👤 Ann-Christin Groh

Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3 Show less

In epithelia, apicobasal cell polarization is closely linked to cell-cell contact formation, both controlled by the conserved Crumbs (CRB) complex, which includes the transmembrane protein Crumbs (CRB3a) and adapter proteins PALS1, PATJ, and LIN7c. In MDCK II cells, a model for cell polarization, depletion of PALS1 - which binds to all CRB components - leads to defective cell polarization and improper distribution of tight junction proteins, resulting in severe epithelial barrier defects in 3D cyst models. This study investigated whether this phenotype is associated with transcriptional changes by analyzing wildtype (WT) and PALS1 knockout (KO) MDCK II cell lines grown under non-confluent conditions and in 3D cyst cultures. Our results indicate that the transition from non-confluent cells to 3D cysts involves numerous differentially expressed genes (DEGs) in both WT and KO cells. Importantly, the analyses revealed significant overlaps between WT and KO cells in their maturation processes, suggesting that most identified DEGs are linked to differentiation from non-confluent to polarized MDCK cells and likely not a result of PALS1 deficiency. Gene Ontology (GO) enrichment and over-representation analyses using REACTOME and KEGG databases confirmed these similarities. In contrast, the direct comparison of WT and KO cells at the two stages showed fewer DEGs and overlaps in associated biological processes and signaling pathways. DEGs associated with the 3D stage, in which the phenotype manifests, contain DEGs and pathways that were predominantly linked to cell cycle linked processes, centromere assembly, or DNA replication. Furthermore, the transcription of genes encoding key junction proteins, additional polarity proteins, and cell-substrate interaction proteins is less affected by the loss of PALS1, indicating that PALS1 influences the transcriptional profiles in epithelial cells as a modulating factor. Show less

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier. Show less

One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans. To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name. BBDF compiled all of the available CLN3 gene and protein data from biological databases, repositories of federally and privately funded projects, patent and trademark offices, science and technology journals, industrial drug and pipeline reports as well as clinical trial reports and with painstaking precision, validated the information together with experts in Batten disease, lysosomal storage disease, lysosome/endosome biology. The finished product is an indexed review of the CLN3 gene and protein which is not limited in page size or number of references, references all available primary experiments, and does not draw conclusions for the reader. Revisiting the experimental history of a target gene and its product ensures that inaccuracies and contradictions come to light, long-held beliefs and assumptions continue to be challenged, and information that was previously deemed inconsequential gets a second look. Compiling the information into one manuscript with all appropriate primary references provides quick clues to which studies have been completed under which conditions and what information has been reported. This compendium does not seek to replace original articles or subtopic reviews but provides an historical roadmap to completed works. Show less

CLN diseases are rare lysosomal storage diseases characterized by progressive axonal degeneration and neuron loss in the CNS, manifesting in disability, blindness, and premature death. We have previously demonstrated that, in animal models of infantile and juvenile forms of CLN disease (CLN1 and CLN3, respectively), secondary neuroinflammation in the CNS substantially amplifies neural damage, opening the possibility that immunomodulatory treatment might improve disease outcome. First, we recapitulated the inflammatory phenotype, originally seen in mice in autopsies of CLN patients. We then treated mouse models of CLN1 and CLN3 disease with the clinically approved immunomodulatory compounds fingolimod (0.5 mg/kg/day) and teriflunomide (10 mg/kg/day) by consistent supply in the drinking water for 5 months. The treatment was well tolerated and reduced T cell numbers and microgliosis in the CNS of both models. Moreover, axonal damage, neuron loss, retinal thinning, and brain atrophy were substantially attenuated in both models, along with reduced frequency of myoclonic jerks in Ppt1 Show less

CLN diseases are mostly fatal lysosomal storage diseases that lead to neurodegeneration in the CNS. We have previously shown that CD8+ T-lymphocytes contribute to axonal perturbation and neuron loss in the CNS of Ppt1(-/-) mice, a model of CLN1 disease. We now investigated the role of the inflammation-related cell adhesion molecule sialoadhesin (Sn) in Ppt1(-/-) and Cln3(-/-) mice, a model of the most frequent form, CLN3 disease. Microglia/macrophages in the CNS of both models showed an upregulation of Sn and markers for proinflammatory M1 polarization and antigen presentation. Sn+ microglia/macrophages associated with SMI32+ axonal spheroids and CD8+ T-lymphocytes. To analyze their pathogenic impact, we crossbred both models with Sn-deficient mice and scored axonal degeneration and neuronal integrity using immunohistochemistry, electron microscopy and optical coherence tomography. Degenerative alterations in the retinotectal pathway of Ppt1(-/-)Sn(-/-) and Cln3(-/-)Sn(-/-) mice were significantly reduced. Ppt1(-/-)Sn(-/-) mice also showed a substantially improved clinical phenotype and extended lifespan, attenuated numbers of M1-polarized microglia/macrophages and reduced expression levels of proinflammatory cytokines. This was accompanied by an increased frequency of CD8+CD122+ T-lymphocytes in the CNS of Ppt1(-/-)Sn(-/-) mice, the regulatory phenotype of which was demonstrated by impaired survival of CD8+CD122- effector T-lymphocytes in co-culture experiments. We show for the first time that increased Sn expression on microglia/macrophages contributes to neural perturbation in two distinct models of CLN disease. Our data also indicate that a rarely described CD8+CD122+ T-cell population can regulate the corresponding diseases. These studies provide insights into CLN pathogenesis and may guide in designing immuno-regulatory treatment strategies. Show less

The neuronal ceroid lipofuscinoses constitute a group of fatal inherited lysosomal storage diseases that manifest in profound neurodegeneration in the CNS. Visual impairment usually is an early symptom and selective degeneration of retinal neurons has been described in patients suffering from distinct disease subtypes. We have previously demonstrated that palmitoyl protein thioesterase 1 deficient (Ppt1-/-) mice, a model of the infantile disease subtype, exhibit progressive axonal degeneration in the optic nerve and loss of retinal ganglion cells, faithfully reflecting disease severity in the CNS. Here we performed spectral domain optical coherence tomography (OCT) in Ppt1-/- and ceroid lipofuscinosis neuronal 3 deficient (Cln3-/-) mice, which are models of infantile and juvenile neuronal ceroid lipofuscinosis, respectively, in order to establish a non-invasive method to assess retinal alterations and monitor disease severity in vivo. Blue laser autofluorescence imaging revealed increased accumulation of autofluorescent storage material in the inner retinae of 7-month-old Ppt1-/- and of 16-month-old Cln3-/- mice in comparison with age-matched control littermates. Additionally, optical coherence tomography demonstrated reduced thickness of retinae in knockout mice in comparison with age-matched control littermates. High resolution scans and manual measurements allowed for separation of different retinal composite layers and revealed a thinning of layers in the inner retinae of both mouse models at distinct ages. OCT measurements correlated well with subsequent histological analysis of the same retinae. These results demonstrate the feasibility of OCT to assess neurodegenerative disease severity in mouse models of neuronal ceroid lipofuscinosis and might have important implications for diagnostic evaluation of disease progression and therapeutic efficacy in patients. Moreover, the non-invasive method allows for longitudinal studies in experimental models, reducing the number of animals used for research. Show less