Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

The neuronal ceroid lipofuscinoses are fatal neurodegenerative disorders in which the visual system is affected early in disease progression. A typical accompanying feature is neuroinflammation, the pathogenic impact of which is presently obscure. Here we investigated the role of inflammatory cells in palmitoyl protein thioesterase 1-deficient (Ppt1(-/-)) mice, a model of infantile neuronal ceroid lipofuscinosis (CLN1 disease, infantile), predominantly focusing on the visual system. We detected an early infiltration of CD8+ T-lymphocytes and observed activation of microglia/macrophage-like cells. To analyse the pathogenic impact of lymphocytes, we crossbred Ppt1(-/-) mice with mutants lacking lymphocytes (Rag1(-/-)), and scored axonal transport, axonal perturbation and neuronal survival. This lack of lymphocytes led to a significant amelioration of disease phenotypes, not only in the retino-tectal system, but also in other regions of the central nervous system. Finally, reconstitution experiments revealed a crucial role of CD8+ T-lymphocytes in pathogenesis. Our study provides novel pathomechanistic insights that may be crucial for developing treatment strategies.

Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1 −/− ) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1 −/− mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1 −/− mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders.infantile Batten disease | adeno-associated virus | spinal cord | brain | combination therapy

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Spinal manifestations of CLN1 disease start during the early postnatal period Aim: To understand the progression of CLN1 disease and develop effective therapies we need to characterize early sites of pathology. Therefore, we performed a comprehensive evaluation of the nature and timing of early CLN1 disease pathology in the spinal cord, which appears especially vulnerable, and how this may affect behaviour. Methods: We measured the spinal volume and neuronal number, and quantified glial activation, lymphocyte infiltration and oligodendrocyte maturation, as well as cytokine profile analysis during the early stages of pathology in Ppt1-deficient (Ppt1 À/À) mouse spinal cords. We then performed quantitative gait analysis and open-field behaviour tests to investigate the behavioural correlates during this period. Results: We detected significant microglial activation in Ppt1 À/À spinal cords at 1 month. This was followed by astrocytosis, selective interneuron loss, altered spinal volumes and oligodendrocyte maturation at 2 months, before significant storage material accumulation and lymphocyte infiltration at 3 months. The same time course was apparent for inflammatory cytokine expression that was altered as early as one month. There was a transient early period at 2 months when Ppt1 À/À mice had a significantly altered gait that resembles the presentation in children with CLN1 disease. This occurred before an anticipated decline in overall locomotor performance across all ages. Conclusion: These data reveal disease onset 2 months (25% of lifespan) earlier than expected, while spinal maturation is still ongoing. Our multidisciplinary data provide new insights into the spatio-temporal staging of CLN1 pathogenesis during ongoing postnatal maturation, and highlight the need to deliver therapies during the presymptomatic period.

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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.