👤 Peter M van Hasselt

Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell-derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients' lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms. Show less

The juvenile variant of Neuronal Ceroid Lipofuscinosis (CLN3 disease/Batten disease) is a rare progressive brain disease in children and young adults, characterized by vision loss, decline in cognitive and motor capacities and epilepsy. Children with CLN3 disease often show disturbed behaviour and emotions. The aim of this study is to gain a better understanding of the behaviour and emotions of children with CLN3 disease and to examine the support that the children and their parents are receiving. A combination of qualitative and quantitative analysis was used to analyse patient files and parent interviews. Using a framework analysis approach a codebook was developed, the sources were coded and the data were analysed. The analysis resulted in overviews of (1) typical behaviour and emotions of children as a consequence of CLN3 disease, (2) the support children with CLN3 disease receive, (3) the support parents of these children receive, and (4) the problems these parents face. For a few children their visual, physical or cognitive deterioration was found to lead to specific emotions and behaviour. The quantitative analysis showed that anxiety was reported for all children. The presented overviews on support contain tacit knowledge of health care professionals that has been made explicit by this study. The overviews may provide a lead to adaptable support-modules for children with CLN3 disease and their parents. Show less

Mucopolysaccharidoses (MPS) are devastating inherited diseases treated with hematopoietic cell transplantation (HCT). However, disease progression, especially skeletal, still occurs in all patients. Secondary inflammation has been hypothesized to be a cause. To investigate whether systemic inflammation is present in untreated patients and to evaluate the effect of HCT on systemic inflammation, dried blood spots (n = 66) of patients with MPS (n = 33) treated with HCT between 2003 and 2019 were included. Time points consisted of pre-HCT and, for patients with MPS type I (MPS I), also at 1, 3, and 10 years of follow-up. Ninety-two markers of the OLINK inflammation panel were measured and compared with those of age-matched control subjects (n = 31) by using principal component analysis and Wilcoxon rank sum tests with correction. Median age at transplantation was 1.3 years (range, 0.2-4.8 years), and median time of pre-HCT sample to transplantation was 0.1 year. Normal leukocyte enzyme activity levels were achieved in 93% of patients post-HCT. Pretransplant samples showed clear separation of patients and control subjects. Markers that differentiated pre-HCT between control subjects and patients were mainly pro-inflammatory (50%) or related to bone homeostasis and extracellular matrix degradation (33%). After 10 years' follow-up, only 5 markers (receptor activator of nuclear factor kappa-Β ligand, osteoprotegerin, axis inhibition protein 1 [AXIN1], stem cell factor, and Fms-related tyrosine kinase 3 ligand) remained significantly increased, with a large fold change difference between patients with MPS I and control subjects. In conclusion, systemic inflammation is present in untreated MPS patients and is reduced upon treatment with HCT. Markers related to bone homeostasis remain elevated up to 10 years after HCT and possibly reflect the ongoing skeletal disease, making them potential biomarkers for the evaluation of new therapies. Show less

Quantifying lymphocyte vacuolization in peripheral blood smears (PBSs) serves as a measure for disease severity in CLN3 disease-a lysosomal storage disorder of childhood-onset. However, thus far quantification methods are based on labor-intensive manual assessment of PBSs. As machine learning techniques like convolutional neural networks (CNNs) have been deployed quite successfully in detecting pathological features in PBSs, we explored whether these techniques could be utilized to automate quantification of lymphocyte vacuolization. Here, we present and validate a deep learning pipeline that automates quantification of lymphocyte vacuolization. By using two CNNs in succession, trained for cytoplasm-segmentation and vacuolization-detection, respectively, we obtained an excellent correlation with manual quantification of lymphocyte vacuolization ( Show less

