👤 Maureen J MacDonald

Alzheimer disease (AD) biomarker and genetic testing results are increasingly disclosed to cognitively unimpaired adults in research and could in the future inform clinical treatment decisions in this population. To assess psychological outcomes after returning 3 categories of amyloid biomarker results as well as apolipoprotein E (APOE) genotypes. This cohort study was a secondary analysis of data collected as part of screening for the multisite AHEAD preclinical AD trial. Participants were individuals aged 55 to 80 years undergoing screening from July 14, 2020, to October 15, 2024. Participants were informed whether they had not-detected, intermediate, or elevated amyloid positron emission tomography levels, as well as their APOE genotype, which were categorized as noncarrier, ε4 heterozygote, or ε4 homozygote. Impact of Events Scale (IES; 15 items to assess intrusive thoughts and avoidance; each item is scored as not at all [0], rarely [1], sometimes [3], or often [5]; total range, 0-75), collected 24 to 72 hours after disclosure, and change in a scale measuring concerns about AD dementia (adapted scale using 6 items in which participants indicated their level of agreement with statements related to their perceived probability of developing AD dementia; items scored as strongly disagree [1] through strongly agree [5]; total range, 6-30), calculated by subtracting the score collected before biomarker testing from 1 collected after biomarker and genetic test results disclosure. Among 3414 included individuals, the mean (SD) age was 68.8 (6.0) years and 2116 (62%) were female. Group mean IES scores were below clinically significant thresholds. Nevertheless, across genetic groups, learning an elevated amyloid result (1184 participants) was associated with higher IES (mean [SD], 10.5 [10.9]) than intermediate amyloid (482 participants; mean [SD] IES, 8.8 [9.8]), and intermediate amyloid was associated with higher scores than not-detected amyloid (1748 participants; mean [SD] IES, 6.5 [8.4]). Across amyloid groups, learning APOE ε4 homozygosity (337 participants) was associated with higher mean (SD) IES (12.7 [11.6]) than heterozygosity (1609 participants; 9.1 [10.2]), and heterozygosity was associated with higher IES than noncarrier status (1468 participants; mean [SD] IES, 6.2 [8.1]). Both types of information were significant in an analysis of covariance model; no interaction effect was observed. In contrast, only biomarker disclosure was associated with differential change in concerns about AD dementia. Those with elevated amyloid showed a mean (SD) increase in concern (0.8 [3.5]), those with intermediate amyloid showed a smaller increase (0.4 [3.7]), and those with not-detected amyloid showed decreased concerns (-1.1 [4.2]). In this cohort study of cognitively unimpaired adults, associations with intrusive thoughts were observed to differ among genetic and biomarker subgroups; such associations were limited to biomarker subgroups for measures of perceived dementia risk. Show less

Exercise and heat stress have been reported to independently provide benefits to brain health. We tested the hypothesis that 8 weeks of post-exercise local heating, passive local heating only, or exercise training only improves cognitive performance compared to a control group. Sixty young, healthy participants (n = 30 female, age: 23 [3] years) were randomised into one of four groups: control (CON), aerobic exercise (EX), local heating (HEAT), or combined heat and exercise (HEATEX). Participants completed supervised sessions three times per week for 8 weeks. Exercise sessions were completed at 70-75% of maximum heart rate on a cycle ergometer, and local heating sessions involved hot water immersion (42°C) of the feet (both 45 min duration). The HEATEX group performed both the EX and HEAT components sequentially in the same session (90 min total duration). Cognitive performance was measured at baseline and at the end of the 8-week intervention using the digit symbol substitution task (DSST) and the Stroop test. There was a main effect of time (P < 0.001) where DSST performance improved; however, there was no group effect (P = 0.089) or time by group interaction (P = 0.119). There was no effect of the interventions on Stroop cost (baseline: 90 [SD: 70] ms; post-intervention: 84 [SD: 70] ms; time by condition interaction P = 0.205). Similarly, there were no effects of the interventions on circulating plasma concentrations of brain-derived neurotrophic factor (interaction P = 0.189). Eight weeks of exercise training and/or local heating is not sufficient to improve cognitive performance in young, moderately fit individuals. Show less

Pre-pregnancy obesity (ppOB) is linked to pregnancy complications and abnormal fetal growth through placental mechanisms, and long non-coding RNAs (lncRNAs) may play an epigenetic role in these processes. We investigated overall and sex-specific associations of pre-pregnancy body mass index (ppBMI), ppOB, and birthweight with placental lncRNA transcripts in two birth cohorts. Study participants were mother-child dyads recruited to the CANDLE (Memphis, TN)( Show less

ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation. Show less

Infants who are not breast-fed benefit from formula with both docosahexaenoic acid (C22:6n3) and arachidonic acid (ARA; C20:4n6). The amount of ARA needed to support immune function is unknown. Infants who carry specific fatty acid desaturase (FADS) polymorphisms may require more dietary ARA to maintain adequate ARA status. The aim of the study was to determine whether ARA intake or FADS polymorphisms alter ARA levels of lymphocytes, plasma, and red blood cells in term infants fed infant formula. Infants (N = 89) were enrolled in this prospective, double-blind controlled study. Infants were randomized to consume formula containing 17 mg docosahexaenoic acid and 0, 25, or 34 mg ARA/100 kcal for 10 weeks. Fatty acid composition of plasma phosphatidylcholine and phosphatidylethanolamine, total fatty acids of lymphocytes and red blood cells, activation markers of lymphocytes, and polymorphisms in FADS1 and FADS2 were determined. Lymphocyte ARA was higher in the 25-ARA formula group than in the 0- or 34-ARA groups. In plasma, 16:0/20:4 and 18:0/20:4 species of phosphatidylcholine and phosphatidylethanolamine were highest and 16:0/18:2 and 18:0/18:2 were lowest in the 34-ARA formula group. In minor allele carriers of FADS1 and FADS2, plasma ARA content was elevated only at the highest level of ARA consumed. B-cell activation marker CD54 was elevated in infants who consumed formula containing no ARA. ARA level in plasma is reduced by low ARA consumption and by minor alleles in FADS. Dietary ARA may exert an immunoregulatory role on B-cell activation by decreasing 16:0/18:2 and 18:0/18:2 species of phospholipids. ARA intake from 25 to 34 mg/100 kcal is sufficient to maintain cell ARA level in infants across genotypes. Show less

The glucagon-like peptide-1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor transduce nutrient-stimulated signals to control beta cell function. Although the GLP-1 receptor (GLP-1R) is a validated drug target for diabetes, the importance of the GIP receptor (GIPR) for the function of beta cells remains uncertain. We demonstrate that mice with selective ablation of GIPR in beta cells (MIP-Cre:Gipr(Flox/Flox); Gipr(-/-βCell)) exhibit lower levels of meal-stimulated insulin secretion, decreased expansion of adipose tissue mass and preservation of insulin sensitivity when compared to MIP-Cre controls. Beta cells from Gipr(-/-βCell) mice display greater sensitivity to apoptosis and markedly lower islet expression of T cell-specific transcription factor-1 (TCF1, encoded by Tcf7), a protein not previously characterized in beta cells. GIP, but not GLP-1, promotes beta cell Tcf7 expression via a cyclic adenosine monophosphate (cAMP)-independent and extracellular signal-regulated kinase (ERK)-dependent pathway. Tcf7 (in mice) or TCF7 (in humans) levels are lower in islets taken from diabetic mice and in humans with type 2 diabetes; knockdown of TCF7 in human and mouse islets impairs the cytoprotective responsiveness to GIP and enhances the magnitude of apoptotic injury, whereas restoring TCF1 levels in beta cells from Gipr(-/-βCell) mice lowers the number of apoptotic cells compared to that seen in MIP-Cre controls. Tcf7(-/-) mice show impaired insulin secretion, deterioration of glucose tolerance with either aging and/or high-fat feeding and increased sensitivity to beta cell injury relative to wild-type (WT) controls. Hence the GIPR-TCF1 axis represents a potential therapeutic target for preserving both the function and survival of vulnerable, diabetic beta cells. Show less

