Biology subjects

Santorelli, F. M.

Publications and source records attributed to Santorelli, F. M..

7 recordsLinked to original sources

bioRxiv2026

Pathological variants in HPDL cause collapse of the neuro-glial unit during human cortical maturation

Biallelic variants in HPDL cause a severe neurological disorder in childhood, but the mechanisms linking early developmental abnormalities to later cortical degeneration remain unclear. Here, using long-term iPSC-derived human cortical cultures derived from four patients, we investigated the late consequences of HPDL deficiency through quantitative immunofluorescence and bulk RNA-seq. We found that HPDL-deficient cortical cultures undergo progressive synaptic impairment, with marked loss of PSD95-positive postsynaptic puncta despite largely preserved general neuronal maturation markers. This phenotype is accompanied by a profound reduction in astrocytes and oligodendrocytes, together with increased neuronal apoptosis and transcriptional dysregulation of genes linked to extracellular matrix organization, cellular stress, and neurodegeneration. Unexpectedly, late-stage mutant cultures also show reactivation of early developmental programs, including aberrant expression of NEUROD4 and other proneural regulators, suggesting instability of cell identity during cortical maturation. Together, these findings support a model in which HPDL deficiency first perturbs cortical developmental timing and later drives collapse of the neuro-glial unit, linking premature neurogenesis to synaptic failure, glial loss, and progressive neurodegeneration.

neuroscience↗

bioRxiv2026

Translational lipidomics reveals BMP and its precursor LPG as biomarkers for CLN5 Batten disease

CLN5 Batten disease, caused by biallelic mutations in CLN5, is a rare, early-onset neurodegenerative lysosomal storage disorder that has no cure and lacks validated biomarkers, hindering accurate diagnosis and assessment of therapeutic response. We recently identified CLN5 as the synthase of bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid crucial for lysosome function and lipid catabolism. This suggested BMP and its precursor lysophosphatidylglycerol (LPG) as clinically relevant biomarkers. It also prompted in vivo confirmation of CLN5 as the biologically relevant lysosomal BMP synthase. Here we show that murine and ovine disease models lacking CLN5 show significant and universal depletion of BMP and elevation of LPG across tissues and brain regions, consistent with the biochemical function of CLN5. Additionally, lysosomal lysates from murine models of CLN5 Batten disease lack the ability to synthesize BMP from its precursor LPG, establishing CLN5 as the main BMP synthase in vivo. Of importance, CLN5 patient-derived fibroblasts show BMP depletion and LPG elevation. Translating these results towards clinical utility, we demonstrate BMP and LPG to be accessible biomarkers for CLN5 Batten disease in both plasma and dried blood spots, enabling early diagnosis and patient screening.

cell biology↗

bioRxiv2025

Stereospecific GPG acylation by CLN8 drives BMP biosynthesis and its loss leads to Batten disease

Loss-of-function mutations in the endoplasmic reticulum membrane protein CLN8 cause Batten disease, a neurodegenerative lysosomal storage disorder1. Together with the lysosomal enzyme CLN5, CLN8 mediates the biosynthesis of bis(monoacylglycero)phosphate (BMP), a phospholipid essential for lysosomal function and distinguished by its unique S,S stereochemistry2,3. However, the role of CLN8 in BMP synthesis has remained unclear. Here we establish that CLN8 is a glycerophosphoglycerol (GPG) acyltransferase that catalyses the stereospecific acylation of S,S-GPG to produce S,S-lysophosphatidylglycerol (LPG), the CLN5 substrate in BMP synthesis. Using cryo-electron microscopy, we resolve structures of the CLN8 homodimer in apo and substrate-bound states at 2.7 [A] resolution, revealing the active site architecture and a ping-pong acyl transfer mechanism. Batten disease-causing missense mutations impair CLN8 enzymatic activity in vitro and reduce BMP levels in a Cln8R24G knock- in mouse, whereas the Cln8mnd mouse frameshift mutation causes complete loss of BMP in vivo. Exogenous S,S-LPG, but not the R,S stereoisomer, restored BMP synthesis in CLN8- deficient cells and mice, and improved neurological phenotypes in cln8 mutant zebrafish. Together, these findings define the enzymatic function of CLN8, elucidate the biochemical basis of CLN8 Batten disease, and establish a proof-of-concept for treating it through stereospecific BMP precursor supplementation.

biochemistry↗

bioRxiv2025

HPDL is critical in human cortical development via regulation of mitochondrial functional properties

