Combination HSCT and intravenous AAV-mediated gene therapy in a canine model proves pivotal for translation of Krabbe disease therapy.

Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (∼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).

Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.

Large animal models contribute to the development of therapies for central and peripheral nervous system dysfunction in patients with lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a group of 70 monogenic disorders characterized by the lysosomal accumulation of a substrate. As a group, LSDs affect ~1 in 5000 live births; however, each individual storage disease is rare, limiting the ability to perform natural history studies or to perform clinical trials. Perhaps in no other biomedical field have naturally occurring large animal (canine, feline, ovine, caprine, and bovine) models been so essential for understanding the fundamentals of disease pathogenesis and for developing safe and effective therapies. These models were critical for the development of hematopoietic stem cell transplantation in α- and ß- mannosidosis, fucosidosis, and the mucopolysaccharidoses; enzyme replacement therapy for fucosidosis, the mucopolysaccharidoses, and neuronal ceroid lipofuscinosis; and small molecule therapy in Niemann-Pick type C disease. However, their most notable contributions to the biomedical field are in the development of gene therapy for LSDs. Adeno-associated viral vectors to treat nervous system disease have been evaluated in the large animal models of α-mannosidosis, globoid cell leukodystrophy, GM1 and GM2 gangliosidosis, the mucopolysaccharidoses, and neuronal ceroid lipofuscinosis. This review article will summarize the large animal models available for study as well as their contributions to the development of central and peripheral nervous system dysfunction in LSDs.

Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy.

Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood-brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.

Cerebrospinal fluid and serum glycosphingolipid biomarkers in canine globoid cell leukodystrophy (Krabbe Disease).

Globoid cell leukodystrophy (GLD, Krabbe disease, Krabbe's disease) is caused by genetic mutations in the gene encoding, galactosylceramidase (GALC). Deficiency of this enzyme results in central and peripheral nervous system pathology, and is characterized by loss of myelin and an infiltration of globoid cells. The canine model of GLD provides a translational model which faithfully recapitulates much of the human disease pathology. Targeted lipidomic analysis was conducted in serum and cerebrospinal fluid (CSF) over the lifetime of GLD affected and normal canines, and in brain tissue at humane endpoint to better understand disease progression and identify potential biomarkers of disease. Psychosine, a substrate of GALC and primary contributor to the pathology in GLD, was observed to be significantly elevated in the serum and CSF by 2 or 4 weeks of age, respectively, and steadily increased over the lifetime of affected animals. Importantly, psychosine concentration strongly correlated with disease severity. Galactosylceramide, glucosylceramide, and lactosylceramide were also found to be elevated in the CSF of affected animals and increased with age. Psychosine and galactosylceramide were found to be significantly increased in brain tissue at humane endpoint. This study identified several biomarkers which may be useful in the development of therapeutics for GLD.

Application of N-palmitoyl-O-phosphocholineserine for diagnosis and assessment of response to treatment in Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder caused by mutations in either the NPC1 or the NPC2 gene. A new class of lipids, N-acyl-O-phosphocholineserines were recently identified as NPC biomarkers. The most abundant species in this class of lipid, N-palmitoyl-O-phosphocholineserine (PPCS), was evaluated for diagnosis of NPC disease and treatment efficacy assessment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) in NPC. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated to measure PPCS in human plasma and cerebrospinal fluid (CSF). A cutoff of 248 ng/mL in plasma provided a sensitivity of 100.0% and specificity of 96.6% in identifying NPC1 patients from control and NPC1 carrier subjects. PPCS was significantly elevated in CSF from NPC1 patients, and CSF PPCS levels were significantly correlated with NPC neurological disease severity scores. Plasma and CSF PPCS did not change significantly in response to intrathetical (IT) HPßCD treatment. In an intravenous (IV) HPßCD trial, plasma PPCS in all patients was significantly reduced. These results demonstrate that plasma PPCS was able to diagnose NPC1 patients with high sensitivity and specificity, and to evaluate the peripheral treatment efficacy of IV HPßCD treatment.