Susceptibility to inherited cryptorchidism in the LE/orl rat may be associated with genetic loci that influence developmental patterning of the gubernaculum by the fetal testis. Cryptorchidism in the LE/orl rat is associated with a unique combination of homozygous minor alleles at multiple loci, and the encoded proteins are co-localized with androgen receptor (AR) and Leydig cells in fetal gubernaculum and testis, respectively. Prior studies have shown aberrant perinatal gubernacular migration, muscle patterning defects and reduced fetal testicular testosterone in the LE/orl strain. In addition, altered expression of androgen-responsive, cytoskeletal and muscle-related transcripts in the LE/orl fetal gubernaculum suggest a role for defective AR signaling in cryptorchidism susceptibility. The long-term LE/orl colony and short-term colonies of outbred Crl:LE and Crl:SD, and inbred WKY/Ncrl rats were maintained for studies. Animals were intercrossed (LE/orl X WKY/Ncrl), and obligate heterozygotes were reciprocally backcrossed to LE/orl rats to generate 54 F2 males used for genotyping and/or linkage analysis. At least five fetuses per gestational time point from two or more litters were used for quantitative real-time RT-PCR (qRT-PCR) and freshly harvested embryonic (E) day 17 gubernaculum was used to generate conditionally immortalized cell lines. We completed genotyping and gene expression analyses using genome-wide microsatellite markers and single nucleotide polymorphism (SNP) arrays, PCR amplification, direct sequencing, restriction enzyme digest with fragment analysis, whole genome sequencing (WGS), and qRT-PCR. Linkage analysis was performed in Haploview with multiple testing correction, and qRT-PCR data were analyzed using ANOVA after log transformation. Imaging was performed using custom and commercial antibodies directed at candidate proteins in gubernaculum and testis tissues, and gubernaculum cell lines. LE/orl rats showed reduced fertility and fecundity, and higher risk of perinatal death as compared with Crl:LE rats, but there were no differences in breeding outcomes between normal and unilaterally cryptorchid males. Linkage analysis identified multiple peaks, and with selective breeding of outbred Crl:LE and Crl:SD strains for alleles within two of the most significant (P < 0.003) peaks on chromosomes 6 and 16, we were able to generate a non-LE/orl cryptorchid rat. Associated loci contain potentially functional minor alleles (0.25-0.36 in tested rat strains) including an exonic deletion in Syne2, a large intronic insertion in Ncoa4 (an AR coactivator) and potentially deleterious variants in Solh/Capn15, Ankrd28, and Hsd17b2. Existing WGS data indicate that homozygosity for these combined alleles does not occur in any other sequenced rat strain. We observed a modifying effect of the Syne2(del) allele on expression of other candidate genes, particularly Ncoa4, and for muscle and hormone-responsive transcripts. The selected candidate genes/proteins are highly expressed, androgen-responsive and/or co-localized with developing muscle and AR in fetal gubernaculum, and co-localized with Leydig cells in fetal testis. The present study identified multiple cryptorchidism-associated linkage peaks in the LE/orl rat, containing potentially causal alleles. These are strong candidate susceptibility loci, but further studies are needed to demonstrate functional relevance to the phenotype. Association data from both human and rat models of spontaneous, nonsyndromic cryptorchidism support a polygenic etiology of the disease. Both the present study and a human genome-wide association study suggest that common variants with weak effects contribute to susceptibility, and may exist in genes encoding proteins that participate in AR signaling in the developing gubernaculum. These findings have potential implications for the gene-environment interaction in the etiology of cryptorchidism. Sequences were deposited in the Rat Genome Database (RGD, http://rgd.mcw.edu/). This work was supported by: R01HD060769 from the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), 2P20GM103446 and P20GM103464 from the National Institute of General Medical Sciences (NIGMS), and Nemours Biomedical Research. The authors have no competing interests to declare. Show less

Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides -546 and -399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors. Show less

There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries. Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets. Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n=10; carbamoyl phosphate synthetase (CPS1) deficiency, n=29; ornithine transcarbamoylase (OTC) deficiency, n=214; citrullinaemia, n=108; argininosuccinic aciduria (ASA), n=80; arginase deficiency, n=23 was reported. The majority of patients (70%; n=327) were aged 0-16y and 30% (n=137) >16y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n=174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n=84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n=97] were prescribed oral energy supplements. Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required. Show less

