Hemanth R. Nelvagal

MTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

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.

Towards a new understanding of NCL pathogenesis

The Neuronal Ceroid Lipofuscinoses (NCLs, Batten disease) are a group of inherited neurodegenerative disorders that have been traditionally grouped together on the basis of certain shared clinical and pathological features. However, as the number of genes that appear to cause new forms of NCL continues to grow, it is timely to reassess our understanding of the pathogenesis of these disorders and what groups them together. The various NCL subtypes do indeed share features of a build-up of autofluorescent storage material, progressive neuron loss and activation of the innate immune system. The characterisation of animal models has highlighted the selective nature of neuron loss and its intimate relationship with glial activation, rather than the generalised build-up of storage material. More recent data provide evidence for the pathway-dependent nature of pathology, the contribution of glial dysfunction, and the involvement of new brain regions previously thought to be unaffected, and it is becoming apparent that pathology extends beyond the brain. These data have important implications, not just for therapy, but also for our understanding of these disorders. However, looking beneath these broadly similar pathological themes evidence emerges for marked differences in the nature and extent of these events in different forms of NCL. Indeed, given the widely different nature of the mutated gene products it is perhaps more surprising that these disorders resemble each other as much as they do. Such data raise the question whether we should rethink the collective grouping of these gene deficiencies together, or whether it would be better to consider them as separate entities. This article is part of a Special Issue entitled: Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease).

Immune cells perturb axons and impair neuronal survival in a mouse model of infantile neuronal ceroid lipofuscinosis

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.

Partial Correction of the CNS Lysosomal Storage Defect in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis by Neonatal CNS Administration of an Adeno-Associated Virus Serotype rh. 10 Vector Expressing the Human CLN3 Gene

Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and other diseases in this category.

Intrathecal enzyme replacement therapy improves motor function and survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are a group of related hereditary lysosomal storage disorders characterized by progressive loss of neurons in the central nervous system resulting in dementia, loss of motor skills, seizures and blindness. A characteristic intralysosomal accumulation of autofluorescent storage material occurs in the brain and other tissues. Three major forms and nearly a dozen minor forms of NCL are recognized. Infantile-onset NCL (CLN1 disease) is caused by severe deficiency in a soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1) and no therapy beyond supportive care is available. Homozygous Ppt1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death around 8 months. Direct delivery of lysosomal enzymes to the cerebrospinal fluid is an approach that has gained traction in small and large animal models of several other neuropathic lysosomal storage diseases, and has advanced to clinical trials. In the current study, Ppt1 knockout mice were treated with purified recombinant human PPT1 enzyme delivered to the lumbar intrathecal space on each of three consecutive days at 6 weeks of age. Untreated PPT1 knockout mice and wild-type mice served as additional controls. Four enzyme concentration levels (0, 2.6, 5.3 and 10.6 mg/ml of specific activity 20 U/mg) were administered in a volume of 80 μl infused over 8 min. Each group consisted of 16-20 mice. The treatment was well tolerated. Disease-specific survival was 233, 267, 272, and 284days for each of the four treatment groups, respectively, and the effect of treatment was highly significant (p<0.0001). The timing of motor deterioration was also delayed. Neuropathology was improved as evidenced by decreased autofluorescent storage material in the spinal cord and a decrease in CD68 staining in the cortex and spinal cord. The improvements in motor function and survival are similar to results reported for preclinical studies involving other lysosomal storage disorders, such as CLN2/TPP1 deficiency, for which intraventricular ERT is being offered in clinical trials. If ERT delivery to the CSF proves to be efficacious in these disorders, PPT1 deficiency may also be amenable to this approach.

Translating preclinical models of neuronal ceroid lipofuscinosis: progress and prospects

ABSTRACT Introduction: The Neuronal Ceroid Lipofuscinoses (NCLs, or Batten Disease) are a group of distinct inherited neurodegenerative Lysosomal Storage Disorders (LSDs) that mainly affect children and young adults. Despite recent advances, these are fatal and profoundly disabling disorders for which there remains a pressing need to devise curative therapies. Areas covered: This article details the advances from new preclinical models of the NCLs, together with progress in developing experimental therapeutic strategies and in translating these advances into clinical trials. Expert opinion: Animal models have been crucial for improving the understanding of the pathological mechanisms in each form, and for testing experimental therapies. With the relative success of some pre-clinical experimental approaches, several clinical trials have been initiated. These have had varying degrees of success, but the recent FDA approval of recombinant enzyme replacement for CLN2 disease, an approach initially tested in mouse and dog models, is a positive step towards the goal of effective therapies for the NCLs. As new data emerges from preclinical models about NCL pathogenesis, it is increasingly likely that a combination of therapies that either target different regions of the body, or multiple disease mechanisms, will be required to effectively treat the NCLs.