Pharmacokinetics and distribution of 2-hydroxypropyl-ß-cyclodextrin following a single intrathecal dose to cats.

2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is an experimental therapy for Niemann-Pick disease type C (NPC) that reduced neuronal cholesterol and ganglioside storage, reduced Purkinje cell death, and increased lifespan in npc1-/- mice and NPC1 cats. In this study, tissue distribution was investigated in normal cats that received a single 120-mg dose of [14 C]-HP-ß-CD (approximately 200 µCi/cat) via the cerebellomedullary cistern (CBMC) and lumbar cistern. One cat was euthanized at each of various time points up to 24 hours postdose for subsequent processing and quantitative whole-body autoradiographic analysis. HP-ß-CD-derived radioactivity absorbed from the CBMC was widely distributed to cat tissues; most tissues were observed to have reached their highest concentration at 1 hour postdose. HP-ß-CD-derived radioactivity penetrated into the deeper parts of the central nervous system with the highest concentration at 4 hours (403 µg Eq/g or 0.28 mM) and remained high (49.7 µg Eq/g or 0.03 mM) at 24 hours. The relatively long half-life (11-30 hours) in cerebral ventricles and the subarachnoid space surrounding the brain and spinal cord might contribute to the efficacy of HP-ß-CD in NPC1 cats. Other tissues with high concentrations of radioactivity were nasal turbinates, pituitary gland, and urinary bladder, while relatively low concentrations were observed in blood and bile.

N-acyl-O-phosphocholineserines: structures of a novel class of lipids that are biomarkers for Niemann-Pick C1 disease.

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H]+ ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

Intrathecal 2-hydroxypropyl-ß-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1-2 trial.

BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterised by progressive neurodegeneration. In preclinical testing, 2-hydroxypropyl-ß-cyclodextrins (HPßCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological manifestations, and increased lifespan in mouse and cat models of NPC1. The aim of this study was to assess the safety and efficacy of lumbar intrathecal HPßCD. METHODS: In this open-label, dose-escalation phase 1-2a study, we gave monthly intrathecal HPßCD to participants with NPC1 with neurological manifestation at the National Institutes of Health (NIH), Bethesda, MD, USA. To explore the potential effect of 2-week dosing, three additional participants were enrolled in a parallel study at Rush University Medical Center (RUMC), Chicago, IL, USA. Participants from the NIH were non-randomly, sequentially assigned in cohorts of three to receive monthly initial intrathecal HPßCD at doses of 50, 200, 300, or 400 mg per month. A fifth cohort of two participants received initial doses of 900 mg. Participants from RUMC initially received 200 or 400 mg every 2 weeks. The dose was escalated based on tolerance or safety data from higher dose cohorts. Serum and CSF 24(S)-hydroxycholesterol (24[S]-HC), which serves as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NNSS) were used to compare disease progression in HPßCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. FINDINGS: Between Sept 21, 2013, and Jan 19, 2015, 32 participants with NPC1 were assessed for eligibility at the National Institutes of Health. 18 patients were excluded due to inclusion criteria not met (six patients), declined to participate (three patients), pursued independent expanded access and obtained the drug outside of the study (three patients), enrolled in the RUMC cohort (one patient), or too late for the trial enrolment (five patients). 14 patients were enrolled and sequentially assigned to receive intrathecal HPßCD at a starting dose of 50 mg per month (three patients), 200 mg per month (three patients), 300 mg per month (three patients), 400 mg per month (three patients), or 900 mg per month (two patients). During the first year, two patients had treatment interrupted for one dose, based on grade 1 ototoxicity. All 14 patients were assessed at 12 months. Between 12 and 18 months, one participant had treatment interrupted at 17 months due to hepatocellular carcinoma, one patient had dose interruption for 2 doses based on caregiver hardship and one patient had treatment interrupted for 1 dose for mastoiditis. 11 patients were assessed at 18 months. Between Dec 11, 2013, and June 25, 2014, three participants were assessed for eligibility and enrolled at RUMC, and were assigned to receive intrathecal HPßCD at a starting dose of 200 mg every 2 weeks (two patients), or 400 mg every two weeks (one patient). There were no dropouts in this group and all 3 patients were assessed at 18 months. Biomarker studies were consistent with improved neuronal cholesterol homoeostasis and decreased neuronal pathology. Post-drug plasma 24(S)-HC area under the curve (AUC8-72) values, an indicator of neuronal cholesterol homoeostasis, were significantly higher than post-saline plasma 24(S)-HC AUC8-72 after doses of 900 mg (p=0·0063) and 1200 mg (p=0·0037). CSF 24(S)-HC concentrations in three participants given either 600 or 900 mg of HPßCD were increased about two fold (p=0·0032) after drug administration. No drug-related serious adverse events were observed. Mid-frequency to high-frequency hearing loss, an expected adverse event, was documented in all participants. When managed with hearing aids, this did not have an appreciable effect on daily communication. The NNSS for the 14 participants treated monthly increased at a rate of 1·22, SEM 0·34 points per year compared with 2·92, SEM 0·27 points per year (p=0·0002) for the 21 patient comparison group. Decreased progression was observed for NNSS domains of ambulation (p=0·0622), cognition (p=0·0040) and speech (p=0·0423). INTERPRETATION: Patients with NPC1 treated with intrathecal HPßCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPßCD. FUNDING: National Institutes of Health, Dana's Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samantha's Search for the Cure Foundation, National Niemann-Pick Disease Foundation, Support of Accelerated Research for NPC Disease, Vtesse, Janssen Research and Development, a Johnson & Johnson company, and Johnson & Johnson.