Neuronal ceroid lipofuscinosis (NCL) is a genetically heterogeneous group of lysosomal diseases that collectively compose the most common Mendelian form of childhood-onset neurodegeneration. It is estimated that ∼8% of individuals diagnosed with NCL by conservative clinical and histopathologic criteria have been ruled out for mutations in the nine known NCL-associated genes, suggesting that additional genes remain unidentified. To further understand the genetic underpinnings of the NCLs, we performed whole-exome sequencing on DNA samples from a Mexican family affected by a molecularly undefined form of NCL characterized by infantile-onset progressive myoclonic epilepsy (PME), vision loss, cognitive and motor regression, premature death, and prominent NCL-type storage material. Using a recessive model to filter the identified variants, we found a single homozygous variant, c.550C>T in KCTD7, that causes a p.Arg184Cys missense change in potassium channel tetramerization domain-containing protein 7 (KCTD7) in the affected individuals. The mutation was predicted to be deleterious and was absent in over 6,000 controls. The identified variant altered the localization pattern of KCTD7 and abrogated interaction with cullin-3, a ubiquitin-ligase component and known KCTD7 interactor. Intriguingly, murine cerebellar cells derived from a juvenile NCL model (CLN3) showed enrichment of endogenous KCTD7. Whereas KCTD7 mutations have previously been linked to PME without lysosomal storage, this study clearly demonstrates that KCTD7 mutations also cause a rare, infantile-onset NCL subtype designated as CLN14. Show less

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells. CbCln6(nclf/nclf) cells and CbCln3(Δex7/8/Δex7/8) cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival. Show less

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors. Show less

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Deltaex7/8) knock-in mice and CbCln3(Deltaex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL) is the result of mutations in the Cln3 gene. The Cln3 knock-in mouse (Cln3Deltaex7/8) reproduces the most common Cln3 mutation and we have now characterized the CNS of these mice at 12 months of age. With the exception of the thalamus, Cln3Deltaex7/8 homozygotes displayed no significant regional atrophy, but a range of changes in individual laminar thickness that resulted in variable cortical thinning across subfields. Stereological analysis revealed a pronounced loss of neurons within individual laminae of somatosensory cortex of affected mice and the novel finding of a loss of sensory relay thalamic neurons. These affected mice also exhibited profound astrocytic reactions that were most pronounced in the neocortex and thalamus, but diminished in other brain regions. These data provide the first direct evidence for neurodegenerative and reactive changes in the thalamocortical system in JNCL and emphasize the localized nature of these events. Show less

Hypertrophic cardiomyopathy (HCM) is one of the most common causes of sudden cardiac death in young adults and is a familial disease in at least 60% of cases. Causative mutations have been identified in several sarcomeric genes, including the myosin binding protein C (MYBPC3) gene. Although numerous causative mutations have been identified, the pathogenetic process is still poorly understood. A large animal model of familial HCM in the cat has been identified and may be used for additional study. As the first spontaneous large animal model of this familial disease, feline familial HCM provides a valuable model for investigators to evaluate pathophysiologic processes and therapeutic (pharmacologic or genetic) manipulations. The MYBPC3 gene was chosen as a candidate gene in this model after identifying a reduction in the protein in myocardium from affected cats in comparison to control cats (P<0.001). DNA sequencing was performed and sequence alterations were evaluated for evidence that they changed the amino acid produced, that the amino acid was conserved and that the protein structure was altered. We identified a single base pair change (G to C) in the feline MYBPC3 gene in affected cats that computationally alters the protein conformation of this gene and results in sarcomeric disorganization. We have identified a causative mutation in the feline MYBPC3 gene that results in the development of familial HCM. This is the first report of a spontaneous mutation causing HCM in a non-human species. It should provide a valuable model for evaluating pathophysiologic processes and therapeutic manipulations. Show less

JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. Show less

Juvenile-onset neuronal ceroid lipofuscinosis (JNCL; Batten disease) features hallmark membrane deposits and loss of central nervous system (CNS) neurons. Most cases of the disease are due to recessive inheritance of an approximately 1 kb deletion in the CLN3 gene, encoding battenin. To investigate the common JNCL mutation, we have introduced an identical genomic DNA deletion into the murine CLN3 homologue (Cln3) to create Cln3( Deltaex7/8) knock-in mice. The Cln3( Deltaex7/8) allele produced alternatively spliced mRNAs, including a variant predicting non-truncated protein, as well as mutant battenin that was detected in the cytoplasm of cells in the periphery and CNS. Moreover, Cln3( Deltaex7/8) homozygotes exhibited accrual of JNCL-like membrane deposits from before birth, in proportion to battenin levels, which were high in liver and select neuronal populations. However, liver enzymes and CNS development were normal. Instead, Cln3( Deltaex7/8) mice displayed recessively inherited degenerative changes in retina, cerebral cortex and cerebellum, as well as neurological deficits and premature death. Thus, the harmful impact of the common JNCL mutation on the CNS was not well correlated with membrane deposition per se, suggesting instead a specific battenin activity that is essential for the survival of CNS neurons. Show less