To help differentiate CLN3 (Batten) disease, a devastating childhood metabolic disorder, from the similarly presenting early-onset Stargardt disease (STGD1). Early clinical identification of children with CLN3 disease is essential for adequate referral, counselling and rehabilitation. Medical chart review of 38 children who were referred to a specialized ophthalmological centre because of rapid vision loss. The patients were subsequently diagnosed with either CLN3 disease (18 patients) or early-onset STGD1 (20 patients). Both children who were later diagnosed with CLN3 disease, as children who were later diagnosed with early-onset STGD1, initially presented with visual acuity (VA) loss due to macular dystrophy at 5-10 years of age. VA in CLN3 disease decreased significantly faster than in STGD1 (p = 0.01). Colour vision was often already severely affected in CLN3 disease while unaffected or only mildly affected in STGD1. Optic disc pallor on fundoscopy and an abnormal nerve fibre layer on optical coherence tomography were common in CLN3 disease compared to generally unaffected in STGD1. In CLN3 disease, dark-adapted (DA) full-field electroretinogram (ERG) responses were either absent or electronegative. In early-onset STGD1, DA ERG responses were generally unaffected. None of the STGD1 patients had an electronegative ERG. Already upon presentation at the ophthalmologist, the retina in CLN3 disease is more extensively and more severely affected compared to the retina in early-onset STGD1. This results in more rapid VA loss, severe colour vision abnormalities and abnormal DA ERG responses as the main differentiating early clinical features of CLN3 disease. Show less

The CLN3 disease spectrum ranges from a childhood-onset neurodegenerative disorder to a retina-only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well-known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity. Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal-associated membrane protein 1 (LAMP-1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis. Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes ( Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies. Show less

CLN3 disease is a disorder of lysosomal homeostasis predominantly affecting the retina and the brain. The severity of the underlying mutations in We present three patients with an identical Contrasting our initial expectations, patients with a start codon variant in Show less

The juvenile form of neuronal ceroid Lipofuscinosis (JNCL) is the most common form within this group of rare lysosomal storage disorders, causing pediatric neurodegeneration. The genetic disorder, which is caused by recessive mutations affecting the CLN3 gene, features progressive vision loss, cognitive and motor decline and other psychiatric conditions, seizure episodes, leading to premature death. Animal models have traditionally aid the understanding of the disease mechanisms and pathology and are very relevant for biomarker research and therapeutic testing. Nevertheless, there is a need for establishing reliable and predictive human cellular models to study the disease. Since patient material, particularly from children, is scarce and difficult to obtain, we generated an engineered a CLN3-mutant isogenic human induced pluripotent stem cell (hiPSC) line carrying the c.1054C → T pathologic variant, using state of the art CRISPR/Cas9 technology. To prove the suitability of the isogenic pair to model JNCL, we screened for disease-specific phenotypes in non-neuronal two-dimensional cell culture models as well as in cerebral brain organoids. Our data demonstrates that the sole introduction of the pathogenic variant gives rise to classical hallmarks of JNCL in vitro. Additionally, we discovered an alteration of the splicing caused by this particular mutation. Next, we derived cerebral organoids and used them as a neurodevelopmental model to study the particular effects of the CLN3 Show less

To delineate timing of motor decline in CLN3 disease. Motor function, assessed by the 6-Minute Walk Test (6MWT), was evaluated repeatedly in 15 patients with CLN3 disease, resulting in 65 test results and during one occasion in 2 control cohorts. One control cohort (n = 14) had isolated visual impairment; a second cohort (n = 12) exhibited visual impairment in combination with neurologic impairments. Based on 6MWT reference values in healthy sighted children, In CLN3 disease, 6MWT scores were already impaired from first testing near diagnosis (mean The 6MWT unveils early onset of motor decline in CLN3 disease. Show less

CLN3 disease is a major cause of childhood neurodegeneration. Onset of visual failure around 6 years of age is thought to precede cognitive deterioration by a few years, but casuistic reports question this paradigm. The aim of our study is to delineate timing of cognitive decline in CLN3 disease. Early neurocognitive functioning in CLN3 disease was analyzed using age at onset of visual and cognitive decline and IQ scores from literature-derived patient descriptions, supplemented with IQ scores and school history from a retrospective referral center cohort. We analyzed protracted and classical CLN3 separately and added a control group of patients diagnosed with juvenile onset macular degeneration (early onset Stargardt disease) to control for possible effects of rapid vision loss on neurocognitive functioning. Onset of cognitive decline at a mean age of 6.8 years (range 2-13 years, n = 19) paralleled onset of visual deterioration at a mean age of 6.4 years (range 4-9 years, n = 81) as supported by an early decline in IQ scores in classical CLN3 disease. Onset and course of vision loss was similar in patients with protracted CLN3. The decreased IQ levels at diagnosis (mean 68.4, range 57-79, n = 9) in the referral cohort were consistently associated with an aberrant early school history contrasting normal school history and cognition in Stargardt disease patients. Cognitive dysfunction is universally present around diagnosis in classical CLN3 disease. Show less