Crowdsourcing reproducible seizure forecasting in human and canine epilepsy.

SEE MORMANN AND ANDRZEJAK DOI101093/BRAIN/AWW091 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE : Accurate forecasting of epileptic seizures has the potential to transform clinical epilepsy care. However, progress toward reliable seizure forecasting has been hampered by lack of open access to long duration recordings with an adequate number of seizures for investigators to rigorously compare algorithms and results. A seizure forecasting competition was conducted on kaggle.com using open access chronic ambulatory intracranial electroencephalography from five canines with naturally occurring epilepsy and two humans undergoing prolonged wide bandwidth intracranial electroencephalographic monitoring. Data were provided to participants as 10-min interictal and preictal clips, with approximately half of the 60 GB data bundle labelled (interictal/preictal) for algorithm training and half unlabelled for evaluation. The contestants developed custom algorithms and uploaded their classifications (interictal/preictal) for the unknown testing data, and a randomly selected 40% of data segments were scored and results broadcasted on a public leader board. The contest ran from August to November 2014, and 654 participants submitted 17 856 classifications of the unlabelled test data. The top performing entry scored 0.84 area under the classification curve. Following the contest, additional held-out unlabelled data clips were provided to the top 10 participants and they submitted classifications for the new unseen data. The resulting area under the classification curves were well above chance forecasting, but did show a mean 6.54 ± 2.45% (min, max: 0.30, 20.2) decline in performance. The kaggle.com model using open access data and algorithms generated reproducible research that advanced seizure forecasting. The overall performance from multiple contestants on unseen data was better than a random predictor, and demonstrates the feasibility of seizure forecasting in canine and human epilepsy.media-1vid110.1093/brain/aww045_video_abstractaww045_video_abstract.

Clinical Improvement of Alpha-mannosidosis Cat Following a Single Cisterna Magna Infusion of AAV1.

