The neuronal ceroid lipofuscinosis family of lysosomal storage diseases, also called CLN1 disease, is characterized by the deficiency of palmitoyl-protein thioesterase 1 (PPT1). In this study, we investigated the impact of PPT1 deficiency on hippocampal GABAergic interneurons (INs) and associated neural network oscillations in a PPT1-KI (CLN1 c.451 C > T (p.R151X)) mouse model. Using a combination of in vivo electrophysiology, immunostaining, and fiber photometry, we observed that PPT1 deficiency led to the activation of caspase 3 in parvalbumin-positive (PV+) INs, an increased activity of pyramidal neurons and theta/gamma oscillation power, and the disruption of theta-gamma cross-frequency coupling (CFC) in the early stage of the CLN1 disease model. In the late stage of the CLN1 disease model, we observed the reduced neuronal activity, extensive neuronal loss including PV+ INs, and the emergence of spontaneous epileptiform discharges and the pathological ripples. Treatment with diazepam partially restored oscillatory coupling and reduced seizure-like activities. Our research indicated that PPT1 deficiency leads to early selective impairment of PV+ INs, triggering overactivation of pyramidal neurons and network dysfunction, which consequently results in seizures and neurodegeneration. This research provides novel insights into the pathogenesis of CLN1 disease and potential therapeutic strategies for the intervention of CLN1 disease by improving the function of inhibitory INs via caspase inhibition.

CLN1 disease is a fatal neurodegenerative condition caused by deficiency in palmitoyl-protein thioesterase 1 (PPT1), for which no disease-modifying therapy exists. The disease affects the entire central nervous system (CNS), necessitating widespread delivery of therapeutics to the brain and spinal cord. Adeno-associated virus (AAV)-based PPT1 gene therapy delivered intrathecally has been tested in mouse models but has shown limited efficacy due to inadequate brain bioavailability. Here, to maximize therapeutic benefit, PPT1 was engineered for improved cross-correction capabilities, packaged in Spark100, a neurotropic AAV capsid, and administered through intracerebroventricular route in neonatal Ppt1-/- mice. This achieved sustained expression of PPT1 protein across the CNS, including key disease-relevant structures, for up to 15 months. It resulted in long-term therapeutic benefits, such as extended lifespan, preserved neurobehavioral function, and prevention of neuropathology, making treated Ppt1-/- mice nearly indistinguishable from wild type. A translatability study in healthy adult sheep, assessing biodistribution of therapeutic in a large and fully developed brain, showed widespread CNS transduction and PPT1 expression with no adverse effects. These studies demonstrate the potential of this approach for treating CLN1 disease and suggest that a similar platform, using a secreted therapeutic protein, might apply to other neurological disorders with broad CNS deficits.

Neurodegeneration is a devastating manifestation in most lysosomal storage disorders (LSDs). Loss-of-function mutations in CLN1, encoding palmitoyl-protein thioesterase-1 (PPT1), cause CLN1 disease, a devastating neurodegenerative LSD that has no curative treatment. Numerous proteins in the brain require dynamic S-palmitoylation (palmitoylation-depalmitoylation) for trafficking to their destination. Although PPT1 depalmitoylates S-palmitoylated proteins and its deficiency causes CLN1 disease, the underlying pathogenic mechanism has remained elusive. We report that Niemann-Pick C1 (NPC1), a polytopic membrane protein mediating lysosomal cholesterol egress, requires dynamic S-palmitoylation for trafficking to the lysosome. In Cln1-/- mice, Ppt1 deficiency misroutes NPC1-dysregulating lysosomal cholesterol homeostasis. Along with this defect, increased oxysterol-binding protein (OSBP) promotes cholesterol-mediated activation of mechanistic target of rapamycin C1 (mTORC1), which inhibits autophagy contributing to neurodegeneration. Pharmacological inhibition of OSBP suppresses mTORC1 activation, rescues autophagy, and ameliorates neuropathology in Cln1-/- mice. Our findings reveal a previously unrecognized role of CLN1/PPT1 in lysosomal cholesterol homeostasis and suggest that suppression of mTORC1 activation may be beneficial for CLN1 disease.

Children with neurodegenerative disease often have debilitating gastrointestinal symptoms. We hypothesized that this may be due at least in part to underappreciated degeneration of neurons in the enteric nervous system (ENS), the master regulator of bowel function. To test this hypothesis, we evaluated mouse models of neuronal ceroid lipofuscinosis type 1 and 2 (CLN1 and CLN2 disease, respectively), neurodegenerative lysosomal storage disorders caused by deficiencies in palmitoyl protein thioesterase-1 and tripeptidyl peptidase-1, respectively. Both mouse lines displayed slow bowel transit in vivo that worsened with age. Although the ENS appeared to develop normally in these mice, there was a progressive and profound loss of myenteric plexus neurons accompanied by changes in enteric glia in adult mice. Similar pathology was evident in colon autopsy material from a child with CLN1 disease. Neonatal administration of adeno-associated virus-mediated gene therapy prevented bowel transit defects, ameliorated loss of enteric neurons, and extended survival in mice. Treatment after weaning was less effective than treating neonatally but still extended the lifespan of CLN1 disease mice. These data provide proof-of-principle evidence of ENS degeneration in two lysosomal storage diseases and suggest that gene therapy can ameliorate ENS disease, also improving survival.

CLN1 disease, one of the most severe forms of neuronal ceroid lipofuscinosis (NCLs or Batten disease), is a rapidly progressing pediatric neurodegenerative disorder caused by mutations in the PPT1 gene. The disease is characterized by lysosomal storage accumulation, an early onset neuroimmune response, motor impairment, and premature death. N-acetyl-L-leucine (NALL), an orally bioavailable modified amino acid, has demonstrated clinical efficacy in Niemann–Pick type C and other lysosomal storage disorders. Here, we assessed the efficacy of chronic NALL treatment in the Ppt1−/− mouse model of CLN1 disease. Mice received NALL (0.1 g/kg/day) in chow either from weaning (1 month, presymptomatic) or from 4 months (symptomatic) until 7 months (normal disease endstage), with additional survival cohorts. NALL treatment did not extend survival in Ppt1−/− mice and produced no significant improvement in gait coordination or rotarod performance, with only minimal improvements in select gait variability parameters in presymptomatically treated mice. Histological analyses revealed no reduction in microglial or astrocyte activation, nor in storage material accumulation, key CLN1 disease-associated phenotypes. These findings indicate that NALL monotherapy has limited therapeutic efficacy in CLN1 disease mice and suggest that its mechanisms of action may not address the underlying pathophysiology of this disorder. The online version contains supplementary material available at 10.1038/s41598-025-32984-x.