Although a disease-modifying therapy for classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) exists, poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2^R207X mice, which carry one of the most common pathogenic mutations in human patients but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities, including spontaneous seizures, providing a robust, quantifiable, and clinically relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord and differed markedly from the staging seen in mouse models of other forms of neuronal ceroid lipofuscinosis. Neonatal administration of adeno-associated virus serotype 9–mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the life span of Cln2^R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging preclinical efficacy of therapeutic interventions for CLN2 disease.

CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1^–/–) and CLN1^R151X sheep to assess how to potentially scale up for translation. In Cln1^–/– mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1^R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.

The cellular etiology of seizures in CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), remains elusive. Given that Cln2 ^R207X/R207X mice display fatal spontaneous seizures and an early loss of several cortical GABAergic interneuron populations, we hypothesized that these 2 events might be causally related. To study the cell-autonomous effects of interneuron-specific TPP1 deficiency, we first generated transgenic mice expressing loxP -flanked lysosomal membrane–tethered TPP1 (TPP1LAMP1 mice) on the Cln2 ^R207X/R207X genetic background, and then crossed TPP1LAMP1 mice with Vgat-Cre mice. These Vgat-Cre; TPP1LAMP1 mice accumulated storage material in cortical and striatal interneurons. Vgat-Cre; TPP1LAMP1 mice also died more readily after pentylenetetrazole-induced seizures, indicating that interneuron-specific TPP1 deficiency renders these mice more susceptible to seizure-induced mortality. We also selectively activated interneurons using designer receptors exclusively activated by designer drugs (DREADDs) in Vgat-Cre; Cln2 ^R207X/R207X mice. Electroencephalogram monitoring revealed that DREADD-mediated activation of interneurons markedly accelerated the onset of spontaneous seizures and seizure-associated death in Vgat-Cre; Cln2 ^R207X/R207X mice, suggesting that modulating interneuron activity can exacerbate epileptiform abnormalities. Taken together, these results provide mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.

CLN1 disease, caused by mutations in the PPT1 gene, is a fatal neurodegenerative lysosomal storage disorder. While central nervous system (CNS) pathology is well documented, the impact on peripheral tissues remains unclear. Having previously described severe spinal cord pathology, we investigated whether PPT1 deficiency also impacts the neuromuscular junction (NMJ) and skeletal muscle, and whether early systemic gene therapy can prevent these disease manifestations. NMJ morphology, terminal Schwann cell (tSC) coverage, and skeletal muscle structure were examined in symptomatic and end-stage Ppt1^−/− mice. Neonatal mice received systemic AAV9-hCLN1 gene therapy via intravenous injection. Untreated Ppt1^−/− mice exhibited pronounced NMJ pathology, including progressive tSC loss, apparently reduced innervation, and increased abnormal acetylcholine receptor clustering. In parallel, we observed skeletal muscle atrophy, with decreased myofiber diameter and reduced myonuclear content, despite preserved sciatic nerve morphology. Systemic AAV9-hCLN1 therapy partially prevented or ameliorated these phenotypes, preserving NMJ innervation and muscle fiber structure. These findings identify peripheral NMJ and muscle abnormalities as previously unrecognized features of CLN1 disease and provide proof-of-concept that early systemic gene therapy can mitigate these effects. Our results highlight the systemic nature of CLN1 pathology and support the need for treatments that address both CNS and peripheral targets for comprehensive disease modification.