Lysosomal storage diseases (LSDs) are debilitating neurometabolic disorders for most of which long-term effective therapies have not been developed. Gene therapy is a potential treatment but a critical barrier to treating the brain is the need for global correction. We tested the efficacy of cisterna magna infusion of adeno-associated virus type 1 (AAV1) expressing feline alpha-mannosidase gene in the postsymptomatic alpha-mannosidosis (AMD) cat, a homologue of the human disease. Lysosomal alpha-mannosidase (MANB) activity in the cerebrospinal fluid (CSF) and serum were increased above the control values in untreated AMD cats. Clinical neurological signs were delayed in onset and reduced in severity. The lifespan of the treated cats was significantly extended. Postmortem histopathology showed resolution of lysosomal storage lesions throughout the brain. MANB activity in brain tissue was significantly above the levels of untreated tissues. The results demonstrate that a single cisterna magna injection of AAV1 into the CSF can mediate widespread neuronal transduction of the brain and meaningful clinical improvement. Thus, cisterna magna gene delivery by AAV1 appears to be a viable strategy for treatment of the whole brain in AMD and should be applicable to many of the neurotropic LSDs as well as other neurogenetic disorders.

Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in ß-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.

Canine and Feline Models of Human Genetic Diseases and Their Contributions to Advancing Clinical Therapies
.

For many lethal or debilitating genetic disorders in patients there are no satisfactory therapies. Several barriers exist that hinder the developments of effective therapies including the limited availability of clinically relevant animal models that faithfully recapitulate human genetic disease. In 1974, the Referral Center for Animal Models of Human Genetic Disease (RCAM) was established by Dr. Donald F. Patterson and continued by Dr. Mark E. Haskins at the University of Pennsylvania with the mission to discover, understand, treat, and maintain breeding colonies of naturally occurring hereditary disorders in dogs and cats that are orthologous to those found in human patients. Although non-human primates, sheep, and pig models are also available within the medical community, naturally occurring diseases are rarely identified in non-human primates, and the vast behavioral, clinicopathological, physiological, and anatomical knowledge available regarding dogs and cats far surpasses what is available in ovine and porcine species. The canine and feline models that are maintained at RCAM are presented here with a focus on preclinical therapy data. Clinical studies that have been generated from preclinical work in these models are also presented.

Mechanism of neuromuscular dysfunction in Krabbe disease.

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher-Thy1.1-yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

Cholesterol homeostatic responses provide biomarkers for monitoring treatment for the neurodegenerative disease Niemann-Pick C1 (NPC1).

Niemann-Pick C1 (NPC1) disease is a rare, neurodegenerative lysosomal cholesterol storage disorder, typified by progressive cognitive and motor function impairment. Affected individuals usually succumb to the disease in adolescence. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has emerged as a promising intervention that reduces lipid storage and prolongs survival in NPC1 disease animal models. A barrier to the development of HP-ß-CD and other treatments for NPC disease has been the lack of validated biochemical measures to evaluate efficacy. Here we explored whether cholesterol homeostatic responses resulting from HP-ß-CD-mediated redistribution of sequestered lysosomal cholesterol could provide biomarkers to monitor treatment. Upon direct CNS delivery of HP-ß-CD, we found increases in plasma 24(S)-HC in two independent NPC1 disease animal models, findings that were confirmed in human NPC1 subjects receiving HP-ß-CD. Since circulating 24(S)-HC is almost exclusively CNS-derived, the increase in plasma 24(S)-HC provides a peripheral, non-invasive measure of the CNS effect of HP-ß-CD. Our findings suggest that plasma 24(S)-HC, along with the other cholesterol-derived markers examined in this study, can serve as biomarkers that will accelerate development of therapeutics for NPC1 disease.

Clinical, electrophysiological, and biochemical markers of peripheral and central nervous system disease in canine globoid cell leukodystrophy (Krabbe's disease).

