Gene therapy ameliorates spontaneous seizures associated with cortical neuron loss in a Cln2R207X mouse model

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 Cln2R207X 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 Cln2R207X 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.


Tissue processing and Nissl staining
Mice were anesthetized using 2% isoflurane anesthesia and transcardially perfused with PBS. The brain and spinal column were dissected out. The brain was bisected in the sagittal plane and spinal column divided into cervical, thoracic and lumbosacral segments. One brain hemisphere and the thoracic segment of cord were frozen and stored at -80°C for biochemical and molecular analysis, while the other brain hemisphere and cervical and lumbo-sacral segments of cord were fixed in 4% paraformaldehyde (PFA) solution for 48 hours and subsequently cryoprotected in 30% sucrose in 50 mM TBS (pH=7.6). Cryoprotected brain and spinal cord tissues were sectioned along the coronal axis at 40µm using a Microm HM430 freezing microtome (Microm International, Germany) equipped with a Physitemp BFS-40MPA freezing stage (Physitemp, Clifton, NJ) prior to histological analysis. 40 µm brain and spinal cord sections were stained for cresyl fast violet (Nissl) to reveal their cytoarchitecture. For mouse tissue, a 1 in 6 series of brain and 1 in 48 series of spinal cord sections were mounted on chrome-gelatin coated microscope slides (ThermoFisher Scientific) and air-dried overnight. All sections were then incubated for at least 45 minutes at 60°C in 0.1% cresyl fast violet and 0.05% acetic acid. Stained sections were then differentiated through a series of graded ethanol solutions (70%, 80%, 90%, 95% and 2x100%) (Fisher Scientific, MA) before clearing in Xylene (Fisher Scientific) and coverslipping with DPX (Fisher Scientific), a xylene-based mountant.

Unbiased stereological counts of neuron number
Nissl-stained neurons with a clearly identifiable nucleus were counted using the optical fractionator method with the following sampling scheme using 100× oil objective (NA 1.4).

Immunohistochemistry
A modified immunofluorescence protocol: a one-in-six series of coronal forebrain sections and a one-in-forty-eight series of coronal spinal cord sections were mounted on 25 x 75 mm microscope slides (Fisher Scientific) and air-dried for 30 minutes before blocking in Tris-buffered saline (TBS) with 4% Triton X-100 (Alfa Aesar) and 15% normal goat serum (Vector) for 1 hour in room temperature. Sections were then incubated for the following primary antibodies in TBS with 4% Triton X-100 and 10% normal goat serum for 2 hours: and 15% normal swine or rabbit serum for 2 hours, followed by 2-hour incubation in Vectastain ABC (avidin-biotin, 1:200, Vector). Immunoreactivity was visualized using 0.05% DAB (Sigma) and 0.005% H2O2, followed by airdrying for 2 hours and coverslipping with DPX mounting medium (Electron Microscopy Sciences).

Primers for RT-qPCR
The sequences of primers (5' to 3') used for RT-qPCR are as follows. GFAP

Quantitative gait analysis
Gait analysis was performed using the CatWalk XT (Noldus Information Technology bv, Wagenigen, Netherlands) semi-automated gait analysis system, as described previously (1,2).
Briefly, mice were trained at least 2 days prior to data analysis and habituated to the room where behavior was performed overnight before testing. Each mouse needed to have run durations ranging from 1 to 5 seconds to be considered compliant.

Neurotransmitter analysis
The concentration of cortical neurotransmitters including glutamate (Glu), glutamine (Gln), and GABA was measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Mouse cortex samples (n=4, Cln2 R207X and WT mice at 3 months of age) were homogenized in water (10 mL/g tissue). All amino acids listed above were extracted from 20μL of homogenate with 200μL of methanol after addition of 20μL of internal standard solution containing Glu-d3 (500μg/mL), Gln-13 C5 (300μg/mL), and GABA-d6 (300μg/mL). A seven-point standard curve was prepared in duplicate. The analysis of amino acids was performed on a Shimadzu 20AD HPLC system and a SIL-20AC autosampler coupled to 4000Qtrap mass spectrometer (AB Sciex) operated in positive multiple reaction monitoring (MRM) mode. Data processing was conducted with Analyst 1.6.3 (Applied Biosystems).
Calibrators that deviate by more than 15% of nominal concentrations were excluded from construction of calibration curve, except that deviation of 20% was acceptable for the lower limit of quantification (LLOQ), which is the low end of calibration curve.