Human brain development is highly regulated by several spatiotemporal processes, which disruption can result in severe neurological disorders. Emerging evidence highlights the pivotal role of mitochondrial function as one of these fundamental pathways involved in neurodevelopment. Our study investigates the role of 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) protein in cortical neurogenesis and mitochondrial activity, since mutations in the HPDL gene are associated with SPG83, a childhood-onset form of hereditary spastic paraplegia characterized by corticospinal tract degeneration and cortical abnormalities. Starting from mutant neuroblastoma cells, we demonstrated that HPDL is essential to mitochondrial respiratory chain supercomplex assembly and cellular redox balance. Moreover, transcriptomic analyses revealed dysregulated pathways related to neurogenesis, implicating HPDL role in early cortical development. To further elucidate the role of HPDL, we generated cortical neurons and organoids from SPG83 patient-derived induced pluripotent stem cells. Mutant cells exhibited premature neurogenesis at early differentiation stages, likely leading to depletion of cortical progenitors, as evidenced by decreased proliferation, slight increase of apoptosis, and unbalanced cortical type composition at later stages. Furthermore, cortical organoids derived from SPG83 patients showed impaired growth, reminding microcephaly observed in severe cases. In addition, mitochondrial morpho-functional characterization in mutant neurons confirmed disruption of OxPhos chain functionality and increased ROS generation rate. Treatment of cortical cells with two antioxidant compounds, could partially revert premature neurogenesis. In conclusion, our findings reveal a critical role for HPDL in coordinating cortical progenitor proliferation, neurogenesis, and mitochondrial function. These insights shed light on a mechanistical understanding of SPG83 pathology and underscore the therapeutic potential of targeting oxidative stress in this and related neurological disorders.

neuroscience↗

bioRxiv2025

Targeted long-read RNA sequencing reveals the complexity of CLN3 transcription and the consequences of the most common 1-kb deletion in patients with juvenile CLN3 disease

Most genes are not yet fully annotated, and the extent of their transcript diversity and the roles and significance of specific isoforms is not understood. This information is therefore lacking for disease genes. The CLN3 gene underlies classic juvenile CLN3 disease, also known as juvenile neuronal ceroid lipofuscinosis, a rare paediatric neurodegenerative disorder. The most common cause of this biallelic disorder is a 1-kb intragenic deletion that removes two internal coding exons (exons 7 and 8). Here, we report findings from the first long-read RNA sequencing targeting CLN3 in blood samples derived from control individuals and from patients clinically and genetically diagnosed with juvenile CLN3 disease. We find that CLN3 transcription is complex, with >80 different transcripts encoding >35 different open reading frames (ORF) of different lengths, and no dominantly expressed transcript. The 1-kb deletion has direct consequences on this. This is consistent across patients, with total loss of some transcripts including those encoding the canonical 438 amino acid protein and other significant smaller isoforms. The highest expressed disease transcripts include those lacking exons 7 and 8 and encoding a 181 amino acid protein isoform, and other novel isoforms that lack additional exons and encode longer ORFs. The different effects on transcription of other CLN3 disease-causing variants are revealed in single patients. Together, these findings confirm the complexity of transcription at the CLN3 locus, reveal the impact of the 1-kb deletion and other variants on isoform abundance, and highlight the importance of understanding the contribution of these isoforms to CLN3 function in health and disease. Moreover, they impact the future design and development of personalised therapeutics and the design and generation of disease models. Finally, they underline the importance of full annotation for disease genes.

genetics↗

bioRxiv2024

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed upon treatment of Spg11 knockout mice with venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat treatment of Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

neuroscience↗

bioRxiv2021

Sacsin cotranslational degradation causes autosomal recessive spastic ataxia of Charlevoix-Saguenay

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is caused by more than 200 different mutations in the SACS gene encoding sacsin, a huge multimodular protein of unknown function. ARSACS phenotypic spectrum is highly variable. Previous studies correlated the nature and position of SACS mutations with age of onset or disease severity, though the effects on protein stability were not considered. In this study, we explain mechanistically the lack of genotype-phenotype correlation in ARSACS, with important consequences for disease diagnosis and treatment. We found that sacsin is almost absent in ARSACS fibroblasts, regardless of the nature of the mutation. We did not detect sacsin in patients with truncating mutations, while we found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense mutations. We excluded SACS mRNA decay, defective translation, or faster post-translational degradation as causes of protein reduction. Conversely, we demonstrated that nascent mutant sacsin protein undergoes preemptive cotranslational degradation, emerging as a novel cause of a human disease. Based on these findings, sacsin levels should be included in the diagnostic algorithm for ARSACS.

genetics↗