Globoid cell leukodystrophy (GLD), or Krabbe's disease, is a debilitating and always fatal pediatric neurodegenerative disease caused by a mutation in the gene encoding the hydrolytic enzyme galactosylceramidase (GALC). In the absence of GALC, progressive loss of myelin and accumulation of a neurotoxic substrate lead to incapacitating loss of motor and cognitive function and death, typically by 2 years of age. Currently, there is no cure. Recent convincing evidence of the therapeutic potential of combining gene and cell therapies in the murine model of GLD has accelerated the requirement for validated markers of disease to evaluate therapeutic efficacy. Here we demonstrate clinically relevant and quantifiable measures of central (CNS) and peripheral (PNS) nervous system disease progression in the naturally occurring canine model of GLD. As measured by brainstem auditory-evoked response testing, GLD dogs demonstrated a significant increase in I-V interpeak latency and hearing threshold at all time points. Motor nerve conduction velocities (NCVs) in GLD dogs were significantly lower than normal by 12-16 weeks of age, and sensory NCV was significantly lower than normal by 8-12 weeks of age, serving as a sensitive indicator of peripheral nerve dysfunction. Post-mortem histological evaluations confirmed neuroimaging and electrodiagnostic assessments and detailed loss of myelin and accumulation of storage product in the CNS and the PNS. Additionally, cerebrospinal fluid psychosine concentrations were significantly elevated in GLD dogs, demonstrating potential as a biochemical marker of disease. These data demonstrate that CNS and PNS disease progression can be quantified over time in the canine model of GLD with tools identical to those used to assess human patients. © 2016 Wiley Periodicals, Inc.

Cerebrospinal Fluid Calbindin D Concentration as a Biomarker of Cerebellar Disease Progression in Niemann-Pick Type C1 Disease.

Niemann-Pick type C (NPC) 1 disease is a rare, inherited, neurodegenerative disease. Clear evidence of the therapeutic efficacy of 2-hydroxypropyl-ß-cyclodextrin (HPßCD) in animal models resulted in the initiation of a phase I/IIa clinical trial in 2013 and a phase IIb/III trial in 2015. With clinical trials ongoing, validation of a biomarker to track disease progression and serve as a supporting outcome measure of therapeutic efficacy has become compulsory. In this study, we evaluated calcium-binding protein calbindin D-28K (calbindin) concentrations in the cerebrospinal fluid (CSF) as a biomarker of NPC1 disease. In the naturally occurring feline model, CSF calbindin was significantly elevated at 3 weeks of age, prior to the onset of cerebellar dysfunction, and steadily increased to >10-fold over normal at end-stage disease. Biweekly intrathecal administration of HPßCD initiated prior to the onset of neurologic dysfunction completely normalized CSF calbindin in NPC1 cats at all time points analyzed when followed up to 78 weeks of age. Initiation of HPßCD after the onset of clinical signs (16 weeks of age) resulted in a delayed reduction of calbindin levels in the CSF. Evaluation of CSF from patients with NPC1 revealed that calbindin concentrations were significantly elevated compared with CSF samples collected from unaffected patients. Off-label treatment of patients with NPC1 with miglustat, an inhibitor of glycosphingolipid biosynthesis, significantly decreased CSF calbindin compared with pretreatment concentrations. These data suggest that the CSF calbindin concentration is a sensitive biomarker of NPC1 disease that could be instrumental as an outcome measure of therapeutic efficacy in ongoing clinical trials.

Mining continuous intracranial EEG in focal canine epilepsy: Relating interictal bursts to seizure onsets.

