disease.

CLN3 is required for the clearance of glycerophosphodiesters from lysosomes.

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.

Serine-Carboxyl Peptidases, Sedolisins: From Discovery to Evolution.

Sedolisin is a proteolytic enzyme, listed in the peptidase database MEROPS as a founding member of clan SB, family S53. This enzyme, although active at low pH, was originally shown not to be inhibited by an aspartic peptidase specific inhibitor, S-PI (pepstatin Ac). In this Perspective, the S53 family is described from the moment of original identification to evolution. The representative enzymes of the family are sedolisin, kumamolisin, and TPP-1. They exhibit the following unique features. (1) The fold of the molecule is similar to that of subtilisin, but the catalytic residues consist of a triad, Ser/Glu/Asp, that is unlike the Ser/His/Asp triad of subtilisin. (2) The molecule is expressed as a pro-form composed of the amino-terminal prosegment and the active domain. Additionally, some members of this family have an additional, carboxy-terminal prosegment. (3) Their optimum pH for activity is in the acidic region, not in the neutral to alkaline region where subtilisin is active. (4) Their distribution in nature is very broad across the three kingdoms of life. (5) Some of these enzymes from fungi and bacteria are pathogens to plants. (6) Some of them have significant potential applications for industry. (7) The lack of a TPP-1 gene in human brain is the cause of incurable juvenile neuronal ceroid lipofuscinosis (Batten's disease).

Pacemaker Implantation in Juvenile Neuronal Ceroid Lipofuscinosis (CLN3)-A Long-Term Follow-Up Study.

It is well documented that deteriorating heart function due to deposition of ceroid lipopigment is a significant co-morbidity in Juvenile Neuronal Ceroid Lipofuscinosis (CLN3 disease) although the exact disease mechanisms remain unknown in any NCL form. An increasing frequency of cardiac conduction disorders including severe bradycardia and sinus arrest is seen in the late teens, as is a left ventricular hypertrophy in the early 20s. Only a few case reports of pacemaker implantation have been published, and so far, no long-term follow-up study exists. As new treatment options emerge, more patients will live longer and the need for pacemaker will likely increase, why knowledge of long-term outcome is needed. In the present study, we present the course of six patients from the original Danish CLN3-heart population study (n = 29) published in 2011 in whom pacemaker implantation was indicated from a cardiac point of view. In two cases, the families deselected pacemaker implantation. In four males, aged 19-29 years, all having a good general condition, a dual-chamber pacemaker (St. Jude Medical™ Accent/Assurity MRI™) was implanted in general anesthesia without any complications. At follow-up 9 years later, three were still alive. According to the parents' opinion they still have a good quality of life, now 26, 30, and 36 years old. Pacemaker treatment is safe and may have great impact on quality of life. However, the medical indication for pacemaker treatment is relative and it is important that various aspects, including the patient's general condition and family preferences, are thoroughly discussed before making the final decision.

Converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system

ABSTRACT PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.

A Novel CLN6 Variant Associated With Juvenile Neuronal Ceroid Lipofuscinosis in Patients With Absence of Visual Loss as a Presenting Feature.

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of autosomal recessive lysosomal storage disorders that are characterized by neurodegeneration, progressive cognitive decline, motor impairment, ataxia, loss of vision, seizures, and premature death. To date, pathogenic variants in more than 13 genes have been associated with NCLs. CLN6 encodes an endoplasmic reticulum non-glycosylated transmembrane protein, which is involved in lysosomal acidification. Mutations in CLN6 cause late-infantile juvenile NCL (JNCL) adult-onset NCL, and Kufs disease. Members from two available families with JNCL were clinically evaluated, and samples were collected from consenting individuals. The molecular investigation was performed by whole-exome sequencing, Sanger sequencing, and family segregation analysis. Furthermore, in silico prediction analysis and structural modeling of the identified CLN6 variants were performed. We report clinical and genetic findings of three patients from two Greek-Cypriot families (families 915 and 926) with JNCL. All patients were males, and the first symptoms appeared at the age of 6 years. The proband of family 926 presented with loss of motor abilities, ataxia, spasticity, seizure, and epilepsy. The proband of family 915 had ataxia, spasticity, dysarthria, dystonia, and intellectual disability. Both probands did not show initial signs of vision and/or hearing loss. Molecular analysis of family 926 revealed two CLN6 biallelic variants: the novel, de novo p.Tyr295Cys and the known p.Arg136His variants. In family 915, both patients were homozygous for the p.Arg136His CLN6 variant. Prediction analysis of the two CLN6 variants characterized them as probably damaging and disease-causing. Structural modeling of the variants predicted that they probably cause protein structural differentiation. In conclusion, we describe two unrelated Cypriot families with JNCL. Both families had variants in the CLN6 gene; however, they presented with slightly different symptoms, and notably none of the patients has loss of vision. In silico prediction and structural analyses indicate that both variants are most likely pathogenic.

CLN3, at the crossroads of endocytic trafficking

The CLN3 gene was identified over two decades ago, but the primary function of the CLN3 protein remains unknown. Recessive inheritance of loss of function mutations in CLN3 are responsible for juvenile neuronal ceroid lipofuscinosis (Batten disease, or CLN3 disease), a fatal childhood onset neurodegenerative disease causing vision loss, seizures, progressive dementia, motor function loss and premature death. CLN3 is a multipass transmembrane protein that primarily localizes to endosomes and lysosomes. Defects in endocytosis, autophagy, and lysosomal function are common findings in CLN3-deficiency model systems. However, the molecular mechanisms underlying these defects have not yet been fully elucidated. In this mini-review, we will summarize the current understanding of the CLN3 protein interaction network and discuss how this knowledge is starting to delineate the molecular pathogenesis of CLN3 disease. Accumulating evidence strongly points towards CLN3 playing a role in regulation of the cytoskeleton and cytoskeletal associated proteins to tether cellular membranes, regulation of membrane complexes such as channels/transporters, and modulating the function of small GTPases to effectively mediate vesicular movement and membrane dynamics.