Quantifying storage material accumulation in tissue sections

The ability to reliably quantify the relative degree of storage burden that results from lysosomal dysfunction is an important goal. Such measurements not only allow an assessment of different stages of disease progression, but also the assessment of therapeutic strategies. Although biochemical methods exist for doing this, retaining the anatomical integrity of tissue samples is an important consideration. This chapter provides practical methodological recommendations for achieving this goal in tissue sections, either by directly visualizing or staining the storage material, and subsequently quantifying it via image analysis.

Progress toward Fulfilling the Potential of Immunomodulation in Childhood Neurodegeneration

In this issue of Molecular Therapy, Groh and colleagues1 have provided further evidence that immunomodulatory approaches may be of value in treating two types of lysosomal storage disorder, CLN1 and CLN3 disease.1 The study builds upon their previous work in mouse models, which revealed an adaptive immune response mediated by sialoadhesin,2,3 showing low-level infiltration of the brain by predominantly CD8+ lymphocytes, and that genetically blocking this infiltration partly slowed disease progression and extended life-span moderately.

27. Therapeutic Efficacy of Intracranial and Intrathecal AAV2/9-PPT1 in Infantile Batten Disease

Background The neuronal ceroid lipofuscinoses (NCLs) are a group of the most common pediatric neurodegenerative lysosomal storage disorders. Infantile NCL (INCL), caused by a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), is characterized clinically by progressive cognitive and motor decline, profound neurodegeneration and neuroinflammation, and accumulation of autofluorescent storage material (AFSM). Infantile NCL murine model recapitulates the human disease. AAV2/5-PPT1 intracranial (IC) delivery delayed the onset of INCL histopathological markers in the forebrain and cerebellum and improved preclinical outcome measures. However, overall disease progression was only partially corrected suggesting peripheral nervous system involvement. In collaboration with Dr. Jon Cooper (Kings College, London), we discovered substantial progressive pathology in the spinal cord: neuronal loss and axon density, significant microgliosis and astrocytosis, and AFSM (Nelvagal H etal, manuscript in prep). These data suggest that the spinal cord could be an important therapeutic target. We hypothesize that IC and intrathecal (IT) gene therapy in combination will significantly improve the lifespan, preclinical outcome measures, and histopathological markers as compared to either therapy alone. Methods We generated five groups (n=10): PPT1-/-, wild type, and PPT1-/- injected with IC, IT, or the combination IC/IT AAV2/9-PPT1. For IC injections, 3-2µl bilateral intracranial injections were performed. For IT injections, one 15µl bolus injection into the lumbar subarachnoid space was performed. The AAV2/9-PPT1 virus was diluted to 1×1012 viral particles/ml. To date, we have collected 3, 5, and 7-month time points for all groups, and have generated a 9-month time point. Samples will be analyzed for PPT1 enzyme activity, AFSM, neuroinflammation and neurohistopathology, spinal cord pathology, and a histochemical stain for PPT1. Lifespan, behavior, and brain weight (gross measure of atrophy) will be analyzed. Significance was determined using a 2-way ANOVA test. Results PPT1-/- mice have a median lifespan of 35.8 weeks and rapid decline in rotarod performance beginning at 5 months. There is a progressive decline in PPT1-/- brain weight beginning at 3 months. IT AAV2/9-PPT1 mice have a median lifespan of 48.4 weeks and have a steady decline in rotarod performance beginning at 7 months. There is a progressive decline in the IT mice brain weight compared to wild-type, reaching significance at 7 months (p<. 001); however, it had significantly less atrophy than PPT1-/- brains until 7 months (p<0.05). IC AAV2/9-PPT1 mice have a median lifespan of 58.5 weeks and a rapid decline in rotarod performance beginning at 9 months. IC AAV2/9-PPT1 mice brain weight are not significantly different than wild-type. To date, at 66 weeks, all IC/IT AAV2/9-PPT1 mice are alive. There is a significant decline in IC/IT mice rotarod performance at 15 months. The IC/IT mice brain weight is not significantly different than wild-type. Data for the enzyme activity, neuroinflammatory markers, histopathology, and histochemical stain will be complete by April 2016. Conclusions To date, these data confirm that targeting the entire CNS will provide a significant step for INCL therapy. The combination therapy significantly increases the lifespan beyond that of an additive benefit. As expected, modifying the gene therapy vector from IC-AAV2/5 to IC-AAV2/9 significantly improved preclinical outcome measures. Lastly, the IT AAV2/9-PPT1 injections suggest that spinal cord disease plays an important role in INCL pathogenesis. These findings could form the basis for an effective therapeutic strategy that incorporates targeting multiple facets of INCL disease.