OBJECTIVE: Brain regions are localized for resection during epilepsy surgery based on rare seizures observed during a short period of intracranial electroencephalography (iEEG) monitoring. Interictal epileptiform bursts, which are more prevalent than seizures, may provide complementary information to aid in epilepsy evaluation. In this study, we leverage a long-term iEEG dataset from canines with naturally occurring epilepsy to investigate interictal bursts and their electrographic relationship to seizures. METHODS: Four dogs were included in this study, each monitored previously with continuous iEEG for periods of 475.7, 329.9, 45.8, and 451.8 days, respectively, for a total of >11,000 h. Seizures and bursts were detected and validated by two board-certified epileptologists. A published Bayesian model was applied to analyze the dynamics of interictal epileptic bursts on EEG and compare them to seizures. RESULTS: In three dogs, bursts were stereotyped and found to be statistically similar to periods before or near seizure onsets. Seizures from one dog during status epilepticus were markedly different from other seizures in terms of burst similarity. SIGNIFICANCE: Shorter epileptic bursts explored in this work have the potential to yield significant information about the distribution of epileptic events. In our data, bursts are at least an order of magnitude more prevalent than seizures and occur much more regularly. Our finding that bursts often display pronounced similarity to seizure onsets suggests that they contain relevant information about the epileptic networks from which they arise and may aide in the clinical evaluation of epilepsy in patients.

Temporal behavior of seizures and interictal bursts in prolonged intracranial recordings from epileptic canines.

OBJECTIVE: Epilepsy is a chronic disorder, but seizure recordings are usually obtained in the acute setting. The chronic behavior of seizures and the interictal bursts that sometimes initiate them is unknown. We investigate the variability of these electrographic patterns over an extended period of time using chronic intracranial recordings in canine epilepsy. METHODS: Continuous, yearlong intracranial electroencephalography (iEEG) recordings from four dogs with naturally occurring epilepsy were analyzed for seizures and interictal bursts. Following automated detection and clinician verification of interictal bursts and seizures, temporal trends of seizures, burst count, and burst-burst similarities were determined. One dog developed status epilepticus, the recordings of which were also investigated. RESULTS: Multiple seizure types, determined by onset channels, were observed in each dog, with significant temporal variation between types. The first 14 days of invasive recording, analogous to the average duration of clinical invasive recordings in humans, did not capture the entirety of seizure types. Seizures typically occurred in clusters, and isolated seizures were rare. The count and dynamics of interictal bursts form distinct groups and do not stabilize until several weeks after implantation. SIGNIFICANCE: There is significant temporal variability in seizures and interictal bursts after electrode implantation that requires several weeks to reach steady state. These findings, comparable to those reported in humans implanted with the NeuroPace Responsive Neurostimulator System (RNS) device, suggest that transient network changes following electrode implantation may need to be taken into account when interpreting or analyzing iEEG during evaluation for epilepsy surgery. Chronic, ambulatory iEEG may be better suited to accurately map epileptic networks in appropriate individuals.

Insights into the Pathogenesis and Treatment of Krabbe Disease.

Krabbe disease (globoid cell leukodystrophy, GLD) is an inherited disease caused by a deficiency in the lysosomal enzyme galactocerebrosidase (GALC). The major galactosylated lipid degraded by GALC is galactosylceramide. However, GALC is also responsible for the degradation of galactosylsphingosine (psychosine), a highly cytotoxic glycolipid. It has been hypothesized that GALC-deficiency leads to psychosine accumulation that preferentially kills oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Krabbe disease has traditionally been considered a white matter disease characterized by the loss and disorganization of myelin, infiltration of multinucleated monocytes/macrophages (globoid cells) and lymphocytes, and dysregulation of pro-inflammatory cytokines and chemokines. However, new studies have revealed unexpected neuronal deficiencies. Infantile Krabbe disease is believed to be the most common and aggressive form. However, juvenile and adult onset forms have been described. Children affected with infantile Krabbe disease present with motor dysfunction, cognitive decline, intractable seizures, and premature death between two to five years of age. Murine, canine, and primate models of GALC deficiency have been described and have played an important role in our understanding of this invariably fatal disease. Although there is no cure for Krabbe disease, hematopoietic stem cell transplantation can slow the progression of disease. Recent pre-clinical data indicate that simulataneously targeting multiple pathogenic mechanisms greatly increases efficacy in the murine model of Krabbe disease. A better understanding of the underlying pathogenesis will identify new therapeutic targets that may further increase efficacy.